Energy and protein

metabolism and nutrition in

sustainable animal production

EAAP publication No. 134

edited by:
James W. Oltjen
Ermias Kebreab
Hélène Lapierre
Energy and protein metabolism and nutrition in
sustainable animal production
EAAP – European Federation of Animal Science

The University of California

Department of Animal Science

The European Association for Animal Production wishes to express its appreciation to the
Ministero per le Politiche Agricole e Forestali and the Associazione Italiana Allevatori for their
valuable support of its activities
Energy and protein
metabolism and nutrition in
sustainable animal production

4th International Symposium on Energy and Protein Metabolism and Nutrition

Sacramento, California, USA
9-12 September 2013

EAAP publication No. 134

edited by:
James W. Oltjen
Ermias Kebreab
Hélène Lapierre

;EKIRMRKIR%GEHIQMG
4 Y F P M W L I V W
 Buy a print copy of this book at
www.WageningenAcademic.com/eaap134

This work is subject to copyright. All rights

are reserved, whether the whole or part
of the material is concerned. Nothing
from this publication may be translated,
reproduced, stored in a computerised
system or published in any form or in any
manner, including electronic, mechanical,
reprographic or photographic, without
prior written permission from the
publisher:
Wageningen Academic Publishers
P.O. Box 220
6700 AE Wageningen
The Netherlands
www.WageningenAcademic.com
[email protected]

The individual contributions in this

ISBN: 978-90-8686-227-6 publication and any liabilities arising from
e-ISBN: 978-90-8686-781-3 them remain the responsibility of the
DOI: 10.3920/978-90-8686-781-3 authors.

The designations employed and the

ISSN 0071-2477 presentation of material in this publication
do not imply the expression of any opinion
whatsoever on the part of the European
Photo cover: Jim Oltjen and Association for Animal Production
Romeo Capell concerning the legal status of any country,
territory, city or area or of its authorities,
or concerning the delimitation of its
First published, 2013 frontiers or boundaries.

The publisher is not responsible for possible

©Wageningen Academic Publishers damages, which could be a result of content
The Netherlands, 2013 derived from this publication.
The 4th EAAP International Symposium on Energy and Protein Metabolism and Nutrition was
organized in Sacramento, California (USA) on 9-12 September 2013. A special symposium as tribute
to the late Professor R. Lee Baldwin was also organized on 12 September 2013.

The symposium was chaired by:

‡ James Oltjen
7KHVFLHQWL¿FSURJUDPPHRIWKHV\PSRVLXPZDVFRFKDLUHGE\
‡ Ermias Kebreab
‡ Hélène Lapierre

,QFRQVXOWDWLRQZLWKWKH,QWHUQDWLRQDO6FLHQWL¿F&RPPLWWHH
‡ Mario Luis Chizzoti (Brazil)
‡ Matteo Crovetto (Italy)
‡ Kristen Johnson (Alternate, USA)
‡ Michael Kreuzer (Secretary, Switzerland)
‡ Hélène Lapierre (Canada)
‡ Jan Erik Lindberg (President, Sweden)
‡ Toyomizu Masaaki (Japan)
‡ Cornelia Metges (Outgoing, Germany)
‡ James Oltjen (USA)
‡ Isabelle Ortigues-Marty (France)

DQGOLDLVLQJZLWKWKH1DWLRQDO6FLHQWL¿F&RPPLWWHH
‡ Ransom Baldwin
‡ Karen Beauchemin
‡ Brian Bequette
‡ John Cant
‡ Theresa Davis
‡ Kees de Lange
‡ Lorraine Doepel
‡ Jeff Firkins
‡ Harvey Freetly
‡ Mark Hanigan (Chair, Baldwin Symposium)
‡ Kris Johnson
‡ Kyle McLeod
‡ John McNamara
‡ Phil Miller
‡ Gordon Murdoch
‡ Daniel Ouellet (Editorial)
‡ Luis Tedeschi

Energy and protein metabolism and nutrition in sustainable animal production 5

Table of contents
Preface 23
James W. Oltjen

Keynotes

Feeding the planet: key challenges 27

M. Herrero

Role of animal products in feeding the planet 35

J.E. Hermansen, M.T. Knudsen and T. Kristensen

Part 1. Energy and protein interactions, ruminants

Challenges in ruminant nutrition: towards minimal nitrogen losses in cattle 47

J. Dijkstra, C.K. Reynolds, E. Kebreab, A. Bannink, J.L. Ellis, J. France and
A.M. van Vuuren

Small intestinal fermentation contributes substantially to starch disappearance in milk-fed

calves 59
M.S. Gilbert, A.J. Pantophlet, J.J.G.C. van den Borne, H.A. Schols and W.J.J. Gerrits

1XWULHQWGLJHVWLRQE\GDLU\FRZVIHGGLHWVUHSODFLQJVWDUFKZLWKQRQIRUDJH¿EHU 
S.D. Ranathunga, M.M. Abdelqader and K.F. Kalscheur

(IIHFWRIGLIIHUHQWGLHWDU\OHYHOVRI4XHEUDFKRWDQQLQH[WUDFWRQQLWURJHQDQG¿EHU
GLJHVWLELOLW\DQGSRVWUXPLQDOPLFURELDOSURWHLQÀRZLQKHLIHUV 
S. Ahnert, A. Susenbeth and U. Dickhoefer

(IIHFWRIIHVFXHWR[LFRVLVRQQLWURJHQDQGHQHUJ\EDODQFHLQ+ROVWHLQVWHHUV 
A.F. Koontz, D.H. Kim, A.P. Foote, L.P. Bush, J.L. Klotz, K.R. McLeod and D.L. Harmon

3HUIRUPDQFHHI¿FLHQF\DQGHVWLPDWHGPDLQWHQDQFHHQHUJ\UHTXLUHPHQWVRIBos taurus
and Bos indicusFDWWOH 
R.D. Sainz, G.D. Cruz, E. Mendes, C.U. Magnabosco, Y.B. Farjalla, F.R.C. Araujo,
R.C. Gomes and P.R. Leme

Energy for maintaining liveweight: an indicator of adaptive abilities of beef cows? 71

A. De La Torre, F. Blanc, E. Recoules, D. Egal and J. Agabriel

Response to high altitude grazing in metabolic traits and performance by yak crossbreds
and yaks 73
S. Barsila, N.R. Devkota, M. Kreuzer and S. Marquardt

Estimates of nutritional requirement of sheep, goats and cattle in tropical and warm
countries: a meta-analysis study 75
N. Salah, D. Sauvant and H. Archimède

Energy and protein metabolism and nutrition in sustainable animal production 7

Net energy and protein requirements for growth of goats kids 77
I.A.M.A. Teixeira, N. St-Pierre, A. Cannas, A.P. Souza, M.H.R. Fernandes and
K.T. Resende

Low protein solid feed enhances nitrogen utilization by urea-N recycling in veal calves 79
H. Berends, J.J.G.C. van den Borne, B.A. Røjen, J. van Baal and W.J.J. Gerrits

Effect of replacing feed grains by food by-product on energy metabolism of lactating cows 81
K. Higuchi, F. Ohtani, Y. Kobayashi, I. Nonaka, K. Yayou, M. Sutoh and O. Enishi

Source of carbohydrate and protein in the diet of recently weaned dairy calves 83
T.M. Hill, J.D. Quigley H.G. Bateman, II, J.M. Aldrich and R.L. Schlotterbeck

6XEVWLWXWLQJEDUOH\E\VRUJKXPHQKDQFHVHI¿FLHQF\RIVWDUFKDQGSURWHLQXWLOL]DWLRQLQODPEV 
M. Yahaghi, J.B. Liang, J. Balcells, R. Valizadeh and M.F. Jahromi

,QÀXHQFHRIGLIIHUHQWJUDVVODQGYHJHWDWLRQW\SHVRQUXPLQDOSURWR]RDDQGDPPRQLDLQ
beef cattle 87
I.D.M. Gangnat, J.O. Zeitz, D. Warner, M. Kreuzer and F. Leiber

Nutritional evaluation of bulls receiving supplements with different protein: carbohydrate

ratios 89
E.E.L. Valente, M.F. Paulino, E. Detmann, S.C. Valadares Filho and M.L. Chizzotti

Energy value of Tifton-85 (Cynodon spp.) for Gir and F1 Holstein × Gir dairy heifers
using the respirometric technique 91
A.L.C.C. Borges, H.F. Lage, R.R. Silva, J.R.M. Ruas, A.U. Carvalho, E.O.S. Saliba and
N.M. Rodriguez

Substitution of corn by mesquite pod meal in pellet diets for lambs: nitrogen compounds
metabolism 93
M.L.A. Pereira, T.C.J. Pereira, H.G.O. Silva, J.F. Cruz, P.J.P. Almeida, A.B. Santos,
E.J. Santos and C.A.M. Peixoto

Excretion of purine derivatives and nitrogen compounds in lactating goats fed other
protein sources 95
M.L.A. Pereira, A.B. Santos, A.J. Del Rei, J.F. Cruz, P.J.P. Almeida, T.C.J. Pereira,
E.J. Santos and C.A.M. Peixoto

Nutritional evaluation and performance of beef cattle fed with crude glycerin diets 97
J.P.I.S. Monnerat, I.M. Oliveira, P.V.R. Paulino, R. Mezzomo, L.H.P. Silva, P.P. Silva and
J.P. Ferreira

The effect of substituting urea for a commercial slow release urea as supplement to sheep
fed a poor quality Eragrostis curvula hay 99
A.M. Mentz, A. Hassen, W.A. Van Niekerk, H. Mynhardt and R. Coertze

(IIHFWRIDSSOLFDWLRQRI¿EURO\WLFHQ]\PHSURGXFWVDWGLIIHUHQWOHYHOVRQin vitro ruminal

fermentation of low quality feeds 101
B. Shenkute, A. Hassen and N.E. Odongo

8 Energy and protein metabolism and nutrition in sustainable animal production

Effect of metabolizable energy intake on energy partitioning into muscle and fat in
Pelibuey ewes 105
A.J. Chay-Canul, J.C. Ku-Vera, A.J. Ayala-Burgos, M.L. Chizzotti, J.G. Magaña-Monforte
and L.O. Tedeschi

Mammary gland development in heifers under different metabolizable protein and

metabolizable energy ratios 107
R.L. Albino, M.I. Marcondes, B.C. Gomes, L.G.R. Pereira, T.E. da Silva and A.S. Trece

(IIHFWRIVWDUFKVRXUFHDQG¿EHUOHYHOLQPL[HGGLHWVRQODFWDWLQJ0XUFLDQR*UDQDGLQD
goat: Substrate oxidation and milk performance 109
C. Fernández, M.C. López and M. Lachica

(IIHFWRIWKHVWDUFKVRXUFHDQG¿EHUOHYHOLQPL[HGGLHWVRQWKHHQHUJ\EDODQFHRIODFWDWLQJ
Murciano-Granadina goats 111
M.C. López, C. Fernández and M. Lachica

The development of the gravid uterus of Santa Inês ewes and ewe lambs under two
nutritional planes 113
L.F.L. Cavalcanti, I. Borges, F.A. Souza, G.L. Macedo Júnior and L.O. Tedeschi

The effect of a limited supply of phenylalanine, threonine, and tryptophan on milk yield
and composition 115
I.H. Iroshan, H. Lapierre and L. Doepel

0HWDEROLFDQGKRUPRQDOSUR¿OHRIEXOOVGXULQJHYDOXDWLRQRIQXWULWLRQDOUHTXLUHPHQWVE\
respirometric technique under different plane of nutrition 117
A.L.C.C. Borges, P.A.D. Vivenza, R.R. Silva, E.J. Facury Filho, H.F. Lage,
P.H.A. Carvalho, P.R.O. Paes and N.M. Rodriguez

Methane emission and digestibility of goats subjected to feed restriction 119

A.R.C. Lima, K.T. Resende, I.A.M.A. Teixeira, T.F.V. Bompadre, R.T.S. Frighetto and
M.H.M.R. Fernandes

(I¿FLHQF\RIXWLOL]DWLRQRIGLHWDU\QLWURJHQIRUPLONSURGXFWLRQE\GXDOSXUSRVHFRZVIHG
increasing levels of Leucaena leucocephala forage mixed with Pennisetum purpureum grass 121
A. Ruiz-González, A.J. Ayala-Burgos, C.F. Aguilar-Pérez and J.C. Ku-Vera

0LON\LHOGDQGFRPSRVLWLRQDQGHI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROLVDEOHHQHUJ\IRU
lactation by Pelibuey ewes 123
J.C. Espinoza-Hernández, A.J. Ayala-Burgos, C.F. Aguilar-Pérez, J.G. Magaña-Monforte
and J.C. Ku-Vera

3URWHLQWXUQRYHUDQGLQIUDUHGWKHUPRJUDSK\LQ1HOORUHEXOOVFODVVL¿HGIRUUHVLGXDOIHHGLQWDNH 
M.L. Chizzotti, D. Heiderich, W.L.B. Aziani, M.M. Ladeira, E.E.L. Valente,
T. Yanagi Junior, F.H.M. Chizzotti, L. Schiassi and D. Lourençoni

Differences in residual feed intake are largely explained by changes in body composition 127
M.L. Nascimento, A.R.D.L. Souza, A.S. Chaves, S.R. de Medeiros, R.R. Tullio,
M.M. de Alencar, A.N. Rosa and D.P.D. Lanna

Energy and protein metabolism and nutrition in sustainable animal production 9

Net protein requirement of pregnancy of goats with single and twin pregnancy 129
C.J. Härter, D.S. Castagnino, L.D. Lima, A.R. Rivera, H.G.O. Silva, K.T. Resende and
I.A.M.A. Teixeira

Effect of gender on net energy and protein requirements for growth of goats 131
A.K. de Almeida, S.P. Silva, D.S. Costa, M.H.M.R. Fernandes, I.A.M.A. Teixeira and
K.T. Resende

Energy utilization for gain of goat kids 135

T.F.V. Bompadre, K.T. Resende, M.H.M.R. Fernandes, O. Boaventura Neto,
A.N. Mendonca, B. Biagioli and I.A.M.A. Teixeira

Protein requirements for growth of male and female Saanen goats kids 137
F.O.M. Figueiredo, R.F. Leite, M.M. Freire, M.H.M.R. Fernandes, K.T. Resende and
I.A.M.A. Teixeira

,QÀXHQFHRIGLIIHUHQWOLSLGVXSSOHPHQWVDQGIUHTXHQFLHVVXSSO\RQPLFURELDOSURWHLQ
synthesis in beef heifers 139
M.C.A. Santana, T.T. Berchielli, G. Fiorentini, R.A. Reis, V.C. Modesto and G.T. Pereira

Part 2. Energy and protein interactions, monogastrics

Exploring the biology of energy and protein utilization in non-ruminant animals to

LPSURYHQXWULHQWXWLOL]DWLRQHI¿FLHQFLHV 
C.F.M. de Lange, C.L. Levesque and H.R. Martínez-Ramírez

Effects of low birth weight and 3 wk feed restriction on energy metabolism in growing pigs 157
R. Krüger, S. Görs, K. Martens, M. Derno, B.U. Metzler-Zebeli, C. Nebendahl,
H.M. Hammon and C.C. Metges

,QÀXHQFHRIIHHGLQJOHYHODQGHQHUJ\VRXUFHRQO\VLQHUHTXLUHPHQWVLQJURZLQJSLJV 
E.M.A.M. Bruininx, W.J.J. Gerrits, I. Eising, P. Vervenne, P. Sakkas, and
J.J.G.C. van den Borne

The use of free amino acids in piglet diets allows the formulation of very low crude
SURWHLQGLHWV 
M. Gloaguen, N. Le Floc’h, Y. Primot, E. Corrent and J. van Milgen

Lysine concentration in dietary protein affects performance and pattern of nutrient

UHWHQWLRQRIZHDQHG,EHULDQSLJOHWV 
R. Barea, L. Lara, J.F. Aguilera and R. Nieto

Effect of processing of oilseed meals on the apparent ileal protein digestibility and
SHUIRUPDQFHLQSLJV 
T.G. Hulshof, P. Bikker and A.F.B. van der Poel

'HWHUPLQDWLRQRIWKHQH[WOLPLWLQJDPLQRDFLGLQ\RXQJSLJOHWV 
A.J.M. Jansman, H. van Diepen, M. Rovers and E. Corrent

10 Energy and protein metabolism and nutrition in sustainable animal production

)HHGLQJORZSURWHLQDPLQRDFLGIRUWL¿HGGLHWVGLGQRWDIIHFWSHUIRUPDQFHDQGFDUFDVV
FRPSRVLWLRQRIJURZLQJ¿QLVKLQJSLJV 
J.K. Htoo, J. Trautwein, J. Gao and G. Dusel

Changes in protein turnover during pregnancy in pigs at amino acid intake in excess of
requirements 173
60RHKQ05D¿L3%3HQFKDU]DQG52%DOO

Effect of reducing dietary energy and protein on growth performance and carcass traits of
broilers 175
C.K. Girish, S.V. Rama Rao and R.L. Payne

Metabolic use of a growing diet for red-legged partridge (Alectoris rufa) chicks 177
M. Lachica, J.F. Aguilera, R. Nieto and I. Fernández-Fígares

The amino acid composition of body protein in broilers is affected by the sulphur amino
acid supply 179
J.A. Conde-Aguilera, C. Cobo-Ortega, S. Tesseraud, M. Lessire, Y. Mercier and
J. van Milgen

Body composition and nutrient partitioning in long term supplementation of betaine and
conjugated linoleic acid in mice 183
J.M. Rodríguez-López, L. González-Valero, M. Lachica and I. Fernández-Fígares

Effect of immunocastration in combination with addition of fat to diet on quantitative

oxidation of nutrients and fat retention in male pigs 185
1%DWRUHN-1REOHW6'XERLV0%RQQHDX0ýDQGHN3RWRNDUDQG(/DEXVVLHUH

Net energy content of dry extruded-expelled soybean meal fed to growing pigs using
indirect calorimetry 187
D.E. Velayudhan, J.M. Heo and C.M. Nyachoti

(IIHFWRIHDUO\VXUJLFDOFDVWUDWLRQDQGLPPXQHFDVWUDWLRQRQSRVWSUDQGLDOQXWULHQWSUR¿OHV
in male pigs 189
N. Le Floc’h, A. Prunier, J. van Milgen, H. Furbeyre and I. Louveau

Determination of the valine requirements for growth in pigs from 8 to 18 kg 191

E. Assadi, J.V. Nørgaard, N. Canibe, B.B. Jensen, B. Nielsen, K. Blaabjerg and
H.D. Poulsen

Ideal isoleucine and valine to lysine ratios in low protein diets for growing pigs 193
C. Wecke, A. Pastor and F. Liebert

Interaction between the valine and tryptophan requirement in young piglets 195
A.J.M. Jansman, H. van Diepen, M. Rovers and E. Corrent

Is high protein diet a good nutrition strategy for broiler chickens reared at heat stress
condition? 197
D.M.B. Campos, M.F. Fernandez-Alarcon, F.A. Souza, W.C.L. Nogueira, F.H. Hada,
P.R.O. Carneiro and M. Macari

Energy and protein metabolism and nutrition in sustainable animal production 11

Evaluation of energy systems in corn and barley based diets and an enzyme complex in
broiler chicks 199
S. Cerrate, J. Caldas, R. Ekmay, J. England and C. Coon

9DULRXV¿EHUIUDFWLRQVDVHQHUJ\VXSSO\WRH[HUFLVLQJKRUVHV 
C. Brøkner, D. Austbø, J.A. Næsset, D. Blache, K.E. Bach Knudsen and A.H. Tauson

Changes in protein turnover during pregnancy in pigs when feeding limiting amounts of
amino acids. 203
60RHKQ05D¿L3%3HQFKDU]DQG52%DOO

0HWDDQDO\WLFDOVWXG\RQWKHSHUIRUPDQFHDQGXWLOL]DWLRQHI¿FLHQF\RIGLIIHUHQW
methionine sources by pigs 205
A. Remus, L. Hauschild, I. Andretta, M. Kipper, C.R. Lehnen and R. Isola

Estimating digestible threonine requirements for growing pigs by meta-analysis 207

R. Isola, L. Hauschild, M.C. Thomaz, N.K. Sakomura and A. Remus

/\VLQHVXSSO\LQ¿QLVKLQJEURLOHUVHIIHFWRQSHUIRUPDQFHVDQGPHDWTXDOLW\ 
M. Lessire, Y. Primot, E. Corrent, Fraysse Pauline, S. Tesseraud and C. Berri

Part 3. Tools and techniques

Proteomic tools help understanding the metabolic adaptation to negative energy balance in
dairy cows 213
B. Kuhla and C.C. Metges

Quantifying subclinical ruminal drinking using a [13C]-[15N2]-urea based method in veal

calves 223
H. Berends, J.J.G.C. van den Borne and W.J.J. Gerrits

Estimation of 24-h energy expenditure in dairy cows using the 13C bicarbonate dilution
method 225
A. Münger, L.D. Kaufmann, S. Görs, C.C. Metges and F. Dohme-Meier

The impact of nutritional, animal and farm management factors on variation in milk urea
content 227
A. Bannink, J.W. Spek, J.L. Ellis and J. Dijkstra

Metabolizable energy of pure stand alfalfa hay estimated from near infrared spectra 229
C. Old, N. Ohanesian, J. Miller, R. Hinders, W. Vogt, J. Oltjen and D. Sapienza

Improving in sacco incubation technique to evaluate starch degradability of fresh and

fermented corn silage for ruminants 231
J. Peyrat, E. Watremez, A. Le Morvan, A. Férard, G. Cabon, P.V. Protin, P. Nozière and
R. Baumont

A titration approach to identify the capacity for starch digestion in milk-fed calves 233
M.S. Gilbert, J.J.G.C. van den Borne, A.J. Pantophlet and W.J.J. Gerrits

12 Energy and protein metabolism and nutrition in sustainable animal production

Application of washed rumen technique for rapid determination of fasting heat production
in steers 235
D.H. Kim, K.R. McLeod, J.L. Klotz, A.F. Koontz, A.P. Foote and D.L. Harmon

Challenge models to study the effect of immune system activation on amino acid
metabolism in pigs 237
E. Kampman-van de Hoek, W.J.J. Gerrits, J.J.G.C. van den Borne,
C.M.C. van der Peet-Schwering, H. van Beers and A.J.M. Jansman

Effect of an enzyme complex and dietary nutrients on endogenous losses of amino acids
in chicks 239
S. Cerrate, K. Vignale, R. Ekmay, J. England and C. Coon

Validation of the oral 13C-bicarbonate tracer technique against indirect calorimetry for the
estimation of energy expenditure in resting dogs 241
C. Larsson, R.B. Jensen, P. Junghans and A-H. Tauson

Validation of the 13C-bicarbonate tracer technique against indirect calorimetry for

estimation of energy expenditure in resting ponies 243
R.B. Jensen, C. Larsson, P. Junghans and A.-H. Tauson

%LRPHWU\DQGELRHOHFWULFDOLPSHGDQFHDQDO\VLVWRHVWLPDWHERG\FRPSRVLWLRQRI¿VK
tambatinga (Colossoma macropomum × Piaractus brachypomus) 245
F.T. Andrade, M.L.T. Abreu, J.B. Lopes, E. Lanferdini and A. Saraiva

(YDOXDWLRQRIWKHLQIUDUHGVSHFWURVFRS\PHWKRGIRUWKHTXDQWL¿FDWLRQRI1$12/,3(
marker in feces of dairy cattle 247
E.O.S. Saliba, N.C. Gonçalves, G.S.S.C. Barbosa, A.L.C.C. Borges, N.M. Rodriguez,
G.R. Moreira and F.A. Silva

A continuous approach to assess methane production rate in ruminants using respiration

chambers 249
L.F.L. Cavalcanti, M.A. Fonseca, J.G.L. Regadas Filho and L.O. Tedeschi

Part 4. Regulation

Mechanisms of regulation of intramuscular fat deposition in porcine muscle by dietary

lysine content 253
M. Katsumata, T. Kyoya, H. Kobayashi, A. Ishida, A. Ashihara and K. Nakashima

,PSDFWRIGLHWDU\ODUJLQLQHVXSSO\GXULQJHDUO\JHVWDWLRQRQP\R¿EHUGHYHORSPHQWLQ
QHZERUQSLJV 
G. Bee, C.E. Pardo and M. Kreuzer

Effect of feed restriction and birth weight on molecular and metabolic response in the
OLYHURISLJV 
C. Nebendahl, R. Krüger, S. Görs, K. Giggel, B.U. Metzler-Zebeli, H.M. Hammon,
E. Albrecht, R. Pfuhl and C.C. Metges

Energy and protein metabolism and nutrition in sustainable animal production 13

(PEU\RWKHUPDOPDQLSXODWLRQKDVORQJODVWLQJHIIHFWVRQHQHUJ\PHWDEROLVPLQFKLFNHQV 
T. Loyau, S. Métayer-Coustard, C. Praud, C. Berri, M.J. Duclos, S. Tesseraud, N. Rideau,
P. Chartrin, C. Hennequet-Antier, N. Everaert, S. Yahav, S. Mignon-Grasteau and
A. Collin

Heat stress-induced overproduction of mitochondrial reactive oxygen species is down-

UHJXODWHGLQOD\LQJW\SHFKLFNHQV 
M. Kikusato and M. Toyomizu

)XQFWLRQDOUROHVRIDTXDSRULQVDQGXUHDWUDQVSRUWHUVLQXUHDÀX[DFURVVWKHUXPLQDOHSLWKHOLXP 
M.E. Walpole, B.L. Schurmann, P. Górka, G.B. Penner, M.E. Loewen and T. Mutsvangwa

Modulation of insulin signaling by n-3 PUFA in chicken liver 271

S. Tesseraud, S. Métayer-Coustard, P. Chartrin, D. Hermier, N. Simon, C. Peyronnet,
M. Lessire and E. Baéza

+RUPRQHDQGPHWDEROLWHOHYHOVGLIIHUEHWZHHQFHUHEURVSLQDOÀXLGDQGSODVPDRI
periparturient dairy cows 273
T. Laeger, H. Sauerwein, C.C. Metges and B. Kuhla

+HSDWLFĮ1DQGȕ2-adrenergic receptors in dairy cows with different fat mobilization

during early lactation 275
H.M. Hammon, U. Kautzsch, C. Reiko, B. Kuhla, M. Röntgen, M. Derno and C.C. Metges

Insulin signaling of glucose uptake in skeletal muscle of lactating dairy cows 277
S.K. Spachmann, U. Schönhusen, B. Kuhla, M. Röntgen and H.M. Hammon

,QÀXHQFHRIDGAT1 polymorphism on response of dairy cows to ruminal supplementation

of linseed oil 279
A.M. van Vuuren, L. Kruijt, H.C.A. Widjaja, A. Klop, A.J. Cnossen, D. Anjema and
J. van Baal

Correlations between plasma ghrelin and parameters of fat metabolism in early lactating
dairy cows 283
S. Börner, M. Derno, H.M. Hammon, M. Röntgen, S. Thanthan, H. Kuwayama and
B. Kuhla

Effects of realimentation after nutrient restriction during early to mid-gestation on

pancreatic digestive enzymes in beef cattle 285
F.E. Doscher, E.A. Nere, R.D. Yunusova, L.E. Camacho, C.O. Lemley, L.D. Prezotto,
K.A. Vonnahme, J.S. Caton and K.C. Swanson

,QKLELWRU\HIIHFWRIDPPRQLDRQXUHDÀX[DFURVVUXPHQHSLWKHOLXPGHSHQGVRQOHYHORI
serosal urea 287
M.E. Walpole, G.B. Penner and T. Mutsvangwa

Effects of starch derived substrates on pancreatic and mucosal enzyme activities in milk-
fed calves 289
A.J. Pantophlet, M.S. Gilbert, J.J.G.C. van den Borne, R.J. Vonk, W.J.J. Gerrits,
A. Pluschke, H.A. Schols, M.G. Priebe and J. Roelofsen

14 Energy and protein metabolism and nutrition in sustainable animal production

Effect of heat stress on intake and metabolism of Bos taurus (Angus) and Bos indicus
(Nellore) 291
E.E.L. Valente, M.L. Chizzotti, C.V.R. Oliveira, M.C. Galvão, S.S. Domingues,
A.C. Rodrigues M.M. Ladeira R.A. Ferreira and F.H.M. Chizzotti

Growth hormone releasing factor and secretion of growth hormone in Iberian and
Landrace gilts 293
J.M. Rodríguez-López, L. González-Valero, M. Lachica and I. Fernández-Fígares

(IIHFWVRIDVLPSOHRUDFRPSOH[VWDUWHUPLFURELRWDRQWKHJDVWULFWUDQVFULSWRPHSUR¿OHRI
caesarean derived piglets 295
D. Priori, M. Colombo, S.J. Koopmans, A.J.M. Jansman, G. Schiavo, P. Trevisi and
P. Bosi

%LWWHUWDVWHUHFHSWRUJHQHVLQSLJV613LGHQWL¿FDWLRQE\XVLQJQH[WJHQHUDWLRQ
semiconductor sequencing 297
L. Fontanesi, F. Bertolini, E. Scotti, G. Schiavo, P. Trevisi, M. Colombo, P.L. Martelli,
R. Casadio and P. Bosi

Post weaning growth and metabolism in F2-offspring of protein restricted mink dams 299
C.F. Matthiesen and A.-H. Tauson

Investigation of the glucose metabolism of the embryonic and neonatal broiler chicks by
injection of insulin 301
L. Franssens, J. Lesuisse, A. Koppenol, Y. Wang, E. Willems, J. Buyse, E. Decuypere and
N. Everaert

Stage of egg production regulates protein turnover and lysine partitioning for broiler breeders 305
R.D. Ekmay, K. Vignale, C. Salas, J. England, S. Cerrate and C.N. Coon

Lipid utilization for egg formation in broiler breeders 307

C. Salas, R. Ekmay, J. England, S. Cerrate and C.N. Coon

The heat-induced production of reactive oxygen species regulates protein content in

cultured chick skeletal muscle cells 309
H. Yoshida, M. Kikusato, K. Souma and M. Toyomizu

Part 5. Modeling / systems biology

Fasting heat production and metabolic body size in non-ruminant growing farm animals 313
J. Noblet, E. Labussière, S. Dubois, C.F.M. de Lange, R. Barea, J. Lasnier, V. Rivera,
M. Warpechowski and J. van Milgen

The evolution of INRA feeding systems for ruminants based on absorbed nutrients and
animal responses 315
P. Nozière, D. Sauvant, J.L. Peyraud and co-workers

Integrative model of the digestive tract including the interactions involved in energy and
protein digestion in ruminants 317
D. Sauvant and P. Nozière

Energy and protein metabolism and nutrition in sustainable animal production 15

(IIHFWVRIGLHWFRPSRVLWLRQGXULQJWKH¿QLVKLQJSHULRGRQSURWHLQDQGOLSLGGHSRVLWLQ
young bulls 319
J. Agabriel, M. Al-Jammas, I. Ortigues-Marty, C. Villetelle, P. Nozière and
F. Garcia-Launay

,QWHJUDWLQJQXWULWLRQDODQGUHSURGXFWLYHPRGHOVWRLPSURYHUHSURGXFWLYHHI¿FLHQF\LQ
dairy cattle 321
S.L. Shields, H. Woelders, M. Boer, C. Stötzel, S. Röeblitz, J. Plöntzke and J.P. McNamara

Use of thermodynamic equations to improve predictions of volatile fatty acid production

by the Molly cow model 323
S. Ghimire, R.A. Kohn, P. Gregorini and M.D. Hanigan

BR-CORTE 1.0: online software for diet optimization on tropical conditions 325
S.C. Valadares Filho, M.L. Chizzotti, P.A.S. Machado, H.F. Amaral and T. Furtado

A structural equation model to analyze energy utilization in lactating dairy cows 327
L.E. Moraes, A.B. Strathe, E. Kebreab, D.P. Casper, J. Dijkstra, J. France and J.G. Fadel

Protein deposition potential and modeling of methionine requirements in homozygous

(Na/Na) and heterozygous (Na/na) naked neck meat type chicken 329
D.R. Khan, C. Wecke and F. Liebert

/LQHDUDQGQRQOLQHDUHVWLPDWHVRIWKHHI¿FLHQF\ZLWKZKLFKPHWDEROL]DEOHHQHUJ\LVXVHG
for maintenance or gain 331
C.A. Old and H.A. Rossow

Responses of laying hens to methionine and cystine intake 333

H.C.P. Bendezu, N.K. Sakomura, L. Hauschild, J. Sato, J.C.P. Dorigam and E.P. Silva

Assessment of ideal dietary amino acid ratios between branched-chain amino acids for
growing chicken 335
A. Pastor, C. Wecke and F. Liebert

Meta-analysis of the response of growing pigs to valine content of the diet 339
J. van Milgen, M. Gloaguen, N. Le Floc’h, L. Brossard, Y. Primot and E. Corrent

Prediction of dry matter intake in dairy calves 341

A.L. Silva, M.I. Marcondes, M.M. Campos, F.S. Machado, M.M.D. Castro and A.S. Trece

Re-evaluation of lysine requirement of broilers based on protein deposition and lysine

HI¿FLHQF\ 
J.C.P. Dorigam, N.K. Sakomura, L. Hauschild and E.P. Silva

Ideal ratio (relative to lysine) of methionine + cystine and threonine for broiler breeders 345
A.R. Troni, N.K. Sakomura, C.F.S. Oliveira, E.P. Silva, F.G.P. Costa and D.C.Z. Donato

Prediction of net hepatic release of glucose using a ‘hybrid’ mechanistic model in

ruminants applied to positive energy balance 347
L. Bahloul, I. Ortigues-Marty, J. Vernet, H. Lapierre, P. Nozière and D. Sauvant

16 Energy and protein metabolism and nutrition in sustainable animal production

Part 6. Products and health

Recent advances in understanding the interactions between nutrients and immunity in

farm animals 353
K.C. Klasing and V.J. Iseri

0LONIDWW\DFLGSUR¿OHLQGDLU\FRZVGXULQJDQHJDWLYHHQHUJ\EDODQFHLQHDUO\ODFWDWLRQ
DQGIHHGUHVWULFWLRQLQPLGODFWDWLRQ 
J.J. Gross, H.A. van Dorland, R.M. Bruckmaier and F.J. Schwarz

7KHHIIHFWRIORQJWHUPEXW\UDWHVXSSOHPHQWDWLRQWRKLJKSURGXFLQJSHULSDUWXULHQWGDLU\FRZV 
L. Vandaele, J. Van Eys, B. D’Heer, B. Ampe and S. De Campeneere

Peripartal energy balance and peripheral blood mononuclear cell activation in normal and
KLJKPRELOL]LQJGDLU\FRZV 
A. Schwarm, T. Viergutz, B. Kuhla, H.M. Hammon and M. Schweigel-Röntgen

Impact of CFA and dietary protein supply on acute phase responses and nitrogen retention
LQSLJV 
E. Kampman-van de Hoek, P. Sakkas, J.J.G.C. van den Borne, W.J.J. Gerrits,
C.M.C. van der Peet-Schwering, H. van Beers and A.J.M. Jansman

1DQRQXWULWLRQDVDPHWKRGRIDQWLFDQFHUWKHUDS\ 
E. Sawosz, M. Grodzik, S. Jaworski, M. Wierzbicki, M. Prasek and A. Chwalibog

Differential expression of innate immune system genes in liver of beef cattle with
divergent phenotypes for RFI 371
.$-RKQVRQ--0LFKDO*(&DUVWHQV$1+DÀDDQG7'$)RUEHV

(IIHFWRIKLJKIDWE\SURGXFWVSHOOHWVLQ¿QLVKLQJGLHWVIRUVWHHUV 
P. Górka, J.J. McKinnon and G.B. Penner

Microbial activity in the large intestine of chickens fed diets containing different sources
of inulin-type fructans 375
07DFLDN0%DUV]F]$7XĞQLR(ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá

Effect of dietary protein and carbohydrates on phenolic compounds formation in the large
intestine of pigs 377
07DFLDN$7XĞQLR0%DUV]F](ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá

Microbial activity in the large intestine of piglets fed diets with different sources of inulin 379
0%DUV]F]07DFLDN$7XĞQLR(ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá

7UDQVJHQLFÀD[LQKLJKIDWGLHWLQKLELWVLQÀDPPDWRU\VWDWHGHYHORSPHQWLQPLFHOLYHU 
00DWXVLHZLF],.RVLHUDG]ND0ĩXNDQG-6]RSD

Part 7. Tissue metabolism

0HFKDQLVPVXQGHUO\LQJYDULDWLRQLQEHHIFDWWOHIHHGHI¿FLHQF\UROHVRIPXVFOHDQG
adipose tissues. 385
R.A. Hill, C.M. Welch, M. McGee, S. Acharya, S. Ji, C.S. Schneider and J.K. Ahola

Energy and protein metabolism and nutrition in sustainable animal production 17

$GLSRVHWLVVXHSUHIHUHQFHVIRUDFHWDWHDQGJOXFRVHE\¿QLVKLQJVWHHUV 
W.A.D. Nayananjalie, T.R. Wiles, D.E. Gerrard, M.A. McCann and M.D. Hanigan

*URZWKUDWHLQEHHIFDWWOHDIIHFWVDGLSRVHJHQHH[SUHVVLRQDQGVNHOHWDOPXVFOH¿EHUW\SH 
P.A. Lancaster, M.A. Vaughn, J.D. Starkey, E.D. Sharman, C.R. Krehbiel and G.W. Horn

First evidence of an insulin-sensitive glucose transporter in chicken: GLUT-12 391

E. Coudert, J. Dupont, J. Simon, E. Cailleau-Audouin, S. Crochet, M.J. Duclos,
S. Tesseraud and S. Métayer-Coustard

,QÀXHQFHRIPLWRFKRQGULDOIXQFWLRQRQIHHGHI¿FLHQF\RIEURLOHUVZLWKDQGZLWKRXWJURZWK
enhancing levels of minerals supplementation during a coccidiosis challenge 393
G. Acetoze, R. Kurzbard, J.J. Ramsey, K.C. Klasing and H.A. Rossow

Expression of amino acid transporter in porcine skeletal muscles during postnatal

development 395
A. Ishida, A. Ashihara, K. Nakashima and M. Katsumata

Rate of rumen epithelial adaptation for sodium and short chain fatty acid absorption 397
B.L. Schurmann, M.E. Walpole, P. Górka and G.B. Penner

5XPLQDQWVSHFL¿FPROHFXODUDQGV\VWHPLFDGDSWDWLRQRIUHQDOHOHFWURO\WHKDQGOLQJWRORZ
N intake 399
S. Starke, C. Cox, K.-H. Südekum and K. Huber

1XWULHQWXWLOL]DWLRQGXULQJLQÀDPPDWLRQGLIIHUVEHWZHHQSLJVVHOHFWHGIRUGLIIHUHQFHVLQ
IHHGHI¿FLHQF\ 
E. Labussière, E. Merlot, J.-N. Thibault, J. Noblet, N. Le Floc’h and J. van Milgen

Differential protein deposition in tissues of growing Iberian and Landrace × Large White
pigs under identical nutritional management 403
R. Nieto, R. Barea, L. Lara, R.A. Márquez and J.F. Aguilera

Intravenous administration of arginine to twin-bearing ewes enhances birth weight and

peri-renal fat stores of female offspring in sheep 405
S. McCoard, F. Sales, N. Wards, Q. Sciascia, M. Oliver, J. Koolaard and
D. van der Linden

Effect of dietary protein concentration and forage type on nitrogen metabolism and
QXWULHQWÀX[DFURVVWKHSRUWDOGUDLQHGYLVFHUDDQGWKHOLYHULQODFWDWLQJGDLU\FRZV 
C.E.S. Barratt, L.A. Crompton, C. Green, D.J. Humphries, R.D. Pilgrim and C.K.
Reynolds

Effect of abomasal amino acid infusion on splanchnic metabolism in postpartum transition

dairy cows 409
M. Larsen, C. Galindo, D.R. Ouellet, G. Maxin, N.B. Kristensen and H. Lapierre

Net portal appearance of amino acids in Iberian compared to Landrace pigs 411
L. González-Valero, J.M. Rodríguez-López, M. Lachica and I. Fernández-Fígares

18 Energy and protein metabolism and nutrition in sustainable animal production

8UHDQLWURJHQDEVRUEHGIURPWKHKLQGJXWLVXVHGHI¿FLHQWO\IRUERG\SURWHLQGHSRVLWLRQLQ
SLJVIHGDGLHWGH¿FLHQWLQQRQHVVHQWLDODPLQRDFLGQLWURJHQ 
W. Mansilla, D. Columbus, J.K. Htoo and C.F.M. de Lange

Supplementation with a leucine pulse during continuous feeding stimulates protein

synthesis and suppresses protein degradation pathways in skeletal muscle of neonatal pigs 415
C. Boutry, S.W. El-Kadi, A. Suryawan, S.M. Wheatley, R.A. Orellana, H.V. Nguyen and
T.A. Davis

Effects of copper nanoparticles on metabolic rate and development of layer embryos 417
L. Pineda, E. Sawosz, K.P. Vadalasetty and A. Chwalibog

$WURJLQDPXVFOHVSHFL¿FXELTXLWLQOLJDVHLVKLJKO\H[SUHVVHGLQWKHVPRRWKPXVFOHRI
the chicken gizzard 419
K. Nakashima, A. Ishida and M. Katsumata

An in ovo 13C-tracer approach to explore liver intermediary metabolism in developing

chicken embryos 421
Q. Hu, U. Agarwal and B.J. Bequette

7KHKLJKIDWGLHWDQGÀD[VHHGFDNHLQÀXHQFHGRQOLSLGPHWDEROLVPLQPLFHVHOHFWHGIRU
body weight 423
M. Matusiewicz, S. Fiedorowicz, K. Fiszdon, I. Kosieradzka and W. Bielecki

%UDQFKHGFKDLQĮNHWRDFLGVLQSODVPDRIJURZLQJFKLFNHQZKHQLVWKHWLPHIRUEORRG
sampling? 425
A. Pastor, A. Sünder and F. Liebert

Lean accretion and protein turnover are enhanced by intermittent bolus feeding in
neonatal pigs 427
S.W. El-Kadi, C. Boutry, A. Suryawan, M.C. Gazzaneo, R.A. Orellana, N. Srivastava,
H.V. Nguyen, M.L. Fiorotto and T.A. Davis

Changes in tissue amino acid composition and protein metabolism in piglets due to a
limiting supply of total sulphur amino acids 429
J.A. Conde-Aguilera, C. Cobo-Ortega, N. Le Floc’h, Y. Mercier and J. van Milgen

Changes in fatty acid composition of intramuscular fat in growing Iberian and Landrace ×
Large White pigs under identical nutritional management 431
I. Seiquer, A. Haro, J.F. Aguilera and R. Nieto

Portal-drained viscera heat production in pigs fed betaine and conjugated linoleic acid
(CLA) supplemented diets 433
M.L. Rojas-Cano, M. Lachica, L. Lara, A. Haro and I. Fernández-Fígares

The effect of dietary carbohydrate composition on net portal appearance of nutrients and
AA liver uptake in dairy cows fed low protein diets 435
G. Cantalapiedra-Hijar, J.M. Rodriguez-Lopez, A. Illovies, F. Messad and
I. Ortigues-Marty

Energy and protein metabolism and nutrition in sustainable animal production 19

Uptake of arterial amino acids by ruminal tissue in Holstein cows under washed rumen
conditions 437
A.C. Storm, M. Aguilar, M.D. Hanigan, N.B. Kristensen and M. Larsen

Uptake of arterial amino acids by ruminal tissue in periparturient Holstein cows 439
M. Larsen, A.C. Storm and N.B. Kristensen

Effects of ethanol on splanchnic nutrient metabolism in sheep at different intake levels 441
T. Obitsu, K. Nishimura, Y. Udaka, T. Sugino and K. Taniguchi

Contribution of amino acids to glucose and lactose synthesis in lactating dairy cows 443
G. Maxin, D.R. Ouellet and H. Lapierre

Contribution of essential amino acids to glucose metabolism and lactose secretion in late
lactation dairy cows 447
H. Lapierre, S. Lemosquet and D.R. Ouellet

Mammary gland from lactating cows responded additively to individual essential amino
acids in casein synthesis rate 449
S.I. Arriola Apelo, L.M. Singer, X. Lin, W.K. Ray, R.F. Helm and M.D. Hanigan

Effect of amino acid supply on whole body and tissue glucose kinetics in postpartum
dairy cows 451
C. Galindo, M. Larsen, D.R. Ouellet, G. Maxin, D. Pellerin and H. Lapierre

(IIHFWVRIPHWDEROL]DEOHSURWHLQVXSSO\RQ1HI¿FLHQF\SODVPDDPLQRDFLGFRQFHQWUDWLRQV
in dairy cows 453
D.R. Ouellet, D. Valkeners and H. Lapierre

Small intestinal, stomach complex, and total gastrointestinal tract masses are decreased
UHODWLYHWRERG\ZHLJKWLQKLJKHI¿FLHQF\VWHHUV 
A.M. Meyer, K.W. Christensen, W.J. Means, S.L. Lake and S.I. Paisley

Whole body oxidative metabolism in dairy cows with a different liver fat content in early
lactation 457
M. Derno, S. Börner, H.M. Hammon, M. Röntgen and B. Kuhla

Lipolytic capacity of visceral adipose tissue in the dairy cow 459

Á. Kenéz, L. Locher, A. Rizk, S. Dänicke, J. Rehage and K. Huber

(IIHFWVRIQXWULHQWUHVWULFWLRQRQOLYHUDQGVPDOOLQWHVWLQHHQHUJ\XVHLQSUHJQDQWEHHIFRZV 
L.D. Prezotto, L.E. Camacho, C.O. Lemley, F.E. Doscher, J.S. Caton, K.A. Vonnahme and
K.C. Swanson

The effect of pregnancy on weight change, visceral organ mass and circulating serum
PHWDEROLWHVLQPDWXUHEHHIFRZV 
K.M. Wood, C.J. Fitzsimmons, S.P. Miller, B.W. McBride and K.C. Swanson

6NHOHWDOPXVFOHIDWW\DFLGR[LGDWLRQLQODFWDWLQJGDLU\FRZVGXULQJHDUO\ODFWDWLRQ 
C. Schäff, H.M. Hammon, M. Röntgen and B. Kuhla

20 Energy and protein metabolism and nutrition in sustainable animal production

7UDQVFULSWRPHSUR¿OHFRPSDULVRQEHWZHHQEHHIDQGGDLU\DGLSRVHSRROHGP51$UHYHDOV
GLIIHUHQFHV 
J.M. Thomson, P. Stothard and J.P. McNamara

Effects of omitting the dry period on plasma progesterone and prolactin during
lactogenesis and on colostrum IgG content in dairy cows during the periparturient period 471
H.A. van Dorland, R.S. Zbinden, G. Remmelin, B. Kemp, A.T.M. van Knegsel and
R.M. Bruckmaier

Part 8. Environmental sustainability

The contribution of animal production to agricultural sustainability 475

M. Doreau, H.P.S. Makkar and P. Lecomte

Environmental, social, and economic footprints of current and past beef production systems 487
K.R. Stackhouse-Lawson, J.O. Reagan, B.J. Isenberg, E.J. Pollak, T. Battagliese,
B. Ulhman, C. Barcan, I. Schulze, J. Silva and C.A. Rotz

Effect of fat supplementation and stage of lactation on methane emission in dairy cows 489
L. Alstrup, M.R. Weisbjerg and P. Lund

Methane emission and protein precipitating ability of condensed tannins from warm-
season perennial legumes 491
H.D. Naumann, L.O. Tedeschi, A.E. Hagerman, B.D. Lambert and J.P. Muir

Short-term dose effects of feeding monensin on methane emissions from lactating

Holstein dairy cattle 493
S.E. Place, Y. Pan, Y. Zhao and F.M. Mitloehner

Methane emission from sheep is related to concentrations of rumen volatile fatty acids 495
C.S. Pinares-Patiño, H. Kjestrup, S. MacLean, E. Sandoval, G. Molano, R. Harland,
S. Hickey, E. Young, K. Dodds, K. Knowler, N. Pickering and J. McEwan

(QHUJ\HI¿FLHQF\DQGPHWKDQHHPLVVLRQE\VKHHSIHGVRUJKXPVLODJHVDWGLIIHUHQW
maturation stage 497
F.S. Machado, N.M. Rodríguez, M.N. Ribas, F.P. Pôssas, L.C. Gonçalves and
L.G.R. Pereira

Methane emission from lactating cows fed diets with different forage base 499
S. Colombini, L. Rapetti, G. Galassi, L. Malagutti, M. Pirondini and G.M. Crovetto

Effect of condensed tannins on methane emission and ruminal microbial populations 501
M. Rira, C. Marie-Magdeleine, H. Archimède, D.P. Morgavi and M. Doreau

Methane emission by cattle supplemented with additives in Brazil 503

A.L.C.C. Borges, M.P. Fonseca, R.R. Silva, H.F. Lage, J.A.S. Rodrigues, L.C. Gonçalves,
I. Borges and E.O.S. Saliba

Effect of paddy rice diets on performance in chickens under thermoneutral and heat stress
conditions 505
F. Nanto, C. Ito, M. Kikusato, S. Ohwada and M. Toyomizu

Energy and protein metabolism and nutrition in sustainable animal production 21

Effect of invertebrates on growth performance and feeding behavior of red-legged
partridge (Alectoris rufa) chicks 509
M. Lachica and I. Fernández-Fígares

Approach to determine the amino acid composition of the natural diet of red-legged
partridge (Alectoris rufa) 511
I. Fernández-Fígares and M. Lachica

Part 9. Baldwin symposium

The life and legacy of Dr. Ransom Leland (‘Lee’) Baldwin V 515
R.L. Baldwin, VI and C.C. Calvert

Application of mathematical modelling in animal nutrition, physiology and energy balance 517
J. France

Contributions of Lee Baldwin to lactation biology 521

J.P. Cant

Modeling animal growth with Lee Baldwin 523

Roberto D. Sainz

Advances in rumen microbiology 527

M.R. Murphy

Author index 529

22 Energy and protein metabolism and nutrition in sustainable animal production

Preface
The 4th EAAP International Symposium on Energy and Protein Metabolism and Nutrition (ISEP) was
held at the Sheraton Grand Hotel in Sacramento, California, USA from September 9th to September
12th, 2013. This followed the 3rd ISEP in Parma (2010), the 2nd ISEP in Vichy (2007), and the 1st
,6(3LQ5RVWRFN:DUQHPĦQGH  7KHVHIROORZWKHSUHYLRXV(QHUJ\6\PSRVLXPDQG3URWHLQ
Symposium which were held separately, all under the auspices of the European Association of
$QLPDO3URGXFWLRQDQGWKH&RPPLVVLRQRQ$QLPDO1XWULWLRQLWLVDOVRWKH¿UVWWLPHWKHV\PSRVLXP
had been held in North America.

As world population increases, demand for food, particularly animal products, is expected to grow
substantially. Because of limited area for expansion of animal agriculture and increased consumer
FRQFHUQIRUWKHHQYLURQPHQWDOLPSDFWRIDQLPDOSURGXFWLRQJDLQVLQDQLPDOHI¿FLHQF\ZLOOKDYHWR
be part of the solution. The 4th International Symposium on Energy and Protein Metabolism and
Nutrition addressed key issues of how energy and protein are utilized and interact in farm animals
from the molecular to the whole animal and even to the herd or group level of organization. Key issues
addressed include energy/protein interactions, methodology such as in vitro and in vivo techniques,
regulation including pre-natal programming and endocrine regulation, modeling/systems biology,
products and health of animals, tissue metabolism, and environmental sustainability in agriculture.
ISEP also included a tribute to the late Professor R. Lee Baldwin of the University of California,
'DYLVDOHDGHULQWKH¿HOG

The 4th Symposium began with the premise that improved understanding of animal energetics and
protein metabolism will be required for sustainable animal production. Over 200 participants, from
27 countries, made theatre and poster presentations; two-page abstracts for contributed papers and
full length papers by invited speakers are contained herein.

$WWHQGHHVDWWKH,6(3KHDUGVLJQL¿FDQWQHZUHVHDUFKZLWKLQYLWHGVSHDNHUVDQGRUDODQGSRVWHU
communications by participants. The Symposium combined fundamental research with applied
research and practical applications. Because energy and protein metabolism and nutrition cannot
be addressed separately, a better and deeper understanding of nutrient metabolism and nutrition can
be achieved only by integrating the outcomes of scientists conducting research on different aspects.

Participants and accompanying persons were housed in one hotel and shared common meals in an
effort to increase networking possibilities and stimulate interactions; they were also treated to a
showcase of California agriculture and hospitality.

We thank all those who helped make this Symposium successful, especially the sponsors and the
International, North American, and Local Organizing Committees. Finally, we would also like to
thank all the participants for making possible a meeting with a great deal of interaction; we hope this
meeting has given us more tools to address questions that need to be answered for a real sustainable
DJULFXOWXUHVFLHQWL¿FDOO\

James W. Oltjen

Energy and protein metabolism and nutrition in sustainable animal production 23

Keynotes
Feeding the planet: key challenges
M. Herrero
&RPPRQZHDOWK6FLHQWL¿FDQG,QGXVWULDO5HVHDUFK2UJDQLVDWLRQ&DUPRG\5RDG6W/XFLD
QLD, Australia; [email protected]

Abstract
The notion of feeding 9 billion people sustainably in the next forty years presents considerable
challenges. Population growth and human dietary changes remain the key drivers of the large increases
in future food demand. While the world food system can respond to meet this demand, there are
challenges to ensure this happens sustainably, equitably and within our conceived limits of a safe
environmental space. This paper discusses the key trends in food production and consumption, and the
key challenges for feeding the planet. Especial attention is given to livestock, a key part of the puzzle
for ensuring sustainable nutritional security and environmental sustainability for future generations.

Introduction
The global food system is experiencing profound changes as a result of anthropogenic pressures. The
ever-increasing human population (to reach 9 billion by 2050) together with changes in consumption
patterns (i.e. increasing demand for livestock products) caused by urbanization, increasing incomes,
and nutritional and environmental concerns, are shaping what we eat, who eats, and how much, more
WKDQHYHU7KHGRXEOHEXUGHQRIQXWULWLRQ RYHUFRQVXPSWLRQDQGXQGHUQXWULWLRQ LVGH¿QLQJUHVHDUFK
agendas, policies and conceptions about food in different ways around the world.

$JDLQVWWKLVEDFNJURXQGLVDJOREDOIRRGV\VWHPWKDWZLOOKDYHWRLPSURYHLWVUHVRXUFHXVHHI¿FLHQF\
DQGHQYLURQPHQWDOSHUIRUPDQFHVLJQL¿FDQWO\LQRUGHUWRHQVXUHWKHVXVWDLQDELOLW\RIJOREDOIRRG
production and consumption within established planetary boundaries of greenhouse gas emissions,
and water and nutrient use amongst others.

Livestock, the largest land use sector on Earth is an important part of this puzzle and many solutions
to the challenges facing how to feed the world sustainably lie in how we manage this sector. This
brief paper aims to discuss some of the key ways in which we could increase the sustainability of
the world food system, and some of the challenges to overcome.

Key challenges: the future demand for food

Table 1 shows the FAO projections of global food consumption to 2050 (Alexandratos and Bruinsma,
2012). The main conclusions from these projections, as well as others (ie. IAASTD 2009), is that a
shift to diets with more animal products and fats, is likely to happen, mostly in the developing world
as a result of increased incomes and urbanization. While the consumption per capita of cereals is
likely to stabilize, population growth will increase the total quantities of both meat (almost doubling)
and cereals (50%) needed to feed the world in 2050.

The supply response of the global agriculture and livestock sectors is likely to be able to accommodate
these demand increases (Alexandratos and Bruinsma, 2012). All recent projections have important
common features: (1) Local production under current yield trends in many parts of the world, like
Sub-Saharan Africa (SSA) and parts of Asia, will not be able to meet local food demand. Hence
increases in food trade are projected to increase in the future in some parts of the world. This is a
key aspect of balancing the food supply and demand equation. (2) While increases in the yields of
crops and livestock have occurred in most regions of the world (apart from SSA), all projections
show a variable increase in cropland and grassland expansion to meet demand (Smith et al., 2010)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 27
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_1, © Wageningen Academic Publishers 2013
7DEOH3URMHFWLRQVRIJOREDOGHPDQGIRUIRRGWR $OH[DQGUDWRVDQG%UXLQVPD 

2005/2007 2050

Population (millions)  

Cereals for food (kg per capita) 158 
Cereals for all uses (kg per capita) 314 330
Meat consumption (kg per capita) 38.7 49.4
Oil crops for food (kg per capita) 12.1 
Oil crops for all uses (kg per capita) 21.9 30.5
Meat production (million tonnes) 258 455
Cereal yields, rice paddy (t/ha) 3.32 4.30
Arable land area (million ha) 1,592 

and (3) also an increase in animal numbers, but with monogastric production (pork and poultry)
growing at faster rates than ruminants (meat especially, and less so for milk). (4) These factors lead
to net increases in greenhouse gas emissions (GHG) from the agricultural and livestock sectors, but
a diminishing trend in the emissions intensities across commodities (GHG per unit of product). (5)
projections of water use show increased pressure on total fresh water resources, notably on blue
ZDWHU LUULJDWLRQ DQGPRGHUDWHLQFUHDVHVLQWKHHI¿FLHQF\RIJUHHQZDWHUXVH &$ 2WKHU
studies have also demonstrated large quantities of reactive nitrogen used and a potential depletion
of phosphorus stocks in the future (Bouwman et al., 2011). Hence, food production can be attained
under current productivity and demand trends, but not necessarily making inroads in the improvement
of our environmental goals.

Several authors (Foley et al., 2011; Garnett and Godfray, 2011; Godfray et al., 2010; Herrero et
al., 2010) have suggested different mechanisms for improving the sustainability of the world food
system. The three most often mentioned are:
1. Increasing productivity (managing the supply side): Increasing agricultural productivity and
overall food production have been the pillar for designing strategies for feeding the world since
the industrial revolution. Notable gains have been made in many parts of the world (developed
FRXQWULHVDQG/DWLQ$PHULFDDQG$VLD 7KHUHLVVLJQL¿FDQWRQJRLQJUHVHDUFKRQKRZWRVXVWDLQDEO\
intensify global food production, how to bridge yield gaps of crops and livestock and how to
LPSURYHYDOXHFKDLQVVRWKDWERWKSURGXFHUVDQGFRQVXPHUVEHQH¿WIURPSRWHQWLDO\LHOGLQFUHDVHV
while using less, the same, or slightly more inputs.
2. Reducing waste in food value chains. This subject has received attention recently (Godfray
et al., 2010), and it has been estimated that food waste can account for up to 40% of losses
relative to food production. Figure 1 (Godfray et al., 2010) shows that in the developing world
WKHVHORVVHVRFFXUPRVWO\GXHWRSRVWKDUYHVWDFWLYLWLHVOLNHGH¿FLHQWKDUYHVWLQJDQGVWRUDJH
methods, pests, export regulations and others. In the developed world this occurs mostly at the
post-consumption stage, due to poor management of product sell-out dates in the value chain
and direct food disposal by consumers (i.e. discarding food from fridges).
3. Consuming more sustainable diets (managing the demand for food): There is evidence that
PRGLI\LQJZKDWZHHDWFRXOGKDYHVLJQL¿FDQWLPSDFWVRQWKHXVHRIUHVRXUFHVOLNHODQGDQG
ZDWHULWFRXOGUHGXFH*+*HPLVVLRQVDQGLWFRXOGKDYHLPSRUWDQWKHDOWKDQGQXWULWLRQDOEHQH¿WV
$ORWRIHPSKDVLVKDVEHHQSXWLQWKHSRWHQWLDOEHQH¿WVRIUHGXFLQJUHGPHDWFRQVXPSWLRQDQG
the promotion of ‘healthy’ diets (Stehfest et al., 2009) (Table 2). These studies have shown that
reductions in livestock consumption could lead to reduced land use change, directly from less
land clearing for raising animals or for producing feed crops. These land sparing gains, in turn
lead to lower GHG emissions in general.

28 Energy and protein metabolism and nutrition in sustainable animal production

)LJXUH0DNHXSRIWRWDOIRRGZDVWHLQGHYHORSHGDQGGHYHORSLQJFRXQWULHV *RGIUD\et al. 

 7KLVLVDVLJQL¿FDQW¿QGLQJ+RZHYHUWKLVVSDFHKDVQRWEHHQH[SORUHGVXI¿FLHQWO\WRSURYLGH
suitable, practical, regional/country guidance for consumers and for policy makers to effect the
necessary changes in local food systems, and to modify consumer behavior. At the global level,
WKLVFRQFHSWKDVRQO\EHHQVWXGLHGVXSHU¿FLDOO\DQGZLWKRXWFRQVLGHULQJLPSRUWDQWGLPHQVLRQV
such as dietary diversity and cultural preferences extending beyond measures of kilocalorie
consumption, and what would be the social and economic impacts of reducing the size of the
livestock sector. These additional dimensions are essential for understanding the biological and
socio-economic implications of diet sustainability on the global food system. We need to go
beyond simplistic recommendations like ‘stop eating meat’ to make this area of research useful,
and provide alternatives and practical guidelines for achieving these kinds of gains. This is of
particular importance for the developing world, where livestock product demand projections
GHPRQVWUDWHWKDWHYHQZLWKVLJQL¿FDQWFRQVXPSWLRQJURZWKFRQVXPSWLRQSHUFDSLWDZLOOUHPDLQ
VLJQL¿FDQWO\ORZHUWKDQLQWKHGHYHORSHGZRUOG ,$$67' 

The implementation of these strategies is not straightforward. There are many challenges and
trade-offs and they are complex because there are competing economic, social and environmental
claims to their implementation. Additionally, human nature, the single biggest ingredient in this
PL[SOD\VDNH\UROHLQGH¿QLQJRXUFKRLFHV7KHVHFKRLFHVDUHQRWDOZD\VLQIDYRURIORQJWHUP
sustainability. More often the attainment of shorter term gains prevails, especially in a world of

7DEOH/DQGXVHHPLVVLRQVLQDQGIRUWKHUHIHUHQFHVFHQDULRDQGIRXUYDULDQWVRIGLHWDU\
FRPSRVLWLRQ 6WHKIHVWet al. 

GtCeq

2000 3.0
2000 – reference 3.3
2050 – no red meat 1.7
2050 – no meat 1.5
2050 – no animal products 1.1
2050 – healthy diet 2.1

Energy and protein metabolism and nutrition in sustainable animal production 29

LQFUHDVLQJUHVRXUFHOLPLWDWLRQVDQGZLWKVLJQL¿FDQWSUHVVXUHVRQOLYHOLKRRGV6RPHRIWKHVHNH\
challenges are explained below.

Closing yield gaps of crops and livestock and increasing adoption rates of key technologies
in the developing world

Closing yield gaps, mainly of crops, has been the strategy of choice for increasing food production
worldwide (Foley et al., 2010; Van Ittersum et al. 6LJQL¿FDQWSURGXFWLYLW\JDLQVKDYHEHHQ
observed in the last 40 years, however in many smallholder systems in the world, low productivity
still prevails (Tittonell and Giller, 2013). In terms of livestock yield gaps, there has been continuous
productivity increases in the developed world or in parts of the developing world where market
orientation has been a thrust for the livestock sector. A notable example, presented by Capper et al.
(2009) demonstrates that milk yields per animal in the US have increased several-fold since the 1940s,
while at the same time reducing the size of the dairy herd considerably in the process. However,
we still lack solid global information on livestock yield gaps to inform the potential productivity
JDLQVIRUGLIIHUHQWSDUWVRIWKHZRUOG7KLVLVDQDUHDWKDWUHTXLUHVVLJQL¿FDQWUHVHDUFKHVSHFLDOO\
for guiding research in the developing world, where most of the potential for increasing yields lies.

In livestock systems, attaining yield gaps has essential pre-requisites. The availability of inputs (high
quality feeds, fertilizers, etc.), services (veterinarians, extension) and in many cases, the development
of markets and their associated value chains need to be developed (McDermott et al., 2010), as these
are key incentives for systems to intensify (Herrero et al., 2010; McDermott et al., 2012). Currently,
adoption of better feeding practices, like improved forages, have shown low adoption rates. For
example Thornton and Herrero (2010) found adoption rates of dual purpose crops, agroforestry
practices and improved pastures in the order of 15-25% of farmers in selected developing regions,
RYHUD\HDUKRUL]RQ,QFUHDVLQJDGRSWLRQUDWHVZLOOUHTXLUHVLJQL¿FDQWSXEOLFDQGSULYDWH
investment and institutional change to be able to not only increase the % of farmer adopting, but
also for reducing the adoption lag times that are often large.

$FKLHYLQJVXVWDLQDEOHLQWHQVL¿FDWLRQZLWKRXWLWVSRWHQWLDOO\SHUYHUVHLQFHQWLYHV

7KHFRQFHSWRIVXVWDLQDEOHLQWHQVL¿FDWLRQVRXQGVWRPDQ\DVDZLQZLQVWUDWHJ\WRLQFUHDVHUHVRXUFH
XVHHI¿FLHQFLHV)URPDOLYHVWRFNSHUVSHFWLYHPRVWZHOOPDQDJHGLQWHQVL¿FDWLRQSUDFWLFHVLQWKH
SDVWKDYHOHGDOVRWRLPSURYHGV\VWHPVSUR¿WDELOLW\ LHSDVWXUHLQWHQVL¿FDWLRQDQGVXSSOHPHQWDWLRQ
LQWKHWURSLFVKDVVLJQL¿FDQWO\LPSURYHGPLONDQGPHDWSURGXFWLRQ $VDUHVXOWIDUPHUVKDYHRIWHQ
increased the size of the operation (more animals, more land use changes) in order to increase even
further the economic returns. This growth in turn has led to increased environmental problems (more
deforestation, increased GHG emissions, land degradation). A critical challenge ahead is how to
UHJXODWHLQWHQVL¿FDWLRQVRWKDWLWLVWUXO\VXVWDLQDEOHDQGRSHUDWHVZLWKLQOLPLWVRISURGXFWLRQJURZWK
protects biodiversity and other ecosystems services, and attains net or near net reductions in the use
of resources. This is of particular importance, as having less animals, but of higher productivity,
VHHPVWREHHVVHQWLDOWRPD[LPL]HWKHHQYLURQPHQWDOEHQH¿WV LHUHGXFWLRQVLQ*+*HPLVVLRQVDQG
land use) of productivity growth in livestock systems (Thornton and Herrero, 2010).

What we eat matters: how far can we go towards modifying human diets in different parts
of the world?

The debate on human diets is dominated by the concerns of the developed world and the middle classes
of middle income countries, on the negative health impacts of livestock product over-consumption
(McMichael et al., 2007), and by the global integrated assessment community interested in reducing
GHG emissions from the agriculture, food and land use sectors (Smith et al. 2013; Stehfest et al.,
2009). At the same time, the discourse in the developing world is changing towards the recognition

30 Energy and protein metabolism and nutrition in sustainable animal production

that achieving nutritional security might be a more important target than just promoting a higher
kilocalorie consumption (Herrero et al., 2013). According to these authors, this does not mean that
we should take as given the projected trajectories of animal consumption proposed by the so called
‘livestock revolution’. They are not inevitable. Part of our responsibility is to challenge these future
trajectories, and ensure that we identify consumption levels for different parts of the world that will
achieve the best compromise between a culturally-appropriate, healthy diet that includes livestock
products (or not), economic growth, livelihoods and livestock’s impacts on the environment. The
other part of the agenda is ensuring that policies geared towards the promotion of healthy sustainable
diets occur throughout the food systems (from the regulatory bodies, the food industry, all the way
to the different value chain actors.

Structural change and competitiveness in the livestock sector

Large parts of the food systems of the developing world have as a starting point the smallholder
PL[HGFURSOLYHVWRFNIDUPHURUWKHYXOQHUDEOHSDVWRUDOLVW6RPHRIWKHVHV\VWHPVSURGXFHDVLJQL¿FDQW
DPRXQWRIIRRGPRVWO\IRUORFDOFRQVXPSWLRQWKH\KDYHVLJQL¿FDQWH[SORLWDEOH\LHOGJDSVLQFURSV
and livestock, and in many cases they have low opportunity costs of labor. If livestock is to be used
as an engine for poverty reduction, it is essential that these producers become market-orientated.
The degree of competitiveness of smallholders against imports from countries that can produce
vast amounts of animal products, at lower production costs, will be a crucial factor to determine
the success of many men and women livestock farmers in the developing world, especially as the
volume of traded livestock products increases due to trade liberalization. Formal and informal markets
will need to ensure the supply of cheaper, locally produced, safe livestock products to adequately
FRPSHWH7KLVLPSOLHVDVLJQL¿FDQWUHGXFWLRQLQWUDQVDFWLRQFRVWVIRUWKHSURYLVLRQRILQSXWV
LQFUHDVHGUHVRXUFHXVHHI¿FLHQFLHVDQGYHU\UHVSRQVLYHLQQRYDWLYHDQGVXSSRUWLQJLQVWLWXWLRQVIRU
WKHOLYHVWRFNVHFWRULQGHYHORSLQJFRXQWULHV )$2 +HQFHLQYHVWPHQWLQGHYHORSLQJHI¿FLHQW
value chains (including market development, service provision, adequate institutional support, etc.)
should be high in the development agenda, to create incentives for smallholders to integrate in the
market economy, formal or informal.

Recently land consolidation has occurred in many parts of Africa, as foreign investors buy large
blocks of land for developing them into large farms. Advocates of large scale farming argue in
IDYRURIWKHKLJKHUHI¿FLHQFLHVRIUHVRXUFHXVHRIWHQIRXQGLQWKHVHV\VWHPVDQGKRZVLPSOHLW
is to disseminate technology and effect technological change. However, we lack comprehensive
information on the impacts of different farming avenues and their future evolutionary pathways on
water cycles, biodiversity, social aspects (nutrition, incomes, employment), coping with production
risks (i.e. climate variability and change, commodity prices) and others.

$WKULYLQJHQYLURQPHQWDOO\UHVSRQVLEOHGLYHUVL¿HGFRPPHUFLDOVPDOOKROGHUVHFWRUWKDWKHOSVIHHG
WKHZRUOGDQGOLIWVSHRSOHRXWRISRYHUW\WRJHWKHUZLWKDODUJHVFDOHHI¿FLHQWOLYHVWRFNVHFWRUWKDW
HI¿FLHQWO\SURGXFHVIRRGZKLOHJHQHUDWLQJHQRXJKHPSOR\PHQWIRUUXUDOSHRSOHVKRXOGFRH[LVW7KH
balance between these ways of farming is likely to be different in different regions of the world, but
understanding where the balance should lie for achieving socially and environmentally goals is still
WKHTXHVWLRQWKDWPHULWVVLJQL¿FDQWUHVHDUFK

The success of paying for environmental services (PES)

3(6VFKHPHVDVDQLQFRPHGLYHUVL¿FDWLRQVWUDWHJ\IRUPLWLJDWLQJFOLPDWHFKDQJHRUIRUSURWHFWLQJ
important regional or global goods that regulate essential biogeochemical cycles have received a
lot of attention recently. However, not many successful examples exist with smallholder livestock
producers or pastoralists (Herrero et al., 2013). Proofs of concept that test how these schemes could
operate in very fragmented systems, with multiple users of the land or in communal pastoral areas

Energy and protein metabolism and nutrition in sustainable animal production 31

are necessary. Research on fair, equitable and robust monitoring and evaluation frameworks and
mechanisms for effecting payments schemes that work under these conditions is necessary. The
SURPLVHRI3(6VFKHPHVDVDPHDQVRIGHOLYHUWKHLQFRPHGLYHUVL¿FDWLRQIRUWKHSRRUDQGQDWXUDO
resource protection necessary to produce food while protecting the world’s ecosystems is yet to be
seen on a large scale.

Institutional and market mechanisms for reaching smallholders

The reality is that livestock production in the developing world is largely fragmented and disorganized.
Under-investment in extension systems and other support services have rendered poor producers
GLVHQIUDQFKLVHGWRDFFHVVNH\VXSSRUWV\VWHPVQHFHVVDU\IRULQFUHDVLQJSURGXFWLYLW\DQGHI¿FLHQF\RU
in cases important safety nets for reducing vulnerability (i.e. to drought/famines). The poorer farmers
are unlikely to be able to respond sustainably to the increased demands for animal products without
increased public investment in innovation and support platforms, as these are essential to foster the
technological change required to increase productivity and link them to markets (McDermott et al.,
2010). More advanced farmers or larger farmers in the developing world are likely to rely on the
private sector for these support services. It is essential that the roles of women in production and
trading of livestock products and in controlling livestock assets are taken into consideration when
designing these institutional mechanisms.

Animal health and food safety: regulation and surveillance in an era of more animals,
higher volumes of food trade and more diseases

Security of animal source foods, health threats to animal assets, and food safety are inextricably
linked. Animal disease threatens livelihoods and economies. For intensive livestock farmers, animal
health costs while a small part of overall farm enterprise costs are a large part of avoidable costs,
and hence a leverage point for increasing productivity. For poor livestock-keepers, animal health
typically represents one of the largest single costs and epidemic animal disease one of the biggest
and most feared risks to livestock-keeping.

Animal disease is also the main obstacle to trade in animals and animal products. Despite recent
attempts at liberalization, sanitary and phytosanitary regulations still allow importing countries to
take a precautionary ‘if in doubt, keep it out’ approach. This denies people-poor but livestock-rich
countries an opportunity to trade their way out of poverty while imposing unpredictable shocks on
all countries for which livestock trade is important (for example the Rift Valley Fever pandemic
has periodically interrupted the lucrative trade in live sheep and goats from the horn of Africa to
the Arabian Peninsula).

We are living in an era of unprecedented ecosystem change and the current upsurge in emerging
disease (75% of which are zoonotic) is predictable. An ecosystems perspective sees farming and
natural systems as containing pools of pathogens with circulate among hosts, vectors and the
HQYLURQPHQW,QV\VWHPVZKLFKDUHVWDEOHFRQQHFWHGDQGGLYHUVL¿HGKRVWVDQGSDWKRJHQFRHYROXWLRQ
favors lowered pathogenicity and less disease. However, anthropogenic incursion into ecosystems
and ecosystem alteration allows pathogens to encounter new hosts and new diseases to emerge. An
H[DPSOHRIHFRV\VWHPSURYLVLRQRIGLVHDVHUHJXODWLRQVHUYLFHVLVWKHÀDUHXSVRIKXPDQVOHHSLQJ
sickness seen in West Africa after African swine fever killed village pigs leading the tsetse vector
of sleeping sickness to shift closer to houses and bite more people.

Food safety is increasingly viewed as inseparable from food security and while intensive agriculture
can produce cheap products it also introduces new health risks for both animals and people. This is
of essential importance to consider how far we go in intensifying livestock production, and the food
V\VWHPLQJHQHUDO,QSDUWLFXODULQWHQVL¿FDWLRQVHOHFWVSDWKRJHQVKDUGWRGHWHFWLQDQLPDOSRSXODWLRQV

32 Energy and protein metabolism and nutrition in sustainable animal production

or (such as Campylobacter spp. in poultry or diarrhoeagenic Escherichia coli in cattle) or survive
conventional treatment (such as antibiotic resistance). The wide geographic scale and large volume
of consolidated food distribution systems means that food-borne diseases can spread rapidly and
affect large numbers of people greatly removed from the point of production.

Can the food system adapt to climate change and mitigate GHG emissions at a fast enough pace?

Climate change is likely to cause severe impacts on livestock systems and on poor vulnerable
producers. According to Herrero et al. (2013), the capacity and speed of adaptation of smallholders
ZLOOSOD\DQGLPSRUWDQWUROHLQGH¿QLQJWKHFRQWULEXWLRQRIOLYHVWRFNWROLYHOLKRRGVXQGHUFOLPDWH
change. At the same time, in a low carbon economy, it will be essential that the sector mitigates
GHG effectively in relation to other sectors. Demonstrating that these options are real with tangible
examples is essential to generate the evidence for increasing the investments in climate change
adaptation and mitigation for the livestock sector. This becomes more imperative as the global food
system prepares to become part of the climate change negotiations.

Conclusions
Feeding 9 billion people is a formidable but attainable task. Doing it sustainably will depend on
several things, including our capacity to reach more equitable and healthy levels of consumption,
our ability to invest in key food production systems of the world, on maintaining well regulated
and economically viable production systems, and on achieving a balance between environmental
and social goals.

The livestock sector, the largest land user on Earth, holds a large stake on how to achieve the balance
between food production, livelihoods and environmental objectives. A mixture of sustainable
LQWHQVL¿FDWLRQSURWHFWLQJELRGLYHUVLW\DQGHFRV\VWHPVVHUYLFHVWRJHWKHUZLWKVWUDWHJLHVDQGSROLFLHV
to reduce animal numbers simultaneously, may yield a suitable compromise for achieving these goals.

References
Alexandratos, N. and Bruinsma, J. 2012. World agriculture to 2030/2050. The 2012 revision. ESA Working Paper
)RRGDQG$JULFXOWXUH2UJDQL]DWLRQRIWKH8QLWHG1DWLRQV5RPH,WDO\S
%RXZPDQ$).OHLQ*ROGHZLMN.9DQGHU+RHN.:%HXVHQ$+:9DQ9XXUHQ'3:LOOHPV-5X¿QR
M.C. and Stehfest, E. 2011. Exploring global changes in nitrogen and phosphorus cycles in agriculture induced
by livestock production for the period 1900-2050. PNAS, doi/10.1073/pnas.1012878108.
CA. 2007. Comprehensive Assessment of Water Management in Agriculture (D. Molden, Editor). Earthscan, London,
UK.
Capper, J.L., Cady, R.A. and Bauman, D.E. 2009. The environmental impact of dairy production: 1944 compared with
-RXUQDORI$QLPDO6FLHQFH
FAO 2009. The State of Food and Agriculture. Livestock in the Balance. Food and Agriculture Organization of the
United Nations, Rome, Italy.
Foley, J.A., Ramankutty, N., Brauman, K. A., Cassidy, E. S., Gerber, J. S., Johnston, M., Mueller, N. D., O’Connell, C.,
Ray, D. K., West, P. C., Balzer, C., Bennett, E. M., Carpenter, S. R., Hill, J., Monfreda, C., Polasky, S., Rockstrom,
J., Sheehan, J., Siebert, S., Tilman, D. and Zak, D. P. M. 2011. Solutions for a cultivated planet. Nature 478, 337-342.
*DUQHWW7DQG*RGIUD\+&-6XVWDLQDEOHLQWHQVL¿FDWLRQQDYLJDWLQJWKURXJKDFRXUVHRIFRPSOH[LWLHV)RRG
and Climate Research Network, University of Oxford, UK.
Godfray H.C.J., Beddington, J.R., Crute, I.R., Haddad, L., Lawrence, D., Muir, J.F., Pretty, J., Robinson, S., Thomas,
S.M. and Toulmin, C. 2010. Food security: the challenge of feeding 9 billion people. Science 327, 812-818.
+HUUHUR0*UDFH'-RKQVRQ11MXNL-(QDKRUR'6LOYHVWUL6DQG5X¿QR07KHUROHVRIOLYHVWRFN
in developing countries. Animal 7 (1), 3-18.

Energy and protein metabolism and nutrition in sustainable animal production 33

Herrero, M., Thornton PK., Notenbaert AM., Wood S., Msangi S., Freeman HA., Bossio D., Dixon J., Peters M., van de
Steeg J., Lynam J., Parthasarathy Rao P., Macmillan S., Gerard B., McDermott J, Seré C. and Rosegrant M. 2010.
Smart investments in sustainable food production: revisiting mixed crop-livestock systems. Science 327, 822-825.
IAASTD. 2009. Agriculture at a Crossroads. International Assessment of Agriculture, Science and Technology
Development. Island Press, 590 p.
0F'HUPRWW--6WDDO6)UHHPDQ+$+HUUHUR0YDQGH6WHHJ-6XVWDLQLQJLQWHQVL¿FDWLRQRIVPDOOKROGHU
systems in the tropics. Livestock Science 130, 95-109.
McMichael, A.J., Powles, J.W., Butler, C.D. and Uauy, R. 2007. Food, livestock production, energy, climate change,
DQGKHDOWK7KH/DQFHW
Smith, P., Gregory, P.J, van Vuuren D.P., Obersteiner, M., Havlik, P.,Rounsevell, M., Woods, J., Stehfest, E. and Bellarby,
-&RPSHWLWLRQIRUODQG3KLORVRSKLFDO7UDQVDFWLRQVRIWKH5R\DO6RFLHW\%
Smith, P., Haberl, H., Popp, A., Erb, K., Lauk, C., Harper, R., Tubiello, F., Siqueira Pinto, A., Jafari, M., Sohi, S.,
Masera, O., Böttcher, H., Berndes, G., Bustamante, M., Ahammad, H., Clark, H., Dong, H., Elsiddig, E.A., Mbow,
C., Ravindranath, N.H., Rice, C.W., Robledo-Abad, C., Romanovskaya, A., Sperling, F., Herrero, M., House, J.I.
and Rose, S. 2013. How much land based greenhouse gas mitigation can be achieved without compromising food
VHFXULW\DQGHQYLURQPHQWDOJRDOV"*OREDO&KDQJH%LRORJ\ '2,JFELQSUHVV 
6WHKIHVW(%RXZPDQ/YDQ9XXUHQ'3GHQ(O]HQ0*-(LFNKRXW%DQG.DEDW3&OLPDWHEHQH¿WV
of changing diet. Climatic Change 95, 83-102.
Thornton P.K. and Herrero M. 2010. The potential for reduced methane and carbon dioxide emissions from livestock
DQGSDVWXUHPDQDJHPHQWLQWKHWURSLFV31$6
7LWWRQHOO3DQG*LOOHU.:KHQ\LHOGJDSVDUHSRYHUW\WUDSV7KHSDUDGLJPRIHFRORJLFDOLQWHQVL¿FDWLRQLQ
$IULFDQDJULFXOWXUH)LHOG&URSV5HVHDUFK
Van Ittersum, M., Cassman, K., Grassini, P, Wolf, J., Tittonell, P. and Hochman, Z. 2013. Yield gap analysis with local
to global relevance – a review. Field Crops Research 143, 4-17.

34 Energy and protein metabolism and nutrition in sustainable animal production

Role of animal products in feeding the planet
J.E. Hermansen, M.T. Knudsen and T. Kristensen
$DUKXV8QLYHUVLW\%OLFKHUV$OOp7MHOH'HQPDUN[email protected]

Abstract
Livestock products contribute today by approximately 13% of calories and 28% of protein to the
global human population with a huge difference between developed, transition and developing
countries. As a consequence of increased consumption per capita and a larger population, it is
projected that global meat consumption will increase by 40% in 2030 and 70% in 2050. This has
large resource use implications and furthermore the foreseen increased production is to happen within
the context of growing scarcity of natural resources and challenges posed by climate change. All
this put challenges on the livestock production systems. The major growth in livestock production
will take place in intensive industrialized systems very much focused on poultry and pork. It will
be important that the feeds can be based on very high yielding new crops that serve both as feed
and other purposes in order to reduce land use requirements. This asks for new knowledge and
technology that can transform the biomass from the high yielding crops to high quality feed. Also,
it will be very important that the energy left in the manure is recovered to substitute fossil energy
or energy produced from new biomass. Still, a major part of dairy and beef production will take
SODFHLQPL[HGV\VWHPVZKLFKLQVRPHSDUWVRIWKHZRUOGDUHUDWKHULQHI¿FLHQWLQWKHLUXVHRIQDWXUDO
UHVRXUFHV6XFKV\VWHPVKDYHWRLQFUHDVHHI¿FLHQF\KDYLQJPRUHRIWKHIHHGFRQVXPHGWUDQVIRUPHG
into food instead of feed requirements for animal maintenance. Food from extensive grazing systems
will in particular be important to support the nutritional needs of populations in food-insecure areas.
The major challenge here is to stop land degradation and restoring the functioning of the grassland,
including grassland role to sequester carbon and contribute to other eco-system services.

Introduction
Although the expressions ‘feeding the planet’ and ‘food security’ may be perceived as two distinct
challenges, it is clear that when considering animal products, they are two sides of the same
coin. Animal products clearly have a role in ensuring food security – food security including the
dimensions: availability, access, stability and utilization for all human communities. At the same
time, however, it is beyond arguing that the ecological costs of animal products typically are much
higher than for plant products. While in a typical Northern European diet it is estimated, that animal
products covers approximately 25% of the calorie coverage of the diet, the animal products are
UHVSRQVLEOHIRUPRUHWKDQRIWKHFDUERQIRRWSULQWRIWKHW\SLFDOGLHW +HUPDQVHQDQG2OHVHQ
2009). At the same time it is well recognized that production of feed for livestock is a major driver
of land use change (Steinfeld et al. SXWWLQJSUHVVXUHRQJOREDOELRGLYHUVLW\DQGHQKDQFH
emissions of carbon dioxide to the atmosphere as a consequences of transforming grassland, forest,
DQGVDYDQQDWRDUDEOHODQG(J81(3  HVWLPDWHVDQLQFUHDVHLQVR\EHDQSURGXFWLRQIURP
square km in 1992 to almost 10.000 square km in 2009. Thus, there are good reasons to focus on the
role of animal products in our diets and in relation to the overall environmental concerns of today.

Several developments in addition tend to aggravate these environmental concerns. Thus, besides
the growth in global population, during the latest 20 years the overall food production has increased
considerable more than the growth in population to ensure a better food supply and this trend is
expected to be maintained (UNEP, 2011). Further, the consumption of meat has increased considerable
more than the population growth. Figure 1 shows the global dietary change towards higher per capita
PHDWFRQVXPSWLRQLQWKHSHULRGIURPWR )$267$7 7KHPHDWFRQVXPSWLRQSHU
FDSLWDLQ(XURSHDQG1RUWK$PHULFDWKRXJKÀDWWHQLQJRXWLVVWLOOFRQVLGHUDEO\KLJKHUWKDQLQ$VLD
and Africa. Recently, the meat consumption per person has shown a larger increase in Asia than in

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 35
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_2, © Wageningen Academic Publishers 2013
 140

120 Northern America

(total)
(kg per capita per year)

100
Meat consumption

Brazil
80 Europe (total)

60 China
World (total)
40
Asia (total)
20
Africa (total)
0
1961
1963
1965
1967
1969
1971
1973
1975
1977
1979
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
2001
2003
2005
2007
2009
Figure 1. Meat consumption per capita per year on average in the world, in Europe, North America,
$VLD$IULFDDQGLQ&KLQDDQG%UD]LO )$267$7 

Africa and especially China has shown a remarkable increase during the last 30 years. Projections
of future meat consumption predict that China by 2030 will have a per capita meat consumption of
approx. 75 kg/person/year and Brazil is predicted to have a per capita meat consumption of almost
90 kg/person/year by 2030 (Msangi and Rosegrant, 2011). Thus, the Internal Food Policy Research
Institute (IFPRI) predicts that over the next decades, virtually all growth in demand for meat will
come from the transition economies and the developing world (IFPRI, 2012).

At the same time, there is competition for land for biomass production for other purposes than
food, in particular energy, which contributes to the overall pressure on land resources. The overall
transformation of land to agricultural land ranged between 5-10 million ha per year during the latest
40 years with a particular increase in the latest years.

All this being said, livestock contribute with around 13% of global calories for human consumption,
but approximately 28% of protein through provision of meat, milk and eggs (FAO, 2011). In the
developed world the numbers are 20 and 48%, respectively. While in the wealthiest part of the world,
the protein coverage is easily met, and actually can be met for the major part by animal protein, this
is not the case in the poorest part of the world. In these regions lack of calories and protein seems
WRJRKDQGLQKDQGDQGKHUHOLYHVWRFNSURGXFWVRIWHQSOD\DVLJQL¿FDQWUROHLQHQVXULQJSURWHLQDV
well as other essential nutrient. Even in small amounts livestock products contribute to ensure the
proper nutrition of poor families due to the content and bioavailability of important micro-nutrients.
In addition, livestock has a role in supporting food security in vulnerable small-holder populations,
through delivering stability of food and income. FAO (2012) estimates that close to one billion of
the world’s poorest people rely on livestock for their livelihoods.

7KXVDQLPDOSURGXFWVKDYHDVLJQL¿FDQWUROHLQIHHGLQJWKHSODQHWEXWWKHSURGXFWLRQLVFKDOOHQJHG
by ecological side-effects which need to be addressed. These side-effects naturally depend on type
of animal production system and how they are managed.

Global livestock production and livestock production systems

Livestock are produced in different ways in different parts of the world. Thus, the contribution to
food supply and food security as well as the ecological impact of production differs markedly among
different livestock products and livestock systems. In Table 1 is shown the global production and the

36 Energy and protein metabolism and nutrition in sustainable animal production

7DEOH&KDQJHVLQJOREDOOLYHVWRFNSURGXFWLRQWRWDODQGSHUSHUVRQWR DIWHU)$2 

Production (million tonnes) Production per person (kg)

 2007   2007 

Pig meat 33.9 100.0 294% 9.8 14.9 152%

Beef and buffalo meat   180%  9.8 93%
Eggs, primary 18.2  353% 5.3  183%
Milk, total 381.8  178% 110.3 102.0 92%
Poultry meat 12.4 88.0 711%  13.2 
Sheep and goat meat  13.1 202% 1.9 2.0 105%

VXSSO\SHUFDSLWDLQFOXVLYHWKHFKDQJHVLQWKH\HDUSHULRGIURPWR)RUDOOSURGXFWV
the production increased by a factor 2-7 in that period, most pronounced for poultry meat but also
very pronounced for pigs and eggs. Also per capita production increased except for beef and milk,
which were almost unchanged. Thus, from a food supply point of view, poultry- and pig products
became much more important during that period. Projections estimate total meat consumption in
RIPLOOLRQWRQ±DQLQFUHDVHRIIURPWKHQXPEHUVJLYHQLQ7DEOH

The increasing demand for meat on the world markets will mainly be supplied by Brazil, USA,
Oceania and Europe. In an international trade perspective, Brazil is expected to play a major role
being a strong net exporter of livestock products (FAO, 2012) and has currently the world’s largest
export of both beef and chicken meat (FAOSTAT, 2013).

Data on what type of production from different production systems are less updated, but Table 2
gives a good picture of the relative importance of the different productions systems for each product.
Grazing systems include both extensive grazing system and intensive grazing systems. The extensive
systems are prevailing in dry areas that are marginal for crop production and sparsely populated
(Southern Africa, central and western Asia, Australia and western North America) and these systems
provides for about 7% of beef production and 12% of sheep and goat meat production. The intensive
grazing systems appear in the temperate zones where high quality forages can be grown (most of
Europe, North America and South America) and account for about 17% of total beef and sheep and
goat meat production.

The mixed farming systems are systems where cropping and livestock production are interlinked
activities and where at least 10% of the total value of production comes from non-livestock activities.
Most of the milk and beef produced comes from such systems – the rain fed systems prevailing

7DEOH*OREDOOLYHVWRFNSURGXFWLRQDYHUDJHE\SURGXFWLRQV\VWHPWRPLOOLRQWRQ DIWHU
)$2 

Grazing Rainfed mixed Irrigated mixed Landless/industrial Total

Beef  29.3 12.9 3.9 

Mutton 3.8 4.0 4.0 0.1 11.9
Pork 0.8 12.5 29.1 52.8 95.2
Poultry meat 1.2 8.0 11.7 52.8 73.7
Milk 72 319 204 - 594
Eggs 0.5  17.1 35.7 58.9

Energy and protein metabolism and nutrition in sustainable animal production 37

in temperate Europe and North America as well as sub-humid regions of tropical Africa and Latin
America, and the irrigated systems prevailing in East and South Asia. It is worth mentioning also,
that approximately 50% of the world’s cereal production in developing countries comes from mixed
systems (Herrero et al., 2010), and as highlighted by Smith et al. (2013) by providing manure and
±LQWKHFDVHRIPDQ\VPDOOKROGHUV\VWHPV±GUDXJKWSRZHUIRU¿HOGRSHUDWLRQVWKHUHE\VXSSRUWLQJ
the production of this staple food.

7KHLQGXVWULDOV\VWHPVDUHGH¿QHGDVWKRVHV\VWHPVWKDWUHFHLYHDWOHDVWRIWKHIHHGIURPRWKHU
enterprises. The major part of poultry are produced in such systems often found near large urban
centres, and thus relying on global feed resources, and slightly more than half of pork production
takes place in such systems.

Ecological impacts related to livestock production

The livestock production is related to a range of ecological impacts affected by the production system
DQGLWVHI¿FLHQF\:KLOHQRWGHVLJQHGIRULWWKHFDUERQIRRWSULQWRIOLYHVWRFNSURGXFWVFDQEHDJRRG
indicator of the overall impact, since often a good correlation exist between the carbon footprint
DQGLPSDFWVOLNHHXWURSKLFDWLRQIRVVLOHQHUJ\XVHDQGDFLGL¿FDWLRQ7KHUHDVRQLVWKDWWKHFDUERQ
footprint aggregates the CO2 emission related to use of fossil energy, the global warming effect of
emission of nitrogenous substances from feeds and animals, and the methane emissions related to
enteric digestion as well as from manure management, both of which are related to overall feed use.
Thus, feed use is a very important aspect when considering the carbon foot print of livestock products
(Hermansen and Kristensen, 2011). This is not to say that the carbon foot print is the only relevant
indicator, but it helps to get an overview in the broad sense. Furthermore, the carbon footprint is a
highly relevant indicator in policy for consideration of combating climate change.

Table 3 shows typical numbers for carbon footprint and land requirements mainly representing
relatively productive Western European systems. Red meat clearly has a higher carbon footprint

7DEOH/DQGUHTXLUHPHQW P2 DQGFDUERQIRRWSULQW NJ&22HT RISURGXFWVSHUNJSURGXFW6RXUFHV

IRUFDUERQIRRWSULQW'H9ULHVDQG'H%RHU  1JX\HQet al.  ,PSRUWDQFHRIL/8&
after Audsley et al.   aNJ&22/m2 DQG6FKPLGWet al.   &22/m2

m2 Carbon footprint without and with accounting

for indirect land use changes (iLUC)

Without iLUC To be added with iLUC

Milk 1-2 ~1 +0.2-1.5

Egg 4-7 4-5 
Broiler 5-7 2-4 +0.8-5
Pork  3-5 +0.9-5
Beef 15-45
– Intensive bulls 12 mo. ~17 ~12 +3-13
– Steers 24 mo. ~23 ~22 +2-10
‘Productive land’ ~13
‘Less productive land’ ~10
– Suckler system ~43 ~25 +2-10
‘Productive land’ ~13
‘Non productive land’ ~30

Meat is given per kg weight of carcass.

38 Energy and protein metabolism and nutrition in sustainable animal production

than ‘industrialized’ poultry and pork. The carbon footprint of the red meat, however, is very must
dependent on how the beef is produced – intensive beef from one year old calves being produced
with the lowest carbon footprint whereas the suckler system results in the highest carbon footprint
of around 27 kg CO2eq per kg carcass. For reference, Desjardins et al. (2012) in a review of carbon
footprint of beef found that beef cattle from Canada, US, EU, and Southern Australia had similar
carbon footprints on average but with large regional differences, whereas the carbon footprint from
cattle grazing under extensive conditions, such as Northern Australia and Brazil, were likely to have
a higher carbon footprint.

In the numbers given above no consideration was given to how the production affects land use
changes. As already mentioned requirements for feed is a main driver of land use changes, which
in turn is a considerable source of CO2 emissions. Thus, it is estimated that such continues land use
changes are responsible for 12% of the world’s yearly CO2 emissions (World Resources Institute,
2009), because huge stocks of carbon in vegetation and soil are released to the atmosphere. Some
attempts have been made to include indirect land use changes in the assessment of the carbon footprint
of the land based products. Eg. Audsley et al. (2009) argue that all land use results in a pressure
on the global (limited) land resources, and consequently that all crops have to carry this burden
proportionally. Audsley et al. (2009) found that on average every cultivated ha of land resulted in
an emission of 1.43 ton of CO2 as a results of indirect land use changes following that occupation.
This corresponds to a load of 140 g CO2 per occupied m2 globally. In contrast, Schmidt et al. (2012)
modeled the marginal effect of including 1 extra ha land for cropping, and found that this effect
results in on average 7.83 t CO2eq per occupied ha (depending on the productivity of the land used)
or as a global average 783 g CO2eq per m2.

In Table 3 we illustrate the impact on the carbon footprint of including this effect of indirect land
use changes using the value from Audsley et al. (2009) as the most conservative estimate and the
results from Schmidt et al. (2012) as the highest estimate. Using the lowest estimate means that the
carbon footprint of most products are increased by 25-30%, while using the highest value increase
the carbon foot print by more than 100% for the non-grazing based systems, and for the grass
based systems the increase is about 50%. The estimates by Schmidt et al. (2012) seems close to
the values found by Laborde (2011), which are used to evaluate the consequences of increased use
of land for energy purpose as recommended for use in the EU. Thus, in a situation where the meat
production does expand rapidly globally, it seems most reasonable to include this high value in the
overall assessment.

In the assessments given in Table 3, we distinguished between use of productive cropland (including
cultivated grassland) and less productive grass land that in fact does not represent a resource for
other biomass production purposes. This separation of land use in the different systems is of course
QRWXQLYHUVDOEXWZDVVSHFL¿FIRUWKHFDVHLQYHVWLJDWHG+RZHYHUWKLVGLVWLQFWLRQKDVDKXJHLPSDFW
on the assessment of the environmental impact as illustrated in Table 3, when indirect land use
changes are included.

Like there is a close connection between land use and impacts on global warming, when taking
indirect land use changes into account, the same is true when considering impacts on biodiversity.
Impacts on biodiversity are challenging to describe satisfactorily. However, some concepts are present
to support assessment of land use related impacts on biodiversity. Thus, De Schryver et al. (2010)
established a framework where the damage to biodiversity was expressed as Potential Disappeared
Fraction (PDF) of vascular species compared to a baseline that would be the natural vegetation of
the area in question. This characterization factor is unit-less and can be aggregated according to
different types of m2 of land occupied for a given product. Based on UK conditions they established
the PDF’s as given in Table 4.

Energy and protein metabolism and nutrition in sustainable animal production 39

It is clear from Table 4 that while use of land for intensive cultivation has a huge negative impact
on biodiversity, use of land for grazing, and in particular grazing on low fertilized land has a much
lower negative impact on biodiversity and may even enhance biodiversity compared to a semi natural
forest. Thus, such aspects need to be brought into the picture when assessing land use requirement
of different livestock products, but as also mentioned the methodology is only in its virgin stage. It
is obvious, that a huge reduction in complexity takes place when reducing the biodiversity aspect to
the species richness of vascular plants, but on the other hand this indicator clearly catches important
elements of biodiversity, since most often there is a correlation between species richness of vascular
plants and species richness of insects and mammals (e.g. birds).

While no doubt the industrial livestock systems to a wide extent is based on feed that compete
with foods for humans directly or indirectly, and in this way is in a land competition situation and
causing land use changes, this is not always the case for other systems. Thus, systems that primarily
depend on grazing compete less with resources that could be used for human food, and such systems
produce about 12% of global milk and 9% of global meat production (FAO, 2011). Considering the
contribution of livestock products to the protein supply, FAO estimated the edible protein output/
input ration for different countries. In countries with huge landless production of monogastrics,
OLNH86$DQG*HUPDQ\WKLVUDWLRQZDVOHVVWKDQ  LQ%UD]LODQG&KLQDDSSUR[LPDWHO\
in India around 4 and in Mongolia and East African countries beyond 10 (FAO, 2011). In a huge
country like India this positive protein balance supplied by smallholder livestock systems amount
WRPLOOWRQZKLFKFDQEHFRPSDUHGWRDSURWHLQµGH¿FLW¶RIOLYHVWRFNLQ86$DQG*HUPDQ\RI
approximately 8.9 mill ton.

The estimations as mentioned above are related with huge uncertainties, but it is important to have
WKLVRYHUDOOSLFWXUHLQPLQGZKHQFRQVLGHULQJWKHHI¿FLHQF\RIOLYHVWRFNSURGXFWLRQLQIHHGLQJWKH
planet. Also, as pointed out by Smith et al. (2013) in most mixed crop-livestock system used by
small holders the main animal feed consists of crop residues. When considering the challenges and
prospects in the future, it is important that these features of such systems are not deteriorated, but
in fact enhanced.

7DEOH'DPDJHWRELRGLYHUVLW\H[SUHVVHGDV3RWHQWLDO'LVDSSHDUHG)UDFWLRQ 3') RIYDVFXODU

VSHFLHVIRUGLIIHUHQWW\SHVRIODQGXVH PRGL¿HGDIWHU'H6FKU\YHUet al 

Type of land use Median Min (2.5%) Max (2.5%)

Baseline (semi-natural forest) 0.00

Grazing organic infertile grassland -0.33  0.13
Grazing organic fertile grassland -0.01 -0.18 0.15
Grazing less intensive fertile grassland  0.14 0.52
Organic arable land  0.15 0.51
Less intensive arable land 0.44 0.31 0.54
Intensive woodland 0.55 0.44 
Grazing intensive fertile grassland   0.72
Intensive tall grassland 0.70  0.77
Intensive arable land 0.79 0.73 0.83

40 Energy and protein metabolism and nutrition in sustainable animal production

Challenges and prospects for different livestock production systems
Taking the point of departure in the different livestock systems presented in Table 2, the challenges
and opportunities seem to differ substantially as also highlighted in the ‘Global Agenda for Action
in support of sustainable livestock sector development’ (FAO, 2013).

Landless/industrial systems

Looking at the trend in production of different types of livestock, the increased demand for meat
globally seems to a wide extent to be covered by pork and poultry products. Today these production
systems are by large based on feed that heavily compete with human food (or produced on land
ZKLFKSRWHQWLDOFRXOGEHXVHGIRUKXPDQIRRG $IXUWKHUH[SDQVLRQLVIRUHVHHQWRKDYHVLJQL¿FDQW
negative consequences in relation to land use changes and the connected effects such as global
warming and reduction of global biodiversity, and to be in sharp competition with the request for
biomass for other purposes. On the other hand given the expected growth in human wealth in the
long term in transition and developing countries, this increased demand will no doubt substantiate,
DQGWKXVWKLVIRUPRIOLYHVWRFNSURGXFWLRQPXVWEHIRUHVHHQWRJHWDVWHDG\PRUHVLJQL¿FDQWUROHLQ
supplying the growing population’s nutritional needs.

,QDGGUHVVLQJWKLVFKDOOHQJHWKHSURGXFWLRQHI¿FLHQF\QHHGVWREHFRQVLGHUHGIXUWKHU/RRNLQJDW
the livestock part in itself, substantial improvements have been obtained in reducing feed use per
kg of meat produced during the last decades. This is still an important issue, but two issues should
be given more focus: the feed stock supply and the waste treatment.

5HJDUGLQJIHHGVWRFNDWWKHPRPHQWVLJQL¿FDQWHIIRUWVDUHSXWKRZEHVWWRXVHWKHE\SURGXFWVIURP
the biofuel industry as feed, like Dried Distillers Grains with Solubles (DDGS), which constitute
DVLJQL¿FDQWIHHGVRXUFH7KLVLVYHU\LPSRUWDQWLQDLPLQJIRUORZHULQJWKHHQYLURQPHQWDOLPSDFW
of the food produced, but the main feed needed still seems to be cereals and soybean, both crops
with a relatively low yield of biomass per unit of land occupied. In temperate areas of the world
mixtures of grass and legumes typically can produce 50% or more of both dry biomass and protein
per ha compared to cereals, and – if legumes are included – typically with less resource use like
fertilizer and pesticides. This biomass it not directly suited for monogastrics, but given the demand
IRUELRPDVVIRUDUDQJHRISXUSRVHVWKHLQWURGXFWLRQRIELRUH¿QHU\SURFHVVHVPD\WUDQVIRUPSDUWRI
this biomass to high quality feed, leaving other parts of the biomass for other high quality products
or biofuel. It is a challenge for animal nutritionists to contribute with insights and options for new
feeds based on very high yielding crops that are not immediately suitable as feed.

Regarding waste treatment, still in many places the manure nutrients (50-70% of the input) are not
HI¿FLHQWO\XVHGIRUSODQWSURGXFWLRQEXWUHSUHVHQWDQHQYLURQPHQWDORYHUORDG)XUWKHUDSSUR[LPDWHO\
RIWKHHQHUJ\LQSXWLVSUHVHQWLQWKHPDQXUH(I¿FLHQWXVHRIWKLVUHVRXUFHIRUELRJDVFRQVWLWXWHV
a major potential for counteracting the emission of greenhouses gasses of producing livestock
products. Thus, we estimated that utilizing manure in the form of slurry from the pig production for
biogas potentially reduced the carbon footprint of the pork meat by 30%, partly as a result of energy
recovery and partly because less a greenhouse gas emissions were released from the manure (Nguyen
et al. 6XFKDQHI¿FLHQWXWLOL]DWLRQUHTXLUHVDJRRGLQIUDVWUXFWXUH+RZHYHULQSDUWLFXODUIRU
the systems in consideration with bigger and bigger enterprises, and mainly systems based on liquid
manure, this should be possible to establish.

Mixed systems

A very huge part of the global food supply is produced in mixed systems, not least for milk and beef.
One can distinguish between large scale mixed systems in temperate areas of the developed world

Energy and protein metabolism and nutrition in sustainable animal production 41

and small scale mixed systems in the developing world. The large scale mixed systems in fact also
often rely to a wide extent on feed that compete with human food, and in the development of such
V\VWHPWRIXUWKHUIXO¿OOWKHLUUROHLQJOREDOIRRGVXSSO\WKHVDPHDVSHFWVDVGHVFULEHGIRUODQGOHVV
systems should be addressed.

Regarding small scale mixed systems, the main challenge is with the words from the Global Agenda
RI$FWLRQLQVXSSRUWRIVXVWDLQDEOHOLYHVWRFNGHYHORSPHQW )$2 WRµ&ORVLQJWKHHI¿FLHQF\
gap’. It is recognized that a number of technological possibilities or production concepts are yet
not fully exploited in such systems. Mixed systems in the developing world is estimated to supply
RIWKHEHHIRIWKHPLONDQGRIWKHODPEPHDW 7DUDZDOLet al., 2011) in these regions,
where the demand for animal products (per capita and not least in total due to increased population)
are expected to increase markedly. Thus, it is very important that the production is better optimized.
Taraweli et al. (2011) pointed out a number of issues to be dealt with to allow a better optimization
of such systems. Main issues are to allow for keeping fewer animals with higher productivity, thus
turning more feed energy into production instead of maintenance. This includes better feeding
and understanding of importance of small feed supplements to local feed sources, use of better
genotypes, and improved multipurpose crops, including legumes. While realizing that such changes
are not easy since livestock has other important roles than as direct food in such communities, it is
IRXQGLPSHUDWLYHWKDWLQWHQVL¿FDWLRQKDVWRKDSSHQZKLFKUHVXOWVLQPRUHIRRGSURGXFHGZLWKRXW
compromising use of natural resources.

Grazing systems

Approximately 50% of the land used for livestock production is extensively used grassland for
grazing. While the supply in absolute terms to global animal based foods are limited, these systems
support nutrition and livelihood of many vulnerable and food insecure people. In fact livestock
provide more food security in arid regions than do crop production (Kratli et al., 2013). Due to
climatic conditions, it is not likely that the biomass yield from such systems can be increased,
except in cases where the land was been partly degraded. It is estimated that at least 20% of such
land are degraded due to inappropriate management resulting in lower biomass yield and high
erosion. Thus the challenge for the extensive grassland based systems is on the one hand to improve
grazing management by adapting the stocking rate to the carrying capacity, and hereby avoid further
GHJUDGDWLRQ,QIDFWEHWWHUJUD]LQJSUDFWLFHPD\DVDUHVXOWVKDYHVXFKDUHDVWRFRQWULEXWHVLJQL¿FDQWO\
to global carbon sequestration (Wilkes et al., 2012).

Conclusion
While the consumption of livestock products seems to have reached a plateau per person in developed
countries, it is increasing rapidly in transition and developing countries. As a consequence of increased
consumption per capita and a larger population, it is forecasted that global meat consumption will
increase considerably. While the livestock sector provides high value food and other social functions,
its resource use implications are however large. Livestock uses the major part of the agricultural
land through grazing and consumption of feed crops, and plays a major role in emissions related to
climate change and reduced biodiversity, which are all major concerns of today.

To overcome the challenges mentioned, it will be very important that the feeds can be based on
very high yielding new crops that serve both as feed and other purposes in order to reduce land use
requirement, and that animal nutritionist are active in exploring these possibilities. For dairy and beef
SURGXFWLRQWDNLQJSODFHLQPL[HGV\VWHPVVRPHRIZKLFKDUHUDWKHULQHI¿FLHQWLQWKHLUXVHLIQDWXUDO
UHVRXUFHVLWLVLPSHUDWLYHWRLQFUHDVHHI¿FLHQF\KDYLQJPRUHRIWKHIHHGFRQVXPHGWUDQVIRUPHGLQWR
food instead of feed requirements for animal maintenance. Also extensive grazing systems need to

42 Energy and protein metabolism and nutrition in sustainable animal production

be adapted to ensure stop of land degradation and enhance the role of grassland to sequester carbon
and contribute to other eco-system services.

References
Audsley, E., Brander, M., Chatterton, J., Murphy-Bokern, D., Webster, C. and A. Williams, 2009. How low can we
go? An assessment of greenhouse gas emissions from the UK food system and the scope for reducing them by
2050. WWF-UK.
De Schryver, A.M., Goedkoop, M.J., Leuven, R.S.E.W. and M.A.J. Huijbregts, 2010. Uncertainties in the application of
the species area relationship for characterisation factors of land occupation in life cycle assessment. International
-RXUQDORI/LIH&\FOH$VVHVVPHQW
Desjardins, R.L., Worth, D.E., Verge, X.P.C., Maxime, D., Dyer, J. and D. Cerkowniak, 2012. Carbon Footprint of
Beef Cattle. Sustainability 4, 3279-3301.
De Vries, M., and I.J.M. de Boer, 2010. Comparing environmental impacts for livestock products: A review of life
cycle assessments. Livestock Science, 128, 1-11.
EC, 2012. EC (draft) Commission staff working document – Impact Assessment on indirect land use change related
to biofuels and bioliquids, 125 p.
FAO, 2011. World livestock 2011 – livestock in food security. Rome, FAO 115 pp.
FAO, 2012. Livestock sector development for poverty reduction: An economic and policy perspective –Livestock’s
many virtues. FAO, Rome.
)$2:RUOGIRRGDQGDJULFXOWXUHLQUHYLHZ,Q7KH6WDWHRI)RRGDQG$JULFXOWXUHS
FAO, 2013. Global Agenda for Action http://www.livestockdialogue.org/.
FAOSTAT, 2013. FAOSTAT. Food and Agriculture Organization of the United Nations (FAO). Online at http://faostat.
IDRRUJVLWHGHIDXOWDVS[DQFRU.
Hermansen, J.E. and Kristensen, T., 2011. Management options to reduce the carbon foot print of livestock products.
Animal Frontiers 1, 13-19.
Hermansen, J.E. and Olsen, J., 2009. (Climate impacts from Danish agriculture and food production (In Danish))
9DQGRJ-RUG
Herrero, M., Thornton, P.K., Notenbaert, A.M., Wood, S., Msangi, S., Freeman, H.A., Bossio, D., Dixon, J., Peters,
M., van de Steeg, J., Lynam, J., Parthasarathy Rao, P., MacMillan, S., Gerard, B., McDermott, J., Seré, C. and
M. Rosegrant, 2010. Smart investments in sustainable food production: Revisiting mixed crop-livestock systems.
Science 327, 822-825.
IFPRI, 2012. The meat of the issue. IFPRI Insights Magazine, Magazine of the International Food Policy Research
,QVWLWXWH2FWREHU2QOLQHDWhttp://insights.ifpri.info/2012/10/the-meat-of-the-issue-2/.
Krätli, S., Huelsebusch, C., Brooks, S. and B. Kaufmann, 2013. Pastoralism: A critical asset for food security under
global climate change. Animal Frontiers 3, 42-50.
Laborde, D., 2011. Assessing the Land Use Change Consequences of European Biofuel Policies. Final Report, October
2011, IFPRI, 111 pp.
Msangi S. and M.W. Rosegrant, 2011. Feeding the future’s changing diets. Implications for agriculture markets,
nutrition, and policy. 2020 Conference: Leveraging Agriculture for Improving Nutrition and Health. February
10-12, 2011, New Delhi, India. 12 p.
Nguyen, T.L.T., J.E. Hermansen, Horsted, K., 2011. Design of innovative pork chains with low environmental load.
'HOLYHUDEOHVXEPLWWHGLQWKHSURMHFW,PSURYLQJWKHTXDOLW\RISRUNDQGSRUNSURGXFWVIRUWKHFRQVXPHUSS
Nguyen, T.L.T., J.E. Hermansen, and L. Mogensen, 2010. Environmental consequences of different beef production
V\VWHPVLQWKH(8-RXUQDORI&OHDQHU3URGXFWLRQ
Nguyen, T.L.T., Hermansen, J.E. and L. Mogensen, 2010. Fossil energy and GHG saving potentials of pig farming in
WKH(8(QHUJ\3ROLF\
Nguyen, T.L.T., Hermansen, J.E. and L. Mogensen, 2011. Environmental assessment of Danish Pork. Internal Report,
Faculty of Agricultural Sciences, Aarhus University, Aarhus, Denmark. 34 pp.
Schmidt, J.H., Reinhard, J. and B. Weidema, 2012. A model of indirect land use change. 8th international conference
on LCA in the agri-foodsector, Rennes, France, 2-4 October 2012.

Energy and protein metabolism and nutrition in sustainable animal production 43

Smith, J., Sones, K., Grace, D., MacMillan, S., Tarawali, S. and M. Herrero, 2013. Beyond milk, meat, and eggs: Role
RIOLYHVWRFNLQIRRGDQGQXWULWLRQVHFXULW\$QLPDO)URQWLHUV
6WHLQIHOG+*HUEHU3:DVVHQDDU7&DVWHO95RVDOHV0DQG&GH+DDQ/LYHVWRFN¶VORQJVKDGRZ±
environmental issues and options. FAO. 390 pp.
Tarawali, S., Herrero, M., Descheemaeker, K., Grings, E. and M. Blummel, 2011. Pathways for sustainable development
of mixed crop livestock systems: Taking a livestock and pro-poor approach. Livestock Science 139, 11-21.
UNEP, 2011. Keeping track of our changing environment. From Rio to Rio +20. United Nations Environmental
3URJUDPPH1DLUREL.HQ\DSSwww.unep.org.
Wilkes, A., Solymosi, K. and T. Tennigkeit, 2012. Options to support grassland restoration in the context of climate
FKDQJHPLWLJDWLRQ5HSRUWFRPPLVVLRQHGE\)$2)UHLEXUJ*HUPDQ\SS
World Resources Institute, 2009. World Greenhouse Gas Emissions: 2005. Online på http://www.wri.org/chart/world-
greenhouse-gas-emissions-2005.

44 Energy and protein metabolism and nutrition in sustainable animal production

Part 1. Energy and protein interactions, ruminants
Challenges in ruminant nutrition: towards minimal nitrogen losses in cattle
J. Dijkstra1, C.K. Reynolds2, E. Kebreab, A. Bannink, J.L. Ellis1, J. France and A.M. van Vuuren
1$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\  $+ :DJHQLQJHQ WKH 1HWKHUODQGV
[email protected]
2Animal Science Research Group, School of Agriculture, Policy and Development, University of
5HDGLQJ5HDGLQJ5*$58QLWHG.LQJGRP
'HSDUWPHQWRI$QLPDO6FLHQFH8QLYHUVLW\RI&DOLIRUQLD'DYLV&$86$
:DJHQLQJHQ85/LYHVWRFN5HVHDUFK32%R[$%/HO\VWDGWKH1HWKHUODQGV
Department of Animal and Poultry Science, University of Guelph, Guelph ON, N1G 2W1, Canada

Abstract
5XPLQDQWVSOD\DNH\UROHLQKXPDQIRRGSURGXFWLRQE\FRQYHUWLQJ¿EHUULFKSODQWUHVRXUFHVWKDW
humans cannot (or choose not to) consume into high-quality food that humans can eat. However,
this conversion causes unavoidable losses of nitrogen (N) in feces and urine from ruminants that
may become an environmental burden, in particular nitrate (NO3-) leaching, ammonia (NH3)
volatilization and nitrous oxide (N2O) emissions. The aim of this paper is to identify the maximal
WKHRUHWLFDO1HI¿FLHQF\DWWKHDQLPDOOHYHODQGWKHFKDOOHQJHVDQGRSSRUWXQLWLHVWRDFKLHYHWKLV
PD[LPDO1HI¿FLHQF\7KLVLVGRQHYLDVWULYLQJIRUWKHORZHVWSRVVLEOH1H[FUHWLRQLQXULQHDQG
feces, and for purposes here, with a focus on dairy cattle. Inevitable N losses in dairy cattle include
losses associated with urinary excretion of urea synthesized from ammonia produced in the rumen;
undigested microbial protein excreted in feces; microbial nucleic acids synthesized in the rumen
and excreted mainly in urine; fecal and urinary excretion resulting from endogenous secretions; and
urinary excretion related to maintenance and milk protein synthesis. The theoretical upper limit of
1XVHHI¿FLHQF\LQDGDLU\FRZSURGXFLQJNJIDWDQGSURWHLQFRUUHFWHGPLONGLV+LJKHU
HI¿FLHQFLHVPD\EHDFKLHYHGEXWWKHVHUHTXLUHPDMRULQSXWVRIKXPDQHGLEOHUHVRXUFHV7KHSUHVHQW
analysis demonstrates there is little or no scope to reduce N losses related to microbial nucleic
acid synthesis, recycling of N to the rumen, intestinal digestion of microbial protein, and animal
PDLQWHQDQFHUHTXLUHPHQWV6WUDWHJLHVWRUHGXFH1ORVVHVDQGLPSURYH1HI¿FLHQF\VKRXOGIRFXVRQ
DQRSWLPDOVXSSO\RIUXPHQGHJUDGDEOH1DQGRSWLPDOHI¿FLHQF\RIXWLOL]DWLRQRIDEVRUEHGDPLQR
DFLGVIRUPLONSURWHLQV\QWKHVLV7RLPSURYH1HI¿FLHQF\LQWHJUDWLRQEHWZHHQSURWHLQDQGHQHUJ\
metabolism is essential, and energy and protein should be considered together rather than as two
GLVWLQFWHQWLWLHV$PDMRUFKDOOHQJHLQVWUDWHJLHVWRRSWLPL]HKLJK¿EHUGLHWVIRUKLJKPLON1HI¿FLHQF\
will be to avoid increases in enteric methane production associated with these dietary strategies.

Introduction
Global consumption of dairy products and beef is projected to rise by well over 50% by 2050
(FAO, 2011). In the face of competing demands for resources, ruminants play a key role in human
food production in converting plant resources that humans cannot (or choose not to) consume into
high-quality food that humans can eat. For dairy cattle, the return on human edible protein inputs
(calculated as the output of human edible protein in products compared with human edible protein
LQSXWZLWKIHHG LVODUJHUWKDQ UDQJHWRLQ¿QLWHLQ¿QLWHLIGLHWFRQWDLQVQRKXPDQHGLEOH
protein) (reviewed by Dijkstra et al., 2013a), indicating that dairy cattle add to the total human
IRRGVXSSO\)RUEHHIFDWWOHSURWHLQHI¿FLHQFLHVRQDKXPDQHGLEOHEDVLVDUHDOVRRIWHQODUJHUWKDQ
EXWDUHPRUHYDULDEOH UDQJHWRLQ¿QLWH WKDQIRUGDLU\FDWWOH7KHDELOLW\RIUXPLQDQWVWRWXUQ
¿EURXVIHHGUHVRXUFHVLQWRHGLEOHDQLPDOIRRGRIKLJKELRORJLFDOYDOXHLVOLNHO\WREHFRPHRIJUHDWHU
VLJQL¿FDQFHLQWHUPVRIJOREDOKXPDQIRRGSURGXFWLRQDVWKHSRSXODWLRQRIWKHSODQHWDQGGHPDQG
for human-edible plant resources increases rapidly.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 47
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_3, © Wageningen Academic Publishers 2013
Despite the positive contribution of ruminants in terms of food security, there is a widespread
SHUFHSWLRQWKDWWKHSURGXFWLRQRIPLONRUPHDWLVDQLQHI¿FLHQWZD\WRXVHQDWXUDOUHVRXUFHV,Q
FDWWOHSURGXFWLRQV\VWHPVVLPSOHRYHUDOOHI¿FLHQF\ DPRXQWRISURGXFWSURGXFHGYHUVXVDPRXQWRI
IHHGFRQVXPHG LVZHOOEHORZ7KHJHQHUDOUHOLDQFHRIFDWWOHRQIRUDJHVDQG¿EURXVFRSURGXFWV
DVIHHGVDQGWKHUROHRIIHUPHQWDWLRQLQWKHLUGLJHVWLRQUHVXOWVLQORZHURYHUDOOHI¿FLHQFLHVWKDQLQ
pig or poultry systems (Reynolds et al 7KHORZRYHUDOOHI¿FLHQF\RIIHHGXWLOL]DWLRQLQFDWWOH
LVDOVRDPDMRUGHWHUPLQDQWRIWKHLUHQYLURQPHQWDOLPSDFWDQGWKHQHHGIRULPSURYHGHI¿FLHQF\LQ
milk or meat production to minimize environmental impact is evident.

Among the main environmental concerns is the emission of nitrogen (N) to the environment. Major
losses of N occur in cattle production systems, including nitrate (NO3-) leaching, ammonia (NH3)
volatilization, and nitrous oxide (N2O) emissions (Steinfeld et al 7KHSULQFLSDOGULYHURI1
losses from cattle is N intake. Variation in dietary N supply will affect urinary N output in particular
and urine N is more susceptible to leaching and volatile losses than fecal N. Thus, reducing urinary
1H[FUHWLRQZLOOUHGXFHHQYLURQPHQWDOLPSDFWVLJQL¿FDQWO\7KHHI¿FLHQF\RIFRQYHUVLRQRIIHHG
N into milk or meat N in cattle varies widely (Figure 1). Such large variation suggests that major
improvements in reducing N excretion in feces and urine are possible. The aim of this paper is to
LGHQWLI\PD[LPDO1HI¿FLHQF\SRVVLEOHDWWKHDQLPDOOHYHODQGWKHFKDOOHQJHVDQGRSSRUWXQLWLHVWR
DFKLHYHWKLVPD[LPDO1HI¿FLHQF\WKURXJKORZHVWSRVVLEOH1H[FUHWLRQLQXULQHDQGIHFHVNHHSLQJ
in mind the role of cattle in utilizing human inedible feed resources. The present paper focuses on
dairy cattle, but the main concepts apply to beef cattle and other ruminants as well.

7KHRUHWLFDOPD[LPXPLQQLWURJHQHI¿FLHQF\
Production of milk or meat causes some avoidable and some inevitable losses of N in feces and
XULQH7KH1XVHHI¿FLHQF\RIDGDLU\FRZLVWKHTXDQWLW\RIIHHG1FRQVXPHGWKDWLVFDSWXUHGLQ
PLONDOVRFDOOHGWKHPLON1HI¿FLHQF\ 01( 9DQ9XXUHQDQG0HLMV  FDOFXODWHGDQ01(
RIWRLQDQLGHDOVLWXDWLRQRIDNJFRZSURGXFLQJNJPLONGZLWKJSURWHLQNJ
milk and fed on a well-balanced diet, with inevitable losses in feces and urine being 115 and 55 g
N/d, respectively. Assuming a daily DM intake of ~18 kg/d, the dietary crude protein (CP) content
in this optimal situation is ~105 g/kg DM. Van Vuuren and Meijs (1987) did not include inevitable
LQHI¿FLHQF\LQUXPHQPLFURELDOSURWHLQV\QWKHVLV 036 WKRXJKDQGDVVXPHGDOOPLFURELDOSURWHLQ
is digested. Therefore the actual maximal MNE may be lower than 0.40 to 0.45.

An updated calculation of the inevitable N losses associated with milk protein production in dairy
FDWWOHLVSUHVHQWHGLQ7DEOH7KHVHXQDYRLGDEOHORVVHVDUHFDOFXODWHGIRUDUHIHUHQFHFRZRINJ
live weight producing 40 kg/d of fat and protein corrected milk (FPCM) with a true protein content
RIJNJ'U\PDWWHULQWDNH '0, LVNJGDQGGLHWQHWHQHUJ\FRQWHQW0-NJ'07RWDO
tract diet digestibility is 0.80 and dietary content of rumen fermentable organic matter (RFOM) 0.55
kg/kg DM. The estimated minimal N losses in feces and urine are 89 and 174 g/d, respectively, and
the theoretical upper limit of MNE is 0.43 (Table 1). Usually, MNE is well below this maximal
attainable value. Calsamiglia et al. (2010) reported MNE in the lowest and highest quartile being
DQGLQD86GDWDVHW Q  DQGDQGLQD(XURSHDQGDWDVHW Q  UHVSHFWLYHO\
7KHPHDQ01(LQWKHODUJHGDWDEDVHSUHVHQWHGLQ)LJXUHLV 6' 7KHFKDOOHQJHLVWKXV
WRLPSURYH01(WREHFORVHUWRWKHWKHRUHWLFDOPD[LPXPHI¿FLHQF\RI$OWKRXJKDQLQFUHDVH
in N intake level increases milk N output, it is not related (P>0.05) to MNE (Figure 1). However,
GLHWDU\1FRQWHQWLVUHODWHGWR01(ZLWKDVORSHRI 6( 7KHVHUHVXOWVLQGLFDWH
WKHVLJQL¿FDQFHRIOHYHORIIHHGLQWDNHRQ1HI¿FLHQF\2FFDVLRQDOO\01(YDOXHVODUJHUWKDQ
KDYHEHHQUHSRUWHG7KHVHKLJKHI¿FLHQFLHVWHQGWREHDVVRFLDWHGZLWKERG\SURWHLQPRELOL]DWLRQ
DVLQGLFDWHGE\WKHUHODWLRQVKLSEHWZHHQPLON1HI¿FLHQF\DQG1EDODQFH )LJXUH $QHJDWLYH1
balance may occur for several days or weeks, in particular in early lactation (Van Knegsel et al.,

48 Energy and protein metabolism and nutrition in sustainable animal production

Ratio of milk N to N intake adjusted for study

Milk nitrogen adjusted for study, g/day

0.35 250

0.30 200

0.25 150

0.20 100

0.15 50

200 400 600 800 200 400 600 800

Nitrogen intake (g/day) Nitrogen intake (g/day)

Ratio of milk N to N intake adjusted for study

Ratio of milk N to N intake adjusted for study

0.30
0.30

0.25
0.25

0.20
0.20

0.15

20 25 30 35 40 45 -200 -100 0 100 200

Nitrogen concentration in diet (gN/kg) Nitrogen Balance (g/d)

)LJXUH8QLYDULDWHUHODWLRQVKLSVEHWZHHQ1LQWDNH JG RU1FRQWHQWLQWKHGLHW JNJ'0 DQG1

RXWSXWLQPLON JG DQGPLON1HI¿FLHQF\ 1RXWSXWLQPLONGLYLGHGE\1LQSXWLQIHHG 7KHUHVSRQVHV
KDYHEHHQDGMXVWHGIRUVWXG\HIIHFW1LWURJHQEDODQFHGDWDRILQGLYLGXDOFRZV Q  IURP.HEUHDE
et al.  7KHHTXDWLRQVGHVFULELQJWKHUHODWLRQVKLSVZHUH0LON1  6(  6(  
1LQWDNH5DWLRRIPLON1WR1LQWDNH  6(  ± 6(  1LQWDNH5DWLR
RIPLON1WR1LQWDNH  6(  ± 6(  1FRQFHQWUDWLRQ5DWLRRIPLON1
WR1LQWDNH  6(  ± 6(  1EDODQFH

2007), but has to be replenished later in the lactation cycle. In the present approach, zero body N
balance is assumed.

The main areas in which N losses in dairy cattle occur include urinary excretion of urea synthesized
from ammonia lost from the rumen; undigested microbial true protein excreted in feces; microbial
nucleic acids synthesized in the rumen and excreted mainly in urine; fecal and urinary excretion
resulting from endogenous secretions; and urinary excretion related to maintenance, milk protein
synthesis and amino acids (AA) absorbed in excess of requirement. These losses are discussed below.

Energy and protein metabolism and nutrition in sustainable animal production 49

7DEOH,QHYLWDEOH1ORVVHV J1GD\ 1RXWSXWLQPLON J1GD\ DQGWKHRUHWLFDOPD[LPXPPLON1
HI¿FLHQF\LQDGDLU\FRZSURGXFLQJNJIDWDQGSURWHLQFRUUHFWHGPLONSHUGD\DQGDWUXHSURWHLQ
FRQWHQWRIJNJ

N in feces N in urine N in milk

,QHI¿FLHQF\UXPHQPLFURELDOSURWHLQV\QWKHVLV 35
Rumen microbial nucleic acids 13 71
Undigested microbial protein 37
Endogenous secretions 39 19
Maintenance 13
Milk  198

Total 89 174 198

0D[LPXPPLON1HI¿FLHQF\ 0.43

Nitrogen losses related to rumen microbial protein synthesis

Sources of N for microbial protein synthesis

Ammonia is the most important source of N for protein synthesis in the rumen. Satter and Slyter
 REVHUYHGWKDWDPPRQLD1FRQFHQWUDWLRQRIaP0LVVXI¿FLHQWWRDFKLHYHPD[LPDO036LQ
FRQWLQXRXVFXOWXUHV,QYLYRKLJKHUDPPRQLD1FRQFHQWUDWLRQV EHWZHHQWRP0 DUHUHTXLUHG
to maximize MPS (Reynal and Broderick, 2005). Because ammonia is washed out from the rumen
ZLWKWKHÀXLGSKDVHDQGLVDEVRUEHGWKURXJKWKHUXPHQZDOOVXFKOHYHOVRIDPPRQLD1WRDFKLHYH
optimal MPS will always result in some losses of N. Assuming in the reference dairy cow a rumen
volume of 80 l, a fractional liquid passage rate of 0.15/h and a fractional ammonia absorption rate
RIKWKHPLQLPDODPPRQLDFRQFHQWUDWLRQRIP0JLYHVULVHWRDORVVRIJGDPPRQLD1
from the rumen. If 75 g urea-N/d is recycled to the rumen (discussed later), the net unavoidable
N-loss is at least 30 g N/d which is excreted as urea-N in the urine.

$OWKRXJKDPPRQLD1PD\EHWKHVROHVRXUFHRI1IRU036EHQH¿WVRIVXSSO\LQJSUHIRUPHG$$
DQGSHSWLGHVRQ036DQG036HI¿FLHQF\DUHZHOOHVWDEOLVKHG%DVHGRQVWRLFKLRPHWULFSULQFLSOHV
Dijkstra et al  FDOFXODWHGWKDWWKHHI¿FLHQF\RIV\QWKHVLVRIPLFURELDO&3ZLWKSUHIRUPHG$$
and peptides, compared with ammonia, is ~20% higher when expressed per unit OM utilized and
~75% higher when expressed per unit carbohydrate utilized. Russell and Sniffen (1984) observed in
YLWURWKDWDGGLWLRQRIWU\SWLFDVHWRDPHGLXPZLWKDPPRQLDDVVROH1VRXUFHLQFUHDVHG036HI¿FLHQF\
SHUXQLWFDUERK\GUDWHDFFRUGLQJWRDSDWWHUQRIGLPLQLVKLQJUHWXUQVZLWKHI¿FLHQF\EHLQJKLJKHU
at the highest level of trypticase addition. However, when expressed per unit OM utilized, microbial
HI¿FLHQF\LPSURYHGE\XSWRDOHYHORIWU\SWLFDVHEHLQJRIWRWDO20ZKHUHDVHI¿FLHQF\
GHFOLQHGZLWKIXUWKHUWU\SWLFDVHDGGLWLRQV0D[LPDO036HI¿FLHQFLHVSHUXQLW20ZKHQSHSWLGH
VXSSO\LVEDODQFHGZLWKUHTXLUHPHQWKDYHEHHQFRQ¿UPHGLQRWKHULQYLWURH[SHULPHQWV HJ%URRNV
et al., 2012). Thus, supply of preformed AA or peptides to rumen microbes in addition to supply
RIDPPRQLDLQLWLDOO\LQFUHDVHVPLFURELDOHI¿FLHQF\EXWDIWHUUHDFKLQJDQRSWLPXPIXUWKHU$$DQG
SHSWLGHDGGLWLRQVGHFUHDVHHI¿FLHQF\$WWKHKLJKHVWPLFURELDOHI¿FLHQF\OHYHODSDUWRIWKH$$DQG
SHSWLGHVVXSSOLHGDUHIHUPHQWHGWRDPPRQLDLQFUHDVLQJUXPHQÀXLGDPPRQLDFRQFHQWUDWLRQVDQG
adding to the net unavoidable N-losses from the rumen of 30 g N/d calculated above.

In a different approach, Bach et al  DQDO\]HGWKHUHODWLRQVKLSEHWZHHQ036DQGHI¿FLHQF\

of N use in the rumen (amount of microbial N produced per unit available N) in continuous culture

50 Energy and protein metabolism and nutrition in sustainable animal production

fermenters using mixed model analysis adjusting for study effect. They observed a quadratic
UHODWLRQVKLSEHWZHHQ1XVHHI¿FLHQF\DQGHI¿FLHQF\RI036ZLWKDQRSWLPXPHI¿FLHQF\RIJURZWK
REWDLQHGDWHI¿FLHQF\RIJPLFURELDO1NJ5)20DQGUXPHQ1XVHHI¿FLHQF\RI )LJXUH 
,QYDULRXVFRQWLQXRXVFXOWXUHVWXGLHVKLJKHU1XVHHI¿FLHQFLHV a ZHUHREWDLQHG7RFDOFXODWH
the lowest level of rumen degradable N (RDN) required and minimal N losses related to microbial
JURZWKWKH1XVHHI¿FLHQF\UHODWLRQVKLSSUHVHQWHGE\%DFKet al. (2005) was arbitrarily increased by
10%. Recycling of urea to the rumen contributes to the supply of RDN, and the relationship between
N intake and urea-N recycled to the gastro-intestinal tract of dairy cattle derived by Reynolds and
Kristensen (2008) was adopted to calculate the contribution of dietary RDN to total RDN required.
Up to 75% of urea-N recycled to the gastro-intestinal tract enters the forestomachs (Lobley et al.,
2000). For the reference cow, the lowest loss of N calculated based on these assumptions was 35
JGDWDQHI¿FLHQF\RIJPLFURELDO1NJ5)20DQGDFDOFXODWHGDSSDUHQWHI¿FLHQF\RIXVH
of dietary N to microbial N of 0.90. This N loss is in broad agreement with the net unavoidable N
loss from the rumen (>30 g/d) calculated before. For the reference cow, to avoid such losses dietary
RDN content should only be 15 g/kg DM or 94 g CP/kg DM. This dietary RDN level is lower
than levels recommended in various evaluation systems [e.g. the NRC (2001) recommendation
on rumen degradable protein is ~95 to 105 g/kg DM] and lower than used in practice. Surplus
rumen degradable protein-N is excreted as urea-N in urine. Urea-N is the most variable component
in urine, contributing from 50 to well over 90% of all N in urine (Dijkstra et al., 2013b). Current
protein evaluation systems have only limited value in providing guidance to reduce these losses, and
drawbacks of these systems are well recognized (e.g. Dijkstra et al., 2007; Hanigan, 2005). Single
protein values for a feed quote static conditions, whereas the protein supply depends on a number
of interrelated factors, including site of digestion and level of energy substrates available. Energy
and protein systems have been developed independently, despite vast evidence of the interactions
between energy- and protein-yielding nutrients. Protein evaluation systems do not predict how
performance, in terms of production and excretion, will change in response to deliberate changes
in feeding strategy. To improve upon current prediction schemes, new feeding systems need to be
based on mechanisms that govern the response of animals to nutrients, by quantitatively describing
metabolite supply at a more detailed level than the current aggregated components.

)LJXUH5HODWLRQVKLSEHWZHHQHI¿FLHQF\RIPLFURELDOFUXGHSURWHLQV\QWKHVLV JPLFURELDO1NJ
IHUPHQWHGRUJDQLFPDWWHU DQG1ORVVIURPWKHUXPHQ JG  VROLGOLQH DQG1HI¿FLHQF\ JPLFURELDO
1JGLHWDU\UXPHQDYDLODEOH1  GDVKHGOLQH LQDUHIHUHQFHFRZSURGXFLQJNJIDWDQGSURWHLQ
FRUUHFWHGPLONG7KHUHODWLRQVKLSEHWZHHQPLFURELDOV\QWKHVLVDQG1HI¿FLHQF\ JPLFURELDO1J
WRWDODYDLODEOH1 ZDVEDVHGRQ%DFKet al  'LHWDU\UXPHQDYDLODEOH1LVWKHGLIIHUHQFH
between total available N and urea-N recycled to the rumen, with the latter based on Reynolds and
.ULVWHQVHQ  6HHWH[WIRUIXUWKHUGHWDLOVRQFDOFXODWLRQV

Energy and protein metabolism and nutrition in sustainable animal production 51

Nitrogen recycling to the rumen

The use of non-protein N (NPN) sources, including ammonia originating from recycled urea, for
MPS in the rumen enables ruminants to survive and produce milk and meat on diets that are based
VROHO\RQ131VRXUFHV 9LUWDQHQ 7KHDELOLW\RIUXPHQPLFURRUJDQLVPVWRV\QWKHVL]HSURWHLQ
from urea recycled to the rumen could, in theory, compensate for some of the potential loss of N
that occurs through degradation of protein in the rumen and metabolism of AA absorbed from the
gut. In order to take advantage of this potential, endogenous N sources must be transferred to the
forestomachs and microbial protein synthesized, digested and absorbed. On diets with a relatively
KLJKUDWLRRIUXPHQIHUPHQWDEOHHQHUJ\WRUXPHQGHJUDGDEOHSURWHLQWKHÀRZRIQRQDPPRQLD1
into the duodenum can be higher than dietary N intake, reducing the amount of N excreted in urine
WKURXJKUHGXFHGDPPRQLDDEVRUSWLRQDQGXUHDV\QWKHVLV7KLVKLJKGXRGHQDOQRQDPPRQLD1ÀRZLV
possible when microbes in the rumen use more ammonia from recycled urea to synthesize microbial
CP, than ammonia produced from fermentation of dietary N sources. Thus the recycling of N to the
rumen and subsequent incorporation of recycled N into microbial protein may well be central to the
success of diets of low N content in dairy production (Calsamiglia et al., 2010).

Cattle are particularly adept at using as much intake N as possible. Depending on dietary N
concentration, between 30 and almost 100% of all urea entering the blood pool is returned to the
gut (Reynolds and Kristensen, 2008). Urinary N excretion still occurs even when 100% of urea is
returned to the gut, but the N is then excreted primarily in forms other than urea, including purine
derivatives (that may act as a marker of MPS), hippuric acid, creatine and creatinine (review Dijkstra
et al., 2013b). The transfer of urea-N from blood to the gut is via saliva and across the gut epithelia.
In general, this transfer is positively related to blood urea concentration and rumen fermentable
energy supply, and negatively to rumen ammonia concentration (Reynolds and Kristensen, 2008).
Although salivary and epithelial urea transport is adaptable to the N-status of the ruminant, when
expressed in absolute amounts, urea-N transport to the gut is little affected by changes in dietary N
content. Røjen et al. (2011a,b) reported that various urea transporters are expressed in bovine rumen
papillae. However, none of the investigated transcripts or proteins correlated with the increased
rumen epithelial urea permeability observed with low dietary N concentration. Reduced N supply
increased the relative extraction of arterial urea-N across the rumen and portal-drained viscera with an
overall shift towards the rumen. Adaptation of epithelial urea-N extraction with decreasing N supply
compensated for decreasing arterial concentration of urea-N, and thus a rather constant transport of
urea across treatments was observed. Thus, cows appear largely unable to up-regulate urea-N transport
VXI¿FLHQWO\WRIXOO\FRPSHQVDWHIRUWKH1UHPRYHGIURPWKHGLHWDQGWRVXEVWDQWLDOO\LQFUHDVH01(
In the present calculations, a linear relationship between N intake and urea-N transfer to the gut
with a slope of 0.12 g urea-N per g dietary N intake (Reynolds and Kristensen, 2008) was adopted.

Microbial nucleic acids

Microbial N is composed mostly of AA-N and nucleic acid-N. Synthesis of microbial nucleic acids
represents an irreversible loss of N excreted largely in urine (Dijkstra et al., 2013b). Pyrimidines of
microbial or endogenous origin undergo ring cleavage, and the major end products of catabolism
DUHȕDPLQRDFLGVDPPRQLDDQG&22. Various derivatives of purine metabolism are present in urine,
viz. allantoin, uric acid, xanthine and hypoxanthine. Some 20-25% of rumen microbial N is present
as nucleic acid-N (Dijkstra et al., 1992; NRC, 2001). There is virtually no scope to reduce losses of
N related to nucleic acid synthesis in the rumen. Nucleic acid contents of rumen microorganisms are
smallest at low fractional growth rates (Bates et al., 1985). However, in general high fractional growth
rates are required to reduce maintenance cost of microbes as a fraction of total energy expenditure
3LUW DQGFRQVHTXHQWO\WRPD[LPL]HHI¿FLHQF\RI0367KHWUXHLOHDOGLJHVWLELOLW\RIQXFOHLF
acids is 0.81 to 0.87 (Storm et al., 1983). Thus, rumen microbial synthesis of nucleic acids leads to
FRQVLGHUDEOHORVVHVRI1SDUWLFXODUO\LQXULQH)RUWKHUHIHUHQFHFRZDVVXPLQJKLJK036HI¿FLHQF\

52 Energy and protein metabolism and nutrition in sustainable animal production

and thus a high nucleic acid N content (25% of total microbial N), these inevitable N losses are 71
and 13 g N/d in urine and feces, respectively.

Digestion of microbial true protein and escape protein

The N-containing compounds entering the small intestine comprise mainly microbial protein and
nucleic acids produced in the rumen and feed protein that has escaped degradation in the rumen.
The digestibility of these fractions in the small intestine determines the amount of N absorbed as
well as that excreted with feces. There is little opportunity to improve the intestinal digestibility of
microbial and most rumen undegraded protein sources. In various protein evaluation systems used
worldwide, true digestibility of microbial true protein in the small intestine is 80-85%. These values
are largely based on digestibility of microbial AA in the small intestine of sheep (Storm et al., 1983).
In dairy cattle, small intestinal digestibility of microbial AA-N was 75-77% (Larsen et al., 2001).
The true digestibility of undegraded escape protein is variable but may be nearly 100% (Vérité and
Peyraud, 1989). For the reference cow, assuming complete digestion of rumen escape feed protein
and 85% of microbial true protein, fecal N losses are 37 g/d.

N losses related to endogenous secretions

Amino acids required for endogenous protein synthesis are related to replacement of sloughed
epithelial cells and release of digestive enzymes (Tamminga, 1992). These losses are not restricted
to the excretion of metabolic fecal N. Reabsorption of AA originating from endogenous losses will
mask its importance, because resynthesis of protein to replace endogenous losses is associated with
VLJQL¿FDQWORVVHVRIHQHUJ\DQG$$(QGRJHQRXVVHFUHWLRQVDUHPDLQO\UHODWHGWRWKHTXDQWLW\RI
DM or OM passing through the intestinal tract or to the undigested DM or OM excreted in feces
(NRC, 2001; Van Duinkerken et al., 2011; Vérité and Peyraud, 1989). Endogenous protein losses
LQFOXGLQJPLFURELDOSURWHLQV\QWKHVL]HGIURPXUHDWUDQVIHUUHGLQWRWKHKLQGJXWPD\YDU\EHWZHHQ
DQGJNJRIIHFDO'0RXWSXW 7DPPLQJD ,QWKHSUHVHQWDSSURDFKHI¿FLHQF\RIXWLOL]DWLRQ
RIDEVRUEHG$$IRUHQGRJHQRXVSURWHLQRIDQGJURVVV\QWKHVLVRIJHQGRJHQRXV1NJIHFDO
DM output are assumed (Van Duinkerken et al., 2011). For the reference cow, inevitable N losses
LQIHFHVDQGXULQHDUHDQGJGUHVSHFWLYHO\,QJHQHUDO¿EHUULFKGLHWVJLYHULVHWRKLJKHU
endogenous losses than diets with high levels of non-structural carbohydrates. A main challenge
in the conversion of human-inedible feed resources into milk or meat in cattle whilst improving
1XVHHI¿FLHQF\LVWRXVH¿EHUULFK\HWKLJKO\GLJHVWLEOHGLHWV)RUH[DPSOHDFKDQJHRIWRWDO'0
GLJHVWLELOLW\IURPWRLPSURYHVPD[LPDO01(IURPWR

N losses related to maintenance and milk protein synthesis

Losses of N related to maintenance requirements are largely unavoidable and include scurf, skin
secretions and hair, and tissue maintenance requirement losses. In various protein evaluation systems,
endogenous losses (already discussed) may or may not be part of maintenance requirements. Except
for endogenous N losses, N losses due to maintenance are relatively small (Tamminga et al., 1992).
For the reference cow, this N loss is 13 g/d and is excreted mainly in urine, with a minor part being
losses in hair, scurf and skin secretions. No substantial improvement in MNE can be achieved by
trying to reduce maintenance requirements.

7KHXWLOL]DWLRQHI¿FLHQF\RIDEVRUEHG$$IRUPLONSURWHLQV\QWKHVLVDVVXPHGLQYDULRXVSURWHLQ
HYDOXDWLRQV\VWHPVLVWR(YHQWKRXJKWKLVLVEHORZWKHWKHRUHWLFDOPD[LPXPHI¿FLHQF\RI
LQH[SHULPHQWVPXFKORZHUHI¿FLHQFLHVDUHXVXDOO\REVHUYHG,QWKH$)5&  DSSURDFK
WKHHI¿FLHQF\RI$$XWLOL]DWLRQIRUPLONSURWHLQZKHQ$$VXSSO\OLPLWVDQLPDOSHUIRUPDQFHLVWKH
UHVXOWRIWZRHI¿FLHQF\IDFWRUV7KH¿UVWIDFWRULVWKHHI¿FLHQF\ZLWKZKLFKDQµLGHDO¶$$PL[WXUHLV
utilized, which is assumed to be an animal characteristic and is set at 0.85. The second factor is the

Energy and protein metabolism and nutrition in sustainable animal production 53

biological value or the extent to which the absorbed AA mixture differs from the ideal one, which
is a characteristic determined mainly by diet composition. The default biological value is 0.80 for
ODFWDWLRQ $)5& 7KHPXOWLSOLFDWLRQRIERWKIDFWRUVUHVXOWVLQDQHI¿FLHQF\FRQYHUVLRQIDFWRU
RI)RUWKHUHIHUHQFHFRZPLQLPDO1ORVVHVLQFRQYHUVLRQRIDEVRUEHG$$WRPLONSURWHLQRFFXU
ZKHQDQLGHDO$$PL[WXUHLVDVVXPHGDQGDUHJG

8VXDOO\IDFWRUVRWKHUWKDQDEVRUEHG$$VXSSO\OLPLWPLONSURWHLQSURGXFWLRQDQGWKHHI¿FLHQF\
of absorbed AA utilization is lower than the theoretical maximum. Based on relationships between
supply of AA absorbed in the small intestine and milk protein yield across a number of experiments,
WKLVHI¿FLHQF\ZDVZKHQWKHORZHVW$$DOORZDQFHJLYLQJWKHJUHDWHVWSURWHLQ\LHOGZDVUHJUHVVHG
&DQW +RZHYHUZKHQWKHGDWDZHUHDGMXVWHGIRUWULDOHIIHFWVWKHPDUJLQDOHI¿FLHQF\DYHUDJHG
RQO\,QDPHWDDQDO\VLVWKHWUDQVIHUHI¿FLHQF\RISRVWUXPLQDOFDVHLQRU$$ZLWKFDVHLQSUR¿OH
LQIXVLRQVRQPLONSURWHLQ\LHOGVKRZHGDTXDGUDWLFSDWWHUQDQGPD[LPXPPDUJLQDOHI¿FLHQF\ZDVVWLOO
only 0.38 (Lapierre et al 7KHVHHVWLPDWHVRI$$XWLOL]DWLRQHI¿FLHQF\UHÀHFWWKHXWLOL]DWLRQRI
LQFUHPHQWVRIPHWDEROL]DEOHSURWHLQ $$ DERYHDQGEHORZVSHFL¿FUHTXLUHPHQWV 5H\QROGV 
DQGKDYHDPDMRULPSDFWRQRYHUDOO01($QHI¿FLHQF\RIDEVRUEHG$$XWLOL]DWLRQIRUPLONSURWHLQ
RI UHIHUHQFHFRZ  SURWHLQHYDOXDWLRQV\VWHPV DQG PD[LPXPLQH[SHULPHQWV 
UHVXOWVLQPD[LPDO01(RIDQGUHVSHFWLYHO\

Intermediary metabolism of AA between the duodenum and the mammary gland explains the
GHFUHDVHGHI¿FLHQF\RIWUDQVIHURIDEVRUEHG$$LQWRPLONSURWHLQDVPD[LPDO\LHOGLVDSSURDFKHG
2QDYHUDJHRIHVWLPDWHGGLJHVWLEOH$$ZDVUHFRYHUHGLQWKHSRUWDOYHLQZLWKWKHORVV  GXH
to endogenous secretions (discussed previously) and oxidation of AA across the gut wall (Lapierre
et al 7KHPDJQLWXGHRIWKLVORVVLVQRWXQLIRUPDPRQJ$$DQGYDULHVEHWZHHQOHVVWKDQ
for histidine to more than 0.90 for some non-essential AA. Next, the liver removes on average 0.45
of portal absorbed AA, again with considerable variation between individual AA. Liver fractional
extraction of AA relative to portal absorption increases at higher AA supply. Finally, the utilization
of post-liver available AA by the mammary gland is relatively high but again variable, which implies
that improvements can be made. A considerable part of variation in AA use by splanchnic tissues
and mammary gland is related to the supply of energy, and thus AA requirements, and further
details can be found in various reviews (Hanigan, 2005; Lapierre et al., 2010). Thus, the use of
FRQVWDQWV WR IRUDEVRUEHG$$XWLOL]DWLRQIRUPLONSURWHLQV\QWKHVLVLQPRVWFXUUHQWSURWHLQ
HYDOXDWLRQV\VWHPVDSSHDUVLQDGHTXDWHLQYLHZRIWKHGHFUHDVLQJHI¿FLHQF\ZLWKLQFUHDVLQJVXSSO\
of absorbed AA observed. Mechanistic models at the organ level that integrate the metabolism of
energy substrates and individual AA enable incorporation of the rapidly expanding experimental
knowledge on post-absorptive AA dynamics, and help to obtain a better quantitative understanding
WRIXUWKHUUHGXFHVSRVWDEVRUSWLYH1ORVVHV8OWLPDWHO\WKHHI¿FLHQF\RIDEVRUEHG$$XWLOL]DWLRQLV
determined by their requirements by the mammary gland, which is modulated by stage of lactation,
genetics, and energy supply (Reynolds, 2001).

Integration: nitrogen losses, energy substrates and methane production

7KHSUHVHQWDSSURDFKKDVFRQ¿UPHGWKDWWKHUHLVOLWWOHRUQRVFRSHWRLPSURYH01(DQGUHGXFH1
losses related to microbial nucleic acids, recycling of N to the rumen, intestinal digestion of microbial
protein, and animal maintenance requirements. Strategies to reduce N losses should focus on an
RSWLPDOVXSSO\RI5'1DQGRSWLPDOHI¿FLHQF\RIDEVRUEHG$$XWLOL]DWLRQIRUPLONSURWHLQV\QWKHVLV
A major challenge here is to achieve such reductions using human inedible feed resources. In theory,
if the reference dairy cow is fed a diet without RFOM (digestion to occur in intestine only) whilst
PDLQWDLQLQJWKHGHIDXOWWRWDOWUDFWGLJHVWLELOLW\RIPD[LPDO01(LQFUHDVHVIURPWR
7KXVWKHXVHRI¿EURXVKXPDQLQHGLEOHIHHGUHVRXUFHVE\PLFURRUJDQLVPVLQWKHIRUHVWRPDFKV
occurs at a cost, which is amongst others unavoidable N loss associated with microbial metabolism
and a reduced MNE.

54 Energy and protein metabolism and nutrition in sustainable animal production

,QVWULYLQJIRURSWLPDOVXSSO\RI5'1DQGRSWLPDOHI¿FLHQF\RIXWLOL]DWLRQRIDEVRUEHG$$WKHSURSHU
supply of energy yielding nutrients is crucial. If the supply of energy to rumen microbes increases
whilst RDN supply does not change, less ammonia-N is formed and lost as urea-N in urine. Similarly,
increased supply of metabolizable energy (ME) may reduce N losses in post-absorptive tissues. For
example, MNE of a low energy, high protein diet improved from 0.20 to 0.24 when either supply of
protein was reduced or supply of energy was increased, and improved to 0.28 with both (Rius et al.,
2010). Data presented in Figure 1 were subjected to multivariate meta-analysis by Kebreab et al. (2010)
and prediction of urinary N output, but not fecal N output, improved when metabolizability (ratio of ME
to gross energy) or ME intake was added as co-variable to a model that already included N intake. The
VORSHVZHUHJXULQH1JGLHW1DQGíJXULQH10-0(LQGLFDWLQJUHGXFHGXULQDU\1RXWSXW
when dietary ME concentration increases. Thus, in attempts to improve MNE, changes in energy supply
have to be considered. In view of an integrated approach to reduce greenhouse gas emissions on farm,
it should be noted that mitigation options aimed at reducing urinary N excretion may result in elevated
methane (CH4) emission depending largely on the type of carbohydrate consumed with grass (Ellis et
al., 2012). Methane production declines if starch or digestible nutrients escaping rumen fermentation
UHSODFHSURWHLQLQWKHGLHWEXWULVHVLIGLHWDU\¿EHUOHYHOVLQFUHDVH'LMNVWUDet al. (2011) estimated an
increase of on average 0.30 g CH4 per g urinary N decrease for various nutritional interventions with
grass silage based diets aimed to improve MNE. Using standard emission factors for direct and indirect
N2O emissions, the estimated N2O emission reduction (in CO2 equivalents) resulting from decreased
manure N output was more than offset by a rise in enteric CH4 production (Dijkstra et al., unpublished).
Moraes et al. (2012) developed a linear programming model to formulate minimum cost diets when
environmental policies are present. In their evaluations, imposing CH4 restrictions increased N losses
from the animal. In view of these results, a major challenge in reducing N losses from dairy cattle is
WR¿QGDQRSWLPDOQXWULWLRQDOEDODQFHZLWKRXWLQFUHDVLQJHQWHULFSURGXFWLRQRI&+4.

,PSURYHPHQWVLQQXWULWLRQJHQHWLFVDQGWHFKQRORJ\KDYHGUDPDWLFDOO\LPSURYHGWKHHI¿FLHQF\RI
milk production in the past decades, particularly in developed countries. Such improvements are
due to increased yield per animal which dilutes the nutrient costs of maintenance. Although feed
HI¿FLHQF\ZLOOVLJQL¿FDQWO\LPSURYHZLWKLQFUHDVHVLQSURGXFWLRQOHYHOPD[LPDODFKLHYDEOH01(
may not improve to a similar extent (Figure 3). For example, an increase in FPCM production from
WRNJ\ULPSURYHVIHHGHI¿FLHQF\E\ IURPWRNJIHHG'0NJ)3&0 
and enteric CH4 production per kg FPCM is lowered as well (Bannink et al., 2011). However,
the maximal MNE improves by only 5% (from 0.40 to 0.42). Thus, to reduce the amount of feed
resources used per unit milk, further production gains may be effective, but diet composition options
(as discussed previously) rather than further production gains may be preferred to improve MNE.

Conclusions
Reducing N output in urine from cattle is critical to reducing NO3 leaching, NH3 volatilization
and N2O emission and achieving an environmentally sustainable production. Large variation in N
HI¿FLHQF\SUHVHQWVDQRSSRUWXQLW\WRPDQLSXODWHGLHWVWRLPSURYH1HI¿FLHQF\*LYHQWKHUROHRI
dairy cattle in conversion of human inedible resources into human edible high quality foods, milk N
HI¿FLHQFLHVKLJKHUWKDQVRPHDUHXQOLNHO\WREHDFKLHYHG7KHUHLVOLWWOHRSSRUWXQLW\WRUHGXFH
N losses related to incomplete digestion of microbial protein, to synthesis of microbial nucleic acids,
and to animal maintenance requirements. Dietary strategies to reduce N losses should focus on an
RSWLPDOVXSSO\RIUXPHQGHJUDGDEOH1DQGRSWLPDOHI¿FLHQF\RIDEVRUEHGDPLQRDFLGXWLOL]DWLRQIRU
milk protein synthesis. Integration between protein and energy metabolism is essential, and energy
and protein should be considered conjointly rather than as two distinct entities. Current protein
HYDOXDWLRQV\VWHPVSUHGLFWUHTXLUHPHQWVIRUGLHWDU\1EXWWKHVHUHTXLUHPHQWVDUHXVXDOO\¿[HGRU
FRPSULVHOLQHDUDSSUR[LPDWLRQVDQGWKHV\VWHPVGRQRWSUHGLFWUHVSRQVHVLQ1HI¿FLHQF\WRGLHWDU\
FKDQJHV$PDMRUFKDOOHQJHLQVWUDWHJLHVWRRSWLPL]HKLJK¿EHUGLHWVIRUKLJKPLON1HI¿FLHQF\ZLOO
be to avoid increases in enteric CH4 production associated with these dietary strategies.

Energy and protein metabolism and nutrition in sustainable animal production 55

)LJXUH5HODWLRQVKLSEHWZHHQPLONSURGXFWLRQOHYHO NJIDWDQGSURWHLQFRUUHFWHGPLON\HDU DQG
IHHGHI¿FLHQF\ NJIHHGNJ)3&0 RUWKHRUHWLFDOPD[LPDOPLON1HI¿FLHQF\ NJ1RXWSXWLQPLONNJ
1LQSXWLQIHHG )HHGHI¿FLHQF\FDOFXODWHGZLWKWKH'XWFKHQHUJ\HYDOXDWLRQV\VWHPDQGPD[LPDO
PLON1HI¿FLHQF\FDOFXODWHGDVGHVFULEHGLQWH[W

Acknowledgements
Partially funded by the Commission of the European Communities (Rednex project FP7-
KBBE-2007-1).

References
AFRC, 1992. Technical committee on responses to nutrients, report no. 9. Nutritive requirements of ruminant animals:
3URWHLQ1XWU$EVWU5HY6HULHV%
Bach, A., S. Calsamiglia and M. D. Stern, 2005. Nitrogen metabolism in the rumen. J. Dairy Sci. 88, E9-E21.
Bannink, A., M.W. van Schijndel and J. Dijkstra, 2011. A model of enteric fermentation in dairy cows to estimate
methane emission for the Dutch National Inventory Report using the IPCC Tier 3 approach. Anim. Feed Sci.
7HFKQRO
%DWHV'%-$*LOOHW6$%DUJRDQG:*%HUJHQ7KHHIIHFWRIVSHFL¿FJURZWKUDWHDQGVWDJHRIJURZWKRQ
QXFOHLFDFLGSURWHLQYDOXHVRISXUHFXOWXUHVDQGPL[HGUXPLQDOEDFWHULD-$QLP6FL
Brooks, M.A., R.M. Harvey, N.F. Johnson and M.S. Kerley, 2012. Rumen degradable protein supply affects microbial
HI¿FLHQF\LQFRQWLQXRXVFXOWXUHDQGJURZWKLQVWHHUV-$QLP6FL
Calsamiglia, S., A. Ferret, C.K. Reynolds, N.B. Kristensen and A.M. van Vuuren, 2010. Strategies for optimizing
QLWURJHQXVHE\UXPLQDQWV$QLPDO
Cant, J.P., 2005. Integration of data in feed evaluation systems. In: Dijkstra, J., J.M. Forbes and J. France (editors),
Quantitative aspects of ruminant digestion and metabolism, 2nd edition. CABI Publishing, Wallingford, UK:

Dijkstra, J., E. Kebreab, J.A.N. Mills, W.F. Pellikaan, S. López, A. Bannink and J. France, 2007. From nutrient
UHTXLUHPHQWWRDQLPDOUHVSRQVHSUHGLFWLQJWKHSUR¿OHRIQXWULHQWVDYDLODEOHIRUDEVRUSWLRQLQGDLU\FDWWOH$QLPDO
1, 99-111.
'LMNVWUD-+'6W&1HDO'(%HHYHUDQG-)UDQFH6LPXODWLRQRIQXWULHQWGLJHVWLRQDEVRUSWLRQDQGRXWÀRZ
LQWKHUXPHQPRGHOGHVFULSWLRQ-1XWU
'LMNVWUD--)UDQFH-/(OOLV$%6WUDWKH(.HEUHDEDQG$%DQQLQND3URGXFWLRQHI¿FLHQF\RIUXPLQDQWV
feed, nitrogen and methane. In: Kebreab, E. (editor), Sustainable animal agriculture. CAB International, Wallingford,
UK, in press.

56 Energy and protein metabolism and nutrition in sustainable animal production

Dijkstra, J., O. Oenema and A. Bannink, 2011. Dietary strategies to reducing N excretion from cattle: implications for
methane emissions. Curr. Opin. Environm. Sust. 3, 414-422.
Dijkstra, J., O. Oenema, J.W. van Groenigen, J.W. Spek, A.M. van Vuuren and A. Bannink, 2013b. Diet effects on
urine composition of cattle and N2O emissions. Animal, in press.
Ellis, J.L., J. Dijkstra, J. France, A.J. Parsons, G.R. Edwards, S. Rasmussen, E. Kebreab and A. Bannink, 2012. Effect
of high-sugar grasses on methane emissions simulated using a dynamic model. J. Dairy Sci. 95, 272-285.
FAO, 2011. World Livestock 2011 – Livestock in Food Security. FAO, Rome.
Hanigan, M.D., 2005. Quantitative aspects of ruminant splanchnic metabolism as related to predicting animal
performance. Anim. Sci. 80, 23-32.
Kebreab, E., A.B. Strathe, J. Dijkstra, J.A.N. Mills, C.K. Reynolds, L.A. Crompton, T. Yan and J. France, 2010. Energy
and protein interactions and their effect on nitrogen excretion in dairy cows. In: Crovetto, G.M. (ed.), 3rd EAAP
international symposium on energy and protein metabolism and nutrition. Wageningen Academic Publishers,
Netherlands: 417-425.
Lapierre, H., C.E. Galindo, S. Lemosquet, I. Ortigues-Marty, L. Doepel and D.R. Ouellet, 2010. Protein supply, glucose
kinetics and milk yield in dairy cows. In: Crovetto, G.M. (editor), 3rd EAAP international symposium on energy
and protein metabolism and nutrition. Wageningen Academic Publishers, Netherlands: 275-285.
/DSLHUUH+'3DFKHFR5%HUWKLDXPH'52XHOOHW&*6FKZDE3'XEUHXLO*+ROWURSDQG*(/REOH\
What is the true supply of amino acids for a dairy cow? J. Dairy Sci. 89, E1-E14.
Larsen, M., T.G. Madsen, M.R. Weisbjerg, T. Hvelplund and J. Madsen, 2001. Small intestinal digestibility of microbial
and endogenous amino acids in dairy cows. J. Anim. Physiol. Anim. Nutr. 85, 9-21.
/REOH\*('0%UHPPHUDQG*=XXU(IIHFWVRIGLHWTXDOLW\RQXUHDIDWHVLQVKHHSDVDVVHVVHGE\UH¿QHG
non-invasive [15N151@XUHDNLQHWLFV%U-1XWU
Moraes, L.E., J.E. Wilen, P.H. Robinson and J.G. Fadel, 2012. A linear programming model to optimize diets in
HQYLURQPHQWDOSROLF\VFHQDULRV-'DLU\6FL
National Research Council, 2001. Nutrient requirements of dairy cattle, 7th revised ed. National Academy Press,
Washington DC, USA.
3LUW6-7KHPDLQWHQDQFHHQHUJ\RIEDFWHULDLQJURZLQJFXOWXUHV3URF56RF/RQGRQ6HU%
Reynal, S.M. and G.A. Broderick, 2005. Effect of dietary level of rumen-degraded protein on production and nitrogen
PHWDEROLVPLQODFWDWLQJGDLU\FRZV-'DLU\6FL
Reynolds, C.K., 2001. Economics of visceral energy metabolism in ruminants: Toll keeping or internal revenue service?
J. Anim. Sci. 80, E74-E84.
5H\QROGV&./$&URPSWRQDQG-$10LOOV,PSURYLQJWKHHI¿FLHQF\RIHQHUJ\XWLOLVDWLRQLQFDWWOH$QLP
3URG6FL
Reynolds, C.K. and N.B. Kristensen, 2008. Nitrogen recycling through the gut and the nitrogen economy of ruminants:
$QDV\QFKURQRXVV\PELRVLV-$QLP6FL((
Rius, A.G., M.L. McGilliard, C.A. Umberger and M.D. Hanigan, 2010. Interactions of energy and predicted metabolizable
SURWHLQLQGHWHUPLQLQJQLWURJHQHI¿FLHQF\LQWKHODFWDWLQJGDLU\FRZ-'DLU\6FL
Røjen, B.A., S.B. Poulsen, P.K. Theil and N.B. Kristensen, 2011b. Effects of dietary nitrogen concentration on messenger
RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating
Holstein cows. J. Dairy Sci. 94, 2587-2591.
5¡MHQ%$3.7KHLODQG1%.ULVWHQVHQD(IIHFWVRIQLWURJHQVXSSO\RQLQWHURUJDQÀX[HVRIXUHD1DQG
renal urea-N kinetics in lactating Holstein cows. J. Dairy Sci. 94, 2532-2544.
Russell, J.B. and C.J. Sniffen, 1984. Effect of carbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen
EDFWHULDLQYLWUR-'DLU\6FL
Satter, L.D. and L.L. Slyter, 1974. Effect of ammonia concentration on rumen microbial protein production in-vitro.
Br. J. Nutr. 32, 199-208.
6WHLQIHOG+3*HUEHU7:DVVHQDDU9&DVWHO05RVDOHVDQG&'H+DDQ/LYHVWRFN¶V/RQJ6KDGRZ
Environmental Issues and Options. FAO, Rome.
Storm, E.R., D.S. Brown and E.R. Ørskov, 1983. The nutritive value of rumen micro-organisms in ruminants. 3. The
digestion of microbial amino and nucleic acids in, and losses of endogenous nitrogen from, the small intestine of
sheep. Brit. J. Nutr. 50, 479-485.
Tamminga, S., 1992. Nutrition management of dairy cows as a contribution to pollution control. J. Dairy Sci. 75, 345-357.

Energy and protein metabolism and nutrition in sustainable animal production 57

Van Duinkerken, G., M.C. Blok, A. Bannink, J.W. Cone, J. Dijkstra, A.M. van Vuuren and S. Tamminga, 2011. Update of
WKH'XWFKSURWHLQHYDOXDWLRQV\VWHPIRUUXPLQDQWVWKH'9(2(%V\VWHP-$JULF6FL&DPE
Van Knegsel, A.T.M., H. van den Brand, J. Dijkstra, W.M. van Straalen, M.J.W. Heetkamp, S. Tamminga and B.
Kemp, 2007. Dietary energy source in dairy cows in early lactation: energy partitioning and milk composition.
-'DLU\6FL
Van Vuuren, A.M. and J.A.C. Meijs, 1987. Effects of herbage composition and supplement feeding on the excretion
of nitrogen in dung and urine by grazing dairy cows. In: Van der Meer, H.G., R.J. Unwin, T.A. van Dijk and G.C.
Ennik (eds.), Animal manure on grassland and fodder crops. Fertilizer or waste? Martinus Nijhoff Publishers,
Dordrecht, the Netherlands: 17-25.
Vérité, R. and J.L. Peyraud, 1989. Protein: the PDI system. In: Jarrige, R. (ed.), Ruminant nutrition: recommended
allowances and feed tables. John Libbey Eurotext, Paris: 33-47.
9LUWDQHQ$,0LONSURGXFWLRQRIFRZVRQSURWHLQIUHHIHHG6FLHQFH

58 Energy and protein metabolism and nutrition in sustainable animal production

Small intestinal fermentation contributes substantially to starch
disappearance in milk-fed calves
M.S. Gilbert1, A.J. Pantophlet2, J.J.G.C. van den Borne1, H.A. Schols and W.J.J. Gerrits1
1$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands; [email protected]
2&HQWUHIRU0HGLFDO%LRPLFV8QLYHUVLW\0HGLFDO&HQWUH*URQLQJHQ32%R[55
Groningen, the Netherlands
/DERUDWRU\RI)RRG&KHPLVWU\:DJHQLQJHQ8QLYHUVLW\32%R[(9:DJHQLQJHQWKH
Netherlands

Introduction
Calf milk replacers commonly contain 40-50% lactose. For economic reasons, starch is of interest
DVDODFWRVHUHSODFHU6PDOOLQWHVWLQDOGLVDSSHDUDQFHRIVWDUFK  ZDVORZHUWKDQWKDWRIJOXFRVH
(85%) when infused in the abomasum of steers (Kreikemeier and Harmon, 1995), indicating that
enzyme activity required for the hydrolysis of starch to glucose limits starch digestion. Which enzyme
system is limiting starch digestion in milk-fed calves is unknown. Portal glucose appearance was only
57% of small intestinal starch disappearance (Kreikemeier and Harmon, 1995). This gap includes
starch fermentation and glucose use by portal drained visceral tissues. In steers, abomasal infusion
of a starch hydrolysate resulted in a linear decrease in ileal pH (Branco et al., 1999), illustrating that
fermentation may be an important contributor to small intestinal starch disappearance.

The objectives were therefore (1) to determine the rate-limiting enzyme for hydrolysis and
disappearance of starch from the intestinal lumen and (2) to quantify starch fermentation in milk-
fed calves.

Material and methods

Forty male calves (224±2.0 kg BW) were fed milk replacer containing either 18% lactose (control)
or 18% of one of 4 corn starch products. The 4 corn starch products differed in the enzymes required
IRUWKHLUFRPSOHWHK\GURO\VLVWRJOXFRVHJHODWLQL]HGVWDUFK ĮDP\ODVHDQGPDOWDVH PDOWRGH[WULQ
ĮDP\ODVHDQGPDOWDVH PDOWRGH[WULQZLWKĮEUDQFKLQJ ĮDP\ODVHPDOWDVHDQGLVRPDOWDVH DQG
maltose (maltase). Calves were adapted to the diets for 15 weeks before the start of the measurements.
All diets included Co-EDTA as an indigestible marker. The corn starch products (1.093 atom% 13C)
differed in natural 13C enrichment from lactose (1.073 atom% 13C) and the remainder of the diet
(1.078 atom% 13C).

Feces were collected quantitatively during 4 days to measure total tract digestibility of the starch
products and to calculate total tract starch fermentation based on fecal 13C excretion (Gerrits et al.,
 %ORRGVDPSOHVZHUHWDNHQDWDQGPLQDIWHUIHHGLQJWRPHDVXUH
13C enrichment in plasma glucose. On the day of blood sampling only, control calves received 13C
enriched lactose (1.092 atom% 13& &DOYHVZHUHVDFUL¿FHGKDIWHUIHHGLQJDQGLOHDOGLJHVWDZHUH
collected to measure ileal digestibility of starch products. Variables were analyzed for treatment
effects by ANOVA.

Results and discussion

$SSDUHQWWRWDOWUDFW “ DQGLOHDO “ VWDUFKGLJHVWLELOLW\GLGQRWGLIIHUEHWZHHQ
starch products. Total tract starch fermentation, estimated from increased fecal 13C excretion, was
not affected by treatment and averaged 478±28 g/d, corresponding to 101% of starch intake (Table
 6WDUFKIHGFDOYHVSURGXFHGPRUHIHFHV J'0G WKDQFRQWUROFDOYHV P<0.001), which

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 59
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_4, © Wageningen Academic Publishers 2013
7DEOH)HFDOFKDUDFWHULVWLFVRIPLONIHGFDOYHVIHGDPLONUHSODFHUFRQWDLQLQJRQO\ODFWRVH &21 
RURIQDWXUDOO\&HQULFKHGVWDUFKSURGXFWV *6JHODWLQL]HGVWDUFK0'PDOWRGH[WULQ0'%
PDOWRGH[WULQZLWKDKLJKGHJUHHRIEUDQFKLQJ07PDOWRVH 

Treatment CON GS MD MDB MT P-value1

Mean ± SE

Number of calves 7 8 7 8 7
Fecal 13C enrichment, atom%  1.0829 1.0832 1.0825 1.0828 <0.001
Fecal dry matter output, g/d “ 259±15 245±19 222±9 225±12 0.003
Fermentation2, g/d - “ 530±91 432±20 431±53 0.422
Fermentation2, % of intake - 98±7 107±9 97±5 104±13 0.925

1 P-value for differences between treatments. When no value for the CON treatment is shown, the P-value applies for
differences between the starch product treatments only.
2 Estimated fermentation of starch products.

FRQVLVWHGRIJVWDUFKJIDWDQGJDVKSHUGD\7KLVOHDYHVJGXQDFFRXQWHGIRUZKLFKLV
hypothesized to be increased undigested microbial mass resulting from starch fermentation. Assuming
a ratio of 5 gram starch fermented for each g of fecal microbial output (Lanzas et al., 2007), this
ZRXOGUHTXLUHJGVWDUFKWREHIHUPHQWHGFRUUHVSRQGLQJWRRIVWDUFKLQWDNH,QWKHFRQWURO
calves, 13C enrichment in plasma glucose increased from 1.083 to 1.089 atom% within 3h after
feeding (P<0.001). In starch-fed calves, 13C enrichment in plasma glucose did not increase relative
WREDVHOLQH DWRP +HQFHDEVRUSWLRQRIVWDUFKGHULYHGJOXFRVHZDVQRWVXI¿FLHQWWROHDG
to a measurable increase in 13C enrichment in plasma glucose.

The combination of the 4 starch products would lead us to deduce the rate-limiting enzyme for
starch digestion in milk-fed calves. Ileal starch digestibility did not differ between starch products,
suggesting that maltase activity limits starch digestion in milk-fed calves. Two methods were used
to quantify starch fermentation; one based on increased fecal DM output and the other on increased
fecal 13&H[FUHWLRQ%DVHGRQWKHVHPHWKRGVVWDUFKIHUPHQWDWLRQZDVWRRIWKHVWDUFK
intake in milk-fed calves. This is in agreement with the absence of a postprandial response in 13C
enrichment of plasma glucose to feeding corn starch products that are characterized by a relatively
high natural 13C enrichment. Nearly 40% of the starch was fermented in the colon. Therefore, an
DGGLWLRQDOWRRIWKHVWDUFKLQWDNHLVIHUPHQWHGEHIRUHWKHFRORQ2YHUDOOWKLVVWXG\VKRZV
that small intestinal fermentation contributes substantially to starch disappearance and that maltase
limits starch digestion in milk-fed calves.

References
Branco, A.F., D.L. Harmon, D.W. Bohnert, B.T. Larson and M.L. Bauer, 1999. Estimating true digestibility of
nonstructural carbohydrates in the small intestine of steers. J. Anim. Sci. 77, 1889-1895.
Gerrits, W.J.J., M.W. Bosch and J.J.G.C. van den Borne, 2012. Quantifying resistant starch using novel, in vivo
methodology and the energetic utilization of fermented starch in pigs. J. Nutr. 142, 238-244.
Kreikemeier, K.K. and D.L. Harmon, 1995. Abomasal glucose, maize starch and maize dextrin infusions in cattle –
6PDOOLQWHVWLQDOGLVDSSHDUDQFHQHWSRUWDOJOXFRVHÀX[DQGLOHDOROLJRVDFFKDULGHÀRZ%U-1XWU
Lanzas, C., C.J. Sniffen, S. Seo, L.O. Tedeschi and D.G. Fox, 2007. A revised CNCPS feed carbohydrate fractionation
VFKHPHIRUIRUPXODWLQJUDWLRQVIRUUXPLQDQWV$QLP)HHG6FL7HFKQRO

60 Energy and protein metabolism and nutrition in sustainable animal production

Nutrient digestion by dairy cows fed diets replacing starch with non-
IRUDJH¿EHU
S.D. Ranathunga, M.M. Abdelqader and K.F. Kalscheur
'DLU\6FLHQFH'HSDUWPHQW6RXWK'DNRWD6WDWH8QLYHUVLW\6'86$
[email protected]

Introduction
Corn starch is used as the main energy source in lactating dairy cow diets. Feeding high levels
of corn starch may be associated with negative health impacts on lactating dairy cows, such as
ruminal acidosis and laminitis along with higher feed costs and lower income from reduced milk
components. Dried distillers grains with solubles (DG), a co-product of the ethanol industry, is an
excellent source of energy. Ranathunga et al. (2010) demonstrated that that incrementally reducing
the amount of starch in a ration from a high of 29% to a low of 20% by adding DG resulted in
similar milk production and composition by lactating dairy cows. The objective of the study was to
HYDOXDWHWKHHIIHFWRIUHSODFLQJVWDUFKIURPFRUQZLWKQRQIRUDJH¿EHUIURP'*DQGVR\EHDQKXOOV
RQWKHQXWULHQWÀRZWRWKHRPDVXPUXPLQDOQXWULHQWGHJUDGDELOLW\WRWDOWUDFWQXWULHQWGLJHVWLELOLW\
and nitrogen partition of lactating dairy cows.

Material and methods

6L[+ROVWHLQFRZVZLWKUXPLQDO¿VWXODZHUHDVVLJQHGWRDPXOWLSOHî/DWLQVTXDUHGHVLJQ7KUHH
diets were formulated: (1) high starch (HS) containing 33% starch with 0% DG; (2) medium starch
(MS) containing 25% starch with 12% DG; and (3) low starch (LS) containing 17% starch with 24%
DG. Ground corn, soybean meal, expeller soybean meal, and an inert fat were replaced by DG and
soybean hulls to formulate diets containing high, medium, and low starch concentrations. Ingredient
and nutrient composition is described in Table 1.

Table 1. Ingredient and nutrient composition of the diets.

Item HS MS LS

Alfalfa hay 25.0 25.0 25.0

Corn silage 25.0 25.0 25.0
Ground corn 31.5 20.5 9.5
DG 0 12.0 24.0
Soybean hulls 0 7.5 15.0
Soybean meal, 44% 8.00 4.00 0
Expellers soybean meal 7.25  0
Ruminally inert fat  0.80 0
Minerals and vitamins  1.57 1.50

DM, % of diet 70.8 71.4 71.9

CP, % of DM 17.2 17.2 17.2
NDF, % of DM 25.4 31.5 
Forage NDF, % of DM 20.5 20.5 20.5
Starch, % of DM 32.4  

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 61
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_5, © Wageningen Academic Publishers 2013
1XWULHQWÀRZWRWKHRPDVXPZDVPHDVXUHGXVLQJRPDVDOVDPSOLQJDQGWKHWULSOHPDUNHUPHWKRG
(CoEDTA, Yb, and indigestible NDF as markers; Reynal and Broderick, 2005). Total tract digestibility
of nutrients and nitrogen excretion data were measured by total collection of feces and urine.

Data were analyzed using the MIXED procedure (SAS, 2001). Orthogonal contrasts were used to
test linear (L), and quadratic (Q) effects.

Results and discussion

As dietary starch concentration decreased from 31% to 18%, DMI increased linearly (3 0.04). As
dietary starch concentration decreased across diets, milk yield (3 0.04), milk and fat percentages
(3 0.04) increased linearly, whereas protein percentages (3 0.01) decreased linearly.

Flow of DM (10.5, 11.2, and 11.8 kg/d) and OM (8.2, 8.8, and 9.1 kg/d) to the omasum increased
OLQHDUO\ZKHQVWDUFKZDVUHSODFHGZLWKQRQIRUDJH¿EHU$YHUDJHÀRZRI1')$')&3DQGVWDUFK
WRWKHRPDVXPZDVQRWDIIHFWHGE\WKHGLHWV DQGNJGUHVSHFWLYHO\ 

Table 2 summarizes the data on nutrient digestion and nitrogen metabolism. Apparent ruminal
GHJUDGDELOLW\RI'020DQG&3ZDVVLPLODUDFURVVWKHGLHWV DQG ZKHUHDVWKH
apparent ruminal degradability of NDF (P<0.01) and ADF (3 0.10) increased linearly when starch
ZDVUHSODFHGZLWKQRQIRUDJH¿EHU7KHUHZDVDWHQGHQF\WRUHGXFHWKHDSSDUHQWUXPLQDOGHJUDGDELOLW\
of starch (P< ZKHQVWDUFKZDVUHSODFHGZLWKQRQIRUDJH¿EHU7RWDOWUDFWGLJHVWLELOLW\RI'0
 20  &3  DQGVWDUFK  ZHUHVLPLODULUUHVSHFWLYHRIWKHGLHWEXWWRWDO
tract digestibility for NDF and ADF increased linearly (P<0.01) with the inclusion of non-forage
¿EHUWRUHSODFHVWDUFK

As dietary starch concentrations were decreased across diets, fecal N excretion tended to increase
linearly (3 0.09) and urinary N excretion increased linearly (3 0.01). However, average N excreted

Table 2. Nutrient digestion and N metabolism.

Item HS1 MS1 LS1 SEM P-value2

Ruminal degradability, %
DM 52.0 49.3 50.3 1.51 NS
NDF 33.7  58.9  L
ADF 51.5 57.1  3.32 L
Starch 94.2 91.9 90.8  LT
Total tract digestibility, %
DM 70.9 71.8 70.3 0.77 NS
NDF 41.1 52.7 59.5 2.50 L
ADF 41.0 50.9 58.2 3.00 L
Starch     NS
Nitrogen metabolism
Fecal N excretion, g/d  185 198 20.1 LT
Urine N excretion, g/d 214 218   L
Milk N, g/d   150 11.5 LT
1HI¿FLHQF\ 25.7 25.7 22.2 1.82 L

1HS = high starch diet; MS = medium starch diet; LS =low starch diet.
2L = linear effect (P<0.05); Q = quadratic effect (P<0.05); LT = linear effect (tendency) (P<0.10);
16 QRQVLJQL¿FDQW

62 Energy and protein metabolism and nutrition in sustainable animal production

via feces and urine as a percentage of total N intake was similar across the diets (28.9 and 35.5%
respectively). There was a tendency to reduce N excreted as true milk protein (3  DQG1
HI¿FLHQF\ 3 0.03) decreased linearly across the diets when starch was replaced with non-forage
¿EHU$OWKRXJKUHSODFLQJGLHWDU\VWDUFKZLWKQRQIRUDJH¿EHUGLGQRWDIIHFWUXPLQDODQGWRWDOWUDFW
digestibility of DM, OM, and CP, it increased ruminal and total tract digestibility of NDF and ADF.

References
Ranathunga, S. D., K. F. Kalscheur, A. R. Hippen, and D. J. Schingoethe, 2010. Replacement of starch from corn with
QRQIRUDJH¿EHUIURPGLVWLOOHUVJUDLQVDQGVR\KXOOVLQGLHWVRIODFWDWLQJGDLU\FRZV-'DLU\6FL
Reynal, S. M., and G. A. Broderick, 2005. Effect of dietary level of rumen-degraded protein on production and nitrogen
PHWDEROLVPLQODFWDWLQJGDLU\FRZV-'DLU\6FL
SAS, 2002. Version 9.3. SAS Institute Inc., Cary, NC, USA.

Energy and protein metabolism and nutrition in sustainable animal production 63

Effect of different dietary levels of Quebracho tannin extract on nitrogen
DQG¿EHUGLJHVWLELOLW\DQGSRVWUXPLQDOPLFURELDOSURWHLQÀRZLQKHLIHUV
S. Ahnert1, A. Susenbeth1 and U. Dickhoefer1,2
1,QVWLWXWHRI$QLPDO1XWULWLRQDQG3K\VLRORJ\&KULVWLDQ$OEUHFKWV8QLYHUVLWDHW]X.LHO.LHO
Germany; [email protected]
2Current address: Institute of Animal Production in the Tropics and Subtropics, University of
+RKHQKHLP6WXWWJDUW*HUPDQ\

Introduction
The effect of condensed tannins (CT) on protein digestion in ruminants appears to be dosage-
dependent. While low to moderate CT concentrations are considered to increase rumen-escape
protein, higher CT intakes may inhibit microbial crude protein (MCP) synthesis and decrease
nutrient digestibility (Barry and McNabb, 1999). Our aim was thus to determine how graded
GRVDJHVRID4XHEUDFKRWDQQLQH[WUDFW 47(WRWDOSKHQROLFFRQWHQW LQÀXHQFH0&3ÀRZDQG
GLJHVWLELOLWLHVRIQHXWUDOGHWHUJHQW¿EHU 1') DFLGGHWHUJHQW¿EHU $') QLWURJHQ 1 DQGDFLG
detergent insoluble N (ADIN).

Material and methods

6L[UXPHQ¿VWXODWHGKHLIHUV “NJERG\ZHLJKW ZHUHRIIHUHGNJJUDVVKD\NJ
FRQFHQWUDWHIHHGDQGJRIDPLQHUDOSUHPL[SHUGD\LQWZRHTXDOPHDOV7KHVWXG\FRPSULVHG
one period without QTE supplementation (Control I) followed by four periods when all animals
UHFHLYHGRU47(RIWKHLUGDLO\GU\PDWWHU '0 LQWDNH+DOIRIWKH47(GRVDJHZDV
LQIXVHGLQWUDUXPLQDOO\DWHDFKIHHGLQJ(YHU\SHULRGFRPSULVHGGRIDGDSWDWLRQDQGGRIWRWDO
XULQHDQGIHFHVFROOHFWLRQ'XULQJWKHFROOHFWLRQSHULRGUXPLQDOÀXLGVDPSOHVZHUHWDNHQIURP
three animals at 1, 2, 4, and 8 h after morning feeding. Subsequent to period 5, QTE dosing was
ceased and urine and feces were collected again for 10 d (Control II) after 14 d of adaptation. Feed
DQGIHFHVVDPSOHVZHUHDQDO\]HGIRU1')$')1DQG$',1FRQFHQWUDWLRQVDQGUXPHQÀXLG
IRUDPPRQLD1FRQWHQW'XRGHQDO0&3ÀRZZDVHVWLPDWHGIURPXULQDU\DOODQWRLQDQGXULFDFLG
excretions according to Chen and Orskov (2003), assuming a constant proportion of purine-N in
PLFURELDO16LJQL¿FDQW P<0.05) treatment effects were determined by ANOVA and regression
analyses by R software 2.12.0.

Results and discussion

Urinary purine derivative excretion was highest with 99 and 103 mM/d at 0% and 1% QTE,
UHVSHFWLYHO\DQGGHFOLQHGZLWKLQFUHDVLQJ47(VXSSOHPHQWDWLRQWRP0GDW47(UHÀHFWLQJ
DGHFUHDVHLQ0&3ÀRZIURP DW47( WRJG DW47(P<0.001; Table 1).
Apparent total tract digestibility of NDF declined linearly with increasing QTE dosage from 71.8%
ZLWKRXW47(WRDW47( 52=0.58; P<0.001). Digestibility of ADF decreased linearly
IURPDW47(WRDW47( 52=0.54; P<0.001). Besides a direct inhibition of
rumen microbes, this lower rumen carbohydrate fermentation may have also caused the decrease
LQ0&3ÀRZ1RFOHDUWUHQGZDVREVHUYHGIRUDSSDUHQWGLJHVWLELOLW\RI$',1 52=0.15; 3 0.021).
$SSDUHQW1GLJHVWLELOLW\GHFUHDVHGOLQHDUO\E\IURPZLWKRXW47(WRDW47(
(R2=0.90; P<0.001). This reduction can be associated with an incomplete post-ruminal dissociation of
tannin-protein complexes, an impaired protein absorption in the small intestine, reduced digestibility
RI¿EHUERXQG1DQGLQFUHDVHGHQGRJHQRXV1ORVVHV5XPHQDPPRQLD1FRQFHQWUDWLRQVZHUH
unaffected by QTE (4.7-5.8 mM/l; 3 0.171), most likely due to a lower ammonia-N incorporation
in MCP that outweighed the decrease in ammonia-N release from protein degradation. However,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 65
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_6, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIGLIIHUHQWOHYHOVRI4XHEUDFKRWDQQLQH[WUDFW 47( RQXULQDU\SXULQHGHULYDWLYH
3' H[FUHWLRQPLFURELDOFUXGHSURWHLQ 0&3 ÀRZDSSDUHQWWRWDOWUDFWGLJHVWLELOLW\RI1')$')
1$',1DQGUXPLQDODPPRQLD1FRQFHQWUDWLRQLQKHLIHUV OHDVWVTXDUHPHDQV6(0Q  

Parameter, Dietary QTE concentration (% daily dry matter intake) SEM P-value
unit
0 1 2 4  0
(Control I) (Control II)

PD, mM/d 99 c,d 103 d 95 b,c 90 b 80 a 98 b,c,d 4 <0.001

MCP, g/d  c,d 334 d 291 b,c  b 213 a 307 b,c,d 21 <0.001
Apparent total tract digestibility, %
NDF 71.8 b 70.0 b 70.7 b  a 59.0 a 71.0 b  <0.001
ADF  b  b,c  b 54.2 a,c  a  b 1.8 <0.001
N  d  c,d  c  b 59.8 a 71.8 d  <0.001
ADIN 12.5 a 9.1 a 11.5 a 1.7 a 5.3 a 35.8 b 3.9 <0.001
Ruminal ammonia-N, mM/l
5.4 5.7 4.7 5.8 5.2  0.4 0.171

1')QHXWUDOGHWHUJHQW¿EHU$')DFLGGHWHUJHQW¿EHU1QLWURJHQ$',1DFLGGHWHUJHQWLQVROXEOHQLWURJHQ6(0
standard error of the mean.

these conclusions are valid only, if the purine content in microbial matter remained constant and
thus the accuracy of MCP estimates were unaffected by the diet.

:KLOHGLHWDU\47(OHYHOVRIDQGPDUNHGO\UHGXFHGQXWULHQWGLJHVWLELOLWLHV0&3V\QWKHVLV
was already affected at 2% QTE in the diet. In conclusion, the supplementation of QTE at low
levels is expected to achieve no negative effects on MCP synthesis and a reduced ruminal protein
degradation may therefore improve post-ruminal protein supply. At dietary levels of QTE<2% of
DM intake it is unlikely that the decrease in MCP might be compensated by an increase in rumen-
escape protein. Furthermore, the decrease in energy supply to the animal and a possible negative
effect on feed intake allow for the use of QTE at very low concentrations only. Further studies are
needed to identify to what extent ruminal protein degradation is reduced and whether post-ruminal
protein digestibility is affected by QTE supplementation.

References
Barry, T.N., and W.C. McNabb, 1999. The implications of condensed tannins on the nutritive value of temperate forages
IHGWRUXPLQDQWV%U-1XWU
Chen X.B., and E.R. Orskov, 2003. Research on urinary excretion of purine derivatives in ruminants: Past, present and
future. International Feed Research Unit, Aberdeen, UK, 34 p.

66 Energy and protein metabolism and nutrition in sustainable animal production

Effect of fescue toxicosis on nitrogen and energy balance in Holstein steers
A.F. Koontz1, D.H. Kim1, A.P. Foote1, L.P. Bush2, J.L. Klotz, K.R. McLeod1 and D.L. Harmon1
1Department of Animal and Food Sciences, University of Kentucky, Lexington, KY, USA;
[email protected]
2Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA
USDA-ARS, Forage-Animal Production Research Unit, Lexington, KY, USA

Introduction
Animals consuming endophyte-infected tall fescue exhibit reduced weight gain and feed intake,
with the most severe effects occurring during summer months. Heat stress and plane of nutrition
have independent effects on metabolic activity (Birkelo et al., 1991; O’Brien et al., 2010) while,
HUJRWDONDORLGVUHGXFHEORRGÀRZWRDQGQXWULHQWDEVRUSWLRQIURPWKHUXPHQ )RRWHet al., 2012).
This research was designed to separate the effects of alkaloid consumption from those of reduced
intake and environmental temperature.

Material and methods

Two experiments were conducted, each using six ruminally cannulated Holstein steers weight-
matched (348±13 and 217±7 kg, respectively) and pair-fed alfalfa cubes to separate the effects of
alkaloid ingestion and energy intake. During each period, one steer per pair was ruminally dosed
twice daily with ground endophyte-infected tall fescue seed (E+; 0.025 mg ergovaline/kg BW/d),
the other with ground endophyte-free tall fescue seed (E-). Fescue seed dosing provided negligible
N and E to the animals. Exp. 1 utilized a two period crossover design, with two temperatures, (22 °C
and 30 °C) within each period. E+ steers were offered feed at 1.5×NEm. On d8 of each temperature
segment, animals were moved to metabolism stalls with indirect calorimetry head-boxes for fasting
heat production (FHP) determination. Rumen contents were removed, the reticulorumen was washed
DQG¿OOHGZLWKDEXIIHU 1D&O 1D+&23=24; KHCO3=30; K2HPO4=2; CaCl2=1.5; MgCl2=1.5
mmol/kg buffer), to which an E+ or E- fescue seed extract was added at 12h intervals to maintain
alkaloid treatment presentation during FHP. After 12 h, heart rate (HR), O2 consumption, CO2
SURGXFWLRQDQGXULQDU\RXWSXWZHUHUHFRUGHGIRUK([SXVHGDIRXUSHULRGFURVVRYHUGHVLJQ
with a 2×2 factorial treatment structure. Factors were endophyte (E+ vs. E-) and energy intake (1.8 ×
NEm, H vs. 1.1 × NEm, L). After 8 d of diet adaptation animals were dosed twice daily with ground
tall fescue seed for the remainder of each period. On d 17-21 total fecal and urinary output were
collected, with animals placed into indirect calorimetry head-boxes during d 20-21. Feed, feces,
and urine samples were analyzed for DM, N, and GE. Heat production was determined using the
%URXZHU  HTXDWLRQ'DWDZHUHDQDO\]HGXVLQJWKH0,;('SURFHGXUHRI6$6ZLWKVWHHUDV
WKHH[SHULPHQWDOXQLWDQLPDODQGSHULRGDVUDQGRPHIIHFWVDQG¿[HGHIIHFWVRIHQGRSK\WHWUHDWPHQW
and temperature (Exp. 1) or intake (Exp. 2).

Results and discussion

There was no difference (P>0.9) in DMI or DMI/kg0.75 between endophyte treatments in both
experiments, and DMI was different (P<0.01) between H and L in Exp. 2 by design.

During Exp. 1, increased temperature decreased intake (P<0.01), but had no effect on other
measurements. There were no interactions of temperature and endophyte treatment. O2 consumption
decreased (3 0.04) and CO2 production tended to be reduced (3 0.07) during E+ treatment. Fasting
heat production (kcal/kg BW0.75) was lower (3 7DEOH LQDQLPDOVUHFHLYLQJ(WUHDWPHQW
at both temperatures.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 67
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_7, © Wageningen Academic Publishers 2013
7DEOH&DORULPHWULFGDWDIURPVWHHUVGRVHGZLWKHQGRSK\WHIUHH ( DQGHQGRSK\WHLQIHFWHG ( 
WDOOIHVFXHVHHGDWƒ&DQGƒ& ([S 

Item Treatment SEM P=

E- E+ Main effects Interaction

22 °C 30 °C 22 °C 30 °C Endophyte (E) temperature (T) E × T

Heat produced  353.7 288.2 288.9 18.81  0.332 
(kJ/kg BW0.75/d)

In Exp. 2, animals on H feeding had higher (P<0.03) water, N, and energy intakes, energy and N
excretion, as well as retained N, DE, ME, and RE. There were no differences (P>0.15) in these values
between endophyte treatments. On average, L fed animals were below maintenance requirements,
based on an average RE of -111 kJ/kg BW.75 (Table 2). Thus the H and L diets show animals above
DQGEHORZPDLQWHQDQFHUHVSHFWLYHO\7KHLQWHUDFWLRQRILQWDNHîHQGRSK\WHZDVVLJQL¿FDQWIRU&22
production (3  DQGWHQGHGWREHVLJQL¿FDQW 3”0.11) for O2 consumption, CH4 production,
and HP. For each of these measures H was greater than L, and the difference between intakes was
greater with E+ treatment.

These data indicate that ingestion of endophyte infected tall fescue seed in pair fed steers reduces
basal metabolism, but does not alter total N or E balance. The reduction in FHP may be offset by
increased urinary and gaseous energy losses, as well as increased heat production by E+ dosed animals
at intakes above maintenance. These results indicate that reduced intake is likely the primary cause
of the reduced weight gain associated with fescue toxicosis.

7DEOH'LJHVWLEOHPHWDEROL]DEOHDQGUHWDLQHGHQHUJ\DQGKHDWSURGXFWLRQ N-NJ%:G RI

VWHHUVGRVHGZLWKHQGRSK\WHIUHH ( DQGHQGRSK\WHLQIHFWHG ( WDOOIHVFXHVHHGIHGDW[1(m
/ DQG[1(m +  ([S 

Treatment SEM P

E- E+ Main effects Interaction

L H L H Endophyte (E) Intake (I) E × I

Digestible energy 572.5  553.9 950.3   <0.0001 
Metabolizable energy  841.9  795.1 50.85 0.381 <0.0001 0.540
Retained energy -123.5  -100.3 97.9 55.55 0.585 0.0002 
Heat production 574.4  542.9  18.47 0.828 <0.0001 0.075

References
Birkelo, C. P., Johnson, D. E. and Phetteplace, H. P. 1991. Maintenance requirements of beef cattle as affected by
VHDVRQRQGLIIHUHQWSODQHVRIQXWULWLRQ-$QLP6FL  
%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV3URFUG6\PS(QHUJ\0HWDE7URRQ6FRWODQG
Foote, A. P., Kristensen, N. B., Klotz, J. L., Kim, D. H., Koontz, A. F., McLeod, K. R., Bush, L. P. and Harmon, D. L.
(UJRWDONDORLGVGHFUHDVHUXPHQHSLWKHOLDOEORRGÀRZ-$QLP6FL 6XSSO  $EVWU 
O’Brien, M. D., Rhoads, R. P., Sanders, S. R., Duff, G. C. and Baumgard, L. H. 2010. Metabolic adaptations to heat
VWUHVVLQJURZLQJFDWWOH'RPHVW$QLP(QGRFULQ  

68 Energy and protein metabolism and nutrition in sustainable animal production

3HUIRUPDQFHHI¿FLHQF\DQGHVWLPDWHGPDLQWHQDQFHHQHUJ\UHTXLUHPHQWV
of Bos taurus and Bos indicus cattle
R.D. Sainz1,2, G.D. Cruz1, E. Mendes2, C.U. Magnabosco2, Y.B. Farjalla, F.R.C. Araujo, R.C.
Gomes and P.R. Leme
1University of California, Davis, CA, USA; [email protected]
2Embrapa, Brasília, DF, Brazil
Aval Serviços Tecnológicos, Goiânia, GO, Brazil
Universidade de São Paulo, Pirassununga, SP, Brazil

Introduction
The NRC (2000) concluded that maintenance requirements of Bos indicus cattle were about 10%
lower than those of Bos taurus cattle, however other reports do not support any difference between
breed types (Tedeschi et al. $UHFHQWIRFXVRQJHQHWLFVHOHFWLRQIRULQFUHDVHGIHHGHI¿FLHQF\
has led to greater efforts to quantify individual feed intakes on farms and research institutes. This
study has combined several of those data sets, including B. indicus and B. taurus cattle, in high and
ORZHI¿FLHQF\FDWHJRULHVLQRUGHUWRUHYLVLWWKHPDLQWHQDQFHLVVXH

Material and methods

Individual data on feed intakes and weight gains from 11 experiments with a mean duration of
“GD\VFRQGXFWHGDWWKH8QLYHUVLW\RI&DOLIRUQLD±'DYLV WULDOV$QJXV+HUHIRUGDQG
crossbreds), the University of São Paulo – Pirassununga (1 trial, Nelore), and three private ranches
in Brazil (Guaporé Pecuária, Pontes e Lacerda – MT, 2 trials, Polled Nelore; Rancho da Matinha,
Uberaba – MG, 2 trials, Nelore; Fazenda Perfeita União, Pirajuí – SP, 2 trials, Guzerá) were pooled
and analyzed. The combined data set comprised 838 animals (127 B. taurus steers and 711 B. indicus
bulls). In each trial, residual feed intakes (RFI) were calculated as the residual of the regression of
dry matter intake (DMI) on average metabolic body weight and average daily gain (ADG). Animals
with RFI below mean -0.5 SD, within 0.5 SD of the mean or above mean -0.5 SD were classed as
Low, Medium and High RFI groups, respectively. In addition, for each trial the dietary metabolizable
DQGQHWHQHUJ\YDOXHVZHUHHVWLPDWHGDQGVWDQGDUG15&  JDLQHTXDWLRQVXVHGWR¿WWKH
PDLQWHQDQFHHQHUJ\UHTXLUHPHQWWRWKHGDWDIRUHDFKDQLPDO =LQQDQG6KHQ 'DWDZHUH
subjected to analysis of variance, with Breed Type and RFI Group as main effects. The interaction
EHWZHHQ%UHHG7\SHDQG5),JURXSZDVQRWVWDWLVWLFDOO\VLJQL¿FDQWIRUDQ\YDULDEOH

Results and discussion

Differences among RFI groups were as expected, with Low RFI cattle having lower DMI, similar
ADG, greater gain:feed, and lower RFI values than High RFI cattle (Table 1). Maintenance energy
requirements were lower in Low vs. High RFI cattle. Dry matter intakes were lower in B. indicus
than in B. taurus cattle, both in absolute terms and relative to body weight. This was accompanied
by lower weight gains and gain:feed. As expected, RFI values did not differ between types, since the
RFI calculation was done within trial. Maintenance energy requirements were lower in B. indicus than
in B. taurus cattle, but this difference was smaller than that among RFI groups of both cattle types.

These results support the conclusion of the NRC (2000) of lower maintenance requirements of B.
indicus cattle as compared to B. taurus cattle. In these data, the difference in maintenance requirements
was about 7%, but this is confounded by the fact the all the B. taurus animals were steers and all the
B. indicus animals were bulls. One would expect a greater difference between animals of the same
sexual condition. On the other hand, the difference in maintenance requirements between the most
DQGOHDVWHI¿FLHQWJURXSVZDVJUHDWHUWKDQLQGLFDWLQJWKDWYDULDWLRQLQPDLQWHQDQFHUHTXLUHPHQWV

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 69
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_8, © Wageningen Academic Publishers 2013
Table 1. Performance of Bos taurus and Bos indicusFDWWOHLQGLIIHUHQWHI¿FLHQF\FDWHJRULHV

Variable Type RFI group SD2 P-value1

B. taurus B. indicus High Medium Low Type RFI group

N 127 711 233  241

Dry matter intake, kg/d 10.34 9.50 10.97   1.77 <0.001 <0.001
Dry matter intake, % of BW 2.51 2.33  2.43 2.22 0.34 <0.001 <0.001
Average daily gain, kg/d 1.511 1.222 1.384 1.349  0.325 <0.001 0.428
Gain:feed 0.147 0.130  0.140 0.149 0.027 <0.001 <0.001
Residual feed intake, kg/d 0.032 0.009 0.882 0.032 -0.853 0.374 0.534 <0.001
0DLQWHQDQFHFRHI¿FLHQW 0.0750   0.0710 0.0597 0.020 0.012 <0.001
Mcal/kg0.75/d

1 Probability of a Type I error.

2 SD, pooled standard deviation.

within breed types is much greater than the variation among breed types. We conclude that there is
JUHDWVFRSHIRUJHQHWLFVHOHFWLRQIRULQFUHDVHGIHHGHI¿FLHQF\LQDOOEHHIFDWWOHEUHHGVDQGWKDW5),
may be used as an indicator trait for maintenance requirement.

References
National Research Council, 2000. Nutrient Requirements of Beef Cattle, 7th Rev. ed., National Academy Press,
Washington, DC.
Tedeschi, L.O., C. Boin, D.G. Fox, P.R. Leme, G.F. Alleoni and D.P.D. Lanna, 2002. Energy requirement for maintenance
DQGJURZWKRI1HOORUHEXOOVDQGVWHHUVIHGKLJKIRUDJHGLHWV-$QLP6FL
=LQQ5$DQG<6KHQ,QWHUDFWLRQRIGLHWDU\FDOFLXPDQGVXSSOHPHQWDOIDWRQGLJHVWLYHIXQFWLRQDQGJURZWK
performance in feedlot steers. J. Anim. Sci. 74, 2303-2309.

70 Energy and protein metabolism and nutrition in sustainable animal production

Energy for maintaining liveweight: an indicator of adaptive abilities of
beef cows?
A. De La Torre1,2, F. Blanc2,1, E. Recoules1,2, D. Egal and J. Agabriel1,2
1,15$805+HUELYRUHV6DLQW*HQqV&KDPSDQHOOH)UDQFH
[email protected]
29HW$JUR6XS805+HUELYRUHV&OHUPRQW)HUUDQG)UDQFH
,15$8(0RQWVG¶$XYHUJQH2UFLYDO)UDQFH

Introduction
Productivity of low input livestock systems partly relies on animals’ ability to cope with changing
environments while achieving productive and reproductive performances. In such conditions,
individuals’ robustness could be estimated by indicators of animal adaptive abilities which account
for energy variations across the productive cycle. In mature suckler cows, the net energy requirements
for production are low (30%) compared to those for maintenance (70%) which complicates the
evaluation of adaptive abilities. The later could be approached by estimating the net energy required
for maintaining liveweight constant (Em). The objective of this study was to (1) estimate in beef cows
having different body reserves at calving the partition of net energy between net energy outputs (Em,
Emilk) and net energy inputs (Eintake + Etissues) and (2) test the relevance of Em as an indicator
of adaptive abilities of beef cows.

Material and methods

Fourty multiparous charolais cows varying in body condition at calving (BCS scale from 0 to 5):
20 thin (T, BCS=2.0±0.04) vs. 20 fat (F, BCS=2.8±0.08) were submitted to a nutritional challenge
consisting in a sequence of two contrasted feeding periods. During period 1 (P1), from calving to
120 days postpartum, cows were reared indoors and were fed the same basal diet according to two
energy levels (Control, C vs. Low, L). Intake expressed in NE for lactation (NEL) averaged 0.57
DQG0-GNJ0.75 for C and L cows. At the end of P1, all cows were turned out to a permanent
SDVWXUHIRUDGD\VSHULRG 3 ,QGLYLGXDOLQWDNHVDWJUD]LQJZHUHHVWLPDWHGIURP¿OOXQLWV\VWHP
(INRA, 2007). During P1 and P2, body weight, BCS and milk production were regularly measured.
Body lipids were assessed by measuring subcutaneous adipose cell diameter at calving and at the end
RI3DQG3(PZDVFDOFXODWHGRYHU3DQG3,WZDVGH¿QHGDVIROORZ(P (LQWDNH± (PLON
+ Etissues), all terms expressed in Net Energy for lactation. Energy in milk was taken at 3.2 MJ/kg
assuming a standard milk composition for beef cows (INRA, 2007). The tissue net energy conversion
rate to milk production was taken as 0.8. We assumed that the net energy value of a kilogram of body
PDVVFKDQJHLVHTXDOWR0-îRIOLSLGV0-[RIIDWIUHHPDVV *DUFLDDQG$JDEULHO
2007). Data were analyzed using PROC MIXED (SAS) including BCS at calving, postpartum energy
OHYHOVDQGGD\VDV¿[HGHIIHFWVDQGLQGLYLGXDODVUDQGRPHIIHFW

Results and discussion

Over the whole experimental period, average milk production and calf body weight gain were similar
between the four groups. Over P1, FL and TL cows lost 43±13 and 25±17 kg compared to FC and
TC cows, respectively (Table 1). L treatment resulted mainly in a fat mass loss, whereas C treatment
induced no (FC) or only a slight (TC) weight gain which was mostly due to an increase in fat mass
(99 MJ). These discrepancies resulted in differences in net energy balance (Figure 1). Decreasing
E intake led to a decrease in Em, which was 25% lower in L vs. C level of energy intake (3).
At the end of P2, FL and TL cows weighed 20 and 10 kg less than FC and TC cows, respectively.
Over P2, Em remained lower in cows previously submitted to energy restriction in comparison to
C cows (3). This result is related to a compensatory rebound which is associated to lower

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 71
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_9, © Wageningen Academic Publishers 2013
Table 1. Effect of body condition at calving and postpartum energy level on milk production, average
GDLO\JDLQRIFDOYHV $'* OLYHZHLJKW /: DQGDGLSRVHFHOOVGLDPHWHU $&' 

Period 1 (indoors) Period 2 (pasture)

FC FL TC TL SEM FC FL TC TL SEM

Milk (kg/day) 8.3 8.7 9.2 7.0     7.1 0.5
ADG (kg/day) 0.8 0.9 0.9 0.7 0.09 1.1 1.2 1.1 1.1 0.10
LW start(kg) ab 852a ab 753b 17.4 827a 790ab 734ab b 17.8
LW end (kg) 834a 810ab ab 719b 17.9 805 824   18.2
ACD change -4.5a b a -10.0b 2.0 7.5 11.2 0.93 3.4 

Figure 1. Effect of body condition at calving and postpartum energy level on energy balance and
SDUWLWLRQGXULQJWKHLQLWLDOLQGRRUV  DQGSDVWXUH  SHULRGV

Em after a period of nutritional restriction (Hornick, 2000). It is the lag time in adaptation of Em
before its gradual increase which gives to the restricted cows the ability to recover weight loss and
condition without affecting milk production. Much of Em variation with energy level is related to
rapid changes in visceral mass and energy expenditures (Freetly et al., 1998; Ortigues et al., 1993).

Thus, the ability of cows to mobilize and recover body reserves under restriction/refeeding periods
provides them the adaptive ability to low net energy for maintaining liveweight constant. The lag time
in adaptation of Em as a result of the feeding restriction is partly the support of cows’ adaptation at
least at short and medium term. The long term effects of adaptation should be further investigated.
Variations in Em can be so interpreted as an indicator of the ability of mature producing cows to
face nutritional constraints.

References
)UHHWO\+-1LHQDEHUDQG7%URZQ%UDQGO(I¿FLHQF\RIHQHUJ\DQGQLWURJHQORVVDQGJDLQLQPDWXUHFRZV
-RXUQDORI$QLPDO6FLHQFH
Garcia F. and J. Agabriel, 2007. Update of feeding recommendations for fattening of culled cows. Development of a
model for the estimation of the composition of weight gain and associated requirements. Productions Animales,
20, 137-149.
Hornick J., C. Van Eename, A. Clinquart, O. Gerard and L. Istasse, 2000. Mecanism of reduced and compensatory
growth. Domestic Animal Endocrinology, 19, 121-132.
INRA 2007. Alimentation des bovins, ovins et caprins. Besoins des animaux – valeurs des aliments, Quae (Ed), Versailles
Ortigues I., M. Petit, J. Agabriel and M. Vermorel, 1993. Maintenance requirements in metabolizable energy of adult
QRQSUHJQDQWQRQODFWDWLQJ&KDURODLV&RZV-RXUQDORI$QLPDO6FLHQFH
SAS, 2009. Release 9.2. SAS Inst. Inc, cary, NC, USA.

72 Energy and protein metabolism and nutrition in sustainable animal production

Response to high altitude grazing in metabolic traits and performance
by yak crossbreds and yaks
S. Barsila1, N.R. Devkota2, M. Kreuzer1 and S. Marquardt1
1 (7+ =XULFK ,QVWLWXWH RI $JULFXOWXUDO 6FLHQFHV  =XULFK 6ZLW]HUODQG
[email protected]
2Institute of Agriculture and Animal Science, Rampur, Chitwan, Nepal

Introduction
Yaks (Bos grunniens) and yak crossbreds are kept in transhumant systems using different pastures along
an altitudinal gradient in the Nepalese Himalayan Mountains. Yaks are known to be well adapted to cope
with low oxygen partial pressure, low temperatures and the harsh mountain environment (Wiener et al.,
 DQGWKHUHVXOWLQJHQHUJ\GH¿FLHQF\/HVVLQIRUPDWLRQLVDYDLODEOHRQWKHDGDSWLYHFDSDELOLWLHV
of yak crossbreds. Crossbreeding is a strategy to obtain higher milk yields especially by heterosis. In
the Eastern Nepalese Himalayan Mountains, two different local breeds are used for crossbreeding.
Bhelang bulls from near Tibet (Bos taurus genotype) are crossed with female yaks, and yak bulls are
crossed with female Nepalese hill cattle (Bos indicus). Female crosses of cattle bulls × yak cows are
locally called Dimjo chauries, while those which are produced using cows and yak bulls are called
Urang chauries (Joshi, 1982). In order to compare the adaptive capacity of these two crossbred types
with that of yaks, an experiment was conducted at two altitudes in the Taplejung District of Nepal.

Material and methods

Six adult lactating females per genotype that calved in 1-2 week intervals from the beginning of
April onwards (B. grunniens × B. indicus Q <î, B. taurus × B. grunniens Q 7î< DQG B.
grunniens Q < ZHUHVHOHFWHG'LIIHUHQWIURPWKHFURVVEUHGVWKH\DNVZHUHDFFRPSDQLHGE\WKHLU
calves and only milked in the morning. The crossbreds were milked twice a day. The experiment
ZDVFRQGXFWHGDWP $XJXVW DQGP 2FWREHU GXULQJZHHNVHDFK7KH¿UVWGD\VZHUH
XVHGDVDGDSWDWLRQSHULRGDQGWKHODVWGD\VZHUHXVHGIRUPHDVXUHPHQWVDQGGDWDVDPSOLQJ'DLO\
measured data included milk yield and milk composition. Milk fat, protein and lactose were analysed
with a portable milk analyser (Lactoscan SA-L, Milkotronic Limited, Nova Zagora, Bulgaria) at every
milking. For the crossbreds, the aliquot was calculated from morning and evening measurements.
In yaks, the amount suckled by the calves was determined by the weigh-suckle-weigh method.
Heart rate was measured in one animal per genotype attaching the device to a different animal
HYHU\GD\3RODU(TXLQH&6;7URWWLQJLQVWUXPHQWV 3RODU(OHFWUR2\.HPSHOH)LQODQG DQG
the corresponding software were used for measurement and analysis of the cardiac data. Only short
HYHQWVRIFRQVHFXWLYHPLQXWHVZKHUHWKHDQLPDOVZHUHVWDQGLQJ ”VWHSVPLQ ZHUHXVHGIRUGDWD
analysis by calculating a mean value from 1-4 measurements/animal. Respiration rate and rectal
temperature were measured in the morning and evening after milking by using a stethoscope and
DWKHUPRPHWHU(DUEORRGVDPSOHVZHUHWDNHQRQWKH¿UVWGD\RIDGDSWDWLRQDQGWKHODVWGD\RIWKH
sampling period and were immediately analysed for haemoglobin, glucose and lactate using point-
of-care devices designed for human application (HemoCue Hb 201+, HemoCue AB, Ångelholm,
Sweden; Accu-Chek Aviva, Roche Diagnostics, Mannheim, Germany and Accutrend Plus, Roche
Diagnostics, Mannheim, Germany).

The Mixed procedure of the SAS program (SAS Institute Inc., Cary, USA, 2009 version 9.3) was
used for statistical analysis. Data on heart rate and milk yield and composition was analyzed with
DOWLWXGHJHQRW\SHDQGLWVLQWHUDFWLRQDV¿[HGHIIHFWVDOWLWXGHDVUHSHDWHGIDFWRUDQGDQLPDOQHVWHG
within genotype as subject. For respiration rate and rectal temperature the model included additionally
the effect of daytime and a random effect was estimated here to account for the two repeated within-
subject factors time and altitude.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 73
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_10, © Wageningen Academic Publishers 2013
Results and discussion
Blood haemoglobin was affected by genotype and altitude (P<0.001). The highest levels were
UHFRUGHGDWWKHHQGRIWKHVWD\DWP DQGJOLQ<7î<DQG<î,UHVSHFWLYHO\ 
Y already started from higher levels (154 g/l) than TxY (145 g/l) and even more than Y×I (119 g/l)
as measured at arrival at 4,700 m. Haemoglobin values decreased (P<0.05) during the stay at 3,000
m for both crossbreds, but were unchanged (P> IRU<$W¿UVWVDPSOLQJDWPEORRG
JOXFRVHOHYHOZDVKLJKHULQ7[< PPROO WKDQLQ<î, PPROO ZLWK<EHLQJLQWHUPHGLDWH
(P>0.05); this had been levelled out until the second sampling at 4,700 m. Across sampling times
DQGJHQRW\SHVJOXFRVHOHYHOVZHUHKLJKHUDWPDVFRPSDUHGWRP DQGPPROO
respectively). In blood lactate, differences between the genotypes were large at 4,700 m being low
LQ< PPROO LQWHUPHGLDWHLQ7î< PPROO DQGKLJKLQ<î, PPROO 7KHYDOXHV
decreased during the stay at 4,700 m in all three genotypes by keeping the same order. At 3,000 m,
no differences were found between the crossbreds and times of measurement (P>0.05). Only the
values measured for yaks were higher (5.5 mmol/l, P< DWWKH¿UVWPHDVXUHPHQW+HDUWUDWH
was overall higher at 4,700 m as compared to 3,000 m (78 and 50 beats/min, respectively). Also
respiration rate and rectal temperature were higher at 4,700 m as compared to 3,000 m. Elevated
levels of these three traits and of blood haemoglobin as response to altitude were also found elsewhere
(Bianca and Näf, 1979). At 4,700 m, milk yield (kg/day) was 2.5 (T×Y), 2.1 (Y) and 2.1 (Y×I). At
3,000 m, Y and Y×I only had a milk yield of 0.8 kg/day while T×Y still yielded 1.2 kg/day. Milk
IDWFRQWHQWZDVKLJKHUDWPEHLQJKLJKHVWLQ<  IROORZHGE\7î<  DQGORZHVW
LQ<î,  $WPPLONSURWHLQFRQWHQWZDVJHQHUDOO\KLJKHU DQGLQ<7î<
and Y×I, respectively, P>0.05) than at 3,000 m.

The initially very high levels of haemoglobin and low levels of lactate and the comparably high
yield and milk protein content illustrate that Y are better adapted to high altitude than any of the
crossbreds. However, T×Y not only showed the best performance among the three genotypes, but
was also close to Y in the variables indicative of adaptation. Having the choice between Y×I and
T×Y, the latter therefore seems to be a better alternative for farmers which have access to very high
altitude pastures.

References
Bianca W. and F. Näf, 1979. Responses of cattle to the combined exposure, to diurnal temperature rhythm (-5 to 25
°C) and to simulated high-altitude (4,000 m). Int. J. Biometeor 23, 299-310.
Joshi, D.D., 1982. Yak and chauri husbandry in Nepal. K. D. Joshi (Publisher), Kathmandu, Nepal. 145 pp.
:LHQHU*+-LDQOLQDQG5-/RQJ HGV 7KH<DN(GLWLRQ5$33XEOLFDWLRQ)$25HJLRQDO2I¿FH
IRU$VLDDQGWKH3DFL¿F%DQJNRN7KDLODQGSS

74 Energy and protein metabolism and nutrition in sustainable animal production

Estimates of nutritional requirement of sheep, goats and cattle in
tropical and warm countries: a meta-analysis study
N. Salah1, D. Sauvant2 and H. Archimède1
1,15$858QLWpGH5HFKHUFKHV=RRWHFKQLTXHV3HWLWERXUJ*XDGHORXSH)UDQFH
[email protected]
2,15$8050RGpOLVDWLRQ6\VWpPLTXH$SSOLTXpHDX[5XPLQDQWVUXH&ODXGH%HUQDUG
3DULV)UDQFH

Introduction
Until now, feeding recommendations for livestock in tropical and warm areas are largely based on
standards produced in temperate area (ARC, 1984; INRA, 1989; NRC, 1985). Nevertheless, energy
and protein requirements of livestock in warm area could differ from those of temperate area. The
objective of this study was to estimate energy and protein requirements of ruminants in tropical and
warm area using a meta-analysis.

Material and methods

Published studies performed with growing sheep, goat and cattle were used for this analysis. To
be included in this meta-analysis, the papers were selected on some criteria. At a minimum, trials
should report data on animal weight gain, chemical composition of diets, intake (organic matter, OM
and crude protein, CP), total tract digestibility (OM and CP) and nitrogen balance. Metabolisable
energy intake (kcal MEI/kg BW) was predicted from digestible OM intake per BW (g DOMI/kg
BW) on the basis of a data base:

MEI/BW = -2.03 + 4.03 DOMI/BW (n=975, R2=0.99, RSD=11.3)

In total, 590 publications representing 2,225 dietary treatments were pooled to be used in the present
study. There were 325 and 1,287; 145 and 544; 119 and 394 publications and treatments for sheep,
goat and cattle respectively. Genotype animals from warm regions were differentiated from those of
temperate regions. The meta-analysis has been performed following recommendations of Sauvant et
al. (2008). Inter-publications regressions of nutrient intake (as explained variables) on average daily
gain (ADG) were performed to estimate requirements for maintenance and growth. The intercept and
the slope of the regression are the estimation of the maintenance and growth requirements respectively.
0RUHRYHUWRWHVWVLPXOWDQHRXVO\WKHLQÀXHQFHVRIVSHFLHVDQGJHQRW\SHVRQHLWKHUWKHLQWHUFHSWRU
the slope of the regressions, analyses of variance-covariance were applied on the parameters. The
study effect was considered random. ME has been used as energy unit. Protein requirements were
estimated regressing Digestible CP intake (DCPI) on ADG or retained nitrogen (Nr). Two units of
BW (BW and BW0.75) were tested because hierarchy between species could differ depending on
unit. Normal distribution of data was observed within and across species and BW units.

Results and discussion

(TXDWLRQLQGLFDWHVWKDW0(IRUPDLQWHQDQFH 0(P RIFDWWOHZDVVLJQL¿FDQWO\KLJKHUWKDQ
those of sheep and goat according to their BW0.75. No differences were noted between genotypes.
&RQFHUQLQJ0(UHTXLUHPHQWIRUJURZWK 0(J GLIIHUHQFHVZHUHQRWVLJQL¿FDQWEHWZHHQVSHFLHV
nor genotypes. When requirements were estimated on the basis of the BW, values were lower with
cattle compared with small ruminants (Equation 2). Moreover, MEm was higher for tropical small
ruminants compared to the temperate ones (Equation 2).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 75
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_11, © Wageningen Academic Publishers 2013
7DEOH,QWHUSXEOLFDWLRQVUHJUHVVLRQVRI(QHUJ\LQWDNH .FDOPHWDEROLFHQHUJ\LQWDNH0(,NJERG\
ZHLJKW%:RU0(,NJPHWDEROLFERG\ZHLJKW0%: RUGLJHVWLEOHFUXGHSURWHLQLQWDNH J'&3, 
RQDYHUDJHGDLO\JDLQV $'*J SHUIRUPHGWRHVWLPDWHUHTXLUHPHQWVIRUPDLQWHQDQFHDQGJURZWK
of warm genotype livestock.

Equation Equation regression

1 0(,0%: (L$'*0%: Q VSHFLHV 52=0.41, RSD=42.2)

Ei = 129.2±4.2 kcal ME /kg MBW for small ruminants, 150.3±4.2 kcal ME/kg MBW
for cattle.
2 0(,%: (L$'*%: Q VSHFLHV 52 56' 
(L “NFDO0(NJ%:IRUVPDOOUXPLQDQWV“NFDO0(NJ%:IRU
cattle.
3 '&3,0%: (L1U0%: Q VSHFLHV 52 56' 
(L “IRUVKHHS“IRUJRDWVDQG“IRUFDWWOH
4 '&3,0%: $'*0%:±$'*0%:2 (n=331, R2=0.41,
RSD=2.1)
5 ADG/MBW = Ei + 9.85 Nr/MBW (n=182, species=3, R2=0.41, RSD=3.74)
(L “IRUVKHHS“IRUJRDWV“IRUFDWWOH

7KHUHJUHVVLRQRI'&3,DJDLQVW1ULQGLFDWHGVLJQL¿FDQWO\KLJKHUSURWHLQPDLQWHQDQFHUHTXLUHPHQW
for sheep compared to goats with an intermediate value for cattle (Equation 3). No differences were
noted between species and genotype for protein growth requirement assimilated as Nr. However, it
is known that Nr leads to an overestimation of growth requirements. The regression of DCPI against
ADG was curvilinear (Equation 4). No differences were noted between species and genotype for
protein maintenance requirements. As the regression is not linear, the marginal DCP requirement/kg
$'*GHFUHDVHVIURPJ'&3NJ$'*ZKHQ$'*LVFORVHWRWRJ'&3NJ$'*ZKHQ
$'* JNJ0%:DQGHTXDOWRJ'&3NJ$'*ZKHQ$'* JNJ0%: FRUUHVSRQGLQJ
to the highest values recorded for growth in the data base).

As a conclusion, in this study we hypothesized that MEm corresponds to ME requirements for

absence of weight gain rather than absence of energy gain. Moreover we have not taken into account
changes in the energy content of BW gain. This approach is different from that used to establish
some international standards (INRA, ARC or NRC) and could partly explain some of the observed
differences. However, the comparisons made in this study, with the same methodology, indicate that
the energy and protein requirement for maintenance and growth estimated for tropical livestock are
higher than values for temperate livestock.

References
ARC, 1984. Nutrient requirements of ruminant livestock.
INRA, 1989. Ruminant nutrition. Recommended Allowances and Feed Tables. Jarrige R. (ed.) Paris, France, 373 p.
15&1XWULHQWUHTXLUHPHQWVRIVKHHSWK5HYLVHGHG:DVKLQJWRQ'&1DWLRQDO$FDGHP\RI6FLHQFHV
Sauvant, D., P. Schmidely, J.J. Daudina and N.R. St-Pierre, 2008. Meta-analyses of experimental data in animal
nutrition. Animal 2, 1203-1214.

76 Energy and protein metabolism and nutrition in sustainable animal production

Net energy and protein requirements for growth of goats kids
I.A.M.A. Teixeira1, N. St-Pierre2, A. Cannas, A.P. Souza1, M.H.R. Fernandes1 and K.T. Resende1
181(63 8QLY (VW 3DXOLVWD 'HS GH =RRWHFQLD  -DERWLFDEDO 63 %UD]LO
[email protected]
27KH2KLR6WDWH8QLYHUVLW\'HSDUWPHQWRI$QLPDO6FLHQFHV&ROXPEXV2+86$
8QLYHUVLWi'HJOL6WXGLGL6DVVDUL'LSDUWLPHQWRGL6FLHQ]H=RRWHFQLFKH6DVVDUL,WDO\

Introduction
Knowledge of nutritional requirements is decisive for a successful nutrition system, since animals
IHGSURSHUO\FRQYHUWPRUHHI¿FLHQWO\WKHQXWULHQWVLQJHVWHGLQSURGXFWV&XUUHQWIHHGLQJV\VWHPVIRU
goats still comprise information extrapolated from sheep and/or cattle. However, the data obtained
with these species should not be used for goats, because of differences between these species, such
as dietary habits, physical activity, milk and carcass composition, different adaptation mechanisms,
among others (NRC, 2007). Therefore, the objective of this study was to determine net energy and
protein requirements for growth of goat kids, using meta-analysis as a statistical tool.

Material and methods

A total of 412 goat kids from nine studies carried out at UNESP (Jaboticabal, Brazil) and UFV
(Viçosa; Brazil) were used. These studies comprise goats of 4 genotypes, 3 genders and in different
composition of mature body weight (BW). The main characteristics of each study are presented
LQ7DEOH,Q¿YHRIWKHVWXGLHV VWXGLHVDQG JRDWNLGVZHUHXQZHDQHGLQSDUWRIWKH
trial. All studies used diet with roughage to concentrate ratio of approximately 50:50. In all studies
individual measurements of intake, digestibility, crude protein and metabolizable energy intake and
body composition (based on comparative slaughter) were available.

Net energy and protein requirements for gain were estimated using animals fed ad libitum.
Logarithmized allometric equations were used to obtain prediction models of protein and energy

Table 1. Description of the studies used in the meta-analysis.

Study N Gender Genotype Body weight1 Body composition2

1 34 Male 1/2 Boer, 1/2 Saanen 15 to 25 18.5; 9.7; 1.9

2 30 Male 1/2 Boer, 1/2 Saanen 5 to15 19.0; 5.4; 1.5
3 41 Male Saanen 5 to 20 17.8; 7.4; 1.7
4 45 Castrated male Saanen 20 to 35 
5 33 Male 3/4 Boer, 1/4 Saanen 20 to 35 18.9; 14.9; 2.3
 18 Male 1/2 local, 1/2 dairy breed 5 to 25 
7 30 Male 1/2 local, 1/2 dairy breed 5 to 15 18.0; 8.9; 2.5
8  Castrated male
23 Female 5 to 15 25.0; 8.2; 2.1
31 Male Saanen
9  Castrated male
33 Female 30 to 45 
32 Male Saanen

1 Body weight (kg) range of the animals from the initial to the slaughter body weight.
2 Average of protein (% empty body weight – EBW), fat (% EBW) and energy (Mcal/kg EBW) body composition of
the animals, respectively.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 77
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_12, © Wageningen Academic Publishers 2013
concentration of empty body weight (EBW), based on the amount of protein and energy in the
empty body. And the derivative of these equations were used to estimate net protein and energy
requirements for gain, according to ARC (1980). Net protein and energy maintenance requirements
were estimated by regressing protein and energy retention on crude protein and metabolizable
energy intake, respectively. The data were analyzed using MIXED procedure of SAS and orthogonal
contrasts were used to test the intercepts and slope estimates in the regression equations among the
main evaluated factors (genotype, gender and body weight range) across studies.

Results and discussion

The parameters of the equation to predict energy and protein concentration of empty body weight are
presented in Table 2. Net protein requirement for gain decreased as body weight increased (P<0.05),
in Saanen goat kids. An opposite pattern was observed with net energy requirement for gain, in which
net energy requirement increased with increasing of body weight (P<0.05). It was also observed
DVLJQL¿FDQWJHQRW\SHHIIHFWLQWKHQHWHQHUJ\UHTXLUHPHQWVIRUJDLQ P<0.05). On the other hand,
there was no gender effect in net protein and energy requirement for gain of goat kids (P>0.05).
At the beginning of the growth stage, net protein requirements for maintenance of Boer crossbred
goat kids were greater compared to Saanen goat kids (P< ,WZDVQRWREVHUYHGVLJQL¿FDQWJHQGHU
effect in the net energy and protein requirement for maintenance in Saanen goat kids (P>0.05).
In conclusion, net requirements for growth of goat kids is not affected by gender, on the other hand
JHQRW\SHLQÀXHQFHGQHWUHTXLUHPHQWVIRUJURZWK

Table 2. Parameters of the regression equations of protein and energy concentration on empty body
ZHLJKW (%: RIJRDWNLGV

Study Protein1 Energy2 P-value3

Intercept Slope Intercept Slope

1 2.38±0.12 0.90±0.10 2.84±0.11 1.37±0.10 <0.0001

2 “ “ 2.94±0.05 “ <0.0001
3 2.20±0.04 1.04±0.04 3.07±0.04 “ <0.0001
4 2.32±0.12 0.94±0.09 2.52±0.11 “ <0.0001
5 “ 1.08±0.09 3.04±0.11 1.23±0.08 <0.0001
 2.18±0.04 1.10±0.04 3.05±0.04 1.19±0.04 <0.0001
7 2.22±0.04 1.04±0.04 “ 1.45±0.04 <0.0001
8 castrated male 2.43±0.05 “ 3.14±0.05 “ <0.0001
8 female “ 0.92±0.05 3.07±0.05 1.30±0.05 <0.0001
8 male 2.40±0.05 1.00±0.05 3.12±0.05 1.22±0.05 <0.0001
9 castrated male “ 1.11±0.11 “ 1.53±0.11 <0.0001
9 female “ 1.14±0.11 “ 1.55±0.11 <0.0001
9 male 2.33±0.19 0.93±0.13 2.50±0.18 “ <0.0001

1 Log protein (g) = b0 + b1 Log EBW (kg).

2 Log energy (kcal) = b0 + b1 Log EBW (kg).
3 P-value for intercept and slope on protein and energy.

References
ARC. Agricultural Research Council, 1980. The nutrient requirements of ruminant livestock. The Gresham Press,
London, England, 351 pp.
NRC. National Research Council. 2007. Nutrient requirements of small ruminants. Sheep, Goats, Cervids and New
World Camelids. National Academy Press, Washington, USA, 292 pp.

78 Energy and protein metabolism and nutrition in sustainable animal production

Low protein solid feed enhances nitrogen utilization by urea-N recycling
in veal calves
H. Berends1, J.J.G.C. van den Borne1, B.A. Røjen2, J. van Baal1 and W.J.J. Gerrits1
1 Animal Nutrition Group, Wageningen University, Wageningen, the Netherlands;
[email protected]
2Department of Animal Health and Bioscience, Aarhus University, Tjele, Denmark

Introduction
Veal calves fed merely milk replacer (MR) are typically known for their low nitrogen (N) utilization
for growth and high urea-N excretion (Gerrits et al 8UHD1UHF\FOLQJIURPEORRGWRWKH
gastrointestinal tract is expected to play a minor role in these preruminant calves, as shown by the
80% recovery of an intravenous pulse dose of [13C]urea in 48-h urine. We have recently shown that
SURYLVLRQRIORZSURWHLQVROLGIHHG 6) LQFUHDVHVWKHHI¿FLHQF\RI1XWLOL]DWLRQIRUSURWHLQJDLQLQ
veal calves, particularly towards the end of the fattening period (Berends et al., 2012). We expect
that in these calves, urea-N recycling could be stimulated by a low protein-to-energy ratio in rumen
contents, and by high blood urea-N concentrations (Reynolds and Kristensen, 2008). Urea-N entry
into the gastrointestinal tract may be mediated through urea-N transporters and aquaglyceroporins
DQGLVSRWHQWLDOO\LQÀXHQFHGE\GLHWDU\IDFWRUV 5¡MHQet al., 2011; Simmons et al., 2009). The
current study was designed to quantify the effect of low-protein SF intake, provided in addition to
MR, on urea-N recycling and expression of genes associated with urea transport in veal calves. We
hypothesized that low protein solid feed provision in addition to MR would contribute substantially
to urea-N recycling and expression of genes associated with urea transport.

Material and methods

Forty-eight Holstein Friesian male calves, 55±0.3 kg of bodyweight (BW), were fed 41 g of MR/
kg BW0.75/d, and assigned to 1 of 4 levels of SF intake: 0, 9, 18, or 27 g of DM of SF/kg BW0.75/d.
The SF mixture was 25% chopped straw, 25% chopped corn silage and 50% concentrate on a DM
EDVLV8UHD1UHF\FOLQJZDVTXDQWL¿HGDW“NJ%:E\LQWUDYHQRXVDGPLQLVWUDWLRQRI>15N15N]
urea for 24 h after a priming dose to enrich the body urea-N pool to approximately 0.15 atom%
H[FHVV&XPXODWLYHKXULQHZDVDQDO\VHGIRUXUHDLVRWRSRPHUVDQGIHFHVIRUWRWDO15N enrichment.
Complete N-balance was performed over a 4 d period. At slaughter, rumen tissue samples were
collected from the ventral caudal site to determine the absolute mRNA levels of urea transporter-B
(UT-B), and aquaporins 3 (AQP3) and 7 (AQP7) by real-time quantitative PCR. Each expression
level was normalized to that of importin 8 (IPO8). RT-qPCR data were analysed by ANOVA with
¿[HGHIIHFW6)LQWDNH8UHD1UHF\FOLQJDQG57T3&5GDWDZHUHDQDO\]HGE\UHJUHVVLRQZLWK6)
intake as regressor.

Results and discussion

Urea entry rate, assumed equal to total urea synthesis, averaged 28.7 g urea-N/d for all calves and
was not affected by SF intake (P>7DEOH 8UHDHQWU\WRWKHJXWZDVHVWLPDWHGJXUHD1G
for calves without SF, and increased with SF intake (0.28±0.09 g urea-N/g DM from SF/kg0.75/d;
P<0.01). Subsequently, the estimated return of urea-N to the ornithine cycle was 1.08 g urea-N/d
for calves without SF and increased with SF intake (0.10±0.02 g urea-N/g DM from SF/kg0.75/d;
P<0.001). Estimated fecal excretion was 0.15 g urea-N/d for calves without SF and increased with SF
intake (0.08±0.01 g urea-N/g DM from SF/kg0.75/d; P<0.001), which is likely undigested microbial
N. As a net result, urea-N used for anabolic purposes was estimated 5.57 g urea-N/d for calves
without SF and increased with SF intake (0.10±0.08 g urea-N/g DM from SF/kg0.75/d; P<0.01).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 79
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_13, © Wageningen Academic Publishers 2013
7DEOH(IIHFWVRILQFUHPHQWDO6)LQWDNH LQJ'0NJG IHGLQDGGLWLRQWRD05GLHWRQXUHD1
WUDQVIHUV LQJXUHD1G EDVHGRQ>N1@XUHDLQIXVLRQVLQYHDOFDOYHV NJ%: 6)LQWDNH
ZDVRUJ'0NJGDQG05LQWDNHZDVLGHQWLFDOIRUDOOFDOYHV J'0NJG 

Response parameter Covariable SF0 level Beta

Estimate SE P-value

Urea entry rate (UER) SF intake 30.41 -0.12 0.098 NS

Urea entry to the GIT (GER) SF intake  0.28 0.088 <0.01
Return to ornithine cycle (ROC) SF intake 1.08 0.10 0.021 <0.001
Loss to feces (UFE) SF intake 0.15 0.08 0.008 <0.001
Re-use for anabolism (UUA) SF intake 5.57 0.10 0.079 <0.01

The increase in urea-N used for anabolic purposes explained 19% of the increase observed in whole
body N retention, as reported by Berends et al. (2012).

7KHP51$H[SUHVVLRQRI87%ZDVLQFUHDVHGPRUHWKDQ¿YHIROGZLWK6) )LJXUH 7KHP51$

expression of AQP3 increased (P<0.05) with SF intake, whereas mRNA expression of AQP7 did
not. In conclusion, an increase in low-protein SF intake was accompanied by an increased gut entry
rate of urea-N, likely to be a substantial portion of the observed increase in N retention. Furthermore,
any SF stimulates expression of mRNA of UT-B in the rumen wall.

5,0
mRNA expression

4,0
SF0
3,0
2,0 SF9
1,0 SF18
0,0 SF27
bUTB bAQP3 bAQP7
)LJXUHP51$H[SUHVVLRQRIE87%E$43DQGE$43LQYHDOFDOYHV ZN 6)LQWDNHZDV
RUJ'0NJ%:GDQG05LQWDNHZDVLGHQWLFDOIRUDOOFDOYHV J'0NJ%:G 
Data are means ± SEM of 12 calves for each treatment.

References
Berends, H., J.J.G.C. van den Borne, S.J.J. Alferink, C.G. van Reenen, E.A.M. Bokkers and W.J.J. Gerrits. 2012.
Low-protein solid feed improves the utilization of milk replacer for protein gain in veal calves. J. Dairy Sci. 95,

*HUULWV:--*+7ROPDQ-:6FKUDPD67DPPLQJD0:%RVFKDQG0:$9HUVWHJHQ(IIHFWRISURWHLQ
and protein-free energy intake on protein and fat deposition rates in preruminant calves of 80 to 240 kg live weight.
J. Anim. Sci. 74, 2129-2139.
Reynolds, C.K. and N.B. Kristensen. 2008. Nitrogen recycling through the gut and the N economy of ruminants: An
DV\QFKURQRXVV\PELRVLV-$QLP6FL((
Røjen, B.A., S.B. Poulsen, P.K. Theil, R.A. Fenton and N.B. Kristensen. 2011. Effects of dietary nitrogen concentration
on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae
from lactating Holstein cows. J. Dairy Sci. 94, 2578-2591.
Simmons, N.L., A.S. Chaudhry, C. Graham, E.S. Scriven, A. Thistlethwaite, C.P. Smith and G.S. Stewart. 2009. Dietary
regulation of ruminal bovine UT-B urea transporter expression and localization. J. Anim. Sci. 87, 3288-3299.

80 Energy and protein metabolism and nutrition in sustainable animal production

Effect of replacing feed grains by food by-product on energy metabolism
of lactating cows
K. Higuchi1, F. Ohtani1, Y. Kobayashi1, I. Nonaka2, K. Yayou, M. Sutoh1 and O. Enishi1
11DWLRQDO,QVWLWXWHRI/LYHVWRFNDQG*UDVVODQG6FLHQFHV,NHQRGDL7VXNXED,EDUDNL
Japan; [email protected]
21DWLRQDO,QVWLWXWHRI.\XV\X2NLQDZD$JULFXOWXUDO5HVHDUFK&HQWHU6X\D.RVKL.XPDPRWR
-DSDQ
1DWLRQDO,QVWLWXWHRI$JURELRORJLFDO6FLHQFHV,NHQRGDL7VXNXED,EDUDNL-DSDQ

Introduction
The lactating cow requires a large amount of energy for milk production. Thus, it is important to
clarify and satisfy the cow’s energy requirement for milk production. Additionally, it is also critical
WRLPSURYHWKHHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQIRUPLONSURGXFWLRQWRUHGXFHERWKFRZ¶VPHWDEROLF
load and feed costs. We have begun work to estimate the impact of roughage to concentrate ratio in
the diet on energy metabolism of the lactating cow at the whole-body and mammary gland level.
,QWKLVH[SHULPHQWWKHHQHUJ\HI¿FLHQF\IRUPLONSURGXFWLRQIURPFRZVIHGGLHWVZKLFKFRQWDLQHG
45% roughage and either feed grains or a by-product were examined.

Material and methods

7KUHH+ROVWHLQODFWDWLQJFRZV¿WWHGZLWKDQXOWUDVRXQGÀRZSUREHDURXQGWKHOHIWH[WHUQDOSXGLF
artery were housed in a temperature and humidity controlled-room. Cows were fed 2 diets: a control
diet (Control, 40% Italian ryegrass silage, 5% alfalfa hay cube and 55% concentrate mix), or a by-
product diet (Bypro, replacing a part of soybean meal, corn and barley of the control diet by beet
pulp and soybean curd residue) for 21-days periods according to a cross over design. The crude
protein content of the Control and Bypro diets were 17.2 and 18.2%, respectively. The feeds were
fed to meet 100% of the total digestible nutrients requirement for a lactating Holstein cow producing
28 kg milk/d. The energy balance trials were conducted using open circuit respiratory chambers
with digestion trial apparatus (Higuchi et al.E 7KHHI¿FLHQF\RIPHWDEROL]DEOHHQHUJ\ 0( 
utilization for lactation (kl) was calculated assuming ME requirement for maintenance (MEm) as
N-NJ%:0.75 (NARO, 2007). The energy expenditure of the udder was calculated from their
R[\JHQFRQVXPSWLRQXVLQJDUWHULRYHQRXVGLIIHUHQFHWHFKQLTXHPHDVXULQJPDPPDU\EORRGÀRZDQG
blood oxygen concentration (Higuchi et al., 2010a). Blood metabolites were analyzed enzymatically
by commercial kits. The data were analyzed according to the following model using GLM procedure
of SAS 9.2: Yijk = μ + Ti + Fj + Ck + eijk; where μ is the overall mean; Ti, Fj, Ck are the effects of the
term, feed, and cow, respectively; and eijk is the residual error.

Results and discussion

7KHUHZHUHQRVLJQL¿FDQWGLIIHUHQFHVEHWZHHQWUHDWPHQWVLQERG\ZHLJKWGU\PDWWHULQWDNHDQG
PLON\LHOG 7DEOH 7KHPLONIDWFRQWHQWZDVVLJQL¿FDQWO\KLJKHULQFRZVJLYHQWKH%\SURGLHW
'U\PDWWHUDQG¿EHUGLJHVWLELOLW\ZHUHVDPHLQERWKGLHWVEXWVWDUFKGLJHVWLELOLW\ZDVKLJKHU
in Control (98.3<95.7%, P<0.05). Gross energy intake tended to be higher in Bypro; however,
energy partitioning for feces, urine, methane, heat production, milk and retention were not different
between treatments. The kl did not differ between treatments. The average of metabolizability was
FDOFXODWHGDVDQGWKLVYDOXHZDVORZHUWKDQWKDW a LQWKHSUHYLRXVVWXG\LQZKLFK
cows receiving 30% roughage diets (Higuchi et al., 2010a). In spite of that the klZRXOGEHa
ZKHQPHWDEROL]DELOLW\LV $5&1$52 WKHDYHUDJHRINlZDVFDOFXODWHGDV
in this study. Agnew and Yan (2000) suggested that increasing dietary proportion of forage increased
0(PEXWKDGQRVLJQL¿FDQWHIIHFWRQNl in lactating dairy cows. Therefore MEm was calculated as

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 81
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_14, © Wageningen Academic Publishers 2013
Table 1. Performance, whole body and mammary gland energy metabolism.

Control Bypro1 SEM

Body weight, kg  594 1

Dry matter intake, kg/day 17.5 17.5 0.0
Milk yield, kg/day 24.4 24.7 0.3
Milk fat, % 4.3 4.4b 0.0
Gross energy intake, kJ/kg BW0.75/day 2, 2,730a 5
Fecal energy, kJ/kg BW0.75/day  883 4
Urinary energy, kJ/kg BW0.75/day 70 77 1
Methane energy, kJ/kg BW0.75/day 182 17 4
Heat production, kJ/kg BW0.75/day 8 858 15
Milk energy, kJ/kg BW0.75/day 41  7
Retained energy, kJ/kg BW0.75/day  74 4
ME intake, kJ/kg BW0.75/day 1,5 1,594 12
kl  7 0.01
0DPPDU\EORRGÀRZOPLQ 4.3 3.7a 0.1
Mammary energy expenditure, kJ/kg BW0.75/day   
Mammary energy uptake, kJ/kg BW0.75/day 705 723a 2
0DPPDU\HQHUJ\HI¿FLHQF\ 0.91 0.92 0.01
Mammary glycerol uptake, mmol/h 10.9 17.1b 1.5

1 Superscript a=P<0.10; b=P<0.05.

¿[HGYDOXHWKXVWKHNl was seemed to show apparently higher value in this study. Mammary energy
H[SHQGLWXUHGLGQRWGLIIHUEHWZHHQWUHDWPHQWV'HVSLWHWKHGHFUHDVHLQPDPPDU\EORRGÀRZLQ
Bypro, mammary energy uptake tended to be higher in the Bypro supplemented cows. The amounts
of nutrient supplies toward the mammary gland for milk production were not different between
treatments; however, mammary glycerol uptake was higher in Bypro. The higher milk fat content
and higher energy uptake of mammary gland in cows given the Bypro diet were due to higher uptake
RIQXWULHQWVPRUHWKDQWKHGHFOLQHLQPDPPDU\EORRGÀRZIRUPLONSURGXFWLRQDWPDPPDU\JODQG

References
Agnew, R.E., and Yan, T. 2000. Impact of recent research on energy feeding systems for dairy cattle. Livest. Prod.
6FL
Agricultural Research Council, 1980. The Nutrient Requirements of Ruminant Livestock. CAB INTERNATIONAL, UK.
Higuchi, K., Y. Kobayashi, I. Nonaka, and O. Enishi, 2010b. A new data acquisition system for respiration trial system
on metabolism laboratory in National Institute of Livestock and Grassland Science. Bull. Natl. Inst. Livest. Grassl.
Sci. (Jpn.), 10, 15-27.
Higuchi, K., F. Ohtani, Y. Kobayashi, I. Nonaka, K. Yayou, M. Sutoh and O. Enishi, 2010a. Mammary energy
consumption and its relation to whole body energy metabolism in lactating cows fed high-concentrate diets. In:
Crovetto G.M.(eds), Energy and protein metabolism and nutrition. Wageningen Academic Publishers, Wageningen,
the Netherlands, EAAP publication No. 127: 231-232.
National Agriculture and Food Research Organization (NARO), 2007. Japanese Feeding Standard for Dairy Cattle,
Japan Livestock Industry Association, Japan.

82 Energy and protein metabolism and nutrition in sustainable animal production

Source of carbohydrate and protein in the diet of recently weaned dairy
calves
T.M. Hill, J.D. Quigley, H.G. Bateman, II, J.M. Aldrich and R.L. Schlotterbeck
1XUWXUH5HVHDUFK&HQWHU3URYLPL1RUWK$PHULFD%URRNYLOOH2+86$[email protected]

Introduction
7KH'DLU\15&  VXJJHVWVWKDWGLJHVWLEOH¿EHULVQHHGHGLQWKHGLHWRIFDOYHV+RZHYHULQRXU
recent research, live body weight average daily gain (ADG) decreased when starch from corn was
UHSODFHGZLWKVRXUFHVRIGLJHVWLEOH¿EHULHVR\EHDQKXOOVZKHDWPLGGOLQJVRUGLVWLOOHU¶VGULHGJUDLQV
with soluble (Hill et al., 2008; Suarez-Mena et al., 2012). Additionally, limited recent research has
not demonstrated consistent ADG responses to manipulating a calf’s diet for metabolizable protein
compared to control diets that were predominately corn and soybean meal (Hill et al., 2007). The
objective was to measure diet digestibility as a potential means to explain differences in performance
when different carbohydrate and protein sources were fed to calves.

Material and methods

:HFRQGXFWHGGWULDOVZLWKZHDQHG+ROVWHLQGDLU\FDOYHV LQLWLDOO\“NJ%:WRG
RIDJH IHGDFRQFHQWUDWHDQGFKRSSHGJUDVVKD\GLHWV(DFKWULDOXVHGFDOYHV FDOYHV
SHQ 'XULQJRIWKHODVWGRIWKH¿UVWWULDODQGRIGRIWKHVHFRQGDQGWKLUGZHHNRIWKH
second trial, fecal samples were taken from the ground of half of the pens. Care was taken to not
sample non-fecal material. Fecal samples were composited by pen. The diet was sampled on the
same days and composited by diet. Acid insoluble ash was used as an internal marker of digestion.
'LJHVWLELOLW\HVWLPDWHVDORQJZLWKGSHUIRUPDQFHZHUHDQDO\]HG

In Trial 1, a textured diet (19% CP) with high starch (52% starch, 13% NDF) based on whole corn
DQGRDWVRUDSHOOHWHGORZVWDUFK VWDUFK1') KLJKGLJHVWLEOH¿EHUGLHWZHUHXVHG SHQV
diet). Within starch level, diets were formulated from all supplemental soybean meal or soybean meal
with blood meal and Alimet® to provide 2 metabolizable protein levels (1 and 1.07% metabolizable
lysine plus methionine). The 4 treatments were analyzed as a completely randomized design with
a 2×2 factorial arrangement with pen as experimental unit. Differences were declared at P<0.05.
Factors were starch, protein, and their interaction.

In Trial 2, all ground, pelleted diets (19% CP) were fed. Diets were based on soybean hulls, wheat
middlings, or corn, which contained increasing concentrations of starch (13, 27, and 42% starch;
1') UHVSHFWLYHO\ SHQVGLHW &RQWUDVWVWDWHPHQWVZHUHFRQVWUXFWHGWRVHSDUDWH
differences in the means (soybean hulls plus wheat middlings vs. corn; soybean hulls vs. wheat
middlings). Differences were declared at P<0.05.

Results and discussion

In Trial 1, intake of OM did not differ (3.0±0.08 kg/d). Digestibility of OM was greater in calves
IHGKLJK  YVORZ  VWDUFKGLHWV 7DEOH 'LJHVWLELOLW\RI1') YV DQGVWDUFK
(0.95 vs. 0.99) were less in calves fed the high vs. low starch diets. Calf ADG and hip width change
ZHUHJUHDWHUIRUKLJKYVORZVWDUFKGLHWV6RXUFHRISURWHLQGLGQRWLQÀXHQFHGLJHVWLELOLW\RU$'*

In Trial 2, intake of OM was not different (2.3±0.10 kg/d). Digestibility of OM was greater in calves
fed corn (0.85) vs. other (0.78) diets (Table 2). Digestibility of NDF was greater for calves fed
soybean hulls vs. wheat middlings. Starch digestibility averaged 0.98 and was not different among
treatments. Calf ADG and hip width change were greater in calves fed corn vs. other diets.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 83
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_15, © Wageningen Academic Publishers 2013
7DEOH'LJHVWLELOLW\RIQXWULHQWVDYHUDJHGDLO\JDLQ $'* DQGKLSZLGWKFKDQJHLQ7ULDO

Starch (ST) High Low SEM P value

Metabolizable protein (MP) Low High Low High ST MP ST×MP

OM digestion, fraction 0.849 0.850 0.802 0.789  0.01 0.34 0.28
NDF digestion, fraction      0.01 0.54 0.12
Starch digestion, fraction 0.951 0.957 0.990 0.987 0.003 0.01  0.18
ADG, kg/d 1.07 1.09 1.00 1.00 0.032 0.02 0.79 0.72
Hip width change, cm  5.9 5.4 5.2 0.22 0.01  

Table 2. Digestibility of nutrients, average daily gain (ADG), and hip width change in Trial 2.

Nutrient Soyhulls (S) Middlings (M) Corn (C) SEM P-value

S+M vs. C S vs. M

OM digestion fraction 0.775  0.858  0.01 0.21

NDF digestion fraction 0.707   0.0313 0.34 0.01
Starch digestion, fraction  0.989 0.970 0.0057 0.13 0.15
ADG, kg/d 0.95 0.93 1.11 0.049 0.01 0.57
Hip width change, cm 5.1 5.0  0.23 0.01 0.44

High starch diets were more digestible and supported more growth in 2 to 4 month old dairy calves
WKDQUHSODFLQJVWDUFKZLWKGLJHVWLEOH¿EHU0DQLSXODWLQJPHWDEROL]DEOHSURWHLQFRPSDUHGWRDFRQWURO
diet that was predominately corn and soybean meal did not alter growth or digestibility.

References
Hill, T.M., J.M. Aldrich, R.L. Schlotterbeck and H.G. Bateman, II, 2007. Protein concentration for starters fed to
transported neonatal calves. Prof. Anim. Sci. 23, 123-134.
Hill, T.M., H. G. Bateman, J.M. Aldrich and R.L. Schlotterbeck, 2008. Effect of feeding different carbohydrate sources
and amounts to young calves. J. Dairy Sci. 91, 3128-3137.
National Research Council, 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC.
Suarez-Mena, F.X., T.M. Hill, A.J. Heinrichs, H.G. Bateman, II, J.M. Aldrich and R.L. Schlotterbeck, 2011. Effects of
including corn distillers dried grains with solubles in dairy calf feeds. J. Dairy Sci. 94, 3037-3044.

84 Energy and protein metabolism and nutrition in sustainable animal production

6XEVWLWXWLQJEDUOH\E\VRUJKXPHQKDQFHVHI¿FLHQF\RIVWDUFKDQG
protein utilization in lambs
M. Yahaghi1, J.B. Liang2, J. Balcells, R. Valizadeh and M.F. Jahromi2
1Northeast Animal Breeding Station, Mashhad, Iran
2,QVWLWXWH RI 7URSLFDO $JULFXOWXUH 8QLYHUVLWL 3XWUD 0DOD\VLD  830 0DOD\VLD
[email protected]
'HSDUWPHQWRI$QLPDO3URGXFWLRQ(76($/OHLGD8QLYHUVLW\/OHLGD6SDLQ
Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Introduction
Use of cereal grains with starch of different degradation rates allows for a more synchronized release
RIHQHUJ\DQG1WRSUHYHQWDUDSLGGURSLQUXPHQS+DQGLQFUHDVHRXWÀRZRIUXPLQDOVWDUFKIRU
PRUHHI¿FLHQWGLJHVWLRQLQWKHVPDOOLQWHVWLQH $EUDPVRQet al., 2005). Yahaghi et al. (2012) reported
WKDWSDUWLDOO\VXEVWLWXWLQJEDUOH\E\VRUJKXPVLJQL¿FDQWO\LQFUHDVHGJURZWKUDWHRIODPEVEHFDXVH
RILQFUHDVHGRXWÀRZRIVWDUFKWRWKHLQWHVWLQHDQGKLJKHUPLFURELDO1 01 \LHOG+RZHYHUZKHQ
barley was completely replaced by sorghum, high proportion of the intestinal starch was undigested
(unpublished data). This follow-up study examined whether extrusion can increase the digestibility
of sorghum starch in the intestine to allow for a higher substitution of barley by sorghum in lambs.
The optimal extrusion condition for sorghum grain (Bicolor L. Moench) was prior-determined as
150 °C/55 bars pressure.

Material and methods

Eighteen newly weaned male Iranian Baluchi lambs (approx. 32 kg) were randomly assigned to
three treatments in a complete randomized design (CRD). Three iso-caloric (10.25 MJ/kg DM) and
LVRQLWURJHQRXV &3JNJ GLHWVFRQVLVWHGRIURXJKDJH DOIDOID WRFRQFHQWUDWHZHUHXVHG
Barley (B) was the main energy grain for the control and was substituted partially (50%, BSE) or
fully (SE) with extruded sorghum. The study consisted of 10-wk feeding, 7-d digestibility and 2-d
JXWDEVRUSWLRQNLQHWLFVWULDOV<WWHUELXP <E DFHWDWHDVÀRZPDUNHUZDVRUDOO\DGPLQLVWHUHG
times a day in the digestibility trial. At end of trial, lambs were euthanized to determine starch and
N disappearances through the gastro-intestinal tract (GIT) as in Yahaghi et al. (unpublished data).
%ULHÀ\EHVLGHUHWLFXORUXPHQDQGDERPDVXPWKHVPDOOLQWHVWLQHZDVVHFWLRQHGLQWRPVHJPHQWV
Digesta samples from the different segments were taken. Yb in the digesta samples (Hart and Polan,
1984), rumen MN (Chen and Gomes, 1995) and starch (Horadagoda et al., 2008) were determined.
7KHSRVWUXPHQÀRZRIQXWULHQWV Nf) (starch and N) in different parts (i) of the GIT was calculated
using the nutrient and Yb concentrations in post rumen samples: Nfi = [N (i, g) × Yb (i, mg)/ Daily
Yb doses (mg/d)]. Data were analysed in CRD using GLM procedure (SAS, 2003) and differences
LQ/6'ZHUHGHFODUHGVLJQL¿FDQWDWP<0.05.

Results and discussion

2XUUHVXOWVVKRZVLJQL¿FDQWO\KLJKHURXWÀRZRIVWDUFKIURPUXPHQLQWKH6E lambs compared to the
other two treatments (Figure 1, left). And 84.9% of the intestinal starch in SE lambs was digested
while only 57.8% of the intestinal starch from untreated sorghum was digested (Figure 1, right),
suggesting higher digestibility of the extruded sorghum starch in the intestine.

Lambs in SEGLHWKDGKLJKHUUXPLQDOWRWDO101DQGGLHWDU\1 7DEOH RXWÀRZVXJJHVWLQJWKDW6E

GLHWLQFUHDVHG01V\QWKHVLVUHVXOWHGLQKLJKHUWRWDO1RXWÀRZ7KHKLJKHUVWDUFKDQG1ÀRZVDQG
their higher digestion rates in the SE lambs had resulted in an additional 80 g/d BW gain, equivalent
to 38% improvement, compared to lambs in the control barley diet.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 85
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_16, © Wageningen Academic Publishers 2013
)LJXUH OHIW 3RVWUXPLQDOVWDUFKÀRZLQODPEVIHGGLHWZLWKEDUOH\ % EDUOH\H[WUXGHGVRUJKXP
%6E DQGH[WUXGHGVRUJKXP 6E  ULJKW 3RVWUXPLQDOVWDUFKÀRZLQODPEVIHHGGLHWVZLWK
EDUOH\ % EDUOH\VRUJKXP %6 DQGVRUJKXP 6  XQSXEOLVKHGGDWD 

7DEOH5XPLQDO1ÀRZLQODPEVIHGEDUOH\ % EDUOH\H[WUXGHGVRUJKXP %6E DQGH[WUXGHG

VRUJKXP 6E GLHWV

Item (g/day) B BSE SE SEM* P<

N intake 45.0 44.0 44.0 0.81 0.38

'XRGHQDO1ÀRZ 45.5b b 53.1a 1.13 0.01

0LFURELDO1ÀRZ b 18.9b 24.1a 1.04 0.01
1RQPLFURELDO1ÀRZ 25.9b b 29.0a  0.02

a,b Means within row with different superscripts differ (P<0.05).

References
$EUDPVRQ6,%UXFNHQWDO//LSVKLW]80RDOHP6=DPZHODQG$$ULHOL6WDUFKGLJHVWLRQVLWHLQÀXHQFHRI
ruminal and abomasal starch infusion on starch digestion and utilization in dairy cows. J. Anim. Sci. 80, 201-207.
Chen, X.B. and M.J. Gomes, 1995. Estimation of Microbial Protein Supply to Sheep and Cattle Based on Urinary
Excretion of Purine Derivatives – An Overview of the Technical Details. International Feed Resources Unit. Rowett
Research Institute, Aberdeen, Scotland, UK, 21 P.
Hart, S. P. and C.E. Polan, 1984. Simultaneous Extraction and Determination of Ytterbium and Cobalt
(WK\OHQHGLDPLQHWHWUDDFHWDWH&RPSOH[LQ)HFHV-'DLU\6FL
Horadagoda, A., W. Fulkerson, I. Barchia, R. Dobos and K. Nandra, 2008. The effect of grain species, processing and time
RIIHHGLQJRQWKHHI¿FLHQF\RIIHHGXWLOL]DWLRQDQGPLFURELDOSURWHLQV\QWKHVLVLQVKHHS-/LYHVW6FL
SAS, 2003. SAS version 9.1: SAS institute Cary, NC.
Yahaghi, M., J.B. Liang, J. Balcells, R. Valizadeh, A.R., Alimon and Y.W. Ho, 2012. Effect of replacing barley with
corn or sorghum grain on rumen fermentation characteristics and performance of Iranian Baluchi lamb fed high
FRQFHQWUDWHUDWLRQV-$QLP3URG6FL

86 Energy and protein metabolism and nutrition in sustainable animal production

,QÀXHQFHRIGLIIHUHQWJUDVVODQGYHJHWDWLRQW\SHVRQUXPLQDOSURWR]RD
and ammonia in beef cattle
I.D.M. Gangnat1, J.O. Zeitz1, D. Warner1,2, M. Kreuzer1 and F. Leiber1
1ETH Zurich, Institute of Agricultural Sciences, Animal Nutrition Group, Zurich, Switzerland;
[email protected]
2Wageningen University, Animal Nutrition Group, Wageningen, the Netherlands

Introduction
Grassland vegetation types, which vary in concentration of nutrients and plant secondary compounds,
PD\LQÀXHQFHUXPLQDOPHWDEROLVPGLIIHUHQWO\5XPLQDOSURWR]RDSOD\DPDMRUUROHLQUXPLQDOSURWHLQ
GHJUDGDWLRQWRDPPRQLDDQGDUHLQYROYHGLQ¿EUHGLJHVWLRQ7KHLUUROHLQDQGUHVSRQVHWRFRQFHQWUDWH
based diets is well known. However, the effects of extensive grass-based diets on the composition
of the protozoa population in the rumen are poorly understood. The present experiment investigated
ZKHWKHUIHHGLQJJUDVVRUKD\KDUYHVWHGIURPGLIIHUHQWYHJHWDWLRQW\SHVLQÀXHQFHVUXPLQDOQLWURJHQ
metabolism and protozoa populations in beef heifers.

Material and methods

7KHH[SHULPHQWZDVFRQGXFWHGZLWK/LPRXVLQKHLIHUV “NJOLYHZHLJKWDWVODXJKWHU 
During the 2 months prior to slaughter, animals were fed ad libitum either fresh grass originating
from a lowland ley (Poa pratensis-Lolium perenne; CG), or fresh biodiverse alpine grass (Crepido
aureae-Festucetum rubrae, Deschampsio cespitosae-Poetum alpinae and Calthion meadows; AG),
or a mixture (1:1 in dry matter (DM); AH) of AG and hay from CG. The dorsal rumen contents were
sampled immediately after slaughter and kept on ice for about 2 h until analysis. Strained ruminal
ÀXLGZDVDQDO\VHGIRUS+DQGDPPRQLD 1+3) concentration using a potentiometer equipped with
the respective electrodes. Protozoa (Isotrichidae, Entodininae, Diplodininae, Ophryoscolecinae and
Blepharocorythidae ZHUHFRXQWHGPLFURVFRSLFDOO\LQWKHVWUDLQHGUXPHQÀXLG1LWURJHQ 1 DQGDVK
were determined in freeze-dried rumen content. Grass samples were taken every week per vegetation
type to be representative of the area to be harvested in the following 7 days. The oven-dried grass
DQGKD\VDPSOHVZHUHDQDO\VHGIRU1DQGDVK DFFRUGLQJWR$2$& QHXWUDOGHWHUJHQW¿EUH
1') DFLGGHWHUJHQW¿EUH $') DQGDFLGGHWHUJHQWOLJQLQ $'/  DFFRUGLQJWR9DQ6RHVWet al.,
1991). The values obtained were pooled to obtain the average diet composition over the last 4 weeks
before slaughter. Data were subjected to analysis of variance testing for the vegetation type effect.

Results and discussion

Over the last 4 weeks of the trial, the N content of the diet was higher in CG than in AG and AH
(29, 18 and 17 g/kg, respectively; P<0.01), whereas ash content was lower in AG and AH than in
&* DQGJNJUHVSHFWLYHO\P<0.01). The NDF content was similar among groups (523
g/kg DM), whereas ADF was higher in AG than CG (323 and 297 g/kg DM, respectively; P<0.05)
and tended to be higher in AG than AH (299 g/kg DM; 3 0.07). The ADL content was higher
in AG than AH (47 and 38 g/kg DM; P<0.05), with CG being intermediate (41 g/kg DM). The
addition of hay, which had a lower content of ADF and ADL than the fresh grass due to harvest at
an earlier vegetation stage, lowered the ADF and ADL contents of the AH diet. The most contrasting
difference between CG and AG, based on the present analysis, was their N content. Additionally,
the proportion of herbs (as opposed to legumes and grasses) was higher in AG (40% in the total
fresh weight; mainly 7DUD[DFXPRI¿FLQDOH, Alchemilla xanthochlora, Crepis aurea) than in CG
KHUEV 7KHUXPLQDOÀXLGS+ZDVVLPLODUDPRQJJURXSV “ &RQFHQWUDWLRQVRI1DQG
ash in ruminal content differed between groups in accordance with the N and ash concentrations
in the corresponding diets (R2=0.81 for both N and ash). Indeed, the N concentration in ruminal

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 87
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_17, © Wageningen Academic Publishers 2013
FRQWHQWZDVKLJKHUIRU&*WKDQ$+DQG$* DQGJNJUHVSHFWLYHO\P<0.001).
5XPLQDOÀXLG1+3FRQFHQWUDWLRQRI&*GLIIHUHGIURPWKDWRI$+DQG$* YVDQG
mmol/l, respectively; SEM=1.30; P<0.001), with the highest values found for CG consistent with
the highest feed N content. Total protozoal counts were higher in CG than AG (3.7×105 and 2.0×105/
ml, respectively; SEM=0.32×104; P<0.05), and was intermediate in the AH group (2.7×105/ml). A
treatment effect on the proportion of some protozoa (sub)families in the entire population was found
(Figure 1). The proportion of the Blepharocorythidae was higher in CG than AH (7.9% and 2.1% of
WRWDOUHVSHFWLYHO\6(0 P<0.05) and tended to be higher in CG than AG (2.9%; 3 0.07). The
proportion of Entodininae was lower in CG than AG (59.9% and 78.0%, respectively; SEM=4.80;
P<0.05), and was intermediate in the AH group (72.8%). The diet had no effect on the proportions
of Diplodininae, Ophryoscolecinae and Isotrichidae on total protozoa (P>0.1).

The present results suggest that an alpine-grass based diet (AG) low in N and containing a high
amount of herbs presumed to be rich in plant secondary compounds decreases both counts of total
rumen protozoa and NH3 concentration compared to a lowland-grass based diet (CG) high in N and
FRPSRVHGRIDIHZFXOWLYDWHGJUDVVVSHFLHV+RZHYHUWKLVGLGQRWLQÀXHQFHWKHJURZWKSHUIRUPDQFH
of the animals (data not shown) suggesting that N was excessively present in CG rather than it
ZDVGH¿FLHQWZLWK$*)XUWKHUPRUHWKHDOSLQHJUDVVLQÀXHQFHGWKHFRPSRVLWLRQRIWKHSURWR]RDO
population in a way that the main species Entodininae was represented in a higher proportion at the
cost of the less dominant Blepharocorythidae.

)LJXUH3URSRUWLRQRISURWR]RD VXE IDPLOLHVLQUXPHQÀXLGRIEHHIKHLIHUVIHGIUHVKORZODQG

FXOWLYDWHGJUDVV &* IUHVKKLJKODQGDOSLQHJUDVV $* RUDPL[WXUH LQ'0$+ RI$*DQG
hay from CG.

References
$VVRFLDWLRQRI2I¿FLDO$QDO\WLFDO&KHPLVWV $2$& 2I¿FLDOPHWKRGVRIDQDO\VLVth edition. Arlington, VA:
AOAC Inc.
9DQ6RHVW3--%5REHUWVRQDQG%$/HZLV0HWKRGVIRUGLHWDU\¿EHUQHXWUDOGHWHUJHQW¿EHUDQGQRQVWDUFK
polysaccharides in relation to animal nutrition. J Dairy Sci. 74, 3583-3597.

88 Energy and protein metabolism and nutrition in sustainable animal production

Nutritional evaluation of bulls receiving supplements with different
protein: carbohydrate ratios
E.E.L. Valente1, M.F. Paulino2, E. Detmann2, S.C. Valadares Filho2 and M.L. Chizzotti1
1Animal Science Departament, Universidade Federal de Lavras, Lavras, Brazil;
PDULRFKL]]RWWL#G]RXÀDEU
2Animal Science Departament, Universidade Federal de Viçosa, Viçosa, Brazil

Introduction
Modern systems of beef cattle production on tropical pasture aim for continuous growth and slaughter
of young animals. Thus strategic supplementation, where supplying limiting nutrients to increase
pasture use, is an important way to increase weight gain across the year (Tonello et al., 2011).
However, the relationship between protein:carbohydrate in a supplement determines the interactive
effects between intake and diet digestibility (Souza et al., 2010). Additionally, the intensity of these
effects is determined by composition of basal diet (pasture) that varies through the year. Thus, the
aim of present work was to evaluate the intake and digestibility of young bulls supplemented with
different protein:carbohydrates ratios grazing tropical pastures from 4 until 18 months of age.

Material and methods

)LIW\¿YHEHHIFDOYHVZLWK“NJRI%:DQGGD\VRIDJHJUD]LQJ6LJQDOJUDVV
(Brachiaria decumbens) pasture were used. The experimental period lasted 430 days encompassing
4 seasons (phases): phase 1 = suckling phase in rainy-dry transition season (112 days); phase 2 =
post-weaning in dry season (84 days); phase 3 = post-weaning in the dry-rainy transition season
(84 days); phase 4 = fattening phase in the rainy season (150 days). The treatments consisted of 5
nutritional plans: control = animals received mineral mixture only; HPHC = high protein and high
carbohydrate supplement; HPLC = high protein and low carbohydrate supplement; LPHC = low
protein and high carbohydrate supplement; LPLC = low protein and low carbohydrate supplement
(Table 1). Approximately 50 and 25% of protein requirement were supplied in high and low protein
supplement, respectively, and about 30 and 15% of total digestible nutrients (TDN) requirement was
supplied in high and low carbohydrate supplement, respectively. Half of the stipulated requirements
were supplied by supplement in the suckling phase due to the milk intake. Every 28 days, the
amount of supplement was adjusted by using the estimated protein and energy requirement by BR-
CORTE (Valadares et al., 2010). The forage intake and digestibility was measured on day 50 of
the experimental period and every 100 days using oxide chrome and indigestible neutral detergent
¿EHUDVPDUNHUV0LONLQWDNHZDVHVWLPDWHGRQGD\VDQGRIWKHH[SHULPHQWDOSHULRGE\
milking the cows after an injection of 2 ml of oxytocin (10 IU/ml; Ocitovet®, Brazil). This study was
carried on using a completely randomized design using a 2×2 + 1 factorial arrangement to evaluate
the nutritional plans (two protein levels, two carbohydrate levels and one control). The variables
were evaluated according to a complete random design in repeated measures over time design by
using mixed models method.

Results and discussion

Dry matter (DM) intake was higher (P<0.05) in dry to rain transitions and rain seasons for all
nutritional plans (Table 2). Non-supplemented animals had lower (P<0.05) intake of DM and TDN
than supplemented animals in all seasons. Although differences on DM intake was not observed
between supplemented animals, the supplements with high carbohydrate (HPHC and LPHC) had
lower (P<0.05) forage intake (P<0.05) in the suckling phase (rain to dry transition season) and in the
rain season. However, the HPHC plan had higher (P<0.05) intake and digestibility of neutral detergent
¿EHU,WFDQEHFRQFOXGHGWKDWVXSSOHPHQWVZLWKKLJKSURWHLQOHYHO VXSSO\LQJ&3UHTXLUHPHQW 

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 89
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_18, © Wageningen Academic Publishers 2013
increase intake and digestibility of diet, particularly when associated with high carbohydrate level
(supplying 30% TDN requirement).

Table 1. Chemical composition of supplement and pasture

Supplement Pasture1

Control HPHC HPLC LPHC Phase 1 Phase 2 Phase 3 Phase 4

Dry matter 87.1 89.5 85.8 87.0  42.5 28.0 21.3
Organic matter 89.3 87.4 88.4 85.8 91.4 92.4 92.4 91.5
Crude protein 29.2 55.3 15.4 29.5 8.8 5.5 12.1 10.7
apNDF2 8.7 10.2 7.4 9.2    
Ether Extract  1.5 3.0 2.4 1.2 1.2 1.5 1.2
CNF3  23.3 57.2   20.7 17.3 17.9

1 Obtained by handle plucked sampling.

21HXWUDOGHWHUJHQW¿EHUFRUUHFWHGIRUDVKDQGSURWHLQ
31RQ¿EURXVFDUERK\GUDWHV

7DEOH,QWDNHRIVXSSOHPHQWWRWDOGU\PDWWHU '0 DQGWRWDOGLJHVWLEOHQXWULHQWV 7'1

Nutritional plan Production phase SE

Phase 1 Phase 2 Phase 3 Phase 4

DM
Control %E 13.14Bb %D D 0.59
HPHC 17.58Ac 19.30Abc 23.10ABa 20.59ab 0.57
HPLC 18.28Ab $E 24.78Aa 21.91a 
LPHC $%E 17.50Ab $%D 19.52b 
LPLC $%F $EF 23.44ABa 18.87ab 0.58
TDN
Control 11.04Ba 7.44Cb 12.43Ba 11.41a 0.39
HPHC 13.91Aa 11.88Ab 14.99Aa 13.18a 0.37
HPLC 13.48ABab 9.85ABc 15.77Aa 13.49b 0.40
LPHC 12.11ABab 10.55ABb 14.19ABa 12.05ab 0.40
LPLC 11.87Bb %F $%D 12.52ab 0.38

References
Souza, M.A., E. Detmann, M.F. Paulino, C.B. Sampaio, I. Lazzarini and S.C. Valadares Filho, 2010. Intake, digestibility
DQGUXPHQG\QDPLFVRIQHXWUDOGHWHUJHQW¿EHULQFDWWOHIHGORZTXDOLW\WURSLFDOIRUDJHDQGVXSSOHPHQWHGZLWK
nitrogen and/or starch. Trop Anim Health Prod 42, 1299-1310.
Tonello, C.L., A.L. Branco, C.Y. Tsutsumi, C.Y. Tsutsumi, L.R. Bueno, R.C. Serrano and S.M. Coneglian, 2011.
Suplementação sobre o desempenho de bovinos de corte em pastagens: época do ano. Semin-Cienc agrar 32,
373-382.

90 Energy and protein metabolism and nutrition in sustainable animal production

Energy value of Tifton-85 (Cynodon spp.) for Gir and F1 Holstein × Gir
dairy heifers using the respirometric technique
A.L.C.C. Borges1, H.F. Lage1, R.R. Silva1, J.R.M. Ruas2, A.U. Carvalho1, E.O.S. Saliba1 and N.M.
Rodriguez1
1Departament of Animal Science of Veterinary School of Federal University of Minas Gerais, Belo
Horizonte, Brazil; [email protected]
2$JULFXOWXUDO5HVHDUFK&RUSRUDWLRQRI0LQDV*HUDLV (3$0,* %UD]LO

Introduction
The NRC (2001) proposes the use of net energy system to express the nutritional requirements of
the animal and to evaluate the energy value of foods. In tropical regions, however, information at
WKLVOHYHORIUH¿QHPHQWLVVWLOOVFDUFHIRUFLQJWKHQXWULWLRQLVWVLQWKRVHDUHDVWRXVHLQWHUQDWLRQDO
system’s data when formulating diets for dairy cattle.

The aim of this study was to compare the energy value of Tifton-85 (Cynodon spp.) hay consumed
by Gir and F1 Holstein × Gir dairy heifers using the calorimetric technique.

Material and methods

The study was conducted at the Veterinary School of the Federal University of Minas Gerais,
located in Belo Horizonte, Brazil. Six Gir and six crossbred F1 Holstein × Gir heifers with average
weight of 450 kg were used. Before the trial, a pre-trial period was conducted in order to adapt
the metabolism of the animals to the experimental diet (composed by Tifton-85 hay and mineral
QXFOHXV WKDWZDVRIIHUHGLQVXI¿FLHQWTXDQWLW\WRDOORZVOLJKWZHLJKWJDLQ JGD\ NHHSLQJWKH
heifers as close as possible to maintenance level. In this period daily food offered and refusals were
weighted on a daily basis and animals were weighted every 15 days. Diet digestibility determination
and calorimetric measurements began when stability in dry matter intake and minimum daily gain
were achieved. In order to express the energy content of hay, energy losses in feces, urine, methane
production and heat increment were discounted. The daily amount of feces produced was determined
E\WRWDOFROOHFWLRQGXULQJ¿YHGD\V7KHWRWDOGDLO\YROXPHRIXULQHZDVHVWLPDWHGDVGHVFULEHGE\
Valadares et al. (1999). Gross energy consumption, fecal and urinary energy losses were determined
by combustion of these samples in adiabatic bomb calorimeter. The daily methane production and
other gases (O2, CO2) needed to calculate the heat production of the animals according to Brouwer
 HTXDWLRQZHUHREWDLQHGE\KRXVLQJWKHDQLPDOIRUDSHULRGRIKLQVLGHDRSHQFLUFXLWV\VWHP
calorimetric chamber as described by Rodríguez et al. (2007). The heat increment was estimated
by difference between the heat production determined from animals fed the experimental diet and
after a 72 h fast. The experimental design was completely randomized. The means were subjected
to analysis using the statistical package SAEG (2007) and means were compared by Fisher’s F test
at 5% probability (P<0.05).

Results and discussion

1RVLJQL¿FDQWGLIIHUHQFHVZHUHIRXQGIRUHYDOXDWLQJWKHHQHUJ\GHQVLW\RIWKHGLHWH[SUHVVHGLQ
different ways between the two genetic groups (P>0.05) as shown in Table 1.

The average value found in the experimental diet sample combustion (gross energy, GE) was 4.43
Mcal/kg DM, which is very similar to the value suggested by AFRC (1993) of 4.4 to 4.5 Mcal/kg
DM to express the gross energy content of ruminant feed.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 91
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_19, © Wageningen Academic Publishers 2013
Table 1. Gross, digestible, metabolizable and net energy values expressed in megacalories per
NLORJUDPVRIGU\PDWWHU 0FDONJ'0 RIWKHH[SHULPHQWDOGLHWEDVHGRQ7LIWRQ Cynodon VSS 
determined in Gir and F1 Holstein × Gir heifers.

Item Genetic group1 Standard error

Gir F1 H×G

Gross energy (Mcal/kg DM) 4.44a 4.42a 0.007

Digestible energy (Mcal/kg DM) a 2.72a 0.052
Metabolizable energy (Mcal/kg DM) 2.18a 2.19a 
Net energy (Mcal/kg DM) 1.41a 1.38a 0.078

1 Values followed by different letters in the same row differ by Fisher’s test (P<0.05).

The digestible energy (DE) had a mean value of 2.7 Mcal/kg DM for both genetic groups, i.e.
approximately 39% of gross energy intake was lost in faeces. The value found has great similarity
with the DE content of Tifton-85 hay proposed by the NRC (2001) for animals fed at maintenance
level, which is 2.5 Mcal/kg DM.

7KHPHWDEROL]DEOHHQHUJ\RIWKHGLHWZDV0FDONJ'0LQGLFDWLQJORVVRIDQGRIWKH
GE consumed as urine and methane, respectively. The net energy (NE) had a mean value of 1.39
0FDONJ'0UHÀHFWLQJWKHORVVRI*(LQWDNHDVKHDWLQFUHPHQWDERXWRIWKH*(LQWDNH7KH
NRC (2001) suggests for animals with intake level three times the maintenance, a ME and NE value
RI7LIWRQKD\RI0FDONJ'0DQG0FDONJUHVSHFWLYHO\7KHSDVVDJHUDWHFDQH[SODLQ
the difference between the values found in this work and proposed by the NRC (2001) for dairy
cows since the increase in consumption is related to increased passage rates of the digesta, leading
to reduced dietary energy utilization by the animal.

Conclusion
The energy value of Tifton-85 hay (Cynodon spp.) was statistically similar for both Gir and crossbreed
F1 Holstein × Gir heifers fed at maintenance level.

Acknowledgements
We would like to thank CNPq, CNPq-INCT, FAPEMIG, CAPES and EPAMIG for their cooperation
in carrying out this work.

References
AFRC, 1993. Agricultural and food research council. Energy and requirements of ruminants. Wallingford, Commonwealth
Agricultural Bureaux International, 159p.
%URXZHU(5HSRUWRI6XE&RPPLWWHHRQ&RQVWDQWVDQG)DFWRUV3URFUG6\PS2Q(QHUJ\0HWDEROLVP
EAAP publication no. 11, p. 441-443.
NRC, 2001. National Research Council. Nutrient requirements of dairy cattle. 7 ed., Washington, D.C.: National
Academic of Sciences, 381p.
Rodríguez, N.M., W.E. Campos, M.L. Lachica, et al., 2007. A calorimetry system for metabolism trials. Arq. Bras.
Med. Vet. Zootec, 59, 495-500.
Valadares, R.F.D., G.A. Broderick, S.C. Valadares Filho, et al., 1999. Effect of replacing alfalfa with high moisture
FRUQRQUXPLQDOSURWHLQV\QWKHVLVHVWLPDWHGIURPH[FUHWLRQRIWRWDOSXULQHGHULYDWLYHV-'DLU\6FL

92 Energy and protein metabolism and nutrition in sustainable animal production

Substitution of corn by mesquite pod meal in pellet diets for lambs:
nitrogen compounds metabolism
M.L.A. Pereira, T.C.J. Pereira, H.G.O. Silva, J.F. Cruz, P.J.P. Almeida, A.B. Santos, E.J. Santos
and C.A.M. Peixoto
State University of Southwest Bahia, Itapetinga, BA, Brazil; [email protected]

Introduction
4XDQWL¿FDWLRQRIWRWDOH[FUHWLRQRIXULQHLVHVVHQWLDOWRGHVFULEHSURFHVVHVVXFKDVQLWURJHQEDODQFH
or even to estimate metabolizable energy and microbial protein synthesis by excretion of purine
derivatives (PD). Total collection of urine for 24 hours over nine days, is laborious, very uncomfortable
for the animals, and can interfere with other variables. For this reason, it is important to develop
methodologies that allow the shortest possible time of urine collection, or even to make the total
urine collection unnecessary. One opportunity is the use of spot urine collection with estimates based
on creatinine excretion. In this context, the present study was conducted to compare the excretion
of urinary metabolites using spot urine collection at intervals of 4 hours with those obtained from
total urine collection lasting 24 hours in lambs.

Material and methods

We used four lambs Santa Inês x Dorper, with body weight (BW) of 23.0±0.5 kg. The animals
were distributed in one 4×4Latin Square, during a four experimental periods of 12 days each. The
animals were fed pelleted diets with levels of substitution of milled corn grain by mesquite pod meal
030  DQG LQWKHQDWXUDOPDWWHU 10 RIWRWDOGLHWZKLFKZDVFRPSRVHG
of 30% alfalfa hay and 70% concentrate. The feed was provided twice daily, at 07:00 h and 15:00
h. On the 12th day of each period, urine samples were collected, every 4 hours, after the supply of
morning feed, over a 24 hour, period during the spontaneous voiding. The remainder was reserved
for obtaining total urine volume each day. At the end of 24 hours, the urine samples collected were
also considered for obtaining the total volume of urine.

The effects of treatment, collection time and treatment × time were performed by variance analysis
and the probabilities of the effects linear, quadratic and cubic of the contrasts were obtained.
Regression analysis was performed according to the level of MPM. All analyzes were performed
XVLQJWKHVWDWLVWLFDOSURJUDP 352&0,;(' RI6$6VWDWLVWLFDOSDFNDJH  

Results and discussion

Table 1 shows that there was no treatment effect on total PD excretion, urea and total nitrogen
obtained from the total collection of urine. These results differed when using the method of spot
urine collection. Based on this methodology, the analysis of variance detected effect of treatment for
urinary excretion of total PD, estimating maximum excretion of PD for the diet with corn replacement
LQ6LQFHWKHUHZDVYDULDWLRQLQJORPHUXODU¿OWUDWLRQUDWH *)5  7DEOH WKHFKDQJHLQWKH
excretion of PD estimated in spot urine samples, as a function of the diet, was not due to the increased
V\QWKHVLVRIERG\3',WLVZRUWKQRWLQJWKDWDVLJQL¿FDQWGLIIHUHQFHZDVGHWHFWHGEHWZHHQGLHWV
for the daily excretion of urea-N in urine when considering all six times of urine collection, which
was not observed with the total collection of urine. It is more accurate and precise to the detect
differences between the experimental diets when the number of urine samples. For urea-nitrogen it
was estimated that minimum excretion happened when the level of MPM was 52.5%, not following
the behavior of GFR. Thus, these results corroborate the fact that the increase in GFR may even
LQFUHDVHWKH¿OWHUHGORDGRIXUHDEXWWKHLUVXEVHTXHQWWXEXODUUHDEVRUSWLRQZDVKLJKHUDWWKLVOHYHO
of MPM. Based on these results, it is concluded that the GFR ranges, concentrations of creatinine

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 93
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_20, © Wageningen Academic Publishers 2013
7DEOH3UREDELOLW\YDOXHV PYDOXH IRUWKHHIIHFWVRIWUHDWPHQWWLPHDQGWUHDWPHQWîWLPH 7U[
7LPH LQWHUDFWLRQRQWKHXULQDU\H[FUHWLRQRIPHWDEROLWHVXVLQJWRWDOXULQHDQGVSRWXULQHFROOHFWLRQ
in lambs fed with replacement levels of milled corn grain by mesquite pod meal in pelleted diets.

Item Total urine (24 hours) Spot urine (4 in 4 hours)

Treatment Treatment Time Treatment × time

Total purine derivatives (g/day) >0.05 0.008  0.718

Total nitrogen compounds (g/day) >0.05   0.711
Urea nitrogen (g/day) >0.05 0.0002 0.437 0.855
PD: C na 0.304 0.554 0.445
TN: C na  0.487 0.800
UN:C na <0.0001 0.388 0.838

PD:C = purine derivatives:creatinine ratio; TN:C = total nitrogen:creatinine ratio; NU:C = urea nitrogen:creatinine ratio,
calculated from the concentration in mg/dl; na = not analyzed.

7DEOH0HDQVRIOHDVWVTXDUHVIRUWKHQLWURJHQPHWDEROLVPZDWHULQWDNHJORPHUXODU¿OWUDWLRQUDWH
DQGXULQHYROXPHXVLQJKRXUWRWDOXULQHFROOHFWLRQLQODPEVIHGUHSODFHPHQWOHYHOVRIPLOOHGFRUQ
grain by mesquite pod meal in pelleted diets

Item Replacement level (% NM) ASE

0 30  90

Urea excretion (g/day)1 19.42  17.08  

Urea nitrogen (g/day)1  7.07 7.97 8.43 1.43
Nitrogen concentration in plasma (ml/dl)1 20.59 20.81 20.83 21.09 1.40
Nitrogen balance (g/day)1 11.98 14.92   4.01
Nitrogen digested (g/day)1  29.80  28.49 4.08
Losses of nitrogen in urine (g/day)1  14.89   0.08
Total purine derivatives (mmol/day)1  79.17 87.37 82.37 
Absorbed purines (mmol/day)1  94.25 104.0  19.07
Microbial protein synthesis (g/day)1 425.3 428.2   
Water intake (g/kg BW)1 98.13 93.47  107.9 10.54
*ORPHUXODU¿OWUDWLRQUDWH POPLQ%: 2 2.07 1.80 2.02  0.23
Urine volume (g/kg BW)1 54.02 43.74 38.45  21.88

1Variance analysis: P>0.05 for linear, quadratic and cubic effects of the contrasts.
2*)5  “  “ 030 ± “ 0302  VLJQL¿FDQFH P<0.0001); ASE=average
standard error.

in the urine can vary over the day, so it is recommended that spot urine samples should be collected
at different sampling times to constitute representing a 24-hour cycle in experiments with lambs.

References
6$6,QVWLWXWH6WDWLVWLFDO$QDO\VLV6\VWHP8VHU¶VJXLGH&DU\6$6,QVWLWXWH

94 Energy and protein metabolism and nutrition in sustainable animal production

Excretion of purine derivatives and nitrogen compounds in lactating
goats fed other protein sources
M.L.A. Pereira, A.B. Santos, A.J. Del Rei, J.F. Cruz, P.J.P. Almeida, T.C.J. Pereira, E.J. Santos and
C.A.M. Peixoto
State University of Southwest Bahia, Itapetinga, BA, Brazil; [email protected]

Introduction
In the attempt to simplify the collection of urine samples are collected four hours after the feeding
in the morning and urinary creatinine has been used as an indicator of the daily urinary production
(Leal et al., 2007). However, there are doubts about possible variation in the excretion of purine
derivatives and nitrogenous wastes as a function of time, which would derail the obtainment of a
spot urine sampling at any hour of the day. The aim of the present experiment was to evaluate the
daily excretions of purine derivatives (PD), urea (U) and total nitrogen (TN) and the ratio of those
with creatinine (C) obtained using the spot urine collection, at intervals 2 h, in lactating goats.

Material and methods

We used four Alpine goats, 94.0±9.0 days in milk, producing 1.7±0.4 kg of milk and weighing (BW)
“NJDWWKHEHJLQQLQJRIWKHH[SHULPHQWGLVWULEXWHGLQDî/DWLQVTXDUHZLWKSHULRGVRI
15 days each. The animals were fed with diets containing the same proportions of mesquite pod meal
and milled corn grain, as energy sources, associated with different protein sources (soybean meal-
SM, cottonseed meal-CM, aerial part hay of cassava-APHC or leucaena hay-LH). Diets consisted of
7LIWRQJUDVVKD\  DQGFRQFHQWUDWH  SUHVHQWHGRI&3LQ'0'LHWVZHUHIHGad
libitumDWDQGZLWKZDWHUFRQVWDQWO\DYDLODEOH2QWKHWKGD\RIHDFKH[SHULPHQWDO
period, urine samples were collected every 2 hours after feeding in the 24 hour period during the
spontaneous voiding. The daily excretion of C can be used as indicator of urine volume in dairy
goats, whose value was 20.34 mg/kg BW/day. The effects of treatment, collection time and treatment
x time interaction were evaluated by variance analysis. Regression analysis was performed for each
variable in function of collection time for each treatment. All analyzes were performed using the
VWDWLVWLFDOSURJUDP 352&0,;(' RI6$6VWDWLVWLFDOSDFNDJH  

Results and discussion

In diets containing SM, the concentration of C was changed quadratically, reaching a minimum at
11.07 hours after morning feeding. The ratio of the concentration of PD and C ranged over 24 hours,
with the peak excretion of PD, which occurred 11.05 hours after morning feeding (Figure 1). However,
IRU&0GLHWDVWKH&FRQFHQWUDWLRQZDVPLQLPDOWRKRXUVDIWHUPRUQLQJIHHGLQJHVWLPDWLQJ
PD[LPXPYROXPHRIXULQHDWKRXUVDQGWKHUDWLREHWZHHQWKH3'DQG&LQKRXUV )LJXUH
1). The APHC diet did not alter any of these metabolic parameters. The LH diet provided variation
of U:C ratio to peak at 7.47 hours after the morning feeding, probably due to higher concentrations
of blood U and their urinary excretion until this time after morning feeding (Figure 1). Both diets
SM and CM affect circadian concentration of C in the urine, being SM diet altered the excretion of
PD due to its increased tissue synthesis and CM diet altered the excretion of PD due to the change in
the volume of urine produced. Moreover, both the metabolism and excretion of PD were not affected
by the use of APHC and LH diets, with the latter affected the liver and renal metabolism of U. Thus,
there is need for further studies to identify the time of spot urine sampling which is representative of
the total 24 h-urine collection in trials for evaluation of urinary excretion of PD, U and TN.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 95
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_21, © Wageningen Academic Publishers 2013
 15 2,5

Purine derivates: creatinine (mmol/L)

14
Derivates of purine (mmol/day)

13 2,0
12
11 1,5
10
9 1,0
8
7 0,5
6
0,0
5
0 2 4 6 8 10 12 14 16 18 20 22 24
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hours)
Time (hours)
Soy bran Ǔ= 7.49919 + 0.333501*T – 0.0150774*T2; R2 = 0.54 Soy bran Ǔ= 0.9848 + 0.04597*T – 0.00211*T2; R2=0.75
Cottonseed bran Ǔ= 9.48068 + 0.519584*T – 0.0237194*T2; R2=0.58 Cottonseed bran Ǔ= 1.25955 + 0.0696528*T – 0.00328036*T2; R2=0.96
Hay from the aerial part of cassava Ǔ= 11.0868 mmol/dia Hay from the aerial part of cassava Ǔ= 1.420386 mmol/L
Hay from leucaena Ǔ= 12.3973 mmol/dia Hay from leucaena Ǔ= 1.604026 mmol/L

15
16
14
13 14

Urea: creatinine (mg/dL)

12 12

11 10
Urea (g/day)

10 8
9 6
8 4
7 2
6 0
0 2 4 6 8 10 12 14 16 18 20 22 24
5
0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours)
Time (hours) Soy bran Ǔ= 9.485119 mg/dL
Soy bran Ǔ= 8.52105 g/day Cottonseed bran Ǔ= 13.25689 mg/dL
Cottonseed bran Ǔ= 11.3074 g/day Hay from the aerial part of the cassava Ǔ= 9.540596 mg/dL
Hay from the aerial part of cassava Ǔ= 8.8339 g/day Hay from leucaena Ǔ= 8.93942 + 0.279144*T – 0.0186825*T2; R2
Hay from Leucaena Ǔ= 7.6732 g/day

12 10
11 9
Total nitrogen: creatinine (mg/dL)

10
8
9
7
Total nitrogen (g/day)

8
7 6
6 5
5 4
4
3
3
2 2
1 1
0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hours) Time (hours)
Soy bran Ǔ= 4.7173 g/day Soy bran Ǔ= 6.022618 mg/dL
Cottonseed bran Ǔ= 8.9745 g/day Cottonseed bran Ǔ= 6.559653 mg/dL
Hay from the aerial part of cassava Ǔ= 3.6396 g/day Hay from the aerial part of cassava Ǔ= 5.404535 mg/dL
Hay from Leucaena Ǔ= 3.9140 g/day Hay from Leucaena Ǔ= 6.066728 mg/dL

)LJXUH([FUHWLRQRISXULQHGHULYDWLYHV 3' XUHD 8 WRWDOQLWURJHQ 71 DQGWKHUDWLRRIWKRVH

ZLWKFUHDWLQLQH & RIODFWDWLQJJRDWVLQIXQFWLRQRIWLPHIRUGLIIHUHQWGLHWV 3UREDELOLW\IRUHIIHFW
RIWUHDWPHQW 7U WLPH 7 DQGWLPH[WUHDWPHQWLQWHUDFWLRQ 7[7U IRUIROORZLQJYDULDEOHV3'
7U7DQG7î7U5DWLR3'&7U77î7U8
7U77î7U5DWLR8&7U77î7U717U
77Uî75DWLR71&7U77î7U

References
Leal, T.L., Valadares, R.F.D., Valadares Filho, S.C., Campos, J.M.S., Detmann, E.,Teixeira, R.M.A., Marcondes, M.I.
9DULDo}HVGLiULDVQDVH[FUHo}HVGHFUHDWLQLQDHGHULYDGRVGHSXULQDVHPQRYLOKDV5%UDV=RRWHF

96 Energy and protein metabolism and nutrition in sustainable animal production

Nutritional evaluation and performance of beef cattle fed with crude
glycerin diets
J.P.I.S. Monnerat1, I.M. Oliveira1, P.V.R. Paulino1, R. Mezzomo2, L.H.P. Silva1, P.P. Silva1 and J.P.
Ferreira1
1Department of Animal Science, Universidade Federal de Viçosa, Brazil; [email protected]
2Department of Animal Science, Universidade Federal Rural da Amazônia, Brazil

Introduction
*O\FHULQLVDE\SURGXFWIURPWKHELRGLHVHOLQGXVWU\WKDWUHVXOWVIURPWUDQVHVWHUL¿FDWLRQRIYHJHWDEOH
oils (Crandall, 2004) and presents high concentrations of glycerol (Dasari et al., 2005). Thus, glycerin
is a potential ingredient that would be included in ruminant diets as an energy source to replace
energetic feedstuffs. However glycerol as a macro ingredient had not have an established estimate
of energy values or these values are not available for typical feeding scenarios. Moreover there are
few studies that have explored some of the attributes and issues pertinent to glycerol as a feed for
beef cattle where the value of glycerol was examined as a replacement for corn grain. Therefore two
WULDOVZHUHFDUULHGRXWWRHYDOXDWHFUXGHJO\FHULQDVIHHGVWXIILQEHHIFDWWOHGLHWV7KH¿UVWH[SHULPHQW
was conducted to determine the energy value of crude glycerin as an ingredient in beef cattle diets.
The second study was developed with the objective of evaluating the effect of replacing corn with
crude glycerin on the intake and total apparent digestibility of diet components and performance of
beef cattle under feedlot conditions.

Material and methods

To determine the energy value of glycerin eight crossbred steers with an average initial weight of
334±121.13 kg were used. The animals were divided in four groups of 2 animals and each group was
fed diets containing two crude glycerin levels (5 and 15% of diet dry matter) during two experimental
periods. The experiment was evaluated according to a two-by-two Latin Square design, with 4
simultaneous squares. Within each square, each crude glycerin level was applied (5 and 15%). Diets
with different crude glycerin levels were independently applied to each latin square according to the
model: Yijkl ȝ6i + Tj + A(i)k + P(i)l + STijİijkl.

:KHUHȝ JHQHUDOPHDQ6i HIIHFWRIWKH/DWLQ6TXDUHL ¿[HG 7j = effect of the crude glycerin

OHYHOM ¿[HG $(i)k = effect of the animal k nested to the Latin Square I (random); P(i)l = effect of
the period l nested to the Latin Square i (random); STij = interaction effect of Latin Square i and
WUHDWPHQWM ¿[HG DQGİijkl = random error.

7KLUW\FURVVEUHHG5HG$QJXVî1HOORUHEXOOVZLWKDQDYHUDJHLQLWLDOZHLJKWRI“NJ
ZHUHXVHG7KHDQLPDOVZHUHDVVLJQHGWRDFRPSOHWHUDQGRPL]HGGHVLJQZLWK¿YHWUHDWPHQWVDQG
six replications per treatments (0, 5, 10, 15 or 20% glycerin inclusion on DM basis) during 84 days.
Comparisons between treatments means were performed in accordance with the following orthogonal
contrasts: linear and quadratic effects for the substitution level of corn ground with crude glycerin
DQGZDVLGHQWL¿HGDVWKHSUREDELOLW\OHYHO

Results and discussion

7KHHQHUJ\YDOXHVIRXQGIRUWKHJO\FHUROZHUHJNJ7'10FDONJ'(DQG0FDO
kg of metabolizable energy. These data suggests that crude glycerin could have higher energetic
potential than maize (858.3 g/kg TDN), sorghum (788.0 g/kg TDN) and wheat bran (715.4 g/kg
TDN) (Valadares Filho et al., 2010). The dry matter intake and nutrient digestibility were unaffected
(P>0.05) by treatments (Table 1). Digestibility is the result of the competition between digestion

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 97
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_22, © Wageningen Academic Publishers 2013
Table 1. Dry matter intake, total apparent digestibility, levels of total digestible nutrients and
performance of beef cattle fed with different levels of crude glycerin.

Item1 Crude glycerin, % of DM SEM P-value

0 5 10 15 20 Linear Quadratic

DMI, kg/d 9.59  9.01  9.39 0.9823 0.9385 0.9134
% of DM
DM       0.0711 0.4177
OM     71.04 4.2337  
CP  71.55     0.8947 
EE 78.58 77.01  81.19 83.03 4.9859 0.0520 0.8305
NDF   47.75 49.52 48.51 7.1839  0.9000
NFC  78.59  82.38 85.38 5.5301 0.0733 0.3230
TDN 71.57 70.82 73.37   4.4098 0.0981 
ADG, kg/d 1.90 2.12 1.85  2.07 0.2532 0.8095 

1 DMI = Dry matter intake; DM = digestibility of dry matter; OM = digestibility of organic matter; EE = digestibility of

HWKHUH[WUDFW&3 GLJHVWLELOLW\RIFUXGHSURWHLQ1')DS GLJHVWLELOLW\RIQHXWUDOGHWHUJHQW¿EHU1)&DS GLJHVWLELOLW\

RIQRQ¿EHUFDUERK\GUDWHV7'1 WRWDOGLJHVWLEOHQXWULHQWV$'* DYHUDJHGDLO\JDLQ

and passage rates. Passage rate is positively correlated with the DMI. Thus, the absence of a crude
glycerin effect on the intake of DM, OM, and all dietary constituents and similarity of the others
ingredients composition in the all diets can explain these results.

In agreement to absence of differences among treatments shown above, animal performance also was
not affected by crude glycerin inclusion (P>0.05, Table 1) although the diets had been formulated
to expected ADG lower than observed. These results are in agreement with results from previous
VWXGLHVHYDOXDWLQJWKHSHUIRUPDQFHRI¿QLVKLQJFDWWOHIHGFUXGHJO\FHULQ 0DFKet al., 2008; Terré
et al., 2011). It can be concluded crude glycerin contains as much energy as starch feedstuffs and
LWVLQFOXVLRQLQGLHWVXSWRRIGU\PDWWHUFDQEHXVHGLQGLHWVIRU¿QLVKLQJEHHIFDWWOHEHFDXVH
it does not lead to detrimental effects on intake and performance.

References
&UDQGDOO/*O\FHURODEXQGDQFHFDXVHIRUFRQFHUQ,QIRUP
Dasari, M.A., P.P. Kiatsimkul, W.R. Sutterlin and G.J. Suppes, 2005. Low-pressure hydrogenolysis of glycerol to
propylene glycol. Appl. Catalysis A 281, 225-231.
Mach, N., A. Bach and M. Devant, 2008. Effects of crude glycerin supplementation on performance and meat quality
RI+ROVWHLQEXOOVIHGKLJKFRQFHQWUDWHGLHWV-$QLP6FL
Terré, M., A. Nudda, P. Casado and A. Bach, 2011. The use of glycerine in rations for light lamb during the fattening
SHULRG$QLPDO)HHG6FLHQFHDQG7HFKQRORJ\
Valadares Filho, S.C., P.V.R. Paulino, M.I. Marcondes and M.L. Chizzotti, 2010. Exigências nutricionais de zebuínos
puros e cruzados. BR-CORTE. 2th ed. UFV: DZO. Viçosa, MG p. 185.

98 Energy and protein metabolism and nutrition in sustainable animal production

The effect of substituting urea for a commercial slow release urea as
supplement to sheep fed a poor quality Eragrostis curvula hay
A.M. Mentz, A. Hassen, W.A. Van Niekerk, H. Mynhardt and R. Coertze
'HSDUWPHQWRI$QLPDODQG:LOGOLIH6FLHQFHV8QLYHUVLW\RI3UHWRULD3UHWRULD6RXWK$IULFD
[email protected]

Introduction
7KH¿UVWOLPLWLQJQXWULHQWIRUVKHHSDWPDLQWHQDQFHUHFHLYLQJSRRUTXDOLW\KD\LVUXPHQGHJUDGDEOH
protein (RDP), and addition of urea can substitute part of the RDP (Köster et al., 1997). However,
urea is highly soluble and can cause rapid increases in rumen ammonia (NH3-N) concentrations.
The aim of this study was to determine whether a slow release nitrogen (N) source (Optigen II)
can be substituted in place of a rapid release N source (urea) without having any negative effects
on intake, digestibility, rumen fermentation and microbial protein synthesis when the sheep are fed
poor quality roughage.

Material and methods

)LYHUXPLQDOO\FDQQXODWHGZHWKHUVZHUHXVHGIRUWKHWULDOLQDîODWLQVTXDUHGHVLJQ7KH¿YH
different treatments were: 100% urea (T1); 75% urea:25% Optigen II (T2); 50% urea:50% Optigen
II (T3); 25% urea:75% Optigen II (T4) and 100% Optigen II (T5), with the same inclusion level of
starch and a mineral premix between treatments. Supplements were infused twice per day directly
into the rumen at 9:00 and 15:00. During the following 9-day experimental period the sheep were
transferred to individual metabolic cages. Feed, orts and water intake were measured, sampled and
pooled within treatment. Urine was collected, sampled and pooled to determine microbial protein
supply (Chen and Gomes, 1992; Chen et al., 'XULQJWKHODVWWKUHHGD\VUXPLQDOÀXLGZDV
collected at six hour intervals, pooled and analysed for VFA and rumen NH3-N concentrations, and
UXPLQDOÀXLGS+ZDVPHDVXUHG7KHGDWDZDVDQDO\VHGXVLQJ$129$ 6$6 IRU/DWLQ
square design. Treatment differences were detected using the F-test.

Results and discussion

A combination of urea and Optigen II at a proportion of 25:75 appeared to be a preferred level of
VXSSOHPHQWDWLRQGXHWRKLJKHURUJDQLFPDWWHU 20, QHXWUDOGHWHUJHQW¿EUH 1') DQGGLJHVWLEOH
organic matter intakes (DOMI) (Table 1). The intake values recorded for the 100% urea and 100%
Optigen II treatment did not differ (P>0.05). No differences (P>0.05) were recorded for organic
PDWWHUGLJHVWLELOLW\ 20' DQGQHXWUDOGHWHUJHQW¿EUH 1') GLJHVWLELOLW\EHWZHHQWKHWUHDWPHQWV
+RZHYHUWKH2SWLJHQ,,WUHDWPHQWKDGDVLJQL¿FDQWO\ P<0.05) lower apparent nitrogen
digestibility, which might have been the result of a slower rumen NH3-N release and higher nitrogen
excretion than the other treatments (data not reported). No differences were observed for pH and
VFA between different treatments (Table 1). The effective degradability of both DM and NDF did
not differ (P>0.05) between treatments (data not reported). Neither were there differences between
treatments for the volatile fatty acid (VFA) production (except butyrate) and total microbial crude
nitrogen (MCN) production.

%DVHGRQWKHUHVXOWVREWDLQHGLWVHHPVWKDWLWFRXOGEHEHQH¿FLDOWRVXEVWLWXWHSDUW XSWR RI

the urea in a balanced supplement with an slow release urea source (optigen II) without affecting
intake, digestibility, rumen fermentation and microbial protein supply to the ruminant receiving poor
quality Eragrostis curvula hay. However, from an economical point of view, urea might still be the
preferred NPN source, because the price of nitrogen from urea is cheaper than Optigen.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 99
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_23, © Wageningen Academic Publishers 2013
Table 1. Effect of slow and rapid release rumen nitrogen on intake, digestibility rumen fermentation
parameters and microbial synthesis.

Parameter Treatments SE

T1 T2 T3 T4 T5

Digestibility (%)
OM 49.18  48.47  48.42 
NDF 57.58 54.15 55.70   1.21
N 91.23a 89.29a 92.52a a 83.75b 1.11
Intake (g/day)
OM b 905.11ab b 973.15a 854.42b 23.45
NDF ab 781.44ab b a 747.21b 
DOMI 450.52ab 438.74b ab a 427.90b 15.71
Intake (g/kgBW0:75)
OM 41.08b 42.88ab 41.91ab 45.33a 40.34b 1.17
NDF ab 37.01ab ab a 35.27b 1.03
DOMI 20.95ab 20.75b 21.30ab 23.21a 20.15b 
Rumen parameter
pH      0.04
NH3-N (mg/ 100 ml) 5.84 4.93 5.00 5.40 4.55 0.55
Total VFA (mmol/l) 87.50 82.82 84.35 82.01 85.49 
Acetate (%)      
Propionate (%)  17.04    
Butyrate (%) 4.85b a 5.50a 5.81a a 0.20
Acetate:Propionate 4.44  4.59 4.89  0.22
Microbial protein synthesis
MCN (g/day) 39.94 41.70 33.18 33.88  3.94
gMCN/gDOMI 0.0441 0.0418 0.0349 0.0375 0.0444 0.003

a,b,cMeans within a row with different superscripts differ (P<0.05).

T1 = 100% Urea; T2 = 75% Urea : 25% Optigen II; T3 = 50% Urea : 50% Optigen II; T4 = 25% Urea : 75% Optigen
II; T5 = 100% Optigen II.

Acknowledgments
The authors would like to acknowledge the national research foundation (NRF) and the international
foundation for science (IFS) for providing research grant to cover the running cost of this study.

References
Chen, X. B. and M.J. Gomes, 1992. Estimation of microbial protein supply to sheep and cattle based on urinary excretion
of purine derivatives-An overview of the technical details, International Feed Resources Unit, Rowett Research
Institute, Bucksburn Aberdeen AB2 9SB, UK Occasional Publication.
Chen, X.B., A.T. Mejia, D.J. Kyle and E.R. Ørskov, 1995. Evaluation of the use of the purine derivative: creatinine
ratio in spot urine and plasma samples as an index of microbial protein supply in ruminants: studies in sheep.
Journal of Agricultural Science, Cambridge 125, 137-143
Köster, H.H., R.C. Cochran, E.C. Titgemeyer, E.S. Vanzant, T.G. Nagaraja, K.K. Kreikemeier and G. St. Jean, 1997.
Effect of increasing proportion of supplemental nitrogen from urea on intake and utilization of low-quality tallgrass-
prairie forage by beef steers. J Anim Sci 75, 1393-1399.

100 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWRIDSSOLFDWLRQRI¿EURO\WLFHQ]\PHSURGXFWVDWGLIIHUHQWOHYHOVRQ
in vitro ruminal fermentation of low quality feeds
B. Shenkute1, A. Hassen1 and N.E. Odongo2
1'HSDUWPHQWRI$QLPDODQG:LOGOLIH6FLHQFHV8QLYHUVLW\RI3UHWRULD3UHWRULD6RXWK$IULFD
[email protected]
2Animal Production and Health Section, International Atomic Energy Agency, Vienna, Austria

Introduction
Plant cell walls typically consist of about 35-50% cellulose, 20-35% hemicelluloses, and 10-25%
OLJQLQLQWKHGU\PDVV 6WLFNOHQ &RQVLGHULQJWKHVHDEXQGDQW¿EUHVRXUFHVVLJQL¿FDQW
improvement in the cell wall digestibility has been achieved over past decades through various
treatment options. Cellulase and xylanase are two major ruminant diet enzyme groups that are
able to respectively break down the cellulose and xylans found in the plant cell wall components
(Beauchemin et al. DQGEURXJKWVLJQL¿FDQWHIIHFWVLQLPSURYLQJGLJHVWLELOLW\+RZHYHU
their optimal level of inclusion is dependent on the diet under consideration, suggesting the need
to determine optimum rate of inclusion of a given enzyme preparation for the various feeds (Yang
et al. 7KLVVWXG\HYDOXDWHVWKHHIIHFWVRI¿EURO\WLFFHOOXODVHDQG[\ODQDVHHQ]\PHVRQLQ
vitro digestibility, gas production and volatile fatty acid production from Eragrostsis curvula and
maize stover.

Material and methods

Feed samples were collected, dried in forced oven, and ground to pass a 1 mm sieve in a Wiley
mill and stored for later analysis. Xylanase activity was assayed using 1% (w/v) birchwood xylan
as a substrate following the procedure described by Bailey et al. (1992) while Endo-glucanase and
Exo-glucanase enzyme activities were assayed following the method described by Wood and Bhat
 5XPLQDOÀXLGZDVFROOHFWHGDQGSUHSDUHGDQDHURELFDOO\XVLQJVWDQGDUGSURFHGXUHVZKLOH
the culture medium was prepared as described in Goering and Van Soest (1970). A semi-automated
system was used to measure gas production as described by Theodorou et al. (1994) for 72 h. Enzyme
VROXWLRQZDVSUHSDUHGEDVHGRQUHTXLUHGGRVHUDWH V IRUVSHFL¿FH[SHULPHQWDOWUHDWPHQWVLQRUGHUWR
deliver the desired amount of enzyme in a 1ml aliquot. About 500 mg of the respective feed sample
was weighed into 120 ml serum bottle, and 1ml of the appropriate enzyme treatment was directly
SLSHWWHGRQWRWKHVXEVWUDWHDQGLQFXEDWHGIRUK7KHQPORIUXPLQDOÀXLGPHGLXP  ZDV
added under a stream of CO2 to each of the serum bottles. Two replicates per run, and a total of four
runs were executed for every sample. Degradability of NDF was measured after terminating the
incubation at 48 h. The data were subjected to the general linear model (GLM) procedure of SAS
(2001). Analysis of variance was conducted, separately for the each feeds according to the following
PRGHO\LMN ȝ)L5MHLMN ZKHUH)LVWKHIHHGDQG5 the experimental run). All multiple
comparisons among means were carried out with Duncan’s multiple-range test

Result and Discussion

The cumulative gas production recorded at various time intervals, DM and NDF degradability were
VLJQL¿FDQWO\ P< LQÀXHQFHGE\DGGLWLRQRIERWKHQ]\PHV 7DEOHDQG *DVSURGXFWLRQ
DQG¿EHUGHJUDGDWLRQLQFUHDVHGZLWKLQFUHDVLQJOHYHOVRIHQ]\PHDSSOLFDWLRQIRUERWKWHVWIHHGV
except the lowest dose rates for the xylanase enzyme that did not differ from the control. There was
continuous degradation of the feeds over time, probably due to continuous effect of enzymes. This
might be partly due to the pre-incubation effect that resulted in stable enzyme-feed complex, which
further increased their resistance for proteolysis and aids during latter fermentation periods (Yang
et al., 1999). Among the enzyme treated feeds, the production of acetate and total VFA increased

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 101
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_24, © Wageningen Academic Publishers 2013
7DEOH7KHHIIHFWVRI&HOOXODVHHQ]\PHVRQJDVSURGXFWLRQ POJ'0 1')GHJUDGDWLRQ  
and production of VFA.

Cellulase dose Poor quality roughage1

rate (mg/g DM)
Eragrostis curvula hay Maize stover

GP48 NDF VFA Acetate propionate GP48 NDF VFA Acetate propionate
deg deg

0 21.9g 33.7f 34.7 23.0 7.29 24.7f 39.4f 57.7  13.9
0.5 22.8f 35.2f 34.9 23.1 7.33 25.3f 40.0f   14.0
1 27.3e 39.7e 49.7 33.4 10.1 e 40.9e  47.3 18.7
2 31.9d d  34.1 10.3 33.2d d 78.2 52.8 15.1
3 32.7c 47.2c 55.3 37.2 10.4 38.2c 49.0c 79.1  14.2
4 b 48.9b 57.2 38.5  40.8b 53.8b 80.0 50.0 19.4
5 41.1a 51.0a 34.7 23.0 7.29 42.2a a 57.7  13.9

10HDQVZLWKGLIIHUHQWOHWWHUV VXSHUVFULSWV ZLWKLQDFROXPQGLIIHUVLJQL¿FDQWO\DWLQGLFDWHGP-value, P<0.05 for each

enzyme.

Table 2. The effects of Xylanase enzymes on gas production (ml/0.5 g DM), NDF degradation (%)
and production of VFA.

Xylanase dose Poor quality roughage1

rate (mg/ g DM)
Eragrostise curvula hay Maize stover

GP48 NDF VFA Acetate Propionate GP48 NDF VFA Acetate Propionate
deg deg

0 21.9f 33.7d 34.7 23.0 7.29 24.7f 39.4d 57.7  13.9
0.25 22.0f 33.9e 44.2 29.4  23.9f 40.4d  50.3 12.0
0.5 22.9e 34.8e 52.1 34.9  f 41.4d 71.1 43.1 17.9
1 d 37.5d 52.9 35.5 9.99 27.1e 45.7c 71.4 44.1 17.5
2 29.8c 41.2c 57.3 37.7 11.2 30.9d 49.1b 71.9 51.5 11.8
3 b 45.7b 58.5 39.2 10.8 b 53.9a 73.0 52.8 
4 a 48.9a 34.7 23.0 7.29 a 55.9a 57.7  13.9

10HDQVZLWKGLIIHUHQWOHWWHUV VXSHUVFULSWV ZLWKLQDFROXPQGLIIHUVLJQL¿FDQWO\DWLQGLFDWHGP-value, P<0.05 for each

enzyme.

with increasing application level of enzymes. In agreement with our result, many authors (Eun and
%HDXFKHPLQ KDYHQRWLFHGLQFUHDVLQJ¿EUHGHJUDGDELOLW\RIGLHWVRUIHHGVWXIIVZLWKHQ]\PH
supplementation. This observed increases in the production of total VFA, acetate and propionate as
ZHOODV¿EUHGLVDSSHDUDQFHFRXOGLQFUHDVHWKHÀRZRIPLFURELDO1DQGPLFURELDOFRORQL]DWLRQRI
WKHVXEVWUDWHUHVXOWLQJLQHQKDQFHG¿EUHGHJUDGDWLRQ

Conclusion
The pre-treatment of these low quality forages with cellulase at 5 mg/g DM and xylanase at 4 mg/g
DM improved in vitro ruminal fermentation and degradability of NDF.

102 Energy and protein metabolism and nutrition in sustainable animal production
Reference
%HDXFKHPLQ.$'&RORPEDWWR'30RUJDYLDQG:=<DQJ8VHRIH[RJHQRXV¿EURO\WLFHQ]\PHVWRLPSURYH
feed utilization by ruminants. J. of Anim. Sci. 81, E37-E47.
(XQ-6DQG.$%HDXFKHPLQ$VVHVVPHQWRIWKHHI¿FDF\RIYDU\LQJH[SHULPHQWDOH[RJHQRXV¿EURO\WLFHQ]\PHV
using in vitro fermentation characteristics. Anim. Feed Sci. Technol., 132, 298-315.
SAS Institute Inc., 2001. SAS/STAT Software, Version 8.2. SAS Institute Inc., Cary, NC, USA.
Sticklen, M.B., 2008. Plant genetic engineering for biofuel production:towards affordable cellulosic ethanol. Nature
reviews Genetics. 9, 433-443.
Theodorou, M.K., B.A. Williams, M.S. Dhanoa, A.B. McAllen and J. France, 1994. A simple gas production method
using pressure transducers to determine the fermentation kinetics of ruminant feed. Anim. Feed Sci. Technol.,
48, 185-197.
Van Soest, P.J., 1982. Nutritional ecology of ruminants. O and B books. Inc. Oregon,USA.
Yang, W.Z., K.A. Beauchemin and L.M. Rode, 1999. Effects of an enzyme feed additive on extent of digestion and
milk production of lactating dairy cows. J. Dairy Sci. 82,391-403.

Energy and protein metabolism and nutrition in sustainable animal production 103
Effect of metabolizable energy intake on energy partitioning into muscle
and fat in Pelibuey ewes
A.J. Chay-Canul1, J.C. Ku-Vera2, A.J. Ayala-Burgos2, M.L. Chizzotti, J.G. Magaña-Monforte2 and
L.O. Tedeschi
1División Académica de Ciencias Agropecuarias, Universidad Juárez Autónoma de Tabasco,
Villahermosa, Tabasco, México; DMFKF#\DKRRFRPP[
2)DFXOWDGGH0HGLFLQD9HWHULQDULD\=RRWHFQLD8QLYHUVLGDG$XWyQRPDGH<XFDWiQ&3
Mérida, Yucatán, México
'HSDUWDPHQWRGH=RRWHFQLD8QLYHUVLGDGH)HGHUDOGH/DYUDV/DYUDV0*%UD]LO
'HSDUWPHQWRI$QLPDO6FLHQFH7H[DV$ 08QLYHUVLW\&ROOHJH6WDWLRQ7;86$

Introduction
,Q UXPLQDQWV VHDVRQDOLW\ DQG SK\VLRORJLFDO FRQGLWLRQV UHVXOW LQ ÀXFWXDWLRQV LQ IHHG LQWDNH
inducing periods of underfeeding and refeeding throughout the year. These seasonal variations
LQGXFHÀXFWXDWLRQVLQOLYHZHLJKWLQJUD]LQJDQLPDOV $WWLet al., 2000; Mahouachi and Atti, 2005;
Kamalzadeh et al., 2009), and consequently in the energy content of the carcass and the whole body.
Nonetheless, in hair sheep breeds there is scarce information relative to carcass composition and
energy changes in the carcass during periods of weight loss and gain. Moreover, it has been reported
that knowledge of body composition of productive animals is of relevance in order to better assess
nutrient requirements. The aim of the present work was to evaluate the effect of metabolizable
energy intake (MEI) on energy partitioning of muscular and adipose tissues in adult Pelibuey ewes.

Material and methods

Twenty four adult non-pregnant, non-lactating Pelibuey ewes with BW of 37.2±4.0 kg and BCS of
2.5±0.12 in a scale 1 to 5, where BCS 1 represents a thin animal and 5 an obese animal as described
by Russell et al.  ZHUHUDQGRPO\DVVLJQHGWRIRXUJURXSVRIVL[DQLPDOVHDFK2QHJURXS
was slaughtered at the beginning of the experiment for baseline measurements of carcass tissues
and energy content. The remaining ewes were randomly allotted to three groups of six animals and
were individually housed in metabolic crates, and fed three levels of MEI: low (L), medium (M)
DQGKLJK + GXULQJGD\VWRDFKLHYHGHVLUDEOHFKDQJHVLQ%:DQG%&60(,ZDV
and 0.532 MJ/kgBW0.75/day for L, M and H levels respectively. At the end of the experiment all
ewes were slaughtered. Carcass was split at the dorsal midline in two equal halves, weighed, and
FKLOOHGDWƒ&IRUK$IWHUUHIULJHUDWLRQFDUFDVVZDVZHLJKHGDQGWKHOHIWKDOIRIWKHFDUFDVVZDV
completely dissected into carcass fat (subcutaneous and intermuscular), muscle and bone, and each
component weighed separately. Dissected tissues of the left carcass were adjusted to the whole carcass.
A proportional sample (about 1 kg) was taken of the muscle and adipose tissue of the carcass from
each animal, ground and stored at -20 °C in plastic containers. Tissue samples were freeze-dried to
determine water content. Dry samples were ground in a hammer mill and analyzed for gross energy.
At the end of the experiment, one ewe from treatment L and another from treatment H were removed
from the experiment due to illness and their data were not included in the analysis. Data of energy
content in tissues of the carcass were analyzed as a completely randomized design using analysis of
YDULDQFHDQGWKH7XNH\WHVWZDVXVHGZKHQVLJQL¿FDQWGLIIHUHQFHVDPRQJWUHDWPHQWVZHUHGHWHFWHG
Statistical tests were carried out with PROC GLM of SAS (SAS, 2002).

Energy contained in muscle and adipose tissues in the carcass showed differences (P<0.05) between
feeding levels (Figure 1). The total energy content of the carcass in the initial group was 129.05
0-7KHSURSRUWLRQVRIHQHUJ\FRQWDLQHGDVPXVFOHDQGIDWZHUHDQGDQGDQG
DQGIRU/0DQG+OHYHOVUHVSHFWLYHO\(QHUJ\EDODQFHVLQWKHFDUFDVV HVWLPDWHGDVWKH
HQHUJ\FRQWHQWLQWKHFDUFDVVRILQLWLDOJURXSPLQXVWKH¿QDOHQHUJ\FRQWHQWLQWKHFDUFDVVRIRWKHU

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 105
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_25, © Wageningen Academic Publishers 2013
 160

140 Muscle
Fat
120

Total energy (MJ)

100

80

60

40

20

0
Initial Low Medium High
Treatments
Figure 1. Energy content in the carcass of adult Pelibuey ewes fed three levels of MEI.

groups and divided between the days in experimentation), were -1.099, -0.515 and 0.285 MJ for L,
0DQG+OHYHOVUHVSHFWLYHO\5HVXOWVLQGLFDWHWKDWHZHVDWWKH/DQG0OHYHOVORVWDQGRI
their initial energy content in the adipose and muscle tissues of the carcass respectively, while the
H level increased its energy content by 14%. Most of the energy stored in the carcass of Pelibuey
ewe is contained in muscle tissues.

References
Atti, N., P. Nozière, M. Doreau, C. Kayouli and F. Bocquier. 2000. Effects of underfeeding and refeeding on offals
weight in the Barbary ewes. Small Rumin. Res. 38: 37-43.
Kamalzadeh, A., W.J. Koops and A. Kiasat. 2009. Effect of qualitative feed restriction on energy metabolism and
nitrogen retention in sheep. South African J. Anim. Sci. 39: 30-39.
Mahouachi, M., Atti, N., 2005. Effects of restricted feeding and re-feeding of Barbarine lams: intake, growth and non-
carcass components. Anim. Sci. 81:305-312.
5XVVHOO$-)'RQH\-0*XQQ5*6XEMHFWLYHDVVHVVPHQWRIERG\IDWLQOLYHVKHHS-$JULF6FL
(Camb.). 72: 451-454.
SAS. 2002. Institute Inc., SAS/STAT. Software, Ver. 9.00, Cary, NC27512-8000. USA.

106 Energy and protein metabolism and nutrition in sustainable animal production
Mammary gland development in heifers under different metabolizable
protein and metabolizable energy ratios
R.L. Albino1, M.I. Marcondes1, B.C. Gomes2, L.G.R. Pereira2, T.E. da Silva1 and A.S. Trece1
1 Departamento de Zootecnia, Universidade Federal de Viçosa, Viçosa-MG, Brazil;
[email protected]
2Empresa Brasileira de Pesquisa Agropecuária, Embrapa Gado de Leite, Juiz de Fora MG, Brazil

Introduction
Mammary development is affected by nutrient intake, which affects the intensity of weight gain.
There are several ways to evaluate mammary development, but almost all involve the slaughter of the
animals. On other hand, other studies have been conducted with ultrasound to evaluate fat deposition
on lean tissue (Wertz et al., 2002). This experiment was designed to evaluate dry matter intake,
performance, and mammary gland development in Holstein heifers under different metabolizable
protein and metabolizable energy ratios.

Material and methods

The experiment was conducted at the Universidade Federal de Viçosa, Brazil, from December2011
WR$SULO7ZHQW\¿YHKHLIHUVZHUHGLYLGHGLQWRWUHDWPHQWV7KHWUHDWPHQWVZHUHFRPSRVHGRI
metabolizable protein and metabolizable energy ratios (MPMER), with 33, 38, 43, 48, 53 gram of
metabolizable protein per Mcal of metabolizable energy (ME). The diet with the 43 ratio was based
on recommendations of the (NRC, 2001), and two levels above and below this recommendation
were also established. All diets were isoenergetic in composition (2.3 Mcal de ME/kg of dry matter),
being used corn silage, ground corn, soybean meal and wheat bran to formulate the diet. The dry
PDWWHULQWDNH '0, ZDVFRQWUROOHGGDLO\DQGDOOKHLIHUVZHUHZHLJKHGLQWKHEHJLQQLQJDQG¿QDORI
the trial to calculated daily weight gain (DWG). Ultrasounds were performed in all quarters of each
heifer, at the end of each month, to evaluate mammary gland development. The software ImageJ®
1,+86$ ZDVXVHGWRDQDO\]HSL[HOVIURPSRLQWVZLWKLQHDFKLPDJH )LJXUH 7KHQWKH
average pixels were used to analyze density on mammary gland image. The pixels are represented
E\DVFDOHRIJUD\VFDOH  EODFN ZKLWH 7KH'0,':*DQGIHHGHI¿FLHQF\ )( ZHUH
analyzed in randomized block design. The mammary gland data were analyzed in a split plot design
with repeated measures. Statistical analysis was done using SAS (SAS Institute, Cary, NC, USA).

Results and discussion

The MPMER did not affect DMI (3  RU':* 3 0.090. However, there was a linear
decreasing effect of the MPMER in FE (3 0.034), where animals with greater protein level were
PRUHHI¿FLHQWIRUJDLQLQJZHLJKW 7DEOH ,WLVOLNHO\WKDWGLHWVZLWKJUHDWHU030(5SHUPLWD
greater deposit of protein, while diets with lower MPMER had the lipid deposition increased. It is
ZHOONQRZWKDWSURWHLQGHSRVLWLVOLQNHGWRDKLJKHUZDWHUGHSRVLWZKLFKUHÀHFWVLQDJUHDWHUGHSRVLW
of lean tissue per kilo of gain.,QUHODWLRQWRWKHPDPPDU\JODQGGDWDWKHUHZHUHVLJQL¿FDQWHIIHFWV
for treatment (3 0.0001), period (3  DQGSHULRGWUHDWPHQWLQWHUDFWLRQ 3 0.01). Throughout
the experiment, the heifers fed diets with high protein (MPMER 48 and 53) showed a reduction in
pixel value. However the heifers fed with low protein (MPMER 33 and 38) showed an increase of
pixel value (Figure 2). It is inferred that images generated with high pixel value are associated with
DJUHDWHUIDWDFFXPXODWLRQLQWKHPDPPDU\JODQGGXHWRJUHDWHUUHÀHFWLRQRIIDWRYHUWKHXOWUDVRXQG
waves (Nishimura et al., 2011). Thus, ultrasound images of heifers submitted to high protein
treatments might be related with a smaller proportion of fat accumulation in the mammary gland,
GXHWKHJUHDWHUUHÀHFWLRQRIVRXQGZDYHV2QWKHRWKHUKDQGKHLIHUVWKDWUHFHLYHGDORZSURWHLQGLHW
might have had a greater proportion of fat in the mammary gland.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 107
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_26, © Wageningen Academic Publishers 2013
Figure 1. Image representing the method used to analyze mammary gland pictures.

7DEOH'U\PDWWHULQWDNHGDLO\ZHLJKWJDLQDQGIHHGHI¿FLHQF\

Itens MP:ME ratio P-value CV

33 38 43 48 53 LINEAR QD

DMI (kg/day) 7.09      0.327 9.57
DWG (kg/day) 1.01 0.94 0.97 1 1.2 0.091  
FE  7.17    0.035 0.128 -

'0, GU\PDWWHULQWDNH':* GDLO\ZHLJKWJDLQ)( IHHGHI¿FLHQF\4& TXDGUDWLF&9 FRHI¿FLHQWRIYDULDWLRQ

125,000
120,000
115,000
110,000
Pixels

105,000
100,000
95,000
90,000
1 2 3 4
Period of evaluation
Figure 2. Pixel value variation according to period-treatment interaction. The symbols are representing
   Ŷ  Ÿ  î DQGJRIPHWDEROL]DEOHSURWHLQSHU0FDORIPHWDEROL]DEOHHQHUJ\ ¼ 

References
National Research Council (NRC). 2001. Nutrient Requirements of Dairy Cattle. 7th ed. National Academy Press,
Washington, DC.
Nishimura, M.;Yoshida, T; EL-khodery, S.; Miyoshi, M.; Furuoka H.; Yasuda, J. and Miyahara, K., 2011. Ultrasound
Imaging of Mammary Glands in Dairy Heifers at Different Stages of Growth. J. Vet. Med. Sci. 73(1): 19-24.
Wertz, A. E., Berger, L. L., Walker, P. M., Faulkner, D. B., McKeith, F. K. and Rodriguez-Zas, S. L, 2002. Early-
weaning and post weaning nutritional management affect feedlot performance, carcass merit, and the relationship
RIWKULEIDWPDUEOLQJVFRUHDQGIHHGHI¿FLHQF\DPRQJ$QJXVDQG:DJ\XKHLIHUV-$1,06&,

108 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWRIVWDUFKVRXUFHDQG¿EHUOHYHOLQPL[HGGLHWVRQODFWDWLQJ
Murciano-Granadina goat: Substrate oxidation and milk performance
C. Fernández1, M.C. López1 and M. Lachica2
1,QVWLWXWHIRU$QLPDO6FLHQFHDQG7HFKQRORJ\8QLYHUVLGDG3ROLWpFQLFDGH9DOHQFLD9DOHQFLD
Spain; [email protected]
2'HSDUWPHQWRI$QLPDO1XWULWLRQ(VWDFLyQ([SHULPHQWDOGHO=DLGtQ&6,&$UPLOOD*UDQDGD
Spain

Introduction
To achieve the maximum milk production potential, dairy ruminants need to ensure high intake of
energy. This might be accomplished by raising the dietary concentration of rapidly degraded non-
¿EHUFDUERK\GUDWHVVXFKDVVWDUFKIURPFHUHDOJUDLQV7KHLQFUHDVHRIVWDUFKFRQFHQWUDWLRQLQGLHWV
for dairy cows, however, can lead to undesirable ruminal fermentation, compromising the nutrient
supply for milk production and composition. The partial replacement of cereal grain with low starch
by-product feeds represents a potential alternative to overcome this limitation. The aim of the present
ZRUNZDVWRGHWHUPLQHWKHHIIHFWRIWKHVRXUFHRIVWDUFK EDUOH\DQGFRUQ DQG¿EHUFRQWHQWRQWKH
substrate oxidation and milk performance of lactating Murciano-Granadina goats.

Material and methods

Twenty Murciano-Granadina goats at mid lactation were used following two crosses over designs.
Four diets were used with alfalfa hay as forage source. Diet BS had 41% of barley grain; in diet BF
WKHEDUOH\JUDLQZDVUHSODFHGE\RI¿EURXVE\SURGXFWVGLHW&6KDGRIFRUQJUDLQDQG
LQGLHW&)WKHFRUQJUDLQZDVUHSODFHGE\RI¿EURXVE\SURGXFWV)RUGLHWVGHWDLOVVHH/ySH]et
al. (2013). The goats were allocated on individual metabolism cages. After 10 d of adaptation, feed
intake, refusal, total fecal and urine output, and milk yield were recorded daily during 5 d period.
Then, heat production from oxidation of nutrients (HPx) was determined during 24 h using a mobile
open-circuit respirometry system connected to a head box. Gas exchange measurements for estimation
of carbohydrate, fat and protein oxidation were used by Brouwer (1958). He developed equations
for calculation of net oxidation of nutrients. Energy associated with the oxidation of protein (OXP),
carbohydrate (OXCHO) and fat (OXF) was calculated by the method described by Chwalibog et al.
(1997) for ruminants. The CO2/CH4 ratio of 3 and 1.7 was used for high grain and high forage diets,
respectively (Fahey and Berger, 1988). The estimation of oxidation CO2 (CO2x) was carried out by
reducing CO2 production with 3 and 1.7 times CH4 production. The calculations were as follows:

+3[ N-  î22 + 5.02 × CO2[±î1XU2;3 î1XUî N-J 2;&+2 

î22 + 4.174 × CO2[±î1XU î N-J 2;)  î22 – 1.719 × CO2x
±î1XU î N-J

Gases were expressed in l/d and urinary nitrogen (Nur) in g/d. The data were analyzed by the MIXED
procedure of SAS.

Results and discussion

In order to estimate the CO2/CH4 ratio of our diets, the theoretical rumen fermentation balance
SURSRVHGE\:ROLQ  ZDVIROORZHGDQGDUDWLRRIDQGZHUHIRXQGIRUVWDUFKULFKDQG
¿EURXVGLHWV6OLJKWO\GLIIHUHQWUHJDUGVWKHDVVXPHGK\SRWKHVLV$OWKRXJKQRVLJQL¿FDQWHIIHFWVZHUH
observed for HP and kl, greater milk energy output in BF and CF and higher tissue energy retention
in BS and CS were found (López et al., 2013). Average milk yield (Table 1) was 2.24 kg/d, and milk
fat content was different (P<0.05) among diets (5.49% in higher starch diets and 7.14% in higher

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 109
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_27, © Wageningen Academic Publishers 2013
7DEOH'DLO\PLON\LHOGV NJG PLONFRPSRVLWLRQ  DQGR[LGDWLRQ N-NJ%: RISURWHLQ
2;3 FDUERK\GUDWH 2;&+2 DQGIDW 2;) DQGWKHLUFRQWULEXWLRQ  WRWKHKHDWSURGXFWLRQ
IURPVXEVWUDWHVR[LGDWLRQ +3[ RIODFWDWLQJ0XUFLDQR*UDQDGLQDJRDWV Q  

BS BF CS CF SEM P-value

Milk yield 2.4 2.2 2.2 2.1 0.91 0.2108

Dry matter 14.9a 15.8ab 15.1a b 0.19 0.0029
Fat 5.5a b 5.5a 7.9b 0.29 
Protein 3.9 3.9 4.1 4.0  
Lactose 4.7 4.7 4.8 4.7 0.04 0.8730
HPx 754 803   7.20 
OXP 100a 101a 97a 138b 4.10 0.0001
OXCHO 512 330 444 334 29.4 0.0704
OXF 142a 373c 235ab 289b 30.1 0.0457
OXP/HPx 13.3a 12.5a 12.4a 18.2b 0.54 0.0001
OXCHO/HPx a 41.5b 57.3a 43.4b 3.85 
OXF/HPx 18.9 45.9 30.3 38.4 3.85 0.0741

a,b,c9DOXHVZLWKLQDURZZLWKGLIIHUHQWVXSHUVFULSWZHUHVLJQL¿FDQWO\GLIIHUHQW P<0.05).

1')GLHWV 7KLVLVSUREDEO\GXHWRWKHKLJKHU1')FRQWHQWLQ¿EURXVWKDQVWDUFKGLHWV YV 

The depression in milk fat upon feeding starch rich diets has been explained by a shift from a high
availability of fat precursors to glucose and by a shift from lipogenesis to gluconeogenesis. Average
HPx was 773 kJ/kg0.75%:GDQGQRWVLJQL¿FDQWGLIIHUHQFHVZHUHIRXQGDPRQJWUHDWPHQWV7KH
OXP was greater (P<0.05) for CF vs. others, probably due to lower microbial protein synthesis. A
tendency of higher OXCHO for starch rich diets was observed (478 kJ/kg0.75 BW/d on average for
diets BS and CS, and 332 kJ/kg0.75 BW/d for diets BF and CF). Differences (P<0.05) were found
for OXF (189 kJ/kg0.75 BW/d on average for diets BS and CS, and 331 kJ/kg0.75 BW/d for diets BF
and CF). Chwalibog et al.  SRLQWHGRXWWKDWZLWKLQFUHDVLQJWKHDPRXQWRI¿EURXVE\SURGXFWV
more fermentation and less glucose was absorbed directly and oxidized as OXCHO, while more
carbohydrate was converted to volatile fatty acids and oxidized as OXF. Therefore, most of the
+3[GHULYHGIURP2;)ZDVLQ¿EURXVGLHWV IRU¿EURXVYVIRUVWDUFKGLHWVRQDYHUDJH 
PHDQZKLOHIRU2;&+2REWDLQHGIRU¿EURXVDQGVWDUFKGLHWVZDVRQDYHUDJHDQGUHVSHFWLYHO\

5HVXOWVVXJJHVWWKDWLQFOXVLRQRI¿EURXVE\SURGXFWVLQWKHGLHWRIPLGODFWDWLRQJRDWVUHGXFH2;&+2
and increases OXF and fat content without compromising milk yield.

References
Brouwer, E. 1958. On simple formulae for calculating the heat expenditure and the quantities of carbohydrate and fat
metabolized in ruminants, from data on gaseous exchange and urine N. Pages 182-194 in Proc. 1th Symposium
on Energy Metabolism. EAAP. Publ. 8. Academic Press, London.
Chwalibog, A., A.H. Tauson and G. Thorbek, 1997. Quantitative oxidation of nutrients in growing calves. Z.
(UQlKUXQJVZLVV
Fahey, G.C. and L.L. Berger, 1988. Carbohydrate nutrition of ruminants. In: Church, D.C. (ed.), The Ruminant Animal.
'LJHVWLYH1XWULWLRQDQG3K\VLRORJ\3UHQWLFH+DOO(QJOHZRRG&OLIIV1-SS
/ySH]0&&)HUQiQGH]DQG0/DFKLFD(IIHFWRIWKHVWDUFKVRXUFHDQG¿EHUOHYHOLQPL[HGGLHWVRQHQHUJ\
balance of lactating Murciano-Granadinas goats. ISEEP.
:ROLQ0-$WKHRUHWLFDOUXPHQIHUPHQWDWLRQEDODQFH-'DLU\6FL

110 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWRIWKHVWDUFKVRXUFHDQG¿EHUOHYHOLQPL[HGGLHWVRQWKHHQHUJ\
balance of lactating Murciano-Granadina goats
M.C. López1, C. Fernández1 and M. Lachica2
1,QVWLWXWHIRU$QLPDO6FLHQFHDQG7HFKQRORJ\8QLYHUVLGDG3ROLWpFQLFDGH9DOHQFLD9DOHQFLD
Spain; [email protected]
2'HSDUWPHQWRI$QLPDO1XWULWLRQ(VWDFLyQ([SHULPHQWDOGHO=DLGtQ&6,&$UPLOOD*UDQDGD
Spain

Introduction
The Spanish ruminant production system (FEDNA, 2009) is based on high use of concentrate
(40-70%) instead of whole forage rations due to the lack of pasture. Goat livestock occupies the
second position in the EU with 30% of the total milk production. By-product feeds have been used
extensively in dairy cattle diets as an economical substitute for corn and barley grain. There is an
increasing interest in the nutritive value of by-product feeds for dairy ruminant diets.

7KHDLPRIWKLVZRUNZDVWRGHWHUPLQHWKHHIIHFWRIWKHVWDUFKVRXUFHDQG¿EHUFRQWHQWRQWKHHQHUJ\
EDODQFHDQGWKHHI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROL]DEOHHQHUJ\ 0( IRUPLONSURGXFWLRQ Nl) of
lactating Murciano-Granadina goats.

Material and methods

Twenty Murciano-Granadina goats at mid lactation (43±0.5 kg BW) were used following two cross
RYHUGHVLJQV)RXULVRSURWHLFDQGLVRHQHUJHWLFGLHWV>JRIFUXGHSURWHLQ &3 NJRIGU\PDWWHU
(DM) and 19.1 MJ of gross energy (GE)/kg DM, respectively] were used, with alfalfa hay as forage
source. The ratio forage:concentrate was 41:59. Diet BS had 41% of barley grain; in diet BF the
EDUOH\JUDLQZDVUHSODFHGE\RI¿EURXVE\SURGXFWVGLHW&6KDGRIFRUQJUDLQDQGLQGLHW
&)WKHFRUQJUDLQZDVUHSODFHGE\RI¿EURXVE\SURGXFWV7KH¿EURXVE\SURGXFWZDVDEOHQG
of 18.5% of soy hulls, 15% of gluten feed and 7.5% of wheat bran. Diet BS and CS had an average
starch content of 27%, and BF and CF of 7%. The diet BS and CS had an average NDF content of
35%, and BF and CF of 47%. The goats were allocated to individual metabolism cages. After 10 d
of adaptation, feed intake, refusal, total fecal and urine output, and milk yield were recorded daily
during 5 d period. Heat production (HP) was determined during 24 h using a mobile open-circuit
UHVSLURPHWU\V\VWHPFRQQHFWHGWRDKHDGER[DQGFDOFXODWHGDFFRUGLQJWR%URXZHU  7KH
retained energy was calculated as ME – HP, the retained energy as tissue as ME – HP – milk energy,
the RQ as CO2/O2, and kl as corrected milk energy/(ME intake – MEm) assuming MEm=401 kJ/
kg0.75 BW/d (Aguilera et al., 1990). The data were analyzed by the MIXED procedure of SAS.

Results and discussion

Greater organic matter (OM) digestibility (P<0.05) was observed for diets higher in starch (BS and
&6 WKDQ¿EURXVGLHWV %)DQG&) 7KHORZHVWYDOXHVLQ&3DQG1')GLJHVWLELOLW\ZDVIRUGLHW&6
7DEOH 7KHVLJQL¿FDQWUHGXFWLRQRI20GLJHVWLELOLW\ZDVSUREDEO\GXHWRWKHLQFUHDVLQJ1')
content of diets BF and CF combined with the greater value of starch for diets BS and CS. NDF
GLJHVWLELOLW\ZDVQXPHULFDOO\JUHDWHUIRU¿EURXVGLHWVSUREDEO\GXHWRWKHVRXUFHRI¿EURXVLQJUHGLHQWV
¿EURXVE\SURGXFWVDQGDOIDOIDKD\ ZKHQFRPSDUHGZLWKVWDUFKULFKGLHWVZKHUHWKHRQO\VRXUFH
RI¿EHUZDVDOIDOIDKD\1RVLJQL¿FDQWGLIIHUHQFHVZHUHIRXQGIRUWKHLQWDNHRI0( N-NJ0.75
%:GDVDYHUDJH ZLWKKLJKHUQXPHULFDOYDOXHVIRU¿EURXVWKDQVWDUFKGLHWV DQGN-
kg0.75%:GRQDYHUDJHUHVSHFWLYHO\ 1RVLJQL¿FDQWGLIIHUHQFHVDPRQJGLHWVZHUHREVHUYHGLQ
HP with an average value of 788 kJ/kg0.75%:G1HLWKHUVLJQL¿FDQWGLIIHUHQFHZDVIRXQGIRU54
values (1.0 on average). The retained energy as milk was greater (P< IRU¿EURXVGLHWV N-

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 111
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_28, © Wageningen Academic Publishers 2013
7DEOH'DLO\LQWDNH NJG GLJHVWLELOLW\  DQGPHWDEROL]DEOHHQHUJ\ 0(N-NJ%: LQWDNH
KHDWSURGXFWLRQUHWDLQHGHQHUJ\HI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROL]DEOHHQHUJ\IRUPLONSURGXFWLRQ
Nl DQG54 &22/O2 RIODFWDWLQJ0XUFLDQR*UDQDGLQDJRDWV Q  XQGHUIRXUGLHWVEDVHGRQ
EDUOH\JUDLQ %6 EDUOH\JUDLQDQG¿EURXVE\SURGXFWV %) FRUQJUDLQ &6 DQGFRUQJUDLQDQG
¿EURXVE\SURGXFWV &) 

BS BF CS CF SEM P-value

Dry matter intake 2.02 2.07 2.03 2.09 0.024 0.7733

Digestibility, %
Organic matter a b a b  0.0280
1HXWUDOGHWHUJHQW¿EHU a a b 49.5a 1.893 0.0032
Starch 99.2  99.1  0.090 0.7598
Crude protein 78.0a 75.8ab b b 0.753 0.0395
ME intake 1414 1439 1430 1443 15.8 
Heat production  819 792 774 7.2 0.0559
Retained energy      
Milk energy 504b 544a 501b 554a 13.9 
Tissue energy 142a b 137a 115ab 17.8 0.0451
kl     0.007 0.0737
RQ   1.03 0.99 0.014 

a,b9DOXHVZLWKLQDURZZLWKGLIIHUHQWVXSHUVFULSWZHUHVLJQL¿FDQWO\GLIIHUHQW P<0.05).

kg0.75 BW/d, as average) than starch diets (503 kJ/kg0.75 BW/d, as average). The retained energy as
tissue was greater (P<0.05) for starch diets (140 kJ/kg0.75%:GDVDYHUDJH WKDQKLJK¿EHUGLHWV
N-NJ0.75 BW/d, as average). Van Knegsel et al. (2007) showed that cows fed a lipogenic diet
partitioned more energy to milk than cows fed a glucogenic diet, with a tendency for higher energy
PRELOL]DWLRQIURPERG\IDW1RVLJQL¿FDQWGLIIHUHQFHVZHUHREVHUYHGDPRQJGLHWVIRUNl RQ
average) and the value was similar than the obtained by Aguilera et al. (1990) with the same goat
breed (kl  

,QWKHSUHVHQWVVWXG\WKHXVHRI¿EURXVE\SURGXFWVDVUHSODFHURIEDUOH\DQGFRUQJUDLQDVVWDUFK
sources, had no effect on ME intake and it increased the retained energy as milk without affect the kl.

References
Aguilera, J.F., C. Prieto and J. Fonollá, 1990. Protein and energy metabolism of lactating Granadina goats. Br. J. Nutr.

%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV,Q%OD[WHU./ HG 3URFHHGLQJVRIWKH7KLUG
EAAP Symposium on Energy Metabolism. Publication No. 11. Academic Press, London, pp. 441-443.
FEDNA, 2009. Nutritional Requirements for Dairy Ruminants. ed. Fundación Española para el Desarrollo de la
Nutrición Animal, Madrid, Spain.
Van Knegsel, A.T.M., H. van den Brand, J. Dijkstra, W.M. van Straalen, M.J. Heetkamp, S. Tamminga and B. Kemp,
2007. Dietary energy source in dairy cows in early lactation: energy partitioning and milk composition. J. Dairy
6FL

112 Energy and protein metabolism and nutrition in sustainable animal production
The development of the gravid uterus of Santa Inês ewes and ewe lambs
under two nutritional planes
L.F.L. Cavalcanti1,2, I. Borges1, F.A. Souza1, G.L. Macedo Júnior and L.O. Tedeschi2
1 Departamento de Zootecnia, Universidade Federal de Minas Gerais, Brazil;
[email protected]
2Department of Animal Science, Texas A&M University, USA
Universidade Federal de Uberlândia, Brazil

Introduction
The Santa Inês is the most common ovine breed in Brazil, and there is a lack of information about the
nutrient requirements for this genotype. As a result of the inhospitable environment where this breed
was established, the metabolism of these animals evolved different strategies to reserve and utilize
QXWULHQWVZKLFKPD\LQÀXHQFHLWVUHTXLUHPHQWV7KHPRVWVLJQL¿FDQWGUDLQRIQXWULHQWVRISUHJQDQW
ewes is the gravid uterus (GU) mainly during the last third of gestation because of the increased
fetus growth rate (Rattray et al., 1974). The objective of this study was to measure the weight and
composition of the GU of Santa Ines ewes and ewe lambs (primiparous ewes).

Material and methods

The animals (38 ewe lambs and 75 ewes) were mated with rams of known fertility and after 45 days
IURPPDWLQJZHUHH[DPLQHGWRFRQ¿UPSUHJQDQF\DQGWRVSOLWWKHHZHVEDVHGRQWKHLUSUROL¿FDF\
(ZHODPEVZLWKWZRIHWXVHVZHUHGLVFDUGHGEHFDXVHRIWKHORZIUHTXHQF\DQGGLI¿FXOW\WRIRUP
groups. The animals were divided into two nutritional planes, ad libitum and restricted (15% less
protein and energy than the non-restricted group). Ewe lambs were slaughtered at 0, 100, 130, and
140 days of pregnancy, and ewes at 0, 90, 110, 130, and 140 days of pregnancy. The gravid uterus
and its content were removed and weighed. Samples were collected and later submitted to dry matter
(DM) and crude protein (CP) analysis. An exponential function (W = W0 × exp(k×t)), where: W
is the weight or CP content (g), W0 is the initial value (g), k is fractional growth rate (1/d), and t
LVWKHWLPHLQGD\VIURPPDWLQJZDV¿WWHGWRWKHGDWD7KHODFNRI¿WZDVDVVHVVHGEDVHGRQWKH
F-test and the necessity of additional parameters for each group due to other factors (e.g. nutritional
plane, category and number of fetuses) was determined. Additionally, an allometric analysis of CP
FRQWHQWYHUVXVWKH*8ZHLJKWZDVSHUIRUPHGE\¿WWLQJWKHIXQFWLRQ&3*8 Dî*8b, where ‘a’ is
a constant (g), CPGU is the content (g) of CP in the gravid uterus (GU), GU is weight (g) and ‘b’ is
the allometric exponent (dimensionless).

Results and discussion

7KH*8ZHLJKWZDVRQO\DIIHFWHGE\SUROL¿FDF\EHLQJIRUVLQJOHJHVWDWLRQ RQHIHWXV GHVFULEHGE\
WKHHTXDWLRQZLWK: “DQGN “UHJDUGOHVVRIQXWULWLRQDOSODQHRUFDWHJRU\
)RUHZHVZLWKWZRIHWXVHVWKHSDUDPHWHUVREWDLQHGZHUH: “DQGN “)RU
WKH&3FRQWHQWLWZDVLPSRVVLEOHWR¿WRQHPRGHOIRUERWKHZHVDQGHZHODPEV7KHVDPHSDWWHUQ
ZDVREVHUYHGIRUWKHSUROL¿FDF\+HQFHRQHHTXDWLRQZDVGHYHORSHGUHJDUGOHVVRIWKHOHYHORI
nutrition. Three functions were obtained: for all ewe lambs (W0=9.320±3.13, k=0.030±.002); for
ewes with single gestation (W0=9.371±4.40, k=0.032±.003) and the last one for ewes with two
IHWXVHV : “N “ DVVHHQDW)LJXUH7KHSURWHLQGHSRVLWLRQRQWKHJUDYLG
uterus in this study was overestimated by the equation of CSIRO (2007) with the same purpose since
DSSUR[LPDWHO\GD\VRISUHJQDQF\HYHQZKHQFRPSDUHGWRWKHHTXDWLRQIRUWZLQJHVWDWLRQ7KLV
may be explained by the difference between Santa Ines animals and the traditionally breeds from
Australia. Accordingly to Robinson and McDonald (1979) the birth weight is affected by breed and
nutritional level, been those factors the main determinants of nutrient deposition rate on gravid uterus.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 113
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_29, © Wageningen Academic Publishers 2013
)LJXUH&UXGHSURWHLQFRQWHQW J RQWKHJUDYLGXWHUXVGXULQJSUHJQDQF\IRUHZHODPEVDQGHZHV
with single or two fetuses.

Severe restrictions of energy (30% or greater) for long time affect the fetus development (Gao et al.,
2009). In this trial the lowest nutritional level was not able to affect the gravid uterus development,
what may be explained by the restriction used in this study that probably was not so severe. This
result was also observed by (Koong et al., 1975) Koong et al. (1975) when they decomposed the k
FRHI¿FLHQWLQWRWKUHHIDFWRUVOHYHORIQXWULWLRQVWDJHRIJHVWDWLRQDQGQXPEHURIIHWXVHV8VLQJD
VWHSZLVHUHJUHVVLRQWHFKQLTXHWKH\FRQFOXGHGWKDWRQO\WKHWZRODVWIDFWRUVLQÀXHQFHGWKHJURZWK
development, and additionally, that the model for which the number of fetuses was directly related
to the initial weight (W0) was more accurate than when related to the growth rate (k), what is quite
similar to the results found in the present study for ewes with one or two fetuses. When we used
the allometric equation, the exponent ‘b’ was 1.132±0.04, what means that the CPGU deposition
is not isometric to the weight increase of GU. These results suggested that, within the range of the
evaluated nutritional planes, the main modulator of GU development and, consequently, of the
nutrient requirements for pregnancy for Santa Ines sheep are pregnancy stage (proportion of variance
H[SODLQHGȦ2  FDWHJRU\ Ȧ2  DQGSUROL¿FDF\ Ȧ2=0.125). Moreover, the protein is
deposited in a faster rate of deposition than the rate of growth of the whole gravid uterus.

References
&6,521XWULHQWUHTXLUHPHQWVRIGRPHVWLFDWHGUXPLQDQWV&RPPRQZHDOWKVFLHQWL¿FDQGLQGXVWULDOUHVHDUFK
organisation Collingwood, 270 pp.
Gao F., Y. Liu, and X. Hou, 2009. Effect of maternal undernutrition during late pregnancy on growth and development
RIRYLQHIHWDOYLVFHUDORUJDQV$VLDQ$XVWUDO-$QLP
Koong, L. J., W. N. Garret, and P. V. Rattray, 1975. A description of the dynamics of fetal growth in sheep. J. Anim.
6FL
Rattray, P. V., W. N. Garret, N. E. East, and N. Hinman. 1974. Growth, development and composition of the ovine
FRQFHSWXVDQGPDPPDU\JODQGGXULQJSUHJQDQF\-$QLP6FL
Robinson, J. and I. McDonald, 1979. Ovine prenatal growth, its mathematical description and the effects of maternal
nutrition. Ann. Biol. Anim. Bioch. 19, 225-234.

114 Energy and protein metabolism and nutrition in sustainable animal production
The effect of a limited supply of phenylalanine, threonine, and
tryptophan on milk yield and composition
I.H. Iroshan1, H. Lapierre2 and L. Doepel1
18QLYHUVLW\ RI &DOJDU\ )DFXOW\ RI 9HWHULQDU\ 0HGLFLQH &DOJDU\ $% 71 1 &DQDGD
[email protected]
2$JULFXOWXUHDQG$JUL)RRG&DQDGD6KHUEURRNH4&-0&&DQDGD

Introduction
In dairy cows, milk and milk component yields respond strongly to deletion of all essential amino
acids (AA) from an infused AA mixture, but do not respond to deletion of non-essential AA (Doepel
and Lapierre, 2010). Lysine and methionine are considered 1st and 2nd limiting AA on corn-based
GLHWV 15& ZKLOHRQJUDVVVLODJHEDVHGGLHWVKLVWLGLQHWHQGVWREH¿UVWOLPLWLQJ 9DQKDWDORet
al., 1999). A large proportion of the research in dairy cows has focused on these three AA. Indeed,
deletion of these 3 AA from an infusion mixture decreased milk protein yield to the negative control
level; however, deletion of the branched-chain AA from the mixture did not alter milk and milk
protein yields (Weekes et al 7RGDWHSKHQ\ODODQLQH 3KH WKUHRQLQH 7KU DQGWU\SWRSKDQ
(Trp) have received minimal attention, but the importance of these AA should not be overlooked.
7KHGLJHVWLYHÀRZVRIWKHVH$$DUHRQDYHUDJHORZHUWKDQHVWLPDWHGUHFRPPHQGDWLRQV 'RHSHOet
al., 2004). Additionally, these AA are usually taken up by the mammary gland in the same amount
as that used for milk protein secretion which then raises the question ‘will milk protein synthesis
decrease if the supply of these AA is limited?’ The objective of this study was to determine how
milk and milk component yields change when Phe, Thre, and Trp supply is limited.

Material and methods

Five 2nd lactation Holstein cows were used in a 5×5 Latin square with 10-day periods. Cows were
fed a diet formulated to supply 100% of the net energy requirement and 70% of the metabolizable
protein (MP) requirement (NRC, 2001). The treatments were abomasal infusions of water (Ctl),
all AA (TAA) at 925 g/day, all AA excluding Thre (No-Thr), all AA excluding Phe (No-Phe), and
all AA excluding Trp (No-Trp). The TAA treatment was estimated to supply, with the diet, 100%
of the MP requirement, and AA composition of the TAA was the same as that of casein. Infusates
were delivered continuously in 15 l/d of water. Arterial (coccygeal vessel) and venous (mammary
vein) blood samples were collected every 2 hours on day 10 between the AM and PM milkings
Q  3ODVPDZDVDQDO\VHGIRUXUHD1DQGȕK\GUR[\EXW\UDWH %+%$ 0LONVDPSOHVIURPWKH
last 3 days of each period were analyzed for lactose, fat, protein, and milk urea-N (MUN). Data
ZHUHDQDO\]HGXVLQJWKH0,;('SURFHGXUHRI6$6ZLWKWUHDWPHQWDVWKH¿[HGHIIHFWDQGSHULRG
and cow as random effects. Treatment differences were determined using pre-planned contrasts,
comparing each treatment to TAA.

Results
Dry matter intake tended to be lower for No-Phe vs. TAA (3 0.10). Relative to TAA, milk yield
tended to be reduced in CTL, No-Phe and No-Thr cows (0.10<P<0.15; Table 1). Milk protein yield
was lower (P<0.01) for CTL and No-Phe than for TAA and tended to be lower (3 0.13) for No-
Thr. Milk fat yield (1128 g/d) and lactose yield (1428 g/d) were not affected by treatment (P>0.15).
MUN was higher (P<0.01) for TAA than Ctl. Plasma urea-N concentration was increased with TAA
compared with CTL, but was lower for TAA than for No-Phe. BHBA arterial concentration and
PDPPDU\JODQGH[WUDFWLRQUDWH 3 0.82) were not affected by treatment. Deletion of Trp
from the infusate did not affect (P>0.15) any measured parameters.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 115
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_30, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIDPLQRDFLGVGHOHWLRQRQGU\PDWWHULQWDNH '0, PLONDQGPLONFRPSRQHQW\LHOG
and metabolite concentrations.1

Parameter Ctl Treatment SEM P-value

No-Phe No-Thr No-Trp TAA Ctl TAA TAA No-Phe TAA No-Thr

DMI, kg/d 17.8 17.0  17.2 18.0   0.10 
Yield, kg/d
Milk 29.9 30.1 30.4 32.8 33.4 2.12 0.10 0.12 0.15
Protein 0.78 0.77 0.84 0.88 0.91 0.04 0.01 0.01 0.13
Fat 1.04 1.08  1.25 1.12 0.07 0.34  0.71
Lactose  1.38 1.39 1.51 1.50 0.09 0.15 0.22 0.25
MUN, mg/dl 8.8 13.5  14.2 14.1 0.94 0.003  0.70
Plasma metabolites, mM
Urea-N  13.2   11.2 0.74 0.001 0.03 
BHBA 0.54 0.49  0.53 0.52 0.04 0.71 0.47 0.22

1 Pre-planned contrast comparing TAA vs. each treatment; TAA vs. No-Trp not shown: all P-values >0.15.

In conclusion, the results of the current study show that a limited supply of Phe and Thr negatively
affects milk and milk protein secretion, with Phe limitation on protein yield being stronger than Thr
limitation. The elevated urea-N with No-Phe relative to TAA supports that milk protein synthesis
ZDVOLPLWHGE\DGH¿FLHQF\RI3KHDQGWKXV$$VXSSO\QRWXVHGIRUPLONSURWHLQVHFUHWLRQDQG
WKHUHIRUHLQH[FHVVZDVFRQYHUWHGWRXUHD'H¿FLHQFLHVRI3KH7KUDQG7USKDGQRHIIHFWRQPLON
fat and lactose yield.

References
Doepel, L. and H. Lapierre, 2010. Changes in production and mammary metabolism of dairy cows in response to
HVVHQWLDODQGQRQHVVHQWLDO$$LQIXVLRQV-'DLU\6FL
Doepel, L., D. Pacheco, J.J. Kennelly, M.D. Hanigan, I.F. López and H. Lapierre, 2004. Milk protein synthesis as a
function of AA supply. J. Dairy Sci. 87, 1279-1297.
National Research Council, 2001. Nutritional requirements of dairy cattle. 7th Revised Edn. National Academy Press,
Washington D.C.
SAS. 2002. SAS System for Mixed Models. SAS Institute Inc., Cary, NC.
Vanhatalo, A. P. Huhtanen, V. Toivonen and T. Varvikko, 1999. Response of dairy cows fed grass silage diets to abomasal
LQIXVLRQVRIKLVWLGLQHDORQHRULQFRPELQDWLRQVZLWK0HWDQG/\V-'DLU\6FL
:HHNHV7/3+/XLPHVDQG-3&DQW5HVSRQVHVWRDPLQRDFLGLPEDODQFHVDQGGH¿FLHQFLHVLQODFWDWLQJGDLU\
cows. J. Dairy Sci. 89, 2177-2187.

116 Energy and protein metabolism and nutrition in sustainable animal production
0HWDEROLFDQGKRUPRQDOSUR¿OHRIEXOOVGXULQJHYDOXDWLRQRIQXWULWLRQDO
requirements by respirometric technique under different plane of nutrition
A.L.C.C. Borges, P.A.D. Vivenza, R.R. Silva, E.J. Facury Filho, H.F. Lage, P.H.A. Carvalho, P.R.O.
Paes and N.M. Rodriguez
Departament of Animal Science of Veterinary School of Federal University of Minas Gerais, Belo
Horizonte, Brazil; [email protected]

Introduction
The knowledge of animal metabolism against different nutritional conditions imposed is of great
importance, since the ratio of nutrients absorbed deposited in the body or that are in full use is
dependent on metabolic and hormonal status of the animal (Payne and Payne, 1987). The objective
RIWKLVZRUNZDVWRVWXG\WKHKRUPRQDODQGPHWDEROLFSUR¿OHRIEXOOVGXULQJHYDOXDWLRQRIWKH
nutritional requirements of energy, by respirometry technique, in different nutritional plans and
undergoing a fasting period.

Material and methods

Fifteen F1-crosbred (Bos taurus indicus) × Holstein (Bos taurus taurus) bulls, with average weight
of 304 kg were used. The animals were randomly assigned into three groups and fed diets to provide
mild (maintenance group), medium (medium gain group) and high (ad libitum group) weight gains,
initially formulated for gains of 0.100 kg, 0.500 kg and 0.900 kg/day. For measurement of fasting
heat production, which is the net energy for maintenance (NEm) through respirometric technique,
the animals are subjected to 72 h of fasting. The respirometric chamber is performed between 48 and
72 h of fasting. Four times during the fasting were evaluated: the beginning of fasting, 24, 48 and
KRIIDVWLQJ,QVXOLQJOXFRVHEHWDK\GUR[\EXW\UDWH %+%ȕ QRQHVWHUL¿HGIDWW\DFLGV 1()$ 
urea, creatinine, total protein, albumin, globulins, triglycerides, aspartate aminotransferase (AST)
and hematocrit were evaluated. The blood samples were collected by venipuncture/coccygeal artery
using a vacuum collection system. The experimental design was a randomized design with a split-
plot scheme, since two factors were evaluated nutritional plan (plot factor) and fasting time (subplot
factor). The data were analysed by the GLM procedures (SAS, 2001).

Results and discussion

7KHIDVWLQJSHULRGRIKZDVFKDUDFWHUL]HGE\GHFUHDVHLQJOXFRVHFRQFHQWUDWLRQGXULQJWKH¿UVW
hours and recovery levels of this component in the maximum time of fasting. This is associated with
mobilization of lipid and protein reserves during fasting, which varied between groups, being higher
in group ad libitum taking these animals the highest values of plasma NEFA and urea, although the
ȕ%+%YDOXHVZHUHVLPLODULQERWKJURXSVEXWLQFUHDVLQJZLWKWKHLQFUHDVHGWLPHRIIDVWLQJ,WLV
suggested that ad libitum group (greater body weight) are more susceptible to the effects of fasting,
LQYLHZRILWVOLNHO\JUHDWHUHQHUJ\UHTXLUHPHQWIRUPDLQWHQDQFHRQFHWKHERG\ZHLJKWKDVVLJQL¿FDQW
LQÀXHQFHRQWKLV 6PLWKDQG%DOGZLQ ,QVLWXDWLRQVRIIRRGGHSULYDWLRQWKH\SUREDEO\KDYH
JUHDWHUHQHUJ\GH¿FLWVLJQDOLQJWKHQHHGWRPRELOL]HWKHERG\LQODUJHUSURSRUWLRQVERG\UHVHUYHV
7KHWDEOHUHSRUWVWKHPHWDEROLFDQGKRUPRQDOSUR¿OHRIEXOOVXQGHUGLIIHUHQWQXWULWLRQDOFRQGLWLRQV

There were no differences in hematocrit values and total protein during fasting, indicating that the
animals were not dehydrated, so there is no interference of hemoconcentration on these parameters.
7KHUHVSRQVHRIDQLPDOVWRIDVWLQJUHYHDOVWKHHI¿FLHQF\RIWKHPDLQWHQDQFHPHFKDQLVPVRIHQHUJ\
within normal in these, ensuring proper physiological condition during the measurement respirometric
chamber, because even the maximum period of fasting, plasma glucose concentrations were within
the reference values proposed by Kaneko et al.  IURPWRPPROO

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 117
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_31, © Wageningen Academic Publishers 2013
7DEOH3ODVPDFRQFHQWUDWLRQVRIJOXFRVHȕ%+%DQG1()$H[SUHVVHGLQPPROOXUHDH[SUHVVHG
LQPJGODQGLQVXOLQH[SUHVVHGLQX8POGXULQJIDVWLQJ DQGK LQJURXSVRIad libitum
gain, medium gain and maintenance.

Component Group Time of fasting period (h)1 Average

0 24 48 72

Glucose (mmol/l) Ad libitum 4.32 a D D 3.84 a 4.00 a

Medium gain 4.11 a 3.82 ab 3.27 ab 3.72 a 3.73 b
Maintenance D 2.92 b 2.89 b 3.58 a 3.25 c
ȕ%+% PPROO Ad libitum 0.391 a 0.419 a D 0.915 a D
Medium gain D 0.375 a 0.554 a 0.923 a 0.530 a
Maintenance 0.398 a 0.422 a 0.704 a 0.930 a D
NEFA (mmol/l) Ad libitum 0.181 a 0.449 a D D D
Medium gain 0.121 a 0.288 a D 0.942 a 0.529 b
Maintenance 0.101 a 0.209 a D D E
Urea (mg/dl) Ad libitum D D 51.07 a 40.43 a 41.94 a
Medium gain 33.03 a 34.98 a 42.22 ab 32.51 a E
Maintenance 33.37 a D 31.15 b D E
Insulin (uU/ml) Ad libitum 14.88 a 8.78 a D D 9.19 a
Medium gain 17.12 a 11.02 a D D D
Maintenance D D 4.88 a D 7.15 b

1 a,b,c (P<0.05).

Acknowledgements
We would like to thank CNPq, CNPq-INCT, FAPEMIG, CAPES and EPAMIG for their cooperation
in carrying out this work.

References
.DQHNR--+DUYH\-:DQG0/%UXVV&OLQLFDOELRFKHPLVWU\RIGRPHVWLFDQLPDOVth ed. Academic Press,
6DQ'LHJRS
3D\QH-03D\QH67KHPHWDEROLFSUR¿OHHG2[IRUI2[IRUG8QLYHUVLW\3UHVV3
Sas, 2001. Release 8.01. Sas Inst. Inc., Cary, Nc, USA.
Smith, N.E, Baldwin, R.L, 1974. Effects of breed, pregnancy and lactation on weight of organs and tissues in dairy
FDWWOH-RXUQDO2I'DLU\6FLHQFH913

118 Energy and protein metabolism and nutrition in sustainable animal production
Methane emission and digestibility of goats subjected to feed restriction
A.R.C. Lima1, K.T. Resende1, I.A.M.A. Teixeira1, T.F.V. Bompadre1, R.T.S. Frighetto2 and M.H.M.R.
Fernandes1
1UNESP Univ Estadual Paulista, Department of Animal Science, Via de Acesso Prof Paulo Donato
&DVWHOODQHVQ-DERWLFDEDO6DR3DXOR%UD]LO[email protected]
2EMBRAPA Environment, Brazilian Agricultural Research Corporation, Laboratório de Ecologia
4XLPLFD5RGRYLD63.P-DJXDULXQD6DR3DXOR%UD]LO

Introduction
Currently, due to environmental and economic issues, ruminant production system has aimed at
PD[LPL]LQJSURGXFWLRQFRQFRPLWDQWZLWKDQLQFUHDVHGHI¿FLHQF\%HFDXVHRIPHWKDQHLVSURGXFHG
in the process of feed energy utilization within the animal, its reduction is usually associated with
improved productivity. Therefore, one of the strategies to optimizing energy use in ruminants is to
minimize enteric methane (CH4) emission during the fermentation process, by reducing dry matter
intake (Shibata and Terada, 2010). Feed restriction is a common practice on goat production in several
SDUWVRIZRUOGDQGFDQEHXVHGWRH[SORLWLQJWKHFRPSHQVDWRU\JDLQUHVXOWLQJLQEHWWHUHI¿FLHQF\
of animal feed energy utilization. Therefore, this study was carried out to evaluate the effect of feed
restriction on methane emission and digestibility.

Material and methods

)LIWHHQGU\QRQODFWDWLQJ1XELDQJRDWV “ ZHUHUDQGRPO\DOORFDWHGLQWRJURXSV EORFNV RI
3 animals receiving the treatments AL: ad libitum intake, MR: moderate restriction (approximately
15% of feed restriction) and SR: severe restriction (approximately 40% of feed restriction). Goats
ZHUHKRXVHGLQLQGLYLGXDOPHWDEROLFSHQV7KHH[SHULPHQWDOGLHW FUXGHSURWHLQ0-*(
/kg DM) consisted of 47% dehydrated corn plant and 53% concentrate. The moderate and severe
feed restriction was determined daily based on the dry matter (DM) intake of the goats in the ad
libitum treatment on the previous day. During a digestion trial, feed intake, feed refusals, feces and
urine were collected for 5 d after 20 d adaptation period. Composites of feeds, feed refusals, and
IHFHVZHUHGULHGDWWRƒ&IRUKDQGJURXQGWKURXJKDPPVFUHHQXVLQJD:LOH\PLOO
Composites of urine were passed through a sieve to remove the large particles, and a subsample
was taken. Gross energy was determined for feeds, feed refusals, feces, and urine using a bomb
calorimeter (Parr Instrument Co.). Measurements of methane emission were performed using the
VXOSKXUKH[DÀXRULGH 6)) tracer technique. Data were analyzed in a randomized complete block
design, using mixed models of SAS.

Results and discussion

*RDWVVXEMHFWHGWRPRGHUDWHDQGVHYHUHUHVWULFWLRQWUHDWPHQWVDWHDSSUR[LPDWHO\DQG
respectively, of those fed ad libitum (Table 1). Methane emission (3 0.01) was greater in goats fed
ad libitum than in goats subjected to moderate and severe feed restriction. However, when methane
energy losses were expressed as a proportion of gross energy intake, there were no differences
between treatments. Digestible energy (11.5 MJ/kg, 3 0.21), metabolizable energy (9.4 MJ/kg,
3 0.53) and metabolizability (qm, 3 0.50) was similar for all treatments. It may be a result of using
equal diet for all treatments. Moreover, the severity of feed restriction imposed was not enough to
FDXVHVLJQL¿FDQWGLIIHUHQFHVLQWKHLQWDNHDPRQJWUHDWPHQWV,QFRQFOXVLRQIHHGUHVWULFWLRQGHFUHDVHG
GDLO\PHWKDQHHPLVVLRQZLWKLQWKHUDQJHRILQWDNHVFRQVLGHUHGKRZHYHULWGLGQRWDIIHFWHI¿FLHQF\
of feed energy utilization.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 119
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_32, © Wageningen Academic Publishers 2013
Table 1. Intake, digestibility and energy utilization of goats subjected to feed restriction.

Variable Feed restriction

NR MR SR P-value SEM

Animals 5 5 5
BW 45.51  45.21 0.47 1.02
DMI (g/day) 824.35 705.39 508.58 0.1041 120.87
DMI (g/kg BW0.75)  43.97 31.39  5.82
OMI (g/day) 791.97a E F 0.013 
Digestibility
DMD (%)   72.80 0.14 2.48
OMD (%) 73.70  71.97 0.13 
ED (%) 71.25  70.85 0.18 3.03
Energy utilization
CH4(g/day) 22.05 18.87 15.43 0.01 1.89
CH4(g/100 g OMI)  2.49 2.77 0.70 0.23
DE (MJ/ kg DM) 11.82 10.99 11.81 0.21 0.495
ME (MJ/ kg DM) 9.75 8.97 9.54 0.53 
q 0.59 0.54 0.57 0.50 0.04
CH4 losses (%) 8.59 8.00 8.89 0.72 0.78

Acknowledgements
FAPESP project number 2008/58351-5 and 2011/14842-8.

References
Shibata M. and F. Terada, 2010. Factors affecting methane production and mitigation in ruminants. Anim. Sci. J. 81, 2-10.

120 Energy and protein metabolism and nutrition in sustainable animal production
(I¿FLHQF\RIXWLOL]DWLRQRIGLHWDU\QLWURJHQIRUPLONSURGXFWLRQE\GXDO
purpose cows fed increasing levels of Leucaena leucocephala forage
mixed with Pennisetum purpureum grass
A. Ruiz-González, A.J. Ayala-Burgos, C.F. Aguilar-Pérez and J.C. Ku-Vera
Department of Animal Nutrition, Faculty of Veterinary Medicine and Animal Science, University of
Yucatan, Merida, Yucatan, Mexico; [email protected]

Introduction
(I¿FLHQF\RIQLWURJHQXWLOL]DWLRQ J1LQSURGXFWJ1LQWDNH(18 E\UXPLQDQWVLVORZZLWK
an average of ~25% (Calsamiglia et al., 2010), but with an ample range of variation (15-40%)
VXJJHVWLQJWKDWLPSURYHPHQWVDUHSRVVLEOH,WLVRISDUDPRXQWLPSRUWDQFHWRLQFUHDVHWKHHI¿FLHQF\
of N utilization in order to improve productive performance (i.e. milk yield) and reduce excretion of
nitrogenous products in urine and feces from ruminants to the environment. Silvopastoral systems
represent a viable means of increasing levels of animal production in the tropics. Usually, high CP
legumes are employed in those systems and the possibility of N loss from the gastrointestinal tract
(Poppi and McLennan, 1995) may constraint productive limits.

Material and methods

Five multiparous crossbred (Bos taurus × Bos indicus) cows with a live-weight of 400±30 kg, were
housed in individual metabolic stalls in a 4×4 Latin square arrangement of treatments (Cochran
and Cox, 1957). Cows were fed increasing levels (0, 15, 30 and 45%; DM basis); of fresh chopped
forage of Leucaena leucocephala mixed with chopped Pennisetum purpureum grass and cows were
supplemented with 2 kg/day of ground corn at the time of milking. Cows were milked manually
RQFHGDLO\ K DQGWKHFDOIKDGUHVWULFWHGDFFHVVWRKLVGDP0LON\LHOG NJ ZDVUHFRUGHGGDLO\
$PLONVDPSOHZDVWDNHQGDLO\GXULQJWKHODVW¿YHGD\VRIHDFKH[SHULPHQWDOSHULRGWRGHWHUPLQH
1FRQWHQWE\WKH.MHOGDKOPHWKRG $2$&,' JURVVHQHUJ\ZDVHVWLPDWHGZLWKWKH
HTXDWLRQSURSRVHGE\7\UUHOODQG5HLG  DQGWKHHI¿FLHQF\RIHQHUJ\LQPLONZDVHVWLPDWHGZLWK
the equation proposed by Yan et al.  (DFKH[SHULPHQWDOSHULRGODVWHGIRUGD\VGD\V
for adaptation and 5 days for measurements of response variables. N intake and N in milk data were
DQDO\]HGZLWK*/0DQGRUWKRJRQDOFRQWUDVWVSHUIRUPHGZLWKWKHVWDWLVWLFDOVRIWZDUH6$6  

Results and discussion

A correlation was found (R2=0.92) between N intake and N in milk. Figure 1 predicts N concentration
in milk to a known CP intake. Nonetheless, N in milk as a percentage of the total N consumed tended

40
N in milk (g/cow/day)

35 y = 0.092x + 16.887
R² = 0.9233 45
30 15 30
0%
25

20

15
50 100 150 200 250
N intake (g/cow/day)
)LJXUH5HODWLRQVKLSEHWZHHQQLWURJHQFRQWHQWLQPLONDQGWRWDOQLWURJHQLQWDNH JFRZGD\ 

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 121
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_33, © Wageningen Academic Publishers 2013
to decrease (P< DV1LQWDNHZDVLQFUHDVHG )LJXUH (I¿FLHQF\RI1XWLOL]DWLRQIRUPLON
SURGXFWLRQDQGWKHHI¿FLHQF\RI0(XWLOL]DWLRQIRUPLONSURGXFWLRQGHFUHDVHG P<0.05) as the level
of N consumed was increased (Table 1). Daily milk yield was 5.1c, 5.3cb and 7.7a kg (SEM=0.3)
for the treatments 0, 15, 30 and 45% Leucaena, respectively. Results agree with Muinga et al. (1992)
ZKRVLJQL¿FDQWO\LQFUHDVHGPLON\LHOG DQGNJG RIFURVVEUHGFRZVIHGDEDVDOUDWLRQ
of Pennisetum purpureum grass supplemented with increasing levels (0, 1 and 2 kg DM/day) of
LeucaenaIRUDJH7KHUHLVVFRSHWRLPSURYHWKHHI¿FLHQF\RI1XWLOL]DWLRQIRUPLONSURGXFWLRQLQ
silvopastoral systems in the tropics where high CP legumes are available for incorporation under
SUDFWLFDOFRQGLWLRQVSDUWLFXODUO\GXULQJWKHGU\VHDVRQZKHQWURSLFDOJUDVVHVDUHGH¿FLHQWLQ1

250 35
N intake (g/cow/day)

30
200
25
150 20
N intake
100 15 (g/cow/day)
10 % N in milk
50
5
0 0
T1 T2 T3 T4
Treatment
Figure 2. Percentage of consumed nitrogen contained in milk.

7DEOH0LONHQHUJ\RXWSXWHI¿FLHQF\RIQLWURJHQXWLOL]DWLRQDQGHI¿FLHQF\RI0(XWLOL]DWLRQIRUPLON
production by crossbred cows fed increasing levels of Leucaena foliage in the ration.

% incorporation of foliage of L. SEM Contrast

leucocephala in the ration (DM basis)

0 15 30 45 L C Q

Milk energy output MJ/d 15.83c 17.71bc 19.55ab 22.10a 1.47 * NS NS

(I¿FLHQF\RI1XWLOL]DWLRQLQPLON 0.29a 0.22b 0.21bc 0.18c 0.04 * NS NS
(I¿FLHQF\RIHQHUJ\LQPLON a b c c 0.10 * NS NS

5RZVZLWKGLIIHUHQWOLWHUDOGLIIHUVLJQL¿FDQWO\ P<0.05).

References
Calsamiglia, S., Ferret, A., Reynolds, C.K., Kristensen, N.B. and van Vuuren, A.M. 2010. Strategies for optimizing
QLWURJHQXVHE\UXPLQDQWV$QLP
Cochran, W. G. and Cox, G. M. 1974. Diseños Experimentales. Edit. Trillas. pp. 145-149.
Muinga, R.W., Thorpe, W. and Topps, J.H. 1992. Voluntary food intake, live-weight change and lactation performance
of crossbred dairy cows given ad libitum Pennisetum purpureum (napier grass var. Bana) supplemented with
leucaena forage in the lowlands semi-humid tropics. Anim. Prod. 55, 331-337.
Poppi, D., and McLennan, S. 1995. Protein and energy utilization by ruminants at pasture. J. Anim. Sci. 73: 278-290.
6$66WDWLVWLFDO$QDO\VLV6\VWHP,QVWLWXWH6$6,QVWLWXWH,QF9HUVLyQ&DU\1&86$
7\UUHOO+)DQG-75HLG3UHGLFWLRQRIWKHHQHUJ\YDOXHRIFRZ¶VPLON-'DLU\6FL
Yan, T., Gordon, F.J., Agnew, R.E., Porter, M.G. and Patterson, D.C. 1997. The metabolisable energy requeriment
IRUPDLQWHQDQFHDQGWKHHI¿FLHQF\RIXWLOL]DWLRQRIPHWDERVDEOHHQHUJ\IRUODFWDWLRQE\GDLU\FRZVRIIHUHGJUDVV
silage-based diets. Lives. Prod. Sci. 51,141-150.

122 Energy and protein metabolism and nutrition in sustainable animal production
0LON\LHOGDQGFRPSRVLWLRQDQGHI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROLVDEOH
energy for lactation by Pelibuey ewes
J.C. Espinoza-Hernández, A.J. Ayala-Burgos, C.F. Aguilar-Pérez, J.G. Magaña-Monforte and J.C. Ku-Vera
Department of Animal Nutrition, Faculty of Veterinary Medicine and Animal Science, University of
<XFDWDQ&30HULGD<XFDWDQ0H[LFR[email protected]

Introduction
In the tropical regions of Mexico the main maternal breed used by sheep producers is the Pelibuey.
7KHUHLVOLWWOHVFLHQWL¿FLQIRUPDWLRQUHODWLYHWRPLONSURGXFWLRQHQHUJ\UHTXLUHPHQWVDQGHI¿FLHQF\RI
utilization of metabolisable energy for milk production at different physiological stages by the Pelibuey
ewe. Nonetheless, the ewe is the main component of the production of lambs (weaners) for fattening
under commercial, practical conditions. This work was carried out to address the above mentioned issues.

Material and methods

The experiment was carried out at the Faculty of Veterinary Medicine and Animal Science, University
of Yucatan, located in South Mexico. Twenty-one multiparous (2-3 lambings), lactating Pelibuey
HZHV “NJ \HDUVROGDQGUHDULQJWZRODPEVHDFKZHUHXVHG(ZHVZHUHUDQGRPO\DOORWWHG
to three treatments of seven ewes each in a completely randomized design, housed in individual
VWDOOVDQGIHGWKUHHOHYHOVRI0(LQWDNH/RZ /0-NJ0.75/day), Medium (M; 1.12 MJ/kg0.75/
GD\ DQG+LJK +0-NJ0.75GD\ GXULQJGD\VDVIURPWKH¿IWKGD\SRVWODPELQJ0LON
yield measurements were carried out during two non-consecutive days each week according to the
WHFKQLTXHRIWKHGRXEOHZHLJKLQJRIODPEV/DPEVZHUHVHSDUDWHGIURPWKHLUGDPV DWK 
minutes before measurements of milk yield, which were carried out ten times during 24 h. Lambs
were allowed to suck their dams for ten minutes to make sure the udder was completely emptied. Milk
was sampled every two non-consecutive days each week obtaining a 100 ml sample per ewe during
WKUHHGDLO\PDQXDOPLONLQJV DQGK )RUFKHPLFDODQDO\VLVRIPLONVDPSOHVZHUH
mixed per treatment per week and a 200 ml aliquot was obtained for fat analysis by the Gerber method
E\/LQJ  SURWHLQZDVHTXLYDOHQWWRWLPHV1FRQFHQWUDWLRQLQPLONDQGZDVGHWHUPLQHG
by the Kjeldahl method, lactose was assayed with a colorimetric technique by copper reduction.
Milk (100 ml) was freeze-dried for DM and GE determination with an adiabatic bomb calorimeter.
(I¿FLHQF\RIXWLOL]DWLRQRI0(IRUODFWDWLRQ kl) was determined with the equation described by Yan
et al.  'DWDRIPLON\LHOGHQHUJ\EDODQFHDQGkl were analyzed as a completely randomized
GHVLJQXVLQJDQDO\VLVRIYDULDQFHDQGWKH7XNH\WHVWZDVXVHGZKHQVLJQL¿FDQWGLIIHUHQFHVDPRQJ
treatments were detected.

Results and discussion

Mean milk yield showed statistical difference (P<0.05) between treatments M and H from L which
may have been due to the effect of ME intake. Fat concentration in milk was not affected (P>0.05)
between treatments. Concentration of crude protein showed difference between treatments, treatment
M gave the lowest concentration (g/kg) relative to treatments H and L. Lactose and GE concentrations
in milk showed statistical differences (P<0.05) between treatments L and M relative to H.

Energy balance was higher (P< IRUWUHDWPHQW$  UHODWLYHWR0  (I¿FLHQF\RI
utilization of ME for lactation was higher (P< IRUWUHDWPHQW0  WKDQIRUWUHDWPHQW$
(0.595). It is concluded that there is a positive relationship between ME intake and milk yield by
Pelibuey ewes rearing twins. However, it seems that there is an inverse relationship between ME
intake and concentrations of fat, protein, lactose and gross energy in milk. ME intake affects the
HI¿FLHQF\RIXWLOL]DWLRQRI0(IRUODFWDWLRQLQWKH3HOLEXH\HZH

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 123
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_34, © Wageningen Academic Publishers 2013
7DEOH0HDQVDQGVWDQGDUGHUURUV 6( IRUOLYHZHLJKW0(LQWDNHPLONSURGXFWLRQDQGFRPSRVLWLRQ
DQGHI¿FLHQF\RI0(XWLOL]DWLRQIRUODFWDWLRQE\3HOLEXH\HZHVIHGWKUHHOHYHOVRI0(

Treatments1 SE

Low Medium High Low Medium High

Live weight
Initial (kg) a 48.14a 43.14a 2.44 2.09 2.89
Weight change (g/d) -185b -139b 58a 0.024 0.024 0.017
Intake
DM intake, (g/kg0.75/d) 49.94c b 149.18a 0.148 0.251 0.757
ME intake (MJ/d) 9.24c 19.92b 27.30a 0.389  1.352
Means of milk yield and composition
Milk yield (kg/d) b a 1.74a 0.137 0.081 0.048
Milk fat (g/kg) a a a 4.239 0.957 1.317
Milk protein (g/kg) a 42.00b 47.00a  0.871 0.849
Milk lactose (g/kg) 43.35a 43.72a 41.23b 0.923 0.528 0.748
Gross energy of milk (MJ/kg) 4.97a 4.25b 4.13b 0.045 0.045 
(I¿FLHQF\RI0(XWLOL]DWLRQIRUODFWDWLRQ
Energy balance (MJ/d) - 0.041b 4.088a - 0.453 0.315
)HHGFRQYHUVLRQHI¿FLHQF\2 - 0.902a 0.702b - 0.058 0.054
*URVVHQHUJHWLFHI¿FLHQF\3 - 0.342a 0.257b - 0.017 0.017
kl 4 - a 0.595b -  0.003

1 a,b,c,: P<0.05 in the same row.

2)HHGFRQYHUVLRQHI¿FLHQF\PLON\LHOGWRWDO'0LQWDNH
3*URVVHQHUJHWLFHI¿FLHQF\JURVVHQHUJ\FRQWHQWRIPLON0(LQWDNH
4 klHI¿FLHQF\RIXWLOL]DWLRQRI0(IRUODFWDWLRQ

Reference
<DQ7&60D\QH7:-.HDG\DQG5($JQHZ(IIHFWVRIGDLU\FRZJHQRW\SHZLWKWZRSODQHVRIQXWULWLRQ
on energy partitioning between milk and body tissue. J. Dairy Sci. 89, 1031-1042.
/LQJ(5$7H[WERRNRI'DLU\&KHPLVWU\9ROWKHG&KDSPDQDQG+DOO/RQGRQ8.

124 Energy and protein metabolism and nutrition in sustainable animal production
3URWHLQWXUQRYHUDQGLQIUDUHGWKHUPRJUDSK\LQ1HOORUHEXOOVFODVVL¿HG
for residual feed intake
M.L. Chizzotti1,2, D. Heiderich2, W.L.B. Aziani2, M.M. Ladeira1,2, E.E.L. Valente2, T. Yanagi Junior,
F.H.M. Chizzotti1, L. Schiassi and D. Lourençoni
1,QVWLWXWR1DFLRQDOGH&LrQFLDH7HFQRORJLDHP&LrQFLD$QLPDO'=28)99LoRVD
MG, Brazil
28QLYHUVLGDGH)HGHUDOGH/DYUDV'HSDUWPHQWRI$QLPDO6FLHQFH'=28)/$/DYUDV
MG, Brazil; PDULRFKL]]RWWL#G]RXÀDEU
8QLYHUVLGDGH)HGHUDOGH/DYUDV'HSDUWPHQWRI$JULFXOWXUDO(QJLQHULQJ'=28)/$
Lavras, MG, Brazil

Introduction
5HVLGXDOIHHGLQWDNH 5), WKHGLIIHUHQFHEHWZHHQDFWXDODQGH[SHFWHGLQWDNHLVDQHI¿FLHQF\WUDLW
that is independent of BW and BW gain. The RFI might be related to a lower protein turnover and a
ORZHUKHDWSURGXFWLRQLQHI¿FLHQWDQLPDOV $UWKXUet al., 2004). The aim of this study was to evaluate
the relationship of RFI with protein turnover and temperature of eyes and skin.

Material and methods

Seventeen Nellore bulls with average body weight of 378 kg were used. The experimental period was
GD\V7KHEXOOVUHFHLYHGD&3DQG0FDORI'(NJRI'0GLHW FKRSSHGHOHSKDQW
JUDVVDQGFRQFHQWUDWH 'U\PDWWHULQWDNHZDVPHDVXUHGGDLO\7KHEXOOVZHUHFODVVL¿HGLQ
low (RFI<-0.5 SD below the mean, n=4), medium (RFI between 0.5 SD the mean, n=9) and high
RFI (RFI>0.5 SD above the mean, n=4) according to Koch et al.  7KHSURWHLQWXUQRYHUZDV
evaluated from urinary excretion of 3-methylhistidine (3-MH). A total urine collection was performed
during 5-d, using funnels adapted to the animals. The 3-MH in urine was determined by HPLC as
ZHOODV7RWDO.MHOGDKO1LWURJHQ2QGD\RIWKHH[SHULPHQWDOSHULRGGHWHUPLQDWLRQVRIH\HDQGVNLQ
temperatures were obtained by infrared thermal image (Fluke Ti 55Ft, Fluke Corporation, USA) and
the pictures were analyzed using the software SmartView 3.0. The maximum eye temperature was
considered as the maximum value into the eyeball and the average eye temperature was considered
as the average temperature in all eyeball area. Similarly, the maximum skin temperature was the
maximum temperature in a similar eye area projected under the eyes and the average skin temperature
was the average temperature in this area. The data were analyzed using GLM and REG procedures
RI6$6  DGRSWLQJVLJQL¿FDQFHOHYHORI

Results and discussion

Table 1 reports the RFI and urinary excretion of N and 3-MH. It was observed a lower (3 0.045)
XULQDU\H[FUHWLRQRI0+ LQPJNJRI%:GD\ LQHI¿FLHQWEXOOV ORZ5), 7KHFRUUHODWLRQEHWZHHQ
RFI value (kg of DM/day) and 3-MH excretion (mg/day) was of 0.45 (3 0.074), indicating that
WKHUHLVDWHQGHQF\RIORZHUPXVFXODUSURWHLQGHJUDGDWLRQLQPRUHHI¿FLHQWEXOOV

There were no differences (P>0.40) on skin or eyeball temperatures among RFI classes. However, it
was found a moderate relation between RFI and maximum eye temperature (r=0.51, 3 0.05) (Figure
1). Low correlations were observed between RFI and maximum skin temperature (r=-0.09, 3 0.73),
average skin temperature (r=0.08, 3 0,77) and average eye temperature (r=0.33, 3 0.20). Heat
LQFUHPHQWPD\FRQWULEXWHWRDQLPDOLQHI¿FLHQF\WKXVDKLJKHUKHDWSURGXFWLRQVKRXOGEHH[SHFWHG
in higher RFI (Montanholi et al., 2009).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 125
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_35, © Wageningen Academic Publishers 2013
7DEOH5HVLGXDOIHHGLQWDNH 5), DQGGDLO\XULQDU\H[FUHWLRQRIPHWK\OKLVWLGLQH 0+ DQG
WRWDO1LQ1HOORUHEXOOVFODVVL¿HGIRU5),

RFI group SEM P-value

Low Medium High

RFI, kg DM/day a b 0.725c 0.103 <0.0001

3-MH, mg/day 97.3a 110.2b 147.4b 13.7 
3-MH,g/kgBW/day 0.201 a 0.232 b 0.312b 0.029 0.0452
N, g/day 215.5 191.0 195.8  0.9283
N,g/kgBW/day 0.432 0.404  0.115 0.9797

a,b,c superscript letters differ at P<0.05.

Figure 1. Relationship between the maximum eye temperature and the residual feed intake in Nellore
bulls.

In conclusion, low RFI Nellore bulls present a lower excretion of 3-MH excretion (protein turnover).
7KHPD[LPXPH\HWHPSHUDWXUHLVPRUHUHODWHGWRIHHGHI¿FLHQF\WKDQWKHDYHUDJHWHPSHUDWXUHRI
eye or skin.

References
$UWKXU3)-$$UFKHUDQG50+HUG)HHGLQWDNHDQGHI¿FLHQF\LQEHHIFDWWOHRYHUYLHZRIUHFHQW$XVWUDOLDQ
UHVHDUFKDQGFKDOOHQJHVIRUWKHIXWXUH$XVWU-([S$JU
.RFK50/$6ZLJHU'&KDPEHUVDQG.(*UHJRU\(I¿FLHQF\RIIHHGXVHLQEHHIFDWWOH-$QLP6FL

Montanholi, Y.R., K.C. Swanson, F.S. Schenkel, B.W. McBride, T.R. Caldwell, S.P. Miller, 2009. On the determination
RIUHVLGXDOIHHGLQWDNHDQGDVVRFLDWLRQVRILQIUDUHGWKHUPRJUDSK\ZLWKHI¿FLHQF\DQGXOWUDVRXQGWUDLWVLQEHHI
bulls. Liv. Sci. 125, 22-30.
SAS, 2003. Release 9.2. SAS Inst. Inc., Cary, NC, USA.

126 Energy and protein metabolism and nutrition in sustainable animal production
Differences in residual feed intake are largely explained by changes in
body composition
M.L. Nascimento1, A.R.D.L. Souza2, A.S. Chaves1, S.R. de Medeiros, R.R. Tullio, M.M. de Alencar,
A.N. Rosa and D.P.D. Lanna1
1University of Sao Paulo/ESALQ, Piracicaba, SP, Brazil; [email protected]
2Universidade Federal Rural de Pernambuco, Recife, PE, Brazil
Embrapa Beef Cattle, Campo Grande-MS, Brazil
Embrapa Cattle Southeast, Sao Carlos-SP, Brazil

Introduction
*HQHWLFVHOHFWLRQRIHI¿FLHQWDQLPDOVFDQUHVXOWLQORZHUFRVWVUHGXFHGHQYLURQPHQWDOLPSDFWDQG
LQFUHDVHGSUR¿WDELOLW\7KHDVVRFLDWLRQVEHWZHHQIHHGHI¿FLHQF\VHOHFWLRQDQGSHUIRUPDQFHDUH
known, however the possible impacts on body composition of Nellore are still unknown. Residual
IHHGLQWDNH 5), GH¿QHGDVWKHGLIIHUHQFHEHWZHHQREVHUYHGDQGSUHGLFWHGLQWDNH SUHGLFWHGIURP
metabolic body weight and daily gain) (Koch et al KDVEHHQSURSRVHGDVDQLQGH[IRUJHQHWLF
selection, however, differences in body composition have been pointed out as a limiting factor in
the use of RFI for cattle selection (Herd and Pitchford, 2011). The objective of this work was to
examine the relationship between RFI and body composition in Nellore steers.

Material and methods

Dataset from three trials in which the animals had similar nutritional history (grazing systems) were
used, totaling 570 steers from 40 bulls (n=341 in trial 1; n=158 in trial 2; n=71 in trial 3). The animals
were fed twice daily with 5% of orts. The diets contained corn silage (trial 1, 40% DM) or sorghum
silage (trial 2 and 3, 40 and 20% DM, respectively); 13,5 (trial 1), 15,4 (trial 2) and 13,2% CP (trial
  WULDO  WULDO DQG0FDO(0SHUNJ'0 WULDO ,QGLYLGXDO'0,ZDVPHDVXUHG
for at least 70 d, considered the minimum period to assess RFI in beef cattle (Arthur, 2001), and
body weight (BW) taken every 14 d.

Fat thickness (FT) and ribeye area (RA) were taken by ultrasound measurements on the 11-12th-rib
DWWKHVWDUWPLGGOHDQG¿QDORIWKHGSHULRG)LQDODQGLQLWLDOHPSW\%:HQHUJ\ZHUHHVWLPDWHGE\
DQHTXDWLRQIRU1HOORUH (%:(QHUJ\ î5$î)752=0.73), the retained
energy was calculated by difference. To estimate the proportion of protein and fat in the gain,
the composition of the fat-free dry matter was held constant. Intramuscular ether extract of the
Longissimus samples were determined.

Average gaily gain (ADG) was estimated by regression between BW and days on feed. RFI was
computed by regression of DMI on mid-test BW0.75 and ADG using mixed models, where effect of
contemporary group (CG) based on feedlot location, year, animal origin and pen type (individual
RUFROOHFWLYH ZDVFRQVLGHUHGUDQGRP7KHDQLPDOVZHUHFODVVL¿HGDVORZPHGLXPDQGKLJK5),
(mean±0.5 SD). High and low classes were compared by MIXED procedure (SAS, 2008) where RFI
FODVVDQG&*ZHUHFRQVLGHUHG¿[HGHIIHFWVVLUHDVUDQGRPDQGLQLWLDODJHDVDFRYDULDWH

Results and discussion

As expected, BW and ADG were not related to RFI class (P>  7DEOH 7KHHI¿FLHQWDQLPDOV
FRQVXPHGOHVVWKDQWKHLQHI¿FLHQWRQHV P<0.05). There was no relationship between RFI
class and ribeye area (P> +RZHYHUHI¿FLHQWDQLPDOV ORZ5), KDGORZHU¿QDOIDWWKLFNQHVV
and intramuscular fat (P< 7KHJUHDWHUHI¿FLHQF\RIWKHDQLPDOVZLWKOHDQHUERG\FRPSRVLWLRQ
is related to better utilization of energy intake for muscle deposition compared to adipose tissue. For

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 127
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_36, © Wageningen Academic Publishers 2013
Table 1. Performance and body composition of Nellore steers with low and high RFI.

Variable RFI classes (N) SE P-value

Low (174) High (187)

RFI, kg/d  +0.985 0.032 <0.0001

Initial BW, kg  349.7 3.42 0.4012
Final BW, kg 439.4 435.9 4.13 
Gain, kg/d 1.18  0.025 0.3759
Feed intake, kg/d 7.77 9.22 0.093 <0.0001
F:G Ratio, DMI/gain 7.11 8.33  <0.0001
Initial ribeye area1, cm2 50.8 50.5 0.592 
Final ribeye area1, cm2    0.9287
Ribeye area change1, cm2 9.25 9.52  
Initial fat thickness1, mm 2.83 2.91 0.095 0.4015
Final fat thickness1, mm 4.88 5.29 0.159 0.0089
Fat thickness change1, mm 2.18  0.132 0.0007
Retained energy, Mcal/d  4.00 0.140 0.0054
% fat of the gain 29.0 32.5 1.735 0.0411
% protein of the gain  14.8 0.382 0.0425
Intramuscular fat, %  3.10 0.458 0.0008

1 Measured by ultrasound.

each gram of protein deposited in muscle tissue, three grams of water are also deposited resulting in
a greater amount of tissue for the same amount of energy intake with lower energy content (Lofgreen
DQG*DUUHW 

8VLQJRXUERG\FRPSRVLWLRQHTXDWLRQZHHVWLPDWHGWKDWHI¿FLHQWDQLPDOVUHWDLQHG0FDOOHVV
energy/day (P< &RQVHTXHQWO\HPSW\ERG\JDLQFRPSRVLWLRQZDVGLIIHUHQWZKHUHHI¿FLHQW
animals had less fat and more protein (P< $VVXPLQJWKHVDPHHVWLPDWHGHI¿FLHQF\RIXVH
of the ME for gain (0.442) according to NRC (1984), the change in estimated body composition,
explained 22.2% of the 1.45 kg/d difference in feed intake between high and low RFI classes. This
effect is greater than the 5% suggested in the literature based on regression analysis.

References
Arthur, P.F.; Archer, J.A.; Johnston, D.J.; Richardson, E.C.; Parnell, P.F. Genetic and phenotypic variance and covariance
FRPSRQHQWVIRUIHHGLQWDNHIHHGHI¿FLHQF\DQGRWKHUSRVWZHDQLQJWUDLWVLQ$QJXVFDWWOH-$QLPDO6FLHQFH
p. 2805-2811, 2001.
+HUG503LWFKIRUG:65HVLGXDOIHHGLQWDNHVHOHFWLRQPDNHVFDWWOHOHDQHUDQGPRUHHI¿FLHQW5HFHQW$GYDQFHV
in Animal Nutrition, Armidale, v.18, p. 45-58, 2011.
.RFK506ZLQJHU/$&KDPEHUV'*UHJRU\.((I¿FLHQF\RIIHHGXVHLQEHHIFDWWOH-$QLPDO6FLHQFH
S
/RIJUHHQ*3DQG:1*DUUHWW$V\VWHPIRUH[SUHVVLQJQHWHQHUJ\UHTXLUHPHQWVDQGIHHGYDOXHVIRUJURZLQJ
DQG¿QLVKLQJEHHIFDWWOH-$QLPDO6FLS
1DWLRQDO5HVHDUFK&RXQFLO1XWULHQWUHTXLUHPHQWVRIEHHIFDWWOHWKHG:DVKLQJWRQ1DWLRQDO$FDGHPLF3UHVV
1984. 90 p.
SAS, 2008. Release 9.2. SAS Inst. Inc., Cary, NC, USA.

128 Energy and protein metabolism and nutrition in sustainable animal production
Net protein requirement of pregnancy of goats with single and twin
pregnancy
C.J. Härter, D.S. Castagnino, L.D. Lima, A.R. Rivera, H.G.O. Silva, K.T. Resende and I.A.M.A.
Teixeira
Faculdade de Ciências Agrárias e Veterinárias, UNESP, Univ Estadual Paulista, Department of
$QLPDO6FLHQFHV-DERWLFDEDO63%UD]LO[email protected]

Introduction
Pregnancy is an important stage in the animal life, because many transformations occur in the whole
body of the pregnant female. However, there is lack of information on nutritional requirements over
the whole pregnancy. Most protein recommendations for goats were obtained from data extrapolated
from cattle and sheep (Sutton and Alderman, 2000). Although ruminants have similar metabolism,
LWLVNQRZQWKDWWKHUHDUHGLIIHUHQFHVEHWZHHQVSHFLHVHVSHFLDOO\UHJDUGLQJWKHHI¿FLHQF\RIIHHG
utilization and body composition. Therefore, the objective of this experiment was to determine net
protein requirement of Oberhasli and Saanen goats with singleton and twin pregnancy.

Material and methods

$WRWDORI2EHUKDVOL “NJ%: DQG6DDQHQ “NJ%: ZHUHXVHG)HPDOHV
of each breed were randomly distributed among the treatments according to the number of fetuses (1
or 2) and gestation period (80, 110 and 140 days), in a completely randomized design with a 2×2×3
factorial arrangementof treatments. The animals were fed ad libitum with a diet formulated to meet
WKHLUUHTXLUHPHQWVDFFRUGLQJWR15&  :KHQWKHIHPDOHJRDWVUHDFKHGWKHVSHFL¿FJHVWDWLRQDO
age they were slaughtered and then there was the withdrawal of the pregnant uterus, mammary
gland and all organs and structures were withdrawn. The entire gravid uterus was weighed and then
VHSDUDWHGLQWRHPSW\XWHUXV XWHUXVSODFHQWDSODFHQWRPHV SODFHQWDOÀXLGDQGIHWXVHVZKLFKZHUH
weighed separately. The mammary gland was weighed after removing the skin. These components
HPSW\XWHUXVSODFHQWDOÀXLGIHWXVHVDQGPDPPDU\JODQG ZHUHJURXQGDQGWKHLUVDPSOHVZHUH
freeze-dried for determination of the contents of dry matter, ash, fat according to AOAC (1995) and
protein by Dumas combustion method (LECO FP-528 LC). The results of growth of gravid uterus
DQGPDPPDU\JODQGZHUH¿WWHGLQWKHQRQOLQHDU*RPSHUW]PRGHO

Y = Wbirth × e-e(ln(-ln(Win/Wbirth)) – b × days of pregnancy)) (1)

using the NLIN procedure of SAS 9.2 (2009) and the daily requirements were found by the
differentiation of the Gompertz :

y = Y × b × ln(Wbirth/Y) (2)

Where: Y = protein accretion (g); Wbirth = weight of protein at birth (g); Win = weight of protein
at beginning of pregnancy (g); e = 2.718282; b = maximum rate of deposition of protein (g/day); y
= protein accretion in g per day.

Results and discussion

7KHSDUDPHWHUVRIWKHHTXDWLRQVWRSUHGLFWWKHSURWHLQDFFUHWLRQLQWKHIHWXVSODFHQWDOÀXLGXWHUXV
and mammary gland are presented in Table 1. In both types of pregnancy, Oberhasli goats presented
heavier fetuses and greater protein accretion throughout pregnancy than Saanen goats. The total
2EHUKDVOLIHWXVHVPDVVDWGD\VRISUHJQDQF\ZDVNJDQGNJLQVLQJOHDQGWZLQSUHJQDQF\
UHVSHFWLYHO\DQGWRWDO6DDQHQIHWXVHVPDVVZDVNJDQGNJLQVLQJOHDQGWZLQSUHJQDQF\

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 129
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_37, © Wageningen Academic Publishers 2013
UHVSHFWLYHO\3URWHLQDFFUHWLRQLQ2EHUKDVOLZDVDQGJZLWKVLQJOHSUHJQDQF\DQG
DQGJZLWKWZLQSUHJQDQF\DWDQGGD\VRISUHJQDQF\UHVSHFWLYHO\
2QWKHRWKHUKDQGSURWHLQDFFUHWLRQLQ6DDQHQZDVDQGJZLWKVLQJOHSUHJQDQF\
DQGDQGJZLWKWZLQSUHJQDQF\DWDQGGD\VRISUHJQDQF\$IWHU
days of pregnancy net protein requirement of female goats with twin pregnancy (g/day) is higher
compared to female goats with single pregnancy. Net protein requirements of Oberhasli goats with
VLQJOHSUHJQDQF\ZHUHJGD\DWDQGGD\VRISUHJQDQF\UHVSHFWLYHO\
and for Oberhasli goats with twin pregnancy were 4.5, 22.1 and 24.0 g/day at 80, 110 and 140 days
of pregnancy, respectively. Net protein requirements of Saanen goats with single pregnancy were
DQGJGD\DQGZLWKWZLQSUHJQDQF\DQGJGD\DWDQG
days of pregnancy, respectively. The litter size determines the largest fraction of the requirements
during pregnancy. In conclusion the Oberhasli goats with twin pregnancy show greater net protein
requirement for pregnancy.

Table 1. Parameters generated by nonlinear equation of Gompertz for estimating net protein
UHTXLUHPHQWV J RISUHJQDQWGDLU\JRDWV

Total protein Rate of protein Protein mass at beginning P-value

mass at birth (g)1 deposition (g/day of pregnancy (g)

Oberhasli with singleton pregnancy

Fetus 1,100 “ 3.94×10-57±4.34×10-55 <0.0001
3ODFHQWDOÀXLG  0.0428±0.0145 3.05×10-29±3.29×10-27 <0.0001
Uterus 500 0.0118±0.0049 14.48±27.94 <0.0001
Mammary gland 200 “ “ <0.0001
Oberhasli with twin pregnancy
Fetus  0.0408±0.0057 5.28×10-72“î-70 <0.0001
3ODFHQWDOÀXLG 50 0.0314±0.0091 2.37×10-14“î-13 <0.0001
Uterus 400 “ 11.30±12.99 <0.0001
Mammary gland 300 0.0145±0.0052 40.27±29.18 <0.0001
Saanen with singleton pregnancy
Fetus 900 0.0375±0.007 3.35×10-53±3.88×10-51 <0.0001
3ODFHQWDOÀXLG 30 0.0292±0.0128 4.17×10-4“ <0.0001
Uterus 200 0.0284±0.0173 10.28±40.70 <0.0001
Mammary gland 205 “ 19.75±78.12 <0.0001
Saanen with twin pregnancy
Fetus 1000 0.0412±0.003 5.30×10±2.54×10 <0.0001
3ODFHQWDOÀXLG 20 0.0411±0.0210 9.78×10-07±2.7×10-05 <0.0001
Uterus 700 0.0117±0.0033 10.49±15.54 <0.0001
Mammary gland 1,200 “ “ <0.0001

1 Value set in the equation to predict the rate of protein deposition and protein mass at beginning of pregnancy.

References
$2$&$VVRFLDWLRQRI2I¿FLDO$QDO\WLFDO&KHPLVWV2I¿FLDO0HWKRGVRI$QDO\VLVth ed. Washington, D.C.
National Research Council. 2007. Nutrient requirements of small ruminants. Sheep, Goats, Cervids and New World
Camelids. National Academy Press. Washington, DC.
Sutton, J. D., Alderman, G. 2000. The energy and protein requirements of pregnant and lactating dairy goats the
$JULFXOWXUHDQG)RRG5HVHDUFK&RXQFLOUHSRUW/LY3URG6FL

130 Energy and protein metabolism and nutrition in sustainable animal production
Effect of gender on net energy and protein requirements for growth of goats
A.K. de Almeida, S.P. Silva, D.S. Costa, M.H.M.R. Fernandes, I.A.M.A. Teixeira and K.T. Resende
Department of Animal Science, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil;
[email protected]

Introduction
Currently, the world is facing challenges related to environmental problems, global warming, and
demography. In this context, goats may take part on an important socio-economic role with its
potential to use marginal land. In addition, these animals adapt easily to various climatic zones,
produce under different systems of husbandry, and successfully convert feed into protein sources.
However, it is crucially important to understand this species’ nutritional requirements, that are
determined by several factors such as management, breed, environment, gender, age and others.
Nutritional requirements for goats have been based on limited data, and are sometimes calculated
by extrapolation of other species needs (ARC, 1980; NRC, 1981). We propose to investigate
Saanen goats, from 30 to 45 kg of body weight (BW), the effect of gender on net protein and energy
requirements for growth.

Material and methods

The experiment was conducted at the Goat facility of Univ Estadual Paulista/Jaboticabal from
-XQHRIWR$SULO:HXVHG6DDQHQJRDWVDPRQJFDVWUDWHGPDOHV Q  LQWDFWPDOHV
(n=14) and females (n=18), of 30.0±1.09 kg of initial body weight and age of 258±53 days. These
animals were randomly allocated into three slaughter groups, conforming body weights, 30, 37.5
and 45 kg. The experimental diet was formulated to meet the species’ nutritional requirements
according to NRC (2007). We offered a mixed diet consisted of dehydrated corn, cracked corn grain,
soybean meal, soybean oil, limestone, and a mineral supplement. The average dry matter intake
during the experiment was 1,030±90 g/d, which corresponded to 2.89% of BW. At slaughter, kids
were stunned by an electric shock, followed by severing of the jugular vein and carotid artery. All
blood was collected and weighed. The digestive tract was weighed before and after it was emptied
in order to determine the empty body weight EBW of animals. The EBW was calculated by BW at
slaughter minus the weight of the contents of the digestive tract, gallbladder and vesicle. The whole
HPSW\ERG\ZDVLQLWLDOO\IUR]HQDWƒ&DQGWKHQFXWLQWRVPDOOSLHFHVDQGJULQG$IWHUJULQGLQJDQG
homogenization, samples were freeze dried for DM determination and performed analysis for fat,
protein and energy contents. The experimental design was completely randomized in a 3×3 factorial
DUUDQJHPHQWZKHUHJHQGHUDQGERG\ZHLJKWZHUHDVVXPHGDV¿[HGHIIHFWVDQGWKHHUURUZDVLLG
1 ı2ij 7RREWDLQHVWLPDWHVRIJDLQFRPSRVLWLRQZHGLYLGHGWKHFDOFXOXVLQSKDVHV7KH¿UVWSKDVH
consisted in obtaining the allometric equations to predict protein and energy concentration from
EBW, according to ARC, 1980, as follows: Log10 (protein and energy contents) = a + [b × log10
(EBW, kg)]; where component amount is protein and energy contents in the EBW. In the second
phase, the equation was differentiated to compute the estimates of gain composition at various EBW:
[Component] = b × 10a × EBW(b-1), where [Component] is protein and energy contents per unit of
empty body weight gain (EWG) (g/kg of gain or kJ/kg of gain). We used the MIXED procedure
(SAS Inst. Inc., Cary, NC, USA), to obtain allometric equations, the CONTRAST option to verify
difference among gender, and the ESTIMATE option to give us the overall intercept and slope.

Results and Discussion

7RRXUNQRZOHGJHRXUVWXG\LVWKH¿UVWWRGHWHUPLQHDQGFRPSDUHHQHUJ\DQGSURWHLQUHTXLUHPHQWVIRU
JURZWKDPRQJIHPDOHPDOHDQGFDVWUDWHGPDOHJRDWVRIWKH6DDQHQEUHHGLQWKH¿QDOSKDVHRIJURZWK
We observed that as animals grow, the content of energy and fat increases, our data presented in Table

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 131
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_38, © Wageningen Academic Publishers 2013
1 demonstrate that females tend to deposit more fat than males. We may also consider that animals
were at different proportion of mature weight: 55.5, 48.4 and 41.5 for females, castrate males and
intact males respectively. This may explain the huge difference in fat content in the EBW and ADG.

Table 2 shows that the proportion of energy increased as BW increased, however the proportion of
protein was nearly constant, suggesting that the fat was the main factor which increased body energy.
Although, this difference did not cause distinction among genders for net energy requirements on
JURZWK6LQFHZHGLGQRW¿QGDGLIIHUHQFHDPRQJJHQGHUVIRUSURWHLQDQGHQHUJ\FRQFHQWUDWLRQVD
general allometric equation was proposed.

$IWHUGLIIHUHQWLDWLRQWKHVHHTXDWLRQVWKHUHWDLQHGHQHUJ\UDQJHGIURPWR0-NJRI(:*
and net ranged from 177 to 182 g/kg of EWG as BW ranged from 30 to 45 kg. Therefore, the net
energy requirements corresponded with the NRC (2007) recommendations, but we have found lower
values for net protein requirements. We have found no differences among gender for Saanen goats
ranging from 30 to 45 kg of BW.

Table 1. Effect of gender on performance and body composition at slaughter for Saanen goats.

Variable Gender P-value

Female Castrate male Intact male

Water, % EBW 52.05±1.08c 57.79±1.02b “a <0.0001

Ash, % EBW 3.93±0.19 3.98±0.18 4.31±0.19 0.32
Protein, % EBW 17.55±0.59 17.08“ ±0.58 
Fat, % EBW 28.42±0.73a 22.41±b 17.14±0.72c <0.0001
ADG, g/d 74.2±7.1b “a “a 0.03

ab Distinct letters in the same row, differ at P<0.05 by least squares means for gender effect.

7DEOH$OORPHWULFHTXDWLRQVDQGUHODWLYHSURSRUWLRQRISURWHLQDQGHQHUJ\LQWKH%:DW
DQGNJ6DDQHQJRDWV

Variable1 Allometric equations Body weight (kg)

30 37 45 RMSE1

EBW,kg EBW, kg = 24.0  37.8 0.99

 “ > “ î%:NJ@
Protein, g/kg of EBW Logprotein, g =   171 0.03
 “ > “ î/RJ(%:NJ@
Energy, MJ/kg of EBW Logenergy, kJ = 10.4 11.9 13.5 0.04
3.24(±0.11) + [1.57(±0.07) × LogEBW, kg]

1506(LVURRWPHDQVTXDUHHUURU7KHHTXDWLRQVZHUHVLJQL¿FDQWP<0.0001.

Conclusion
We conclude that females represented greater fat accumulation in the carcass, although this difference
was not enough to cause distinct energy requirements for growth. Now we can consider that there
are no differences among gender for protein and energy requirements for growth of Saanen goats
at this phase.

132 Energy and protein metabolism and nutrition in sustainable animal production
Acknowledgements
FAPESP process number: 2010/02482-4.

References
Agricultural Research Council, 1980. ARC The nutrient requirement of ruminant livestock. London: 351p.
NRC, 2007. National Research Council. Nutrient requirement of small ruminant. Washington, 292 p.

Energy and protein metabolism and nutrition in sustainable animal production 133
Energy utilization for gain of goat kids
T.F.V. Bompadre, K.T. Resende, M.H.M.R. Fernandes, O. Boaventura Neto, A.N. Mendonca, B.
Biagioli and I.A.M.A. Teixeira
UNESP Universidade Estadual Paulista, Department of Animal Science, Via de Acesso Prof Paulo
'RQDWR&DVWHOODQHVQ-DERWLFDEDO6DR3DXOR%UD]LO[email protected]

Introduction
The total energy body content is based on protein and fat deposition in growing animals, which can
EHVWURQJO\LQÀXHQFHGE\JHQGHU,QWKLVUHJDUGWKHDFFUHWLRQUDWHRISURWHLQDQGIDWFRXOGEHKHOSIXO
IRUXQGHUVWDQGLQJWKHHI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROL]DEOHHQHUJ\ 0( WRQHWHQHUJ\ 1( 
for growth (kg 3UHYLRXVVWXGLHVLQFDWWOHKDYHIRXQGGLIIHUHQFHVLQWKHSDUWLDOHI¿FLHQF\RIXVHRI
metabolizable energy (ME) to net energy (NE) for growth between gender, which was 0.54 for bulls,
0.47 for steers and 0.54 for heifers (Chizzotti et al., 2007). However there is a lack of information
in this regard with goats. Therefore, this study was conducted to evaluate the effect of the gender
RQIDWDQGSURWHLQGHSRVLWLRQLQWKHJDLQDQGRQHQHUJ\HI¿FLHQF\IRUJURZWKLQ6DDQHQJRDWNLGV

Material and methods

A total of 48 Saanen goat kids (18 intact males, 12 castrated males and 18 females) with initial body
ZHLJKW %: RI“NJZHUHXVHG6L[QRQFDVWUDWHGPDOHVDQGIHPDOHVZHUHVODXJKWHUHGDW
beginning of the experiment (baseline animals) to estimate the initial body composition. The reminder
was randomly allocated into groups (blocks) of 3 animals of the same gender, subjected to 3 levels
of intake: ad libitum, 75 and 50% of ad libitum intake. The 75 and 50% of ad libitum intake levels
were determined daily, based on dry matter intake of the kids fed ad libitum on the previous day. The
entire group was slaughtered when the animal fed ad libitum reached 15.2±0.4 kg BW and 101±5
days old. Empty bodies were weighted, ground, mixed and sub sampled for chemical analyses (fat,
protein, ash and gross energy). Initial body composition was determined using equations developed
IURPWKHERG\FRPSRVLWLRQRIWKHEDVHOLQHJRDWNLGV /RIJUHHQDQG*DUUHW 'LHWDU\0(
estimated from a metabolism trial, was 7.8 MJ/kg DM. The data were analyzed using mixed models
of SAS (SAS Inst. Inc., Cary, NC).

Results and discussion

Table 1 shows body composition of intact male, castrated male and female Saanen goat kids. The
equation to predict rate of fat gain on the empty body weight gain (EWG) differed between genders
(3 0.02), showing that females had higher fat deposition than entire and castrated males goat kids
(Equation 1 and 2). On the other hand, rate of protein gain was not affected by gender (3  DQG
a general equation was proposed (Equation 3).

)DWJDLQ IHPDOHV  ““(:*506(  

)DWJDLQ PDOHVDQGFDVWUDWHGPDOHV  ““(:*506(  

Protein gain (all) = 1.57±0.88 + 0.22±0.01 EWG, RMSE = 2.38 (3)

where fat and protein gain, g/d; and EWG = empty weight gain, g/d.

The equations to predict percentage of fat (FIG) in the gain from retained energy concentration
(REc) also differed among genders (P<0.001), suggesting that retained energy as fat had the same

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 135
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_39, © Wageningen Academic Publishers 2013
pattern for females and castrated males and differed for males (Equation 4 and 5). The percentage
RISURWHLQ 3,*(TXDWLRQ LQWKHJDLQIURP5(FZDVQRWDIIHFWHGE\JHQGHU 3 0.10).

),* PDOHV  ““5(F506(  

FIG (females and castrated males) = -12.53±3.81 + 2.52±0.35 REc, RMSE = 2.44 (5)

3,* DOO  “±“5(F506(  

where FIG = fat in gain as % of EWG, PIG= protein in gain as % of EWG, REc= concentration of
retained energy, MJ/kg EWG.

'HVSLWHWKHJUHDWHUIDWJDLQRIIHPDOHVWKHHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQIRUJURZWK Ng), calculated

as the slope of regression of retained energy (KJ/kg0.75 of EBW) on ME intake (KJ/kg0.75 of EBW)
above maintenance, was not affected by gender (Equation 7).

5( ““0(,506(  

where RE= retained energy (KJ/kg0.75 of EBW) and MEI= ME intake (KJ/kg0.75 of EBW).

2XUVWXG\LQGLFDWHGWKDWWKHHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQIRUJURZWKGRHVQRWGLIIHUDPRQJJHQGHU
for Saanen goat kids, in early life.

Table 1. Effect of gender on body composition of Saanen kids.

Gender P-value

Male Castrated Female

Water, % EBW “ “ 59.92±0.85 0.74

Ash, % EBW “ 5.81±0.19 5.75±0.20 0.97
Protein, % EBW 25.79±0.47 25.11±0.55 24.39±0.59 0.18
Fat, % EBW “ 8.27±0.72 “ 0.29
Energy, MJ/kg EBW 9.05±0.21 8.79±0.25 9.02±0.29 0.70
1 EBW = empty BW.

Acknowledgements
FAPESP project number 2008/58351-5 and 2012/07177-0.

References
Chizzotti, M.L., S.C. Valadares Filho, L.O. Tedeschi, F.H.M. Chizzotti and G.E. Carstens, 2007. Energy and protein
requirements for growth and maintenance of F1 Nellore x Red Angus bulls, steers, and heifers. J. Anim. Sci. 85,
1971-1981.
/RIJUHHQ*3DQG:1*DUUHW$V\VWHPIRUH[SUHVVLQJQHWHQHUJ\UHTXLUHPHQWVDQGIHHGYDOXHVIRUJURZLQJ
DQG¿QLVKLQJEHHIFDWWOH-$QLP6FL

136 Energy and protein metabolism and nutrition in sustainable animal production
Protein requirements for growth of male and female Saanen goats kids
F.O.M. Figueiredo, R.F. Leite, M.M. Freire, M.H.M.R. Fernandes, K.T. Resende and I.A.M.A. Teixeira
Department of Animal Science, UNESP Universidade Estadual Paulista, Jaboticabal, SP, Brazil;
IHUQDQGD¿JGR#\DKRRFRPEU

Introduction
Gender plays an important role on deposition rates of different body tissues and due to the variation
in the composition of gain, net protein requirements can differ between males and females. Souza
et al. (2012) reported that bulls had greater protein requirements than castrated males, which were
greater than females at the same age because intact males showed greater lean tissue deposition
in the body than castrated males and females. In dairy goats, only a few studies dealing with the
effect of gender on protein requirements have been published. Thus, the objective of this study was
to determine the net protein requirements for growth of intact males, castrated males, and females
Saanen goat kids using comparative slaughter technique.

Material and methods

Net protein requirements for growth were obtained using 20 castrated males, 20 intact males and 18
IHPDOHV6DDQHQJRDWNLGVDWDQDYHUDJHLQLWLDOERG\ZHLJKW %: RI “ NJLQDFRPSOHWHO\
randomized design. The animals were fed ad libitum and slaughtered at targeted BW of 15, 23 and
NJ6HYHQFDVWUDWHGPDOHVLQWDFWPDOHVDQGIHPDOHVZHUHVODXJKWHUHGDWWKHEHJLQQLQJRIWKH
H[SHULPHQWDWDQDYHUDJH%:RI “ NJDQGDQLQLWLDODJHRI “ GD\V$QRWKHU
FDVWUDWHGPDOHVLQWDFWPDOHVDQGIHPDOHVZHUHVODXJKWHUHGZKHQWKH\UHDFKHG “ 
NJ%:DW “ GD\VRIDJH/DVWO\DQRWKHUDQLPDOVRIHDFKJHQGHUZHUHVODXJKWHUHG
ZKHQWKH\UHDFKHG “ NJ%:DW “ GD\VRIDJH

The experimental diet was formulated to meet or exceed the requirements of growing goat kids,
according to NRC (2007), and consisted of dehydrated, whole corn plant (Zea mays), cracked corn
grain, dehulled, solvent extracted soybean meal, soybean oil, limestone and a mineral supplement
IHGDVDWRWDOPL[HGGLHW'HK\GUDWHGFRUQSODQWVFRQVLVWHGRIZKROHFRUQSODQWV WRPRLVWXUH 
chopped when the kernel milk line was approximately two-thirds of the way down the kernel, and
dehydrated.

Protein body composition was estimated using direct chemical analyses of the whole empty body.
A factorial approach was used to estimate protein requirements for growth.

Simple linear regression analyses were conducted to express empty body weight (EBW) as a function
of BW. The protein content of animals was summarized by allometric regressions of the logarithm
of observed protein content on the logarithm of empty body weight:

LogProt = a + b × LogEBW (1)

where Log10Prot is the log of the total amount of body protein (g), Log10EBW is the log of empty
body weight (EBW, in kg), and a and b are regression parameters.

7KH¿UVWGHULYDWLYHRI (TXDWLRQ ZLWKUHVSHFW(%:\LHOGVHVWLPDWHVRIWKHFRPSRVLWLRQRIWKH

gain at various EBW:

NPg = b × 10a × EBW (b-1) (2)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 137
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_40, © Wageningen Academic Publishers 2013
where NPg is the net protein requirement for gain (g/ kg EBW gain).

All statistical analyses were conducted using the MIXED procedure of SAS, version 9.2.

Results and discussion

*HQGHUGLGQRWVLJQL¿FDQWO\DIIHFWWRWDOERG\SURWHLQ P<0.05). Therefore, only results aggregated
across genders are presented (Table 1).

7KH¿UVWGHULYDWLYHRIWKHHTXDWLRQIRU/RJ10Prot with respect to EBW yields estimates of net protein

requirements for growth:

NPg = 184.83 × EBW -0.03 (3)

From Equation 3, NPgGHFUHDVHGIURPWRJNJRI(%:JDLQEHWZHHQDQGNJ

EBW in Saanen goat kids. Although the decrease in NPg as EBW increases is small, it is in accord
with previous studies that showed a decrease in rate of protein deposition and an increase of rate of
fat deposition as body weight increases (Owens et al., 1993). We did not observe an effect of gender
on rate of EBW protein accretion between 15 and 30 kg BW in Saanen goats.

Table 1. Equations for estimating protein body composition of female, male and castrated male
6DDQHQJRDWNLGVEHWZHHQDQGNJRIERG\ZHLJKW1

Traits Regression equations BW2

15 23 30 RMSE3

Empty body EBW = 11.3 17.9 23.7 0.37

weight  “ > “ [%:NJ@
Total body Log10Prot = 177.2 174.8 173.3 0.01
protein  “ > “ [/RJ(%:NJ@

1 BW is body weight (kg); EBW is empty BW (kg); Log10Prot is the log of the total amount of body protein (g);
Log10EBW is the log of EBW (kg).
2 Values in the table are the predicted EBW and total body protein accretion rates for the 3 values of BW.
3 RMSE is root mean square error.

Acknowledgements
FAPESP project number 2008/58351-5 and 2012/07177-0.

References
1DWLRQDO5HVHDUFK&RXQFLO1XWULHQWUHTXLUHPHQWVRIVPDOOUXPLQDQWV6KHHSJRDWVFHUYLGVDQGQHZZRUOG
camelids. Washington, D.C.: National Academy Press.
Owens, F.N.; Dubeski, P.; Hanson, C.F., 1993. Factors that alter growth and development of ruminants. J. Anim. Sci.,
71:3138-3150.
Souza, E. J. O., Valadares Filho, S. C., Guim, A., Valadares, R. F. D., Marcondes, M. I., Ferreira, M. A., Prados, L. F.,
Benedet, P. B., 2012. Protein requirements for females of Nellore, Nellore × Angus and Nellore × Simmental fed
RQWZRIRUDJHFRQFHQWUDWHUDWLRV5HYLVWD%UDVLOHLUDGH=RRWHFQLD %UD]-$QLP6FL 

138 Energy and protein metabolism and nutrition in sustainable animal production
,QÀXHQFHRIGLIIHUHQWOLSLGVXSSOHPHQWVDQGIUHTXHQFLHVVXSSO\RQ
microbial protein synthesis in beef heifers
M.C.A. Santana1, T.T. Berchielli2, G. Fiorentini, R.A. Reis2, V.C. Modesto2 and G.T. Pereira2
1EMATER, Goiânia, GO, Brazil
2Department of Animal Science, Universidade Estadual Paulista, UNESP, Jaboticabal, Brazil;
[email protected]

Introduction
Increasing the energy density of the diet by adding lipid sources is a nutritional strategy that is used
IRU¿QLVKLQJFDWWOHZKLFKOHDGVWRVDWLVIDFWRU\SHUIRUPDQFH7KHXVHRIOLSLGVLQUXPLQDQWGLHWVKDV
EHHQUHFRPPHQGHGEHFDXVHE\UHGXFLQJPHWKDQRJHQHVLVWKHHQHUJ\HI¿FLHQF\LVLPSURYHG7KHUH
are several ways to provide lipids to the diet, which includes grain, oil and calcium salts. According
to Allen (2000) the main problem of using lipids rich in unsaturated fatty acids (UFAs) in diets for
UXPLQDQWVLVWKHLUHIIHFWRQLQWDNHDQGFRQVHTXHQWO\RQUXPLQDOGLJHVWLELOLW\RIWKH¿EURXVIUDFWLRQ
Also, diets rich in cereal grains cause a pH decrease in the rumen, which inhibits lipolysis and
biohydrogenation. To avoid the negative effects just described, the lipid supplement in the form of
protected fat has been recommended for ruminants because it is considered an inert ruminal fat source
+DUYDWLQHDQG$OOHQ +RZHYHUWKHXVHRIIDWVXSSOHPHQWDWLRQLVPRUHW\SLFDOIRUIHHGORW
cattle than for grazing cattle. In addition, there are only few data on effects of fat supplementation on
grazing cattle (Brokaw et al., 2001; Krysl et al., 1991). Likewise, reducing the fat supplementation
frequency for grazing cattle, due to great territorial extension in some cases, may enhance logistic
management in the farm. Thus, the objective of this study was to evaluate the effects of different
lipid sources supplemented daily or thrice weekly on microbial protein synthesis of grazing heifers.

Material and methods

The experiment was carried out during two months. The design used was completely randomized in
DîIDFWRULDODUUDQJHPHQWDQDO\]HGXVLQJ$129$6LJQL¿FDQFHZDVVHWDWP<0.05 and differences
among means were determined using a t-test. The lipid supplement treatments were, soybean grains
(SG), soybean oil (SO) and calcium salts (ML) (MEGALAC- E Arm & Hammer, Rio de Janeiro,
Brazil) and two supply frequencies; daily (D) or 3 days of week (Monday, Wednesday and Friday)
FDOOHGµDOWHUQDWH¶ $ 7KHVXSSOHPHQWVFRQWDLQHGRQDYHUDJHRIFUXGHSURWHLQRI
HWKHUH[WUDFWDQGRIQHXWUDOGHWHUJHQW¿EHU:LWKUHVSHFWWRWKHVXSSOHPHQWDWLRQIUHTXHQF\
animals fed daily were supplemented at 0.75% of BW, whereas animals fed thrice a week were
supplemented at 1.75% of BW. Spot urine samples were collected from six heifers 4 h after feeding,
HLWKHUZKHQKHLIHUVXULQDWHGVSRQWDQHRXVO\RUIROORZLQJYDJLQDOVWLPXODWLRQ7KHVDPSOHVZHUH¿OWHUHG
through 4 layers of cheesecloth and 50 ml was immediately frozen at -15 °C as stock material. One
aliquot (10 ml) of urine sample (by each animal) was diluted with 40 ml of 0.018 mM H2SO4 and
stored at -15 °C for subsequent analysis. Commercial kits were used to analyze urine for creatinine.
Allantoin and uric acid in urine samples were measured as described by Chen and Gomes, (1992).
7KHPLFURELDOHI¿FLHQF\ZDVREWDLQHGXVLQJWZRVHSDUDWHPHWKRGVWKHPLFURELDOSURGXFWLRQ &3
g/day) was divided by the TDN according to NRC (2001) calculation, or microbial production was
divided by the intake of fermentable organic matter in the rumen (CP/ MOFerm), noting that the
lipid fraction was disregarded from the MOFerm.

Results and discussion

Microbial N synthesis estimated by urinary purine derivative excretion was similar between lipid
VXSSOHPHQWVDQGVXSSO\IUHTXHQFLHVVWXGLHGDVVKRZQRQ7DEOH7KHHI¿FLHQF\RIPLFURELDO
V\QWKHVLVZDVQRWLQÀXHQFHGE\WKHIRUPRIWKHSURYLVLRQRIOLSLGVXSSOHPHQWDQGIUHTXHQFLHVRI

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 139
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_41, © Wageningen Academic Publishers 2013
7DEOH(IIHFWVRIOLSLGVVXSSOHPHQWVOHYHOVDQGVXSSO\IUHTXHQFLHVRQWKHPLFURELDO1 01 V\QWKHVLV
DQGHI¿FLHQF\RI01V\QWKHVLV (06 XVLQJXULQDU\SXULQHGHULYDWLYH 3' H[FUHWLRQ

Supplements and frequencies SEM

SG SO ML D A

Microbial N, g/d 54.07 57.82  53.78  1.59

Microbial CP, g/kgW0.75 4.91 4.58 4.44 4.53 4.70 0.11
TDN, kg/d  3.08   2.81 0.15
Intake, kg/d 5.5 5.9 5.4   0.15
RDTOM, kg/d 4.30  4.22 4.29 4.42 0.14
EMS, g CP/100 g TDN  12.80 13.47 12.91  0.94
EMS, g CP/kg RDTOM   80.88 78.91 81.11 4.08

SG = soybean grain; SO = soybean oil; ML = MEGALAC- E; D = daily; A = alternately; RDTOM = ruminally

degradable true OM.

supplementation, with mean values of 13.11 MCP g/100 g TDN, which is similar to the NRC (2001)
recommendation of 13 g of MCP/100 g of TDN.

7KHDYHUDJHVREVHUYHGIRUWKHLQJHVWLRQRIGU\PDWHULDOZHUHYHUL¿HGDWNJ'0GD\DQGIRU
IHUPHQWDEOHRUJDQLFPDWHULDONJ'0GD\ZKLOHWKHDSSDUHQWGLJHVWLELOLW\FRHI¿FLHQWZDV
observed to be 45.4%. The supplementation frequency can be reduced regardless of lipid source
without negatively impact microbial protein synthesis.

References
Allen, M.S, 2000. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. Journal of Dairy
6FLHQFHYS
Brokaw, L., B. W. Hess, and D. C. Rule. 2001. Supplemental soybean oil or corn for beef heifers grazing summer
pasture: effects on forage intake, ruminal fermentation, and site and extent of digestion. J. Anim. Sci. 79, 2704-2712.
Chen, X. B., M. J. Gomes., 1992. Estimation of microbial protein supply to sheep and cattle based on urinary excretion
of purine derivatives an overview of technical details. International Feed Research Unit. Aberdeen, UK: Rowett
Research Institute (Occasional publication), 21.
+DUYDWLQH.-$OOHQ06)DWVXSSOHPHQWVDIIHFWIUDFWLRQDOUDWHVRIUXPLQDOIDWW\DFLGELRK\GURJHQDWLRQDQG
SDVVDJHLQGDLU\FRZV-RXUQDORI1XWULWLRQYS
.U\VO/-0%-XGNLQVDQG95%RKPDQ,QÀXHQFHRIUXPLQDORUGXRGHQDOVR\EHDQRLOLQIXVLRQRQLQWDNH
ruminal fermentation, site and extent of digestion, and microbial protein synthesis in beef heifers consuming grass
KD\-$QLP6FL
National Research Council – NRC, 2001. Nutrient of requirements of dairy cattle. 7. ed. Washington, D.C.: National
$FDGHPLF3UHVV

140 Energy and protein metabolism and nutrition in sustainable animal production
Part 2. Energy and protein interactions, monogastrics
Exploring the biology of energy and protein utilization in non-ruminant
DQLPDOVWRLPSURYHQXWULHQWXWLOL]DWLRQHI¿FLHQFLHV
C.F.M. de Lange1, C.L. Levesque2 and H.R. Martínez-Ramírez1
1Department of Animal and Poultry Science, University of Guelph, Guelph, ON, N1G 2W1, Canada;
[email protected]
26RXWK'DNRWD6WDWH8QLYHUVLW\%URRNLQJV6'86$

Abstract
In this review, bioavailability of energy and amino acid, and the biological processes that contribute
WRQXWULHQWXWLOL]DWLRQHI¿FLHQF\LQQRQUXPLQDQWDQLPDOVDUHGLVFXVVHG.H\OLPLWDWLRQVRIXVLQJ
digestible energy, metabolizable energy, net energy and standardized ileal amino acid digestibility
YDOXHVLQGLHWIRUPXODWLRQDUHKLJKOLJKWHG$PRGHOOLQJV\VWHPEDVHGRQQXWULHQWÀRZVLVVXJJHVWHG
WRLPSURYHDFFXUDF\RIUHSUHVHQWLQJHQHUJ\XWLOL]DWLRQ,QQXWULHQWÀRZPRGHOVGLHWDU\HQHUJ\
sources are characterized based on content and enzymatic digestibility or fermentability of energy
yielding nutrients, while the use of nutrients for the various body functions is explicitly represented.
In various species animal performance is sensitive to synchronized supply of nutrients; in these cases
the dynamics of nutrient absorption should be considered. In regards to amino acid utilization, the
HIIHFWRIPLFURELDOIHUPHQWDWLRQLQWKHXSSHUJXWDQGPHWDEROLFLQHI¿FLHQF\DVVRFLDWHGHQGRJHQRXV
JXWDPLQRDFLGORVVHVUHPDLQWREHTXDQWL¿HGPRUHDFFXUDWHO\(VSHFLDOO\LQKHDWWUHDWHGLQJUHGLHQWV
some amino acids may be absorbed in a form that renders them unavailable for metabolism by the
animal. Maintenance energy requirements contribute substantially to total energy requirements, and
are best expressed as requirements at the cellular level in ATP equivalents. Between animal variability
LVDNH\GHWHUPLQDQWRILQHI¿FLHQF\RIQXWULHQWXVHLQJURXSVRIDQLPDOV9DULRXVDSSURDFKHVWKDWPD\
EHSXUVXHGWRLPSURYHHI¿FLHQFLHVRIXVLQJSURWHLQDQGRWKHUHQHUJ\\LHOGLQJQXWULHQWVDUHSUHVHQWHG

Introduction
The high feed energy costs, rapidly increasing demand for food animal protein, and concerns about the
environmental impact of animal production are important incentives to improve animal production and
QXWULHQWXWLOL]DWLRQHI¿FLHQFLHV 15&0RXJKDQ 7KHXWLOL]DWLRQRIGLHWDU\ELRDYDLODEOH
energy and protein (i.e. amino acids) for retention in consumable animal proteins involves digestion,
DEVRUSWLRQDQGSRVWDEVRUSWLYHPHWDEROLVP7KHVHELRORJLFDOSURFHVVHVDUHDOOLQÀXHQFHGE\IDFWRUV
associated with the animal (species and genotype, gender, physiological state, health status) and the
environment (diet composition, thermal and physical environment; e.g. NRC, 1994, 2011, 2012).
Energy yielding nutrients, including amino acids, also serve as precursors for bioactive compounds
WKDWLQÀXHQFHJHQHH[SUHVVLRQDQGHQGRFULQHIXQFWLRQDQGPD\DVVXFKDOVREHXVHGWRPDQLSXODWH
SURGXFWLRQHI¿FLHQFLHVLQFOXGLQJPHWDEROLFGLVRUGHUVDQGJXWKHDOWK $YHURXVet al., 2003; Jonker
et al., 2012; Zijlstra et al., 2012). Optimum dietary energy and protein intake will, therefore, vary
between groups of animals, and an understanding of the biology of energy and protein utilization is
required for optimizing energy and nutrient utilization for different groups of animals.

In this short review, bioavailability of energy and amino acids, and the biological processes that
FRQWULEXWHWRQXWULHQWXWLOL]DWLRQHI¿FLHQF\LQQRQUXPLQDQWDQLPDOVDUHGLVFXVVHG0HDQVWRLPSURYH
HI¿FLHQFLHVDQGDUHDVIRUIXUWKHUUHVHDUFKDUHLGHQWL¿HG7KHHPSKDVLVZLOOEHRQSLJVSULPDULO\
EHFDXVHPDQ\RIWKHELRORJLFDOSURFHVVHVWKDWFRQWULEXWHWRQXWULHQWXWLOL]DWLRQKDYHEHHQTXDQWL¿HG
DQGLQWHJUDWHGLQPDWKHPDWLFDOPRGHOVWKDWDOORZXVWRUHODWHQXWULHQWLQSXWVWRUDWHVDQGHI¿FLHQFLHV
of pork production in a quantitative manner (e.g. Kyriazakis, 1999; Van Milgen et al., 2008; NRC,
2012). Many of the concepts also apply to other species and references to other species are made
where appropriate.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 143
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_42, © Wageningen Academic Publishers 2013
One of the biggest challenges in energy and protein nutrition is the prediction and manipulation of
voluntary feed intake. This complex topic is beyond this contribution; for reviews on this topic the
reader is referred elsewhere (e.g. Kyriazakis, 1999; Torrallardona and Roura, 2009; Richards et al.,
2010; Westerterp-Plantenga et al., 2012).

Energy and amino acid bioavailability in feed ingredients

Accurate estimates of bioavailability of energy and nutrients in feed ingredients are required to
properly assess dietary requirements of animals, which are independent of dietary feed ingredient
composition, and to assign nutritional values to alternative feed ingredients. For effective feed
formulation estimates of bioavailability should be additive among feed ingredients. Given the
complexities and expenses associates with accurately determining nutrient bioavailability, measures
of digestibility or metabolizability are widely used in diet evaluation and feed formulation (Columbus
and De Lange, 2012; Moughan et al., 2000; NRC, 1994, 2011, 2012). However, apparent faecal
GLJHVWLELOLW\ZKLFKLVWKHVLPSOHVWPHDVXUHRIQXWULHQWGLJHVWLELOLW\LQPDPPDOVDQG¿VKLVFRQIRXQGHG
with enteric fermentation and endogenous gut nutrients, primarily (mucin-)protein, losses from the
KRVW0RUHRYHUWKHSRVWDEVRUSWLYHHI¿FLHQF\RIQXWULHQWXWLOL]DWLRQVKRXOGEHFRQVLGHUHGHVSHFLDOO\
when assigning bioavailable energy contents to feed ingredients and when evaluating bioavailability
of amino acids in feed ingredients that contain (heat) damaged protein.

Digestible energy (DE) and metabolizable energy (ME) systems are still widely used to assess
bioavailability of energy in feed ingredients for non-ruminant animals, largely because experimental
procedures to establish DE and ME values are relatively simple (Birkett and De Lange, 2001a;
Emmans, 1994; Noblet and van Milgen, 2004; NRC, 1994, 2011, 2012). A key limitation of DE and
0(V\VWHPVLVWKHUHODWLYHO\SRRUSUHGLFWLRQRIIHHGHI¿FLHQF\DQGRWKHUDVSHFWVRIDQLPDOSHUIRUPDQFH
HVSHFLDOO\ZKHQIHHGLQJGLHWVZLWKH[WUHPHIDW¿EHURUSURWHLQFRQWHQWV HJ%LUNHWWDQG'H/DQJH
2001a; Noblet and van Milgen, 2004). A more mechanistic approach to represent energy utilization,
and thus to assign bioavailable energy contents to feed ingredients, is to characterize the content and
bioavailability of energy yielding nutrients (Birkett and De Lange, 2001a; Boisen and Verstegen,
1998; Noblet and van Milgen, 2004). This approach will require an extensive data base of enzymatic
(ileal) digestibility and fermentability (which may be estimated from fecal minus ileal digestibility)
of the dietary energy yielding nutrients (starch, sugars, protein, fat; CVB, 2003), including effects of
feed ingredient processing, interactions among nutrients, and the animal’s physiological state on these
measures of digestibility (Moughan et al., 2000; Noblet et al., 1993; Noblet and van Milgen, 2004).

6WDQGDUGL]HGLOHDOGLJHVWLELOLW\ 6,' FRHI¿FLHQWVDUHXVHGURXWLQHO\WRHVWLPDWHWKHELRDYDLODELOLW\RI

amino acids in feed ingredients for pigs (Stein et al., 2007; NRC, 2012) and have been considered
for poultry (Locatelli et al 7KH6,'YDOXHVUHÀHFWWKDWDPLQRDFLGVWKDWGLVDSSHDUIURPWKH
hindgut are not available for metabolism by the host – hence the term ’ileal digestibility’ – and are
corrected for the basal ileal endogenous amino acid losses – hence the term ‘standardized’. Basal ileal
endogenous amino acid losses represent the difference between amino acids of endogenous origin that
are secreted into the upper gut and the amounts reabsorbed prior to the distal ileum of animals that are
IHGKLJKO\GLJHVWLEOHSURWHLQVRXUFHVDQGGLHWVWKDWDUHGHYRLGRI¿EHUVDQGDQWLQXWULWLRQDOIDFWRUVWKDW
LQGXFHDGGLWLRQDORUVSHFL¿FHQGRJHQRXVJXWDPLQRDFLGORVVHV 6WHLQet al., 2007). The SID values
DUHFRUUHFWHGIRUWKHEDVDOLOHDODPLQRDFLGORVVHVRQO\ODUJHO\EHFDXVHLWLVWHFKQLFDOO\WRRGLI¿FXOW
WRURXWLQHO\PHDVXUHWRWDOLOHDOHQGRJHQRXVDPLQRDFLGORVVHV EDVDOORVVHVSOXVGLHWVSHFL¿FORVVHV
Stein et al., 2007). Until methodology is in place to routinely measure total ileal endogenous amino
DFLGORVVHVDQGWKHVHORVVHVKDYHEHHQTXDQWL¿HGIRUDZLGHUDQJHRIIHHGLQJUHGLHQWV6,'FRHI¿FLHQWV
will provide the basis for estimated bioavailability of amino acids in feed ingredients (Columbus and
'H/DQJH 7RLPSURYHSURWHLQXWLOL]DWLRQGLHWDU\IDFWRUVWKDWLQGXFHVSHFL¿FHQGRJHQRXVJXW
amino acid losses should be considered (Tamminga et al WRJHWKHUZLWKPHWDEROLFLQHI¿FLHQFLHV
associated with synthesis and re-absorption of endogenous gut losses (Columbus and De Lange, 2012).

144 Energy and protein metabolism and nutrition in sustainable animal production
In some instances SID values underestimate bioavailability of amino acids (Columbus and De Lange,
2012). This can occur when amino acids are digested and absorbed in a chemical form that renders
them unavailable for metabolism, which applies in particular to lysine that is present in heat treated
feed ingredients (Rurtherfurd and Moughan, 2012). Another example is the negative impact of
IHUPHQWDEOH¿EHULQWDNHRQWKUHRQLQHELRDYDLODELOLW\ /LEDR0HUFDGRet al.,=KXet al., 2005),
ZKLFKPD\EHDWWULEXWHGWRWKHLPSDFWRI¿EHULQGXFHGHQWHULFIHUPHQWDWLYHDPLQRDFLGFDWDEROLVP
and the use of high threonine containing mucus proteins as nitrogen and energy sources for enteric
microbes (Libao-Mercado et al.,)XOOHU 7KHODWWHUKLJKOLJKWVWKHSRWHQWLDOLPSDFWRI
enteric fermentation in the upper gut of non-ruminants on amino acid utilization. As recently reviewed
by Fuller (2012), enteric fermentation also occurs in the upper gut of non-ruminant animals and
contributes to de novo microbial amino acid synthesis, as well as fermentative catabolism of amino
acids. The impact of enteric fermentation in the upper gut of pigs on the net amino acid supply to
WKHKRVWUHPDLQVWREHTXDQWL¿HG

(QHUJ\DQGQXWULHQWDYDLODELOLW\LQIHHGLQJUHGLHQWVHVSHFLDOO\KLJK¿EHUFRQWDLQLQJFRSURGXFWV
from food and biofuel industries, may be improved by additional processing, pre-treatment with
HQ]\PHVRUPLFURELDOLQRFXODQWVDLPHGDWLPSURYLQJ¿EHUGLJHVWLELOLW\ =LMOVWUDet al., 2010; Yáñez
et al., 2011) and reducing effects of anti-nutritional factors on nutrient digestibility and endogenous
gut nutrient losses (Gilani et al., 2012; Sun et al., 2008; Tamminga et al., 1995).

Post-absorptive energy and nutrient use

In animals absorbed nutrients are required to satisfy requirements for body maintenance functions
and production in the form of whole body protein deposition, whole body lipid deposition, growth
of products of conception, milk production and so on. Production of nutrients that are retained in
the animal’s body or secreted with milk represents the balance between synthesis and degradation,
ERWKRIZKLFKUHTXLUHHQHUJ\DQGDUHDVVRFLDWHGLQHI¿FLHQFLHVRIQXWULHQWXVH *LOOet al., 1989;
0LOOLJDQDQG6XPPHU 

Largely through genetic selection impressive progress has been made in improving the animals’
production potentials (e.g. Kyriazakis, 1999; NRC, 2012). In fact, in many instances the animals’
energy intake capacity limits expression of the animals’ production potential (NRC, 2012). Recently, we
have become more aware of the negative effect of environmental stressors, and especially subclinical
OHYHOVRIGLVHDVHRQDQLPDOSURGXFWLRQHI¿FLHQF\ HJ5DNKVKDQGHKDQG'H/DQJH:LOOLDPV
et al., 1997). Therefore, environmental stressors should be minimized and various approaches to
reduce the animal’s susceptibility to environmental stressors should be pursued, including genetic
selection of animals and diet manipulations. The latter is addressed by Dr. Klasing at this symposium.

The classical partitioning of nutrient intake in growing animals (Kielanowski, 1971) requires
estimates of maintenance nutrient requirements and nutrient retention of body protein and body lipid
deposition, as is presented for ME in Equation 1. In this equation MEm represents maintenance energy
requirements, REl and REp energy retention in the form body lipid and body protein, respectively,
DQGNODQGNSWKHFRUUHVSRQGLQJPDUJLQDOHI¿FLHQF\RIXVLQJ0(LQWDNH7KLVFODVVLFDODSSURDFKKDV
been adopted for various animal species and has been expanded to include other aspects of animal
production (NRC, 1994, 2011, 2012).

ME = MEm + (1/kl) × REl + (1/kp) × Rep (1)

Clearly for the factorial estimation of energy and nutrient requirements animal production levels
VKRXOGEHGHWHUPLQHGDQGLQFUHDVHVLQSURGXFWLRQOHYHOVZLOOJHQHUDOO\LQFUHDVHHQHUJHWLFHI¿FLHQF\
DQGIHHGHI¿FLHQF\EXWWKHVHZLOOQRWEHIXUWKHUGLVFXVVHG,QWKHUHPDLQGHURIWKLVVKRUWUHYLHZ

Energy and protein metabolism and nutrition in sustainable animal production 145
QXWULHQWUHTXLUHPHQWVIRUERG\PDLQWHQDQFHIXQFWLRQVDQGSRVWDEVRUSWLYHHI¿FLHQFLHVRIQXWULHQW
use are discussed.

Maintenance energy requirements

Accurate estimates of maintenance requirements are critical for energy, simply because of the large
contribution of maintenance energy requirements to total energy requirements, especially in non-
lactating reproducing animals (NRC, 1994, 2011, 2012).

Even though the concept of maintenance energy requirements is rather convenient for the factorial
estimation of dietary energy requirements (Equation 1), its relevance for highly productive animals
can be questioned. It may be more appropriate to interpret maintenance energy requirements in
producing animals as support costs for production, which means that maintenance energy requirements
FRQWULEXWHWRWKH PDUJLQDO LQHI¿FLHQF\RIXVLQJHQHUJ\LQWDNHIRUSURGXFWLRQ /DEXVVLqUHet al.,
2011). This interpretation has implications for the estimate of ‘maintenance’ energy requirements
that should be independent of the animal’s performance level and feeding regime (Birkett and De
Lange, 2001a). In the case of growing animals, this is supported by the (statistical) interdependency
EHWZHHQHVWLPDWHVRI0(PDQGPDUJLQDOHI¿FLHQFLHVRIXVLQJ0(LQWDNHIRU5(ODQG5(S %LUNHWW
and De Lange, 2001a; Noblet et al., 1999), and the empirical observations that growing animals fed
at maintenance energy requirements, i.e. at zero body energy balance, will initially gain body protein
(and BW) and mobilize body lipid (e.g. Close et al., 1982; Kyriazakis et al., 1993). Moreover, and
as discussed later, estimates of MEm will vary with diet nutrient composition. Therefore, it is more
appropriate to express maintenance energy requirements as net energy (NEm; i.e. MEm corrected
for heat increment of feeding), or ATP equivalents (Birkett and De Lange, 2001a,b; Boisen and
Verstegen, 1998; Van Milgen et al., 2001; Figure 1).

HP

MEm 1-k g

HPfast
HIm(diet) 1-k m
HIm(body) HP0

NEm
Useful maintenance
energy
ME intake
MEm
)LJXUH6FKHPDWLFUHSUHVHQWDWLRQRIFRPSRQHQWVRIKHDWSURGXFWLRQDVLQÀXHQFHGE\PHWDEROL]DEOH
HQHUJ\LQWDNH$EEUHYLDWLRQV+,P ERG\ KHDWLQFUHPHQWRIPRELOL]LQJERG\QXWULHQWVWRSURYLGH
QHWHQHUJ\UHTXLUHPHQWVIRUPDLQWHQDQFH+,P GLHW KHDWLQFUHPHQWRIXVLQJPHWDEROL]DEOHHQHUJ\
LQWDNHWRSURYLGHQHWHQHUJ\UHTXLUHPHQWVIRUPDLQWHQDQFH+3KHDWSURGXFWLRQ+3H[WUDSRODWHG
heat production at zero energy intake; HPfast, heat production during fasting; kgPDUJLQDOHI¿FLHQF\
of using metabolizable energy intake for body energy retention; kmUHODWLYHHQHUJHWLFHI¿FLHQF\RI
using body and dietary nutrients for supplying useful energy for body maintenance functions; ME
intake, metabolizable energy intake; MEm, metabolizable energy requirements for maintenance;
1(PQHWHQHUJ\UHTXLUHPHQWVIRUPDLQWHQDQFH HTXLYDOHQWWR$73UHTXLUHPHQWVIRUPDLQWHQDQFH
IXQFWLRQVDWWKHWLVVXHOHYHO DGMXVWHGIURP%LUNHWWDQG'H/DQJHD 

146 Energy and protein metabolism and nutrition in sustainable animal production
7KHHVWLPDWHRI1(PZLOOLQÀXHQFHGLUHFWO\WKHDEVROXWH1(YDOXHVDQGWRDOHVVHUH[WHQWWKH
relative NE value of feeds and feed ingredients (Noblet et al., 2013; this publication). Unfortunately,
NEm cannot be determined directly. Traditionally, basal metabolic rate, representing equilibrium
heat production (HP) of a fasting and resting animal has been used to estimate NEm (Figure 1).
Basal metabolic rate cannot be measured directly in experiments either, and is generally estimated
by measurements of fasting heat production (HPfast), which approaches basal metabolic rate
asymptotically in time (Van Milgen et al., 1998). However, the extrapolated plateau HPfast is not
a good estimate for NEm either, since it represents both the energy requirements for basic body
maintenance functions, which may be interpreted as ATP requirements at the cellular level, as well
as heat increment associated with generating ATP from body nutrient stores (Figure 1). Moreover,
SODWHDX+3IDVWLVLQÀXHQFHGE\WKHSUHYLRXVIHHGLQJUHJLPH 'H/DQJHet al., 7KHXVHRISODWHDX
+3IDVWDVDQHVWLPDWHRI1(PLVQRWFRQVLVWHQWZLWKWKHGH¿QLWLRQRI1(SURG %LUNHWWDQG'H/DQJH
2001a). It is more appropriate to represent NEm as kb ×SODWHDX+3IDVWZKHUHNELVWKHHI¿FLHQF\
of converting energy retained in body nutrients to useful energy contained in ATP (Figure 1). In a
similar manner, NEm may be calculated as kd ×0(PZKHUHNGLVWKHHI¿FLHQF\RIXWLOL]LQJ0(
LQWDNHIRUJHQHUDWLQJ$737KHYDOXHNP NGNELVVLPSO\WKHUHODWLYHHI¿FLHQF\RIXVLQJERG\DQG
dietary nutrients to generate NEm. As an alternative to experimental measurement of (plateau) HPfast,
Noblet and van Milgen (2004) estimated HPfast by extrapolating a linear regression of HP against
ME intake above MEm to zero ME intake (HP0). The slope in this regression is determined by kg
RQO\ )LJXUH ZKHUHNJLVWKHPDUJLQDOHI¿FLHQF\RIXVLQJ0(LQWDNHIRUHQHUJ\UHWHQWLRQLQ%:
JDLQ$VVXFK+3KDVQRGLUHFWUHODWLRQVKLSWR+3IDVWDQG1(PDVWKHVHDUHLQÀXHQFHGE\NENG
or km, while there is apparently no relationship between km and kg (Birkett and De Lange, 2001a).

,QVXI¿FLHQWTXDQWLWDWLYHLQIRUPDWLRQLVGHHPHGDYDLODEOHWRDFFXUDWHO\HVWLPDWHHQHUJ\ $73 
requirements for the energy demanding body maintenance functions, such as body protein and body
lipid turnover, maintenance of electrolyte gradients across cell membranes (e.g. sodium and potassium
pumping), muscle tension, etc. (Baldwin, 1995; Gill et al0LOOLJDQDQG6XPPHUV 
Therefore, estimates of maintenance energy requirements may be related to the size of metabolically
active tissues (Koong et al., 1982; Van Milgen et al., 1998; Yen et al., 1997) or metabolic BW, which
may be regarded as a simple predictor of size of metabolically active tissues (Noblet et al., 2013;
this publication).

There are differences in maintenance energy requirements between species, especially for growing
DQLPDOVDQGWKHVHDUHEULHÀ\GLVFXVVHGE\1REOHWet al. (2013; this publication). Within species
there is between animal variability in estimated maintenance energy requirements, and this has been
the basis for genetic selection based on residual feed intake (Kennedy et al., 1993). The latter is
GH¿QHGDVWKHGLIIHUHQFHEHWZHHQHVWLPDWHGPDLQWHQDQFHHQHUJ\UHTXLUHPHQWVRILQGLYLGXDODQLPDOV
within a population versus the population mean, whereby a negative value implies that an animal is
HQHUJHWLFDOO\PRUHHI¿FLHQW2EYLRXVO\WKLVDSSURDFKLVVHQVLWLYHWRWKHPRGHOWKDWLVXVHGWRHVWLPDWH
maintenance energy requirements and environmental conditions. The physiological basis for residual
feed intake has been reviewed recently (Young and Dekkers, 2012), and may be attributed to variation
LQGLJHVWLYHFDSDFLW\DFWLYLW\OHYHOVIHHGLQJEHKDYLRXULPPXQHV\VWHPVWLPXODWLRQ LQÀDPPDWLRQ
and oxidative stress), as well as various aspects of metabolism which will be discussed later.

Maintenance amino acid requirements

Traditionally, maintenance amino acid requirements have been related to the metabolic BW of pigs
[i.e. (BW)0.75; NRC, 1994, 2011, 2012]. This traditional approach fails to represent the biological
processes that contribute to maintenance amino acid requirements and account for factors affecting
them. According to Moughan (1999) the main factors that constitute maintenance amino acid
requirements are basal endogenous gut amino acid losses, integument amino acid losses, and
minimum amino acid catabolism. The latter represents amino acid losses associated with basal whole

Energy and protein metabolism and nutrition in sustainable animal production 147
protein turnover and contributes to the minimum urinary nitrogen excretion. These concepts have
been adopted in NRC (2012) for estimating amino acid requirements of pigs. Basal endogenous
gut amino acid losses, in both the upper and lower gut, may be related to feed dry matter intake
and are the single largest contributor to maintenance amino acid requirements of pigs. Integument
losses (e.g. skin, hair) are quantitatively not that important for pigs and may be related to BW0.75.
$FFRUGLQJWR15&  LQVXI¿FLHQWTXDQWLWDWLYHLQIRUPDWLRQLVDYDLODEOHWRJHQHUDWHUHDVRQDEOH
estimates of minimum amino acid catabolism. For this reason, NRC (2012) applied the post-absorptive
LQHI¿FLHQF\ LQHYLWDEOHDPLQRDFLGFDWDEROLVPGLVFXVVHGEHORZ RIXVLQJLQWDNHRI6,'DPLQRDFLGV
for covering endogenous gut and integument amino acid losses to account for minimum amino acid
catabolism. Under some conditions the use of some amino acids for the production of non-protein
compounds may be quantitatively important. The latter applies, for example, to the use of tryptophan
for synthesis of serotonin and kynurenine or cysteine for synthesis of glutathione. The synthesis rates
RIWKHVHFRPSRXQGVUHPDLQWREHTXDQWL¿HGDFFXUDWHO\DQGPD\EHHOHYDWHGXQGHUVRPHFRQGLWLRQV
IRUH[DPSOHGXULQJLQÀDPPDWLRQ /H)ORF¶Ket al., 2009; Rakhshandeh and De Lange, 2011).

Even in animals at moderate levels of production the contribution of maintenance amino acids
requirements to total amino acids requirements is relatively small (NRC, 2012). Therefore, there is
OLPLWHGRSSRUWXQLW\WRLPSURYHHI¿FLHQFLHVRIDPLQRDFLGXWLOL]DWLRQE\PDQLSXODWLQJPDLQWHQDQFH
UHTXLUHPHQWV([FHSWLRQVDUHWRDYRLGIHHGLQJGLHWVWKDWLQGXFHODUJHDPRXQWVRIVSHFL¿FHQGRJHQRXV
gut amino acid losses (Tamminga et al., 2005), or exposing animals to stressors that induce synthesis
of non-protein compounds that require amino acids, such as tryptophan (Le Floc’h et al., 2009).

3RVWDEVRUSWLYHHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQ
,WLVZHOOHVWDEOLVKHGWKDWHQHUJHWLFHI¿FLHQFLHVLQDQLPDOVDUHLQÀXHQFHGE\GLHWDU\VRXUFHRIHQHUJ\
yielding nutrients (i.e. starch, sugars, protein, fat) and the purpose for which energy is used (i.e.
maintenance, body lipid and protein deposition, milk production, etc.) (Black, 1995; De Lange and
Birkett, 2001a,b; Van Milgen et al., 2001). For example, direct incorporation of dietary lipids into
ERG\OLSLGKDVDQHQHUJHWLFHI¿FLHQF\RIDERXWZKLOHWKHXVHRIOLSLGDVDQHQHUJ\VRXUFHIRU
JHQHUDWLQJ$73LVDERXWHI¿FLHQWDQGGHQRYRHQGRJHQRXVOLSLGV\QWKHVLVIURPGLJHVWLEOHVWDUFK
LVDERXWHI¿FLHQWLQSLJV %ODFN $VUHYLHZHGE\%LUNHWWDQG'H/DQJH D WKH
HVWLPDWHGHQHUJHWLFHI¿FLHQFLHVLQJURZLQJSLJVRIXVLQJ0(LQWDNHIRUERG\SURWHLQGHSRVLWLRQ NS
UDQJLQJIURPWR LVORZHUWKDQWKDWRIERG\OLSLGGHSRVLWLRQ NOUDQJLQJIURPWR 
YDULDELOLW\RIWKHVHHVWLPDWHVUHÀHFWGLIIHUHQFHVLQGLHWDU\HQHUJ\VRXUFHVDFURVVVWXGLHVPDWKHPDWLFDO
models used to generate these estimates, assumptions about maintenance energy requirements,
DQGPD\EHEHWZHHQDQLPDOYDULDELOLW\LQPHWDEROLFHI¿FLHQFLHV(QHUJHWLFLQHI¿FLHQF\ LHKHDW
LQFUHPHQWRIIHHGLQJ UHÀHFWVHQHUJ\FRVWVRIIHHGLQJHVWLRQDQGGLJHVWLRQQXWULHQWDEVRUSWLRQDQG
heat losses associated with metabolic pathways involved in utilization of absorbed energy yielding
nutrients for maintenance and production (e.g. Baldwin, 1995; Birkett and De Lange, 2001c; Van
Milgen, 2002; Figure 2).

+HDWLQFUHPHQWRIIHHGLQJLVDFFRXQWHGIRULQ1(V\VWHPV,QWKHVHV\VWHPV1(LVGH¿QHGDV0(
intake minus heat increment of feeding and estimated as energy retained in animal products plus
NEm. In NE systems for pigs (CVB, 2003; Noblet and Van Milgen, 2004; NRC, 2012) and poultry
(Pirgozliev and Rose, 1999) diet NE values are estimated from nutrient compositions. In these
empirical NE systems diet effects on energy utilization are represented, while animal effects on the
use of energy are not considered or it is assumed that the relative use of energy from various sources
for the various purposes is rather constant. A more mechanistic approach to representing diet and
DQLPDOHIIHFWVRQHQHUJ\XWLOL]DWLRQLVWRPDWKHPDWLFDOO\UHSUHVHQWQXWULHQWDQGPHWDEROLWHÀRZVRI
energy yielding nutrients (Birkett and De Lange, 2001a,b; Boisen and Verstegem 1998; De Lange
DQG%LUNHWW0DFKLHOVDQG+HQNHQ3HWWLJUHZet al.,1992; Van Milgen et al., 2001; Figure
 1XWULHQWÀRZPRGHOVDUHPRUHFRPSOH[WKDQWKHFXUUHQWHPSLULFDO1(V\VWHPVDVWKH\UHTXLUH

148 Energy and protein metabolism and nutrition in sustainable animal production
 Feed intake & diet composition

HP HP

Absorbed nutrients Undigested material

LC fatty acids Glucose V fatty acids Amino acids (N)

Urinary
METABOLISM Excretion
HP (Glucose, acetyl CoA, ATP)
HP

Lipid deposition Protein deposition

Basal ATP requirements

)LJXUH1XWULHQWDQGPHWDEROLWHÀRZVUHSUHVHQWLQJXWLOL]DWLRQRIHQHUJ\\LHOGLQJQXWULHQWVIRUERG\
SURWHLQDQGERG\OLSLGDFFUHWLRQLQJURZLQJDQLPDOV DGMXVWHGIURP%LUNHWWDQG'H/DQJHE 
$EEUHYLDWLRQV$73 DGHQRVLQHWULSKRVSKDWH&R$ FRHQ]\PH$+3 KHDWSURGXFWLRQ/&
)DWW\$FLGV ORQJFKDLQIDWW\DFLGV9)DWW\$FLGV YRODWLOHIDWW\DFLGV

an understanding of nutrient partitioning in animals, and will yield estimates of useful (net) energy
for a feed ingredient or diet that varies with the animal’s physiological state. The latter makes it
GLI¿FXOWWRLQWHJUDWHVXFKPRGHOVZLWK OHDVWFRVW GLHWIRUPXODWLRQ7RRYHUFRPHWKLVGLI¿FXOW\WKH
concept of ‘effective ME’ is used in NRC (2012) to integrate the use of empirical NE systems (to
UHSUHVHQWGLHWHIIHFWVRQHQHUJHWLFHI¿FLHQFLHV ZLWKWKHXVHRIHQHUJ\IRUYDULRXVSXUSRVHV (TXDWLRQ
1), whereby diet ‘effective ME’ content is calculated from the diet NE content with a typical and
¿[HGFRQYHUVLRQ HJIRUJURZLQJ¿QLVKLQJSLJV 7KLVVLPSOL¿FDWLRQUHGXFHVWKHFRQQHFWLRQ
between dietary energy source and use of energy by the animal somewhat.

$EHQH¿WIURPXVLQJPRUHPHFKDQLVWLFPRGHOVRIHQHUJ\XWLOL]DWLRQLVWKDWWKH\DOORZIRUDPRUH
V\VWHPDWLFDSSURDFKWRLGHQWLI\PHDQVWRLPSURYHHQHUJHWLFHI¿FLHQFLHVDQGJXLGHWKHGHVLJQRI
experiments that are targeted towards better understanding of diet and animal effects on key aspects
RIHQHUJ\XWLOL]DWLRQ )LJXUH 1XWULHQWÀRZUHSUHVHQWDWLRQVRIHQHUJ\XWLOL]DWLRQFDQEHIXUWKHU
UH¿QHGE\PDNLQJWKHPG\QDPLFDQGLQFOXGLQJVDWXUDWLRQNLQHWLFVIRUNH\PHWDEROLFSDWKZD\V
However, in growing animals where the number of alternative pathways for individual nutrient is
rather small this may be of limited value. In reproducing animals (e.g. lactating and egg laying) the
inclusion of dynamic aspects is likely to be more critical, for representing the potential mobilization
of body nutrient stores to support production (e.g. Baldwin, 1995; Pettigrew et al.,1992; Birkett
and De Lange, 2001a,b). The representation of amino acid utilization can be integrated in nutrient
ÀRZVPRGHOVWRLPSURYHXQGHUVWDQGLQJRILQWHUDFWLRQVEHWZHHQHQHUJ\DQGSURWHLQXWLOL]DWLRQ HJ
Kyrizakis et al., 1999; Van Milgen et al., 2008; Wies et al., 2004).

Dynamics of nutrient digestion and absorption affect the rate of appearance of nutrients that are
DYDLODEOHIRUPHWDEROLVPDQGPD\EHLQÀXHQFHGWRV\QFKURQL]HVXSSO\RIGLIIHUHQWQXWULHQWVWR
the animal. The importance of considering the dynamics of nutrient absorption has been shown
in broiler chickens (Weurding et al., 2003), veal calves (Van den Borne et al DQGJURZLQJ
pigs (Van den Borne et al., 2007). For example, when feeding different types of starch of similar

Energy and protein metabolism and nutrition in sustainable animal production 149
GLJHVWLELOLW\WREURLOHUFKLFNVPRUHVORZO\GLJHVWHGVWDUFK\LHOGHGKLJKHUHI¿FLHQFLHVRIDPLQRDFLG
utilization for growth (Weurding et al., 2003). It was hypothesized that rapid digestible starch reduced
glucose supply to the lower part of the digestive tract, forcing intestinal tissue to use increased
amounts of amino acids as energy source, and reducing amino acid availability to support growth.
$OWHUQDWLYHO\G\QDPLFVRIQXWULHQWDEVRUSWLRQPD\LQÀXHQFHWKHDQLPDOV¶HQGRFULQRORJ\DQGWKHUHE\
the physiological control of nutrient utilization (Zijlstra et al., 2012). Feeding frequency and feed
ingredient choice may thus be used to manipulate post-absorptive nutrient utilization.

$VPHQWLRQHGHDUOLHUEHWZHHQDQLPDOYDULDWLRQLQHQHUJHWLFHI¿FLHQFLHVPD\LQSDUWEHDWWULEXWHGWR
metabolic processes. An important contributor to maintenance energy requirements, or support costs
IRUSURGXFWLRQLVERG\SURWHLQWXUQRYHU 0LOOLJDQDQG6XPPHUV*LOOet al., 1989). It may thus
be hypothesized that differences in the ratio between protein synthesis and body protein deposition
EHWZHHQDQLPDOVFRQWULEXWHWRGLIIHUHQFHVLQHQHUJHWLFHI¿FLHQFLHVRIERG\SURWHLQGHSRVLWLRQ
However, when comparing growing pigs of two very different genotypes, Landrace and Iberian,
under similar conditions this ratio did not differ, while rather large differences were observed in
whole body protein deposition (Rivera-Ferre et al ,WLVRILQWHUHVWWRQRWHWKDWEHWDDGUHQHUJLF
agonists, such as Ractopamine (PayleanTM) increase protein deposition by increasing protein synthesis
and reducing protein degradation (Kim and Sainz, 1992), and thereby reducing the marginal energy
cost of body protein deposition. Effects of diet, the environment and the animal on the relationship
between protein synthesis and protein deposition remains to be explored further. Another aspect of
HQHUJ\PHWDEROLVPWKDWLVRILQWHUHVWLVPLWRFKRQGULDOUHVSLUDWLRQHI¿FLHQF\ZKLFKKDVEHHQVWXGLHG
in various species, including poultry (Bottje and Carstens, 2009; Ojano-Dirian et al., 2004) and pigs
(Lefaucheur et al., 2011). Mitochondria account for about 90% of whole body oxygen consumption
and generate ATP through oxidative phosphorylation, which is driven by electron gradients across
the inner mitochondrial membrane. Leakage of electrons across the membrane, which is regulated
by so-called uncoupling proteins will reduce ATP yield, increase heat production and therefore
UHGXFHHQHUJHWLFHI¿FLHQFLHV -HåHNet al., 2004). Activity of uncoupling proteins appears related to
R[LGDWLYHVWUHVVZKLFKVXJJHVWVDOLQNZLWKLQÀDPPDWLRQDQGWKHUHIRUHWKHDQLPDOV¶DELOLW\WRFRSH
with a disease challenge. Initial observations in broiler chickens suggest a relationship between
UHVSLUDWRU\DFWLYLW\LQPXVFOHWLVVXHDQGIHHGHI¿FLHQF\ %RWWMHDQG&DUVWHQV2MDQR'LULDQ
et al., EXWWKLVQHHGVWREHFRQ¿UPHG

3RVWDEVRUSWLYHHI¿FLHQF\RIDPLQRDFLGXWLOL]DWLRQ
7KHPD[LPXPPDUJLQDOHI¿FLHQF\RIXWLOL]LQJDYDLODEOHDPLQRDFLGLQWDNHRYHUDQGDERYHPDLQWHQDQFH
amino acid requirements for amino acid retention in body protein deposition is determined largely
by inevitable amino acid catabolism (Moughan, 1999). When obtaining estimates of inevitable
catabolism, care should be taken to differentiate it from preferential catabolism, which occurs when
energy intake limits the expression of the animals body protein deposition capacity, and catabolism
of amino acids that are supplied in excess of requirements (De Lange et al., 2001, 2012). The latter
is of concern when animals are fed constant dietary levels of amino acids over a wide BW range,
because dietary amino acid requirements – expressed as dietary levels – generally decline with
increasing BW. Estimates of inevitable catabolism of lysine and threonine in growing pigs have
been obtained from observations on individual pigs and in closely controlled serial slaughter studies
(NRC, 2012), and are about 25% of available amino acid intake. According to NRC (2012) the rate
of inevitable amino acid catabolism is rather constant over a wide range of amino acid intake levels
(Figure 3), appears to be largely independent of BW, and increases slightly with improvements in
SLJSHUIRUPDQFHSRWHQWLDO,WVKRXOGEHQRWHGWKDWWKHHI¿FLHQF\RIDPLQRDFLGXWLOL]DWLRQIRUERG\
protein utilization is always lower in groups of pigs than in individual pigs, which can be attributed
largely to between-animal variability in amino acid requirements (De Lange et al., 2012). In fact,
LWFDQEHDUJXHGWKDWWKHPDLQFRQWULEXWRUWRWKHGLPLQLVKLQJPDUJLQDOHI¿FLHQF\RIDPLQRDFLG
utilization for body protein deposition is between animal variability rather than a marginal increase

150 Energy and protein metabolism and nutrition in sustainable animal production
A B

)LJXUH2EVHUYHGHIIHFWVRIWKUHRQLQHLQWDNHRQZKROHERG\SURWHLQGHSRVLWLRQ 3')LJXUH$ 
DQGHVWLPDWHGUDWHVRIWKUHRQLQHFDWDEROLVP )LJXUH% 5HVXOWVZHUHREWDLQHGLQDVHULDOVODXJKWHU
study, using individually housed pigs that were fed casein and cornstarch based diets; dietary protein
VXSSO\ZDVDGMXVWHGZHHNO\WRPHHWWKHSLJV¶HVWLPDWHGWKUHRQLQHUHTXLUHPHQWV DGMXVWHGIURP'H
/DQJHHWDO 

in the rate of inevitable amino acid catabolism (Figure 3). Therefore, between animal variability
within groups of animals should be considered when establishing the optimum dietary amino acid
level for groups of animals (Pomar et al., 2003; Morel et al., 2008), and in NRC (2012) adjustments
ZHUHPDGHWRWKHLQHI¿FLHQF\RIXWLOL]LQJDYDLODEOHDPLQRDFLGLQWDNHRYHUDQGDERYHPDLQWHQDQFH
amino acid requirements to account for between animal variability.

7KHUDWHRILQHYLWDEOHFDWDEROLVPLVLQÀXHQFHGE\DQLPDODQGGLHWDU\IDFWRUV$VPHQWLRQHGHDUOLHU
the rate of inevitable amino acid catabolism declines with increasing body protein deposition
capacity (NRC, 2012). Observed antagonisms among amino acids that share common catabolic
SDWKZD\VZKHUHE\DQH[FHVVLYHVXSSO\RIRQHDPLQRDFLGUHGXFHVWKHXWLOL]DWLRQHI¿FLHQF\RIRWKHU
amino acids, has in some instances been attributed to increased amino acid catabolism (Langer et
al., 2000). This applies in particular to the branch-chained amino acids and has been observed in
chickens (Peganova and Eder, 2003), growing pigs (Langer et al., 2000) and sows (Perez-Laspiur et
al., 2009). However, in other instances amino acid antagonisms have been attributed to competition
for amino acid transporters, as illustrated by reduced uptake of tryptophan in the brain of pigs fed
large amounts of neutral amino acids (Henry et al., 1992), and thereby increasing catabolism.
7KHVHH[DPSOHVLOOXVWUDWHWKDWHI¿FLHQFLHVRIXVLQJDPLQRDFLGVWKDWOLPLWDQLPDOSURGXFWLRQPD\
be reduced, as well as animal productivity, by feeding diets with (severely) imbalanced amino acid
SUR¿OHV *DUOLFN 

Conclusions and implications

Key limitations of using digestible energy, metabolizable energy, and standardized ileal amino
acid digestibility values in diet formulation are inaccurate predictions of the animals’ performance
response, especially when feeding diets with extreme nutrient compositions or heat treated ingredients.
These limitations are becoming more critical with increased usage of co-products from the food and
biofuel industries in animal diets. Energy (and protein) utilization is represented more accurately,
LPSURYLQJSUHGLFWDELOLW\RIDQLPDOUHVSRQVHVZKHQLWLVEDVHGRQQXWULHQWDQGPHWDEROLWHÀRZ
PRGHOV,QQXWULHQWÀRZPRGHOVWKHGLHWDU\FRQWHQWVDQGDYDLODELOLW\RIHQHUJ\\LHOGLQJQXWULHQWV
and the use of nutrients by the animals for the various body functions can be represented. Nutrient
ÀRZUHSUHVHQWDWLRQVRIHQHUJ\XWLOL]DWLRQFDQEHIXUWKHUUH¿QHGE\PDNLQJWKHPG\QDPLFDQG
including saturation kinetics for key metabolic pathways. Maintenance energy requirements contribute
substantially to total energy requirements, and are best expressed as net energy requirements at the
FHOOXODUOHYHOLQ$73HTXLYDOHQWV%HWZHHQDQLPDOYDULDELOLW\LVDNH\GHWHUPLQDQWRILQHI¿FLHQF\
of nutrient use in groups of animals, and may be attributed to differences in performance potentials,
animal activity, digestive capacity and immune system activity. Various approaches can be used

Energy and protein metabolism and nutrition in sustainable animal production 151
DVDPHDQVWRLPSURYHHI¿FLHQFLHVRIXVLQJSURWHLQDQGRWKHUHQHUJ\\LHOGLQJQXWULHQWVLQFOXGLQJ
improving performance potentials, feeding more closely to nutrient requirements for targeted levels
RIDQLPDOSHUIRUPDQFHUHGXFLQJWKHQHJDWLYHHIIHFWVRIGLHWDU\¿EHUDQGDQWLQXWULWLRQDOIDFWRUV
on nutrient utilization, reducing the ratio between protein synthesis and protein deposition, and
LPSURYLQJPLWRFKRQGULDOUHVSLUDWLRQHI¿FLHQF\

References
Averous, J., A. Bruhat, S. Mordier and P. Fafournoux, 2003. Recent advances in the understanding of amino acid
regulation of gene expression. J. Nutr. 133, 2040S-20445S.
Baldwin., R.L. 1995. Modeling Ruminant Digestion and Metabolism. Chapman & Hall, New York.
Birkett, S. and K. de Lange, 2001a. Limitations of conventional models and a conceptual framework for a nutrient
ÀRZUHSUHVHQWDWLRQRIHQHUJ\XWLOL]DWLRQE\DQLPDOV%ULW-1XWU
%LUNHWW6DQG.GH/DQJHE&DOLEUDWLRQRIDQXWULHQWÀRZPRGHORIHQHUJ\XWLOL]DWLRQE\JURZLQJSLJV%ULW
-1XWU
Black, J.L. 1995. Modelling energy metabolism in the pig. Pages 87-102 in P.J. Moughan, M.W.A. Verstegen and
M. Visser-Reyneveld (Editors) Modelling growth in the pig. Wageningen Pers, Wageningen, The Netherlands.
Boisen, S. and M.W.A. Verstegen, 1998. Evaluation of feedstuffs and pig diets. Energy or nutrient-based evaluation
systems? II. Proposal for a new nutrient-based evaluation system. Acta Agric. Scand. 48, 95-102
%RWWMH:*DQG*(&DUVWHQV$VVRFLDWLRQRIPLWRFKRQGULDOIXQFWLRQDQGIHHGHI¿FLHQF\LQSRXOWU\DQGOLYHVWRFN
VSHFLHV((
&ORVH:+)%HUVFKDXHUDQG5+HDYHQV7KHLQÀXHQFHRISURWHLQHQHUJ\YDOXHRIWKHUDWLRQDQGOHYHORIIHHG
LQWDNHRQWKHHQHUJ\DQGQLWURJHQEDODQFHRIWKHJURZLQJSLJ%ULW-1XWU
&ROXPEXV'DQG&)0GH/DQJH(YLGHQFHIRUYDOLGLW\RILOHDOGLJHVWLELOLW\FRHI¿FLHQWVLQPRQRJDVWULFV
%ULW-1XWU
CVB (Centraal Veevoeder Bureau), 2003. Veevoedertabel (Table of Feeding Value of Animal Feed Ingredients). Centraal
Veevoeder Bureau, Lelystad, The Netherlands.
De Lange, C.F.M. and S. H. Birkett, 2005. Characterization of the useful energy content in swine and poultry feed
LQJUHGLHQWV&DQ-$QLP6FL
'H/DQJH&)0$0*LOOLVDQG*-6LPSVRQ,QÀXHQFHRIWKUHRQLQHLQWDNHRQZKROHERG\SURWHLQGHSRVLWLRQ
DQGWKUHRQLQHXWLOL]DWLRQLQJURZLQJSLJVIHGSXUL¿HGGLHWV-$QLP6FL
'H/DQJH&)0-YDQ0LOJHQ-1REOHW6'XERLVDQG6%LUNHWW3UHYLRXVIHHGLQJOHYHOLQÀXHQFHVSODWHDX
heat production following a 24 h fast in growing pigs. Brit. J. Nutr. 95, 1082-1087.
'H/DQJH&)0&/HYHVTXHDQG%-.HUU$PLQRDFLGQXWULWLRQDQGIHHGHI¿FLHQF\3DJHVLQ-)
3DWLHQFH HG )HHGHI¿FLHQF\LQSLJV:DJHQLQJHQ$FDGHPLF3UHVV
Emmans, G.C. 1994. Effective energy: A concept of energy utilization applied across species. Brit. J. Nutr. 71, 801-821.
Fuller, M.F. 2012. Determination of protein and amino acid digestibility in foods including implications of gut microbial
DPLQRDFLGV\QWKHVLV%ULW-1XWU66
Garlick, P.J. 2004. The nature of human hazards associated with excessive intake of amino acids. J. Nutr. 134,
66
Gilani, G.S., C. W. Xiao and K.A. Cockell, 2012. Impact of antinutritional factors in food proteins on the digestibility
of protein and the bioavailability of amino acids and on protein Quality. Brit. J. Nutr. 108, S315-332.
Gill, M., J. France, M. Summers, B. McBride and L. Milligan, 1989. Simulation of the energy costs associated with
protein turnover and Na+/K+ transport in growing lambs. J. Nutr. 119, 1287-1299.
Henry, Y., B. Seve, B. Colleaux, P. Ganier, C. Saligaut and P. Jégo, 1992. Interactive effects of dietary levels of tryptophan
and protein on voluntary feed intake and growth performance in pigs, in relation to plasma free amino acid and
hypothalamic serotonin. J. Anim Sci. 70, 1873-1887.
-HåHN30åiüFNRYi05ĤåLüND(ĝNRELVRYiDQG0-DEĤUHN0LWRFKRQGULDOXQFRXSOLQJSURWHLQV±)DFWV
and fantasies Phys. Res. 53, S199-S211.
-RQNHU503.-(QJHOHQDQG1(3'HXW]5ROHRIVSHFL¿FGLHWDU\DPLQRDFLGVLQFOLQLFDOFRQGLWLRQV%ULW
J. Nutr. 108, S139-S148.

152 Energy and protein metabolism and nutrition in sustainable animal production
Kennedy, B.W., J.H. van der Werf and T.H. Meuwissen, 1993. Genetic and statistical properties of residual feed intake.
J. Anim. Sci. 71, 3239-3250.
Kielanowski, J. 1971. Energy requirements of the growing pig. In Pig Production, Pages 183-201 in D.J.A. Cole
(Editors) Proceedings of the Eighteenth Easter School in Agricultural Science, Nottingham, 1971. Penn State
University Press, University Park, PA.
Kim, Y.S. and R.D. Sainz, 1992. Beta adrenergic agonists and hypertrophy of skeletal muscle. Life Sci. 50, 3970407.
Koong, L-J, J. Nienaber, J. Pekas and J-T. Yen, 1982. Effects of plane of nutrition on organ size and fasting heat
SURGXFWLRQLQSLJV-1XWU
Kyriazakis, I. (Editor) 1999. Quantitative Biology of the Pig, I. Kyriazakis, ed. Wallingford, UK: CAB International,
Wallingford, Oxon, UK.
Kyriazakis, I., D. Dotas and G. Emmans, 1993. The effect of breed on the relationship between feed composition and
WKHHI¿FLHQF\RISURWHLQXWLOL]DWLRQLQSLJV%ULW-1XWU
Labussière E., J. van Milgen J., C. de Lange and J. Noblet, 2011. Maintenance energy requirements of growing pigs
DQGFDOYHVDUHLQÀXHQFHGE\IHHGLQJOHYHO-1XWU
Langer, S., P.W.D. Scislowski, D.S. Brown, P. Dewey and M.F. Fuller, 2000. Interactions among the branched-chain
amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid
concentrations and branched-chain keto-acid dehydrogenase activity. Brit. J. Nutr.83, 49-58.
Lefaucheur, L., B. Lebret, P. Ecolan, I. Louveau, M. Damon, A. Prunier, Y. Billon, P. Sellier and H. Gilbert, 2011.
Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs.
-$QLPDO6FL
Le Floc’h, N., L. Lebellego, J.J. Matte, D. Melchior and B. Sève, 2009. The effect of sanitary status degradation and
GLHWDU\WU\SWRSKDQFRQWHQWRQJURZWKUDWHDQGWU\SWRSKDQPHWDEROLVPLQZHDQLQJSLJV-$QLP6FL
/LEDR0HUFDGR$-6/HHVRQ6/DQJHU%-0DUW\DQG&)0GH/DQJH(I¿FLHQF\RIXWLOL]LQJLOHDOGLJHVWLEOH
lysine and threonine for whole body protein deposition in growing pigs is reduced when dietary casein is replaced
E\ZKHDWVKRUWV-$QLP6FL
Locatelli, M.L., V. Ravindran and A. Lemme, 2004. Standardized ileal amino acid digestibility in broiler nutrition. J.
Anm. Sci. 82 (Suppl. 1), 433-433
0DFKLHOV0$0DQG$0+HQNHQ$G\QDPLFVLPXODWLRQPRGHOIRUJURZWKRIWKH$IULFDQ&DW¿VK&ODULDV
JDULHSLQXV %XUFKHOO ,(IIHFWRIIHHGLQJOHYHORQJURZWKDQGHQHUJ\PHWDEROLVP$TXDFXOWXUH
0LOOLJDQ/DQG06XPPHUV7KHELRORJLFDOEDVLVRIPDLQWHQDQFHDQGLWVUHOHYDQFHWRDVVHVVLQJUHVSRQVHVWR
nutrients. Proc. Nutr. Soc. 45, 185-193.
Morel, P.C.H., G.R. Wood and D. Sirisatien, 2008. Effect of genotype, population size and genotype variation on
optimal diet determination for growing pigs. Acta Horticulturae 802, 287-292.
Moughan, P.J. 1999. Protein metabolism in the growing pig. Pages 299-331 In I. Kyriazakis (Editor) Quantitative
Biology of the Pig. CAB International, Wallingford, Oxon, UK.
Moughan, P.J., M.W.A. Verstegen and M. Visser-Reyneveld (Editors), 2000. Feed evaluation – principles and practice.
Wageningen Pers, Wageningen, The Netherlands.
0RXJKDQ3- (GLWRU 'LHWDU\SURWHLQIRUKXPDQKHDOWK%U-1XWU66
NRC (National Research Council), 1987. Predicting feed intake of food producing animals. The National Academies
Press, Washington, DC.
NRC (National Research Council), 1994 Nutrient requirements of poultry. The National Academies Press, Washington,
DC.
NRC (National Research Council), 2010. Toward sustainable agricultural systems in the 21st century. The National
Academies Press, Washington, DC.
15& 1DWLRQDO5HVHDUFK&RXQFLO 1XWULHQWUHTXLUHPHQWVRI¿VKDQGVKULPS7KH1DWLRQDO$FDGHPLHV3UHVV
Washington, DC.
NRC (National Research Council), 2012. Nutrient requirements of swine. The National Academies Press, Washington,
DC.
Noblet, J., C. Karege, S. Dubois and J van Milgen, 1999. Metabolic utilization of energy and maintenance requirements
LQJURZLQJSLJVHIIHFWRIVH[DQGJHQRW\SH-$QLP6FL
Noblet, J., X.S. Shi and S. Dubois, 1993. Metabolic utilization of dietary energy and nutrients for maintenance energy
requirements in sows: basis for a net energy system. Brit. J. Nutr. 70, 407-419.

Energy and protein metabolism and nutrition in sustainable animal production 153
Noblet J. and J. van Milgen, 2004. Energy value of pig feeds: Effect of pig body weight and energy evaluation system.
J. Anim. Sci. 82 (E. Suppl.), E229-E238.
Ojano-Dirain, C. P., M. Iqbal, D. Cawthon, S. Swonger, T. Wing, M. Cooper and W. Bottje, 2004. Determination of
PLWRFKRQGULDOIXQFWLRQLQEURLOHUVZLWKORZDQGKLJKIHHGHI¿FLHQF\3RXOWU\6FL
Peganova, S. and K. Eder. 2003. Interactions of various supplies of isoleucine, valine, leucine and tryptophan on the
performance of laying hens. Poultry Sci. 82, 100-105.
Perez-Laspiur, J., J. L. Burton, P. S. D. Weber, J. Moore, R. N. Kirkwood and N. L. Trottier, 2009. Dietary protein
intake and stage of lactation differentially modulate amino acid transporter mRNS abundance in porcine mammary
WLVVXH-1XWU
Pettigrew, J.E., M. Gill, J. France and W.H. Close. 1992. A mathematical integration of energy and amino acid metabolism
RIODFWDWLQJVRZV-$QLP6FL
Pirgozliev, V. and S.P. Rose, 1999. Net energy systems for poultry feeds: A quantitative review. World’s Poultry Sci.
-
Pomar, C., I. Kyriazakis, G.C. Emmans, and P.W. Knap, 2003. Modeling stochasticity: Dealing with populations rather
WKDQLQGLYLGXDOSLJV-$QLP6FL((
5DNKVKDQGHK$DQG&)0GH/DQJH,PPXQHV\VWHPVWLPXODWLRQLQWKHSLJ3DJHVLQ5-YDQ%DUQHYHOG
(Editor) Manipulating pig production XIII. Australasian Pig Science Association Inc. Werribee, Victoria, Australia.
Richard, M.P., R.W. Rosebrough, C.N. Coon and J.P. McMurtry, 2010. Feed intake regulation for the female broiler
breeder: In theory and in practice. Poultry Sci. 19, 182-193.
5LYHUD)HUUH0*-)$JXLOHUD-)DQG51LHWR'LIIHUHQFHVLQZKROHERG\SURWHLQWXUQRYHUEHWZHHQ,EHULDQ
DQG/DQGUDFHSLJVIHGDGHTXDWHRUO\VLQHGH¿FLHQWGLHWV-$QLP6FL
Rutherfurd, S.M. and P.J. Moughan, 2012. Available versus digestible dietary amino acids. Brit. J. Nutrition. 108,
66
Stein, H., M. Fuller, P.J. Moughan, B. Seve and C.F.M. de Lange, 2007. Amino acid availability and digestibility in
pig feed ingredients: Terminology and application. J. Anim. Sci. 85, 172-180.
Sun, P, D. Li, B. Dong, S. Qiao, and X. Ma, 2008. Effects of soybean glycinin on performance and immune function
LQHDUO\ZHDQHGSLJV$UFK$QLP1XWU
7DPPLQJD6+6FKXO]H-YDQ%UXFKHPDQG-+XLVPDQ7KHQXWULWLRQDOVLJQL¿FDQFHRIHQGRJHQRXV1ORVVHV
along the gastro-intestinal tract of farm animals. Arch. Anim. Nutr. 48, 9-22.
Torrallardona, D. and E. Roura (Editors), 2009. Voluntary feed intake in pigs. Wageningen, The Netherlands: Wageningen
Academic Publishers.
9DQGHQ%RUQH--*&-:6FKUDPD-:+HHWNDPS0:$9HUVWHJHQDQG:--*HUULWV6\QFKURQL]LQJWKH
DYDLODELOLW\RIDPLQRDFLGVDQGJOXFRVHGHFUHDVHVIDWUHWHQWLRQLQKHDY\SUHUXPLQDQWFDOYHV-1XWU
Van den Borne, J.J.G.C., J.W. Schrama, J.W. Heetkamp, M.W.A. Verstegen and W.J.J. Gerrits, 2007. Synchronizing
WKHDYDLODELOLW\RIDPLQRDFLGVDQGJOXFRVHLQFUHDVHVSURWHLQUHWHQWLRQLQSLJV$QLPDO
9DQ0LOJHQ-0RGHOOLQJELRFKHPLFDODVSHFWVRIHQHUJ\PHWDEROLVPLQPDPPDOV-1XWU
Van Milgen, J., Bernier, J.F., Lecozler, Y., Dubois, S. and J. Noblet, 1998. Major determinants of fasting heat production
and energetic cost of activity in growing pigs of different body weight and breed/castration combination. Brit. J.
Nutr. 79, 1-9.
9DQ0LOJHQ--1REOHWDQG6'XERLV(QHUJHWLFHI¿FLHQF\RIVWDUFKSURWHLQDQGOLSLGXWLOL]DWLRQLQJURZLQJ
pigs. J. Nutr. 131, 1309-1318.
Van Milgen, J., J. Noblet, A. Valancogne, S. Dubois and J.Y. Dourmad, 2008. InraPorc: a model and decision support
tool for the nutrition of growing pigs. Anim. Feed Sci. and Techn. 143, 387-405.
Westerterp-Plantenga, M.S., S.G. Lemmens and K.R. Westerterp, 2012 Dietary protein – its role in satiety, energetics
weight loss and health. Brit. J. Nutr. 108, S-105-112.
Weurding, R. E., H. Enting and M.W.A. Verstegen, 2003. The effect of site of starch digestion on performance of
broiler chickens. Anim. Feed Sci. Techn. 110, 175-184.
Weis, R.N., S.H. Birkett, P.C.H. Morel and C.F.M. de Lange, 2004. Independent effects of energy intake and body
weight on physical and chemical body composition in growing entire male pigs. J. Anim. Sci. 82, 109-121.
Williams, N.H., T.S. Stahly and D. R. Zimmerman, 1997. Effect of chronic immune system activation on the rate,
HI¿FLHQF\DQGFRPSRVLWLRQRIJURZWKDQGO\VLQHQHHGVRISLJVIHGIURPWRNJ-$QLP6FL

154 Energy and protein metabolism and nutrition in sustainable animal production
Yáñez, J.L., E. Beltranena, M. Cervantes and R.T. Zijlstra, 2011. Effect of phytase and xylanase supplementation or
particle size on nutrient digestibility of diets containing distillers dried grains with solubles cofermented from
wheat and corn in ileal-cannulated grower pigs. J. Anim. Sci. 89, 113-123.
<HQ7-2[\JHQFRQVXPSWLRQDQGHQHUJ\ÀX[RISRUFLQHVSODQFKQLFWLVVXHV3DJHVLQ-3/DSODFH
C. Fevrier and A. Barbeau. (Editors). Digestive Physiology in pigs. EAAP Publication no. 88. INRA, France.
Young, J.M. and J.C.M. Dekkers, 2012. The genetic and biological basis of residual feed intake as a measure of feed
HI¿FLHQF\3DJHVLQ-)3DWLHQFH (GLWRU )HHGHI¿FLHQF\LQ6ZLQH:DJHQLQJHQ$FDGHPLF3XEOLVKHUV
Wageningen, The Netherlands.
Zhu, C.L., M. Rademacher and C.F.M. de Lange, 2005. Increasing dietary pectin level reduces utilization of digestible
threonine intake, but not lysine intake, for body protein deposition in growing pigs. J. Anim. Sci. 83, 1044-1053.
=LMOVWUD575-KD$':RRGZDUG-)RXKVHDQG7$7*YDQ.HPSHQ6WDUFKDQG¿EHUSURSHUWLHVDIIHFW
their kinetics of digestion and thereby digestive physiology in pigs. J. Anim. Sci. 2012, 90 (Suppl. 4), 49-58.
Zijlstra, R.T., A. Owusi-Asiedu and P.H. Simmins, 2010. Future of NSP-degrading enzymes to improve nutrient
utilization of co-products and gut health in pigs. Livest. Sci. 134, 255-257.

Energy and protein metabolism and nutrition in sustainable animal production 155
Effects of low birth weight and 3 wk feed restriction on energy
metabolism in growing pigs
R. Krüger, S. Görs, K. Martens, M. Derno, B.U. Metzler-Zebeli, C. Nebendahl, H.M. Hammon and
C.C. Metges
,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 
'XPPHUVWRUI*HUPDQ\[email protected]

Introduction
Intrauterine growth retardation (IUGR) has been shown to affect intestinal growth, resulting in
impaired feed utilization, growth performance, and development of skeletal muscle (Quiniou et al.,
2002; Wu et al.0LFKLHOVet al., 2013). This leads to altered muscle and fat proportions (Powell
and Aberle, 1980; Rehfeldt et al. 7HPSRUDU\IHHGUHVWULFWLRQ )5 LQSLJVLVDVVRFLDWHGWR
physiological changes with respect to energy expenditure (EE) and nutrient oxidation pattern during
and after FR compared to adequate dietary conditions (Chwalibog et al., 2004). We explored possible
changes in energy metabolism of IUGR pigs before, during and after a 3 wk period of FR, compared
to pigs with normal birth weight.

Material and methods

After weaning (d28) female German Landrace pigs with low (L; 0.8-1.2 kg; n=22) or normal birth
weight (N; 1.3-1.8 kg; n=24) were fed ad libitum until the age of d 79 according to recommendations.
7KHGLHWFRQVLVWHGRIFRPPHUFLDOVWDUWHUIHHG  RDWÀDNHV  DQGVXFURVH  6XEVHTXHQWO\
half of L and N pigs were continued on ad libitum feeding (control C; LC and NC) with grower feed
 RDWÀDNHV  DQGVXFURVH  XQWLOG7KHUHPDLQLQJSLJVZHUHIHGUHVWULFWLYHO\
(R; LR and NR) from d 80-100 (57% of ad libitum intake of C; grower feed) before they were refed
ad libitum from d 101-130.

Body weight (BW) and feed intake (FI) were monitored. Using indirect calorimetry energy
expenditure (EE; kJ/kg BW), respiratory quotient (RQ), resting metabolic rate (RMR; kJ/kg
BW), carbohydrate oxidation (COX; g/kg BW, g/kg BW/kg FI), and fat oxidation (FOX;
g/kg BW, g/kg BW/kg FI) were calculated on the basis of 24h O2 consumption and 24h CO2
SURGXFWLRQ(QHUJ\HI¿FLHQF\ 4 ZDVFDOFXODWHGDVWKHTXRWLHQWRI(( 0- DQGHQHUJ\LQWDNH
(MJ) to compare LC and NC pigs. Measurements were performed 4 d before (T1) and 4 d after
(T2a) beginning of FR, 4 d before (T2b) and 4 d after (T3a) end of FR, and 25 d after beginning of
refeeding (T3b). Statistical analyses were performed with SAS using PROC MIXED procedure and
Tukey-Kramer post-hoc test (P” 

Results and discussion

)URPELUWKWRG/SLJVZHUHOLJKWHUWKDQ1SLJV GYVNJ3 0.012), although FI was
permanently higher in L pigs (41 vs. 38 g/kg BW on d129; 3  EXWHQHUJ\HI¿FLHQF\ 4YDOXH 
between L and N pigs did not differ.

LC pigs generally exhibited lower FOX (i.e. higher fat synthesis) than NC pigs, irrespective of
age (LC vs. NC: -3.5 vs. 0.0 g/kg BW; 3 0.02). Independent of birth weight FR resulted in
increased FOX at T2a and T2b to cover energy demands (T2a, R vs. C: 5.7 vs. 0.7, P<0.001; T2b,
R vs. C: 4.4 vs. -0.1 g/kg BW, 3 0.025), accompanied by decreased COX as a consequence of
reduced carbohydrate intake (T2a, R vs. C: 45 vs. 59, P<7E5YV&YVJNJ%:,
P<0.001). The RMR (T2b) decreased in order to defend BW (Figure 1). When refed (T3a) EE and
RMR of R pigs increased up to the levels of C pigs, whereas COX in R pigs exceeded the level of

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 157
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_43, © Wageningen Academic Publishers 2013
C pigs (R vs. C: 87 vs. 72 g/kg BW, 3 0.001) due to higher FI after FR (41 vs. 43 g feed/kg
BW, C vs. R; 3 0.03). In R pigs COX related to FI was also higher (T3a, R vs. C: 118 vs. 98 g
feed/kg BW, P<0.001), suggesting glucose utilization to provide NADPH for fat synthesis. At
the same time FOX in R pigs decreased below the level of C pigs, indicating replenishment of fat
depots (T3a, R vs. C: -10 vs. -4 g/kg BW, 3 0.001).

While FR acutely affected components of energy metabolism, IUGR pigs had higher COX and lower
FOX values (both also in relation to FI) compared to N pigs. The magnitude of FR and refeeding
effects in N and L pigs was similar. However, both FR and IUGR induced changes of components
of energy metabolism were not persistent after realimentation.

1400 1050 B. RMR

A. EE

kJ/kg BW 0.62
1350 1000
kJ/kg BW 0.62

1300
950
1250
1200 900
1150 850
1100
1050 800
1000 750
T1 T2a T2b T3a T3b T1 T2a T2b T3a T3b
)LJXUHK(( $ DQG505 % EHIRUH 7 GXULQJ 7DDQG7E DQGDIWHU 7DDQG7E )5
LQSLJVZLWKQRUPDORUORZELUWKZHLJKWFRPSDUHGWRDGOLELWXPIHGSLJV/60HDQV“6(Q 
1&ņŶņ15Ƒ/&²Ÿ²/5¨

Acknowledgements
The study was supported by the German Ministry of Education and Research within the
framework of BioMed-Ernährungsforschung – für ein gesundes Leben, Modul: Biomedizinische
Ernährungsforschung‘ (Vision Epifood, FKZ 0315397B).

References
Chwalibog, A., A.H. Tauson, and G. Thorbek, 2004. Energy metabolism and substrate oxidation in pigs during feeding,
starvation and refeeding. J. Anim. Physiol. Anim. Nutr. 88, 101-112.
Michiels, J., M. De Vos, J. Missotten, A. Ovyn, S. De Smet, and C. Van Ginneken, 2013. Maturation of digestive
function is retarded and plasma antioxidant capacity lowered in fully weaned low birth weight piglets. Br. J. Nutr.
  
Powell, S.E. and E.D. Aberle, 1980. Effects of birth weight on growth and carcass composition of swine. J. Anim.
6FL  
Quiniou, N., J. Dagorn, and D. Gaudré, 2002. Variation of piglets’ birth weight and consequences on subsequent
SHUIRUPDQFH/LYHVWRFN3URG6FL  
5HKIHOGW&DQG*.XKQ&RQVHTXHQFHVRIELUWKZHLJKWIRUSRVWQDWDOJURZWKSHUIRUPDQFHDQGFDUFDVVTXDOLW\
in pigs as related to myogenesis. J. Anim. Sci. 84 (Suppl.), E113-E123.
:X*):%D]HU-0:DOODFHDQG7(6SHQFHU%RDUGLQYLWHGUHYLHZ,QWUDXWHULQHJURZWKUHWDUGDWLRQ
,PSOLFDWLRQVIRUWKHDQLPDOVFLHQFHV-$QLP6FL

158 Energy and protein metabolism and nutrition in sustainable animal production
,QÀXHQFHRIIHHGLQJOHYHODQGHQHUJ\VRXUFHRQO\VLQHUHTXLUHPHQWVLQ
growing pigs
E.M.A.M. Bruininx1,2, W.J.J. Gerrits1, I. Eising1, P. Vervenne2, P. Sakkas1, and J.J.G.C. van den
Borne1
1$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands; [email protected]
2$JUL¿UP,QQRYDWLRQ&HQWHU32%R[+$$SHOGRRUQWKH1HWKHUODQGV

Introduction
In swine nutrition, minimizing the oxidation of imbalanced amino acids is an important issue in
optimizing the use of feed resources. Numerous titration studies have been conducted to determine
the optimum lysine-to-energy ratio, increasingly using within-animal titration approaches. Whether
or not amino acid requirements depend on the level of feed intake has been subject of debate, with
some studies indicating lower lysine requirements with increasing starch intake (Kampman-Van de
Hoek et al., 2013), whereas others found no change with feeding level (Bikker et al., 1994).

Starch and sugars are the predominant energy sources in most swine diets, but dietary fat can also
EHDQLPSRUWDQWHQHUJ\VRXUFH%HQH¿FLDOHIIHFWVRIKLJKIDWOHYHOVRQWKHGLJHVWLRQDQGXWLOL]DWLRQ
of protein in pigs have been reported. Protein digestibility increased (Li and Sauer, 1994) and
GLJHVWLEOH1ZDVUHWDLQHGPRUHHI¿FLHQWO\ %HUVFKDXHUet al., 1983) in response to an iso-energetic
exchange of starch by fat. These interactions of energy source with protein metabolism indicate
that the optimum lysine-to-energy ratio may depend on the energy source used in swine diets. In
addition, it is important to establish whether such relationship would depend on feed intake. The
aim of the current study was therefore to determine the effects of energy source (lard vs. starch)
and feeding level on lysine requirements in growing pigs using a novel within-animal amino acid
titration technique (Kampman-Van de Hoek et al., 2013).

Material and methods

In a 2×2 factorial design, 28 crossbred boars were assigned to one of two feeding levels (FL, 2.2
vs. 2.7 × metabolizable energy requirements for maintenance; MEm, 458 kJ ME/(kg0.75 × d)) and to
one of two diets differing in major dietary energy source (ES, starch vs. lard). Starch and fat were
iso-energetically exchanged on a digestible energy (DE) basis, i.e. 250 g corn starch was substituted
for 104 g lard. A within-animal lysine titration technique was used to assess the responses to changes
in lysine-to-energy ratio. After a 14-d adaptation period to housing and feeding conditions, the
apparent ileal digestible lysine (AIDLys) supply to pigs was decreased from 1.74 to 0.53 g/MJ DE in
9 equidistant steps of 3 d each with a step size of 15 mg/MJ DE. Urine was quantitatively collected
in 24-h intervals and was analyzed for N. Feces were collected over a period of 27 d to calculate
apparent total tract digestibility. Retention of digestible N was expressed relative to digestible N
LQWDNHLQGLFDWLQJWKHHI¿FLHQF\RIGLJHVWLEOH1XWLOL]DWLRQIRUSURWHLQGHSRVLWLRQ$OLQHDUSODWHDX
PRGHO .RRSVDQG*URVVPDQ ZDV¿WWHGWRWKH1UHWHQWLRQGDWDDJDLQVW$,'/\VLQWDNH LQJ
MJ DE) for each pig individually. Data were also analyzed by linear regression procedures. When the
non-linear model described the response of N retention to changes in AIDLys intake better (P<0.1)
than the linear model, lysine requirement could be estimated. Effects of FL, ES and their interaction
on parameter estimates were tested by the GLM procedure of SAS.

Results
The linear-plateau model described the response of N retention to changes in AIDLys intake better
(P<0.1) than the linear model for 25 out of 28 pigs. The estimated AIDLys requirement for these 25

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 159
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_44, © Wageningen Academic Publishers 2013
pigs varied from 0.75 to 0.98 g AIDLys/MJ DE but was not affected by FL or ES (Table 1). Other
FXUYH¿WWLQJSDUDPHWHUHVWLPDWHVZHUHQRWDIIHFWHGE\)/RU(6

Apart from a tendency for starch digestibility there were no interactions between ES and FL. Total
tract digestibility of energy was 2.2%-units higher (P<0.001; data not shown) for the starch diet
than for the fat diet, whereas it tended (3 0.07) to be 0.8%-units lower in the 2.7 × MEm group
than in the 2.2 × MEm group. Total tract protein digestibility tended (3 0.10; data not shown) to
be 0.9%-units lower in the 2.7 × MEm than in the 2.2 × MEm group, but did not differ between the
starch and fat diet. The conversion of digestible energy into body weight gain tended (3 0.07; data
QRWVKRZQ WREHOHVVHI¿FLHQWLQSLJVIHGWKHIDWGLHW

7DEOH(IIHFWVRIIHHGLQJOHYHO YVî0(m DQGHQHUJ\VRXUFH ODUGYVVWDUFK RQSDUDPHWHU

HVWLPDWHV 6(0 IURPDOLQHDUSODWHDXPRGHOGHVFULELQJWKHUHODWLRQEHWZHHQ$,'/\VVXSSO\ J
0-'( DQG1UHWHQWLRQ RIGLJHVWLEOH1LQWDNH LQJURZLQJSLJV

Feeding level P-value Energy source P-value

2.2 × MEm 2.7 × MEm Lard Starch

No. of pigs 13 12 13 12
Intercept, % 11.2±2.0 11.8±2.1 0.821 11.7±2.0 11.3±2.1 0.895
Slope1 “ “  “ “ 0.737
Transition point2 0.84±0.02 “ 0.403 0.85±0.02 0.85±0.02 0.938
Plateau, % “ “ 0.355 “ “ 0.949

1 The slope represents the incremental N retention (% of digestible N intake) per g AIDLys/MJ DE.
2 The transition point represents the AIDLys requirement in g/MJ DE.

Discussion and conclusions

In line with a study in female pigs (Bikker et al., 1994) our results show that the lysine requirements,
expressed as AIDLys relative to DE intake, do not depend on the level of feed intake in growing
male pigs. Although other studies (Berschauer et al., 1983; Bruininx et al., 2011) have indicated
that post-absorptive effects may contribute to an increased N utilization for growth when feeding
high-fat diets, the current study shows that dietary N utilization and lysine requirements in growing
pigs are not affected by energy source.

References
Bikker, P., M.W.A. Verstegen, R.G. Campbell, and B. Kemp, 1994. Digestible lysine requirement of gilt with high
genetic potential for lean gain, in relation to the level of energy intake. J. Anim. Sci. 72, 1744-1753.
Berschauer, F., U. Ehrensvard, G. Gaus, and K.H. Menke, 1983. Untersuchungen über ernährungphysiologische
:LUNXQJHQYRQ)XWWHUIHWWHQLQ5DWLRQHQIUZDFKVHQGH6FKZHLQH$UFK7LHUHUQDKU
Bruininx, E.M.A.M., J.J.G.C. van den Borne, E. van Heugten, J. van Milgen, M.W.A. Verstegen, and W.J.J. Gerrits,
2011. Oxidation of dietary stearic, oleic and linoleic acids in growing pigs follows a biphasic pattern. J. Nutr.

Kampman-van de Hoek, E., W.J.J. Gerrits, C.M.C. van der Peet-Schwering, A.J.M. Jansman, and J.J.G.C. van den
Borne, 2013. A simple amino acid dose-response technique to quantify amino acid requirements of individual
meal-fed pigs. Submitted to J. Anim. Sci.
Koops, W., and M. Grossman, 1993. Multiphasic allometry. Growth Dev. Aging. 57, 183-192.
Li, S., and W.C. Sauer, 1994. The effect of dietary fat content on amino acid digestibility in young pigs. J. Anim. Sci.
72, 1737-1743.

160 Energy and protein metabolism and nutrition in sustainable animal production
The use of free amino acids in piglet diets allows the formulation of very
low crude protein diets
M. Gloaguen1,2, N. Le Floc’h1,2, Y. Primot, E. Corrent and J. van Milgen1,2
1,15$8053(*$6(5HQQHV)UDQFH[email protected]
2$JURFDPSXV2XHVW8053(*$6(5HQQHV)UDQFH
$MLQRPRWR(XURO\VLQHVDV3DULV&HGH[)UDQFH

Introduction
Reducing the dietary crude protein (CP) content using free amino acids (AA) enables improving the
HI¿FLHQF\RIQLWURJHQXWLOL]DWLRQZKLOHPDLQWDLQLQJSHUIRUPDQFHRISLJVDVORQJDV$$UHTXLUHPHQWV
are met. Valine, Ile, His and Leu requirements have been recently estimated (Barea et al., 2009; Van
Milgen et al., 2012; Gloaguen et al., 2012), which makes it possible to formulate diets with a very low
CP content. The objective of this study was to evaluate to what extent the CP content of diets can be
reduced by substituting soybean meal by wheat, barley, and free AA, without affecting performance.

Material and methods

A trial was conducted to measure performance of 84 pigs allotted to six levels of dietary CP. Four
GLHWVZHUHEDVHGRQFHUHDOVDQGVR\EHDQPHDODQGSURYLGHGDQG&37ZR
additional low-CP diets without soybean meal were formulated which provided 13.0 and 14.0%
CP. For the 13.0% CP diet, L-Arg, L-Pro and L-Gly were added to provide the same level of SID
3UR/\VDQG *O\6HU /\VDVWKH&3GLHW/*OXWDPDWHZDVDGGHGDVD1VRXUFHWRDWWDLQ
13.0% CP. In the 14.0% CP diet, L-Glu was added to attain 14.0% CP. All diets were formulated
to provide 1.00% standardized ileal digestible (SID) Lys. Different levels of AA supplementation
ZHUHXVHGWRPDWFKWKHUHTXLUHPHQWRI6,'7KU/\V   0HW&\V /\V  7US/\V  
9DO/\V  ,OH/\V  /HX/\V  +LV/\V  3KH/\V   3KH7\U /\V
(95%), and Arg:Lys (42%). Diets were formulated to provide 9.2 MJ NE/kg, 4.0 g digestible P/kg,
2.9% Ca/digestible P, and an electrolyte balance of 180 mEq/kg.

Six-week-old barrows and female piglets (Pietrain × (Large White × Landrace)) were used. At 5 wk
of age, pigs were blocked by sex, BW, and origin (siblings or half-siblings) and housed individually.
3LJOHWVZLWKLQDEORFN Q  ZHUHDOORWWHGWRWKHGLIIHUHQWGLHWDU\WUHDWPHQWV7KHSUHVWDUWHUGLHW
was gradually replaced by the experimental diets at 10 days post-weaning so that from 12 days
post-weaning onwards, pigs were offered the experimental diets only. Pigs had free access to water
and feed. The experiment lasted 21 d. Pigs were weighed at the beginning and at the end of the
experimental period after an overnight fast for calculation of average daily gain (ADG). Feed intake
and feed refusals were measured weekly for calculation of average daily feed intake (ADFI).

Results and discussion

'LHWDU\&3OHYHOGLGQRWLQÀXHQFH$'),/RZHULQJWKHGLHWDU\&3FRQWHQWIURPWRKDG
QRHIIHFWRQ$'* 7DEOH +RZHYHU$'*DQGIHHGHI¿FLHQF\ZHUHORZHUIRUSLJVIHGWKH
CP diet compared to pigs fed the other diets (P<0.05). In diets formulated without soybean meal,
IHHGHI¿FLHQF\ZDVORZHULQSLJVRIIHUHGWKH&3GLHWWKDQLQWKRVHRIIHUHGWKH&3GLHW
(P< 7KHDGGLWLRQRI/*OXWRDYHU\ORZ&3GLHWUHVWRUHGIHHGHI¿FLHQF\WROHYHOVREVHUYHG
with higher CP contents. The results of this study show that the CP content of a cereal-soybean
meal diet, containing 1.00% SID Lys and supplemented with free Lys, Thr, Met, and Trp, can be
decreased by 4 percentage units with the addition of free Val, Ile, Leu, His, and Phe without affecting
performance in 10-20 kg pigs. Soybean meal can be totally replaced with cereals and free AA if the
CP content of the diet is at least 14%. Therefore, SID levels of 51% Ile:Lys, 100% Leu:Lys, 32%

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 161
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_45, © Wageningen Academic Publishers 2013
Table 1. Effect of reducing dietary CP content on performance of pigs.

CP, % Cereals – SBM – AA Cereals – AA Statistics

  13.5 11.8 13.0 14.0 RSD2 P-value

BWi, kg 12.7 12.7   13.0 12.8  

BWf, kg 22.2a 22.2a 21.9a 20.1b 21.8a 22.3a 1.7 <0.01
ADFI, g/d  775 779 734 810 782  0.55
ADG, g/d 450a 454a 442a 358b 420a 451a  <0.01
G:F, g/g 0.59a 0.59a 0.57a 0.49b 0.52b 0.58a  <0.01

a-b Within a row, means without a common superscript differ (P<0.05); RSD = residual standard deviation.

+LV/\V3KH/\V 3KH7\U /\VDQG$UJ/\VDSSHDUWREHVXI¿FLHQWWRPD[LPL]H

JURZWK7KH&3GLHWUHVXOWHGLQDUHGXFHGIHHGHI¿FLHQF\7KHUHGXFHGSHUIRUPDQFHPD\EH
GXHWRDQ1GH¿FLHQF\EHFDXVHWKHDGGLWLRQRI/*OXWRUHDFK&3UHVWRUHGIHHGHI¿FLHQF\$Q
1GH¿FLHQF\PD\DOVRH[SODLQWKHUHGXFHGIHHGHI¿FLHQF\REVHUYHGZLWKWKHVR\EHDQPHDOEDVHG
GLHWZLWK&37KHUHVXOWVDOVRLQGLFDWHWKDWWKHHI¿FLHQF\RIXWLOL]LQJ$$LVQRWORZHUIRUIUHH
AA compared with protein-bound AA in pigs offered feed ad libitum. In agreement with our results,
Le Bellego et al. (2001) showed that the N utilization is not affected in low CP diets when at least
two meals are fed.

In conclusion, the use of free Val, Leu, Ile, His, and Phe enables a 4 percentage unit reduction in
CP content in diets for piglets and soybean meal can be totally replaced with cereals and free AA.
However, in these pigs at least 14% CP level is required to ensure the N supply. At very low CP
levels, N is a potentially limiting factor for growth in piglets.

References
Barea, R., L. Brossard, N. Le Floc’h, Y. Primot, D. Melchior and J. van Milgen, 2009. The standardized ileal digestible
valine-to-lysine requirement ratio is at least seventy percent in postweaned piglets. J. Anim. Sci. 87, 935-947.
Gloaguen M., Le Floc’h N., Primot Y., Corrent E. and van Milgen J., 2012. Response of piglets to the standardized
ileal digestible isoleucine, histidine and leucine supply in cereal-soybean meal-based diets. Animal, FirstView, 1-8.
Le Bellego, L., J. van Milgen, S. Dubois, and J. Noblet. 2001. Energy utilization of low-protein diets in growing pigs.
J. Anim. Sci. 79, 1259-1271.
Van Milgen, J. et al. 2012. Meta-analysis of the response of growing pigs to the isoleucine concentration in the diet.
$QLPDO

162 Energy and protein metabolism and nutrition in sustainable animal production
Lysine concentration in dietary protein affects performance and pattern
of nutrient retention of weaned Iberian piglets
R. Barea, L. Lara, J.F. Aguilera and R. Nieto
,QVWLWXWHRI$QLPDO1XWULWLRQ(VWDFLyQ([SHULPHQWDOGHO=DLGtQ6SDQLVK&RXQFLOIRU6FLHQWL¿F
5HVHDUFK &6,& &DPLQRGHO-XHYHVVQ$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
7KHDPLQRDFLGFRPSRVLWLRQRIGLHWDU\SURWHLQVSHFL¿FDOO\WKHHVVHQWLDODPLQRDFLGFRPSRVLWLRQ
LVDNH\IDFWRUWKDWDIIHFWVWKHHI¿FLHQF\RIXVHRIGLHWDU\SURWHLQDQGWKHUHIRUHJURZWKDQGSURWHLQ
retention. The balance among essential amino acid for maintenance and production functions has
been established in pigs, with some variations according to the physiological state of the animal
%6$6 3LJJHQRW\SHLVRQHWKHIDFWRUVWKDWPLJKWLQÀXHQFHVXFKEDODQFHDOWKRXJKOLPLWHG
information is available in the literature concerning this issue. The Iberian pig is an obese, slow-
growing breed. In previous work we have shown that Iberian pig requirements for total protein differ
markedly from those of pigs of conventional genotypes (Nieto et al., 2012), although dietary protein
was formulated following the amino acid pattern (g amino acid/kg crude protein) established for
conventional pigs. The present work aimed at establishing the dietary lysine (Lys) requirements (g
Lys/kg crude protein) for optimum performance of post-weaned Iberian piglets.

Material and methods

Sixty six castrated male Iberian piglets of 10 kg bodyweight (BW) and 40 days of age were used.
3LJOHWVZHUHIHGRQHRIVL[H[SHULPHQWDOGLHWVZKLFKGLIIHUHGLQWKHLU/\VFRQWHQW 
and 80 g Lys/kg crude protein). L-Lys. HCl was added in increasing concentration to a basal diet
FRQWDLQLQJFRUQ  EDUOH\  DQGVR\EHDQPHDO  DWWKHH[SHQVHRIFRUQVWDUFK'LHWV
were isonitrogenous and isoenergetic (170 g CP and 14.1 MJ ME per kg dry matter). At the start of the
trial, six piglets were slaughtered to estimate initial body composition. Ten piglets were allocated into
each experimental diet. They were fed twice daily ad libitum and BW was recorded weekly. Piglets
were individually housed in an environmentally controlled room (27±1.5 °C) until they reached 25
kg BW when six pigs from each dietary treatment were slaughtered. At slaughter blood, carcass and
organs were collected and weighed separately. The carcass was ground, homogenized and freeze-
dried for analysis. All analyses have been performed in duplicate. Treatment effects were assessed by
analysis of variance using the GLM procedure of SAS. Orthogonal polynomial contrasts were used
to determine linear and quadratic effects of Lys addition on performance, carcass nutrient retention
and blood metabolites. The experimental protocol was approved by the CSIC Bioethical Committee.

Results and discussion

Average daily feed intake was not affected by Lys supply (ADFI, 890 g dry matter/d; P>0.05).
Both, daily gain (ADG, g/d) and gain-to-feed ratio (ADG:ADFI) increased linearly with increasing
Lys concentration (Table 1; P< DQGVKRZHGQRVLJQL¿FDQWTXDGUDWLFHIIHFWV/LQHDUHIIHFWV
of increasing Lys concentration on dietary CP were also detected for ADG expressed per unit of
ME intake (P<0.001). Carcass gain composition showed that protein increased (linearly, P<0.001;
quadratically, P<0.05) with increasing dietary Lys concentration, reaching maximum values (141
WRJSURWHLQNJFDUFDVVJDLQ ZLWKGLHWVSURYLGLQJWRJ/\VNJ&3&DUFDVVZDWHUUHWHQWLRQ
followed a similar pattern as protein, whereas fat concentration in carcass gain decreased linearly
(P<0.01) and tended to decrease quadratically (3 0.078) on increasing dietary Lys, with maximum
YDOXHVIRUGLHWVSURYLGLQJDQGJ/\VNJGLHWDU\&31RHIIHFWRIGLHWDU\/\VZDVREVHUYHGIRU
ash carcass gain. Plasma urea concentration tend to decrease with increasing dietary Lys (3 
and 3 0.10 for linear and quadratic effects, respectively). No effect of dietary Lys on the other

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 163
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_46, © Wageningen Academic Publishers 2013
blood parameters analysed (glucose, creatinine and triglycerides) was detected. The results obtained
suggests that optimum growth and carcass protein retention in Iberian piglets can be achieved when
dietary protein supply contain at least 70 g Lys/kg CP.

7DEOH(IIHFWRIO\VLQHFRQFHQWUDWLRQRIGLHWDU\SURWHLQRQSLJOHWSHUIRUPDQFHFDUFDVV && QXWULHQW

retention and plasma urea concentration.

g Lys/kg dietary crude protein P-value1

55   70 75 80 SEM Lin Q

Average daily gain, g 387 433 435   473 8 0.001 0.249
Gain:feed, g/g 0.450 0.489 0.489 0.512 0.522 0.521 0.005 0.001 0.177
Gain:ME intake, g/MJ 31.8 34.5 34.5    0.4 0.001 0.177
CC protein gain, g/kg 125 132   141 141 1 0.001 0.044
CC water gain, g/kg 547 580 598 597 590  4 0.001 0.200
CC fat gain, g/kg 300  239 231 242 214 5 0.001 0.078
CC energy gain, MJ/kg 14.9 13.5 12.7 12.7 13.0 11.9 0.2 0.001 0.128
CC ash gain, g/kg   27.7    0.8 0.549 0.772
Plasma urea, mg/100 ml 24.2  19.1 18.9 20.5 18.7   0.100

1 Contrast: Lin=linear effects; Q=quadratic effects.

Acknowledgements
)XQGLQJE\WKH6SDQLVK0,1(&2 JUDQWVUHI$*/DQG$*/ LVJUDWHIXOO\
acknowledged.

References
BSAS. 2003. Nutrient Requirement Standards for Pigs. Brit. Soc. Anim. Sci., Penicuik, UK, 28 pp.
Nieto, R., L. Lara, R. Barea, R. García-Valverde, M.A. Aguinaga, J.A. Conde-Aguilera and J.F. Aguilera. 2012.
Response analysis of the Iberian pig growing from birth to 150 kg body weight to changes in protein and energy
supply. J. Anim. Sci. 90, 3809-3820.

164 Energy and protein metabolism and nutrition in sustainable animal production
Effect of processing of oilseed meals on the apparent ileal protein
digestibility and performance in pigs
T.G. Hulshof1,2, P. Bikker1 and A.F.B. van der Poel2
1:DJHQLQJHQ85/LYHVWRFN5HVHDUFK(GHOKHUWZHJ3+/HO\VWDGWKH1HWKHUODQGV
[email protected]
2$QLPDO1XWULWLRQ*URXS:DJHQLQJHQ8QLYHUVLW\'H(OVW:':DJHQLQJHQWKH1HWKHUODQGV

Introduction
Feed ingredients, e.g. oil seed by-products, generally have undergone several processing steps before
inclusion in animal diets. Processing of feed ingredients and diets can result in conformational changes
of protein structure, the formation of Maillard reaction products and cross-links between amino
acids (Bender, 1972). These changes affect protein quality and the latter two reactions especially
reduce the amount of the essential amino acid lysine (Mauron, 1990). The effects on protein quality
may result in an impaired pig performance presumably related to a decrease in protein, amino acid
and lysine digestibility as found for processed diets (González-Vega et al., 2011). The aims of this
research were to determine the effects of processing of two oil seed by-products, i.e. soybean meal
(SBM) and rapeseed meal (RSM), on apparent ileal crude protein, amino acid and lysine digestibility
and performance in growing pigs and to derive criteria to evaluate protein quality in heat treated
ingredients. First results of ileal crude protein digestibility are presented here.

Material and methods

Ten barrows (initial BW 24.8±0.28 kg) were suited with a SICV cannula (Mroz et al. DQG
individually housed in metabolism cages. Four experimental diets (Table 1) were used, consisting
of a basal N-free diet supplemented with 35% (un)processed SBM or RSM in combination with 7
or 5% of the sugar rich compound XyligTM, respectively. SBM and RSM were used as purchased
or toasted after mixing with XyligTM, in order to induce changes in protein quality. The RSM diets
were supplemented with synthetic lysine, threonine and tryptophan to levels corresponding to the
levels in the SBM diets. In all diets Cr2O3 was added as indigestible marker to calculate the apparent
ileal crude protein digestibility (AID CP). The experiment consisted of a cross-over design with three
periods of 14 days. Feed was provided twice a day at 2.8 times maintenance energy. Ileal chyme
was collected during 12 hours on days 9 and 11 of each period and analysed for CP, amino acids
and Cr2O3. The gain:feed (GF) ratio was calculated as measure for nutrient utilization. The proc
PL[HGSURFHGXUHLQ6$6  ZDVXVHGWRWHVWIRUWKH¿[HGHIIHFWVRIIHHGLQJUHGLHQWSURFHVVLQJ

7DEOH1XWULHQWFRPSRVLWLRQ JNJ RIWKHH[SHULPHQWDOGLHWVFRQWDLQLQJHLWKHUXQSURFHVVHGRU

WRDVWHGVR\EHDQPHDO 6%0 RUUDSHVHHGPHDO 560 

Nutrient SBM RSM

Unprocessed Toasted Unprocessed Toasted

Dry matter 888 895 888 893

Ash 45  39 40
Crude protein  170 121 125
&UXGH¿EUH 12 11 43 47
Crude fat 30 27 32 35
Starch 423 423 433 
Sugars 142  135 129

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 165
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_47, © Wageningen Academic Publishers 2013
treatment, and their interaction on AID CP and GF ratio, with period and animal in the random
statement. PYDOXHVZHUHDVVXPHGWREHVLJQL¿FDQW

Results and discussion

The results of the AID CP and GF ratio are given in Table 2. The interaction between feed ingredient
DQGSURFHVVLQJWUHDWPHQWZDVQRWVLJQL¿FDQWIRU$,'&3 3 0.40) and GF ratio (3  LQGLFDWLQJ
that processing has similar effects on both characteristics for SBM and RSM diets. The animals fed
WKH560GLHWVKDGDVLJQL¿FDQWO\ORZHU$,'&3WKDQWKHDQLPDOVIHGWKH6%0GLHWV7KLVDJUHHVZLWK
other studies indicating that RSM in general has a lower AID CP than SBM (Grala et al., 1998). The
W\SHRIIHHGLQJUHGLHQWGLGQRWLQÀXHQFHWKH*)UDWLRGHVSLWHWKHORZHU$,'&3FRQWHQWSUHVXPDEO\
because of the supplementation of the RSM diets with synthetic lysine, threonine and tryptophan.
3URFHVVLQJRIWKH6%0DQG560UHGXFHGWKH$,'&3DVZHOODVWKH*)UDWLR7KHORZHUHI¿FLHQF\
of nutrient utilization for tissue deposition as indicated by the reduction in GF ratio might be caused
by the reduction in protein digestibility. In conclusion, additional processing of SBM and RSM, i.e.
mixing with a sugar rich compound and toasting, reduced the crude protein digestibility and nutrient
utilization as indicated by the GF ratio. The effects of individual amino acids will be further examined.

7DEOH(IIHFWRIIHHGLQJUHGLHQWLHVR\EHDQPHDO 6%0 RUUDSHVHHGPHDO 560 DQGSURFHVVLQJ

WUHDWPHQWLHQRQHRUWRDVWLQJRQDSSDUHQWLOHDOFUXGHSURWHLQGLJHVWLELOLW\ $,'&3 DQGJDLQIHHG
*) UDWLRLQSLJV

SBM RSM SEM P-value

Unprocessed Toasted Unprocessed Toasted Feed Processing Interaction

ingredient treatment

AID CP 78.4c a a b  <0.0001 <0.0001 0.40

GF ratio 0.48a 0.43ac 0.45a 0.38bc 0.024 0.10 0.02 

a,b,cGLIIHUHQWVXSHUVFULSWVLQWKHVDPHURZLQGLFDWHVLJQL¿FDQWO\ P<0.05) different values.

Acknowledgements
7KHDXWKRUVJUDWHIXOO\DFNQRZOHGJHWKH¿QDQFLDOVXSSRUWIURPWKH:DJHQLQJHQ85µ,323&XVWRPL]HG
1XWULWLRQ¶SURJUDPPH¿QDQFHGE\:DJHQLQJHQ85WKH'XWFK0LQLVWU\RI(FRQRPLF$IIDLUV
$JULFXOWXUH ,QQRYDWLRQ:,$6$JUL¿UP,QQRYDWLRQ&HQWHU25))$$GGLWLYHV%9$MLQRPRWR
Eurolysine s.a.s and Stichting VICTAM BV.

References
Bender, A.E., 1972. Processing damage to protein food: a review. J. Food Technol. 7, 239-250.
González-Vega, J.C., B.G. Kim, J.K. Htoo, A. Lemme and H.H. Stein, 2011. Amino acid digestibility in heated soybean
PHDOIHGWRJURZLQJSLJV-$QLP6FL
Grala, W., M. Verstegen, A. Jansman, J. Huisman and P. van Leeusen, 1998. Ileal apparent protein and amino acid
GLJHVWLELOLWLHVDQGHQGRJHQRXVQLWURJHQORVVHVLQSLJVIHGVR\EHDQDQGUDSHVHHGSURGXFWV-$QLP6FL
0DXURQ-,QÀXHQFHRISURFHVVLQJRQSURWHLQTXDOLW\-1XWU6FL9LWDPLQRO66
0UR]=*&0%DNNHU$:-RQJEORHG5$'HNNHU5-RQJEORHGDQG$YDQ%HHUV$SSDUHQWGLJHVWLELOLW\
of nutrients in diets with different energy density, as estimated by direct and marker methods for pigs with or
without ilea-cecal cannulas. J. Anim. Sci. 74, 403-412.
SAS, 2008. Release 9.2. SAS Inst. Inc., Cary, NC, USA.

166 Energy and protein metabolism and nutrition in sustainable animal production
Determination of the next limiting amino acid in young piglets
A.J.M. Jansman1, H. van Diepen1, M. Rovers2 and E. Corrent
1:DJHQLQJHQ 85 /LYHVWRFN 5HVHDUFK 32 %R[   $% /HO\VWDG WKH 1HWKHUODQGV
[email protected]
22UIID$GGLWLYHV%99LHUOLQJKVWUDDW/&:HUNHQGDPWKH1HWKHUODQGV
$MLQRPRWR(XURO\VLQHVDV5XHGH&RXUFHOOHV3DULV&HGH[)UDQFH

Introduction
After lysine, methionine/cysteine, threonine, tryptophan, and valine it is not clear which essential
amino acid becomes limiting in low protein diets for post weaning piglets. This is related to a
scarcity of information on requirement for other essential amino acids in piglets. Isoleucine, leucine
and histidine are among the candidates to be the next limiting amino acid (Chung and Baker, 1992;
Barea et al., 2009). The aim of the present study was to identify the next limiting amino acid after
lysine, methionine/cysteine, threonine, tryptophan, and valine in diets for post weaning piglets in a
performance study over a period of 4 weeks in the post-weaning period.

Material and methods

Six dietary treatments were evaluated in eight replicates (pens) with eight piglets (barrows) per
pen, weaned at an age of about 28 d. A basal experimental diet with a low crude protein content
(140 g/kg) was formulated based on maize, wheat, barley, soybean meal and whey powder. The
diet was supplemented with free L-lysine, DL-methionine, L-threonine, L-tryptophan, L-valine and
L-phenylalanine to meet the requirement values for these amino acids (10.4 g/kg standardized ileal
GLJHVWLEOH 6,' /\VJNJ6,'PHWKLRQLQHF\VWHLQHJNJ6,'WKUHRQLQHJNJ6,'
WU\SWRSKDQJNJ6,'YDOLQHDQGJNJ6,'SKHQ\ODODQLQHWUHDWPHQW, 7KHEDVDOGLHWZDV
VXSSRVHGWREHGH¿FLHQWLQLVROHXFLQHKLVWLGLQHDQGOHXFLQHIRUSRVWZHDQLQJSLJOHWV JNJ6,'
LVROHXFLQHJNJ6,'KLVWLGLQHDQGJNJ6,'OHXFLQHDQGUHODWLYHWR6,'/\V
respectively). Treatment II received a diet composed of the basal diet supplemented with 1.2 g/kg
/LVROHXFLQHJNJ/KLVWLGLQHDQGJNJ/OHXFLQHXSWRWKHDVVXPHGUHTXLUHPHQWOHYHOVRI
53, 32 and 101% of the content of SID lysine (NRC, 1998). In three other dietary treatments the
supplementation of either L-isoleucine, L-histidine or L-leucine was omitted in the respective diets
for treatments III, IV and V resulting in diets containing 79, 81, and 88% of the assumed requirement
value for isoleucine, histidine and leucine on a SID basis in the respective diets. A reference treatment
9, ZDVLQFOXGHGIHGDGLHWZLWKDFUXGHSURWHLQFRQWHQWRIDERXWJNJVXI¿FLHQWLQDOOHVVHQWLDO
amino acids. Except for amino acids, the diets were formulated to be nutritionally complete (CVB,
15& ,QWKHZHHNDIWHUZHDQLQJWKHGLHWIRUWUHDWPHQW9,ZDVJLYHQWRSLJOHWVLQDOO
treatments. The experimental diets in pelleted form were fed over a subsequent period of four weeks.
Feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were determined over
2-weeks periods.

Results and discussion

2YHUZHHNDQGRIWKHH[SHULPHQWDOSHULRG),%:*DQG)&5ZHUHVLJQL¿FDQWO\DIIHFWHG
by treatment (P<0.05) (Table 1). Compared to treatment I, FI and BWG were numerically higher in
treatment II in weeks 1-2 and 3-4 (P>0.05) and FCR numerically improved in weeks 3-4 (P>0.05).
BWG and FCR were improved for treatment VI compared to treatments I and II in week 1-2 (P<0.05)
and compared to treatment I in week 3-4 (P<0.05).

Over week 1-2, omitting free L-isoleucine supplementation in treatment III resulted in a lower
FI and BWG compared to treatment II (P<0.05), while the FCR was not different. Omitting the

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 167
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_48, © Wageningen Academic Publishers 2013
7DEOH)HHGLQWDNHERG\ZHLJKWJDLQDQGIHHGFRQYHUVLRQUDWLRRYHUZHHNVDQGRIWKH
H[SHULPHQWDOSHULRG ZHHNV 

FI (kg/d) BWG (g/d) FCR

wk 1-2 wk 3-4 wk 1-2 wk 3-4 wk 1-2 wk 3-4

I 0.599abc 1.133ab 410ab ab b bc

II bc 1.154ab 428b 715bc b ab
III a 1.191b 395a 712bc 1.459b c
IV abc 1.107a ab a 1.455b bc
V 0.597ab 1.187b 410ab 727c b abc
VI c 1.174b c 735c 1.355a 1.599a
P 0.023 0.059 <0.001 0.003 <0.001 0.079
LSD 0.031  29 35 0.052 0.055

a,b,c Values with a different superscript in the same column within a factor differ at P<0.05.

supplementation of L-histidine and L-leucine in week 1-2 did not affect performance compared to
treatment II. The reduction in feed intake and body weight gain when omitting the supplementation
RI/LVROHXFLQHLQWKH¿UVWWZRZHHNVLQWUHDWPHQW,,,FRPSDUHGWRWKHDSSDUHQWDEVHQFHRIDUHVSRQVH
to the omission of supplementation of L-histidine or L-leucine in treatments IV and V suggest that
isoleucine was more limiting than histidine and leucine in piglets of this age. Over week 3-4, omitting
the supplementation of L-histidine (treatment IV) reduced BWG compared to treatment II (P<0.05),
while omitting the supplementation of L-isoleucine and L-leucine did not affect performance over
this period. The former suggest that, in contrast to the results in week 1-2, the dietary supply of
histidine was more limiting in week 3-4 compared to the supply of isoleucine and leucine.

It is concluded that amino acid requirements of piglets change during the post weaning period
resulting in isoleucine being the sixth limiting amino acid after lysine, methionine/cysteine, threonine,
tryptophan, and valine over the period of weeks 5-7 of age (2-3 weeks after weaning) and histidine
being the sixth limiting amino acid during weeks 8-9 of age (4-5 weeks after weaning), when using
a diet based on wheat, barley, maize and soybean meal.

References
Barea R., L. Brossard, N. Le Floc’h, Y. Primot, and J. van Milgen, 2009. The standardized ileal digestible isoleucine-
WRO\VLQHUHTXLUHPHQWUDWLRPD\EHOHVVWKDQ¿IW\SHUFHQWLQHOHYHQWRWZHQW\WKUHHNLORJUDPSLJOHWV-RXUQDORI
Animal, Science 87, 4022-4031.
Chung, T.K., D.H. Baker, 1992. Ideal amino acid pattern for 10-kilogram pigs. J. Anim. Sci. 70, 3102-3111.
&9%>5HTXLUHPHQWVIRUDPLQRDFLGVLQSLJOHWVDQGSLJV@,Q'XWFK&9%UHSRUW
NRC, 1998. Nutrient Requirements of Swine. National Research Council. National Academy Press, Washington, US.

168 Energy and protein metabolism and nutrition in sustainable animal production
)HHGLQJORZSURWHLQDPLQRDFLGIRUWL¿HGGLHWVGLGQRWDIIHFW
SHUIRUPDQFHDQGFDUFDVVFRPSRVLWLRQRIJURZLQJ¿QLVKLQJSLJV
J.K. Htoo1, J. Trautwein2, J. Gao1 and G. Dusel2
1(YRQLN,QGXVWULHV$*5RGHQEDFKHU&KDXVVHH+DQDX*HUPDQ\[email protected]
28QLYHUVLW\RI$SSOLHG6FLHQFHV%LQJHQ%HUOLQVWUDVVH%LQJHQDP5KHLQ*HUPDQ\

Introduction
Lowering crude protein (CP) level and balancing with supplemental amino acids (AA) in pig diets
KDVHQYLURQPHQWDODQGHFRQRPLFEHQH¿WV+RZHYHUUHGXFHGSHUIRUPDQFHZDVREVHUYHGZKHQ&3
level was reduced greater than 4%-units in diets for growing pigs although diets were balanced to be
adequate in standardized ileal digestible (SID) essential AA (EAA; Guay et al 7KHFRQWHQW
RIQLWURJHQ 1 LQORZ&3GLHWVVRPHWLPHVPD\EHFRPHLQVXI¿FLHQWIRUHQGRJHQRXVV\QWKHVLVRI
all non-essential AA (NEAA), and this may one of the possible reasons for reduced performance.
Thus, the current experiments were conducted to evaluate the effects of balancing low CP diets
with supplemental AA and maintaining optimal essential AA:total N ratio on performance, carcass
composition and N retention of 24 to 111 kg pigs.

Material and methods

A 83-d trial was conducted with 180 TOPIGS pigs [initial body weight (BW) of 24.4 kg] with 2 pigs
per pen and 18 pens per treatment. Pigs were assigned to 5 diets based on corn, barley, wheat, soybean
PHDO 6%0 DQGUDSHVHHGPHDOIRUHDFKRIWKHJURZHU 6,'/\VG JURZHU 6,'
/\VG ¿QLVKHU 6,'/\VG DQG¿QLVKHU 6,'/\VG SKDVHV
The high CP diet (diet 1) and low CP (diet 5; without SBM) were initially produced without inclusion
of free AA. Diets 2, 3 and 4 were produced by blending diets 1 and 5 in varying proportions (75, 50
and 25% of diet 1). All diets were analyzed for AA and CP contents, and free AA were added to meet
requirements for EAA. All diets contained similar EAA and net energy contents in each phase. Gly,
Pro, Arg and Glu were added in diets 4 and 5 of grower 1 and grower 2 diets to balance the EAA:total
N ratio to be 50% (Table 1). The step-wise reductions of CP level in the diets are shown in Table 2.

Table 1. Dietary treatments.

1 Conventional grains-SBM based diet (Control)

2 Reduced CP diet (-25% SBM inclusion) + EAA (Lys, Met, Thr)
3 Low CP diet (-50% SBM inclusion) + EAA (Lys, Met, Thr, Trp)
4 Low CP diet (-75% SBM inclusion) + EAA + NEAA (EAA:total N of 50%)
5 Low CP diet (without SBM) + EAA + NEAA + Arg in Grower 1 and 2 (EAA:total N ratio of 50%)

7DEOH$QDO\]HGFUXGHSURWHLQFRQWHQW  1 in experimental diets.

Diets Grower 1 Grower 2 Finisher 1 Finisher 2

1 21.4 (20.4) 21.3 (20.2) 17.0 (17.3)  

2 19.4 (19.8) 19.5 (19.5)   14.8 (15.5)
3 17.5 (18.9) 17.8 (18.1) 14.9 (15.7) 13.7 (13.9)
4 15.5 (18.2)   13.9 (14.8) 12.5 (12.7)
5     12.9 (13.9) 11.4 (12.4)

1 Dietary crude protein contents in parentheses are ‘with added free amino acids’.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 169
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_49, © Wageningen Academic Publishers 2013
Finisher 2 diets were fed until the pigs reached slaughter BW of approximately 117 kg, and 10
pigs per treatment except diet 3 (9 pigs) were selected for carcass assessment. On d 95, blood was
FROOHFWHGIURPSLJVSHUWUHDWPHQWWRGHWHUPLQHSODVPDXUHD1 381 FRQFHQWUDWLRQ$GGLWLRQDOO\
DG1EDODQFHWULDOZDVFRQGXFWHGE\IHHGLQJGLHWVRURI¿QLVKHUSKDVHWRSLJV LQLWLDO%:
of 111.0 kg; 3 pigs per treatment) to measure N retention. All data were analyzed by ANOVA using
GLM procedure of SAS with pen as the experimental unit.

Results and discussion

During each phase and the overall 83-d period, average daily feed intake (ADFI), average daily gain
$'* DQGIHHGFRQYHUVLRQUDWLR )&5 DVZHOODV¿QDO%:ZHUHQRWGLIIHUHQWDPRQJWUHDWPHQWV
(P>0.05; Table 3). The PUN concentration (d 95) decreased linearly (P<0.001) as dietary CP
decreased and was lowest for pigs fed diet 5 which indicates a reduced need for deamination of
excess AA (Table 4). The dressing %, lean yield and back fat thickness were not affected by the
CP levels. Daily N retention was not affected (P>0.05) but total N excretion was reduced by 32%
(P< E\ORZHULQJWKH&3OHYHOLQ¿QLVKHUGLHWIURPWR 7DEOH ,QFRQFOXVLRQ
lowering dietary CP level by replacing SBM with supplemental AA is possible to maintain optimal
pig performance when diets are balanced for adequate SID AA and NE, and maintained an EAA:total
N ratio of approximately 50%.

7DEOH(IIHFWRIGLHWDU\SURWHLQOHYHOVRQSLJSHUIRUPDQFH GWR 

Items Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 SEM P-value

BW at d 83, kg 100.0 100.4 98.5 98.1 98.0 0.943 0.171

ADFI, g/d 2,218 2,132    39.80 0.085
ADG, g/d  915 893 888   
FCR, g/g 2.41 2.35 2.43  2.33 0.039 0.324

7DEOH(IIHFWRISURWHLQOHYHOVRQSODVPDXUHD1OHYHO RQG DQGFDUFDVVTXDOLW\

Items Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 SEM P-value

Plasma urea N, mg/dl 15.41a 13.91ab 10.82bc 9.95bc 8.75c  <0.001
BW at slaughter, kg 117.7 117.8 118.3  117.2 0.553 0.904
Dressing, % 77.2  77.4 78.0  0.438 0.425
Lean meat, % 77.2  77.4 78.0  0.438 0.425
Back fat, cm 0.87 0.90 1.10 0.99 1.00 0.090 0.412

7DEOH(IIHFWRIGLHWDU\SURWHLQOHYHOVRQ1UHWHQWLRQLQNJSLJV

Items Finisher 2 diets SEM P-value

'LHW &3 Diet 5 (12.4% CP)

N intake, g/d 51.3 38.9  <0.001

N in urine, g/d 32.8 22.2  0.011
Total N excretion, g/d 40.4 29.3 1.171 0.003
N retained, g/d 10.9  1.150 0.458

170 Energy and protein metabolism and nutrition in sustainable animal production
Reference
*XD\)60'RQRYDQDQG1/7URWWLHU%LRFKHPLFDODQGPRUSKRORJLFDOGHYHORSPHQWVDUHSDUWLDOO\LPSDLUHG
in intestinal mucosa from growing pigs fed reduced-protein diets supplemented with crystalline amino acids. J.
$QLP6FL

Energy and protein metabolism and nutrition in sustainable animal production 171
Changes in protein turnover during pregnancy in pigs at amino acid
intake in excess of requirements
S. Moehn1, 05D¿L2, P.B. Pencharz2 and R.O. Ball1
1'HSDUWPHQWRI$JULFXOWXUH)RRGDQG1XWULWLRQDO6FLHQFH8QLYHUVLW\RI$OEHUWD(GPRQWRQ7*
3&DQDGD[email protected]
25HVHDUFK,QVWLWXWH+RVSLWDOIRU6LFN&KLOGUHQ7RURQWR0*;&DQDGD

Introduction
A series of experiments were completed to determine the amino acid (AA) requirements of pigs for
threonine, lysine, tryptophan and isoleucine (Moehn et al., 2011) in early and late pregnancy. The
objective of the present analysis was to determine which factors affected protein turnover during
pregnancy in pigs when the intake of test AA was above the requirement.

Material and methods

,QHYHU\H[SHULPHQWWRVRZVHDFKUHFHLYHGGLHWVZLWKJUDGHGOHYHOVRIWHVW$$LQERWKHDUO\
and late gestation. Protein turnover was determined using a stochastic model based on the 13C
enrichment in expired CO2 and plasma free phenylalanine (Phe) after oral L[1-13C]Phe dosing.
Results of all experiments were combined by expressing AA intake as a fraction of the mean
requirement determined in each experiment. The observations (n=199 for Phe oxidation [OX] and
UHWHQWLRQIRU3KHÀX[ERG\SURWHLQEUHDNGRZQ>%@DQGV\QWKHVLV>6@ FRYHUHG$$LQWDNHV
EHWZHHQDQGWLPHVWKHUHTXLUHPHQW2WKHUSDUDPHWHUVRIQXWULWLRQDQGVRZJURZWK
and physical characteristics were as described for limiting AA intake in the companion paper. Data
was evaluated using the mixed model procedure of SAS with pig nested in experiment as random
variable. The start model was the same as used for limiting AA intake, and was reduced iteratively
E\GHOHWLQJDOOQRQVLJQL¿FDQWWHUPVXQWLODOOSDUDPHWHUVOHIWZHUHDWP<0.05.

Results
The resulting models explained a high degree of the variation in the data with r2=0.78 (S) to r2 
(OX). Oxidation and Phe retention reacted oppositely in that OX decreased (P<0.02) with increasing
gestational age, Phe intake and maternal BW gain, while Phe retention increased (P<0.03). Oxidation
increased (P<0.01) with increasing BW and with an interaction between gestational age and Phe
intake, while Phe retention decreased (P<0.05). Sow age, alone and in interaction with BW, ME
intake and gestational age minimized Phe OX in the 3rd parity (P<0.03). Phe retention decreased
(3  ZLWKVRZSDULW\

Flux and B decreased (P<0.01) with increasing gestational age and increased with increasing
maternal gain. Flux and B responded quadratically (P<0.02) to increasing test AA intake in excess of
UHTXLUHPHQWVZLWKLQFUHDVHVDSSDUHQWDWWHVW$$LQWDNHVRYHUWLPHVWKHUHTXLUHPHQW$QLQWHUDFWLRQ
between test AA intake and gestational age increased (P< ÀX[DQG%ZKLOHDQLQWHUDFWLRQ
between test AA intake and maternal BW gain decreased (P< ÀX[DQG%3URWHLQV\QWKHVLV
increased (3 0.005) with increasing Phe retention and for sows with greater ME intake (3 0.08)
while an interaction between ME intake and Phe retention decreased S (3 0.011). Subsequent litter
size did not affect Phe kinetics.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 173
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_50, © Wageningen Academic Publishers 2013
Discussion
Discussion of protein metabolism in pregnant sows must take into account the competing demands
for nutrients of maternal and conceptus growth, especially when conceptus growth accelerates in
late pregnancy (NRC, 2012).

The increase in S with increasing Phe retention in sows with greater ME intake indicates that energy
LQWDNHOLPLWHG6%HFDXVHOLWWHUVL]HZDVQRWDVLJQL¿FDQWIDFWRUIRU6WKHOLPLWDWLRQRI6RFFXUUHG
for the whole of maternal body and conceptus.

,QFUHDVLQJVRZPDWHUQDO%:JDLQUHGXFHG2;DQGLQFUHDVHGÀX[DQG%5HGXFWLRQRI2;VDYHV$$
which can be utilized for increased Phe retention together with AA derived from increased B towards
the end of pregnancy, as indicated by the interaction between test AA intake and gestational age.
This agrees with the observed decrease in OX and increase in Phe retention when sows approached
parturition, and is indicative of the accelerated fetal growth in late pregnancy (NRC, 2012).

Test AA requirements have been determined over 3-week periods and represent a mean value for these
periods. At test AA intakes above requirement, no effect of AA intake on Phe kinetics was expected,
based on Salter et al. (1990) who found that lysine intake in excess of requirements had no effect on
6DQG%LQJURZLQJSLJV+RZHYHUÀX[DQG%LQSUHJQDQWVRZVLQFUHDVHGDWWHVW$$LQWDNHDERYH
requirement, and with an interaction between test AA intake and gestational age. This difference in
response of pregnant sows compared to growing pigs may be caused by the exponential increase
in fetal growth as sows approach parturition with concomitant increases in AA requirements. Thus,
WKHREVHUYHGLQFUHDVHRIÀX[DQG%ZLWKLQFUHDVLQJWHVW$$LQWDNHFDQEHUHJDUGHGDVUHÀHFWLRQRI
increased requirements.

Flux and B decreased when increased AA supply interacted with maternal growth. The reduction of
B in the presence of increased test AA indicates that B may be used to supply AA from body tissues
when the AA need of conceptus increases. This occurred mainly in late pregnancy when the rapid
growth of fetuses increased towards parturition, causing greater demands for AA, as shown by the
LQWHUDFWLRQRIWHVW$$LQWDNHDQGJHVWDWLRQDODJH7KLVLQFUHDVHLQÀX[DQG%PD\LQGLFDWHDQLQFUHDVHG
VXSSO\RI$$WRWKHFRQFHSWXVVLQFHWKHLQWHUDFWLRQRI$$ZLWKPDWHUQDOJURZWKUHGXFHGÀX[DQG%

In conclusion, Phe kinetics at AA intake above the requirement were driven by interactions among
sow and conceptus growth with test AA intake and stage of gestation. Changes occur predominantly
in late pregnancy where the linear increase in conceptus growth increases AA requirements, leading
to decreased OX and increased B to satisfy the greater AA need. Thus, Phe kinetics and pregnant
sow requirements should be regarded as dynamic processes rather than discrete values.

References
Moehn, S., C. Levesque, R. Samuel and R.O. Ball. 2011. New energy and amino acid requirements for gestating sows.
$GYDQFHVLQ3RUN3URGXFWLRQ
NRC. 2012. Nutrient Requirements of Swine (11th ed.). Nat. Acad. Press. Washington, DC
Salter, D.N., A. Montgomery, A. Hudson, D.B. Quelch and R.J. Elliott. 1990. Lysine requirements and whole-body
SURWHLQWXUQRYHULQJURZLQJSLJV%U-1XWU

174 Energy and protein metabolism and nutrition in sustainable animal production
Effect of reducing dietary energy and protein on growth performance
and carcass traits of broilers
C.K. Girish1, S.V. Rama Rao2 and R.L. Payne1
1+HDOWKDQG1XWULWLRQ(YRQLN 6($ 3WH/WG6LQJDSRUH[email protected]
2Project Directorate on Poultry, ICAR, Hyderabad, India

Introduction
Dietary energy in excess of maintenance and production requirements of broilers will lead to
increased fat deposition and reduced carcass quality. Low protein diets balanced with supplemental
amino acids (AAs) can reduce the need for dietary energy required to break down dietary intact
protein and for excretion of the excesses of AAs. Additionally, feeding low protein diets will lead to
increased nitrogen utilization while reducing nitrogen excretion. Two experiments were conducted
to evaluate the effects of reducing dietary metabolizable energy (ME) and crude protein (CP) at
constant levels of standardized ileal digestible (SID) AA contents on growth performance and carcass
traits of broiler chickens.

Material and methods

([SHULPHQW ([SW ZDVFRQGXFWHGLQEURRGHUEDWWHULHVZKLOH([SWZDVFRQGXFWHGLQÀRRU
pens. The same dietary treatments were used for both experiments, and the diets were arranged
in a 3×3 factorial design with 3 levels (high, medium and low) of ME and CP in a completely
UDQGRPL]HGGHVLJQ(DFKGLHWZDVRIIHUHGWRUHSOLFDWHVRIELUGVLQ([SWRUUHSOLFDWHVRI
birds in Expt 2. Day-old Cobb 400 commercial broilers were used in both experiments, and the diets
were fed ad libitum from 0 to 42 d of age in mash form. Diets were formulated to meet Evonik’s
$$UHFRPPHQGDWLRQVIRUVWDUWHU G JURZHU G DQG¿QLVKHU G SKDVHVZLWKWKH
exception of the Ile:Lys ratio, which was reduced by 3% to test the current recommendation. The high
CP diet was supplemented with DL-Methionine (DL-M), L-Lysine-HCl (L-Lys) and L-Threonine
/7KU WRUHÀHFWFXUUHQWLQGXVWU\SUDFWLFH7KHQWKHGLHWDU\&3OHYHOZDVUHGXFHGE\DQGRI
high CP while maintaining the minimum AA concentrations. As a result, the medium CP diet was
supplemented with DL-M, L-Lys, L-Thr and L-Valine (L-Val), and the low CP diet was supplemented
with DL-M, L-Lys, L-Thr, L-Val and L-Ile. The concentration of the high, medium, and low ME diets
were 2950, 2850 and 2750 kcal/kg, 3050, 2950 and 2850 kcal/kg, and 3150, 3050 and 2950 kcal/
NJLQWKHVWDUWHUJURZHUDQG¿QLVKHUGLHWVUHVSHFWLYHO\6LPLODUO\WKHFRQFHQWUDWLRQVRI&3GXULQJ
WKHUHVSHFWLYHSKDVHVZHUHDQGDQGDQGDQG

Body weight (BW) and feed intake (FI) were recorded at 11, 21, 28, 35 and 42 d of age, and body
weight gain (BWG) and feed conversion ratio (FCR) were calculated. At the end of experiment,
one bird from each pen weighing close to the average body weight (± 5%) of the respective pen
was selected for carcass trait measurements. Carcass variables included ready to cook yield (RTC),
breast weight (BrW) and abdominal fat (AF). Data were subjected to factorial analyses (Snedecor
and Cochran, 1980) to study the effect of interaction between ME and CP and also independent
HIIHFWRIHDFKPDLQIDFWRU6LJQL¿FDQWGLIIHUHQFHDPRQJGLIIHUHQWWUHDWPHQWPHDQVZHUHFRPSDUHG
using Duncan’s multiple range test (Duncan, 1955) at P<0.05.

Results and discussion

In Expt 1, there were interactions (P<0.01) between ME and CP on BWG and FI but not for FCR
at d 42 (Table 1). Conversely, in Expt 2, there were interactions (P<0.05) on BWG, FI and FCR in
ELUGVUHDUHGRQWKHÀRRUSHQV%LUGVIHGORZ0(LQEDWWHULHVKDGKLJKHU P<0.01) FCR compared
with those fed medium or high ME, but there was no effect of reducing dietary CP on FCR. Birds

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 175
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_51, © Wageningen Academic Publishers 2013
7DEOH'LHWDU\HIIHFWVRQJURZWKSHUIRUPDQFHDQGFDUFDVVWUDLWVRIEURLOHUVDWG

Diets Expt 1 (Battery brooder) Expt 2 (Floor pen)

BWG FI FCR RTC BrW AF BWG FI FCR RTC BrW AF

kg kg kg/kg kg kg kg/kg
(g/kg live BW) (g/kg live BW)

HME.HCP 1.90abcd 3.02abc 1.59 804 221 13.7 2.03bc 3.77cd ab 788  15.0
HME.MCP 1.92abc 2.92bc 1.52  213 15.5 2.12bc cde 1.72b 797 234 
HME.LCP 1.92abc 3.03abc 1.57  215 17.3 2.15b 3.89bc 1.81ab 775 233 17.8
MME.HCP 2.02a 3.14ab 1.55   9.9 2.33a 4.29a 1.85ab 774 225 21.7
MME.MCP 1.79cd 2.80c 1.57 759 217 13.2 2.03bc 3.48e 1.73b 782  17.4
MME.LCP bcd 2.94bc 1.58  218 11.8 2.07bc 3.71cde 1.79ab   12.5
LME.HCP bcd 2.95bc 1.59 749 214 8.5 2.11bc 3.74cde 1.78ab  250 10.2
LME.MCP 1.98ab 3.20a  750   2.13b 4.07ab 1.91a   14.5
LME.LCP 1.77d 2.88c   211 12.4 1.94c 3.55de 1.83ab   14.8
Main effects
HME 1.92 2.99 b 779a  15.5a 2.10  1.80 787 238 
MME 1.89  1.57b b 214 b 2.14 3.83 1.79 773 229 17.2
LME 1.87 3.01 a 752b 213 9.2b  3.79 1.84 772 237 13.2
HCP 1.93 3.04 1.58 773 214 10.7b  3.93a 1.83 782 240a 
MCP 1.90 2.97 1.57 758 215 11.8ab 2.09 3.73b 1.79 782 239a 
LCP 1.85 2.95 1.59  215 13.8a 2.05 3.71b 1.81  225b 15.0

BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio; RTC = ready to cook yield; BrW = breast
weight; AF = abdominal fat; HME = high ME; MME = medium ME; LME = low ME; HCP high CP; MCP medium
CP; LCP low CP.
a,b,c,d,e0HDQVKDYLQJFRPPRQVXSHUVFULSWLQDFROXPQGRQRWYDU\VLJQL¿FDQWO\

UHDUHGLQÀRRUSHQVKDGUHGXFHG),ZKHQGLHWDU\&3ZDVGHFUHDVHGWRPHGLXP&3OHYHOEXWIXUWKHU
reduction to low CP had no effect. Further, there was no effect of reducing dietary ME on FI. There
was no interaction (P>0.05) of ME and CP levels on carcass parameters of broilers either in batteries
RUÀRRUSHQV1HYHUWKHOHVVUHGXFLQJGLHWDU\0(UHGXFHG P<0.01) the relative weights of RTC and
AF in broilers raised in battery brooders, but BrW was not affected. Conversely, reducing dietary
CP did not affect RTC and BrW but AF was higher (3 0.032) in birds fed low CP compared with
WKRVHIHGKLJK&37KH%U:RIEURLOHUVIHGORZ&3LQÀRRUSHQVZDVORZHU 3 0.041) compared
with those fed high or medium CP while there was no effect of reducing dietary ME on carcass traits.

The results suggest that the energy required for protein deposition, as indicated by breast weight, is
lower than that for total body weight gain. These experiments also demonstrate that dietary energy
can be reduced when the ratio of supplemental AAs to intact protein increases, such as in the case
of a low protein diet balanced with supplemental AAs.

References
Duncan, D. B., 1955. Biometrics 11:1-55.
Snedecor, G. W. and W. G. Cochran, 1980. Statistical Methods. Iowa State University Press, Ames.

176 Energy and protein metabolism and nutrition in sustainable animal production
Metabolic use of a growing diet for red-legged partridge (Alectoris rufa)
chicks
M. Lachica, J.F. Aguilera, R. Nieto and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
Red-legged partridge (Alectoris rufa) has an enormous ecological and game importance in Spain and
is the favorite game species. Intensive breeding is used to restock areas, to re-establish or to maintain
the population. More than 4 million birds/yr are released. Paradoxically, there is a lack of information
about its protein and energy requirements, which has led to extrapolation of existing knowledge from
other partridge species to A. rufa. The aim of this study was to determine the metabolic utilization
RIDVSHFL¿FFRPPHUFLDOGLHWIRUJURZLQJSDUWULGJHFKLFNV)RUWKLVSXUSRVHWKHIHHGFRQYHUVLRQ
UDWLR )&5 WKHHI¿FLHQF\RIXWLOL]DWLRQRIGLHWDU\0(IRUPDLQWHQDQFH km), and the partition of
retained energy (RE) as protein (REprot) and fat (REfat) were obtained.

Material and methods

Twenty four chicks were allocated by pairs in metabolic cages and fed ad libitum with a commercial
starter diet (12.0 MJ/kg ME and 280 g/kg CP) for partridge chicks. Feeding, balance and slaughter
trials began at 3 wk of age (70 g average BW) and ended at 7 wk. Initial body composition was
determined in 4 chicks. Feed intake and BW were recorded weekly. Along the third experimental
wk, total excreta was collected to determine energy metabolizability and N balance. Subsequently,
a group of 10 chicks was moved to respirometry chambers for 24 h to obtain heat production (HP)
from gas exchange measurements. Then, at the end of the fourth wk, 1 chick (210 g average BW) per
SDLUZDVNLOOHGIRUGHWHUPLQLQJWKH¿QDOERG\FRPSRVLWLRQ7KHUHPDLQLQJELUGVZHUHGLYLGHGLQ
groups of 5 and HP measured again at a ME intake (MEI) slightly above the theoretical maintenance
level, and after 12 h fasting. The RQ (CO2/O2) was also determined.

The DM content of feed, excreta and carcass was determined by standard procedures (AOAC, 1990)
and total N by the Kjeldahl procedure. The gross energy (GE) was measured in an isoperibolic bomb
calorimeter. The MEI was determined as GE content of the ingested feed minus corresponding excreta.
Metabolizability was obtained as the ME/GE ratio. The RE and REprot (retained N××23.8 kJ/g)
ZHUHFDOFXODWHGDVWKHGLIIHUHQFHEHWZHHQWKHWRWDOERG\HQHUJ\DQGSURWHLQLQWKHLQLWLDODQG¿QDO
killed group. The REfat was the difference between RE and REprot. The linear equation ER (kJ/kg0.75
and d) = -a + b×MEI (kJ/kg0.75DQGG ZDVXVHGWRREWDLQWKHHI¿FLHQF\ b) of utilization of ME for
maintenance (km). ANOVA-I was used to determine the effect of age on FCR. Tukey multiple range
WHVWZDVXVHGWRDVFHUWDLQWKHVWDWLVWLFDOVLJQL¿FDQFHRIGLIIHUHQFHV P<0.05).

Results and discussion

7KHGLHW0(FRQWHQWZDV0-NJDQGWKHDYHUDJHPHWDEROL]DELOLW\“7DEOHVKRZV
PDLQUHVXOWV7KH)&5UDQJHGIURPIRUWKHSHULRGZNWRDWZNEHLQJVLJQL¿FDQWO\
greater (P<0.05) the last one. Under similar experimental conditions, Hermes et al. (1984) with A.
graeca and Ozek et al. (2003) with A. chukar chicks obtained FCR values that ranged, respectively,
DQGIRUZN$OVR2]HN  ZLWKA. chukar reported FCR values that
UDQJHGIRUDQGZNUHVSHFWLYHO\DQGIRUDQGZNUHVSHFWLYHO\
2]HN $VWKH0(OHYHOLQWKHSUHVHQWVWXG\ZDVVLPLODUWRWKHDIRUHPHQWLRQHGVWXGLHV
the greater FCR could indicate an inadequate CP/ME ratio, particularly in the older chicks. As a
precocial bird, A. rufa chicks may need to maintain homeothermy faster than other partridges, in an

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 177
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_52, © Wageningen Academic Publishers 2013
7DEOH%RG\ZHLJKW %:J IHHGLQWDNH JG DQGIHHGFRQYHUVLRQUDWLR )&5 RIAlectoris rufa
chicks fed ad libitumZLWKDVWDUWHUFRPPHUFLDOGLHW PHDQ“VWDQGDUGHUURU 

3-4 wk 4-5 wk ZN ZN

BW1 97.1±4.35 “ “ 208.7±5.82

Intake “ “ “ 18.4±0.45
FCR 3.15±0.342a 2.55±0.100a 2.97±0.074a 4.48±0.334b

1 Measured at the end of every wk period; initial BW=71.3±1.98 g; n=20.

a,b9DOXHVZLWKLQDURZZLWKXQOLNHVXSHUVFULSWOHWWHUZHUHVLJQL¿FDQWO\GLIIHUHQW P<0.05).

environment that shows a big diurnal temperature gradient, as usually occurs in the Iberian Peninsula.
The average RE was 35.0±2.13 kJ/d, with and average REprot and REfatRI“DQG“
N-GUHVSHFWLYHO\7KHUHWDLQHGSURWHLQ&3LQWDNHUDWLRZDV$YHUDJH0(,ZDV“
N-GDQGWKHFDOFXODWHG+3ZDVN-G :NJ%: 3LV  REWDLQHGDQG
:NJ%:IRUA. chukar chicks of 1, 4 and 14 wk, respectively. Marjoniemi et al. (1995) and
Pis (2001) reported values of 21.5 and 17.0 W/kg BW, respectively, for Perdix perdix chicks. Our
value 12.3 W/kg BW for chicks from 4 to 7 wk may indicate greater energy needs for maintenance
WKDQRWKHUSDUWULGJHVSHFLHVRIWKHVDPHDJH7KHJURVVHI¿FLHQF\IRU0(XWLOL]DWLRQZDVDVORZDV
0.193, as a result of the moderate feed intake with respect to the energy needs for maintenance. The
kmZDV“Ad libitum, 0.5×ad libitum and fasting RQ values averaged 0.9±0.03, 0.7±0.04
DQG“UHVSHFWLYHO\

Compared with other partridge species, Alectoris rufa shows lower growth rate and poorer FCR.
The present study provides evidence that the protein and energy requirements differ from those of
other species and are probably slightly higher for maintenance and growth, suggesting a need for
adjusting the diet formulation.

Acknowledgements
7KLVUHVHDUFKZDVVXSSRUWHGE\JUDQWQR$*5IURP-XQWDGH$QGDOXFtD 6SDLQ 

References
$2$&2I¿FLDO0HWKRGVRI$QDO\VLVWKHG$UOLQJWRQ9$
Hermes J.C., A.E. Woodard, P. Vohra and R.L. Snyder, 1984. The effect of light intensity, temperature, and diet on
JURZWKLQ5HG/HJJHGSDUWULGJH3RXOW6FL
Marjoniemi, K., E. Hohtola, A. Putaala and R. Hissa, 1995. Development of temperature regulation in the grey partridge
Perdix perdix:LOGOLIH%LRO
Ozek, K., 2004. Effect of energy level in the diet on body weight, feed consumption and feed conversion ratio at early
growth period in the chukar partridge (Alectoris chukar chukar UDLVHGLQFORVHGFRQ¿QHPHQW5HYXH0pG9pW

2]HN.7KHRSWLPXPSURWHLQFRQWHQWLQKLJKHQHUJ\VWDUWHUGLHWIRU&KXNDU3DUWULGJH Alectoris chukar chukar).
Int. J. Poult. Sci. 5, 522-525.
Ozek, K., O. Yazgan and Y. Bahtiyarca, 2003. Effects of dietary protein and energy concentrations on performance and
carcase characteristics of chukar partridge (Alectoris chukar UDLVHGLQFDSWLYLW\%ULW3RXOWU\6FL
Pis, T., 2001. Development of thermoregulation in hand-reared grey partridges (Perdix perdix). Game Wildl. Sci. 18,
509-520.
Pis, T., 2003. Energy metabolism and thermoregulation in hand-reared chukars (Alectoris chukar). Comp. Biochem.
3K\V$

178 Energy and protein metabolism and nutrition in sustainable animal production
The amino acid composition of body protein in broilers is affected by the
sulphur amino acid supply
J.A. Conde-Aguilera1,2, C. Cobo-Ortega1,2, S. Tesseraud, M. Lessire, Y. Mercier and J. van Milgen1,2
1,15$8053(*$6(6DLQW*LOOHV)UDQFH[email protected]
2$JURFDPSXV2XHVW8053(*$6(5HQQHV)UDQFH
,15$855HFKHUFKHV$YLFROHV1RX]LOO\)UDQFH
$GLVVHR)UDQFH6$6$QWRQ\)UDQFH

Introduction
,QSRXOWU\GLHWVWKHVXOSKXUFRQWDLQLQJDPLQRDFLGV $$ 0HWDQG&\VDUHFRQVLGHUHGWKH¿UVW
limiting AA for protein deposition. In the factorial approach, AA requirements are determined using
WKH$$FRPSRVLWLRQRIUHWDLQHGSURWHLQWKHPD[LPXPHI¿FLHQF\RI$$XWLOL]DWLRQIRUJURZWKDQG
the maintenance AA requirement. It is then assumed that the relative contribution of these traits to the
$$UHTXLUHPHQWLVFRQVWDQWUHVXOWLQJLQDFRQVWDQWLGHDO$$SUR¿OH+RZHYHUWKHUHDUHLQGLFDWLRQV
that the AA composition of body protein is affected by genotype, age, gender, and the protein and
AA contents of the diet. Broiler studies aiming to assess the response to the total sulphur AA (TSAA)
supply mostly focus on performance, carcass yield, and the chemical composition of the whole animal
RUEUHDVWPHDW+RZHYHUDVVKRZQLQSLJVGLIIHUHQWWLVVXHVPD\UHVSRQGGLIIHUHQWO\WRDGH¿FLHQW
TSAA supply. The objective of this study was to quantify the changes in chemical body composition
DQGPHDWTXDOLW\RIEURLOHUVUHFHLYLQJGLHWVHLWKHUGH¿FLHQW 76$$ RUVXI¿FLHQW 76$$ LQ76$$

Material and methods

Eighteen birds (Ross PM3 strain) were used in a comparative slaughter study. Six chickens were
VODXJKWHUHGDWGRIDJHDQGWKHRWKHUVZHUHGLYLGHGLQKRPRJHQRXVJURXSVRIELUGVHDFKWR
received either TSAA- or TSAA+ for 35 d and were slaughtered thereafter. Diets were offered slightly
below the anticipated ad libitum intake, and were formulated so that the nutrient composition (other
than TSAA) met or exceeded the NRC recommendations. Diets were changed weekly to account for
the change in nutrient requirements during the experiment. The supply of Met and TSAA in TSAA-
was about 34% and 22% below those of TSAA+, respectively. At slaughter, birds were divided into
blood, feathers, carcass, Pectoralis major muscle (PM) and offal for analysis.

Results and discussion

Performance and tissue weight gain were not affected by the TSAA supply (Table 1). In birds receiving
diet TSAA-, protein gain was lower in the carcass (P<0.01), empty body (3  DQG30 3 0.10;
Table 2). Lipid gain in birds receiving TSAA- was 78% greater in PM (P<0.001), 28% greater in
abdominal fat (P<0.05) and 10% greater in the carcass (3 0.10). This increased lipid content in
TSAA- birds can be explained through the deamination of AA not used for protein deposition and
for which the carbon-chains can be used for lipid deposition. In PM, reducing the TSAA supply
resulted in a tendency for an increase in the redness value (a*; 3 0.10). The AA composition of
tissues and tissue gain was affected by the TSAA supply, but the Met and Cys concentrations were
changed only in the offal (3 0.08). This contrasts with a study with pigs, who responded to a strong
0HWGH¿FLHQF\E\UHGXFLQJWKHSURWHLQFRQWHQWLQWKHERG\DQGWKH0HWFRQWHQWLQERG\DQGPXVFOH
proteins (Conde-Aguilera et al., 2010). Also, birds receiving diet TSAA- had a greater Ser content in
the empty body, carcass, and PM (P< ZKLFKPD\UHÀHFWDQDGDSWDWLRQRIPHWK\OPHWDEROLVP
In contrast, these birds had lower contents of Lys and Glu in the empty body, of Phe, Tyr, Gly, and
Glu in PM, and of Ala in the offal (P<0.05). Differences in the AA composition of body protein
could be the consequence of changing proportions of different types of proteins in the body. Our
VWXG\LQGLFDWHVWKDWDOWKRXJKFKLFNHQVFRSHZLWKDQ$$GH¿FLHQF\SUHGRPLQDQWO\E\FKDQJLQJWKH

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 179
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_53, © Wageningen Academic Publishers 2013
protein and lipid content in the body, the AA composition is also affected. This questions therefore
WKHXVHRIDFRQVWDQWLGHDO$$SUR¿OHLQSRXOWU\QXWULWLRQ

7DEOH3HUIRUPDQFHDQGPHDWTXDOLW\WUDLWVLQEURLOHUVRIIHUHG76$$RU76$$IURPWRGRIDJH

TSAA- TSAA+ RSD P-value

Final BW (g) 2,589  100 0.48

Average daily gain (g/d)  70.3 2.1 0.11
Gain-to-feed ratio (g/g)    0.10
Meat quality traits
pH (24 h)   0.03 0.15
Lightness (L*) 51.2 51.4 3.0 0.94
Redness (a*)  -0.15 0.74 0.10
Yellowness (b*)  13.2 1.4 0.59
Abdominal fat (g/kg BW) 19.8 15.5 3.1 0.04

7DEOH&RPSRVLWLRQRIWKHHPSW\ERG\DQGWLVVXHZHLJKWJDLQLQEURLOHUVRIIHUHG76$$RU76$$
IURPWRGRIDJH1.

Empty body Carcass

TSAA- TSAA+ RSD TSAA- TSAA+ RSD

Weight gain (g/d)    49.7 51.1 2.3

% of EBW gain 74.3 74.7 1.4
Composition
1î JNJ † 199 3 185** 191 3
Lipid (g/kg) 141 130 12 134† 121 12
Lys   0.10  7.87 0.11
Met 2.29 2.30    0.05
Cys 2.01  0.08 1.08 1.10 0.02
Phe 3.95   3.74 3.80 
Tyr 3.10 3.13  3.12 3.15 0.04
Ser 4.42* 4.20 0.12 3.39**  0.11
Gly 7.30 7.33 0.20   0.30
Ala   0.08   0.13
Glu  12.82  13.20 13.40 0.22
Pro   0.11 5.32 5.20 

180 Energy and protein metabolism and nutrition in sustainable animal production
Table 2. Continued.

PM Offal

TSAA- TSAA+ RSD TSAA- TSAA+ RSD

Weight gain (g/d) 11.1 11.5 1.1 12.3 12.5 0.7

% of EBW gain   1.2 18.4 18.3 1.1
Composition
1î JNJ 230† 237 7  157 8
Lipid (g/kg) *** 9.1 1.5 224 212 18
Lys  8.98  5.98  0.42
Met 2.88 2.85 0.13 2.24† 2.42 0.17
Cys 1.19 1.20 0.08 1.20 1.29 0.11
Phe * 4.25 0.12 3.81 3.78 0.32
Tyr * 3.81 0.11 3.02 3.02 0.29
Ser * 3.22 0.09 3.83  0.32
Gly 4.34* 4.50 0.15 11.13  
Ala  5.75 0.22 *  0.37
Glu 14.00* 14.80 0.59 11.90 12.40 0.93
Pro 3.73 3.72 0.11 7.38† 7.98 0.55

16LJQL¿FDQWOHYHOLQGLFDWHGZLWKLQDURZDQGWLVVXHFRPSRQHQWE\
P” P” P”†P”(%: 
HPSW\ERG\ZHLJKW7KH$$FRPSRVLWLRQLVH[SUHVVHGDVJUDPVSHUJUDPVRI1

References
&RQGH$JXLOHUD-$5%DUHD1/H)ORF¶K//HIDXFKHXUDQG-YDQ0LOJHQ$VXOIXUDPLQRDFLGGH¿FLHQF\
changes the amino acid composition of body protein in piglets. Animal 4:1349-1358.

Energy and protein metabolism and nutrition in sustainable animal production 181
Body composition and nutrient partitioning in long term
supplementation of betaine and conjugated linoleic acid in mice
J.M. Rodríguez-López, L. González-Valero, M. Lachica and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQL¿JDUHV#HH]FVLFHV

Introduction
Betaine (N, N, N-trimethylglycine) and conjugated linoleic acid (CLA) have the potential to alter
growth and body composition in different animal models (Park et al., 1997; Fernández-Fígares
et al., 2008). The effects of these substances are usually evaluated using relatively short studies.
Therefore, the present study was conducted to evaluate the effects of long term dietary betaine and
CLA supplementation on body composition and nutrients deposition in mice.

Material and methods

Male ICR (CD-1) mice, weighing 14-18 g, were group housed (5 mice per cage, 4 cages per treatment)
and fed ad libitum Teklad global 14% protein rodent diet containing either no added betaine or CLA
(Control), 0.5% betaine, 1% CLA, or 0.5% betaine + 1% CLA for 90 days. Water was freely available.
An additional group of 5 mice was slaughtered at the beginning of the experiment to obtain the initial
body composition. Feed intake and spillage was recorded every other day and individual weight
gain was determined weekly. When mice were slaughtered, carcasses were chemically analysed for
water, protein, lean, fat, mineral and energy content. Increases in protein, energy, fat and minerals
ZHUHFDOFXODWHGDVWKHGLIIHUHQFHEHWZHHQWKH¿QDOPHDVXUHGFRPSRVLWLRQRIH[SHULPHQWDOPLFH
and the composition assessed from the initial slaughter group. The treatment effect was assessed
by ANOVA according to a completely randomized design with mouse as experimental unit using
WKH*/0SURFHGXUH7UHDWPHQWPHDQVZHUHFRPSDUHGE\7XNH\¶VSURFHGXUHDQGVLJQL¿FDQFHZDV
set at P<0.05.

Results and discussion

Although studies of CLA effects on body composition are common, CLA effect on nutrient accretion
has been studied to a lesser extent. In the present study, long term dietary CLA and betaine + CLA
intake markedly affected carcass composition increasing protein, protein:fat ratio, water and minerals
and decreasing fat (P<0.001) (Table 1). In a similar way, the composition of gain and carcass
nutrient deposition of mice fed CLA diet were also affected, so that protein, water and minerals
increased whereas fat and energy decreased (P<0.001) compared to control and betaine groups.
Betaine decreased water content of gain and water deposition compared to Control (P<0.001),
and showed a synergistic increase in protein:fat ratio when used with CLA (P<0.001). Similarly,
growing pigs fed with betaine + CLA diets had greater protein deposition than betaine, CLA or
control pigs (Fernández-Fígares et al., 2008). The decrease in body fat could be due to increased
energy expenditure and energy loss in excreta (Terpstra et al., 2002). CLA decreased body fat and
increased lean body mass and ash body content in mice (Park et al., 1997). Nevertheless, mice fed
0.5% CLA high fat diet decreased body fat with no change in protein content (Javadi et al., 2007).
Enhanced carcass mineral content and deposition may indicate increased bone formation which
may protect against loss of bone mass with aging. In that sense, CLA increased markers of bone
formation in mice (Watkins et al., 2001). We have found no reports on the effects of betaine on
body composition in laboratory animals. In feed restricted growing pigs, betaine increased protein
deposition and decreased carcass fat content (Fernández-Fígares et al., 2002).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 183
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_54, © Wageningen Academic Publishers 2013
7DEOH(IIHFWVRIGLHWDU\EHWDLQH  &/$  DQGEHWDLQH&/$RQPLFHFDUFDVVFRPSRVLWLRQ
composition of gain and the rates of carcass deposition of protein, water, fat, minerals and energy
IRUDSHULRGRIGD\V

Control Betaine CLA Betaine+CLA SEM

Carcass composition, g/kg

Protein 194a 197a 217b 214b 2.0
Water a a b 721b 7.0
Fat 110a 115ª b b 
Minerals 373a a 415b 404b 
Protein:fat 2.3a 2.2a 7.0b 8.8c 0.5
Composition of gain, g/kg
Protein 192a a 228b 224b 3.3
Water a 595b c 704d 9.9
Fat a a 47b 31b 0.1
Minerals 37a 37a 44b 42b 1.0
Deposition, mg/d
Protein 54a 51a b b 1.7
Water 170a 152b 187c 204d 5.8
Fat 48a 42a 13b 9b 3.8
Minerals 10a 10a 12b 12b 0.4
Total energy, kJ/d 3.2a 2.9a 2.0b 1.9b 0.2

abcd:LWKLQDURZYDOXHVZLWKGLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\ P<0.001).

Overall, long term dietary supplementation of CLA increased protein and mineral and decreased fat
deposition in growing mice while betaine had a minor effect.

Acknowledgements
7KLVVWXG\ZDV¿QDQFHGE\JUDQW$*/IURP0LQLVWU\RI6FLHQFHDQG,QQRYDWLRQ

References
Fernández-Fígares, I., J.A. Conde-Aguilera, R. Nieto, M. Lachica and J.F. Aguilera, 2008. Synergistic effects of betaine
DQGFRQMXJDWHGOLQROHLFDFLGRQJURZWKDQGFDUFDVVFRPSRVLWLRQRIJURZLQJ,EHULDQSLJV-$QLP6FL
Fernández-Fígares, I., D. Wray-Cahen, N.C. Steele, R.G. Campbell, D.D. Hall, E. Virtanen, and T.J. Caperna, 2002.
Effect of dietary betaine on nutrient utilization and partitioning in the young growing feed-restricted pig. J. Anim.
Sci. 80, 421-428.
Javadi, M., M.J. Geelen, H. Everts, A.G. Lemmens and A.C. Beynen, 2007. Body composition and selected blood
SDUDPHWHUVLQPLFHIHGDFRPELQDWLRQRI¿EUHDQGFRQMXJDWHGOLQROHLFDFLG-$QLP3K\VLRO$QLP1XWU
492-497.
Park Y., K.J. Albright, W. Liu, J.M. Storkson, M.E. Cook and M.W. Pariza, 1997. Effect of conjugated linoleic acid
on body composition in mice. Lipids 32, 853-858.
Terpstra, A.H.M., A.C. Beynen, H. Everts, S. Kocsis, M.B. Katan and P.L. Zock, 2002. The decrease in body fat in
mice fed conjugated linoleic acid is due to increases in energy expenditure and energy loss in the excreta. J. Nutr.
132, 940-945.
Watkins, B.A., H.E. Lippman, L. Le Bouteiller, Y. Li and M.F. Seifert, 2001. Bioactive fatty acids: role in bone biology
and bone cell function. Prog. Lipid. Res. 40, 125-148.

184 Energy and protein metabolism and nutrition in sustainable animal production
Effect of immunocastration in combination with addition of fat to diet on
quantitative oxidation of nutrients and fat retention in male pigs
N. Batorek, J. Noblet, S. Dubois, M. Bonneau, 0ýDQGHN3RWRNDU1,2 and E. Labussiere
1$JULFXOWXUDO,QVWLWXWHRI6ORYHQLD+DFTXHWRYDXOLFD/MXEOMDQD6ORYHQLD
28QLYHUVLW\RI0DULERU)DFXOW\RI$JULFXOWXUHDQG/LIH6FLHQFHV3LYROD+RþH6ORYHQLD
,15$8053HJDVH6DLQW*LOOHV)UDQFH[email protected]
$JURFDPSXV2XHVW8053HJDVH5HQQHV)UDQFH

Introduction
Immunocastration (IC) has been studied as one of the possible replacements for surgical castration
of male pigs to block reproductive function and eliminate boar taint. It has a positive effect on
performance (Batorek et al., 2012) but after IC, increased feed intake (FI) and fat deposition indicate
changes at the metabolic level. The objective of the study was to determine the effects of IC and
increase of dietary fat on energy (E) metabolism and relationships between digested nutrients and
WKHLU¿QDORXWSXWVIRUFDWDEROLFDQGPHWDEROLFSURFHVVLQIDWWHQLQJSLJV

Material and methods

Digestibility and N and E balance in respiration chambers were determined during 8-9 days in 2
subsequent stages prior and after IC with Improvac® (mean body weight BW: 85 and 135 kg) in 4
replicates (2 groups of 2 littermates each). Pigs were fed at 90% of predicted ad libitum ME intake
and were allocated to one of 2 groups: Group 1 was fed low fat diet (LF; standard diet with addition
of 0.5% of rapeseed oil; 2.5% DM of fat) during stage 1 and 2 and group 2 was fed LF during
stage 1 and high fat diet (HF; diet enriched in fat by addition of corn and linseed oils to balance the
C18:3:C18:2 fatty acids (FA) ratio between diets; 8.9% DM of fat) during stage 2. Additionally,
pigs received a test-meal containing 13&Q)$DQG 13CO2 production was measured (on 0,
DQGGD\VDIWHULQJHVWLRQ LQRUGHUWRFDOFXODWHWKHUDWHRIR[LGDWLRQRIWKLV)$7KHth day
was a fasting day with feed deprivation. Heat production (HP) and respiratory quotient (RQ) were
FDOFXODWHGDFFRUGLQJWR%URXZHU  DQGFRPSRQHQWVRI+3 9DQ0LOJHQet al. 1997) were used
to calculate heat increment (HI) as the sum of HP due to physical activity and thermic effect of
feeding. Oxidation of nutrients and their contribution to fat retention (RF) were calculated according
to Chwalibog et al. (1992). Effect of treatment group, stage and their interaction was tested by the
GLM procedure (SAS Inc., Cary, NC, USA).

Results and discussion

To mimic the increase in voluntary FI seen in IM after V2, the ME intake was increased between
stages (P<0.01; Table 1). Total RE increased with stage (P<0.01) and was mainly retained as fat
(P<0.01) in accordance with increased lipogenesis during stage 2 (P<0.01). RF synthesized from
carbohydrates (RF(CHO)) increased with stage (P<0.01; Figure 1), but on HF diet the proportion
of RF originating from dietary fat increased at the expense of RF(CHO) (P<0.01). This explains
the decreased HP (P<0.01; Table 1) and increased RE and RE as fat (P<0.01) on HF diet, because
WKHXWLOL]DWLRQRI0(RULJLQDWLQJIURPOLSLGVLQVWHDGRIFDUERK\GUDWHVLVHQHUJHWLFDOO\PRUHHI¿FLHQW
and results in lower HP (Van Milgen et al., 2001). Nevertheless, the decrease in HI (% ME intake)
with HF during stage 2 was only numerical (Table 1). The RQ was always above 1 and it increased
with stage (P<0.01; Table 1), but it was lower on HF diet (P<0.01), indicating that there was more
oxidation and less synthesis of lipids on HF diet in stage 2. In contrary, production of 13CO2 because
of oxidation of dietary 13&Q)$LQFUHDVHGZLWKVWDJHEXWZDVKLJKHURQ/)GLHWIURPGD\
RQZDUGV )LJXUH LQGLFDWLQJWKDWGLHWDU\SURYLVLRQRIWKLVHVVHQWLDO)$ZDVLQVXI¿FLHQWIRUSLJV
on LF diet to satisfy their minimal oxidation rate.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 185
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_55, © Wageningen Academic Publishers 2013
7DEOH5HVXOWVRIHQHUJ\EDODQFH N-NJRI%:SHUGD\ DQGUHVSLUDWRU\TXRWLHQWLQJURZLQJ
LPPXQRFDVWUDWHGSLJVEHIRUH VWDJH DQGDIWHU VWDJH VXFFHVVIXOLPPXQRFDVWUDWLRQ

Stage 1 Stage 2 r.s.d P-value1

Group 1 2 1 2 S G S×G
Diet LF LF LF HF

Energy balance (kJ/kg of BW per day)

ME intake 2,270a 2,277a 2,449b 2,492b 19 <0.01 0.32 0.10
Heat production ab 1,370b 1,371ab 1,327a 17 0.33 0.77 <0.01
Retained energy as fat a a b 914c 30 <0.01 0.48 <0.01
Total retained energy 924 a 907a 1,078b c 20 <0.01  <0.01
Lipogenesis 559 a 513a b b 38 <0.01  
Heat increment (% of ME) 23.3 23.8 27.5 24.7 2.9 0.13 0.59 0.30
Respiratory quotient 1.10a 1.09a 1.14b 1.07a 0.01 0.14 0.04 <0.01

r.s.d.=residual standard deviation; ME = metabolizable energy; LF = low fat diet; HF = high fat diet.
1 Tested for effects of stage (S), group (G) and their interaction (S×G).

a-c Least squares means within a row with different superscripts differ (P<0.05).

RF(CHO)*** RF(P) RF(F)***

Stage1 b b
Stage2 Group1 c a
Stage2 Group2 a c

0 20 40 60 80 100
RF (%)
)LJXUH3HUFHQWDJHRIWRWDOUHWDLQHGIDW 5) RULJLQDWLQJIURPGLHWDU\FDUERK\GUDWHV 5) &+2 
SURWHLQV 5) 3 DQGIDW 5) )  P<

Day 1 Day 2 Day 5 Fasting day

Stage1
Stage2 Group1
Stage2 Group2
1 6 11 16
13
CO2(% of 13C18:2 ingested)

Figure 2. Excreted CO2 as percentage of consumed &GXULQJWKHVWQGWKDQGIDVWLQJ

day in respiration chamber.

References
%DWRUHN10ýDQGHN3RWRNDU0%RQQHDXDQG-YDQ0LOJHQ0HWDDQDO\VLVRIWKHHIIHFWRILPPXQRFDVWUDWLRQ
RQSURGXFWLRQSHUIRUPDQFHUHSURGXFWLYHRUJDQVDQGERDUWDLQWFRPSRXQGVLQSLJV$QLPDO
%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV,Q(QHUJ\PHWDEROLVPSURFHHGLQJVRIWKHUG
V\PSRVLXPKHOGDW7URRQ6FRWODQG0D\./%OD[WHU HG /RQGRQ$FDGHPLF3UHVV
Chwalibog A., K. Jakobsen, S. Henckel and G. Thorbek, 1992. Estimation of quantitative oxidation and fat retention
IURPFDUERK\GUDWHSURWHLQDQGIDWLQJURZLQJSLJV-$QLP3K\VLRO$QLP1XWU
9DQ0LOJHQ--1REOHWDQG6'XERLV(QHUJHWLFHI¿FLHQF\RIVWDUFKSURWHLQDQGOLSLGXWLOL]DWLRQLQJURZLQJ
pigs. J. Nutr. 131, 1309-1318.
SAS. SAS/STAT 9.1 user’s guide, 2004. SAS Inst. Inc., Cary, NC, USA.

186 Energy and protein metabolism and nutrition in sustainable animal production
Net energy content of dry extruded-expelled soybean meal fed to
growing pigs using indirect calorimetry
D.E. Velayudhan, J.M. Heo and C.M. Nyachoti
8QLYHUVLW\RI0DQLWRED:LQQLSHJ0%571&DQDGDGHHSDNYHW#JPDLOFRP

Introduction
Feed is the single most expensive input in commercial pork production and at least 50% of this cost
FDQEHDWWULEXWHGLQVXSSO\LQJHQHUJ\WRWKHDQLPDOWKXVPDNLQJHQHUJ\¿QDQFLDOO\WKHPRVWYLWDO
component. Swine diets can be formulated on a variety of energy systems such as the digestible
energy (DE), the metabolizable energy (ME) and the net energy (NE) systems of which the NE
system provides more accurate estimates of the energy available to the animal. Energy values of
protein-rich feeds are often overestimated when expressed on a DE or ME system (Noblet et al.,
1994). These discrepancies in measurement of available dietary energy have a drastic effect on the
economics of pig production and there is, therefore, an ongoing interest in adopting the NE system.
The most commonly used protein source in livestock diets is soybean meal (SBM), but it also contains
certain antinutritional factors which depress animal growth performance. Studies show that such
DQWLQXWULWLRQDOIDFWRUVDUHUHGXFHGVLJQL¿FDQWO\GXULQJPHDOSURFHVVLQJ 3HULOODet al., 1997). One
such process is the combination of extrusion with expelling which produces a SBM product called
dry extruded-expelled SBM (DESBM). However, published data pertaining to the energy values
of DESBM for grower pigs are limited. The aim of this study was to determine the NE content of
DESBM in growing pigs using either an indirect calorimetry (IC) or published prediction equations.

Material and methods

7ZHQW\IRXUJURZLQJEDUURZV LQLWLDO%: “NJ ZHUHDOORWWHGLQDFRPSOHWHO\UDQGRPL]HG
GHVLJQWRGLHWDU\WUHDWPHQWVWRJLYHUHSOLFDWHVSHUWUHDWPHQW'LHWDU\WUHDWPHQWVZHUHDFRUQ
soybean meal basal diet (Diet A), a diet containing Diet A and DESBM in a 80:20 ratio with a constant
crude protein content compared with the basal diet (Diet B), a diet with 80:20 ratio of basal diet
and DESBM with a constant corn:soybean meal ratio (Diet C) and a diet with simple substitution
of basal diet with DESBM in 80:20 ratio (Diet D). Pigs were fed in metabolism crates for a period
RIGDWNFDO0(NJ%:/d to determine DE and ME contents by total collection method.
7KHUHDIWHUSLJVZHUHPRYHGLQWRDQLQGLUHFWFDORULPHWHUIRUDKSHULRGZKHUH22 consumption
and CO2 production were measured to determine heat production (HP) and fasting heat production
(FHP). The energy content of DESBM was calculated using the difference method (Woyengo et
al., 2010) by subtracting the NE contribution of the basal diet from the NE of the diets containing
20% DESBM.

Mixed procedure of SAS (Software release 9.2; SAS Institute, Cary, NC, USA) was used to analyse
the data. The individual pig was considered as the experimental unit.

Results and discussion

Table 1 details the energy and heat production values for the 4 dietary treatments as determined using
the IC method. The study demonstrated that the values obtained with the IC method were consistently
greater than those obtained with prediction equations. The NE content of DESBM was calculated
WREHDQGNFDONJ'0IRUGLHWV%&DQG'UHVSHFWLYHO\XVLQJ,& P<0.0001).
Respective values obtained with published equations (Noblet et alD ZHUHDQG
NFDONJ'07KHGLVFUHSDQF\EHWZHHQWKHGHWHUPLQDWLRQWHFKQLTXHXVHGZDVDERXWZKHQ
diets were formulated with a constant protein content or corn:soybean meal ratio (1.0% and 0.7%,
respectively), however, this was 4.1% when diet was formulated with simple substitution technique.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 187
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_56, © Wageningen Academic Publishers 2013
Table 1. Energy and heat production values for diets.

Item Dietary treatments SEM P-value

A B C D

DE, kcal/kg of DM 3,481  3,472 3,473 9.40 

ME, kcal/kg of DM 3,392 3,378    0.431
HP, kcal/kg of DM1 2,007 1,930 2,038 2,000 53.01 
FHP, kcal/kg of DM 1,547 1,424 1,508  53.99 0.513
RE, kcal/kg of DM2 1,385 1,448 1,348  52.85 
NE, kcal/kg of DM3 2,932 2,872 2,855 2,853  <0.0001

1 HP = 3.87 × O2 + 1.20 × CO2 -1.43 × urinary N; where HP heat production (kcal); O2 = oxygen consumption (L);
CO2 = carbon dioxide production (L).
2 RE = ME – HP; where RE = retained energy (kcal/d), ME = metabolisable energy (kcal/d).
3 NE = (RE +FHP)/DMI; where NE= net energy (kcal/kg DM); FHP fasting HP (kcal/d); DMI = dry matter intake (kg/d).

Conclusion
The results from the present study shows that the NE values of DESBM obtained with the IC
method were higher than those obtained with prediction equations for all the three dietary designs;
the disparity being least when formulated with a constant corn:soybean meal ratio. So for routine
NE determination, diets should be formulated to contain a constant ration of other energy yielding
components.

References
Ayoade, D.I., E. Kiarie, M.A. Trinidade Neto and C.M. Nyachoti, 2012. Net energy of diets containing wheat-corn
distillers dried grains with solubles as determined by indirect calorimetry, comparative slaughter, and chemical
composition methods. J. Anim. Sci. 90, 4373-4379.
Noblet, J., H. Fortune, C. Dupire, and S. Dubois, 1993. Digestable, metabolisable and net energy values for 13 feedstuffs
for growing pigs: effect of energy system. Anim. Feed Sci. Technol. 42, 131-149.
Noblet, J., H. Fortune, X. S. Shi, and S. Dubois, 1994. Effect of body weight on net energy value of feeds for growing
SLJV-$QLP6FL
Noblet, J., X.S. Shiand, and S. Dubois, 1993. Metabolic utilization of dietary energy and nutrients for maintenance
energy requirements in sows: basis for a net energy system. Br. J. Nutr. 70, 407-419.
Noblet, J., H. Fortune, X. S. Shi, and S. Dubois, 1994. Prediction of net energy value of feeds for growing pigs. J.
Anim. Sci. 72, 344-354.
Woyengo, T. A., E. Kiarie and C.M. Nyachoti, 2010. Energy and amino acid utilization in expeller-extracted canola
meal fed to growing pigs. J. Anim. Sci. 88, 1433-1441.

188 Energy and protein metabolism and nutrition in sustainable animal production
Effect of early surgical castration and immune castration on
SRVWSUDQGLDOQXWULHQWSUR¿OHVLQPDOHSLJV
N. Le Floc’h1,2, A. Prunier1,2, J. van Milgen1,2, H. Furbeyre1,2 and I. Louveau1,2
1,15$8053(*$6(6DLQW*LOOHV)UDQFH[email protected]
2$JURFDPSXV2XHVW8053(*$6(5HQQHV)UDQFH

Introduction
Rearing of entire males (EM) or immunologically castrated male (IC) pigs are two alternatives to
VXUJLFDOFDVWUDWLRQ0DOHSLJVHDWOHVVDQGH[KLELWKLJKHUJURZWKSHUIRUPDQFHDQGIHHGHI¿FLHQF\
than early surgically castrated pigs (SC). Late castration by immunization against gonadotrophin-
releasing hormone (GnRH) is a relevant strategy to maintain growth performance and prevent the
accumulation of molecules responsible for boar taint in meat (Millet et al., 2011). Indeed, IC pigs are
VLPLODUWR(0XQWLOLPPXQL]DWLRQLVHIIHFWLYH'HVSLWHODUJHGLIIHUHQFHVLQIHHGHI¿FLHQF\EHWZHHQ
EM and SC pigs, the mechanisms involved in these differences have been poorly investigated (Claus
et al., 7KHFXUUHQWVWXG\ZDVXQGHUWDNHQWRGHWHUPLQHZKHWKHUGLIIHUHQFHLQIHHGHI¿FLHQF\
between SC, IC and EM pigs can be explained by difference in nutrient utilization after a meal by
PHDVXULQJSRVWSUDQGLDOSUR¿OHVRISODVPDQXWULHQWDQGKRUPRQHFRQFHQWUDWLRQV

Material and methods

0DOH3LpWUDLQî /DUJH:KLWHî/DQGUDFH SLJV6&(0DQG,&ZHUH¿WWHGZLWKDMXJXODU
catheter under general anaesthesia at 14 weeks of age. Pigs were fed ad libitum a growing diet (CP
1(0-NJ6,'/\V 7KUHHSRVWSUDQGLDOSODVPDQXWULHQWSUR¿OHVZHUHHVWDEOLVKHG
RQHDFKSLJDWDQGZHHNVRIDJH7KHVHSHULRGVZHUHFKRVHQWRVXUURXQGWKHVHFRQG
injection of Improvac®WR,&SLJVWKH¿UVWSHULRGEHLQJEHIRUHWKHGHFUHDVHLQWHVWLFXODUKRUPRQHV
the other periods being during and after the decrease in testicular hormones in IC pigs. On each test
day, blood samples were collected prior to the test meal (400 g), after pigs were fasted overnight,
and for 4 hours after the test meal. Plasma concentrations of glucose, urea, amino acids and insulin
were determined on each blood samples. Data were analyzed as repeated measured using the MIXED
procedure of SAS. The model included the period (age effect), the experimental group (SC, IC and
EM), the time and their interactions.

Results and Discussion

7KHSRVWSUDQGLDOQXWULHQWSUR¿OHVZHUHHVWDEOLVKHGDWDFRQVWDQWDJHDWDQDYHUDJHERG\ZHLJKWRI
DQGNJIRUSHULRGVDQGUHVSHFWLYHO\3ODVPDLQVXOLQFRQFHQWUDWLRQVGLGQRWGLIIHU
between the different periods and between IC, SC and EM pigs (data not shown). The average
glycaemia (Table 1) did not differ between periods in SC and EM pigs whereas it decreased with
age in IC pigs. During period 1, average glycaemia was lower in SC than in the EM and IC pigs but
the time-related variations of glycaemia did not differ between the 3 groups (Figure 1). After the
second injection of Improvac (periods 2 and 3), average glycaemia was greater in EM pigs than in IC
and SC pigs. During period 3, the peak of glucose measured 50 min after the meal distribution was
lower in SC and IC pigs compared with EM pigs. Thereafter, glycaemia remained higher than basal
values in EM pigs until 180 min whereas it did not differ from basal concentrations in SC and IC pigs.

In both IC and SC pigs, the plasma urea concentrations increased with age whereas those of AA
decreased. In EM pigs, both plasma urea and AA concentrations decreased with age. Urea was the
highest in SC pigs whereas it did not differ in IC and EM during the three periods.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 189
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_57, © Wageningen Academic Publishers 2013
Table 1. Effects of surgical castration and immune castration on average plasma glucose, urea, and
total AA concentrations.

Period Experimental groups Statistics

SC IC EM Group Period Group × Period

Glucose P1 1,132x 1,191a,y 1,193y

mg/l P2 1,095x 1,120b,x 1,183y 0.0009 0.0003 0.21
P3 1,092x 1,103b,x 1,178y
Urea P1 253a,x 204a,y 227a,x,y
mg/l P2 289b,x 209a,y 231a,y <0.0001 0.0008 <0.0001
P3 304c,x 239b,y 213b,y
Total AA P1 5,030a a a
μmol/l P2 4,905a 4,527b 4,774b 0.42 <0.0001 0.17
P3 4,545b 4,557b 4,532c

x,y P<0.05 between experimental groups (on a same line).

a,b,c P<0.05 between periods (on a same column).

)LJXUH3ODVPDJOXFRVHFRQFHQWUDWLRQVPHDVXUHGGXULQJWKHKRXUVIROORZLQJWKHPHDOGLVWULEXWLRQ

,QVXPPDU\SODVPDJOXFRVHSUR¿OHVZHUHDIIHFWHGE\LPPXQHFDVWUDWLRQHDUOLHUWKDQXUHDDQG
$$SUR¿OHV7KHDQDO\VLVRISRVWSUDQGLDOJO\FDHPLDLQGLFDWHVDJUHDWHUJOXFRVHFOHDUDQFHLQERWK
castrated groups, which is not in accordance with data reported in rats (Holmang et al., 1992). Our
results suggest that IC pigs would keep the advantages of EM pigs in terms of nitrogen metabolism
during the experimental period.

References
Claus, R., M. Lacorn, K. Danowski, M.C. Pearce and A. Bauer, 2007. Short-term endocrine and metabolic reactions
EHIRUHDQGDIWHUVHFRQGLPPXQL]DWLRQDJDLQVW*Q5+LQERDUV9DFFLQH
Holmang, A and P. Bjorntorp, 1992. The effects of testosterone on insulin sensitivity in male rats. Acta Physiol Scand

Millet S., K. Gielkens, D. De Brabander and G.P.J. Janssens, 2011. Considerations on the performance of immunocastrated
male pigs. Animal 5, 1119-1123.

190 Energy and protein metabolism and nutrition in sustainable animal production
Determination of the valine requirements for growth in pigs from 8 to 18 kg
E. Assadi1, J.V. Nørgaard1, N. Canibe1, B.B. Jensen1, B. Nielsen2, K. Blaabjerg1 and H.D. Poulsen1
1'HSDUWPHQWRI$QLPDO6FLHQFH$DUKXV8QLYHUVLW\7MHOH'HQPDUN[email protected]
2&KU+DQVHQ$6%¡JH$OOp+¡UVKROP'HQPDUN

Introduction
3LJGLHWVDUHVXSSOHPHQWHGZLWKFU\VWDOOLQHDPLQRDFLGV $$ WREDODQFHWKH$$SUR¿OH7KLVLV
necessary to avoid poor nitrogen (N) utilization, urinary N excretion, and low growth rates. Valine
(Val) is one of the branched-chain AA which cannot be synthesized by the animal and therefore is
OLVWHGDVLQGLVSHQVDEOH$$,Q(XURSHDQJURZHUGLHWV9DOLVRIWHQWKH¿IWKOLPLWLQJ$$DIWHUO\VLQH
/\V PHWKLRQLQH 0HW WKUHRQLQH 7KU DQGWU\SWRSKDQ 7US DQGDSSHDUVWREHDGH¿FLHQWQXWULHQW
in pig feedstuffs in regard to the minimum requirement for pigs. The objective of this study was to
determine the requirements of Val that supports the maximum animal performance.

Material and methods

To determine the requirements of Val, 120 individually penned pigs with average weight of 9.4±1.4
kg were used in two periods. The experiment started 4-5 days after weaning at 28 days. The pigs
ZHUHDOORWWHGWRRQHRIWKUHHH[SHULPHQWDOGLHWVZLWKDQGRI6,'9DO/\VUDWLR
The composition of experimental diets is shown in Table 1.

Each period of the experiment ran for 21 days and average daily gain (ADG), average daily feed
intake (ADFI) and feed conversion ratio (FCR) were recorded at the end of each week. Blood samples
were taken 3 hours after feeding from the jugular vein at day 18 of the experiment and analyzed for
plasma Val, urea and Lys content.

The data were analyzed by the MIXED procedure (SAS, 2011) considering the effect of treatments.

Results and discussion

The performance parameters showed that the ADFI for the 21 day period tended to be higher
(P< LQWKH9DO/\VUDWLRFRPSDUHGWRWKHORZHUDQGXSSHU9DO/\VUDWLRV YV
DQGNJLQWKHDQG9DO/\VUDWLRVUHVSHFWLYHO\ 7KH$'*ZDVQRWGLIIHUHQW
DPRQJWKHGLHWDU\WUHDWPHQWVGXULQJWKHGD\SHULRG DQGNJLQWKH
and 0.71:1 Val:Lys ratios, respectively). The FCR also was not affected by increasing AA levels in
WKHGLHWVGXULQJWKHH[SHULPHQWDOSHULRG DQGNJLQWKHDQG
9DO/\VUDWLRVUHVSHFWLYHO\ 7KHGDWDRQEORRGSDUDPHWHUV )LJXUH VKRZHGDVLJQL¿FDQWLQFUHDVH

Plasma Val Plasma urea Plasma Lys

0.3 0.3
c b
b 4 ab a
0.2 a 0.2
2
0.1 0.1

0 0 0
0.63:1 0.67:1 0.71:1 0.63:1 0.67:1 0.71:1 0.63:1 0.67:1 0.71:1

)LJXUH7KHSODVPD9DOXUHDDQG/\VFRQWHQWV PPROO LQWKHWKUHHGLHWDU\9DO/\VUDWLRVGXULQJ

WKHRYHUDOOSHULRG GD\ 7KHKLJKHU9DOOHYHOLQWKHGLHWLQFUHDVHGWKHSODVPD9DOFRQWHQW
P< DQGGHFUHDVHGWKHSODVPDXUHDFRQWHQW P< VLJQL¿FDQWO\

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 191
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_58, © Wageningen Academic Publishers 2013
Table 1. Composition of three experimental diets with increasing Val:Lys ratios.

Diet composition,% SID1 Val:Lys

  0.71:1

Wheat   

Soy protein concentrate 17.5 17.5 17.5
Barley 10.0 10.0 10.0
Fat 2.50 2.50 2.50
Limestone, 38% Ca 1.85 1.85 1.85
Monocalcium phosphate 1.05 1.05 1.05
Salt   
Vit-min premix2 0.400 0.400 0.400
Microgrits3 0.070 0.070 0.070
Phytase4 0.020 0.020 0.020
DL-methionine 0.181 0.181 0.181
L-glutamic acid 5 0.150 0.105 0.059
L-histidine   
L-isoleucine   
L-leucine 0.147 0.147 0.147
L-lysine HCl 0.531 0.531 0.531
L-phenylalanine 0.039 0.039 0.039
L-threonine   
L-tryptophan   
L-valine 0.032 0.077 0.123

1 SID = standardized ileal digestible valine to lysine ratio.

2 Provided the following per kg of diet: 10,000 IU vitamin A, 2,000 IU vitamin D3, 94 IU vitamin E, 2.4 mg vitamin
K3, 2.4 mg vitamin B1, 4.8 mg vitamin B2, 2.4 mg vitamin B, 0.02 mg vitamin B12PJ'SDQWKRWHQLFDFLGPJ
QLDFLQPJELRWLQPJ)H )H ,, VXOSKDWH PJ&X &X ,, VXOSKDWH PJ=Q =Q ,, R[LGH PJ0Q
(Mn(II) oxide), 0.3 mg KI, 0.3 mg Se (Se-selenite).
3 Jadis Additiva, Haarlem, the Netherlands.
4 Natuphos 5000 (100 g/t) (BASF, Ludwigshafen, Germany).
5 Included to compose isonitrogenic diets.

in the plasma Val (P<0.001) and a decrease in the plasma urea content (P<0.04) among the dietary
treatments. The results of the current study were in agreement with the estimates of the optimum
DQG6,'9DO/\VUDWLRIRU$'*DQG$'),UHVSHFWLYHO\IRXQGE\:LOWDIVN\et al. (2009).
Gaines et al.  DOVRUHSRUWHGWKDWSHUIRUPDQFHLQFUHDVHGXQWLODUDWLRRIDQGWKHSODVPDXUHD
content decreased quadratically as the Val:Lys ratio increased in the diet. It can be concluded from
WKHFXUUHQWVWXG\WKDWWKH6,'9DO/\VUDWLRVXSSRUWVPRUH$'),DQG$'*RISLJVFRPSDUHG
WRDQG9DO/\VUDWLRV

References
Gaines A. M., D. C. Kendall, G. L. Allee, J. L. Usry, and B. J. Kerr. 2011. Estimation of the standardized ileal digestible
YDOLQHWRO\VLQHUDWLRLQWRNLORJUDPSLJV-$QLP6FL
Wiltafsky, M. K., B. Schmidtlein, and F. X. Roth. 2009. Estimates of the optimum dietary valine to lysine ratio for
HLJKWWRWZHQW\¿YHNLORJUDPVRIERG\ZHLJKWSLJV-$QLP6FL

192 Energy and protein metabolism and nutrition in sustainable animal production
Ideal isoleucine and valine to lysine ratios in low protein diets for
growing pigs
C. Wecke, A. Pastor and F. Liebert
Division of Animal Nutrition Physiology, Department of Animal Sciences, Georg-August-University,
.HOOQHUZHJ*RHWWLQJHQ*HUPDQ\[email protected]

Introduction
Isoleucine (ILE) or valine (VAL) are considered to be potentially limiting the dietary protein quality
in protein reduced pig diets supplemented with L-lysine·HCl, DL-methionine, L-threonine and
L-tryptophan. Consequently, validated dietary ratios of both amino acids (AA) are necessary to
ensure optimal feed protein utilization in fast growing pigs. Therefore, the present study with growing
IDWWHQLQJSLJVZDVFRQGXFWHGWRGHULYH$$HI¿FLHQF\GDWDRI,/(DQG9$/DVUHODWHGWRO\VLQH /<6 
in low protein diets to conclude ideal amino acid ratios (IAAR) of ILE, VAL and LYS, respectively.

Material and methods

N balance studies (n=7) in modern genotype barrows (German Landrace x Piétrain, 55±1 kg average
%: ZHUHFRQGXFWHGWRPHDVXUH1XWLOL]DWLRQDQG$$HI¿FLHQF\RISURWHLQUHGXFHGGLHWV J&3
NJ EDVHGRQEDUOH\  ¿HOGSHDV  VR\EHDQRLO  PLQHUDOVYLWDPLQVDQGDPL[WXUH
of crystalline AAs, respectively. The individual dietary AA ratios related to lysine were above or
in line with current recommendations of GfE (2008), except the AA under study. Principles of
diet dilution technique were applied to achieve individual limiting position of the AA under study.
Additionally, the LYS diluted diet was supplemented with L-glutamic acid (GLU) to examine effects
RILQFUHDVHGGLVSHQVDEOH$$VXSSO\RQHI¿FLHQF\RIO\VLQHDVWKHUHIHUHQFH$$)RUDQDO\VHVRI1
balance data, an exponential N utilization model was used (Liebert and Wecke, 2008). The model
parameter application ran according to the procedure as described by Wecke and Liebert (2010).
%HFDXVHWKH$$HI¿FLHQF\SDUDPHWHUbc-1 summarizes the digestion, absorption and post-absorptive
utilization processes of the limiting AA and is indirectly related to the physiological requirement
SHUXQLWSURWHLQGHSRVLWLRQWKHUHFLSURFDOUHODWLRQVKLSEHWZHHQ/<6HI¿FLHQF\ DVUHIHUHQFH DQG
REVHUYHGHI¿FLHQF\RILQGLYLGXDO$$XQGHUVWXG\ZDVXWLOL]HGWRGHULYHWKH,$$5VDFFRUGLQJWR
Samadi and Liebert (2008):

IAAR = bcLys-1 : bcLAA-1

Statistical analyses ran with software package IBM SPSS Statistics 19.

Results and discussion

7DEOHVXPPDUL]HVWKHREWDLQHGPRGHOSDUDPHWHURISURWHLQTXDOLW\ E DQG$$HI¿FLHQF\ EF-1).
$FFRUGLQJWRH[SHFWDWLRQDQGZLWKUHIHUHQFHWRWKH/<6GLOXWHGGLHWZLWKHOHYDWHGVXSSO\RI,/( 
JNJ DQG9$/ JNJ VLJQL¿FDQWO\ORZHU P<0.05) data for protein quality were observed due to
DSSOLFDWLRQRIWKH,/(DQG9$/GLOXWHGGLHWV/LNHZLVHWKHLQGLYLGXDO$$HI¿FLHQF\RIWKHUHJDUGLQJ
$$GLOXWHGGLHWZDVVLJQL¿FDQWO\LPSURYHG'XHWRWKHREVHUYHGVLJQL¿FDQWHIIHFWVRQSURWHLQTXDOLW\
DQG$$HI¿FLHQF\GDWD,WKHLQGLYLGXDOSRVLWLRQDVOLPLWLQJ$$ /$$ ZDVFRQ¿UPHG,QDGGLWLRQ
QRVLJQL¿FDQWHIIHFWRI*/8VXSSOHPHQWDWLRQWRWKH/<6GLOXWHGGLHWRQPRGHOSDUDPHWHUb and
bc-1 ZDVREVHUYHG,QFRQVHTXHQFHQRGH¿FLHQF\RIQRQHVVHQWLDO$$ 1($$ FRXOGEHH[SHFWHG
Therefore, the applied dietary EAA:NEAA ratio (47:53) can be stated as adequate.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 193
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_59, © Wageningen Academic Publishers 2013
7DEOH0RGHOSDUDPHWHURISURWHLQTXDOLW\DQG$$HI¿FLHQF\DQGGHULYHG,$$5

Diet1 ILE VAL LYS LYS (+GLU)

LAA content (g/kg) 3.75 4.77 9.05 9.05

cLAA JJ1 2.81 3.58  
Protein quality (b) 424b±8 434b“ 555a±12 513a±10
,/(HI¿FLHQF\ bc-1) 151a±3 103c±4 133b±3 b±3
9$/HI¿FLHQF\ bc-1) 87c±2 121a±4 114b±2 ab±2
/<6HI¿FLHQF\ bc-1) b±1 b±2 83a±2 84a±2
IAAR 55  100

1 Model parameters b resp. bc-1 in original were multiplied by factor 10.

%DVHGRQWKHVHFRQGLWLRQVWKHUHODWLRQVKLSEHWZHHQKLJKHVW/<6HI¿FLHQF\ DVUHIHUHQFH DQG

REVHUYHGPD[LPDOHI¿FLHQF\GDWDIRUERWK,/(DQG9$/ZDVXWLOL]HGWRGHULYHWKH,$$57KHUHVXOWV
are summarized in Table 1.

7KHGHULYHGUDWLR/<6,/(9$/ LVLQOLQHZLWKFXUUHQWUHFRPPHQGDWLRQVRI15&
 EDVHGRQWRWDO$$UHTXLUHPHQWGDWD  %6$6  UHFRPPHQGDWLRQVDUHVOLJKWO\
higher for the proportion of standardized ileal digestible ILE (58), but very similar for standardized
ileal digestible VAL (70). Generally, the yielded ratios of ILE and VAL to LYS in the present study
are higher than current German recommendations (GfE, 2008), assuming an IAAR based on AA
FRPSRVLWLRQRIWKHERG\SURWHLQJDLQ  LQJURZLQJIDWWHQLQJSLJV

References
BSAS (British Society of Animal Science), 2003. Nutrient Requirement Standards for Pigs. (Authors: Whittemore, C.
T., M. J. Hazzledine and W. H. Close). BSAS, Penicuik, UK.
GfE (Gesellschaft für Ernährungsphysiologie), 2008. Recommendations for the Supply of Energy and Nutrients
to Pigs. Committee for Requirement Standards of the Society of Nutrition Physiology. DLG-Verlags-GmbH,
Frankfurt a. M., Germany.
Liebert, F. and C. Wecke, 2008. Models for further developing the evaluation of protein and amino acids as well as for
predicting performance from energy and amino acids intake. In Recommendations for the Supply of Energy and
Nutrients to Pigs. Committee for Requirement Standards of the Society of Nutrition Physiology. DLG-Verlags-
GmbH, Frankfurt a. M., pp. 219-230.
NRC (National Research Council), 2012. Nutrient Requirements of Swine. 11th rev. ed., The National Academies
Press, Washington, D.C.
Samadi and F. Liebert, 2008. Modelling the optimal lysine to threonine ratio in growing chickens depending on age
DQGHI¿FLHQF\RIGLHWDU\DPLQRDFLGXWLOLVDWLRQ%ULW3RXOW6FL
Wecke, C. and F. Liebert, 2010. Optimal dietary lysine to threonine ratio in pigs (30-110 kg BW) derived from observed
GLHWDU\DPLQRDFLGHI¿FLHQF\-$QLP3K\VLROD$QLP1XWUHH

194 Energy and protein metabolism and nutrition in sustainable animal production
Interaction between the valine and tryptophan requirement in young
piglets
A.J.M. Jansman1, H. van Diepen1, M. Rovers2 and E. Corrent
1:DJHQLQJHQ 85 /LYHVWRFN 5HVHDUFK 32 %R[   $% /HO\VWDG WKH 1HWKHUODQGV
[email protected]
22UIID$GGLWLYHV%99LHUOLQJKVWUDDW/&:HUNHQGDPWKH1HWKHUODQGV
$MLQRPRWR(XURO\VLQHVDVUXHGH&RXUFHOOHV3DULV&HGH[)UDQFH

Introduction
After lysine, methionine plus cysteine, threonine and tryptophan, valine is the next limiting amino
acid in most common diets for post weaning piglets, in particular in low protein diets. To be able
to formulate low protein diets it is essential to have adequate information on the requirement for
valine in young piglets and the interaction with the requirements for other amino acids. It is known
that an interaction exist between tryptophan and branched-chain amino acids (BCAA; Val, Leu and
Ile) as these amino acids share a common transport mechanisms across membranes (Henry et al.,
1992; Langer and Fuller, 2000). This suggests that the response towards dietary supplementation
of L-valine could be affected by the dietary level of tryptophan. The aim of the present study was
to evaluate the interaction between the requirement for valine and tryptophan in young piglets in a
performance study in the post weaning period (from about 8 to 24 kg body weight).

Material and methods

Five experimental treatments (I to V) were evaluated each receiving a different experimental diet.
$YDOLQHDQGWU\SWRSKDQGH¿FLHQWORZSURWHLQEDVDOGLHW J&3SHUNJJ6,'7USSHUNJ
7.0 g SID Val per kg; treatment I) was used supplemented with 1.0 g/kg L-valine (treatment II) or
0.5 g/kg L-tryptophan (treatment III) or both (treatment IV). A reference treatment was included
receiving a diet with a crude protein content of 192 g CP/kg and 2.5 g SID Trp per kg and 8.0 g/
kg SID Val (treatment V). The diets were based on barley, maize, wheat, soybean meal and peas as
PDLQLQJUHGLHQWVDQGFRQWDLQHGJ6,'/\VSHUNJ(DFKRIWKH¿YHWUHDWPHQWVZDVHYDOXDWHG
in eight replicates (pens with eight male piglets). Feed intake (FI), body weight gain (BWG) and
feed conversion ratio (FCR) were measured as the response criteria over an experimental period
of four weeks, starting one week post weaning. Except for amino acids, the diets were formulated
WREHQXWULWLRQDOO\FRPSOHWH &9%15& ,QWKH¿UVWZHHNDIWHUZHDQLQJWKHGLHWIRU
treatment V was given to all piglets.

Results and discussion

2YHUZHHNV%:*DQG)&5ZHUHVLJQL¿FDQWO\DIIHFWHGE\WKHGLHWDU\WUHDWPHQWV P<0.05).
Compared to treatment II (only supplemented with L-valine), BWG was improved (+14%) in
treatment IV, receiving the L-valine and L-tryptophan supplemented diet (P<0.05). The FCR over
this period was improved in treatment IV compared to treatments I, II and III (P<0.05). FI over weeks
1-2 did not differ between treatments (P>0.05) but tended to be lower in treatment II compared to the
other treatments. Over the complete experimental period (0-4 weeks; Table 1), FI was numerically
lower in treatment II (only supplemented with L-valine) compared to the other treatments. BWG in
treatment II was lower compared to all other treatments (P<0.05). BWG was numerically highest
in treatment IV (supplemented with both L-valine and L-tryptophan). Over weeks 0-4, FCR was
VLJQL¿FDQWO\ORZHULQWUHDWPHQW,9FRPSDUHGWRDOORWKHUWUHDWPHQWV P<0.05).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 195
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_60, © Wageningen Academic Publishers 2013
Table 1. Feed intake, body weight gain and feed conversion ratio over the experimental period
ZHHNV 

FI (kg/d) % of I BWG (g/d) % of I FCR % of I

I &3JNJ 0.838 100 559 b 100 1.498b 100

II I + Val 0.790 94 530 a 95 1.492b 100
III I + Trp 0.842 101 b 101 1.493b 100
IV I + Val + Trp 0.847 101 582 b 104 1.455a 97
V CP, 192 g/kg 0.842 100 b 102 1.482b 99
P-value 0.058 0.005 0.042
LSD1 0.042 25.8 

a,b Values with a different superscript in the same column within a factor differ at P<0.05.
1/6' OHDVWVLJQL¿FDQWGLIIHUHQFH P<0.05).

The study revealed an interaction between the dietary supply of tryptophan and valine with regard
to the growth performance of post weaning piglets. Supplementation of 1.0 g/kg L-valine to a low
WU\SWRSKDQGLHW JNJ6,'7US ZLWKJNJ&3GHFUHDVHGIHHGLQWDNHDQGERG\ZHLJKWJDLQ
while supplementation of 1.0 g/kg L-valine to a diet with an adequate tryptophan level (2.4 g/kg
SID Trp) increased performance and improved feed conversion ratio in piglets over the period of
weeks 2 to 5 post weaning. The former emphasizes the value of the ideal protein concept in diet
IRUPXODWLRQ &KXQJDQG%DNHU DQGWKXVWKHQHHGIRUSURSHUO\EDODQFLQJDOOHVVHQWLDODPLQR
acids in practical diets in order to achieve maximum performance in post weaning piglets using
low protein diets.

References
Chung, T.K., and D.H. Baker, 1992. Ideal amino acid pattern for 10-kilogram pigs. J. Anim. Sci. 70, 3102-3111.
&9%>5HTXLUHPHQWVIRUDPLQRDFLGVLQSLJOHWVDQGSLJV@,Q'XWFK&9%GRFXPHQWDWLRQUHSRUW
Henry, Y., B. Sève, Y. Colléaux, P. Ganier, C. Saligaut, and P. Jégo, 1992. Interactive effects of dietary levels of
tryptophan and protein on voluntary feed intake and growth performance in pigs, in relation to plasma free amino
acids and hypothalamic serotonin. J. Anim. Sci. 70:1873-1887.
Langer, S., and M. F. Fuller, 2000. Interactions among the branched chain amino acids and their effects on methionine
utilization in growing pigs: Effects of nitrogen retention and amino acid utilization. Br. J. Nutr. 83:43-48.
NRC, 1998. Nutrient Requirements of Swine. National Research Council. National Academy Press, Washington, US.

196 Energy and protein metabolism and nutrition in sustainable animal production
Is high protein diet a good nutrition strategy for broiler chickens reared
at heat stress condition?
D.M.B. Campos, M.F. Fernandez-Alarcon, F.A. Souza, W.C.L. Nogueira, F.H. Hada, P.R.O. Carneiro
and M. Macari
Department of Animal Morphology and Physiology, Sao Paulo State University, Jaboticabal, SP,
Brazil; [email protected]

Introduction
It is well known that heat stress, in growing broiler chicks, impair the performance. Ain Baziz et
al  UHSRUWHGWKDWRIWKHLPSDLUPHQWLQWKHSHUIRUPDQFHZDVGXHWRUHGXFWLRQLQIHHG
consumption, but the last 47% was due to the direct effect of ambient temperature, per se. One of the
approaches that have been proposed to avoid the effect of heat stress in broilers is related to the protein
content in the diet, since protein metabolism is deeply involved in the caloric increment, as compared
ZLWKIDWRUFDUERK\GUDWH$FFRUGLQJWR*RQ]DOH](VTXHUUDDQG/HHVRQ  WKH$UJ/\VUDWLR0HW
source and time to exposure to heat stress affect protein utilization in hyperthermic birds. Thus, this
study was conducted aiming to verify if the increase of protein content in the diet, maintaining the
Arg:Lys ratio, affects broiler chicks performance when reared at different environmental conditions.

Material and methods

It was used seven hundred and twenty male chicks from Cobb™ strain. The experiment was performed
GXULQJWKHEURLOHUFKLFNV¿QLVKLQJSHULRG WRGD\VRIDJH IROORZLQJDSURWHLQOHYHO 
and 22%) × 3 rearing temperature schedules [thermoneutral ad libitum (TN-AL), cyclic heat stress
&+6KDWWKHUPRQHXWUDOLW\DQGKDWƒ& DQGWKHUPRQHXWUDOSDLUIHG 713) ZLWK&+6@
in a factorial design, with four replicates of 20 birds each (total of 9 treatments). From 1-21 days of
DJHWKHELUGVZHUHIHGGLHWFRQWDLQLQJRIFUXGHSURWHLQ &3 DQGNFDO0(NJ'XULQJWKH
experimental period, the diets had the same nutritional and energy levels (3,185 kcal AMEn/kg, 0.7%
Ca, 0.33% available P, 0.20% Na, 1.08-1.10% Lys, 0.49-0.54% Met, 0.79% Met+Cys, 0.70-0.75%
Thr, 0.20-0.25% Trp, 1.53-1.50% Arg, 1.39 Arg:Lys, 0.45-0.50 Met:Lys), except for crude protein
content. Feed intake (FI), feed conversion (FC), daily weight gain (WG), viability (V), productive
HI¿FLHQF\LQGH[ 3(, :*î9  )&î FDUFDVV\LHOG &< EUHDVW % DQGWLJKWGUXPVWLFN 7' 
yield were measured. The data were submitted to analysis of variance (General Linear Model, SAS)
DQGVLJQL¿FDQFHZDVFRPSDUHGE\7XNH\¶VWHVWDWSUREDELOLW\

Results and discussion

The FI was lower and FC was better for 22% CP, in comparison to other levels of CP (Table 1).
Some authors reported the same results (Temim et al., 2000; Widyaratne and Drew, 2011), suggesting
WKDWWKHJURZWKHI¿FLHQF\ZDVHQKDQFHGE\WKHLQFUHDVHGFUXGHSURWHLQRIWKHGLHWLQKHDWVWUHVVRU
thermoneutrality temperature in fast-growing strains.

The treatment C-HS presented worse performance (FI and WG) compared to TN-AL (Table 1).
These results could be related to the chicken attempt to reduce the impact of extra heat load due to
VWUHVV7KHUHGXFWLRQRIJURZWKHI¿FLHQF\FDXVHGE\&+6DIIHFWHG&<OHDGLQJWREHWWHUUHVXOWVLQ
TN-AL treatment. Even under a cyclic heat stress, which allows the chickens to recover, it could
be noticed a negative effect in performance and CY, as found by Gonzalez-Esquerra and Leeson
(2005) and Temim et al. (2000).

There was no effect in V due to the CP treatments or temperature schedules. But, it was observed a
VLJQL¿FDQWLQWHUDFWLRQEHWZHHQWKHPDLQIDFWRUVIRU3(,7KHELUGVIHG&3LQ&+6FRQGLWLRQ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 197
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_61, © Wageningen Academic Publishers 2013
presented better performance than those fed 18.91% CP (Table 2). Since PEI takes into account WG,
V and FC, slightly changes in these parameters was able to promote this improvement. Based on
those results, the increase in the crude protein diet could be a feasible nutritional strategy to reduce
the effects of cyclic thermal stress during the broilers’ growth period.

7DEOH3HUIRUPDQFHRIEURLOHUFKLFNHQVIURPWRGD\VROGVXEPLWWHGWRWKUHHGLHWDU\SURWHLQ
levels and reared under different ambient temperatures.1

CP% FI (kg) FC WG (g) V (%) PEI CY (%) B (%) TD (%)

18.91 A 1.57A  95.00  72.84 39.78 28.14

21.00 3.84A A  95.41  72.41 39.15 
22.00 3.74B 1.52B 102.89 98.33  71.97 38.95 28.92
Temp.
CY-HS C 1.49B 101.38B 95.42  71.52B 38.91 29.3A
TN-PF B 1.59A 100.92B   72.18AB 39.58 22.9B
TN-AL, 22 °C A A A   73.51A 39.40 AB
P-value
CP% 0.0001 0.0092 0.9929 0.1153 0.0105 0.3852  0.0882
Temp. 0.0128 0.0001 0.0114   0.0123  0.0052
CP%*Temp. 0.1438 0.3211  0.4011 0.0120   0.9072
CV 2.52 2.54 4.13 4.27   2.12 1.47

1 Means followed by same uppercase letters in column do not differ by Tukey test (P>0.05).

7DEOH7HPSHUDWXUHDQG&3LQWHUDFWLRQRQWKHELUGV¶3(,IURPWRGD\VROG1

Temp. CP%

18.91 21 22

C-HS 570.84B AB 710.89A

TN-PF 585.30A A A
TN-AL A A A

1 Means followed by the same uppercase letters in row do not differ by Tukey test (P>0.05).

References
$LQ%D]L]+*HUDHUW3$3DGLOKD-&)6*XLOODXPLQ&KURQLF+HDW([SRVXUH(QKDQFHV)DW'HSRVLWLRQ
DQG0RGL¿HV0XVFOHDQG)DW3DUWLWLRQLQ%URLOHU&DUFDVVHV3RXOW6FL.75, 505-513.
Gonzalez-Esquerra, R., and S. Leeson.2005. Effects of Acute Versus Chronic Heat Stress on Broiler Response to
'LHWDU\3URWHLQ3RXOW6FL
*RQ]DOH](VTXHUUD5DQG6/HHVRQ(IIHFWRI$UJLQLQH/\VLQH5DWLRVDQG6RXUFHRI0HWKLRQLQHRQ*URZWK
DQG%RG\3URWHLQ$FFUHWLRQLQ$FXWHO\DQG&KURQLFDOO\+HDW6WUHVVHG%URLOHUV3RXOW6FL
Temim, S., Chagneau, A. M.; Guillaumin, S., Michel, J., Peresson, R., S. Tesseraud. 2000. Does Excess Dietary Protein
Improve Growth Performance and Carcass Characteristics in Heat-Exposed Chickens? Poult. Sci. 79, 312-317.
Widyaratne, G. P., and M. D. Drew. 2011. Effects of protein level and digestibility on the growth and carcass
FKDUDFWHULVWLFVRIEURLOHUFKLFNHQV3RXOW6FL

198 Energy and protein metabolism and nutrition in sustainable animal production
Evaluation of energy systems in corn and barley based diets and an
enzyme complex in broiler chicks
S. Cerrate1, J. Caldas, R. Ekmay2, J. England and C. Coon
1$YLDJHQ,QFRUSRUDWHG+XQWVYLOOH$/86$
2'HSDUWPHQWRI$QLPDO6FLHQFH&RUQHOO8QLYHUVLW\,WKDFD1<86$
3RXOWU\6FLHQFH'HSDUWPHQW8QLYHUVLW\RI$UNDQVDV)D\HWWHYLOOH$586$[email protected]

Introduction
The extensive use of an NE system has not been accepted by the poultry industry because NE feed
evaluation is laborious, values have a high variability and the process is expensive. Productive energy
3( GHYHORSHGE\)UDSVDQG&DUO\OH  ZDVXVHGFRPPHUFLDOO\IURPDQGZDV
unfortunately related to NE. The methodology for the determination of PE is considered the cause
IRUWKHKLJKYDULDELOLW\'H*URRWH  XVHGRQHSODQHRIIHHGLQWDNHDORQJZLWKDQLQGHSHQGHQW
NEm and showed similar variability between ME and NE energy systems. High energy feed costs
have caused the poultry industry to dramatically increase the use of exogenous feed enzymes during
the past 5 years. The objectives of this study were to compare the ME and NE systems for variability,
predicted performance and to determine the extra energy content due to an enzyme complex.

Material and methods

Twenty mash diets from 5 ingredients (corn, soybean meal, pro-plus, barley and poultry fat) and Rovabio®
Max at 0.05% (xylanase, B-glucanase, and phytase) were fed to male chicks from 1 to 21 d old. Feed
intake was adjusted for each diet to supply similar ME intake per day. There were 3 groups of diets which
KDGH[FHVVOHYHOVRIIDW DQG(( SURWHLQ DQG&3 RU¿EHU DQG
QHXWUDOGHWHUJHQW¿EHU DVLQFUHDVLQJSRXOWU\RLO6%03UR3OXVDQGEDUOH\UHVSHFWLYHO\(DFKGLHWDU\
WUHDWPHQWZDVIHGWRUHSOLFDWHÀRRUSHQVRIPDOHDQGWUDQVIHUUHGWRGLJHVWLELOLW\FDJHV FKLFNV
cage) and mixed with 0.5% of titanium oxide (TiO2 $WGRIDJHDQGGRIDJHELUGVSHUÀRRUSHQ
were randomly collected to measure protein and fat gain using the Dual Energy X-ray Absorptiometry
(DEXA) scanner (Lunar Prodigy from GE®). Dn = Nutrientdiet – Nutrientexcreta × (TiO2diet / TiO2excreta);
Dn = nutrient digestibility. ME, kcal/kg = GEdiet – GEexcreta × (TiO2diet / TiO2excreta) – 8.22 × (Ndiet –
Nexcreta × (TiO2diet / TiO2excreta)). NE, kcal/kg = (NEm + NEg)/FI; where: NEm = 90 × 1.15 × BWx0.75,
1(J NFDOJîIDWJDLQNFDOJ[SURWHLQJDLQ+3 0(±1(J+, 0(±1(

Results and discussion

7KHODUJHUFRHI¿FLHQWVIRU1')LQGLJHVWLELOLW\HTXDWLRQVWRLPSURYHSURWHLQ 7DEOH(TXDWLRQ
1.1 and 1.2), fat (Equation 2.1 and 2.2), and starch (Equation 3.1 and 3.2) digestibility from chicks
IHGFDUERK\GUDVHVLPSO\WKDW¿EHUGLJHVWLRQKHOSVWRUHOHDVHWKHQXWULHQWV7KHUHZDVLQWHUDFWLRQ
for ME and enzyme factors, where the extra ME due to carbohydrases were 45 and 213 kcal/kg
for corn and barley based diets, respectively (data not shown). On the other hand, the extra NE due
to carbohydrases was 113 kcal/kg for all types of diets. There was similar variability between ME
&9  DQG1(V\VWHPV &9   3  (QHUJHWLFHI¿FLHQFLHV 1(0( RI0(IURP
FRHI¿FLHQWVRISURWHLQIDWDQGFDUERK\GUDWHVWRSUHGLFW0( (TXDWLRQ DQG1( (TXDWLRQ ZHUH
DQGUHVSHFWLYHO\7KH1(LQWDNHZDVDEHWWHUSUHGLFWRURI%:*UDWKHUWKDQ0(
intake which indicates that NE system is a superior energy system for feed formulation (Figure 1).

In conclusion, the energy evaluation systems (ME vs. NE) affected the hierarchy among digestible
nutrients. The NE intake was a better predictor of BWG than the ME intake and both energy systems
showed similar variability. The extra ME contents due to carbohydrases were 45 and 213 kcal/as-fed
for corn and barley based diets respectively and the extra NE from carbohydrases was 113 kcal/kg
as-fed for all types of diets.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 199
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_62, © Wageningen Academic Publishers 2013
7DEOH5HJUHVVLRQHTXDWLRQVRIGLJHVWLELOLW\FRHI¿FLHQWRIQXWULHQWV '&1'0 0( NFDONJ
DVIHG DQG1( NFDONJDVIHG IURPGLHWDU\DQGGLJHVWHGQXWULHQWV '0 

No. Enzyme Equations n R2 SEM

1.1 No DC CP = 0.921(0.204)a>&3@  a>((@  45 0.988 7.3

b[NDF]x + 1.101(0.180)a[Starch]
1.2 Yes '&&3   a[CP] + 0.930(0.189)a[EE] + 45 0.988 3.9
0.494(0.241)a>1')@  a[Starch]
2.1 No '&((   a[CP] + 0.524(0.329)a[EE]x + 45 0.993 7.0
  b[NDF]
2.2 Yes DC EE = 1.591(0.214)a[CP] – 0.108(0.378)a[EE]x + 45 0.991 8.1
  a[NDF]
3.1 No '&VWDUFK   a – 0.48(0.228)b[NDF] 45 0.095 3.7
3.2 Yes DC starch = 75.2(3.90)a  xa[NDF] 45  4.0
4 Pooled 0(   >G&3@  >G((@  >G&+2@ 80 0.999 49
5 Pooled 0(   >G&3@  >G((@  >G&+2@ 80  132

a,b Values within columns of same type of nutrients and with or without carbohydrases having superscript letters differ

VLJQL¿FDQWO\ P< DFFRUGLQJWRFRQ¿GHQFHLQWHUYDOxP-value >0.05.

)LJXUH/LQHDUUHJUHVVLRQVEHWZHHQERG\ZHLJKWJDLQDQG0(, $ RU1(, % 

References
'H*URRWH*(QHUJHWLFHYDOXDWLRQRIXQVWDELOL]HGDQGVWDELOL]HG¿VKPHDOVLQWHUPVRIPHWDEROL]DEOHHQHUJ\
and net energy for maintenance and growth. Feedstuffs, Minneap.,   
Fraps, G.S., and E.C. Carlely, 1939. The utilization of the energy of feed by growing chicks. Texas Agricultural
Experiment Station Bulletin. 571.

200 Energy and protein metabolism and nutrition in sustainable animal production
9DULRXV¿EHUIUDFWLRQVDVHQHUJ\VXSSO\WRH[HUFLVLQJKRUVHV
C. Brøkner1, D. Austbø2, J.A. Næsset2, D. Blache, K.E. Bach Knudsen and A.H. Tauson1,2
18QLYHUVLW\ RI &RSHQKDJHQ 'HSDUWPHQW RI 9HWHULQDU\ &OLQLFDO DQG $QLPDO 6FLHQFHV 
Frederiksberg C, Denmark; [email protected]
21RUZHJLDQ8QLYHUVLW\RI/LIH6FLHQFHV'HSDUWPHQWRI$QLPDODQG$TXDFXOWXUDO6FLHQFHV
Ås, Norway
8QLYHUVLW\RI:HVWHUQ$XVWUDOLD)DFXOW\RI1DWXUDODQG$JULFXOWXUDO6FLHQFHV3HUWK$XVWUDOLD
$DUKXV8QLYHUVLW\'HSDUWPHQWRI$QLPDO6FLHQFH7MHOH'HQPDUN

Introduction
Horses have evolved to eat forages, which primarily are fermented in the hindgut. Results of previous
VWXGLHVLQGLFDWHWKDW¿EHUULFKLQJUHGLHQWVFDQEHXVHGDVRQO\GLHWIRUH[HUFLVLQJKRUVHV -DQVVRQ
et al., 2012). However, the knowledge on the concomitant pattern of changes in blood metabolite
FRQFHQWUDWLRQVGXHWR¿EHUULFKGLHWVKRUVHVLVOLPLWHG7KLVH[SHULPHQWWKHUHIRUHDLPHGDWVWXG\LQJ
the post-prandial blood plasma response in horses fed diets of different content of soluble and
LQVROXEOH¿EHUIUDFWLRQVZKLOHH[HUFLVLQJGDLO\

Material and methods

Four trotter horses with an average body weight of 544 kg were used in a 4×4 Latin square design
with a sequence of 24 days adaptation followed by 2 sampling days. The feed rations consisted of
only timothy hay (H), hay and molassed sugar beet pulp (Betfor®) combined with either whole oats
(OB) or whole barley (BB), hay and a loose chaff based concentrate (Equigard®) (M). The daily
UDWLRQVZHUHGLYLGHGLQWRWKUHHPHDOVPRUQLQJDWDPDIWHUQRRQDWSPDIWHUHQGRIVDPSOLQJ
and night at 10 pm. The morning concentrate meals were formulated to contain a maximum of 2 g
VWDUFKNJ%:7KHH[SHULPHQWDOGLHWVZHUHIRUPXODWHGWREHLVRHQHUJHWLFDQGWRIXO¿OOWKH'DQLVK
feeding standards for medium work level (1.5× maintenance). Each horse was subjected to a standard
exercise test at the end of each experimental period on an indoor high speed treadmill.

Blood was drawn via jugular vein puncture into heparinized vacutainer tubes at time 0 before the
morning meal and again at 3 and 9 h post feeding. Blood plasma was analyzed for metabolites and
UHJXODWRU\KRUPRQHV)HHG¿EHUDQGVWDUFKFRQWHQWZDVDQDO\]HGDVQRQVWDUFKSRO\VDFFKDULGH 163 
soluble non-cellulosic polysaccharides (S-NCP), insoluble non-cellulosic polysaccharide (I-NCP)
and starch as described by Brøkner et al. (2012). Nutrient intake (kg DM) of the diets H, OB, BB, M
UHVSHFWLYHO\ZDVDVIROORZV16361&3,1&3
starch 0.02, 0.95, 1.09, 0.21. Heart beats were recorded by use of portable polar equine training system.

Results were analyzed statistically by use of SAS version 9.2 (SAS Institute Inc, 1999) and results
ZHUHFRQVLGHUHGVLJQL¿FDQWO\GLIIHUHQWZKHQP<0.05 and a tendency when P<0.10.

Results and discussion

7KHGLHWKLJKHVWLQVROXEOH¿EHU 0 UHVXOWHGLQWKHKLJKHVWSODVPDJOXFRVHDQGLQVXOLQFRQFHQWUDWLRQV
LQWKHPRUQLQJDIWHUQHDUO\KRXUVIDVW )LJXUH 7KLVFDQEHDVFULEHGWRWKHJOXFRQHRJHQLFSURSHUWLHV
of propionic acid through hepatic gluconeogenesis (Jose-Cunilleras and Hinchcliff, 2004). Only the
FRQFHQWUDWLRQ 7DEOH RISODVPDEHWDK\GUR[\EXW\UDWH %+%$ ZDVVLJQL¿FDQWO\DIIHFWHGE\GLHW
(P<0.001) and highest on the hay only diet. The hay diet therefore seemed to result in a negative
HQHUJ\EDODQFHEHFDXVHWKHVLJQL¿FDQWO\KLJKHU%+%$FRQFHQWUDWLRQDQGQXPHULFDOO\KLJKHUSODVPD
QRQHVWHUL¿HGIDWW\DFLGV 1()$ FRQFHQWUDWLRQLQGLFDWHGWKDWWKHKRUVHVZHUHPRELOL]LQJIDW7KLV
implies that the horses were working harder than medium work level. However, the blood lactate

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 201
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_63, © Wageningen Academic Publishers 2013
)LJXUH7KHSRVWSUDQGLDOLQWHUDFWLRQEHWZHHQGLHWDQGWLPHRQEORRGSODVPDJOXFRVH $ DQGLQVXOLQ
% LQKRUVHVIHGGLIIHUHQWGLHWV PHDQRIVDPSOHV +D\ + RDWVDQG%HWIRU® 2% EDUOH\DQG
Betfor® %% (TXLJDUG®DFKDIIEDVHGFRQFHQWUDWH 0 7KHOHWWHUVLQGLFDWHWKHOHYHORIVLJQL¿FDQFH
P” ZKHUHWKHXSSHUFDVHA-B indicate effect of time and the lower casea-c indicate effect of diets.

7DEOH(IIHFWRIGLHWDQGWLPHRQEORRGSODVPD%+%$ /60HDQPPROO DQG1()$ /60HDQ—PROO 1

Diet Time P-value

H OB BB M SE 0 3 9 SE Diet Time

BHBA 0.22a 0.17b 0.17b 0.18b <0.01 0.19a 0.17b 0.19a <0.01 <0.01 <0.01
NEFA  47.0 34.7 31.5 9.2 45.4a b 52.7a 8.8 0.24 <0.01
Whole blood after training, mmol/l
Lactate 2.8 2.9 2.1 2.1 0.8 - - - - 0.8 -

17KHOHYHORIVLJQL¿FDQFHZDVFKRVHQZKHQP-value <0.05. The values in rows with different superscripts differ

VLJQL¿FDQWO\+ KD\2% RDWVDQG%HWIRU%% EDUOH\DQG%HWIRU0 FKDIIEDVHGFRQFHQWUDWH1()$ QRQHVWHUL¿HG
fatty acids; BHBA = beta-hydroxybutyrate.

immediately after training ranged from 2.1-2.9 mmol/l which shows that the anaerobic threshold
had not been reached, and that the horses were working aerobically (Jose-Cunilleras and Hinchcliff,
2004) equivalent to medium work level. The negative energy balance could also be explained by
different energy intake even though the experimental diets were formulated to be iso-energetic. It
is possible that the energy content in the hay was overestimated.

,QFRQFOXVLRQWKLVUHVXOWVVXJJHVWHGWKDW¿EHUEDVHGGLHWVFRXOGIXO¿OOWKHHQHUJ\UHTXLUHPHQWVIRU
horses at medium work level. However, there were indications that the horses on the hay only diet
were in negative energy balance. Since the horses were not working harder than medium level and
the experimental diets were iso-energetic, these results indicate that the feed evaluation system for
KRUVHVQHHGVIXUWKHUUH¿QHPHQWLQRUGHUWRPRUHDFFXUDWHGHWHUPLQHWKHHQHUJ\FRQWHQWLQGLHWVDQG
in particular diets of high content of insoluble non-starch polysaccharides like hay.

References
Brøkner, C., K.E. Bach Knudsen, I. Karaman, K.L. Eybye and A.H. Tauson. 2012b. Chemical and physicochemical
FKDUDFWHUL]DWLRQRIYDULRXVKRUVHIHHGLQJUHGLHQWV$QLP)HHG6FL7HFK
Jansson, A., M. Saastamoinen and J. E. Lindberg. 2012. Forage feeding system. In: Saastamoinen, M., M.J. Santos and
N. Miraglia, (eds.) Forages and grazing in horse nutrition. EAAP series no. 132. Wageningen Academic Publishers,
Wageningen, the Netherlands, pp 289-303.
Jose-Cunilleras, E. and K.W. Hinchcliff. 2004. Carbohydrate metabolism in exercising horses. Equine Comp. Exercise
Physio. 1, 23-32.
SAS Institute Inc. 2008: SAS/STAT User’s Guide, Version 9.2 Cary, NC, USA.

202 Energy and protein metabolism and nutrition in sustainable animal production
Changes in protein turnover during pregnancy in pigs when feeding
limiting amounts of amino acids.
S. Moehn1, 05D¿L2, P.B. Pencharz2 and R.O. Ball1
1'HSDUWPHQWRI$JULFXOWXUH)RRGDQG1XWULWLRQDO6FLHQFH8QLYHUVLW\RI$OEHUWD(GPRQWRQ7*
3&DQDGD[email protected]
25HVHDUFK,QVWLWXWH+RVSLWDOIRU6LFN&KLOGUHQ7RURQWR0*;&DQDGD

Introduction
A series of experiments were completed to determine the amino acid (AA) requirements of pigs
for threonine (Thr), lysine, tryptophan (Trp) and isoleucine (Moehn et al., 2011) in early and late
pregnancy. The objective of the present analysis was to determine which factors caused changes
in protein turnover during pregnancy in pigs when the intake of test AA was below requirements.

Material and methods

,QHYHU\H[SHULPHQWWRVRZVHDFKUHFHLYHGGLHWVZLWKJUDGHGOHYHOVRIWHVW$$LQERWKHDUO\DQG
late gestation. Protein turnover was determined using a stochastic model based on the 13C enrichment
in expired CO2 and plasma free phenylalanine (Phe) after oral L[13C]Phe dosing. To combine the
results of all experiments, test AA intake was expressed as a fraction of the requirement determined
LQHDFKH[SHULPHQWHJWKH7KULQWDNHRIJGHTXDOHGWLPHVWKHUHTXLUHPHQWRIJG
RUWKH7USLQWDNHRIJGHTXDOHGWLPHVWKHUHTXLUHPHQWRIJG7KHREVHUYDWLRQV Q 
IRUR[LGDWLRQ>2;@DQG3KHUHWHQWLRQQ IRUÀX[SURWHLQEUHDNGRZQ>%@DQGV\QWKHVLV>6@ ZHUH
made on 23 sows in their 2nd to 4th parity between day 23 and 110 of pregnancy. Sow body weight
(BW) at each study day ranged from 153.8 kg to 294.0 kg. Maternal and total gain in pregnancy
ZDVEHWZHHQDQGNJDQGDQGNJUHVSHFWLYHO\,QWDNHVRIWHVW$$UDQJHGIURP
0.290 to 0.998 of requirement, Phe intake was constant within sows but varied among experiments
between 8.52 and 15.40 g/d and metabolizable energy (ME) from 510 to 791 MJ/kg0.75. Litter size
was between 4 and 20 piglets born alive. Data were evaluated using the mixed model procedure
of SAS with pig nested in experiment as a random variable. The start model contained the above
parameters, including 2-way and 3-way interactions. The test AA was included in the model without
LQWHUDFWLRQV7KHVWDUWLQJPRGHOZDVUHGXFHGLWHUDWLYHO\E\GHOHWLQJDOOQRQVLJQL¿FDQWWHUPVXQWLO
all parameters left were at P<0.05.

Results
The resulting models explained a high degree of the variation in the data with r2=0.82 (S) to r2=0.92
(Ox, g/d). Of the feed-related factors, only AA intake had an effect on Phe kinetics, while ME intake
SURYHGQRQVLJQL¿FDQWSUREDEO\EHFDXVHRIWKHVPDOOYDULDELOLW\LQ0(GXHWRWKHUHVWULFWHGIHHGLQJ
regimen. Increasing AA intake decreased Phe Ox linearly (3 0.001), which led to increased Phe
retention (3  EHFDXVH6EUHDNGRZQ % DQG3KHÀX[ZHUHQRWDIIHFWHG,QFUHDVLQJ%:
LQFUHDVHG3KHÀX[2[6DQG% P<0.01) but decreased Phe retention. Increasing gestational age
decreased Ox and B linearly (P< DQGKDGTXDGUDWLFHIIHFWVRQÀX[DQG6 P<0.02), which
UHDFKHGPLQLPDRQGD\DQGRIJHVWDWLRQUHVSHFWLYHO\3KHQ\ODODQLQHUHWHQWLRQLQFUHDVHGOLQHDUO\
throughout pregnancy (3 0.001). Phenylalanine retention increased with increasing maternal BW
gain (3 0.001) and decreased (3 0.001) with sow age. For the other parameters of Phe kinetics, sow
DJHLQWHUDFWHGZLWKPDWHUQDO%: IRUÀX[6DQG% RUWRWDOVRZ%:JDLQ IRU2[ 3KHQ\ODODQLQH
OX was less affected by maternal BW gain in older than in younger sows (3 0.015). Flux, S and B
were greatest in parity 3, and were most affected by maternal BW gain in the 3rd parity. Increasing
litter size increased B (3  ÀX[DQG6 P<0.01) but not Ox and Phe retention.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 203
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_64, © Wageningen Academic Publishers 2013
Discussion
The effect of fetal growth on protein metabolism becomes apparent in the increase B, and to a lesser
GHJUHHRIÀX[DQG6ZLWKLQFUHDVLQJOLWWHUVL]H7KHLQFUHDVHLQ%ZRXOGVXSSO\DGGLWLRQDO$$IRU
conceptus growth when AA intake is limiting, while the increase in S represents the greater protein
growth with larger litters and maternal growth. The data could not distinguish between maternal
and conceptus gain for Phe retention and oxidation. The increase in Phe retention was in agreement
with greater total sow BW gain. As expected, increasing the limiting AA intake decreased Ox and
increased Phe retention. The lack of impact on S and B, however, was unexpected because growing
SLJVKDYHVKRZQWRLQFUHDVHÀX[6DQG%LQUHVSRQVHWRLQFUHDVLQJ$$LQWDNH )XOOHUet al., 1987;
Salter et al., 1990). This indicates a qualitatively different response of protein kinetics in pregnancy.
Conversely, the decrease in OX and B with progress of pregnancy and concomitant Phe retention
can be explained by the increased AA requirement for fetal growth in the latter part of pregnancy.
7KLVZDVDLGHGE\WKHUHVSRQVHRIÀX[DQG6WKDWDOVRLQFUHDVHGWRZDUGVWKHHQGRISUHJQDQF\
after reaching a minimum in mid gestation. In contrast to sows given limiting AA intake, S in non-
malnourished women was either not affected by stage of pregnancy (Kalhan et al., 1998) or elevated
in mid pregnancy (Willommet et al., 1992).

In keeping with diminishing maternal growth, Phe retention decreased as sows aged. The response
RIÀX[6DQG%WRPDWHUQDOJDLQZDVPD[LPL]HGLQWKHrd parity. Although sow BW increased from
2nd to 4th parity, maternal gain was similar in the 2nd and 3rd parity, and only dropped off in the 4th.
This may have created a maximum for metabolic demand in the 3rd parity.

In conclusion, beside AA intake, Phe kinetics were affected by the stage of pregnancy and the
maternal and conceptus gain. Reduction of OX and B in late pregnancy was the main response to
accommodate the rapid fetal growth in late pregnancy when test AA intake was limiting.

References
Fuller, M.F., P. J. Reeds, A. Cadenhead, B. Seve, and T. Preston. 1987. Effects of the amount and quality of dietary
protein on nitrogen metabolism and protein turnover of pigs. Br. J. Nutr. 58: 287-300.
Kalhan, S.C., K.Q. Rossi, L.L. Gruca, D.M. Super, and S.M. Savin. 1998. Relation between transamination of branched-
chain amino acids and urea synthesis: evidence from human pregnancy. Am. J. Physiol.275 (Endocrinol. Metab.
38: E423-E431.
Moehn, S., C. Levesque, R. Samuel and R.O. Ball. 2011. New energy and amino acid requirements for gestating sows.
$GYDQFHVLQ3RUN3URGXFWLRQ
Willommet, L., Y. Schutz, R. Whitehead, E. Jéquier and E.B. Fern. 1992. Whole body protein metabolism and resting
HQHUJ\H[SHQGLWXUHLQSUHJQDQW*DPELDQZRPHQ$P-3K\VLRO (QGRFULQRO0HWDE ((
Salter, D.N., A. Montgomery, A. Hudson, D.B. Quelch and R.J. Elliott. 1990. Lysine requirements and whole-body
SURWHLQWXUQRYHULQJURZLQJSLJV%U-1XWU

204 Energy and protein metabolism and nutrition in sustainable animal production
0HWDDQDO\WLFDOVWXG\RQWKHSHUIRUPDQFHDQGXWLOL]DWLRQHI¿FLHQF\RI
different methionine sources by pigs
A. Remus1, L. Hauschild1, I. Andretta2, M. Kipper2, C.R. Lehnen2 and R. Isola1
1Universidade Estadual Paulista, UNESP, Jaboticabal, São Paulo, Brazil; [email protected]
2Universidade Federal de Santa Maria, UFSM, Santa Maria, Rio Grande do Sul, Brazil

Introduction
Methionine (Met) is the second limiting amino acid in swine diets. The utilization of a synthetic
amino acid for supplying this is a consolidated practice. However, the ideal level of this amino acid
to optimize various criteria is still very controversial. Part of the discrepancy between studies may
EHOLQNHGWRLQWHUH[SHULPHQWDOYDULDELOLW\LQÀXHQFLQJWKHUHVXOWV7KLVPHWDDQDO\VLVLVSUHVHQWHGDV
a viable alternative by providing techniques that allow control of the variability, uncovering results
that would not be noticeable in a smaller population. This study focused on the animal response to
ingestion of different levels of methionine.

Material and methods

7KHGDWDEDVHXVHGIRUWKHPHWDDQDO\VLVFRQVLVWHGRIDUWLFOHVSXEOLVKHGLQMRXUQDOVIURPWR
 PRGH 7KLVVWXG\FRQWHPSODWHGSLJVZLWK QXUVHU\SKDVHRIVWXGLHV WR
NJRIERG\ZHLJKW JURZLQJ¿QLVKLQJSKDVHRIVWXGLHV 7KHPHWDEROLFZHLJKWUDWLRZDV
NJ PRGHNJ 7KHIROORZLQJFULWHULDZHUHXVHGIRUVHOHFWLRQRIWKHDUWLFOHV  GLIIHUHQWOHYHOV
of methionine in the diets; (2) all other amino acids (AA) were set at 100% of their ideal level; (3)
presentation within the article of the nutritional composition of diets. Four treatments were considered,
with methionine ratios of: 0.22% basal diet (mode: 0.23%), 0.20% of L-methionine (mode: 0.19%),
0.32% of DL-methionine (mode: 0.30%) and 0.38% (mode: 0.28%) of DL-methionine hydroxy
analogue (MHA). The mean composition of diets was: 2,410 kcal of metabolizable energy (mode:
3,152) and 18.57% crude protein. The composition of total AA was: 1.23% of lysine, 0.23% of
WU\SWRSKDQRIWKUHRQLQHRIPHWKLRQLQHSOXVF\VWHLQHDQGRIF\VWHLQHRI
calcium and 0.79% of phosphorus. A coding category was established for the methionine source. In
addition to this coding category, three other moderating encoding categories were used: (1) general
FRGL¿FDWLRQ  LQWHUFRGL¿FDWLRQ  LQWUDFRGL¿FDWLRQ7KHFULWHULDXWLOL]HGIRUWKHGH¿QLWLRQRI
dependent and independent variables followed the proposals described in the literature (Lovatto et
al., 2007; Sauvant et al., 2008). Step 1, the data were analyzed graphically. With this analysis it was
SRVVLEOHWRJHQHUDWHWKHFRUUHODWLRQK\SRWKHVLVIRUWKHGH¿QLWLRQRIWKHVWDWLVWLFDOPRGHO6WHSWKH
correlation analysis was performed. The statistical model used was covariate adjustment, and all
YDULDEOHVZHUHDGMXVWHGIRUPHWDEROLFERG\ZHLJKW UDLVHGWRWKHSRZHU 7KHDGMXVWPHQWZDV
performed to make possible analyzation and to compare pigs with different physiological states (i.e.
level of maturity). Step 3, the variance-covariance analysis was carried out using the ‘general linear
model’ procedure. The Stundent-Newman-Keuls multi comparison test using Statistica software was
used for comparisons of means.

Results and discussion

7KHHI¿FLHQF\RIGLIIHUHQWVRXUFHVRI0HWZDVGHWHUPLQHGE\UHJUHVVLRQRIWKHDYHUDJHGDLO\JDLQ
$'* DVDIXQFWLRQRIPHWKLRQLQHLQWDNH7KHEHVWHI¿FLHQF\ZDVREVHUYHGLQSLJVIHGGLHWV
supplemented with L-methionine (L-met). These pigs had a gain of 1.44 g/g methionine intake
adjusted for BW (L-met = 0.1324 + 1.445×X, R2 = 90.1%). The worst result was observed
for piglets supplemented with MHA (MHA= 0.2814 + 0.7184×X, R2= 58.2%). The results on
performance and plasmatic urea are presented in Table 1. The methionine intake adjusted for BW
was 33% (P<0.001) higher when pigs received DL-methionine diets (DL-met), in contrast with basal

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 205
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_65, © Wageningen Academic Publishers 2013
Table 1. Performance of pigs receiving different methionine sources adjusted for metabolic body
ZHLJKW UDLVHGWRWKHSRZHU 

Basal L-met DL-met HMA P-value

ADG, kg 0.595a 0.785b c 0.554a <0.001

0.03* 0.04 0.01 0.02
ADFI, kg 1.027a 1.033a 1.119a 0.781b <0.001
0.09 0.13 0.04 
G:F, kg a a 0.701a 0.847b <0.001
0.03  0.02 0.03
Plasma urea N, mg/dl a 7.82b 5.49c 12.55a <0.001
1.59 3.44 0.75 1.89
Methionine intake, g/d 0.398a 0.391a 0.588b 0.392a <0.001
0.04 0.08 0.02 0.04

6XSHUVFULSWV61.PXOWLFRPSDULVRQWHVWDWFRQ¿GHQFHLQWHUYDOV 6WDQGDUGGHYLDWLRQ

diet (BD) (P<0.001). The average daily feed intake (ADFI) was 24% higher (P<0.001) for pigs
fed diets supplemented with L-met or DL-met when compared to groups fed with basal or MHA
GLHWV7KHIHHGHI¿FLHQF\UDWLRZDVEHWWHU P<0.001) when the pigs were fed diets supplied
with L-met compared with MHA. The ADG of piglets supplemented with L-met was 29% higher
(P<0.001) in contrast with MHA and 12% higher in contrast with DL-met. On the regression of
$'*ZLWKPHWKLRQLQHLQWDNHWKHEHVWHI¿FLHQF\ZDVREVHUYHGIRUSLJVIHGGLHWVVXSSOHPHQWHGZLWK
L-methionine (Y = 0.1324 + 1.445×X, R2=0.90).

For the nitrogen (N) balance there was no observed difference (P>0.05) among treatments. Plasmatic
1ZDVDQH[FHSWLRQWRWKLVDWOHVVLQSLJVVXSSOHPHQWHGZLWK'/PHWDQGOHVVLQSLJV
supplemented with L-met in contrast with pigs receiving MHA. Pigs supplemented with MHA had
plasmatic concentration of N similar to pigs fed with basal diet.

Conclusion
L-methionine and DL-methionine improved the performance of pigs in contrast with pigs supplemented
with DL-methionine hydroxy analogue. It is possible that both sources of methionine can be equal
options for supplementation in pigs diets.

References
/RYDWWR3$/HKQHQ&5$QGUHWWD,&DUYDOKR$'DQG+DXVFKLOG/0HWDDQiOLVHHPSHVTXLVDVFLHQWt¿FDV
HQIRTXHHPPHWRGRORJLDV5HYLVWD%UDVLOHLUDGH=RRWHFQLDYS
Sauvant, D., Schmidely, P., Daudin, J.J., and St-Pierre, N.R., 2008, Meta-analyses of experimental data in animal
nutrition: animal, v. 2, p. 1203-1214.

206 Energy and protein metabolism and nutrition in sustainable animal production
Estimating digestible threonine requirements for growing pigs by meta-
analysis
R. Isola, L. Hauschild, M.C. Thomaz, N.K. Sakomura and A. Remus
Universidade Estadual Paulista, UNESP, Jaboticabal, São Paulo, Brazil; [email protected]

Introduction
7KUHRQLQHLVWKHVHFRQGRUWKLUGOLPLWLQJDPLQRDFLGLQSLJGLHWVDQGFDQEHFRPHWKH¿UVWZKHQ
supplemented with synthetic lysine. The utilization of a synthetic amino acid for supplying this is
a consolidated practice. However, the ideal level of this amino acid to optimize various criteria is
still very controversial. Part of the discrepancy between studies may be linked to inter-experimental
YDULDELOLW\LQÀXHQFLQJWKHUHVXOWV7KLVPHWDDQDO\VLVLVSUHVHQWHGDVDYLDEOHDOWHUQDWLYHE\SURYLGLQJ
techniques that allow control of the variability, uncovering results that would not be noticeable in
a smaller population. This study focused on the animal response to ingestion of different levels of
threonine.

Material and methods

7KHGDWDEDVHXVHGIRUWKHPHWDDQDO\VLVFRQVLVWHGRIDUWLFOHVSXEOLVKHGLQMRXUQDOVVLQFH
to 2007. This study contemplated 1,187 pigs in the weight range of 5 kg (nursery phase) to 124
NJ ¿QLVKLQJSKDVH 7RVHOHFWWKHDUWLFOHVZHXVHGWKHIROORZLQJFULWHULDWKHDGGLWLRQRIGLIIHUHQW
levels of dietary digestible threonine (SDThreo) with all other amino acids set at 100% of its ideal
OHYHO7KHDYHUDJHFRPSRVLWLRQRIWKHGLHWVZDVNFDORIGLJHVWLEOHHQHUJ\ '( DQG
crude protein, and the total composition of the amino acids lysine 1.04%, 0.21% tryptophan, 0.35%
PHWKLRQLQHWKUHRQLQHDQGPHWKLRQLQHF\VWHLQH

)RUWKHGH¿QLWLRQRIGHSHQGHQWDQGLQGHSHQGHQWYDULDEOHVWKHFULWHULDGHVFULEHGLQWKHOLWHUDWXUHZDV
used (Lovatto et al., 2007; Sauvant et al., 2008). Analysis of the database followed three steps. Step
1, the graphic analysis. With this analysis it was possible to generate the correlation hypothesis for
WKHGH¿QLWLRQRIWKHVWDWLVWLFDOPRGHO6WHSWKHFRUUHODWLRQDQDO\VLVZDVSHUIRUPHG)LQDOO\LQVWHS
3, analysis of variance-covariance procedure by the ‘general linear model’ was performed, from
which the prediction equations were generated. The variables measured by the intake SDThreo were
feed intake, weight gain, feed conversion and plasma urea. The statistical model used for covariate
adjustment, with the result for plasma urea were adjusted for metabolic body weight (raised to 0.75
power) to enable the analysis and comparison of pigs with different physiological status (at different
stages, ages). The data were analyzed by regression analysis and considered only levels below the
maximum response for weight gain (BWG). A factorial equation was determined to estimate the
demand for SDThreo (SDThreo = 0.049×BW0.75%:* IRUGLIIHUHQWERG\ZHLJKWV7KH¿UVW
component (0.049×BW0.75) is the maintenance and was extracted from the literature. The second is
WKHUHTXLUHPHQW6'7KUHRIRUJURZWK7KHFRHI¿FLHQWZDVHVWLPDWHGE\WKHUHJUHVVLRQRI6'7KUHR
ingested destined for growth (SDThreoG = SDThreo ingested total – SDThreo maintenance) due
WRWKH%:*,QUHJUHVVLRQDQDO\VLVZDVXVHG0LQLWDE

Results and discussion

,QWKHUHJUHVVLRQRIZHLJKWJDLQ %:* 7KU±7KU 52=91.3%),
GDLO\IHHGLQWDNH ), 7KU±7KU 52=91.5%), feed conversion (FC =
±7KU7KU 52  DQGSODVPDXUHDQLWURJHQ 1 
Thr Thr ** 2 + 0.1398, R2=78.7%) versus SDThreo consumption increased linearly (P<0.05) and
quadraticly (P<0.05) for these variables. The regression of SDThreoG according to BWG showed
DJRRG¿W 6'7KUHR* î%:*52=0.95). Each gram of weight gain required an intake

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 207
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_66, © Wageningen Academic Publishers 2013
 0 1 2 3 4
A. Feed Intake, kg C. Body weight gain, kg
6.0 FI= - 0.6099 + 2.885 Thr- 0.3569 Thr**2 2.5 BWG = - 0.01287 + 0.9020 Thr - 0.09671 Thr**2
R2 = 91.5% R2 = 91.3%
2.0
4.5
1.5
3.0
1.0
1.5
0.5
0.0
B. Feed Conversion D. Plasma urea N, mg/100ml/BWM
FCR= 0.6908 - 0.2628 Thr + 0.04996 Thr**2 Plasma urea N= 4.583 - 1.463 Thr + 0.1398Thr**2
0.7 R2 = 60.4% 5 R2 = 78.7%

0.6 4
0.5
3
0.4
2
0.3
1
0 1 2 3 4
Thr intake, g
)LJXUH5HJUHVVLRQRIIHHGLQWDNH $ IHHGFRQYHUVLRQ % ZHLJKWJDLQ & DQGSODVPDXUHD ' 
due to the intake of threonine.

of 0.0185 g SDThreo to meet the potential growth of the animals. This parameter represents an
HI¿FLHQF\RIXWLOL]DWLRQRIRI6'7KUHRIRUZHLJKWJDLQ%DVHGRQWKLVHTXDWLRQDQGWKHGDWDIRU
consumption and weight gain for a high genetic potential animal was used in the Brazilian Tables
of recommended requirements for swine it was estimated requirements of SDThreo for pigs with
ZHLJKWVRIDQGNJ)RUWKHVHZHLJKWVWKHHVWLPDWHGYDOXHVZHUH
0.552 and 0.453 SDThreo% of the diet.

Conclusion
The factorial equation structured based on data from the meta-analysis study allowed us to determine
the response of pigs at different weights to threonine intake and determine the optimal level of
threonine in the diet based on variables available on farm.

References
/RYDWWR3$/HKQHQ&5$QGUHWWD,&DUYDOKR$'DQG+DXVFKLOG/0HWDDQiOLVHHPSHVTXLVDVFLHQWt¿FDV
HQIRTXHHPPHWRGRORJLDV5HYLVWD%UDVLOHLUDGH=RRWHFQLDYS
Sauvant, D., Schmidely, P., Daudin, J.J., and St-Pierre, N.R., 2008, Meta-analyses of experimental data in animal
nutrition: animal, v. 2, p. 1203-1214.

208 Energy and protein metabolism and nutrition in sustainable animal production
/\VLQHVXSSO\LQ¿QLVKLQJEURLOHUVHIIHFWRQSHUIRUPDQFHVDQGPHDWTXDOLW\
M. Lessire1, Y. Primot2, E. Corrent2, Fraysse Pauline2, S. Tesseraud1 and C. Berri1
1,15$855HFKHUFKHV$YLFROHV1RX]LOO\)UDQFH[email protected]
2$MLQRPRWR(XURO\VLQHVDVUXHGH&RXUFHOOHV3DULV&HGH[)UDQFH

Introduction
In poultry production, there is a constant need for updating birds’ requirements especially protein and
amino acids, because of economical purposes, genetic improvements and also because parameters of
interest are changing from the classical ones i.e. body weight, feed conversion ratio, to more recent
ones, i.e. meat yield and processing quality (Berri et al., 2008; Dozier et al., 2010). In chickens,
WKHVHODVWFKDUDFWHULVWLFVDUHVWURQJO\LQÀXHQFHGE\WKHXOWLPDWHS+RIWKHPHDWZKLFKGHSHQGVRQ
muscle glycogen content (Le Bihan-Duval et al., 2008). Recent observations suggest the possibility of
modulating these characteristics by varying the lysine intake of broilers (Jlali et al., 2012). Our objective
ZDVWRGHWHUPLQHWKHGLJHVWLEOHO\VLQHUHTXLUHPHQWVRIPDOH5RVVEURLOHUVGXULQJWKH¿QLVKLQJSHULRG
taking into account birds growth, feed conversion, edible parts yields, adiposity and meat quality.

Material and methods

Twelve experimental diets were computed using the same raw materials, from the same batches,
which were analyzed previously. They presented 4 digestible lysine levels: 7.0, 8.5, 10.0 and 11.5
JNJDQGDPLQRDFLGVSUR¿OHVDQGRIWKHRSWLPXPDPLQRDFLGVSUR¿OHPHQWLRQHG
by Mack et al. (1999). Dietary protein content followed lysine content. On the opposite ME value
and other nutrients contents were independent of lysine content. As a consequence, the ratio ME/
protein decreased with the increase of lysine.

ELUGVZHUHUDLVHGZLWKWKHVDPHGLHWVWLOOWKH\ZHUHGD\VROGWKHQZLWKVLPLODUZHLJKW
ZHUHUHWDLQHG7KHîGLHWDU\WUHDWPHQWVZHUHJLYHQWRWKHVHELUGVGLYLGHGLQÀRRUSHQV P2),
ZLWKSHQV ELUGVSHQ IRUHDFKWUHDWPHQW

$WGD\VRIDJHELUGVZHUHLQGLYLGXDOO\ZHLJKWHGDQGIHHGFRQVXPSWLRQZDVFDOFXODWHGSHUSHQ
In each pen 4 birds were chosen around the mean of the pen and slaughtered. Weight of abdominal
fat, breast meat, and tight and drumsticks were measured. Breast meat quality was estimated by
measuring pH, drip loss, and color. Lysine requirements were calculated using quadratic broken
line models (Pesti et al., 2009).

Results and discussion

Feed consumption decreased with the increase of dietary lysine content. On the opposite, weight gain
increased according to a quadratic equation. Without other limiting amino acids digestible lysine
requirements can be estimated as 10.5 and 11.3 g/kg for levels 100 and 110% respectively. The
corresponding optimum weight gains were 1,580 and 1,592 g (Figure 1). Feed conversion ratio was
improved when lysine content increased. The optimum value was estimated at 12.7 g/kg for levels 100
DQGZLWKDQRSWLPXPHTXDOWR:KHQWKHRWKHUDPLQRDFLGVDUHOLPLWLQJ  UHVXOWV
IROORZHGWKHVDPHWUHQGVEXWWKHHI¿FDF\RIO\VLQHZDVUHGXFHG'LJHVWLEOH/\VLQHUHTXLUHPHQWLV
globally lower for all the performances criteria than for quality criteria, as already observed by Leclercq
(1998). Breast meat yield increased (from 19.3 to 20.2%) and abdominal fat percentage decreased (from
3.2 to 2.2%) with increasing levels of lysine and thus total protein content of feed. Strong effects of
DPLQRDFLGVSUR¿OHDQGO\VLQHFRQWHQWZHUHREVHUYHGRQWKHXOWLPDWHS+7KHORZHVWYDOXHVRIXOWLPDWH
pH (5.8 on average) were obtained in the case of a high intake of amino acids relative to lysine (110%).
7KHKLJKHVWYDOXHV RQDYHUDJH FRUUHVSRQGHGWRWKHORZHVWDPLQRDFLGWRO\VLQHUDWLR  7KHVH

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 209
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_67, © Wageningen Academic Publishers 2013
 Weight gain (g)
Profile Requirement Optimum

FCR
Profile Requirement Optimum

Digestible Lysine (g/kg) Digestible Lysine (g/kg)

Profile Requirement Optimum

% Breast meat/BWG
% Abdominal

Profile Requirement Optimum

Digestible Lysine (g/kg) Digestible Lysine (g/kg)

Figure 1. Evolution of performances and yields according to the digestible lysine and other amino
acids concentrations.

S+YDULDWLRQVKDGDVLJQL¿FDQWLPSDFWRQWKHFRORUDQGZDWHUUHWHQWLRQRIPHDWZLWKWKHPRVWDFLG
PHDWVKDYLQJWKHKLJKHVWOXPLQDQFH/ DQGGULSORVVGXULQJVWRUDJH / DQGGULSORVV LQ
average). These results highlight the importance of integrating meat quality criteria in addition to the
VWDQGDUGSURGXFWLRQFULWHULDZKHQGH¿QLQJGLHWDU\DPLQRDFLGVUHTXLUHPHQWVRIEURLOHUV

Conclusion
Optimizing the lysine content of broiler diets, with an optimum content of the other amino acids
DSSHDUVWREHHVVHQWLDOLQRUGHUWRLPSURYHHI¿FLHQF\DQGTXDOLW\RIWKHSURGXFWLRQ7RWKLVPRGHOLQJ
performances in relation with nutrient content seems to be a solution. In this study, digestible lysine
requirements, with a same mathematical model, varied from 10.4 to 13.7 g/kg for broilers according to
WKHHVVHQWLDODPLQRDFLGVSUR¿OHVXVHGDQGWKHFULWHULDRIUHVSRQVH%XWWKLVWULDOVKRZVWKDWSHUIRUPDQFHV
could be improved (body weight) using amino acids concentrations higher than classical ones, thus
LQGLFDWLQJDODFNRINQRZOHGJHRQ¿QLVKLQJEURLOHUVUHTXLUHPHQWRULQUDZPDWHULDOVGLJHVWLELOLW\XVHG

References
Berri, C., J. Besnard, and C. Relandeau, 2008. Increasing dietary Lysine Increases Final pH and Decreases Drip Loss
of Broiler Breast Meat. Poult. Sci. 87, 480-484.
Dozier, W.A., A. Corzo, M.T. Kidd, P.B. Tillman, J.P. McMurtry, and S.L. Brandon, 2010. Digestible lysine requirements
of male broilers from 28 to 42 days of age. Poult. Sci. 89, 2173-2182.
Jlali, M., V. Gigaud, S. Métayer-Coustard et al. 2012. Modulation of glycogen and breast meat processing ability by
nutrition in chickens: Effect of crude protein level in 2 chicken genotypes. J. Anim. Sci. 90, 447-455.
Le Bihan-Duval, E., M. Debut, C.M. Berri, et al. 2008. Chicken meat quality: genetic variability and relationship with
JURZWKDQGPXVFOHFKDUDFWHULVWLFV%0&*HQHWLFVS
/HFOHUFT%6SHFL¿FHIIHFWVRIO\VLQHRQEURLOHUSURGXFWLRQFRPSDULVRQZLWKWKUHRQLQHDQGYDOLQH3RXOW6FL
77 : 118-123.
Mack, S., D. Bercovici, G. De Groote et al.,GHDODPLQRDFLGSUR¿OHDQGGLHWDU\O\VLQHVSHFL¿FDWLRQIRUEURLOHU
FKLFNHQVRIWRGD\VRIDJH6%U3RXOW6FL
Pesti, G.M., D. Vedenov, J. A. Cason, and L. Billard, 2009. A comparison of methods to estimate nutritional requirements
IURPH[SHULPHQWDOGDWD%U3RXOW6FL

210 Energy and protein metabolism and nutrition in sustainable animal production
Part 3. Tools and techniques
Proteomic tools help understanding the metabolic adaptation to negative
energy balance in dairy cows
B. Kuhla and C.C. Metges
,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 
'XPPHUVWRUI*HUPDQ\[email protected]

Abstract
High-yielding dairy cows have enormous energy and nutrient requirements for milk production
ZKLFKDUHJHQHUDOO\QRWPHWE\DVXI¿FLHQWIHHGLQWDNHUHVXOWLQJLQDQHJDWLYHHQHUJ\EDODQFH
(NEB) characterized by mobilisation of body reserves. It is still controversial whether during early
ODFWDWLRQWKHPRELOL]DWLRQRIERG\UHVHUYHVFDXVHVLQVXI¿FLHQWIHHGLQWDNHRULQVXI¿FLHQWIHHGLQWDNH
causes mobilization of body reserves. In order to distinguish between cause and effect, we designed
feed-restriction studies modelling NEB as well as follow-up studies on periparturient dairy cows
and examined metabolic adaptation processes during NEB. To this end, 2D-gel based proteomic
approaches coupled with MALDI-TOF-MS and MALDI-TOF-TOF analyses are often used for the
LQYHVWLJDWLRQRIFKDQJHVLQSURWHLQH[SUHVVLRQSRVWWUDQVODWLRQDOPRGL¿FDWLRQV 370V DQGSURWHLQ
LGHQWL¿FDWLRQZKLOHVXEVHTXHQW:HVWHUQ%ORWVDUHDSSOLHGWRFRQ¿UPWKHH[LVWHQFHDQGH[SUHVVLRQRI
LQGLYLGXDOSURWHLQV3URWHRPLFSUR¿OLQJLQWLVVXHVREWDLQHGIURPIUHTXHQWOLYHUDQGPXVFOHELRSVLHVRU
from slaughter provides insight into regulatory mechanisms at the translational and posttranslational
level. We were able to demonstrate that phosphorylation of the adenosine monophosphate-activated
protein kinase (AMPK), a cellular energy key sensor, is increased in hypothalamus and liver but not in
skeletal muscle during NEB. Muscle tissue in early lactation showed reduced abundance of muscular
cytoskeletal proteins and enzymes involved in glycogen synthesis, fatty acid degradation, and TCA
cycling, while the expression of enzymes involved in glycolysis, lactate and ATP production was
increased. The functional characterisation of the hepatic oxidative metabolism is of particular interest
because of its role to provide substrates for the mammary gland and its involvement in the control
of feed intake. While feed restriction down-regulated hepatic proteins associated with fatty acid
oxidation, early lactation expression of enzymes participating in fatty and amino acid degradation,
TCA cycling, ATP production, and oxidative stress defence was increased. The integration of
proteome data with corresponding plasma metabolite and hormone concentrations allowed us to
propose an inter-organ crosstalk model in which hepatic and skeletal muscle metabolism in early
lactating cows supports gluconeogenesis for milk production while hepatic oxidation of fatty acids
interferes with the control of feed intake in the brain.

Introduction
In high-producing dairy cows the transition from late pregnancy to early lactation is characterized
by an enormous increase in nutrient and energy demands of the mammary gland in order to express
its genetic potential of milk synthesis. These requirements are generally met by an increase of feed
intake, mobilization of body fat, protein and glycogen reserves, and the saving of fuels in peripheral
organs of lesser priority such as in muscle and liver. The concerted control of body tissue metabolism
thus supports the partitioning of macronutrients towards the mammary gland but in parallel also poses
a challenge to the metabolism of peripheral organs. Although feed intake normally increases from
late pregnancy to early lactation the magnitude of this increase does not match the increase of energy
VHFUHWLRQE\PLON7KHJDSEHWZHHQLQVXI¿FLHQWHQHUJ\LQWDNHDQGLQFUHDVHGHQHUJ\UHTXLUHPHQWVLV
accompanied by the mobilisation of body reserves and results in a transient NEB in early lactation.

:KHWKHULQVXI¿FLHQWHQHUJ\LQWDNHGXULQJHDUO\ODFWDWLRQFDXVHVWKHPRELOL]DWLRQRIERG\UHVHUYHV
RUWKHPRELOL]DWLRQRIERG\UHVHUYHVXQGHUOLHVWKHLQVXI¿FLHQWLQFUHDVHRIIHHGLQWDNHUHPDLQVD
controversial issue (Ingvartsen and Andersen, 2000; Hammon et al., 2009; Weber et al., 2013). Feed

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 213
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_68, © Wageningen Academic Publishers 2013
withdrawal, feed restriction or feeding an energy-diluted ration to mid or late lactating dairy cows
(Gross et al., 2011; Loor et al., 2007) are frequently used models to study metabolic adaptations to
1(%7KHVHH[SHULPHQWDOPRGHOVDOORZWRGLIIHUHQWLDWHEHWZHHQFDXVH LQVXI¿FLHQWHQHUJ\LQWDNH 
and effect (metabolic adaptation), which is generally not possible from association studies by simply
monitoring cows during the transit period. A major difference between transiently occurring NEB
in early lactation and nutrition-induced NEB is, however, that during early lactation dairy cows are
fed ad libitum and thus obviously experience satiety which is not the case when feed is withdrawn.
Hence, comparative studies involving feed/energy restriction/withdrawal and ad libitum feeding
of early lactating cows enable the differentiation between metabolic adaptations to NEB during
hunger or satiety, respectively. Only recently, Allen et al. (2009) summarized experimental evidences
suggesting that in early lactation the delay in reaching a proportionately adequate feed intake is due
to increased vagus nerve activity driven by an activated hepatic oxidative metabolism.

Even during periods of NEB, the mammary gland is highly supplied with macronutrients, primarily
glucose, amino acids and fatty acids serving as important precursors maintaining milk production.
While glucose originates predominantly from gluconeogenesis and glycogenolysis of the liver,
VNHOHWDOPXVFOHSURWHLQLVEURNHQGRZQWR\LHOGDPLQRDFLGVDQGVPDOOHUSHSWLGHVDQGQRQHVWHUL¿HG
fatty acids (NEFA) are mobilized from adipose tissue depots and enter the blood stream. Therefore,
changed plasma metabolite concentrations together with an altered intermediary metabolism in liver,
skeletal muscle, adipose tissue and mammary gland contribute to a major extent to the metabolic
DGDSWDWLRQWR1(%7KHLQWHUPHGLDU\PHWDEROLVPRISHULSKHUDORUJDQVLVQRWRQO\LQÀXHQFHGE\
substrate availability and product concentration but is also regulated by the endocrine and autonomous
nervous system in which the hypothalamus-pituitary axis plays a pivotal role controlling energy
balance.

$SSOLFDWLRQVRIWUDQVFULSWRPLFVDQGWKHPHDVXUHPHQWVRIP51$DEXQGDQFHLGHQWL¿HGGLIIHUHQWLDOO\
expressed key enzymes, nuclear and transcription factors in the mammary gland (Bionaz et al.,
2012), adipose tissue (Sumner-Thomson et al., 2011), and liver (Loor et al., 2005, 2007) decoding
metabolic pathways activated or deactivated in periparturient dairy cows. Although a number of these
WUDQVFULSWLRQDOFKDQJHVGRREYLRXVO\UHÀHFWWKHFRQFHQWUDWLRQRIWKHUHVSHFWLYHSURWHLQRUHYHQLWV
activity (Li et al., 2012a), signal transduction and distinct metabolic pathways such as adipose tissue
lipolysis (Locher et al. DUHH[FOXVLYHO\WULJJHUHGWKURXJKSRVWWUDQVODWLRQDOPRGL¿FDWLRQV

3URWHRPLFSUR¿OLQJLVDWRROWKDWRIIHUVWKHRSSRUWXQLW\WRLGHQWLI\FKDQJHVLQSURWHLQH[SUHVVLRQDW
translational and posttranslational (e.g. phosphorylation, glycosylation, etc.) level and thus enables
us to gain further insight into regulatory mechanisms. Proteomic approaches require a combination
of stringent separation technologies (2-dimensional gel electrophoresis (2D-GE) or high-performance
liquid chromatography (HPLC)), high-resolution mass spectrometry (e.g. MALDI-TOF-MS) and
SRZHUIXOELRLQIRUPDWLFWRROV8OWLPDWHO\WKHTXDQWLW\DQGTXDOLW\RIWKHSURWHLQLGHQWL¿FDWLRQGHSHQGV
on the quality of the protein databases of farm animals which is consistently improving (Lippolis
and Reinhardt, 2008). The proteomic technology allows analysing >1000 different proteins in tissues
RUERG\ÀXLGVLQRQHVLQJOHH[SHULPHQW7KLVSURYLGHVWKHUHVHDUFKHUZLWKLQIRUPDWLRQRQFHOOXODU
processes in response to a certain challenge and allows to identify previously unknown interactions
between different proteins (Lippolis and Reinhardt, 2008), First steps in uncovering the proteome of
bovine tissues, plasma and red blood cells were made a decade ago (Talamo et al., 2003) in which a
IHZPRVWKLJKO\DEXQGDQWSURWHLQVZHUHVHSDUDWHGDQGLGHQWL¿HG+RZHYHU:HVWHUQEORWVRURWKHU
LPPXQHEDVHGDVVD\VVXFKDV5,$RU(/,6$DUHVWLOORIWHQXVHGWRFRQ¿UPUHODWLYHH[SUHVVLRQ
GLIIHUHQFHVRIVHOHFWHGSURWHLQVLQFOXGLQJWKHLUSRVWWUDQVODWLRQDOPRGL¿FDWLRQVSURYLGHGWKDWVSHFL¿F
antibodies are available. Thus, the rapidly developing proteome methodology based on progressed
mass spectrometry techniques together with established immunoassays and plasma metabolite
SUR¿OLQJKDYHFRQWULEXWHGDORWWRWKHXQGHUVWDQGLQJRIPHWDEROLFDGDSWDWLRQWR1(%LQGDLU\FRZV

214 Energy and protein metabolism and nutrition in sustainable animal production
The aim of this paper is to give an overview on the current state of knowledge in regard to the
metabolism of the periparturient dairy cow based on new information gathered by our group and
others using proteomic tools.

Hypothalamus and pituitary

The hypothalamus-pituitary axis is the most important control and regulatory system involved in
maintaining energy homeostasis or homeorhesis. The hypothalamus receives an array of extracellular
humoral and neural information, integrates these input signals to secrete hypothalamic-releasing
hormones, which in turn stimulate or inhibit the secretion of pituitary hormones. In turn released
pituitary hormones may affect metabolic processes in peripheral tissues, thus forming a feedback
control loop.

,QD¿UVWDWWHPSWWRGLVVHFWPHFKDQLVPSRWHQWLDOO\LQYROYHGLQWKHFRQWURORIHQHUJ\KRPHRVWDVLVRI
dairy cows, we investigated how energy restriction caused by feeding a diet with reduced energy
density as compared to ad libitum feeding alters the hypothalamic proteome in early-lactating
cows (Kuhla et al., 2007). By applying a 2D-GE-based proteomic approach, we found that the
glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the TCA-cycle
enzyme aconitase-2 were down-regulated in the restricted group, suggesting an adaptation of brain
metabolism in response to diminished substrate availability. On the other hand, ubiquitin carboxy-
terminal hydrolase-L1, and heat shock protein 70 kDa-protein-5 were found to be up-regulated
SRLQWLQJWRJUHDWHUSURWHRO\VLVLQWKHK\SRWKDODPXVGXULQJWLPHVRIHQHUJ\GH¿FLHQF\ .XKODet al.,
2007). In feed restricted dairy cows, abundance of [Cu-Zn]-superoxide dismutase was also greater
than in ad libitum fed counterparts (Kuhla et al., 2007), indicating increased production of reactive
oxygen species (ROS), which in turn are increasingly considered as a mitochondrial energy sensor in
the brain (Horvath et al., 2009). A cytosolic energy sensor is the adenosine monophosphate-activated
protein kinase (AMPK). This intracellular ‘fuel gauge’ becomes phosphorylated (activated) by
$03ZKHQWKH$73OHYHOLVORZ,QRXUSURWHRPLFDSSURDFKZHIXUWKHULGHQWL¿HGXSUHJXODWLRQRI
5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (Kuhla et
al., 2007), an enzyme directly involved in AMP production, and Western blot analysis revealed that
the ratio of phosphorylated AMPK and total AMPK was elevated in energy restricted dairy cows
(Kuhla et al., 2011a). To conclude, we found that the brain adapts to energy restriction not only by
adapting its intermediary metabolism but also by activating cell signalling pathways triggering for
the maintenance of energy balance. However, we were not able to detect alterations in the expression
of hypothalamic-releasing hormones that would suggest an altered secretion of pituitary hormones.

Nonetheless, the pituitary gland accumulates and secretes nine pituitary hormones [e.g growth
hormone (GH), prolactin (PRL), luteinizing hormone (LH), thyrotropin (TH), Pro-opiomelanocortin
(POMC], which all may carry one or more PTMs or which occur with different peptide lengths.
Different PTMs or peptide lengths may exert opposing physiological functions, for example, dimeric
but not monomeric growth hormone (GH) and prolactin (PRL) activate various cellular pathways
(Langenheim et al. IXOOOHQJWK*+DQG35/SURPRWHDQJLRJHQHVLVEXWWKHLUSURWHRO\WLF
fragments acquire anti-angiogenic properties (Corbacho et al., 2002), or GH peptides with different
lengths possess either insulin-potentiating or anti-insulin properties (Lewis et al., 2000). We asked
the question if the expression of the different pituitary hormone variants differ in response to feed
restriction. Since pituitary hormone antibodies established for RIAs or ELISAs are generally not
suitable to distinguish between different hormone forms, the only method by which this question
can be answered is proteomics. In extracts of bovine pituitary gland, the abundance of all pituitary
KRUPRQHYDULDQWVLGHQWL¿HG *+ 35/ /+ 7+ 320&  GLGKRZHYHUQRWGLIIHULQ
response to feed restriction despite total plasma PRL but not total plasma LH or GH concentrations
ZHUHVLJQL¿FDQWO\UHGXFHGLQWKHIHHGUHVWULFWHGJURXS .XKODet al. 2010). Also pituitary total LH
and total PRL determined by RIA analysis did not differ between ad libitum and feed restricted

Energy and protein metabolism and nutrition in sustainable animal production 215
GDLU\FRZVVXJJHVWLQJWKDWWKHKRUPRQHSRROVL]HLQWKHSLWXLWDU\ZDVVXVWDLQHGDIWHUKIHHG
restriction. It rather seemed that the hormone secretion machinery participated in the adaptation to
feed restriction because expression of annexin A5 and secretogranin V (neuroendocrine polypeptide
7B2), both stimulating the release of hormones into the blood stream, were increasingly expressed
after feed restriction.

Serum, plasma and blood

The sensitivity of 2D-gel-based proteomic approach is generally too low for the detection of proteinous
or peptide hormones in plasma or serum. However, by using a proteomic approach based on agarose
gel electrophoresis, Piccione et al.  LGHQWL¿HGVHYHUDOVHUXPPDUNHUVWKDWZHUHDOWHUHGGXULQJ
WKHSHULSDUWXULHQWSHULRGRIGDLU\FRZV)RUH[DPSOHDEXQGDQFHRIĮJOREXOLQVLQFOXGLQJDFXWHSKDVH
SURWHLQVZHUHLQFUHDVHGLQHDUO\ODFWDWLRQZKLOHȕDQGȖJOREXOLQVZHUHORZHVWDEXQGDQWDURXQG
parturition, indicating increased metabolic stress and a challenged immune system during this time
(Piccione et al., 2011). In regard to the latter, Lippolis et al.  LGHQWL¿HGPRUHWKDQSURWHLQVLQ
neutrophils that were differentially expressed between day 28 before and 2-3 days after parturition by
labelling proteins with isobaric tags for relative and absolute quantitation (iTRAQ). Altered expressions
of High Mobility Group Box proteins, histones, myeloperoxidase, proteins involved in arachidonic
acid metabolism or with antimicrobial activity were indicative of immunosuppression and reduced
neutrophil functionality in effectively combating bacteria in early lactation.

Further efforts have been made in identifying cows affected by metabolic diseases typically occurring
during NEB. Subjecting plasma samples from cows with or without milk fever to 2D differential
in-gel electrophoresis (2D-DIGE), Xia et al., (2012) recently described upregulation of serpin
peptidase inhibitor (angiotensin) and endopin 2B (involved in neural regulation) and downregulation
RI¿EULQRJHQDOEXPLQDQG,J*VLQFRZVDIIHFWHGE\PLONIHYHU+RZHYHUDSURWHLQRXVVHUXP
ELRPDUNHUVSHFL¿FDOO\SUHGLFWLQJWKHGHJUHHRI1(%KDVQRWEHHQLGHQWL¿HGVRIDU,WLVFRQFHLYDEOH
that such a biomarker could be found in neutrophils because a large number of metabolic proteins
LQYROYHGLQWKHJHQHUDWLRQRI1$'3+DQG$73KDYHEHHQLGHQWL¿HGLQDSURWHRPLFVXUYH\ /LSSROLV
and Reinhardt, 2005), but the experimental evidence is still missing.

Muscle

The skeletal muscle represents the major part of body proteins and some of these proteins may be
degraded around parturition. This results in the release of creatinine, 3-methyl histidine, and most
proteinogenic amino acids into circulation to be used for milk protein synthesis. However, the
degradation of skeletal muscle protein is a relatively slow process that begins around parturition and
continuously progresses at least by the 4th week after parturition (Kuhla et al., 2011b). Unexpectedly,
the muscular fat content was lowest around parturition, suggesting that fatty acid degradation in
muscle tissue is a much earlier event in the adaptation to NEB. Also, muscle glycogen reserves were
H[KDXVWHGZLWKLQWKH¿UVWWZRZHHNVDIWHUSDUWXULWLRQDQGZHUHDOPRVWUHSOHQLVKHGDIWHUWKHth week
of lactation. These observations suggest an early mobilisation of glucose and fatty acids followed
by a somewhat delayed mobilisation of amino acids to be either directly used for milk synthesis
or for hepatic anabolic processes in early lactation. To study the intermediary metabolic pathways
underlying the mobilisation of energy reserves from skeletal muscle, we applied semitendinosus
muscle biopsies to 2D-GE-based proteomics. In early lactation, we found two increasingly expressed
SURWHLQVFORVHO\DVVRFLDWHGZLWKIDWW\DFLGGHJUDGDWLRQQDPHO\HOHFWURQWUDQVIHUÀDYRSURWHLQDQG
retinal dehydrogenase 1, in early lactation. This upregulation occurred in parallel with the post
partum plasma NEFA peak and the nadir of muscle fat content, suggesting a stimulated breakdown
of triglycerides and increased fatty acid oxidation in the muscle during early lactation. However,
we were not able to identify any enzyme participating in beta-oxidation.

216 Energy and protein metabolism and nutrition in sustainable animal production
Furthermore, there was increased expression of glycolytic enzymes such as fructose bisphosphate
aldolase A, GAPDH, triosephosphate isomerase, enolase, pyruvate kinase and lactate dehydrogenase,
but also reduced expression of the glycogenesis enzyme UTP-glucose-1-phosphate uridylyltransferase
when compared to the ante partum period. The expression of TCA cycle enzymes (aconitase 2,
malate dehydrogenase) was also lowered in week 4 post partum, whereas the abundance of ATP
homeostasis regulating enzymes (creatine kinase and ATP synthase) was upregulated as compared
WRODWHSUHJQDQF\2XU¿QGLQJVOHGXVWRFRQFOXGHWKDWLQHDUO\ODFWDWLRQ D PXVFOHS\UXYDWHLV
shunted towards lactate and less so towards acetylCoA and (b) that ATP production via the TCA
cycle is decreased (Figure 1). The increased skeletal muscle lactate production likely supports
hepatic gluconeogenesis to which lactate contributes up to 25% in early lactation (Aschenbach et
al., 2010). In Western blot studies we further noticed reduced expression of muscle GLUT4 during
early lactation (Kuhla et al.E %DVHGRQWKHVH¿QGLQJVZHVXJJHVWHGWKDWWKHGHFRXSOHG&RUL
cycle in early lactation supports hepatic gluconeogenesis and thereby glucose partitioning to the
mammary gland (Figure 1).

)LJXUH$FWLYDWLRQRIPDMRUPHWDEROLFSDWKZD\VGXULQJ1(%LQHDUO\ODFWDWLRQDVLGHQWL¿HG
by proteomic approaches. Metabolic and neuronal pathways are in grey while metabolites and
PDFURQXWULHQWÀRZVDUHGLVSOD\HGLQEODFN$PLQRDFLGVSULPDULO\GHULYHGIURPF\WRVNHOHWDOSURWHLQV
and lactate which originates from muscle glycogenolysis and anaerobic glycolysis, are released from
skeletal muscle and utilized by the liver as glucoplastic precursors or by the mammary gland for milk
protein synthesis or free AA in milk. Due to prevailing hypoglycaemia in early lactation, glucose is not
taken up by the muscle decoupling the Cori cycle. Fatty acids, predominantly released from adipose
tissue, serve as substrate for generating energy in muscle and liver in addition to be directly used
IRUPLONIDWSURGXFWLRQ7KHLQFUHDVLQJO\DFWLYDWHGKHSDWLFIDWW\DFLGR[LGDWLRQSDWKZD\VLQÀXHQFH
VLJQDOOLQJRIWKHYDJXVQHUYHDQGWKXVSUHYHQWVXI¿FLHQWLQFUHDVHRIIHHGLQWDNHLQHDUO\ODFWDWLRQ

Energy and protein metabolism and nutrition in sustainable animal production 217
There were also numerous indications for an increased proteasomal activity in muscle tissue
during early lactation: Reduced expression of heat shock protein beta-1 and alpha-crystallin (both
VWDELOL]LQJP\R¿EULOODUSURWHLQV XSUHJXODWLRQRIEULGJLQJLQWHJUDWRUSURWHLQ ZKLFKDFWLYDWHV
caspase-independent processes), reduced abundance of Dj-1 protein (which promotes cell growth)
and upregulation of phosphatidylethanolamine-binding protein 1 (a suppressor of cell development
and growth) and reduced expression of major cytoskeletal proteins. It appears likely that the observed
increased proteasomal activity entails the release of amino acids into the circulation serving either
as substrates for milk protein synthesis or in the case of glucoplastic amino acids as precursors for
hepatic gluconeogenesis.

Liver

Hepatic gluconeogenesis and glycogenolysis are highly active during early lactation. Because
the majority of glucose is used for lactose production in the mammary gland, fatty acids become
an important energy source and are intensively mobilized from adipose tissue during this period.
However, excessive NEFA concentrations are increasingly oxidized by the liver but only to a certain
extent (Grum et al. EHFDXVHRIWKHUHGXFHGDYDLODELOLW\RIR[DORDFHWDWH&RQVHTXHQWO\DFHW\O
&R$GHULYHGIURP1()$R[LGDWLRQZLOOSDUWLDOO\EHFRQYHUWHGWRNHWRQHERGLHVRUUHHVWHUL¿HGWR
triacylglycerides (TG) resulting in the development of fatty liver. The hepatic lipid metabolism has
been suggested to be involved in the control of energy balance. For example, increased hepatic
fatty acid oxidation leads to increased hepatic ATP production which signals satiety to the brain via
YDJDODIIHUHQWVDQGWKXVSUHYHQWVVXI¿FLHQWLQFUHDVHRIIHHGLQWDNHGXULQJHDUO\ODFWDWLRQ $OOHQet
al., 2009). Accordingly, the degree of fatty liver is inversely correlated with feed intake and energy
balance (Hammon et al., 2009; Weber et al., 2013). To examine how the extent of lipid mobilization
LQÀXHQFHVKHSDWLFR[LGDWLYHIDWPHWDEROLVPZHUHWURVSHFWLYHO\DVVLJQHGSHULSDUWXULHQW+ROVWHLQFRZV
either to a group with a high or a low liver fat content post partum (Schäff et al., 2012). Feed intake
expressed as kg dry matter intake per kg body weight was lower in cows with the higher liver fat
content. Liver biopsies taken on day -34, -17, +3, +18, and +30 relative to parturition were applied
to 2D-GE-based proteomics and Western blot analysis. In early lactation we found an increased
SKRVSKRU\ODWLRQRI$03.DQGLQFUHDVHGH[SUHVVLRQRIȕR[LGDWLYHHQ]\PHVLQERWKJURXSV)RUWKH
medium-chain acyl-CoA dehydrogenase, the extent of upregulation was greater in cows developing
WKHKLJKHUOLYHUIDWFRQWHQW+RZHYHUIRUWKHIXUWKHUGRZQVWUHDPORFDWHGȕR[LGDWLYHHQ]\PHVVKRUW
FKDLQVSHFL¿FDF\O&R$GHK\GURJHQDVHHQR\O&R$K\GUDWDVHK\GUR[\DF\O&R$GHK\GURJHQDVH
2, and 3-ketoacyl-CoA thiolase as well as 2,4-dienoyl-CoA reductase 1 were lower expressed in cows
with the higher liver fat content post partum (Schäff et al., 2012). Thus, it seems that cows with the
KLJKHUIDWPRELOLVDWLRQKDYHDQLQFUHDVHGDFWLYDWLRQEXWGLPLQLVKHGFRPSOHWHȕR[LGDWLRQRIIDWW\
acids which has also been observed at the level of mRNA (Li et al., 2012b) and enzyme activity
(Murondoti et al. OHYHO)DLOXUHRIFRPSOHWHPLWRFKRQGULDOȕR[LGDWLRQVHHPVWRUHVXOWLQD
proportionally augmented degradation of fatty acids in peroxisoms and microsomes since we found
LQFUHDVHGH[SUHVVLRQRIWKHSHUR[LVRPDOHQR\O&R$K\GUDWDVHFDWDODVHHOHFWURQWUDQVIHUÀDYRSURWHLQ
and aldehyde dehydrogenase 1 in cows with the higher liver fat content (Schäff et al., 2012).

7KHORZHUH[SUHVVLRQRIPLWRFKRQGULDOȕR[LGDWLYHHQ]\PHVLQFRZVGHYHORSLQJWKHKLJKHUOLYHU
IDWFRQWHQWVKRXOGFRQ¿QHWKHSURGXFWLRQRIDFHW\O&R$$QDOWHUQDWLYHVRXUFHRIDFHW\O&R$LV
the breakdown of ketogenic amino acids. Indeed, we found much lower plasma concentrations of
leucine particularly during early lactation in cows with the higher liver fat content. Also, the observed
increase of serine hydroxymethyltransferase, methylmalonate semialdehyde dehydrogenase, and
glycine C-acetyltransferase expression in cows with the higher liver fat content refer to a prevailing
catabolism of amino acids whose carbon skeletons enter the TCA cycle via pyruvate. Increased
production of pyruvate and phosphoenolpyruvate as indicated by increased expression of L-lactate
GHK\GURJHQDVHDQGĮHQRODVHUHIHUWRDIXUWKHULQFUHDVHGDQDSOHURVLVZKLOHWKHKLJKHUH[SUHVVLRQRI
fumarase points to a greater TCA cycling in cows with the higher liver fat content (Figure 1). The

218 Energy and protein metabolism and nutrition in sustainable animal production
TCA cycle generates NADH which serves as precursor for oxidative phosphorylation and generation
of ATP. Enzymes involved in oxidative phosphorylation like cytochrome c oxidase, ATP-synthase
and pyrophosphatase were increased in early lactation and higher expressed in cows with the high
as compared to the low liver fat, referring to elevated mitochondrial respiration (at least via complex
IV) and ATP production in fatty liver. Intensive mitochondrial oxidation and peroxisomal fatty acid
degradation confronts the liver with high amounts of superoxide radicals and H2O2 which can be
GHWR[L¿HGE\&X=QVXSHUR[LGHGLVPXWDVHDQGSHUR[LUHGR[LQ7KHJUHDWHUH[SUHVVLRQVRIWKH
latter two enzymes in cows with the higher liver fat content indicate an elevated oxidative stress
defence. Moreover, the NADPH-dependent glutathione system was negatively affected in cows
with fatty liver because enzymes involved in the NADPH-producing pentose phosphate cycle
(e.g. transketolase and triposephosphate isomerase) were lower expressed particularly during early
lactation. Furthermore, the hepatic glutathione synthesis requires Cys, Glu, and Gly, but plasma
Cys concentrations were lower in cows with fatty liver indicating an impaired NADPH-dependent
glutathione system and presumably a reduced ability to defend against elevated oxidative stress in
these animals (Schäff et al., 2012).

7RVXPXSWKLVSDUWGXULQJHDUO\ODFWDWLRQOLPLWHGPLWRFKRQGULDOEXWLQFUHDVHGSHUR[LVRPDOȕR[LGDWLRQ
DQGLQFUHDVHGȦR[LGDWLRQRIIDWW\DFLGVZDVDFFRPSDQLHGE\DQLQFUHDVHGGHJUDGDWLRQRIDPLQR
acids. This indicates stimulated anaplerotic reactions and higher TCA cycling followed by increased
mitochondrial respiration in fatty liver (Figure 1). The simultaneously increased production of reactive
oxygen species as also described in plasma of early lactating dairy cows (Bernabucci et al., 2005),
WRJHWKHUZLWKDFRQ¿QHGDYDLODELOLW\RIDQWLR[LGDWLYHVWUHVVHQ]\PHVOHDGVWRR[LGDWLYHVWUHVVIXUWKHU
affecting health and immune function. Activation of these oxidative metabolic pathways may provide
the link for activating the vagus nerve and signalling for disproportionately low feed intake (Figure 1).

In order to distinguish between hepatic oxidative pathways activated during NEB in early lactation
DQGWKRVHDFWLYDWHGLQUHVSRQVHWRLQVXI¿FLHQWHQHUJ\LQWDNHHDUO\ODFWDWLQJGDLU\FRZVZHUHHLWKHU
GHSULYHGIURPIHHGIRUKRXUVRUIHGad libitum before liver tissue sampling (Kuhla et al., 2009).
Feed deprivation increased liver fat content accompanied by augmented AMPK phosphorylation
and down regulation of enzymes associated with fatty acid oxidation (acyl-CoA dehydrogenase and
WKLRODVH 7KHVHUHVXOWVLQGLFDWHWKDWGRZQUHJXODWLRQRIKHSDWLFȕR[LGDWLRQGRHVQRWRQO\VXSSRUWWKH
development of fatty liver but is also involved in signalling hunger to the brain (Allen et al., 2009).

Mammary gland

The liver and the mammary gland have complementary metabolic roles during lactation. In a 2D-DIGE
approach, in which liver and mammary gland tissue from early lactating cows were differentially
labelled, expression differences between tissues (ratio >2) revealed major complementary metabolic
pathways in these tissues (Rawson et al., 2012). While – as expected – enzymes involved in
JOXFRQHRJHQHVLVDQGȕR[LGDWLRQRIIDWW\DFLGVZHUHDEXQGDQWO\H[SUHVVHGLQOLYHUHQ]\PHVIRU
fatty acid (acetyl CoA carboxylase and fatty acid synthase) and lactose (UDPG-pyrophosphorylase
DQGĮODFWDOEXPLQ V\QWKHVLVZHUHRQO\GHWHFWHGLQPDPPDU\JODQG )LJXUH 2QWKHRWKHUKDQG
enzymes involved in the urea cycle or in propionate metabolism were not evident in the mammary
gland (Rawson et al., 2012).

0LONIDWFRQWHQWGUDPDWLFDOO\LQFUHDVHVIURPFRORVWUXPWRPDWXUHPLONDQGGHFOLQHVZLWKLQWKH¿UVW
week of lactation. Reinhardt and Lippolis (2008) investigated protein changes in milk fat globule
membranes using iTRAQ. Mucin 1 and 15, adipophilin, butyrophilin, and xanthine dehydrogenase
and additional proteins associated with various aspects of lipid transport synthesis and secretion
such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty
acid binding protein were upregulated in milk obtained on day 7 compared with colostrum (day 1).
In contrast, apolipoproteins A1, C-III, E, and A-IV were lower in milk compared with colostrum.

Energy and protein metabolism and nutrition in sustainable animal production 219
These data provided new insight into the type of energy and nutrient secretion involved in NEB of
early lactation.

In conclusion, we have demonstrated in this paper that the use of proteomics approaches provides
a new quality of information on the networking of cellular processes during the transition between
pregnancy and lactation in the dairy cow.

References
Allen M.S., B.J. Bradford, and M. Oba, 2009. Board Invited Review: The hepatic oxidation theory of the control of
feed intake and its application to ruminants. J. Anim Sci. 87, 3317-3334.
Aschenbach J.R., N.B. Kristensen, S.S. Donkin, H.M. Hammon, and G.B. Penner, 2010. Gluconeogenesis in dairy
FRZV7KHVHFUHWRIPDNLQJVZHHWPLONIURPVRXUGRXJK,8%0%/LIH
%HUQDEXFFL8%5RQFKL1$/DFHWHUDDQG1DUGRQH,QÀXHQFHRIERG\FRQGLWLRQVFRUHRQUHODWLRQVKLSV
EHWZHHQPHWDEROLFVWDWXVDQGR[LGDWLYHVWUHVVLQSHULSDUWXULHQWGDLU\FRZV-'DLU\6FL
Bionaz M., K. Periasamy, S.L. Rodriguez-Zas, R.E. Everts, H.A. Lewin, W.L. Hurley, and J.J. Loor, 2012. Old and
new stories: revelations from functional analysis of the bovine mammary transcriptome during the lactation cycle.
3/R62QHH
Corbacho A.M., G. Martinez De La Escalera, and C. Clapp, 2002. Roles of prolactin and related members of the
prolactin/ growth hormone/placental lactogen family in angiogenesis. J. Endocrinol. 173, 219-238.
Gross J., H.A. van Dorland, F.J. Schwarz, and R.M. Bruckmaier, 2011. Endocrine changes and liver mRNA abundance
of somatotropic axis and insulin system constituents during negative energy balance at different stages of lactation
in dairy cows. J. Dairy Sci. 94, 3484-3494.
*UXP'(-.'UDFNOH\56<RXQNHU':/D&RXQWDQG--9HHQKXL]HQ1XWULWLRQGXULQJWKHGU\SHULRG
DQGKHSDWLFOLSLGPHWDEROLVPRISHULSDUWXULHQWGDLU\FRZV-'DLU\6FL
Hammon H.M., G. Stürmer, F. Schneider, A. Tuchscherer, H. Blum, T. Engelhard, A. Genzel, R. Staufenbiel, and W.
Kanitz, 2009. Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-
\LHOGLQJGDLU\FRZVZLWKKLJKDQGORZIDWFRQWHQWLQOLYHUDIWHUFDOYLQJ-'DLU\6FL
Horvath T.L., Z.B. Andrews, and S. Diano, 2009. Fuel utilization by hypothalamic neurons: roles for ROS. Trends
Endocrinol. Metab. 20, 78-87.
Ingvartsen K.L., and J.B. Andersen, 2000. Integration of metabolism and intake regulation: a review focusing on
periparturient animals. J. Dairy Sci. 83, 1573-1597.
Kuhla B., D. Albrecht, S. Kuhla, and C.C. Metges, 2009. Proteome analysis of fatty liver in feed deprived dairy cows
reveals interaction of fuel sensing, calcium, fatty acid and glycogen metabolism. Physiol. Genomics. 37, 88-98.
Kuhla B., D. Albrecht, R. Bruckmaier, T. Viergutz, G. Nürnberg, and C.C. Metges, 2010. Proteome and radio immuno
assay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows. Proteomics. 10,
4491-4500.
Kuhla B., S. Görs, and C.C. Metges, 2011a. Hypothalamic Orexin A expression and the involvement of AMPK and
33$5JDPPD6LJQDOLQJLQ(QHUJ\5HVWULFWHG'DLU\&RZV$UFKLY7LHU]XFKW$UFKLYHV$QLPDO%UHHGLQJ

Kuhla B., Kuhla S., P.E. Rudolph, D. Albrecht, and C.C. Metges, 2007. Proteomics analysis of hypothalamic response
WRHQHUJ\UHVWULFWLRQLQGDLU\FRZV3URWHRPLFV
Kuhla B., G. Nürnberg, D. Albrecht, S. Görs, H.M. Hammon, and C.C. Metges, 2011b. Involvement of skeletal
muscle protein, glycogen and fat metabolism in the adaptation on early lactation of dairy cows. J. Proteome Res.

/DQJHQKHLP-)'7DQ$0:DONHUDQG:<&KHQ7ZRZURQJVFDQPDNHDULJKWGLPHUVRISURODFWLQDQG
JURZWKKRUPRQHUHFHSWRUDQWDJRQLVWVEHKDYHDVDJRQLVWV0RO(QGRFULQRO
Lewis U.J., Y.N. Sinha, and G.P. Lewis, 2000. Structure and properties of members of the hGH family: a review.
Endocr. J. 47, S1-8.
/L;;/L*%DL+&KHQ4'HQJ=/LX/=KDQJ*/LXDQG=:DQJD(IIHFWVRIQRQHVWHUL¿HGIDWW\
acids on the gluconeogenesis in bovine hepatocytes. Mol. Cell. Biochem. 359, 385-388.

220 Energy and protein metabolism and nutrition in sustainable animal production
Li P., X.B. Li, S.X. Fu, C.C. Wu, X.X. Wang, G.J. Yu, M. Long, Z. Wang, and G.W. Liu, 2012b. Alterations of fatty
DFLGȕR[LGDWLRQFDSDELOLW\LQWKHOLYHURINHWRWLFFRZV-'DLU\6FL
/LSSROLV-'%'3HWHUVRQ%XUFKDQG7$5HLQKDUGW'LIIHUHQWLDOH[SUHVVLRQDQDO\VLVRISURWHLQVIURP
neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows. Vet. Immunol.
,PPXQRSDWKRO
Lippolis J.D., and T.A. Reinhardt, 2005. Proteomic survey of bovine neutrophils. Vet. Immunol. Immunopathol. 103,

/LSSROLV-'DQG7$5HLQKDUGW&HQWHQQLDOSDSHU3URWHRPLFVLQDQLPDOVFLHQFH-$QLP6FL
Locher L.F., N. Meyer, E.M. Weber, J. Rehage, U. Meyer, S. Dänicke, and K. Huber, 2011. Hormone-sensitive lipase
protein expression and extent of phosphorylation in subcutaneous and retroperitoneal adipose tissues in the
periparturient dairy cow. J. Dairy Sci. 94, 4514-4523.
Loor J.J., H.M. Dann, R.E. Everts, R. Oliveira, C.A. Green, N.A. Guretzky, S.L. Rodriguez-Zas, H.A. Lewin, and
-.'UDFNOH\7HPSRUDOJHQHH[SUHVVLRQSUR¿OLQJRIOLYHUIURPSHULSDUWXULHQWGDLU\FRZVUHYHDOVFRPSOH[
DGDSWLYHPHFKDQLVPVLQKHSDWLFIXQFWLRQ3K\VLRO*HQRPLFV
Loor J.J., R.E. Everts, M. Bionaz, H.M. Dann, D.E. Morin, R. Oliveira, S.L. Rodriguez-Zas, J.K. Drackley, and H.A.
Lewin, Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy
FRZV3K\VLRO*HQRPLFV
Murondoti A., R. Jorritsma, A.C. Beynen, T. Wensing, and M.J. Geelen, 2004. Unrestricted feed intake during the
dry period impairs the postpartum oxidation and synthesis of fatty acids in the liver of dairy cows. J. Dairy Sci.

Schäff C., S. Börner, S. Hacke, U. Kautzsch, D. Albrecht, H.M. Hammon, M. Röntgen, and B. Kuhla, 2012. Increased
anaplerosis, TCA cycling, and oxidative phosphorylation in the liver of dairy cows with intensive body fat
mobilization during early lactation. J. Proteome Res. 11, 5503-5514.
Sumner-Thomson J.M., J.L. Vierck, and J.P. McNamara, 2011. Differential expression of genes in adipose tissue of
¿UVWODFWDWLRQGDLU\FDWWOH-'DLU\6FL
Talamo F., C. D’Ambrosio, S. Arena, P. Del Vecchio, L. Ledda, G. Zehender, L. Ferrara, and A. Scaloni, 2003. Proteins
IURPERYLQHWLVVXHVDQGELRORJLFDOÀXLGVGH¿QLQJDUHIHUHQFHHOHFWURSKRUHVLVPDSIRUOLYHUNLGQH\PXVFOHSODVPD
DQGUHGEORRGFHOOV3URWHRPLFV
Piccione G., V. Messina, A. Schembari, S. Casella, C. Giannetto, and D. Alberghina, 2011. Pattern of serum protein
fractions in dairy cows during different stages of gestation and lactation. J. Dairy Res. 78, 421-425.
Rawson P., C. Stockum, L. Peng, B. Manivannan, K. Lehnert, H.E. Ward, S.D. Berry, S.R. Davis, R.G. Snell, D.
McLauchlan, and T.W. Jordan, 2012. Metabolic proteomics of the liver and mammary gland during lactation. J.
Proteomics. 75, 4429-4435.
Reinhardt T.A., and J.D. Lippolis, Developmental changes in the milk fat globule membrane proteome during the
transition from colostrum to milk. 2008. J. Dairy Sci. 91, 2307-2318.
Sejersen H., M.T. Sørensen, T. Larsen, E. Bendixen, and K.L. Ingvartsen, 2012. Liver protein expression in dairy cows
with high liver triglycerides in early lactation. J. Dairy Sci. 95, 2409-2421.
Weber C., C. Hametner, A. Tuchscherer, B. Losand, E. Kanitz, W. Otten, S.P. Singh, R.M. Bruckmaier, F. Becker,
W. Kanitz, and H.M. Hammon, 2013. Variation in fat mobilization during early lactation differently affects feed
LQWDNHERG\FRQGLWLRQDQGOLSLGDQGJOXFRVHPHWDEROLVPLQKLJK\LHOGLQJGDLU\FRZV-'DLU\6FL
Xia C., H.Y. Zhang, L. Wu, C. Xu, J.S. Zheng, Y.J. Yan, L.J. Yang, and S. Shu, 2012. Proteomic analysis of plasma from
cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry.
5HV9HW6FL

Energy and protein metabolism and nutrition in sustainable animal production 221
Quantifying subclinical ruminal drinking using a [13C]-[15N2]-urea
based method in veal calves
H. Berends, J.J.G.C. van den Borne and W.J.J. Gerrits
$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\  $+ :DJHQLQJHQ WKH 1HWKHUODQGV
[email protected]

Introduction
Ruminal drinking (RD) occurs in calves when ingested milk or milk replacer enters the reticulorumen
LQVWHDGRIWKHDERPDVXPDQGFDQEHFDXVHGE\DIDLOXUHRIWKHUHWLFXODUJURRYHUHÀH[RUE\EDFNÀRZ
of milk replacer (MR) from the abomasum. In a clinical case, RD often results in chronic maldigestion
(Stocker et al., 1999), ruminal acidosis, lack of appetite and recurrent bloat (Van Weeren-Keverling
Buisman et al., 1990), and consequently a reduced growth rate. Although clinical cases of RD have
been described extensively (Herrli-Gygi et al.*HQWLOHet al., 2004; Van Weeren-Keverling
Buisman et al., 1990), data on subclinical RD are scarce, partly due to methodological issues. Recent
studies have shown that subclinical RD can be substantial in veal calves. Between 21 and 35% of
an orally supplied dose of Co-EDTA was recovered in the rumen at slaughter (Suárez et al., 2007;
Berends et al., 2012). Assuming that nutrients from MR are subject to fermentation in the rumen,
subclinical RD may reduce post-absorptive availability of nutrients and result in lower growth rates
in calves. Nonetheless, it is unknown if RD is constant over time (i.e. between meals or days), and
measuring recovery at slaughter does not allow repetitive measurements within one animal. Among
RWKHUVVROLGIHHG 6) SURYLVLRQWRYHDOFDOYHVPD\DOWHUWKHUHWLFXODUJURRYHUHÀH[DQGWKXV5',Q
order to study subclinical RD and related factors, there is a need for a method to quantify RD that
GRHVQRWUHTXLUHFDOYHVWREHVDFUL¿FHG,QWKLVVWXG\VXFKDPHWKRGZDVGHYHORSHGDQGDSSOLHGWR
assess the effect of SF intake on RD.

Material and methods

Forty-eight Holstein Friesian male calves, 55±0.3 kg BW and 53±0.7 d of age at start of the
experiment, were fed 41 g MR/kg BW0.75 per d. Crude protein and fat level of milk replacer were
212 and 192 g/kg DM, respectively. Calves were assigned to 1 of 4 levels of SF intake: 0, 9, 18, or
27 g DM SF/kg BW0.75 per d. The SF mixture consisted of 25% chopped wheat straw, 25% chopped
FRUQVLODJHDQGQRQSHOOHWHGFRQFHQWUDWHRQD'0EDVLV5'ZDVPHDVXUHGDW“NJ
BW by combining oral administration of [13C]urea and intravenous administration of [15N15N]urea
(Figure 1). Incomplete recovery of an oral dose of [13C]urea, provided with MR, in 48-h urine can be

)LJXUH6FKHPDWLFUHSUHVHQWDWLRQRIÀX[HVRIXUHDLVRWRSHVDIWHUDVLQJOHGRVHRUDODGPLQLVWUDWLRQ
RI>&@XUHDDQGKLQWUDYHQRXVDGPLQLVWUDWLRQRI>N1@XUHD DGDSWHGIURP/REOH\et al. 

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 223
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_69, © Wageningen Academic Publishers 2013
explained by RD or by re-entry of absorbed [13C]urea into the gastrointestinal tract, both resulting in
fermentation of urea. To correct for the gut entry rate of previously absorbed [13C]urea, we infused
[15N15N]urea for 24 h (adapted from Marini and Van Amburgh, 2003) and analysed cumulative
XULQHRYHUKIRUXUHDLVRWRSRPHUVDQGIDHFHVIRUWRWDO 15N enrichment. After monomolecular
degradation of urea into N2, isotopomers (14N14N, 15N14N, 15N15N) were analysed using GC-C-
IRMS as described by Marini and Van Amburgh (2003). Infusion of [15N15N]urea was conducted
two days after oral administration of [13C]urea.

Results and discussion

Preliminary results show that recovery of [13C]urea, provided with MR, in urine averaged 73±0.3%
for calves without SF and decreased (1% per g SF/ kg BW0.75 per d; P<0.05) with increasing SF
intake. The gut entry rate averaged 18% of the urea entry rate for calves without SF and increased
with 1.4% per g SF/ kg BW0.75 per d (P<0.05). When corrected for gut entry, the proportion of [13C]
urea absorbed from MR averaged 93±9% and was not affected by SF intake. As a result, subclinical
5'ZDVHVWLPDWHGDW“/EHLQJRIWKH05SURYLGHG,QWRWDORIWKHFDOYHVKDG
subclinical RD of 10% of ingested milk or more but this varied substantially between calves (SEM:
9%). Negative estimates for RD (as a % of ingested milk) were obtained for 15 out of 38 calves: 9
FDOYHVUDQJLQJEHWZHHQDQGDQGFDOYHVUDQJLQJEHWZHHQDQG 

,WFDQEHFRQFOXGHGWKDWTXDQWL¿FDWLRQRIVXEFOLQLFDOUXPLQDOGULQNLQJE\WKHGXDOWUDFHUPHWKRG
(i.e. [13C] and [15N151@XUHD QHHGVUH¿QLQJDQGUHTXLUHVFRPSDULVRQWRDJROGHQVWDQGDUGOLNHWKH
Cobalt recovery. The method could be improved when the fate of orally dosed [13C]urea and the
gut entry rate are studied simultaneously, to avoid effects of variation between and within days.
Ruminal drinking in the current study was generally lower than in previous studies (Suárez et al.,
2007; Berends et al. ZKLFKPD\EHLQÀXHQFHGE\WKH05OHYHOZKLFKZDVORZHULQWKH
current study. Alternative methods that may provide quantitative and repeatable insight in ruminal
drinking may include ultrasonography to measure abomasal volume (Marshall et al., 2005), and/or
WKHDFHWDPLQRSKHQDEVRUSWLRQWHVW 6KDUL¿et al., 2009).

References
Berends, H., J.J.G.C. van den Borne, S.J.J. Alferink, C.G. van Reenen, E.A.M. Bokkers, and W.J.J. Gerrits. 2012.
Low-protein solid feed improves the utilization of milk replacer for protein gain in veal calves. J. Dairy Sci.
  
+HUUOL*\JL0+0+DPPRQ<=ELQGHQ$6WHLQHUDQG-:%OXP5XPLQDOGULQNHUVHQGRFULQHDQG
metabolic status and effects of suckling from a nipple instead of drinking from a bucket. J. Vet. Med. A53, 215-224.
/REOH\*('0%UHPQHUDQG*=XXU(IIHFWVRIGLHWTXDOLW\RQXUHDIDWHVLQVKHHSDVDVVHVVHGE\UH¿QHG
non-invasive [15N151@XUHDNLQHWLFV%U-1XWU
Marini, J.C., and M.E. Van Amburgh. 2003. Nitrogen metabolism and recycling in Holstein heifers. J. Anim. Sci. 81,
545-552.
Marshall., T.S., P.D. Constable, S.S. Crochik, and T. Wittek. 2005. Determination of abomasal emptying rate in suckling
FDOYHVE\XVHRIQXFOHDUVFLQWLJUDSK\DQGDFHWRPLQRSKHQDEVRUSWLRQ$-95  
6KDUL¿.:*UĦQEHUJ66RURRUL00RKULDQG06$KUDUL.KD¿$VVHVVPHQWRIWKHDFHWRPLQRSKHQDEVRUSWLRQ
WHVWDVDGLDJQRVWLFWRROIRUWKHHYDOXDWLRQRIWKHUHWLFXODUJURRYHUHÀH[LQODPEV$P-9HW5HV  
6WRFNHU+/XW]+DQG35XVK&OLQLFDOKDHPDWRORJLFDODQGELRFKHPLFDO¿QGLQJVLQPLONIHGFDOYHVZLWK
chronic indigestion. Vet. Rec. 145, 307-311.
Suárez, B. J., C.G. Van Reenen, N. Stockhofe, J. Dijkstra, and W.J.J. Gerrits. 2007. Effect of roughage source and roughage
to concentrate ratio on animal performance and rumen development in veal calves. J. Dairy Sci. 90, 2390-2403.
Van Weeren-Keverling Buisman, A., J.M.V.M. Mouwen, T. Wensing, and H.J. Breukink. 1990. Intraruminal
administration of milk in the calf as a model for ruminal drinking: Morphological and enzymatical changes in the
jejunal mucosa. Vet. Res. Commun. 14(2), 129-140.

224 Energy and protein metabolism and nutrition in sustainable animal production
Estimation of 24-h energy expenditure in dairy cows using the 13C
bicarbonate dilution method
A. Münger1, L.D. Kaufmann1, S. Görs2, C.C. Metges2 and F. Dohme-Meier1
1$JURVFRSH/LHEHIHOG3RVLHX[5HVHDUFK6WDWLRQ$/332%R[3RVLHX[6ZLW]HUODQG
[email protected]
2/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 :LOKHOP6WDKO$OOHH'XPPHUVWRUI
Germany

Introduction
The interest in pasture-based dairy production systems increases because they can be economically
attractive and environmentally friendly (Peyraud et al., 2010). However, high yielding dairy cows
are not able to cover their nutritional demands just by grazing, and they need additional energy to
express their genetic potential for milk production (Kolver and Muller, 1998). So far available data
about energy requirements of grazing dairy cows are limited due to the lack of suitable measurement
methods. The 13C bicarbonate dilution technique combined with an automatic blood sampling system
DOORZVWKHHVWLPDWLRQRIHQHUJ\H[SHQGLWXUH (( LQJUD]LQJFRZVRYHUDKSHULRG .DXIPDQQet
al ,QRUGHUWRHYDOXDWHLI((PHDVXUHGIRUKFDQEHH[WUDSRODWHGWRKDQH[SHULPHQW
was conducted recording CO2SURGXFWLRQSK\VLFDODFWLYLW\DQGIHHGLQJEHKDYLRUGXULQJWKUHHK
periods per d in dairy cows grazed full-time.

Material and methods

7KHH[SHULPHQWZDVFDUULHGRXWZLWK+ROVWHLQFRZV ERG\ZHLJKW %: “NJGD\V
in milk: 73±12 d; milk yield: 44±4.8 kg/d) and consisted of a 2-wk adaptation period and a 2-wk
experimental period. The cows were equally divided between the two experimental weeks so that
each cow passed a period of 7-d where data was collected. Cows were on pasture from 07:30 to
14:30 h, 17:00 to 22:30 h and from 23:15 to 04:30 h. Between these grazing periods they were kept
in a free-stall barn for preparation of the measurements, feeding of supplement and milking at 05:10
DQGK7KHVXSSOHPHQWFRQVLVWHGRIDFHUHDOEDVHGFRQFHQWUDWHLQDPRXQWVWRPHHWSUHGLFWHG
QXWULHQWUHTXLUHPHQWVDQGZDVRIIHUHGLQWZRHTXDOPHDOVDWDQGKDIWHUPLONLQJXVLQJ
weighing troughs (Insentec B.V., Marknesse, the Netherlands). For each cow milk yield and milk
FRPSRQHQWFRQWHQWVZHUHGHWHUPLQHGGDLO\DQGLQWDNHRQSDVWXUHZDVTXDQWL¿HGE\WKHGRXEOHDONDQH
technique during the respective experimental week. The CO2 production of each cow was determined
on 1 d from 07:00 to 13:00 h (measurement period (MP) A), from 15:00 to 21:00 (MP B) and from
23:00 to 05:00 h (MP C) using the 13C bicarbonate dilution method (Junghans et al., 2007). After
administration of the tracer (0.7 mg NaH13CO3/kg BW) into the jugular vein, blood was sampled with
an automatic blood sampling system on pasture. The blood sampling procedure and the calculation
of the EE were described by Kaufmann et al. (2011). Physical activity of the cows was recorded
over 7 d using pedometers and feeding and rumination behavior was investigated over 5 d using a
behavior recorder. Differences in EE, physical activity and eating and rumination were analyzed
XVLQJDOLQHDUPL[HGPRGHOZLWKPHDVXUHPHQWSHULRGDV¿[HGHIIHFWDQGFRZDVUDQGRPHIIHFW7KH
relationship between EE and physical activity and EE and eating and rumination, respectively, were
examined using Spearman correlation. Estimation of the 24 h EE and the comparison which MP
was the most reliable to estimate 24-h EE were done by regression analysis.

Results and discussion

During the experimental weeks cows produced daily on average 40.8±4.0 kg of milk with 4.1±0.5% of
fat, 3.2±0.2% of protein and 4.7±0.1% of lactose. Total dry matter intake amounted to 19.0±1.5 kg/d
FRPSRVHGRI“NJ'0GKHUEDJHDQG“NJ'0GFRQFHQWUDWH(QHUJ\H[SHQGLWXUHZDV

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 225
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_70, © Wageningen Academic Publishers 2013
lower (P<0.01) in MP A (270 kJ/kg BW0.75) compared to MP B (322) and C (317). Concomitantly,
cows spent more time walking (P<0.001) in MP C (143 min) than in MP A (72 min) and B (73 min).
This was surprising. In general grazing cows are more active at daytime than at night because they
eat more and ruminate less during the day (Gibb et al., 7KLVZDVFRQ¿UPHGE\WKHSUHVHQW
results. However, because of the preparation of the measurements cows had to be moved to the barn
also during MP C, the night period. Furthermore, in contrast to MP A and B during which cows
walked just once from the barn to the pasture, in MP C cows had to cover this distance twice (barn-
pasture, pasture-barn). This might explain the difference in EE between MP A and C. However, no
correlations (P>0.05) existed between EE and physical activity. The clear difference in EE between
MP A and B was also astonishing but might be caused by the different activities cows did in these
periods. While MP A consisted almost solely of time on pasture, in MP B milking and concentrate
feeding were included.

The EE for 24 h was 1204 kJ/kg BW0.75 and was estimated based on the data of the three MP with a
prediction uncertainty of 12%. The value seems plausible because it is about 20% higher compared
to data of grass-fed cows determined in respiration chambers (Bruinenberg et al., 2002). We reported
previously that EE of cows grazing full time was about 20% higher compared to cows fed herbage
of the same quality in a free-stall barn (Kaufmann et al., 2011).

Further regression analysis showed a low standard error of estimate for each MP when used in
conjunction with chosen behavior and activity data to determine EE for 24 h. Differences among
SHULRGVZHUHPLQRUDVZHOO7KHUHIRUHLWFDQEHFRQFOXGHGWKDWHDFKRIWKHWKUHHKPHDVXUHPHQW
periods can be used to estimate EE of grazing cows for 24 h.

References
Bruinenberg, M.H., Van der Honing, Y., Agnew, R.E., Yan, T., van Vuuren, A.M., Valk, H., 2002. Energy metabolism
of dairy cows fed on grass. Livest. Prod. Sci. 75, 117-128.
Gibb, M. J., C. A. Huckle, R. Nuthall, Rook, A. J., 1997. Effect of sward surface height on intake and grazing behaviour
by lactating Holstein-Friesian cows. Grass Forage Sci. 52, 309-321.
Junghans, P., Voigt, J., Jentsch, W., Metges, C.C., Derno, M., 2007. The 13C bicarbonate dilution technique to determine
energy expenditure in young bulls validated by indirect calorimetry. Livest. Sci. 110, 280-287.
Kaufmann, L.D., Münger, A., Rérat, M., Junghans, P., Görs, S., Metges, C.C., Dohme-Meier, F. 2011. Energy expenditure
of grazing cows and cows fed grass indoors as determined by the 13C bicarbonate dilution technique using an
automatic blood sampling system. J. Dairy Sci. 94, 1989-2000.
Kolver, E. S., and L. D. Muller. 1998. Performance and nutrient intake of high producing Holstein cows consuming
pasture or a total mixed ration. J. Dairy Sci. 81:1403-1411.
Peyraud, J.L., Van den Pol-van Dasselaar, A., Dillon, P., Delaby, L. 2010. Producing milk from grazing to reconcile
economic and environmental performances. In: Schnyder, H., Isselstein, J., Taube, F., Auerswald, K., Schellberg,
J., Wachendorf, M., Herrmann, A., Gierus, M., Wrage, N. and A. Hopkins (eds.) Grassland in a changing world,
Organising Committee of the 23th General Meeting of the EGF and Arbeitsgemeinschaft Grünland und Futterbau
GHU*HVHOOVFKDIWIU3ÀDQ]HQEDX9RO

226 Energy and protein metabolism and nutrition in sustainable animal production
The impact of nutritional, animal and farm management factors on
variation in milk urea content
A. Bannink1, J.W. Spek1,2, J.L. Ellis2 and J. Dijkstra2
1:DJHQLQJHQ85/LYHVWRFN5HVHDUFK:DJHQLQJHQ8532%R[$%/HO\VWDGWKH
Netherlands; [email protected]
2:DJHQLQJHQ8QLYHUVLW\:DJHQLQJHQ85:':DJHQLQJHQWKH1HWKHUODQGV

Introduction
Urea excreted in urine is the primary source of undesirable nitrogen (N) emissions from dairy
systems, and it accounts for most of the variation in N excretion. Urea is formed by hepatocytes
from ammonia generated by catabolism of amino acids or absorbed from the gastrointestinal tract.
A minor fraction (<1%) of urea N transported through blood ends up in milk and therefore milk urea
N content (MUN; mg N/dl milk) is an indicator of the amount of urea N excreted in urine. Such
information may be useful to optimize protein nutrition and reduce N emissions in farming practice.
A strong relationship exists between MUN and N excreted in urine, with R2 values typically ranging
between 0.7 and 0.8 (Schröder et al., 2005). However, such high R2 values apply for the total range
of observed MUN and N excretion data. For the narrow range of MUN that is relevant for use as
an indicator in practice a much lower R2 values applies (<0.3). The main part of variation in MUN
has then to be attributed to other factors than urea N excreted in urine (UUN, g N/d). The aim of the
SUHVHQWZRUNZDVWRH[SORUHDQGREWDLQDEHWWHUTXDQWLWDWLYHXQGHUVWDQGLQJRIWKHLQÀXHQFHRIWKHVH
other factors on MUN, to let MUN become a more useful indicator of UUN.

Urea dynamics, milk urea and N excretion

The MUN as a concentration (mg N/dl) depends on urea N concentration in blood plasma (PUN;
mg N/dl blood plasma). Any nutritional parameter that affects PUN dynamics hence also affects
MUN. Therefore, quantifying variation in MUN that is unrelated to variation in UUN requires a
TXDQWLWDWLYHXQGHUVWDQGLQJRI381G\QDPLFVDQGTXDQWL¿FDWLRQRIWKHLQÀXHQFHRIQXWULWLRQDO
management and animal factors on these dynamics.

1XWULWLRQDOIDFWRUVLQÀXHQFLQJ381G\QDPLFVDUHW\SHRIFDUERK\GUDWHVDQGSURWHLQVIHGDQGWKHLU
site of digestion, affecting the dynamics of ammonia entry in blood plasma from the rumen and amino
acid catabolism. Also water dynamics and kidney function affect PUN dynamics. Adding salt to the
GLHWLQFUHDVHXULQHYROXPHDQGORZHUV381E\WRXQLWSHUNJDGGLWLRQDOXULQH 6SHNet al.,
2012a; unpubl.). Water restriction may have an even stronger effect (Burgos et al., 2001; Spek et al.,
 5HQDOUHDEVRUSWLRQRIXUHDWREORRGIURPJORPHUXODU¿OWUDWHPD\YDU\ZLWKZDWHUG\QDPLFV
and protein intake (Thornton, 1970; Røjen et al., 2011; Spek et al. unpubl.). Finally, PUN may be
affected by urea recycling to the gastrointestinal tract. Based on 13C labelled urea infusions, Spek et
al. XQSXEO HVWDEOLVKHGZLWKDQLQFUHDVHG1LQWDNHDLQFUHDVHRI381DQGDLQFUHDVHRI
urea entry rate in blood plasma but no effect on urea recycling, whereas salt addition and increased
urine volume decreased PUN by 17% and decreased urea recycling by 9%.

Next to nutritional factors, management factors such as the moment and frequency of milking and
feeding cause variation in MUN. Net urea transfer depends on urea concentration gradient between
plasma and milk, and changes in MUN follow those in PUN with a time lag because of the time
involved with urea transfer between milk and blood and between milk compartments in the udder.
Hence, MUN is not necessarily the integral of changes of PUN in time. Spek et al. (2012b) used
several infusion protocols with 13C and 15N stable isotope labelled urea to demonstrate urea transfer
dynamics. After injection of labelled urea in the udder cistern, enrichment of subsequent fractions of
PLONFROOHFWHGZLWKPLONLQJ IURP¿UVWPLONWRDOYHRODUPLON YDULHGGHSHQGLQJRQWKHWLPHSHULRG

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 227
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_71, © Wageningen Academic Publishers 2013
between infusion and milking and between various fractions of milk collected. Within half an hour,
substantial fractions of labelled urea in milk were transported from cistern to alveoli, and from milk to
blood. About a third of isotope injected in the mammary gland was recovered in blood after 2 hours.

Also animal related factors may affect the relationship between PUN and UUN such as body weight.
Many animal factors might be covered already by nutritional factors such as feeding behaviour
(dominance) and capacity, or milk production capacity. Genetic factors are involved as MUN appears
to be a heritable characteristic. In a study of Šebek et al. (unpubl.) breeding values for MUN ranged
IURPWRPJGOIRUPHDVXUHPHQWZHHNVRIFRZVLQIHHGLQJWULDOV%UHHGLQJ
YDOXHRI081ZDVQRWUHODWHGWRHI¿FLHQF\RI1XWLOL]DWLRQREVHUYHG+HQFHXQNQRZQKHULWDEOH
animal factors affect MUN but are unrelated to variation in N utilization and hence UUN at identical
1LQWDNHLOOXVWUDWLQJWKHH[LVWHQFHRIIDFWRUVWKDWVKRXOGEHTXDQWL¿DEOHLQSULQFLSOH

Modelling urea dynamics

The dynamics of urea exchange between blood and milk and between udder milk compartments,
in combination with the dynamics of urea (ammonia) entry in blood plasma, of milk synthesis and
secretion (milking), and of urea reabsorption and urea excretion in urine by the kidneys, may cause
MUN to vary independently of UUN. To quantify the urea dynamics in blood plasma, milk, and
kidneys, a mechanistic model was constructed consisting of four state variables representing urea
SRROVLQEORRGNLGQH\ JORPHUXODU¿OWUDWH DQGPLON)OX[HTXDWLRQVDUHGHVFULEHGE\0LFKDHOLV
Menten kinetics or mass action forms. Model inputs are the dynamics of the absorption of ammonia,
the digestion of protein and cations from the gastrointestinal tract, and of milk synthesis and milking.
Model outputs are PUN, MUN, urine volume and UUN. The model is used to improve quantitative
understanding of how nutritional and management factors contribute to variation in MUN that is
unrelated to UUN.

References
Burgos, M.S., M. Senn, F. Sutter, M. Kreuzer and W. Langhans. 2001. Effect of water restriction on feeding and
metabolism in dairy cows. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, 418-427.
5¡MHQ%$3.7KHLODQG1%.ULVWHQVHQ(IIHFWVRIQLWURJHQVXSSO\RQLQWHURUJDQÀX[HVRIXUHD1DQGUHQDO
urea-N kinetics in lactating Holstein cows. J. Dairy Sci. 94, 2532-2544.
6FKU|GHU--$%DQQLQNDQG5.RKQ,PSURYLQJWKHHI¿FLHQF\RIQXWULHQWXVHLQFDWWOHRSHUDWLRQV,Q3IHIIHU
E. and A.N. Hristov (eds.) Nitrogen and phosphorus nutrition of cattle. CAB International, Wallingford, United
Kingdom, pp 255-279.
Spek, J.W., A. Bannink, G. Gort, W.H. Hendriks and J. Dijkstra, 2012a. Effect of sodium chloride intake on urine
volume, urinary urea excretion, and milk urea concentration in lactating dairy cattle. J. Dairy Sci. 95, 7288-7298.
Spek, J.W., J. Dijkstra, J.J.G.C. van den Borne and A. Bannink, 2012b. Short communication: Assessing urea transport
IURPPLONWREORRGLQGDLU\FRZV-'DLU\6FL
6SHN-:-'LMNVWUD*YDQ'XLQNHUNHQDQG$%DQQLQN$UHYLHZRIIDFWRUVLQÀXHQFLQJPLONXUHDFRQFHQWUDWLRQ
and its relationship with urinary urea excretion in lactating dairy cattle. J. Agric. Sci. 151, 407-423.
Thornton, R., 1970. Factors affecting the urinary excretion of urea nitrogen in cattle. I. Sodium chloride and water
loads. Austr. J. Agric. Res. 21, 131-144.

228 Energy and protein metabolism and nutrition in sustainable animal production
Metabolizable energy of pure stand alfalfa hay estimated from near
infrared spectra
C. Old1, N. Ohanesian1, J. Miller1, R. Hinders1, W. Vogt1, J. Oltjen1 and D. Sapienza2
1&DOLIRUQLD&KDSWHU$PHULFDQ5HJLVWU\RI3URIHVVLRQDO$QLPDO6FLHQWLVWV &D$53$6 6DFUDPHQWR
CA, USA; DQWHORSHUDQFK#JPDLOFRP
2Sapienza Analytica LLC, Slater, IA, USA

Introduction
Since the 1950s, alfalfa hay sold in California has been priced based on total digestible nutrient (TDN)
FRQWHQWHVWLPDWHGIURPHLWKHUPRGL¿HGFUXGH¿EHU 0H\HUDQG/RIJUHHQ RUDFLGGHWHUJHQW¿EHU
(ADF). Residues from either procedure can vary in chemical composition for alfalfa hay harvested
throughout the California growing season, which begins in February in extreme southern California
and ends in October. Since energy based feeding standards are those by which others are judged,
CaARPAS undertook this study to determine if metabolizable energy (ME) content of pure stand
alfalfa hay could be determined via near infrared (NIR) spectrophotometry.

Material and methods

Approximately 200 samples of pure stand alfalfa hay were collected throughout the alfalfa growing
regions of California and western Nevada. Of these, nine were selected as representative of
compositional variability in ADF and N content. Samples were cubed and sent to the USDA Dairy
Forage Research Center Madison, WI, USA) for evaluation in a metabolism study. Upon receipt,
cubes were reground and fed as pellets. Energy losses in feces and urine were determined for each
KD\IHGWRZHWKHUODPEV Q  DWHLWKHU 5 RU $/ RIERG\ZHLJKW*DVHRXVHQHUJ\ *( ORVVHV
were estimated utilizing a combined in-silico and in-vitro method. It was recognized that observed
urinary energy (UE) losses exceeded expected, as part of a well balanced diet fed to lactating dairy
cows. In order to approximate ME under actual conditions GE and UE losses were estimated as
RIGLJHVWLEOHHQHUJ\ '( ¿UVWE\WKHHTXDWLRQ0( '( 0( 0HWKDQHHQHUJ\ORVVHV
were estimated from digestible carbohydrates (in vitro, 0.51 moles CH4 per 180 g NDF fermented
and 0.45 moles CH4 per 180 g non-structural carbohydrate fermented) plus measured UE, adjusted
so that determined (GE + UE) losses averaged 18% of DE (ME2). Samples of all hays were oven
GULHG ƒ& DQGJURXQG PP SULRUWREHLQJSODFHGLQD0RGHO1,5VSHFWURSKRWRPHWHU
)RVV1,56\VWHPV86$ 5HÀHFWDQFHYDOXHVIRUȜIURPWRQPZHUHDQDO\]HGXVLQJWKH
chemometric utility UNSCRAMBLER (CAMO Software, Norway) to determine NIR prediction
models for TDN (AL only), DE and ME (1 and 2) at either level of intake.

Results and discussion

Digestible energy determined at R (mean=2.84, range 2.45 to 3.33 Mcal/kg) was greater (P<0.001)
WKDQ'(GHWHUPLQHGDW$/ PHDQ UDQJHWR0FDONJ 0HWDEROL]DEOHHQHUJ\HVWLPDWHG
at R (mean (ME1 and ME2)=2.19, range (ME1) 1.87 to 2.74 Mcal/kg; range (ME2) 1.87 to 2.77
Mcal/kg) was greater (P<0.001) than ME estimated at AL. Average estimates for ME (AL) were
2.19 Mcal/kg (ME1 and ME2), the range for ME1 was 2.19 to 2.74 Mcal/kg and for ME2, 2.19
WR0FDONJ7'1 $/RQO\ DYHUDJHGDQGUDQJHGIURPWR5HVSRQVHYDULDEOHV
(DE, ME and TDN) were predicted from spectral analyses with a minimum R2 of 0.87 (ME2 R)
and a maximum of 0.94 (DE AL and ME1 AL) (Table 1). Meyer and Lofgreen (1959) reported an
R2RIIRUWKHUHODWLRQVKLSEHWZHHQ7'1DQGPRGL¿HGFUXGH¿EHU FUXGH¿EHUSOXVVLOLFD ZKLOH
Bath (1985) reported an identical R2 for the relationship between TDN and ADF; both studies used
wether lambs. In the former study 40 samples of alfalfa hay were evaluated while in the latter nine
were evaluated.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 229
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_72, © Wageningen Academic Publishers 2013
Table 1. NIR prediction statistics

Item R2 Standard error of calibration

DE (R) Mcal/kg 0.92 0.09

DE (AL) Mcal/kg 0.94 0.07
ME1 (R) Mcal/kg 0.92 
ME1 (AL) Mcal/kg 0.94 
ME2 (R) Mcal/kg 0.87 0.08
ME2 (AL) Mcal/kg 0.92 0.07
TDN (%) 0.90 1.3

Analyses of these data indicates that NIR spectra of pure stand alfalfa hays may be used to predict
TDN, DE, and ME content When compared to the system currently used in California to predict alfalfa
quality, accuracy is improved, although further testing is required to make the system functional.

References
%DWK'/$FFXUDF\RIWKHPRGL¿HGFUXGH¿EHUDQGDFLGGHWHUJHQW¿EHUWHVWVWRSUHGLFW7'1RIDOIDOIDKD\3URF
Fifteenth California Alfalfa Symp. 45-49
Meyer, J.H. and G.P. Lofgreen. 1959. Evaluation of alfalfa hay by chemical analyses. J. Animal Sci. 1233-1242

230 Energy and protein metabolism and nutrition in sustainable animal production
Improving in sacco incubation technique to evaluate starch
degradability of fresh and fermented corn silage for ruminants
J. Peyrat1, E. Watremez1, A. Le Morvan1, A. Férard2, G. Cabon2, P.V. Protin2, P. Nozière1 and R. Baumont1
1,15$ 805 +HUELYRUHV 6LWH GH 7KHL[  6DLQW*HQqV &KDPSDQHOOH )UDQFH
[email protected]
2$59$/,6,QVWLWXWGX9pJpWDO6WDWLRQH[SpULPHQWDOHGHOD-DLOOLqUH/D&KDSHOOH6DLQW
Sauveur, France

Introduction
Corn silage is the main forage fed to ruminants and represents the most important energetic supply
to animals of high level of production (dairy cows and fattening animals). Global methods to predict
organic matter digestibility of corn silage were developed in the 1990’s (Aufrère et al., 1992). To improve
the understanding and the prediction of corn energetic substrates degradation in the rumen, one problem
lies in the lack of reference method to evaluate the rate and extent of corn silage starch degradation in
the rumen. This issue could lead to errors in the choice of feed supplements in diet formulation. The
aim of the present experiment was to determine the most adapted experimental in sacco procedure
among current in sacco incubation technique to evaluate degradability of fresh corn and corn silage.

Material and methods

Eight corn samples (two genotypes with different types of starch; two stages of maturity, harvested at 27%
DM and 42% DM, respectively; two methods of conservation: fresh and fermented) were placed in nylon
bags according to the 3 following conditioning methods: dried and 1 mm ground (D1, usual conditioning
for in sacco technique; Michalet-Doreau et al., 1987), dried and 4 mm ground (D4, a conditioning that is
supposed to reduce particle loss through the pores of the bags; Philippeau and Michalet-Doreau, 1997)
and fresh-ground (FG, a conditioning which simulates corn arriving in the rumen after chewing). For each
RIWKHWUHDWPHQWVWKHNLQHWLFVRIVWDUFKGLVDSSHDUDQFHZDVPHDVXUHGLQWKHUXPHQRIWKUHH¿VWXODWHG
FRZVZLWKWZRUHSOLFDWHVSHUFRZDQGDWIROORZLQJLQFXEDWLRQWLPHVDQGK7KHQ
the effective degradability (ED) was calculated with the step by step method (Kristensen et al., 1982),
DVVXPLQJDSDUWLFOHRXWÀRZUDWHRIK(IIHFWVRIJHQRW\SHPDWXULW\FRQVHUYDWLRQPHWKRGVDPSOH
FRQGLWLRQLQJDQGWKHLULQWHUDFWLRQVRQGHJUDGDWLRQSDUDPHWHUVZHUHDQDO\VHGDV¿[HGHIIHFWVDQGDQLPDO
as random effect with the MIXED procedure of SAS (9.1 version, 2002-2003).

Results and discussion

Table 1 reports the effects of conservation method and of sample conditioning on starch degradability.
%HVLGHVLQWHUDFWLRQRIPHWKRGDQGVDPSOHFRQGLWLRQLQJKDGDVLJQL¿FDQWHIIHFWRQVWDUFKGHJUDGDELOLW\

7DEOH6WDUFKGHJUDGDELOLW\ Q  IURPFRUQ IUHVKDQGVLODJH FRQGLWLRQHGLQGLIIHUHQWZD\V

GULHGDQGPPJURXQGGULHGDQGPPJURXQGIUHVKJURXQG 0HDQV“VWDQGDUGHUURUVDUHSUHVHQWHG
DQGVWDWLVWLFDOO\FRPSDUHGZLWKLQHDFKHIIHFW &DQG6 

Conservation of corn (C) Sample conditioning (S)

Fresh Silage Dried and 4 mm Dried and 1 mm Fresh-ground

ground (D4) ground (D1) (FG)

2 h degradability (%) “a “b 32.5±0.42a 53.8±0.42b 55.1±0.42c

Effective degradability (%) “a 74.9±0.43b 58.7±0.44a 72.7±0.44b 74.5±0.44c

a,b,c P<0.05.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 231
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_73, © Wageningen Academic Publishers 2013
(IIHFWLYHGHJUDGDELOLW\ZDVVLJQL¿FDQWO\KLJKHU P<0.01) for corn silage than for fresh corn, regardless
RIWKHVDPSOHFRQGLWLRQLQJPHWKRG7KHHIIHFWLYHGHJUDGDELOLW\RI'VDPSOHVZDVVLJQL¿FDQWO\
higher than that of D4 (P<0.01) and lower than FG samples (P<0.01). This was mainly related to
GLIIHUHQFHVLQWKHGHJUDGDWLRQDWKWKDWZDVVLJQL¿FDQWO\KLJKHUZLWK'WKDQZLWK' P<0.01)
and lower with D1 than with FG samples (P<0.05).

6WDJHRIPDWXULW\DOVRDIIHFWHGVLJQL¿FDQWO\VWDUFKGHJUDGDELOLW\(IIHFWLYHGHJUDGDELOLW\RIHDUO\
VWDJHFKDUDFWHUL]HGE\'0  ZDVVLJQL¿FDQWO\KLJKHU P<0.01) than that of advanced
VWDJHFKDUDFWHUL]HGE\'0  7KLVGHFUHDVHRIHIIHFWLYHGHJUDGDELOLW\ZLWKVWDJHVRI
maturity were consistent for the two genotypes, the conservation and the sample conditioning methods
VLJQL¿FDQWHIIHFWRILQWHUDFWLRQJHQRW\SHîVWDJHîFRQVHUYDWLRQîVDPSOHFRQGLWLRQLQJ P<0.01).

1RVLJQL¿FDQWGLIIHUHQFHZDVIRXQGEHWZHHQWKHWZRJHQRW\SHVRQVWDUFKGHJUDGDELOLW\ 
3  DOWKRXJKJHQRW\SH&KDGDGLIIHUHQWVWDUFKW\SHWKDQJHQRW\SH%

This trial shows high differences between starch degradability of fresh and fermented corn. This
probably can be explained by partial hydrolysis of starch during fermentation process in silage
(Jurjanz and Monteils, 2005). Thus, the use of fresh samples to evaluate corn silage degradability
will underestimate starch degradability although it is more convenient for large varieties trials than
using fermented samples.

The D1 sample conditioning, frequently used in the in sacco studies for evaluation of nitrogen value,
OHDGVWRKLJKHVWLPDWHVRIVWDUFKGHJUDGDELOLW\EHFDXVH¿QHJULQGLQJJHQHUDWHVKLJKORVVHVRIVWDUFK
particles through the pores of nylon bags (Philippeau and Michalet-Doreau, 1997). Similar high
GHJUDGDELOLW\YDOXHVZHUHREWDLQHGZLWKWKH)*VDPSOHV+RZHYHUWKLVPHWKRGZDVPRUHGLI¿FXOW
in its application than the two others, probably because homogeneous sampling of fresh material is
PRUHGLI¿FXOWWKDQRIGU\VDPSOHV7KH'VDPSOHFRQGLWLRQLQJOHDGVWRORZHUHVWLPDWHVRIVWDUFK
degradability, in particular for short incubation times, and the reproducibility was similar for both
D4 and D1 conditions.

The expected decrease in starch degradability when the stage of maturity increases (Philippeau and
Michalet-Doreau, 1997; Jensen et al. LVFOHDUO\FRQ¿UPHGLQWKHSUHVHQWZRUN

To conclude, our results show a difference in starch degradability between fresh and ensiled corn
DQGTXDQWLI\WKHLQÀXHQFHRIGLIIHUHQWVDPSOHFRQGLWLRQLQJDQGFRQ¿UPVWKHHIIHFWRIPDWXULW\VWDJH
From this study, the most suitable method to evaluate starch degradability of corn silage appears to
be the use of silage samples dried and ground at 4 mm.

References
Aufrère, J., D. Graviou, C. Demarquilly, J. Andrieu, J.C. Emile, R. Giovanni and P. Maupetit, 1992. Estimation of
RUJDQLFPDWWHUGLJHVWLELOLW\RIZKROHPDL]HSODQWVE\ODERUDWRU\PHWKRGV$QLP)HHG6FL7HFKQRO
Jensen, C., M.R. Weisbjerg, P. Norgaard, T. Hvelplund, 2005. Effect of maize silage maturity on site of starch and NDF
digestion in lactating dairy cows. Anim. Feed. Sci. Technol. 118 (3), 279-294.
Jurjanz, S and V. Monteils, 2005. Ruminal degradability of corn forages depending on the processing method employed.
Anim. Res. 54, 3-15.
Kristensen, E.S., P.D. Moller and T. Hvelplund, 1982. Estimation of the effective protein degradability in the rumen of
FRZVXVLQJWKHQ\ORQEDJWHFKQLTXHFRPELQHGZLWKWKHRXWÀRZUDWH$FWD$JU6FDQG
Michalet-Doreau, B., R. Vérité and Chapoutot, P, 1987. Méthodologie de mesure de la dégradabilité in sacco de l’azote
des aliments dans le rumen. Bull. Tech. CRZV Theix, 1987.
3KLOLSSHDX&DQG%0LFKDOHW'RUHDX,QÀXHQFHRIJHQRW\SHDQGVWDJHRIPDWXULW\RIPDL]HRQUDWHRIUXPLQDO
VWDUFKGHJUDGDWLRQ$QLP)HHG6FL7HFKQRO
SAS, 2002-2003. Release 9.1. SAS Inst. Inc., Cary, NC, USA.

232 Energy and protein metabolism and nutrition in sustainable animal production
A titration approach to identify the capacity for starch digestion in milk-
fed calves
M.S. Gilbert1, J.J.G.C. van den Borne1, A.J. Pantophlet2 and W.J.J. Gerrits1
1$QLPDO1XWULWLRQ*URXS:DJHQLQJHQ8QLYHUVLW\32%R[$+:DJHQLQJHQWKH
Netherlands; [email protected]
2&HQWUHIRU0HGLFDO%LRPLFV8QLYHUVLW\0HGLFDO&HQWUH*URQLQJHQ32%R[55
Groningen, the Netherlands

Introduction
Calf milk replacers commonly contain 40-50% lactose. For economic reasons, starch is of interest
as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. Ileal
GLVDSSHDUDQFHRIVWDUFKZDVRQO\LQFDOYHVZKHUHDVODFWRVHGLVDSSHDUHGIRU &RRPEH
and Smith, 1974). This indicates that the activity of enzymes required for the hydrolysis of starch to
glucose limits starch digestion in milk-fed calves. It is however unknown which enzyme system is
limiting the rate of starch hydrolysis in the intestinal lumen of calves. In addition, a maximum may
exist for the daily quantity of starch that can be hydrolyzed and absorbed. Potentially, enzyme systems
may also adapt to the starch fed. Both may be subject to considerable inter-individual variation.

Incomplete intestinal starch hydrolysis and absorption leads to fermentation, subsequently reducing
ileal and fecal dry matter (DM) content and pH (Huber et al.5R\.UHLNHPHLHUet al.,
1991). We used this relation between fermentation and fecal DM content and pH in a titration study
where lactose was stepwise exchanged for one of 4 starch products (SPs). These SPs differed in the
enzymes required for their complete hydrolysis to glucose. The objectives were (1) to determine
which enzyme system limits starch digestion in milk-fed calves and (2) to determine the maximum
inclusion level of SPs in the milk replacer.

Material and methods

Forty calves (104±0.5 kg BW) were assigned to either a control diet (with lactose as the only source
of carbohydrates) or one of the 4 titration strategies, in which lactose was gradually exchanged for
one of 4 SPs. The 4 SPs differed in the enzymes required for their complete hydrolysis to glucose:
JHODWLQL]HGVWDUFK ĮDP\ODVHDQGPDOWDVH PDOWRGH[WULQ ĮDP\ODVHDQGPDOWDVH PDOWRGH[WULQZLWK
ĮEUDQFKLQJ ĮDP\ODVHPDOWDVHDQGLVRPDOWDVH DQGPDOWRVH PDOWDVH 'LHWDU\LQFOXVLRQOHYHO
of each SP was increased by 3% per week at the expense of lactose. This increase in inclusion level
RI63VZDVVWRSSHGZKHQIHFDO'0FRQWHQWGURSSHGEHORZFRUUHVSRQGLQJWRRIWKH
average starting fecal DM content, and remained below this value for 3 consecutive weeks. Calves
were fed 52 g of milk replacer per kg metabolic BW per day.

Fecal samples were collected from the rectum each week from each calf to determine DM content
DQGS+7KHPD[LPXPFDSDFLW\RIVWDUFKGLJHVWLRQRIDQLQGLYLGXDOFDOIZDVGH¿QHGDVWKHLQÀHFWLRQ
point in the relation between inclusion level of the SP and fecal DM content or pH. Differences in
WUHDWPHQWDYHUDJHVRIWKHVHLQÀHFWLRQSRLQWVFDQEHXVHGWRLGHQWLI\WKHUDWHOLPLWLQJHQ]\PHV\VWHP
in starch digestion. The maximum capacity of starch digestion was estimated for each calf using
non-linear regression of fecal DM content and pH against the inclusion level of SP.

Results and discussion

For control calves, fecal DM content and pH did not change over time (mean fecal DM content
15.4±0.3%,3 0.481; mean fecal pH 7.5±0.1, 3  $OWKRXJKZHK\SRWKHVL]HGWKDWLQÀHFWLRQ
points could be estimated to identify the rate-limiting enzyme system in starch digestion, the fecal

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 233
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_74, © Wageningen Academic Publishers 2013
DM content and pH already responded to changes in SP inclusion at very low inclusion levels of SPs.
+HQFHIRURXWRI63IHGFDOYHVOLQHDUUHJUHVVLRQSURYLGHGDEHWWHU¿W P<0.05) of the data (SP
inclusion level vs. fecal DM content or pH) than nonlinear regression. Treatment effects on the slope
parameters (fecal DM content or pH against inclusion level) were subsequently analyzed statistically.

Slopes for SP-fed calves were lower (fecal DM content, 3 0.004; fecal pH, P<0.001) than for control
calves and did not differ among SPs (Table 1). Fecal DM content dropped by 0.21% (±0.03%) and
fecal pH dropped by 0.12 (±0.01) for every % increase in dietary SP. The gradual decrease in fecal
DM content and pH with increasing inclusion of SPs indicates that the maximum inclusion level
GRHVQRWPDUNDQLQÀHFWLRQSRLQWIURPHQ]\PDWLFK\GURO\VLVWRIHUPHQWDWLRQEXWWKDW63VDUHDOUHDG\
(partly) fermented at lower inclusion levels.

For the current study, SPs were selected on the enzymes required for their hydrolysis. The combination
of SPs would lead us to deduce the rate-limiting enzyme for starch digestion in milk-fed calves.
As both fecal DM content and pH responded sensitively to increasing inclusion of any of the SPs,
the hydrolysis of maltose to glucose by maltase is probably rate-limiting. Therefore, maltose will
probably induce intestinal fermentation in starch-fed calves. Surprisingly, the fecal DM content and
pH already decreased at low inclusion levels of SPs. Consequently, a maximum capacity of starch
GLJHVWLRQFRXOGQRWEHLGHQWL¿HGDQGIHUPHQWDWLRQRIWKH63VZDVOLNHO\VXEVWDQWLDO

In conclusion, fecal DM content and pH gradually decreased with incremental inclusion of SPs
in calf milk replacer, independent of SP characteristics. This indicates that maltase limits starch
digestion in milk-fed calves and that fermentation may contribute substantially to total tract starch
disappearance in milk-fed calves.

7DEOH6ORSHVRIWKHUHODWLRQEHWZHHQLQFOXVLRQOHYHORIVWDUFKSURGXFWV *6JHODWLQL]HGVWDUFK
MD, maltodextrin; MDB, maltodextrin with a high degree of branching; MT, maltose, exchanged
DWWKHH[SHQVHRIODFWRVH DQGIHFDO'0FRQWHQWRUS+LQPLONIHGFDOYHV

Treatment Control GS MD MDB MT P-value

Mean ± SE

Number of calves 7 8 8 8 8
Slopes1
Fecal DM, % 0.04±0.02a -0.19±0.05ab “ab -0.23±0.03b -0.28±0.09b 0.004
Fecal pH -0.01±0.01a -0.12±0.01b -0.12±0.02b -0.13±0.02b -0.10±0.02b <0.001

1 Slopes represent the change in fecal DM content or pH per 2.3 days (for control treatment) or per one % increase of
a starch product (for starch product treatments).
ab0HDQVZLWKGLIIHUHQWVXSHUVFULSWVLQWKHVDPHURZGLIIHUVLJQL¿FDQWO\ P<0.05).

References
Coombe, N.B. and R.H. Smith, 1974. Digestion and absorption of starch, maltose and lactose by the preruminant calf.
Br. J. Nutr. 31, 227-235.
+XEHU-7$'0F*LOOLDUG56$OOHQDQG1/-DFREVRQ8WLOL]DWLRQRIFDUERK\GUDWHVLQWURGXFHGGLUHFWO\
into omaso-abomasal area of stomach of cattle of various ages. J. Dairy Sci. 44, 321-330.
Kreikemeier, K.K., D.L. Harmon, R.T. Brandt, T.B. Avery and D.E. Johnson, 1991. Small intestinal starch digestion
in steers – Effect of various levels of abomasal glucose, corn starch and corn dextrin infusion on small intestinal
GLVDSSHDUDQFHDQGQHWJOXFRVHDEVRUSWLRQ-$QLP6FL
5R\-+%'LDUUKRHDRIQXWULWLRQDORULJLQ3URF1XWU6RF

234 Energy and protein metabolism and nutrition in sustainable animal production
Application of washed rumen technique for rapid determination of
fasting heat production in steers
D.H. Kim1, K.R. McLeod1, J.L. Klotz2, A.F. Koontz1, A.P. Foote1 and D.L. Harmon1
1Department of Animal and Food Sciences, University of Kentucky, Lexington, KY, USA;
GRK\XQJNLP#JPDLOFRP
2USDA-ARS, Forage-Animal Production Research Unit, Lexington, KY, USA

Introduction
Traditional measurement of maintenance energy requirements in cattle uses estimates of fasting
heat production (HP) made during the third and fourth day of fasting, when the respiratory quotient
54 KDVIDOOHQWRDSSUR[LPDWHO\ %OD[WHU +RZHYHUUHVXOWVYDU\ZLWKWKHOHQJWK GD\V 
RIIDVWLQJ %OD[WHUDQG:DLQPDQ ,QDGGLWLRQWKLVDSSURDFKKDVOLPLWDWLRQVVSHFL¿FDOO\WKH
actual severity of stress and decline of physical activity induced by the extended fasting period.
It was hypothesized that using the washed rumen technique, in conjunction with accurate fasting
HP estimates from short-term energy assessment, would more rapidly emulate a fasting state of
metabolism, and as a result, provide a more robust measure of fasting HP compared with the
traditional methodologies while still excluding most of the energy required for digestion, related
tissue deposition, and activity. Therefore, these experiments were conducted to validate the use of the
washed rumen technique for a rapid measurement of fasting HP and RQ compared with traditional
fasting methodologies.

Material and methods

In Exp. 1, sixteen Holstein steers were divided into two groups of 8 for a comparison of measurements
made following feeding (0-24 h for both groups) and fasting (24-48 h; Fasted; 8 steers BW=237±17
kg) or using the washed rumen model (24-48 h; WR; 8 steers BW=322±30 kg). The experiment was
conducted after steers were adapted to a cubed alfalfa-based diet at 1.5×NEm for 10 d. Steers were
placed into individual head-boxes and respiratory gas exchange was continuously measured on 2
consecutive d to determine HP and RQ under the fed state (d 11, all steers) and during fasting (d
12, 8 steers) or using the washed rumen technique (d 12, 8 steers). On the day of the washed rumen
SURFHGXUHWKHUHWLFXORUXPHQZDVHPSWLHGZDVKHGDQGUH¿OOHGZLWKEXIIHU 1D&O 1D+&23=24;
KHCO3=30; K2HPO4=2; CaCl2=1.5; MgCl2=1.5 mmol/kg of buffer; aerated with a mixture of 75%
N2 and 25% CO2) prior to measurement of gas exchange.

,Q([SVL[+ROVWHLQVWHHUV “NJ ZHUHXVHGLQDUHSOLFDWHGî/DWLQ6TXDUHGHVLJQZLWK

21 d periods, to determine the effects of prior alimentation on RQ and HP under unwashed (fed
state; d 20) and washed rumen (WR; d 21) conditions. Treatments offered were a cubed alfalfa-
based diet fed at 1.0, 1.5, and 2.0 × NEm. Respiratory gas exchange and washed rumen procedures
were conducted as described for Exp. 1. Jugular blood samples were collected prior to the morning
feeding and every 4 h for a subsequent 24-h period on d 20 and 21. The HP and RQ for each steer
were averaged over day and within each hour, and then analyzed using the MIXED procedures of
SAS (SAS Inst. Inc., Cary, NC, USA).

Results and discussion

Exp.1, mean hourly RQ, and daily HP were lower for the WR steers than Fasted steers on average
from 8 to 24 h after removal of rumen contents (P<DQG UHVSHFWLYHO\ )LWWLQJ54GDWD
REWDLQHGGXULQJIDVWLQJWRDRQHSKDVHGHFD\HTXDWLRQVKRZHGWKDWSODWHDXZDVDFKLHYHGDW“
and 0.72±0.01, and time to plateau was 9 and 8 h, for Fasted and WR steers, respectively. Mean RQ
DIWHU:5ZHUHDQG 6(0  IRUWLPHVHJPHQWVWRKWRKDQGWR

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 235
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_75, © Wageningen Academic Publishers 2013
KUHVSHFWLYHO\0HDQIDVWLQJ+3DIWHU:5ZDVDQG 6(0 N- KÂNJ0.75)) for
WLPHVHJPHQWVWRKWRKDQGWRKUHVSHFWLYHO\7KHUHZHUHQRVLJQL¿FDQWGLIIHUHQFHV
in RQ or fasting HP (3 0.225 and 3 UHVSHFWLYHO\ EHWZHHQWKHWLPHVHJPHQWVRIWR
h and 17 to 24 h post-rumen washing. In contrast, both RQ and HP differed (3 0.090 and 0.081,
respectively) across these same time segments for the Fasted group.

Exp. 2, heat production for intakes of 1.0, 1.5 and 2.0 × NEm were 479.2, 587.1 and 713.4 (SEM=4.0
N- GÂNJ0.75 UHVSHFWLYHO\$IWHUUXPHQZDVKLQJIDVWLQJ+3ZDVDFKLHYHGDWDQG
6(0 N- GÂNJ0.75)) for 1.0, 1.5 and 2.0 × NEm prior to fasting, respectively. The plateau RQ
ZHUHDQGIRULQWDNHVRIDQGî1(m during the fed state, respectively. The
RQ were 0.72, 0.71 and 0.71 for WR at 1.0, 1.5 and 2.0 × NEm intakes prior to fasting, respectively.
Mean daily plasma insulin and glucose concentrations were lower for WR than fed state (Table 1,
P< ZKHUHDVFRUWLVRODQGQRQHVWHUL¿HGIDWW\DFLG 1()$ ZHUHKLJKHUIRU:5 P<0.001).
+RZHYHUWKH1()$ZDVQRWDERYHWKUHVKROGOHYHOVIRUDVHYHUHHQHUJ\GH¿FLWWKDWFDQEHLQGXFHG
from prolonged fasting.

In conclusion, these studies demonstrate that a fasting state can be rapidly achieved by using the
washed rumen technique as opposed to the traditional fasting methodologies without a severe energy
GH¿FLW7KLVDSSURDFKPD\SURYLGHDQDOWHUQDWLYHWRWKHWUDGLWLRQDOKIDVWLQJWLPH$SSO\LQJWKH
washed rumen technique may be a more rapid and less stressful means to predict energy required
for maintenance in cattle.

Table 1. Comparison of plasma hormones and metabolites1.

Item Fed state WR SE P-value2

1.0 1.5 2.0 1.0 1.5 2.0 W E W×E

NEm NEm NEm NEm NEm NEm

Cortisol, μg/dl 0.75  1.10 1.08 1.55 1.84 0.13 <0.001 <0.001 NS
Insulin, μIU/ml  4.73  1.79 2.05  0.39 <0.001 <0.001 <0.001
Glucose, mg/dl 78.31  74.09    1.78 <0.001 NS 
NEFA, mmol/l 0.17 0.10 0.09 0.55 0.53 0.53 0.03 <0.001 NS NS
BHBA, mmol/l 0.49 0.50 0.54 0.49 0.52 0.54 0.02 NS 0.09 NS

1 Data are presented as least squared means of animals were fed alfalfa cubes at levels of 1.0, 1.5 and 2.0 × NE based
m
on the BW (Fed state) and incubation of ruminal buffer of 15 kg in the reticulorumen up to 24 h (WR).
2 W, washing effect; E, energy effect; W × E, their interaction.

References
%OD[WHU./7HFKQLTXHVLQHQHUJ\PHWDEROLVPVWXGLHVDQGWKHLUOLPLWDWLRQV3URF1XWU6RF
%OD[WHU./DQG)::DLQPDQ7KHIDVWLQJPHWDEROLVPRIFDWWOH%U-1XWU

236 Energy and protein metabolism and nutrition in sustainable animal production
Challenge models to study the effect of immune system activation on
amino acid metabolism in pigs
E. Kampman-van de Hoek1,2, W.J.J. Gerrits2, J.J.G.C. van den Borne2, C.M.C. van der Peet-
Schwering1, H. van Beers and A.J.M. Jansman1
1'HSDUWPHQWRI$QLPDO1XWULWLRQ:DJHQLQJHQ85/LYHVWRFN5HVHDUFK32%R[$%
Lelystad, the Netherlands; [email protected]
2$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands
6'D$XWRULWHLW'LHUJHQHHVPLGGHOHQ<DOHODDQ&08WUHFKWWKH1HWKHUODQGV

Introduction
In response to (non-)pathogenic challenges, the immune system of pigs can be activated. During
immune system activation, there is a competition for amino acids (AA) between body protein
deposition (growth) and immune function (Sandberg et al., 2007). As a consequence, feed intake
and growth are impaired (Williams et al., 1997) and body protein degradation increases to release
AA, e.g. for the production of acute phase proteins (APP) (Reeds et al., 1994). Knowledge about the
impact of immune system activation on AA metabolism of pigs is limited. The aim of the present study
was to compare the use of Complete Freund’s Adjuvant (CFA) and turpentine oil (TO) to activate
the immune system and to study the effect of immune system activation on AA metabolism in pigs.

Material and methods

Pigs of 30 kg body weight were challenged with either four iv CFA infusions (three on day 0 and
one on day 1 (n=3)), a single sc TO injection (n=3), or iv and sc saline as a control (control, n=2).
Pigs were fed at a restricted feed intake of 2.7 × the estimated ME requirements for maintenance.
Plasma APP concentrations (C-reactive protein (CRP), pigMAP, haptoglobin and albumin) were
GHWHUPLQHGLPPHGLDWHO\EHIRUHDQGDWGD\DQGDIWHUWKHFKDOOHQJH:KLWHEORRGFHOOFRXQWV
and feed intake were determined daily and a 4-d nitrogen (N)-balance was performed (day 0 to 3).
At day 2, a mixture of 7 universally 13C-labelled essential AA (Lys, Met, Trp, Ile, Leu, Val, Phe,
Tyr) was infused iv as a bolus to study AA dilution kinetics, while feeding the pigs hourly portions.
Plasma 13C AA enrichments were measured by isotope ratio mass spectrometry coupled to a gas
FKURPDWRJUDSK)RUHDFKSLJDQG$$DGRXEOHH[SRQHQWLDOPRGHOZDVXVHGWR¿WGDWDRIWKH 13C
AA enrichment in plasma:

E(t) = a1 × exp (b1 × t) + a2 × exp (b2 × t) (1)

where E(t) is the predicted 13C enrichment in plasma AA (APE) at time t (min), and a1, b1, a2, and
b2 are parameter estimates from which the irreversible loss rate (ILR, i.e. the sum of disappearance
RI$$IURPWKHSODVPDSRROWRZDUGVSURWHLQV\QWKHVLVDQG$$R[LGDWLRQLQ—PRONJ%:ÂK ZDV
calculated:

,/5 G DEDE î 

At 9 d after the start of the challenge, pigs were euthanized after which autopsy was performed
with special emphasis to TO injection sites, lung, spleen, liver, and kidney. Data were analyzed
E\$129$ZLWKFKDOOHQJHDV¿[HGHIIHFWRUE\DPL[HGPRGHOZLWKWKHHIIHFWRIFROOHFWLRQGD\
included as repeated measures.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 237
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_76, © Wageningen Academic Publishers 2013
Results and discussion
One day after the start of the challenge serum haptoglobin concentrations increased 9 fold (3 0.03)
in CFA compared to control pigs, but was not increased in TO pigs. Serum CRP concentrations
increased three fold in CFA pigs (3  DQGLQ72SLJV 3 0.03) compared to control pigs.
Two days after the start serum haptoglobin increased 7 fold (3 0.02) in both CFA and TO serum
FRPSDUHGWRFRQWUROSLJ0$3LQFUHDVHGIROG P<0.01) only in TO, and CRP increased 4 fold in TO
(3 0.01) compared to control. In agreement with our results, haptoglobin and pigMAP concentrations
LQFUHDVHGXSWRIROGDQG&53LQFUHDVHGXSWRIROGLQUHVSRQVHWREDFWHULDODQGSDUDVLWLFLQIHFWLRQV
RULQÀDPPDWLRQLQSLJV +HHJDDUGet al., 2011). CFA increased (P<0.05) eosinophil counts at day
1 and 2. Feed intake was reduced at day 0 and 1 in CFA pigs and at day 1 in TO pigs, but not in
control pigs. N retention as % of N intake was similar between groups. The ILR for Lys, Met, Trp,
Ile, Leu, Val, Phe and Tyr was numerically lower in CFA pigs compared to control pigs, with the
most pronounced numeric difference in ILR for Tyr, followed by Leu, Met, and Phe (Figure 1). In
TO pigs, the ILR were almost similar to the control pigs (Figure 1). Autopsy revealed a 1.5 fold
JUHDWHUUHODWLYHOXQJZHLJKWRI&)$SLJVWKDQFRQWUROSLJVDQGLQ¿OWUDWLRQRIJUDQXORPDWRXVFHOOV
in lung (CFA pigs) or at the TO injection sites (TO pigs). In conclusion, the patterns of change in
ILR of AA indicate a change in AA utilization in pigs with an activated immune system as induced
by CFA administration. TO administration led to less pronounced differences in ILR. Possibly, this
change in utilization is due to repartitioning of AA to protein synthesis for the immune system at the
expense of synthesis for net body protein deposition (Sandberg et al., 2007). Further studies will be
performed to quantify the effect of immune system activation on AA metabolism and N retention.

Figure 1. Effect of CFA or TO administration on the irreversible loss rate of plasma amino acids by
intravenous bolus infusion of universally labeled C-amino acids in growing pigs.

Reference
Heegaard, P., Stockmarr, A., Pineiro, M., Carpintero, R., Lampreave, F., Campbell, F., Eckersall, P., Toussaint, M.,
Gruys, E., Sorensen, N. 2011. Optimal combinations of acute phase proteins for detecting infectious disease in
pigs. Vet. Res. 42: 50.
Reeds, P. J., C. R. Fjeld, and F. Jahoor. 1994. Do the differences between the amino acid compositions of acute-phase
DQGPXVFOHSURWHLQVKDYHDEHDULQJRQQLWURJHQORVVLQWUDXPDWLFVWDWHV"-1XWU
Sandberg, F. B., G. C. Emmans, and I. Kyriazakis. 2007. The effects of pathogen challenges on the performance of
QDwYHDQGLPPXQHDQLPDOVWKHSUREOHPRISUHGLFWLRQ$QLPDO
Williams, N. H., T. S. Stahly, and D. R. Zimmerman. 1997. Effect of level of chronic immune system activation on the
JURZWKDQGGLHWDU\O\VLQHQHHGVRISLJVIHGIURPWRNJ-$QLP6FL

238 Energy and protein metabolism and nutrition in sustainable animal production
Effect of an enzyme complex and dietary nutrients on endogenous losses
of amino acids in chicks
S. Cerrate1, K. Vignale, R. Ekmay2, J. England and C. Coon
1$YLDJHQ,QFRUSRUDWHG+XQWVYLOOH$/86$
2'HSDUWPHQWRI$QLPDO6FLHQFH&RUQHOO8QLYHUVLW\,WKDFD1<86$
3RXOWU\6FLHQFH'HSDUWPHQW8QLYHUVLW\RI$UNDQVDV)D\HWWHYLOOH$586$[email protected]

Introduction
Inconsistencies in expected performance have been noted from feeding exogenous enzymes. Several
explanations may exist but it is possible that when feeding diets with exogenous enzymes a metabolic
feedback system limits or regulates endogenous digestive enzyme expression, synthesis, and secretion.
Labeled amino acids may be an advantage over other methods for measuring endogenous losses
because the isotope technique may be used with practical diets. Endogenous amino acid secretions
KDYHEHHQVKRZQWREHDIIHFWHGE\GLHWDU\SURWHLQW\SHVRIIDWVDQG¿EHU 6LULZDQet al., 1989;
Danicke et al., 2000) and digestible amino acid requirements should be corrected for the endogenous
amino acid losses. The objectives of the present study were to determine endogenous amino acid losses
IURPEURLOHUVIHGYDULRXVOHYHOVRISURWHLQIDWDQG¿EHUZLWKDQGZLWKRXWDGGHG5RYDELR0D[TM.

Material and Methods

Twenty mash diets from 5 ingredients (corn, soybean meal, pro-plus, barley and poultry fat) and
Rovabio® Max at 0.05% (xylanase, B-glucanase, and phytase) were fed to male chicks from 1 to
GROG'LHWDU\WUHDWPHQWVKDGH[FHVVOHYHOVRIIDW DQG(( SURWHLQ DQG
&3 RU¿EHU DQGQHXWUDOGHWHUJHQW¿EHU RYHUDFRQWUROGLHW(DFKGLHWZDVIHGWR
UHSOLFDWHÀRRUSHQVRIFKLFNVDQGWUDQVIHUUHGWRGLJHVWLELOLW\FDJHV FKLFNVFDJH DQGPL[HG
with 0.5% of titanium oxide (TiO2). One chick per cage was orally infused with 15N-threonine,
15N-cysteine, 15N-methionine, 15N-lysine, and 15N-leucine from 17 to 21 days of age at 2% of dietary
DPLQRDFLGVUHTXLUHPHQWV$WGRIDJHKDIWHUWKHODVWLQIXVLRQLOHDOGLJHVWDRI¿YHQRQODEHOHG
chicks per cage were sampled and from labeled chicks ileal digesta and wing blood sample were taken.

AIDn (apparent ileal digestibility) = AAdiet – AAileal × (TiO2diet/TiO2ileal)

ELA (endogenous losses of amino acids) = AAileal × EFR × (TiO2diet/TiO2ileal)

()5 HQGRJHQRXVÀRZUDWH  $3(ileal/APEplasma

APE (atom percent excess)ileal or plasma = APElabeled – APEnonlabeled; APE = 15N/ (15N+14N)

Results and discussion

The extra apparent ileal digestible threonine, methionine+cysteine, lysine, and protein from the
added enzymes were 0.04, 0.03, 0.04, and 0.89%, respectively (data not shown). The endogenous
losses (EL) of 15N-threonine and 15N-cysteine were reduced by Rovabio MaxTM (Table 1). The
(/RI&3XVLQJWKH¿YHODEHOHGDPLQRDFLGVZLWKDSUR¿OHRIDPLQRDFLGFRPSRVLWLRQRIPXFLQ
secretion was reduced by Rovabio MaxTM7KHHQGRJHQRXVÀRZUDWH ()5 DQG(/RI15N-leucine
ZHUHLQÀXHQFHGE\WKHGLHWDU\QXWULHQWV )LJXUH ZKHUHWKH(/RI15N-leucine was increased with
LQFUHDVLQJGLHWDU\IDWSURWHLQRU¿EHU

EFR(CPm)=EFRThr[()5Cysx0.28+EFRMethx0.02+EFRLysx0.08+EFRLeu[

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 239
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_77, © Wageningen Academic Publishers 2013
EFR(CPp)=EFRThrx0.24+EFRCysx0.08+EFRMethx0.05+EFRLysx0.24+EFRLeux0.39

Smaller EL of threonine and cysteine from feeding carbohydrase diets suggests that mucin secretions
DUHGHFUHDVHGEHFDXVHWKUHRQLQHDQGF\VWHLQHDUHWKH¿UVWDQGWKLUGPRVWDEXQGDQW$$IRXQGLQPXFLQ
When the extra spared endogenous losses of amino acids due to carbohydrases were compared to
their requirements, dietary requirements for threonine, cysteine, and crude protein were reduced by
4.3, 15.2, and 4.2%, respectively. Variation of EL of 15N-leucine due to dietary nutrients indicates
that pancreatic secretions are the main regulator of these losses. Leucine is known to be the 3rd most
abundant AA in pancreatic protein secretion (Corring and Jung, 1972).

7DEOH(QGRJHQRXVORVVHV (/JNJ'0,/60HDQV RIODEHOHGDPLQRDFLGVDQGFUXGHSURWHLQ

Treatments Thr Cys Meth Lys Leu CPm1 CPp1

Enzymes
No 0.838a 1.775a 0.831  1.153 33.1a 33.2
Yes 0.544b 1.198b 0.730 2.429 1.055 24.7b 
SEM  0.122 0.080 0.417  2.1 
P-value 0.004 0.002 0.374 0.201 0.323 0.007 
Diets
SEM 0.151 0.273 0.178 0.933 0.154 4.7 5.9
P-value 0.752  0.473 0.358 0.0005 0.592 0.239

1&3PDQG&3SZHUHFDOFXODWHGIURPSUR¿OHRIDPLQRDFLGFRPSRVLWLRQRIPXFLQDQGSDQFUHDWLFVHFUHWLRQVUHVSHFWLYHO\
a,b P”

)LJXUH(QGRJHQRXVÀRZUDWH ()5$3(ideal/APEplasma/60($16 DQGHQGRJHQRXVORVVHV (/

JNJ'0,/60($16 RIOHXFLQH

References
Corring, T., and J. Jung, 1972. The amino acid composition of pig pancreatic juice. Nutrition Reproduction International

Danicke, S., W. Bottcher, H. Jeroch, J. Thielebein, and O. Simon, 2000. Replacement of soybean oil with tallow in
rye-based diets without xylanase increases protein synthesis in small intestine of broilers. J. Nutr. 130, 827-834.
6LULZDQ3:/%U\GHQDQG()$QQLVRQ(IIHFWVRIGLHWDU\¿EUHDQGSURWHLQOHYHOVRQHQGRJHQRXVSURWHLQ
secretions in chickens. Proc. Nutr. Soc. Aust. 14, 143.

240 Energy and protein metabolism and nutrition in sustainable animal production
Validation of the oral 13C-bicarbonate tracer technique against indirect
calorimetry for the estimation of energy expenditure in resting dogs
C. Larsson1, R.B. Jensen1, P. Junghans2 and A-H. Tauson1
1Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg,
Denmark; [email protected]
2Leibniz Institute for Farm Animal Biology, Department of Nutritional Physiology ‘Oskar Kellner’,
Dummerstorf, Germany

Introduction
To be able to provide guidelines for appropriate nutrition of dogs, a reliable and feasible method
for investigations of their energy expenditure (EE) would be very valuable. The 13C-bicarbonate
tracer technique (13C-BTT) has been used to assess EE in several species of animals (Junghans et
al., 2007; Lachica and Aguilera, 2003) and in humans (Junghans et al., 2008). This stable isotope
method is based on the 13C kinetics in breath CO2 after administration of 13C labeled sodium
bicarbonate (NaH13CO3). After tracer administration, the ratio between the 13C and 12C in spot
VDPSOHVRIH[SLUHGDLUFROOHFWHGRYHUDVXI¿FLHQWSHULRGRIWLPHFDQEHXVHGWRHVWLPDWHWKH&22
production rate (RCO2) and thereafter the EE. By using oral administration of the tracer the method
can be completely non-invasive, making it an appropriate method for studies in dogs. The aim of this
study was to validate the oral 13C-BTT (o13C-BTT) against indirect calorimetry, the ‘gold standard’
for the estimation of EE. The hypothesis is that the o13C-BTT can be used as a minimal restrictive
and non-invasive method to obtain reliable estimates of EE in dogs under near natural conditions.

Material and methods

Eight privately owned dogs of different breeds were included in this experiment. All dogs were
of normal body weight (BW) and in the weight range of 15-35 kg. Each dog was measured twice
under resting conditions, where the dog was, after an over-night fast, kept in a respiration chamber
WHPSHUDWXUH ƒ&KXPLGLW\  IRUDSSUR[LPDWHO\KRXUV7KH&22 production and
O2 consumption were measured continuously by means of an open-air-circuit respiration unit.
Simultaneously, the ratio between 13C and 12C in expired CO2 was measured online using an infrared
isotope analyzer, every 3rd minute after an oral dose of NaH13CO3 (5 mg/kg BW, incorporated in a
small piece of liver paté), making comparison between the two methods possible.

The CO2 production was estimated using the area under the 13CO2 enrichment-time curve, the dose
of 13C administrated and a recovery factor (RF) for 13C in breath CO2, as described by Elia (1991).
7KH((ZDVFDOFXODWHGDFFRUGLQJWRWKHHTXDWLRQRI%URXZHU  %\XVLQJERWKPHWKRGVLW
was possible to calculate the RF and also the RQ for each measurement. The average values of 0.72
and 0.78 for all measurements, was then used as the estimates for RF and RQ, respectively, in the
calculations of RCO2 and EE when using the 13C-BTT. The values of EE was calculated in kJ/kg
BW0.75/ h, and then standardized to 24 hours (i.e. kJ/kg BW0.75/ d). Data were statistically analysed
using the MIXED procedure of SAS®.

Results and discussion

7KHUHZHUHVLJQL¿FDQWGLIIHUHQFHVLQ(( P<0.001) between the dogs with LS-means, calculated
from the measurements of both methods, ranging from 304 kJ/kg BW0.75/d for dog D, to 511 kJ/kg
BW0.75/d for dog A. This was also expected due to the large variations between the dogs, i.e. breed,
%:DQGDJH+RZHYHUWKHUHZHUHQRVLJQL¿FDQWGLIIHUHQFHV P>0.05) between the two methods used
for estimation of EE in the present study. The results from this study, shown for each measurement in
Figure 1, suggest that the o13C-BTT can be used as a non-invasive method to obtain reliable estimates

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 241
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_78, © Wageningen Academic Publishers 2013
of EE in different types of dogs during resting conditions, and that the estimates of RF=0.72 and
RQ=0.78 can be used when estimating the RCO2 and EE. However, whether the o13C-BTT gives
reliable estimates of EE, and the estimates used for RF and RQ are affected, when used under other
circumstances, e.g. stress or physical activity, needs to be further investigated.

600
EE (kJ/kg BW 0.75/d)

500
400
300
13C-BTT
200
IC
100
0
A A B C C D D E E F F G G H H
Dog
)LJXUH7KHLQGLYLGXDOYDOXHVRIHQHUJ\H[SHQGLWXUH ((LQN-NJ%:G HVWLPDWHGIURPHDFK
PHDVXUPHQWRIHLJKWGRJV $WR+ XQGHUUHVWLQJFRQGLWLRQVE\XVLQJWKHRUDOC-bicarbonate tracer
WHFKQLTXH R&%77 DQGLQGLUHFWFDORULPHWU\ ,& 

References
%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV,Q%OD[WHU./ HGLWRU 3URFHHGLQJVRIWKHrd
EAAP Symposium on Energy Metabolism. Academic Press, London. UK, 441-443.
Elia, M., 1991. Estimation of short-term energy expenditure by the labeled bicarbonate method. In: Whitehead R.G.
and A. Prentice (editors), New techniques in nutritional research. Academic Press New York, USA, 207-227.
Junghans P., Jentsch W. and M. Derno, 2008. Non-invasive 13C bicarbonate tracer technique for measuring energy
H[SHQGLWXUHLQPHQ±DSLORWVWXG\H63(1(XUH-&OLQ1XWU0HWDE  HH
Junghans, P., J. Voigt, W. Jentsch, C.C. Metges, and M. Derno, 2007. The 13C bicarbonate dilution technique to determine
energy expenditure in young bulls validated by indirect calorimetry. Livest. Sci. 110, 280-287.
Lachica, M. and J.F. Aguilera, 2003. Estimation of energy needs of the free-ranging goat with particular reference
to the assessment of its energy expenditure by the 13C-bicarbonate method. Small Ruminant Res. 49, 303-318.

242 Energy and protein metabolism and nutrition in sustainable animal production
Validation of the 13C-bicarbonate tracer technique against indirect
calorimetry for estimation of energy expenditure in resting ponies
R.B. Jensen1, C. Larsson1, P. Junghans2 and A.-H. Tauson1
1Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg,
Denmark; [email protected]
2Leibniz Institute for Farm Animal Biology, Department of Nutritional Physiology ‘Oskar Kellner’,
Dummerstorf, Germany

Introduction
Knowledge on the energy requirements of horses is essential for optimal feeding practice. The ‘gold
standard’ for measuring energy expenditure (EE) is indirect calorimetry where the horse has to be
FRQ¿QHGWRDUHVSLUDWLRQFKDPEHU$OWHUQDWLYHO\WKH13C-bicarbonate tracer technique (13C-BTT) can
be used to measure EE (Elia, 1991). After administration of a dose of 13C labeled sodium bicarbonate,
the ratio between the 13C and 12&LQVSRWVDPSOHVRIH[SLUHGDLUFROOHFWHGRYHUDVXI¿FLHQWSHULRGRI
time can be used to estimate the CO2 production rate (RCO2) and EE. Collection of breath samples
can be done from the respiration chamber or it can be done under free living conditions with breath
bags and a small mask. Combining measurements in the respiration chamber with the 13C-BTT
makes it possible to compare the CO2 production measured with both techniques. Most studies in
other species have used intravenous (IV) administration of 13C-bicarbonate (Urschel et al., 2009;
Junghans et al., 2007). However, oral administration of 13C-bicarbonate in a single bolus could make
the 13C-BTT totally non-invasive. The hypothesis is that the 13C-BTT with IV and oral administration
of the tracer in a single bolus can be used for estimation of RCO2 and EE in sedentary ponies.

Material and methods

Four 3 to 4 years old Shetland ponies weighing 178±48 kg were used in the experiment and they
were fed hay (nutritional composition in % of dry matter: dry matter: 85%, ash: 4%, NDF: 72%,
$')OLJQLQFUXGHSURWHLQVXJDUV WZLFHDGD\DWKDQGK aJ
dry matter/kg body weight daily). Water was available ad libitum at all time. The ponies were trained
to get used to the respiration chambers, and they were placed in the chambers in the morning 1 h
before measurements started. The RCO2 and EE were estimated at 4 occasions (twice with oral and
twice with IV administration of 13C-bicarbonate) with at least one day of rest between measurements.
The ponies were kept in the respiration chambers for approximately 11 hours where CO2 production
and O2 consumption were measured continuously by means of an open-air-circuit respiration unit
WHPSHUDWXUH ƒ&KXPLGLW\ YROXPH P3). Simultaneously, the ratio between 13C
and 12C in expired CO2 was measured online for 11 hours using an infrared isotope analyzer, before
EDVHOLQH DQGHYHU\PLQXWHDIWHUDQRUDORU,9GRVHRI13C-bicarbonate (2,5 mg/kg body weight;
98 atom % 13C, Sigma Aldrich, St. Louis, USA), making comparison between the two methods and
WZRDGPLQLVWUDWLRQURXWHVSRVVLEOH7KHFKDPEHUZDVRSHQHGEULHÀ\ZKHQWKHRUDOGRVHZDVJLYHQ
and with the IV dose the pony was removed, injected and replaced in the chamber. The ponies were
fed their morning hay immediately after the oral or IV dose of 13C-bicarbonate was given. It was
possible to measure 13CO2ZLWKLQPLQDIWHUDGPLQLVWUDWLRQRIWKHLVRWRSHDQGEDVHOLQHZDVUHDFKHG
after approximately 8 hours.

The area under the enrichment-time curve (AUC), the dose of 13C-bicarbonate (D) and a recovery
factor (RF) of 13C were used to calculate RCO2 as described by Elia (1991). Energy expenditure and
the respiratory quotient (RQ) were calculated from CO2 production and O2 consumption according
WR%URXZHU  7KH13C-BTT only estimates CO2 production, and the mean RQ value was used
to estimate EE for this method.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 243
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_79, © Wageningen Academic Publishers 2013
Data was statistically analyzed using the MIXED procedure in SAS®. To study the effect of
administration route of 13C-bicarbonate (IV or oral) on the RQ-value and the RF, route was included
DV¿[HGHIIHFWDQGSRQ\DVUDQGRPHIIHFWLQWKHPRGHO7RVWXG\WKHHIIHFWRIPHWKRG ,&RU13C-BTT)
on CO2SURGXFWLRQDQG((PHWKRGZDVLQFOXGHGDV¿[HGHIIHFWDQGSRQ\DVUDQGRPHIIHFWLQWKH
PRGHO(IIHFWVZHUHFRQVLGHUHGVLJQL¿FDQWLIP<0.05.

Results and discussion

There was no effect (P>0.05) of administration route of 13C-bicarbonate on the RQ, however, there
was an effect of administration route (3 0.03) on the RF (Table 1). The same RQ and the two
different RF were then used to calculate the CO2 production for the 13C-BTT (RQ and RF values
are shown in Table 1). The estimated RF found in this experiment is in agreement with results of
other experiments (Junghans et al. 2007).

7KHUHZDVQRVLJQL¿FDQWGLIIHUHQFH P>0.05) between methods when estimating CO2 production

and EE. The results show that the 13C-BTT can be used to estimate EE with oral administration
of 13C-bicarbonate in sedentary ponies. However, the 13C enrichment-time curves were smoother
when IV administration of the tracer was used, and that it might be easier to model and calculate
$8&ZKHQPHDVXUHPHQWVDUHSHUIRUPHGZLWK,9DGPLQLVWUDWLRQXQGHU¿HOGFRQGLWLRQVZLWKVSRW
sampling of breath air instead of continuous sampling of breath samples in the respiration chamber.

7DEOH(IIHFWVRIDGPLQLVWUDWLRQURXWH ,9RURUDO RUPHWKRG ,&RU &%77 IRUHVWLPDWLRQRI

CO2SURGXFWLRQDQGHQHUJ\H[SHQGLWXUHLQVHGHQWDU\SRQLHV /6PHDQDQGVWDQGDUGGHYLDWLRQ 

Administration route SD P-value

IV Oral Route

Respiratory quotient 0.80 0.79 0.03 NS

Recovery factor (%)   0.03 0.03

Method SD P-value

IC 13C-BTT Route Method R×M

CO2 production (l/d) 782 793 21 NS NS NS

Energy expenditure (kJ/kg0.75/d) 433 442 34 NS NS NS

References
%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV,Q%OD[WHU./ HGLWRU 3URFHHGLQJVRIWKHrd
EAAP Symposium on Energy Metabolism. Academic Press, London. UK, 441-443.
Elia M., 1991. Estimation of short-term energy expenditure by the labeled bicarbonate method. In: Whitehead R.G.
and A. Prentice (editors), New techniques in nutritional research. Academic Press New York, USA, 207-227.
Junghans P., J. Voigt, W. Jentsch, C.C. Metges and M. Derno, 2007. The 13C bicarbonate dilution technique to determine
energy expenditure in young bulls validated by indirect calorimetry. Livest. Sci. 110, 280-287.
Urschel, K.L., T.L. Smith, R.B. Drake, P.A. Harris and R.J. Geor, 2009. Using [13C]Sodium bicarbonate to measure
FDUERQGLR[LGHSURGXFWLRQLQKRUVHVDWUHVW-(TXLQH9HW6FL

244 Energy and protein metabolism and nutrition in sustainable animal production
Biometry and bioelectrical impedance analysis to estimate body
FRPSRVLWLRQRI¿VKWDPEDWLQJD Colossoma macropomum × Piaractus
brachypomus)
F.T. Andrade1, M.L.T. Abreu2, J.B. Lopes1, E. Lanferdini2 and A. Saraiva
18)3,'HSDUWDPHQWRGH%LRItVLFDH)LVLRORJLD7HUHVLQD 3, %UD]LO
28)/$'HSDUWDPHQWRGH=RRWHFQLD/DYUDV 0* %UD]LOPDUYLR#G]RXÀDEU
8)9'HSDUWDPHQWRGH=RRWHFQLD9LoRVD 0* %UD]LO

Introduction
Studies of body composition of livestock involve direct methods (proximate analyzes, carcass
dissection) and indirect methods (densitometry, dilution, bioelectrical impedance and conductance,
x-ray absorptiometry, computed tomography, magnetic resonance, etc.). The bioelectrical impedance
DQDO\VLV %,$ KDVVHOGRPEHHQXVHGLQ¿VK7KHXVHRI%,$WRHVWLPDWHERG\FRPSRVLWLRQRI¿VK
LVMXVWL¿HGEHFDXVHWKHWHFKQLTXHLVUDSLGUHOLDEOHFKHDS 'XQFDQ QRQOHWKDO %RXUGDJHV
 WKHH[HFXWLRQLVUHODWLYHO\HDV\DQGVLPSOHDQGLWDOORZV¿HOGVWXGLHV (OOLV 7KLVVWXG\
ZDVFRQGXFWHGZLWKWKHREMHFWLYHWRDVVHVVWKHXVHRI%,$WRHVWLPDWHERG\FRPSRVLWLRQDQG¿OOHW
\LHOGVRI¿VKWDPEDWLQJD Colossoma macropomum × Piaractus brachypomus).

Material and methods

7ZRKXQGUHGDQGWHQMXYHQLOHWDPEDWLQJD¿VKHVZLWKGD\VRIDJHLQLWLDOZHLJKWRI“
JDQGWRWDOOHQJWKRI“FPZHUHXVHGLQWKHH[SHULPHQWWKDWHQGHGZKHQ¿VKHVUHDFKHG
GD\V“JDQG“FPWRWDOOHQJWKVUHVSHFWLYHO\)LIWHHQ¿VKHVUDQGRPO\
chosen, were anesthetized and subjected to fourteen biweekly assessments (BIA and biometric).
Electrodes and signal detectors (hypodermic needles Delta 20-5) injected one inch deep in well-
GH¿QHGSRLQWVRQWKH¿VKHVDOORZHGWKHPHDVXUHPHQWVRI%,$YDULDEOHVHOHFWULFDOUHVLVWDQFH 5 DQG
electric reactance (Xc), in series. Fish biometry data determined were: weight (W), standard length
6/ DQGYROXPH 9 $IWHUHXWKDQDVLD¿VKHV¶ZKROHFDUFDVVZLWKKHDGVNLQDQG¿OOHW :& DQG
¿OOHWZLWKVNLQ )6 ZHUHZHLJKHG7KH:&VDPSOHVZHUHJURXQGDQGKRPRJHQL]HGWRDQDO\]HIDW
and protein contents. Data of biometry and BIA or just BIA were regressed against the proximate
analysis to develop the models. The GLM procedures (SAS Inst. Inc., Cary, NC, USA) were used and
Mallow’s Cp statistical was calculated to test the adequacy of the number of variables in the models.
Data from proximate analysis of fat and protein expressed as percentage of dry matter or grams of
ZKROH¿VKZHUHFRUUHODWHGZLWKWKHSUHGLFWHG UHFRYHULQJFDOFXODWLRQV XVLQJWKHEHVWPRGHOVZLWK
variables from biometry and BIA or just from BIA.

Results and discussion

The BIA variables (R and Xc) and its parameters (Z, AF and IC) and the biometric variables
increased linearly with age (P<0.001). Protein decreased (P<0.05) and fat increased (P<0.05)
linearly according to age. Fillet with skin yield exhibited a positive linear effect (P<0.05) with age.

The models for prediction of carcass protein and fat on dry matter basis or in grams and skinless
¿OOHW\LHOGDFFRUGLQJWRWKHGDWDRI%,$H[KLELWHGGHWHUPLQDWLRQFRHI¿FLHQWV 52 RIIRU¿OOHW
with skin yield and 0.943 for fat on dry matter basis.

The best models for carcass protein and fat prediction, in grams or dry-matter basis, and skinless
¿OOHW\LHOGDUHSUHVHQWHGLQ7DEOH

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 245
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_80, © Wageningen Academic Publishers 2013
7DEOH(TXDWLRQV EHVWPRGHOV WRHVWLPDWHIDWSURWHLQDQG¿OOHWZLWKVNLQLQWDPEDWLQJD

Component N Data of BIA and/or biometry P-value F-value R2

Fat on dry matter basis, %1 14 Id, R, CITL, W and SL <0.0001 71.05 0.978
Protein on dry matter basis, %2 14 Id, R, CITL, W and SL <0.0504  
Fat, g 3 14 Id, PA, W and SL <0.0001 740.17 0.997
Protein, g 4 14 Id, R, CIEEI, W and SL <0.0001 73.19 0.979
Fillet with skin yield, g5 14 Id, PA, W and SL <0.0415 3.91 

Id = age in days post-hatch; R = electrical resistance; W = weight; SL = standard length; PA = phase angle; CITL =
composition index using total length and reactance; CIEEI = composition index using the separation between electrodes
detectors and reactance; n = number of assessments during the experiment.
1\ ±,G5&, ±:6/
TL
2\ ,G±5±&, + 0.0318951W – 1.3784901SL.
TL
3\ ±,G±3$:±6/
4\ ±,G5±,&
EEI:6/
5\ ,G3$±:±6/

The correlation among the proximate analysis values and the analogs predicted using the best
PRGHOVZLWKELRPHWULFDQG%,$YDULDEOHVVWXGLHGDOVRH[KLELWHGKLJKFRHI¿FLHQWVIRUIDWRQ
dry matter basis, 0.839 for protein on dry matter basis, 0.998 for fat in grams, and 0.989 for protein
LQJUDPVRIZKROH¿VK

,GHQWLFDODQDO\VLVIRU¿OOHWZLWKVNLQ\LHOGUHVXOWHGLQORZHUFRUUHODWLRQFRHI¿FLHQW  

In conclusion, for the hybrid tambatinga, bioelectrical impedance analysis allows the estimation of
¿OOHWZLWKVNLQ\LHOGDQGERG\FRPSRVLWLRQ IDWDQGSURWHLQRQGU\PDWWHUEDVLVRULQWRWDOPDVVRI
¿VK DQGWKHEHVWPRGHOVLQFOXGHELRPHWULFDQG%,$YDULDEOHVRUSDUDPHWHUV

References
Bourdages, C., 2011. Use of bioelectrical impedance analysis (BIA) to predict water and energy content of juvenile
Rainbow Trout (Oncorhynchus mykiss). Master of Science dissertation, Faculty of Science of the University of
Ontario Institute of Technology, Oshawa, Ontario.
Duncan, M.B., 2008. The Use of BIA for Estimating the Body Composition of Various Fish Species. Master of Science
dissertation, Faculty of Virginia Polytechnic Institute and State University, Blacksburg, Virginia.
(OOLV.-6HOHFWHGERG\FRPSRVLWLRQPHWKRGVFDQEHXVHGLQ¿HOGVWXGLHV-1XWU66

246 Energy and protein metabolism and nutrition in sustainable animal production
(YDOXDWLRQRIWKHLQIUDUHGVSHFWURVFRS\PHWKRGIRUWKHTXDQWL¿FDWLRQRI
NANOLIPE marker in feces of dairy cattle
E.O.S. Saliba1, N.C. Gonçalves1, G.S.S.C. Barbosa2, A.L.C.C. Borges1, N.M. Rodriguez1, G.R.
Moreira1 and F.A. Silva1
1Departamento de Zootecnia, Universidade Federal de Minas Gerais, Brazil; [email protected]
2Campus Florestal, Universidade Federal de Viçosa, Brazil

Introduction
LIPE® is an external marker obtained from lignin isolated from Eucaliptus grandis and enriched
with phenolic groups that is used to estimate dry matter intake and digestibility (Saliba et al., 2003).
Recently, methodological improvements in obtaining LIPE® led to the production of a newer and
better version of this marker. Its particles have now reached the nano scale and for this reason it
was called NANOLIPE (Saliba et al., 2012). The Fourier Transform Infrared Spectroscopy (FT-IR)
LVXVHGIRUWKHTXDQWL¿FDWLRQRI1$12/,3(LQIHFHV+RZHYHURQHRIWKHNH\SRLQWVWRHQVXUHWKH
quality of the results is to assess the linearity of the calibration curves used in the determinations
(Thompson, 2005). Therefore, the aim of this study was to statistically evaluate the linearity of the
FT-IR method in quantifying NANOLIPE in feces of dairy cattle.

Material and methods

A calibration curve was established in fecal matrix. For this purpose, known amounts of the
NANOLIPE marker were added in feces of dairy cattle, which have been previously dried at 55
°C for 72 h and ground by a Willey mill type (1 mm particle size). Then, for the calibration and
TXDQWL¿FDWLRQRIWKHPDUNHUD9DULDQ® 800 FT-IR Scimitar Series spectrometer was used.

The working range of the method was chosen according to the known average concentration of the
PDUNHULQURXWLQHVDPSOHV7KXVIRXUOHYHOVRIFRQFHQWUDWLRQLQWKLVUDQJHZHUHGH¿QHG
0.15, 0.20 mg of NANOLIPE per g of fecal dry matter. The calibration samples were independently
prepared by three different analysts under reproducible conditions. The determination was performed
DWUDQGRPUHDGWRPLQLPL]HSRVVLEOHHUURUVDWVSHFL¿FOHYHOV

7KHZDYHQXPEHURIFP-1 was chosen because it resulted in the greatest linearity between

the response (infrared energy absorption) and the concentration of NANOLIPE. This wave number
ZDVWKHVDPHXVHGIRUTXDQWL¿FDWLRQ/,3(® in the initial studies of Saliba et al. (2003).

Statistical tests were performed to determine three assumptions related to the residuum i.e. it has to
follow normal distribution, be independent and homoscedastic. Those tests were: Ryan-Joiner test,
Durbin-Watson test and Levene’s test, respectively, according to Thompson (2005). Besides, the
analysis of variance of the regression equation created allows the evaluation of the FT-IR for the
TXDQWL¿FDWLRQRI1$12/,3(PDUNHULQIHFHVRIGDLU\FDWWOH

Results and discussion

7KHUHVXOWVREWDLQHGE\WKH)7,5VSHFWURVFRS\LQWHUPVRIWKHSHDNKHLJKWDWFP-1 according
to the concentration of NANOLIPE in the feces are shown in Table 1.

The results obtained by the statistical tests previously referred to (Ryan-Joiner, Levene and Durbin-
Watson) are shown in Table 2.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 247
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_81, © Wageningen Academic Publishers 2013
7DEOH$YHUDJHSHDNKHLJKWDWFP-1 in different fecal concentrations of NANOLIPE
measured by FT-IR spectroscopy.

Fecal concentration of NANOLIPE (mg/g)

0.050 0.100 0.150 0.200

Peak height 0.181  0.271 0.313

7DEOH6WDWLVWLFDOHYDOXDWLRQRI)7,5VSHFWURVFRS\IRUWKHTXDQWL¿FDWLRQRI1$12/,3(LQIHFHV
RIGDLU\FDWWOH WRPJJ 

Parameter Analytical curve Results

Normality (R) 0.9750 The residuum follows the normal distribution

Critical value 0.9248
Homoscedasticity (tL) -0.70 The residuum is homoscedastic
Critical value
Independency (d)  The residuum is independent
Į >0.10

5 5\DQ-RLQHUFRUUHODWLRQFRHI¿FLHQWW/ /HYHQH¶V7VWDWLVWLFG 'XUELQ:DWVRQVWDWLVWLFĮ VLJQL¿FDQFH

The analysis of variance showed that the ratio between the root mean square (RMS) of the regression
DQGWKHUHVLGXHZDVIDUH[FHHGLQJWKHFULWLFDOYDOXHIRU)RI FRQ¿GHQFH 
LQGLFDWLQJWKDWUHJUHVVLRQZDVVWDWLVWLFDOO\VLJQL¿FDQW7KHUDWLREHWZHHQWKH506RIODFNRI¿WDQG
SXUHHUURUZDVZKLFKLQGLFDWHVWKHJRRGQHVVRI¿WFRPSDUHGWRWKHFULWLFDOYDOXHRI)2,4 which
LV FRQ¿GHQFH 7KHFRUUHODWLRQFRHI¿FLHQWRIVXSSRUWHGE\VWDWLVWLFDOHYDOXDWLRQ
is excellent, proving the method to be useful for quantifying the NANOLIPE in feces of dairy cattle.

References
Saliba, E. O. S.; Barbosa, G. S. S. C.; Rodriguez, N. M.; Couto Filho, C. C. C.; Moreira, G.R.; Gonçalves, N. C.;
Silva, F. A., 2012. Utilization of nanotechnology to the development of a marker of fecal output in dairy cattle.
In: Doreau, M. (editor), Proceedings of the British Society of Animal Science and the Association of Veterinary
Teaching and Research Work. Advances in Animal Biosciences, Cambridge, Volume 3: 293.
6DOLED(265RGULJXH]103LOR9HORVR'3XUL¿HGOLJQLQH[WUDFWHGIURP(XFDO\SWXVJUDQGLV 3(/, 
used as an external marker in digestibility trails in various animal species. In: Paim, N., Barcelos, J.O.J, Neto,
J.B., Ferreiro, L., Franke, Berbardi, M.L., Dall’Agnol, M. (editors), Proceedings of the 9th World Conference on
Animal Production. UFRGS, Porto Alegre: 221-222.
Thompson, M., 2005. Is my calibration linear? In: Thompson, M. (editor). Analytical Methods Committee Technical
Brief. The Royal Society of Chemistry, Lodon, AMCTB n. 3: 1-2.

248 Energy and protein metabolism and nutrition in sustainable animal production
A continuous approach to assess methane production rate in ruminants
using respiration chambers
L.F.L. Cavalcanti, M.A. Fonseca, J.G.L. Regadas Filho and L.O. Tedeschi
18QLYHUVLGDGH)HGHUDOGH0LQDV*HUDLV$Y$QW{QLR&DUORV%HOR+RUL]RQWH
Brazil; [email protected]
28QLYHUVLGDGH)HGHUDOGH9LoRVD$Y3HWHU+HQU\5ROIV9LoRVD%UD]LO
7H[DV$ 08QLYHUVLW\.OHEHUJ&HQWHU&ROOHJH6WDWLRQ7;86$

Introduction
The methane production from ruminants is one of the most discussed topics in animal production
VLQFHLWKDVEHHQLGHQWL¿HGDVDNH\SUREOHPUHJDUGLQJZRUOGFOLPDWHFKDQJH0HWKDQHSURGXFWLRQ
FDQEHTXDQWL¿HGZLWKRSHQFLUFXLWUHVSLUDWLRQFKDPEHUV8VXDOO\WKHFDOFXODWLRQRIWRWDOPHWKDQH
production is the mean concentration measured along the trial multiplied by the total volume of air
exchanged (l/period), resulting in an average value that does not contemplate the production rates
variation of methane due to the pattern of ruminal fermentation (Dauncey et al., 1978; Aguilera and
3ULHWR5RGULJXH]et al., 2007). Other authors (Brown et al., 1984) have provided a different
approach for gas production calculations in which they included a differential term that is more
appropriate for detecting rapid changes in metabolic rate, such as the result of physical activity.
However, for continuous modeling processes, such as methane production, that approach might not
be suitable. This work evaluates the methane production along the measurement time by modeling
the cumulative gas curve to generate gas production rates that can be used to compare factors, such
period within a day.

Material and methods

A dataset composed by 10 respirometry trials using Angus steers, with 48 h sampling duration, were
JURXSHGDQGWKHPHWKDQHLQVWDQWFRQFHQWUDWLRQRYHUWLPHZDVPXOWLSOLHGE\WKHLQVWDQWDLUÀX[DQG
summed to obtain the cumulative gas curves. Animals were fed at 2% of their body weight (DM
EDVLV DUDWLRQFRQWDLQLQJ0FDONJRIPHWDEROL]DEOHHQHUJ\DQGFUXGHSURWHLQWZLFH
daily (7:30 h and 17:30 h). The feeding times were used as a reference to subset each trial resulting
in four periods by animal (Figure 1). The logarithm of the cumulative production values in each of

)LJXUH([DPSOHRILQVWDQWDQHRXVDQGDFFXPXODWHGSURGXFWLRQRIPHWKDQHLQDQDVVD\RI
KRXUV)WR)UHSUHVHQWWKHIRXUIHHGLQJWLPHV)DQG)DWKDQG)DQG)DWK
bI to bIV represent the four accumulated curve sections on which linear regression was performed
and the slopes were compared.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 249
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_82, © Wageningen Academic Publishers 2013
WKHVHSHULRGVZDVXVHGWR¿WDOLQHDUUHJUHVVLRQ7KHVORSHVREWDLQHGZHUHVXPPDUL]HGDQGDQDO\]HG
as a completely randomized design with repeated measures using a mixed model methodology. This
step allowed us to model the matrix of covariance assuming the spatial power structure, which was
chosen due to the asymmetric time of feeding and the best Akaike´s Information Criterion. The
effect of period was decomposed into orthogonal polynomials of linear, quadratic, and cubic degrees.

Results and discussion

The cumulative curve obtained for each animal presented a 4-pool behavior in which the moment
of acceleration pulses were highly correlated with the feeding times. When we split by period, the
data assumed a curvilinear behavior with a higher rate at the very beginning of each curve, as seen in
Figure 1. Whereas, when the data was linearized through a logarithmic transformation it had a better
OLQHDUUHJUHVVLRQJRRGQHVVRI¿WDVVKRZQE\WKHJUHDWHUYDOXHRIWKHFRHI¿FLHQWRIGHWHUPLQDWLRQ
(average of 0.9913).

The cubic regression model was adopted (P<0.05) to represent the rates of production of each
period during the trial time resulting in the following equation: CH4r ±WW2
±W3, where CH4r is the rate of methane production (l/min) in the time t (hour). The cubic
behavior can be explained by the fact that in those trials, the morning meals were 1.2 times bigger
than the afternoon´s, which increased the intake of fermentable organic matter in the morning period.
7KHUHIRUHWKH¿UVWDQGWKLUGIHHGLQJWLPHVUHVXOWHGLQDJUHDWHUVORSHIRUPHWKDQHSURGXFWLRQ7KH
methodology proposed in this study is capable of evaluating rates of methane production rather
than the total volume as commonly used by other techniques when using respirometry trials. Our
DSSURDFKFDQEHIXUWKHUPRGL¿HGIRUFRPSDULVRQVRIRWKHUIDFWRUVWKDWFDQDIIHFWPHWKDQHSURGXFWLRQ
rates such as different feeds, diets, and breeds. Furthermore, nonlinear functions other than linear
regressions can be used to access the production rate and their parameters might be correlated to
biological phenomena.

References
$JXLOHUD-)DQG&3ULHWR'HVFULSWLRQDQGIXQFWLRQRIDQRSHQFLUFXLWUHVSLUDWLRQSODQWIRUSLJVDQGVPDOO
UXPLQDQWVDQGWKHWHFKQLTXHVXVHGWRPHDVXUHHQHUJ\PHWDEROLVP$UFK$QLP1XWU
Brown, D., T.J. Cole, M.J. Dauncey, R.W. Marrs and P.R. Murgatroyd, 1984. Analysis of gaseous exchange in open-
circuit indirect calorimetry. Med. Biol. Eng. Comput. 22: 333-338.
Dauncey, M.J., P.R. Murgatroyd and T.J. Cole, 1978. A human calorimeter for the direct and indirect measurement of
KHQHUJ\H[SHQGLWXUH%ULW-1XWU
Rodriguez, N.M., W.E. Campos, M.L. Lachica, I. Borges, L.C. Gonçalves, A.L.C.C. Borges and E.O.S. Saliba, 2007.
A calorimetry system for metabolism trials. Arq. Bras. Med. Vet. Zoo. 59: 495-500.

250 Energy and protein metabolism and nutrition in sustainable animal production
Part 4. Regulation
Mechanisms of regulation of intramuscular fat deposition in porcine
muscle by dietary lysine content
M. Katsumata, T. Kyoya, H. Kobayashi, A. Ishida, A. Ashihara and K. Nakashima
1$52,QVWLWXWHRI/LYHVWRFNDQG*UDVVODQG6FLHQFH7VXNXED-DSDQ[email protected]

Abstract
Feeding a low-lysine diet to pigs promotes the deposition of intramuscular fat (IMF). However, the
mechanisms underlying fat accumulation and marbling in porcine muscle remain unclear. As such,
the aim of this study was to elucidate the manner in which dietary lysine regulates IMF deposition in
¿QLVKLQJSLJV/RZGLHWDU\O\VLQHOHYHOVLQFUHDVHGWKHYROXPHEXWQRWWKHQXPEHURILQWUDPXVFXODU
adipocytes in the longissimus dorsi muscle. This observation indicates that lipogenesis in already
existing adipocytes plays a pivotal role in IMF accumulation, as opposed to the differentiation of
preadipocytes into mature adipocytes. We also found that a low-threonine diet did not affect IMF
content in either longissimus dorsi or rhomboideus muscle. Thus, dietary shortages among the various
LQGLVSHQVDEOHDPLQRDFLGVPD\GLIIHUHQWLDOO\LQÀXHQFH,0)GHSRVLWLRQ)XUWKHUPRUHLQVXOLQVLJQDOLQJ
and triacylglyceride levels were enhanced in cultured adipocytes by cellular exposure to low vs.
high concentrations of lysine, suggesting that insulin might similarly contribute to the nutritional
modulation of IMF deposition in vivo.

Introduction
The production of pork with marbling is particularly important in East Asian countries (e.g. Japan
and Korea) due to the eating habits of these regions. For instance, the consumption of fresh pork is
two-fold higher than the consumption of processed pork in Japan. For this reason, we recently turned
our attention to the nutritional regulation of intramuscular fat (IMF) deposition in porcine muscle.

There are two major approaches for the production of pork with an elevated IMF content: (1) breeding
RIKLJK,0)SLJVDQG  QXWULWLRQDOUHJXODWLRQRI,0)GHSRVLWLRQ7KH¿UVWDSSURDFK\LHOGHGWZR
well-known porcine lines that accumulate high amounts of IMF in the longissimus dorsi muscle, the
synthetic ‘TOKYO-X’ line and the ‘Shimofuri-Red’ Duroc line, both developed in Japan (Hyodo
7DNDVDNLet al., 2005; Suzuki et al., 2005). High IMF loin chops derived from these lines
FRQVLVWHQWO\UHFHLYHJRRGUHYLHZVLQWKHPDUNHWIHWFKLQJSULFHVWKDWDUHDSSUR[LPDWHO\WR
more than those of ordinary pork with lower amounts of IMF. Nonetheless, nutritional regulation
can be advantageous compared with specialized breeding because one does not need to rear pigs
KDYLQJDVSHFL¿FJHQHWLFEDFNJURXQG

A number of studies on the promotion of IMF deposition via nutritional regulation were conducted
over the past two decades. A typical tactic was to reduce the amount of protein in the diets (Castell
et al., 1994; Goerl et al., 1995). Proceedings from a recent ISEP 2007 indicated that elevated activity
of stearoyl-CoA-desaturase, a key lipogenic enzyme, led to the enhancement of porcine IMF content
in response to reduced dietary protein levels (Doran et al., 2007). Another tactic has been to add a
single amino acid to the diet. For example, the addition of leucine or arginine increased the IMF
content in porcine muscle (Hyun et al.7DQet al., 2009).

In contrast, our present line of attack involves the reduction of lysine levels in the porcine diet. We
previously reported that a low dietary lysine levels up-regulated GLUT4 mRNA expression and
affected glucose metabolism and substrate oxidation predominantly in the muscle (Katsumata et al.,
2001, 2003). Given that the oxidative capacity of muscle affects IMF content (Goto et al., 1994),
we went on to show that the shortage of a single amino acid, lysine, augmented IMF deposition in
the pig (Katsumata et al., 2005, 2012). However, the mechanisms underlying this process are still

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 253
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_83, © Wageningen Academic Publishers 2013
unknown. Therefore, the aim of this paper was to investigate the manner in which dietary lysine
UHJXODWHV,0)GHSRVLWLRQLQ¿QLVKLQJSLJV

Effects of low dietary levels of lysine on the volume and number of

porcine intramuscular adipocytes
IMF content is mainly dependent on the volume and/or the number of intramuscular adipocytes. The
volume and number of intramuscular adipocytes and the IMF content continuously increased in the
trapezius and semitendinous muscles of pigs until the pigs reach 24 weeks of age (Lee and Kaufman,
1974). Thus, this study investigated the effects of low dietary levels of lysine on the volume and
number of intramuscular adipocytes in porcine longissimus dorsi muscle. The longissimus dorsi
muscle formed the focus of this work, as this muscle is the most important source of pork in Japan.

Duroc × (Large white × Landrace) crossbred pigs aged 10 weeks were employed in this work. The
pigs were assigned to one of two diets: a control diet (n=12) or a low-lysine diet (LL, n=12). The
//GLHWZDVGHVLJQHGVXFKWKDWWKHFRQFHQWUDWLRQRIO\VLQHZDVDSSUR[LPDWHO\RIWKHGLHWDU\
requirement given by the NRC (1998). The concentrations of the other indispensable amino acids
for the pig met the NRC requirements. The control diet was designed to meet the NRC requirements
for all the indispensable amino acids. Half of the pigs assigned to each group were fed the control
or the LL diet for 4 weeks, while the remaining six pigs were fed the control or the LL diet for 8
weeks. Longissimus dorsi muscle specimens were obtained at 4 and 8 weeks after the beginning of
WKHH[SHULPHQW,QDGGLWLRQPXVFOHVSHFLPHQVZHUHREWDLQHGIURPSLJVDWWKHEHJLQQLQJRIWKH
experiment.

The IMF content of the longissimus dorsi muscle of the LL group was already higher than that of the
control group at 4 weeks (P<0.05; 3.8% for the LL group vs. 2.0% for the LL group). At 8 weeks,
the magnitude of the difference in the IMF content was further enhanced (P<IRUWKH//
group vs. 3.3% for the control group). Serial transverse cross sections through the longissimus dorsi
PXVFOH ȝPWKLFN ZHUHPDGHE\XVLQJDFU\RVWDWDWƒ&7KHVHFWLRQVZHUHVWDLQHGZLWK2LO
Red-O to detect triacylglyceride. The volume of the intramuscular adipocytes was calculated by using
a prediction equation, as described elsewhere (Chikuni et al., 1985). As expected from the results of
IMF content, the adipocyte volumes in the LL group were higher at 4 and 8 weeks (Figure1, P<0.05),
but no differences were observed in adipocytes numbers (Figure 1). These results are inconsistent with
those of Lee and Kaufman (1974). Although the exact reasons for this inconsistency are unknown,
it can potentially be explained by differences in the breeds of the pigs and/or the types of muscle

A B
(x103ȝm3) Control LL (cells/cm2) Control LL
2,500 3,000
*
2,000 2,500
2,000
1,500
* 1,500
1000
1000
500 500
0 0
10 wk 14 wk 18 wk 10 wk 14 wk 18 wk
Figure 1. Effect of low dietary levesl of lysine on the volume and number of intramuscular adipocytes
LQWKHSRUFLQHORQJLVVLPXVGRUVLPXVFOH $ $GLSRFXWHYROXPHDQG % DGLSRF\WHQXPEHUDUHVKRZQ
(DFKEDUUHSUHVHQWVPHDQ“6( Q   6LJQL¿FDQWO\GLIIHUHQWIURPWKHFRQWUROJURXS P< 

254 Energy and protein metabolism and nutrition in sustainable animal production
employed in the two studies. As far as the longissimus dorsi muscle is concerned, we can infer that
the accumulation of IMF in the LL group resulted from the hypertrophy of matured adipocytes. This
hypothesis is supported by our previous data, which was presented at the ISEP 2010 and showed that
33$5ȖP51$OHYHOVZHUHKLJKHULQWKHlongissimus dorsi and rhomboideus muscles of pigs fed with
DORZO\VLQHGLHWZKHUHDV&(%3ĮP51$OHYHOVZHUHQRWDIIHFWHG .DWVXPDWDet al., 2010). Since
&(%3ĮLVDPDVWHUUHJXODWRURIDGLSRF\WHGLIIHUHQWLDWLRQWKHUHVXOWLPSO\WKDWORZOHYHOVRIGLHWDU\
lysine do not provoke the differentiation of porcine intramuscular preadipocytes into adipocytes.

Effect of low dietary levels of threonine vs. lysine on porcine IMF

deposition
The question has been raised as to whether dietary reduction of indispensable amino acids other
than lysine would likewise promote IMF deposition in the pig, taking into account the low levels of
dietary protein enhanced the fat content in porcine muscle (Castell et al., 1994; Goerl et al., 1995).
Therefore, we next focused on threonine, another major limiting amino acid in the pig diet, because
feeding a low-lysine diet and a low-threonine diet both enhanced the abundance of GLUT4 mRNA
in porcine skeletal muscle (Katsumata et al., 2003, 2004). Hence, porcine muscle might respond
similarly to a shortage of either dietary lysine or threonine.

Duroc × (Large white × Landrace) crossbred pigs (age 10 weeks) were assigned to one of four groups
(lysine control (LC), low lysine (LL), threonine control (TC), and low threonine (LT)). The LL and
LT diets were designed to yield concentrations of lysine and threonine that were approximately 70%
of the required concentrations set by the Japanese Feeding Standard for Swine (NARO, 2005). All
pigs were fed with the experimental diets for 8 weeks. At the end of the experiment, specimens of
serum, longissimus dorsi muscle, and rhomboideus muscle were collected.

7KHPDLQ¿QGLQJVRIWKLVH[SHULPHQWZHUHWKDWDOWKRXJKWKHVKRUWDJHRIO\VLQHLQWKHGLHWHQKDQFHG
IMF content in the longissimus dorsi muscle and the rhomboideus muscle, the shortage of threonine
GLGQRW )LJXUH 7KHUHIRUHRXUUHVXOWVIDLOHGWRFRQ¿UPWKHK\SRWKHVLVWKDWDVKRUWDJHRIWKUHRQLQH
would also enhance IMF content in porcine muscle, and instead indicate that amino acids have
individual, albeit not necessary predictable, effects on the deposition of IMF in the pig.

/\VLQHKDVTXDQWL¿DEOHDQWLREHVLW\DFWLRQV)RUH[DPSOHUHFHQWZRUNVKRZHGWKDWGLHWDU\O\VLQH
reduced the body fat mass in rats by promoting fatty acid oxidation and by inhibiting fatty acid

5 x
a
4.5 xy
yz
4
3.5 z
IMF content (%)

LC
3 b b
2.5 b LL
2 TC
1.5
LT
1
0,5
0
l. dorsi rhomboideus

Figure 2. Effect of low dietary levels of lysine and threonine on IMF content in the porcine longissimus
dorsi and rhomboideusPXVFOH(DFKEDUUHSUHVHQWVWKHOHDVWVTXDUHPHDQ“WKHSRROHG6( Q  
DDQGE P< [\DQG] P< 

Energy and protein metabolism and nutrition in sustainable animal production 255
synthesis (Kobayashi et al., 2011). Thus, it is conceivable that the shortage of lysine in our study
resulted in the reduced oxidation of fatty acids and accelerated deposition of fat in the muscle.
Therefore, the differential effects of lysine vs. threonine shortage on IMF accumulation might be
explained, at least in part, by contradictory effects of each amino acid on fatty acid metabolism.

7KHVKRUWDJHRIGLHWDU\O\VLQHDQGWKUHRQLQHERWKWHQGHGWRLQÀXHQFHVHUXPLQVXOLQFRQFHQWUDWLRQV
(3 0.079, Table 1). The average insulin concentration of the LC and the TC group was 1.52 ng/ml
while that of the LL and the LT group was 0.85 ng/ml. In contrast, dietary amino acid levels did not
affect serum glucose concentrations (Table 1). These results suggest that the response of peripheral
tissues to insulin might be larger in the LL and LT groups than in the control group. Lipogenesis
occurs downstream of insulin signaling. Dietary protein deprivation increases the strength of insulin
signaling in the liver and muscle of rats (Toyoshima et al., 2004, 2010). Hence, the lysine shortage-
promoted deposition of IMF in porcine muscle might be attributable to enhanced insulin signaling.
In support of this hypothesis, insulin receptor substrate-1 (IRS-1) mRNA levels were higher for the
LL group than for the LC group (data not shown). We are currently investigating the impact of low
dietary lysine on the phosphorylation of IRS-1 in porcine muscle.

Some details of the threonine vs. lysine experiment are described elsewhere (Kobayahsi et al., 2012).

Table 1. Effect of low dietary lysine and threonine levels on growth performance and serum insulin
DQGJOXFRVHFRQFHQWUDWLRQV'DWDDUHH[SUHVVHGDVOHDVWVTXDUHPHDQVDQGSRROHG6( Q  

LC LL TC LT Pooled SE AA1

Feed intake (g/d) 2,831 2,945   101

Live body weight gain (g/d) 1,018 953 988 823 31 **
)HHGHI¿FLHQF\ 0.37 0.32  0.31 0.01 **
Insulin (ng/ml) 1.43   1.07 0.34 3 0.079
Glucose (mg/ml) 114 102 114 120 11

1 Effects of dietary amino acid levels. **=P<0.01.

Effect of lysine concentration on triacylglyceride accumulation in 3T3-

L1 preadipocytes
Feeding a low-lysine diet might result in a reduced supply of lysine to peripheral tissues. In this
case, the enhanced deposition of IMF in the pig could perhaps be ascribed to the decreased levels
of lysine reaching intramuscular adipocytes. However, living, intact animals do not readily lend
themselves to the evaluation of this hypothesis. For instance, low dietary levels of lysine also reduce
the concentrations of IGF-1, while concomitantly increasing the concentrations of glucocorticoids
(Katsumata et al., 2002; Ishida et al., 2011). These altered hormone concentrations could similarly
affect fat accretion in porcine muscle, and thus, we were unable to test whether a decline in the lysine
supply directly promoted IMF deposition in vivo. To overcome this issue, we conducted in vitro
experiments to explore the link between lysine concentration and triacylglyceride accumulation in
cultured 3T3-L1 preadipocytes. Although the 3T3-L1 preadipocyte is of murine rather than porcine
origin, we employed these cells in our work because they represent the most commonly used cell
line for studies on adipogenesis.

The concentration of lysine was set at 921 nmol/ml in standard culture medium made with DMEM and
)%6 FRQWUROPHGLXP /RZO\VLQHPHGLDZHUHSUHSDUHGZLWKO\VLQHFRQFHQWUDWLRQVVHWDWQPRO
ml (×0.5 of the concentration in control medium), 230 nmol/ml (×0.25), and 115 nmol/ml (×0.125).
7KHQPROPOFRQFHQWUDWLRQZDVVLPLODUWRWKHFRQFHQWUDWLRQRIO\VLQHLQWKHSODVPDRISLJVIHG

256 Energy and protein metabolism and nutrition in sustainable animal production
a diet meeting the requirement of lysine, whereas the 115 nmol/ml concentration was similar to the
concentration of lysine in the plasma of pigs fed a low-lysine diet. Eight days after the induction of
SUHDGLSRF\WHGLIIHUHQWLDWLRQE\H[SRVXUHWRDGH¿QHGDGLSRJHQLFFRFNWDLOWULDF\OJO\FHULGHOHYHOV
were measured in the mature adipocytes. As shown in Figure 3, triacylglyceride levels were higher
for cells cultured in ×0.125 medium than for cells cultured in control medium. These observations
demonstrate that the differentiation of 3T3-L1 preadipocytes in low-lysine media promoted fat
deposition in the mature adipocytes.

As mentioned above, insulin signaling might play an important role in the regulation of IMF
deposition in porcine muscle. We next measured insulin receptor, IRS-1, and IRS-2 mRNA levels
in mature adipocytes (Figure 4). Interestingly, mRNAs levels were higher for adipocytes cultured
in ×0.125 medium than for adipocytes cultured in control medium. Furthermore, the degree of
IRS-1 tyrosine phosphorylation was elevated for adipocytes cultured in ×0.125 medium (Figure
5), indicating that insulin signaling is involved in the increased deposition of triacylglycerides in
3T3-L1 cell-derived adipocytes cultured in low-lysine media.

1.8
x
1.6
TG content (μg/×104 cells)

1.4 xy
y
1.2 y Control
1
x 0.5
0.8
ab a x 0.25
0.6
b bc
0.4 x 0.125

0.2
0
0.25 0.05
Concentrations of insulin in the media (μg/ml)
)LJXUH(IIHFWRIO\VLQHFRQFHQWUDWLRQRQWULDF\OJO\FHULGHFRQFHQWLQ7/DGLSRF\WHV(DFKEDU
UHSUHVHQWVWKHPHDQ“6( Q  DEDQGF P< [DQG\ P< 

2.5

2 *
* *
Relative expression

1.5
Control
1 x0.125

0.5

0
IR IRS-1 IRS-2
)LJXUH(IIHFWRIO\VLQHFRQFHQWUDWLRQRQLQVXOLQUHFHSWRU ,5 ,56DQG,56P51$OHYHOVLQ
7/DGLSRF\WHV(DFKEDUVUHSUHVHQWVWKHPHDQ“6( Q  0HDQVIURPWKHFRQWUROFHOOVDUH
H[SUHVVHGDV6U51$OHYHOVZHUHXVHGDVLQWHUQDOVWDQGDUGV 6LJQL¿FDQWO\GLIIHUHQWIURPWKH
FRQWUROJURXS P< 

Energy and protein metabolism and nutrition in sustainable animal production 257
 4

IRS-1/IRS-1 (arbitrary units)

Tyrosine phosphorylation of
*
3.5
3
2.5
Control
2
x0.125
1.5
1
0.5
0
)LJXUH(IIHFWRIO\VLQHFRQFHQWUDWLRQRQW\URVLQHSKRVSKRU\ODWLRQRI,56LQ7/DGLSRF\WHV
(DFKEDUUHSUHVHQWVWKHPHDQ“6( Q  0HDQVIURPWKHFRQWUROFHOOVDUHH[SUHVVHGDV 
6LJQL¿FDQWO\GLIIHUHQWIURPWKHFRQWUROJURXS P< 

Conclusions
Low level of dietary lysine affected the volume but not the number of intramuscular adipocytes in the
porcine longissimus dorsi muscle, suggesting that the promotion of lipogenesis in already existing
adipocytes contributed to the dietary lysine shortage-enhanced deposition of IMF. Conversely, low
dietary threonine level had no impact on IMF accumulation; thus the assorted indispensable amino
acids exert differential effects on the buildup of IMF in the pig. The differential effects of lysine
vs. threonine shortage might be due to disparities in the ability of each amino acid to modulate
fatty acid metabolism. Moreover, the current results suggest that insulin signaling is involved in
the regulation of IMF levels by dietary lysine. Further elucidation of the ability of lysine and other
LQGLVSHQVDEOHDPLQRDFLGVWRLQÀXHQFHLQVXOLQVLJQDOLQJLVH[WUHPHO\LPSRUWDQWDQGZLOOIRUPWKH
basis of our future work, given that insulin signaling has an abundance of critical roles in the control
of global metabolism.

Acknowledgements
A part of this work was supported by the Program for Promotion of Basic and Applied Research for
Innovation in Bio-Oriented Industry.

References
Castell, A.G., R.L. Cliplef, L.M. Poste-Flynn and G. Butler, 1994. Peformance, carcass and pork characteristics of
castrates and gilts self-fed diets differing in protein and lysine: energy ratio. Can. J. Anim. Sci. 74, 519-528.
Chikuni, K., M. Jimbu, S. Ozawa, T. Koishikawa, M. Yoshitake and N. Yano, 1985. Relation among back fat thickness,
fat cell size and fatty acid composition of swine. Jpn. J. Swine Hus. Res. 22, 181-187.
Doran, O., F.M. Whittington, K.G. Hallet and J.D. Wood, 2007. Effect of a reduced protein diet on expression of
lipogenic enzymes in relation to intramuscular fat formation in the pig. In: Ortigues-Marty, I., N. Miraux and
W. Brand-Williams (editors), Energy and protein metabolism and nutrition. Wageningen Academic Publishers,
:DJHQLQJHQWKH1HWKHUODQGV($$3SXEOLFDWLRQ1R
Goerl, K.F., S.J. Eilert, R.W. Mandingo, H.Y. Chen and P.S. Miller, 1995. Pork characteristics as affected by two
SRSXODWLRQVRIVZLQHDQGVL[FUXGHSURWHLQOHYHOV-$QLP6FL
Goto, T., H. Iwamoto, Y. Ono, S. Nishimura, K. Matsuo, Y. Nakanishi, R. Umetsu and H. Takahara, 1994. Comparative
VWXG\RQWKHUHJLRQDOFRPSRVLWLRQRI¿EHUW\SHVLQ0/RQJLVVLPXV7KRUDFLVZLWKGLIIHUHQWPDUEOLQJVFRUHVIRU
-DSDQHVH%ODFNVWHHUV$QLP6FL7HFKQRO -SQ 
Hyun, Y., M. Ellis, F.K. McKeith and D.H. Baker, 2003. Effect of dietary leucine level on growth perfoemance, and
FDUFDVVDQGPHDWTXDOLW\LQ¿QLVKLQJSLJV&DQ-$QLP6FL

258 Energy and protein metabolism and nutrition in sustainable animal production
Hyun, Y., J.D. Kim, M. Ellis, B.A. Peterson, D.H. Baker and F.K. McKeith, 2007. Effect of dietary leucine and lysine
OHYHOVRQLQWUDPXVFXODUIDWFRQWHQWLQ¿QLVKLQJSLJV&DQ-$QLP6FL
+\RGR,$FDVHVWXG\IRULQFUHDVLQJLQWUDPXVFXODUIDWFRQWHQWRISRUN-$QLP%UHHG
Ishida, A., T. Kyoya, K. Nakashima and M. Katsumata, 2011. Muscle protein metabolism during compensatory growth
ZLWKFKDQJLQJGLHWDU\O\VLQHOHYHOVIURPGH¿FLHQWWRVXI¿FLHQWLQJURZLQJUDWV-1XWU6FL9LWDPLQRO
Katsumata, M., S. Kawakami, Y. Kaji, R. Takada and M.J. Dauncey, 2001. Low lysine diet selectively up-regulates
muscle GLUT4 gene and protein expression during postnatal development. In: Chwalibog, A. and K. Jakobsen
(editors), Energy metabolism in animals. Wageningen Pers, Wageningen, the Netherlands, EAAP publication
No. 103, 237-239.
Katsumata, M., S. Kawakami, Y. Kaji, R. Takada and M.J. Dauncey, 2002. Differential regulation of porcine hepatic
,*),P51$H[SUHVVLRQDQGSODVPD,*),FRQFHQWUDWLRQE\DORZO\VLQHGLHW-1XWU
Katsumata, M., M. Matsumoto and Y. Kaji, 2003. Effects of a low lysine diet on glucose metabolism in skeletal
muscle of growing pigs. In: Souffrant W.B. and C.C. Metges (editors), Progress in research on energy and protein
metabolism. Wageningen Academic Publishers, Wageningen, the Netherlands, EAAP publication No. 109, 187-190.
Katsumata, M., S. Kawakami, Y. Kaji and R. Takada, 2004. Circulating levels of insulin-like growth factor-1 and
associated binding proteins in plasma and mRNA expression in tissues of growing pigs on a low threonine diet.
Anim. Sci. 79, 85-92.
Katsumata, M., S. Kobayashi, M. Matsumoto, E. Tsuneishi and Y. Kaji, 2005. Reduced intake of dietary lysine promotes
accumulation of intramuscular fat in the Longissimus dorsiPXVFOHVRI¿QLVKLQJJLOWV$QLP6FL-
Katsumata, M., A. Ishida, T. Kyoya and K. Nakashima, 2010. Effects of dietary lysine levels on expression of
adipogenesis related genes in muscle of growing pigs. In: Crovetto G.M. (editor), Energy and protein metabolism and
QXWULWLRQ:DJHQLQJHQ$FDGHPLF3XEOLVKHUV:DJHQLQJHQWKH1HWKHUODQGV($$3SXEOLFDWLRQ1R
Katsumata, M., T. Kyoya, A. Ishida, M. Ohtsuka and K. Nakashima, 2012. Dose-dependent response of intramuscular
fat accumulation in longissimus dorsiPXVFOHRI¿QLVKLQJSLJVWRGLHWDU\O\VLQHOHYHOV/LYHVW6FL
Kobayashi, H., Y. Hirabayashi, T. Ueda and H. Murakami, 2011. Amino acid loses your weights? Effects of dietary
O\VLQHRQOLSLGDQGFDUERK\GUDWHPHWDEROLVP$PLQRVDQ.HQN\‫܆‬
Kobayashi, H., A. Ishida, A. Ashihara, K. Nakashima and M. Katsumata, 2012. Effects of dietary low level of threonine
DQGO\VLQHRQWKHDFFXPXODWLRQRILQWUDPXVFXODUIDWLQSRUFLQHPXVFOH%LRVFL%LRWHFKQRO%LRFKHP
Lee, Y.B. and R.G. Kauffmann, 1974. Cellular and enzymatic changes with animal growth in porcine intramuscular
adipose tissue. J. Anim. Sci. 38, 532-537.
National Agriculture and Bio-Oriented Research Orgnaization, 2005. Japanese Feeding Standard for Swine, Japan
Livestock Industry Association, Tokyo.
National Research Council, 1998. Nutrient Requirements of Swine, 10th edn. National Academy Press, Washington, DC.
Suzuki, K., H. Kadowaki, T. Shibata, H. Uchida and A. Nishida, 2005. Selection for daily gain, loin-eye area, back
fat thickness and intramuscular fat based on desired gains over seven generations of Duroc pigs. Livest. Prod.
Sci. 97, 193-202.
Takasaki, S., K. Iizuka, A. Suzuki and Y. Itoh, 2005. Chemical, physical and mastication properties of pork from the
new ‘Tokyo X’ breed. J. Cook. Sci. Jpn. 38, 404-409.
Tan, B., Y. Yin, Z. Liu, X. Li, H. Xu, X. Kong, R. Huang, W. Tang, I. Shinzato, S.B. Smith and G. Wu, 2009. Dietary
/DUJLQLQHVXSSOHPHQWDWLRQLQFUHDVHVPXVFOHJDLQDQGUHGXFHVERG\IDWPDVVLQJURZLQJ¿QLVKLQJSLJV$PLQR
$FLGV
Toyoshima, Y., Y. Ohne, S. Takahashi, T. Noguchi and H. Kato, 2004. Dietary protein deprivation decreases the serine
phosphorylation of insulin receptor substrate-1 in rat skeletal muscle. J. Mol. Endocrinol. 32, 519-531.
Toyoshima, Y., R. Tokita, Y. Ohne, F. Hakuno, T. Noguchi, S. Minami, H. Kato and S. Takahashi, 2010. Dietary protein
deprivation upregulates insulin signaling and inhibits gluconeogenesis in rat liver. J. Mol. Endocrinol. 45, 329-340.

Energy and protein metabolism and nutrition in sustainable animal production 259
,PSDFWRIGLHWDU\ODUJLQLQHVXSSO\GXULQJHDUO\JHVWDWLRQRQP\R¿EHU
development in newborn pigs
G. Bee1, C.E. Pardo1,2 and M. Kreuzer2
1'HSDUWPHQWRI$QLPDO6FLHQFH$JURVFRSH3RVLHX[6ZLW]HUODQG[email protected]
2(7+=XULFK,QVWLWXWHRI$JULFXOWXUDO6FLHQFHV=XULFK6ZLW]HUODQG

Introduction
During early pregnancy when placental growth is fastest, the level of arginine and its precursor
RUQLWKLQHLVHOHYDWHGLQSRUFLQHDPQLRWLFDQGDOODQWRLFÀXLG :Xet al 7KLVDEXQGDQFHLV
associated with a high syntheses rate of nitric oxide and polyamine in the porcine placentae (Self et
al., 2004). Wu et al. (2004) provided evidence showing that both nitric oxide and polyamines play
key roles in angiogenesis, which is a critical event during placental growth and fetal development
(Town et al., 2005). These observations led to the hypothesis that arginine is important for placental
and fetal development. Recently, Bérard and Bee (2010) showed that supplementing the gestation
GLHWZLWKODUJLQLQHSRVLWLYHO\DIIHFWHGSULPDU\P\R¿EHUK\SHUSODVLDLQVHPLWHQGLQRVXVPXVFOHV
670 RIGROGIHWXVHV%HFDXVHSULPDU\¿EHUVVHUYHDVDVFDIIROGIRUWKHIRUPDWLRQRIVHFRQGDU\
¿EHUVZHK\SRWKHVL]HGWKDWRIIVSULQJIURPVRZVIHGH[WUDDUJLQLQHEDUHWKHSRWHQWLDOIRUJUHDWHU¿EHU
K\SHUSODVLDDQGXOWLPDWHO\PRUHHI¿FLHQWSRVWQDWDOJURZWK(VSHFLDOO\ORZELUWKZHLJKWSLJVGLVSOD\
ORZP\R¿EHUQXPEHUV7KXVWKHJRDORIWKLVH[SHULPHQWZDVWRHYDOXDWHLIDUJLQLQHVXSSOHPHQWDWLRQ
of the dams has a positive effect on muscle development of such piglets.

Material and methods

Five intact (I), 5 unilaterally hysterectomized-ovariectomized (HO; model for crowded intrauterine
environment) and 5 unilaterally ovariectomized (OL; model for uncrowded intrauterine environment)
VRZVZHUHVXEMHFWHGWRDFURVVRYHUGHVLJQLQWHUPVRIGLHWV SDULWLHVDQG )URPGD\WRGD\
of gestation they were either offered 25 g/d of l-arginine (Arg) in addition to the standard gestation
diet or they received the corresponding control diet (C) which was supplemented with 43 g/d of
l-alanine. At farrowing, litter characteristics were assessed. From each litter, 2 female and 2 male
SLJOHWVZLWKWKHORZHVWDQGPHGLXPELUWKZHLJKWZHUHVDFUL¿FHG,QWHUQDORUJDQVDQGWKH670ZHUH
collected and weighed. Histological analyses of muscles were performed using mATPase staining
after pre-incubation at pH 4.5 and 10.2. The data were analyzed with the mixed procedure (SysStat
13), considering the surgical treatment of the sows, the early gestation diet type, gender and the
DQGZD\LQWHUDFWLRQVDV¿[HGIDFWRUV

Results and discussion

As expected, the number of total piglets born and piglets born alive differed (P<0.05) among sow
JURXSVDQGZDVORZHVWLQ+2  JUHDWHVWLQ,  DQGLQWHUPHGLDWHLQ2/ 
10.0). Similarly, birth weight of offspring born alive differed (P<0.05) between the sow groups and
ZDVOLJKWHVWLQ+2 NJ KHDYLHVWLQ2/ NJ DQGLQWHUPHGLDWHLQ, NJ 7KHODUJHVW
variability (standard deviation) in litter birth weight was observed in HO followed by I and OL
VRZV DQGNJUHVSHFWLYHO\ ([FHSWIRUELUWKZHLJKWYDULDELOLW\ZKLFKZDVORZHU
(P<0.03) in offspring from sows fed Arg compared to C (±0.23 vs. ±0.27 kg), litter characteristics
were not affected by Arg supply.

Compared to OL, progeny from OH and I sows had lower (P<0.05) relative weights of liver (2.90
vs. 2.33; 2.30), kidney (0.84 vs. 0.73; 0.72) and STM (2.30 vs. 2.09; 1.95) and a greater (P<0.05)
EUDLQWROLYHUUDWLR YV 7KHODWWHULVDOVRNQRZQDVEUDLQVSDULQJDQGLVLQGLFDWLYH
of intrauterine growth retardation. Independent of the sow group, brain-to-liver ratios (1.12 vs. 1.22)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 261
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_84, © Wageningen Academic Publishers 2013
tended (P<0.08) to be lower in offspring from Arg compared to C sows, which suggest that l-arginine
supplementation could alleviate the effect of intrauterine growth retardation.

6XSSO\LQJ$UJWR,DQG2/EXWQRW2+VRZVLQVWHDGRI&UHVXOWHGLQJUHDWHUP\R¿EHUK\SHUSODVLD
and, consequently, a larger muscle area (Figure 1). These differences resulted mainly from a greater
P\R¿EHUQXPEHULQWKHOLJKWSRUWLRQ YVî5 RIWKHPXVFOH7KHFXUUHQW¿QGLQJFRQ¿UPV
previous assumptions that arginine supply during early gestation positively affects muscle formation
and growth potential.
A B

7 100

Cross- sectional muscle area (mm2)

bc bc ab c a bc abc ab ab c a bc
6
Myofiber number (x105)

80
5

4 60

3 40
2
20
1

0 0
OH OL I OH OL I
C C
Arg Arg
)LJXUH7RWDOP\R¿EHUQXPEHU SDQHO$ DQGFURVVVHFWLRQDODUHD SDQHO% RIWKHVHPLWHQGLQRVXV
PXVFOHLQRIIVSULQJERUQIURPXQLODWHUDOO\K\VWHUHFWRPL]HGRYDULHFWRPL]HG 2+ XQLODWHUDOO\
RYDULHFWRPL]HG 2/ DQGLQWDFW , VRZVIHGHLWKHUDFRQWUROGLHW & RUDODUJLQLQH $UJ VXSSOHPHQWHG
GLHWIURPGD\WRGD\RIJHVWDWLRQ/HDVWVTXDUHPHDQVZLWKLQHDFKJUDSKZLWKGLIIHUHQW
VXSHUVFULSWGLIIHU P< (UURUEDUVUHSUHVHQWVWDQGDUGHUURURIOHDVWVTXDUHPHDQV

References
Bérard, J. and G. Bee, 2010. Effects of dietary L-arginine supplementation to gilts during early gestation on foetal
VXUYLYDOJURZWKDQGP\R¿EHUIRUPDWLRQ$QLPDO
Self, J. T., T. E. Spencer, G. A. Johnson, J. Hu, F. W. Bazer, and G. Wu, 2004. Glutamine synthesis in the developing
porcine placenta. Biol. Reprod. 70, 1444-1451.
Town, S. C., J. L. Patterson, C. Z. Pereira, G. Gourley, and G. R. Foxcroft, 2005. Embryonic and fetal development in
DFRPPHUFLDOGDPOLQHJHQRW\SH$QLP5HSURG6FL
:X*):%D]HU-0:DOODFHDQG7(6SHQFHU%2$5',19,7('5(9,(:,QWUDXWHULQHJURZWK
UHWDUGDWLRQ,PSOLFDWLRQVIRUWKHDQLPDOVFLHQFHV-$QLP6FL
Wu, G., F. W. Bazer, T. A. Cudd, C. J. Meininger, and T. E. Spencer, 2004. Maternal Nutrition and Fetal Development.
-1XWU

262 Energy and protein metabolism and nutrition in sustainable animal production
Effect of feed restriction and birth weight on molecular and metabolic
response in the liver of pigs
C. Nebendahl1, R. Krüger1, S. Görs1, K. Giggel1, B.U. Metzler-Zebeli1, H.M. Hammon1, E. Albrecht2,
R. Pfuhl2 and C.C. Metges1
1/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\,QVWLWXWHVRI1XWULWLRQDO3K\VLRORJ\'XPPHUVWRUI
Germany; [email protected]
2/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\0XVFOH%LRORJ\DQG*URZWK'XPPHUVWRUI
Germany

Introduction
Intrauterine growth restriction (IUGR) in pigs has been shown to increase body fatness and decrease
meat quality (Rehfeldt et al. 0RUHRYHU,8*5ZDVUHSRUWHGWROHDGWRDOWHUHGPHWDEROLF
and hypothalamic-pituitary-adrenal (HPA) axis function later in life (Poore et al., 2003). Previous
experiments indicated that caloric restriction in early postnatal life may affect liver lipid metabolism
(Garg et al., 2013). In our study, the following questions were addressed at the molecular and
SK\VLRORJLFDOOHYHOLQSLJV  $UHWKHUHGLIIHUHQFHVLQWKHKHSDWLFWUDQVFULSWLRQDOSUR¿OHEHWZHHQ
ORZDQGQRUPDOELUWKZHLJKW"  $UHWKHVHHIIHFWVUHÀHFWHGRQWKHWLVVXHFRPSRVLWLRQOHYHO"  
&RXOGWKHSRVVLEOHELUWKZHLJKWGHSHQGHQWHIIHFWVEHPRGL¿HGE\IHHGUHVWULFWLRQ"  $UHWKHVH
effects persistent?

Material and methods

Liver tissue of female juvenile pigs (n=42; German Landrace) of low (0.8-1.1 kg, U) and normal (1.4-
NJ1 ELUWKZHLJKWVZDVXVHGWRVWXG\WUDQVFULSWLRQDODQGPHWDEROLFUHVSRQVHVWRIHHGUHVWULFWLRQ
5RIFRQWUROGLHW>&0-0(NJ@DJHG IROORZHGE\VXEVHTXHQWUHIHHGLQJ>
MJ ME/kg] until 131 d of age. Overall, four groups (NC, NR, UC, UR) were included in our study to
analyze effects related to birth weight (U vs. N) and/or feed restriction (R vs. C). For whole genome
H[SUHVVLRQVWXGLHVRQSRUFLQHVSHFL¿F$JLOHQW[.PXOWLSOH[DUUD\VDQGWKHDQDO\VLVRIOLSLG
droplets stained with oil red O, liver tissue was taken from animals after overnight fasting (18 h) at
ages 75 d (before feed restriction, T1), 98 d (at the end of a 3 weeks feed restriction period, T2) and
131 d (after 5 week of refeeding, T3). Liver studies were performed on three animals per group at
each time point. For four-group comparisons, Two-Way Analysis of Variance (ANOVA) was used.
When normality test passed (3”0.05), Bonferroni-test was used. Not-normally distributed data were
log-transformed to obtain normality and to allow ANOVA analysis. After quantile normalization of
microarray data, statistical analysis for pairwise comparisons was performed with an unpaired t-test
with unequal variance (Welch-test). All results are depicted as means ± SEM, and were considered
VLJQL¿FDQWO\GLIIHUHQWZKHQP-values were 3”0.05. Statistics were done with SigmaPlot 11.0 software.

Results and discussion

$WG 7 KHSDWLFJHQHH[SUHVVLRQDQDO\VLV )ROG&KDQJH•3” LGHQWL¿HGJHQHV
(95 up- and 99 down-regulated) involved in lipid metabolism to be differentially expressed in U
vs. N animals.

2LOUHG2VWDLQHGOLYHUVHFWLRQVVKRZHGIROGDQGIROGLQFUHDVHVLQWKHWRWDOPHDQDUHDDQG
number of lipid droplets (LDs) in U versus N, respectively (3”0.01). The mean LD size (μm2) was
increased by 24.9% (Figure 1).

However, 3-weeks feed restriction (T2) reduced the total mean area of LDs by 58.3 and 72.7% in
U and N animals, respectively (3”0.01). Additionally, 451 (311 up- and 140 down-regulated) and

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 263
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_85, © Wageningen Academic Publishers 2013
)LJXUH(IIHFWVRIORZ $ DQGQRUPDO % ELUWKZHLJKWRQOLYHUOLSLGGURSOHW /' FRXQWDQG
IRUPDWLRQLQSLJVDWGRIDJH0LFURVFRSLFLPDJHVVKRZKLJKHUWRWDOPHDQDUHDDQGQXPEHUVRI
OLSLGGURSOHWVDVZHOODVPHDQ/'VL]HLQORZ $ YVQRUPDO % ELUWKZHLJKWSLJV

340 (121 up- and 219 down-regulated) genes were found to be differentially regulated in the liver
of N and U after feed restriction, respectively. The differentially regulated genes were allocated to
functional pathways associated with e.g. protein metabolism. Free arginine concentration was found
to be 17% higher in liver samples of UR vs. NR animals (3”0.05).

The second objective of our study was to explore whether the observed responses to feed restriction
DQGELUWKZHLJKWSHUVLVWDIWHUZHHNVRIUHIHHGLQJ 7 ,QWRWDOORQJWHUPUHJXODWHGJHQHV 
XSDQGGRZQUHJXODWHG ZHUHLGHQWL¿HG,QJHQHUDOUHIHHGLQJLQGXFHGWKHUHFRYHU\RIWRWDOPHDQ
LD area in U and N animals, respectively. However, in feed-restricted U animals, the mean LD size
(μm2) was still lower by 23.3% as compared to age-matched controls. In contrast, the mean LD size
was increased in N (+24.7%, 3”0.01). Finally, the data suggest that feed restriction programmed
juvenile female pigs with low birth weight for an increased rate of hepatic lipolysis in later life.

Acknowledgment
The study was supported by BMBF of Germany in the framework of ‚Ernährungsforschung – für ein
gesundes Leben, Modul: Biomedizinische Ernährungsforschung‘ (Vision Epifood, FKZ 0315397B).

References
Garg M., Thamotharan M., Dai Y., Lagishetty V., Matveyenko A.V., Lee W.N. and Devaskar S.U., 2013. Glucose
intolerance and lipid metabolic adaptations in response to intrauterine and postnatal calorie restriction in male
adult rats. Endocrinol 154, 102-113.
Poore, K.R. and Fowden, A.L., 2003. The effects of birth weight hypothalamo-adrenal axis function in juvenile and
DGXOWSLJV-3K\VLRO
5HKIHOGW&DQG.XKQ*&RQVHTXHQFHVRIELUWKZHLJKWIRUSRVWQDWDOJURZWKSHUIRUPDQFHDQGFDUFDVVTXDOLW\
in pigs as related to myogenesis. J Anim Sci 84, 113-123.

264 Energy and protein metabolism and nutrition in sustainable animal production
Embryo thermal manipulation has long-lasting effects on energy
metabolism in chickens
T. Loyau1, S. Métayer-Coustard1, C. Praud1, C. Berri1, M.J. Duclos1, S. Tesseraud1, N. Rideau1,
P. Chartrin1, C. Hennequet-Antier1, N. Everaert2, S. Yahav, S. Mignon-Grasteau1 and A. Collin1
1,15$855HFKHUFKHV$YLFROHV1RX]LOO\)UDQFH[email protected]
2'HSDUWPHQWRI%LRV\VWHPV.8/HXYHQ.DVWHHOSDUN$UHQEHUJ/HXYHQ%HOJLXP
,QVWLWXWHRI$QLPDO6FLHQFH$527KH9ROFDQL&HQWHU32%R[%HW'DJDQ,VUDHO

Introduction
Broiler chickens have limited capacities to sustain high temperatures. However, thermal manipulation
(TM) during embryogenesis has been shown to lower their body temperature at hatch and to improve
thermotolerance until market age (Piestun et al., 2008). This thermotolerance acquisition could
partly be due to changes in sensible heat loss, but also in metabolic rate, especially in energy and
protein metabolisms of birds. The aim of this study was to evaluate the long-lasting effects of TM
during embryogenesis, when coupled or not with a heat challenge at slaughter age (d 34), on plasma
metabolites and hormones, cell signaling and the expression of genes involved in muscle metabolism.

Material and methods

(JJVZHUHLQFXEDWHGLQFRQWUROFRQGLWLRQV &ƒ&5HODWLYH+XPLGLW\5+ RUH[SRVHG
WRWKHUPDOPDQLSXODWLRQ 70KGDWƒ&DQG5+ IURPGD\WRRIHPEU\RJHQHVLV
After rearing in standard conditions, half of each group stayed at control ambient temperature (21 °C;
C and TM) and the other half was submitted to heat challenge (Ch; 32 °C for 5 h; CCh for control
chickens submitted to heat challenge and TMCh for TM chickens submitted to heat challenge) at
34 d of age. At this age, blood was withdrawn from the brachial vein, chickens were slaughtered
and the fast-twitch glycolytic Pectoralis major (PM) muscle was removed from 9 to 11 male broiler
chickens and snap frozen. The expression of genes involved in energy metabolism was analyzed by
T573&5DQGQRUPDOL]HGZLWKȕDFWLQF\WRFKURPH%DQG6XVLQJ*HQRUP® software. Plasma
insulin and thyroid hormone concentrations were measured by RIA. Plasma uric acid, triglyceride
and glucose concentrations were measured enzymatically. Cell signaling was explored by western-
EORWXVLQJSKRVSKRVSHFL¿FDQWLERGLHVGLUHFWHGDJDLQVWNLQDVHVLQYROYHGLQSURWHLQFDUERK\GUDWH
and/or energy metabolism as described by Boussaid-Om Ezzine et al. (2010). Data were analyzed
using the general linear model procedure of SAS, considering incubation and heat challenge within
incubation, i.e.Ch(Incubation), as main effects.

Results and discussion

Incubation conditions did not affect plasma uric acid, triglyceride, triiodothyronine, thyroxine
DQGLQVXOLQFRQFHQWUDWLRQV+RZHYHULWKDGVLJQL¿FDQWORQJODVWLQJHIIHFWVRQWK\URLGKRUPRQH
metabolism by lowering by 40 to 50% the muscle expressions of deiodinases D3 and D2 in TM
animals as compared to controls. TM also decreased the expression of the transcription factor
3*&ĮLQYROYHGLQPLWRFKRQGULDOELRJHQHVLVDQGPHWDEROLFDFWLYDWLRQ :DOWHUDQG6HHEDFKHU
 &RQVLVWHQWO\HQHUJ\SURGXFWLRQSDWKZD\VZHUHPRGL¿HGE\LQFXEDWLRQFRQGLWLRQVDWƒ&
as indicated by decreased muscle citrate synthase and hexokinase 1 expressions in TM compared to
control chickens (Figure 1). Moreover, UCP3 was overexpressed in TMCh group as compared to TM
and C chickens, possibly related to a limitation of oxidative stress in these animals (Mujahid et al.,
 7KLVRYHUH[SUHVVLRQZDVDVVRFLDWHGZLWKDKLJKHUJO\FHPLD “PPROO DVFRPSDUHG
WRRWKHUJURXSV IURP“WR“PPROO 

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 265
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_86, © Wageningen Academic Publishers 2013
Figure 1. Relative expression of genes involved in muscle energy metabolism. C: control broilers,
&&KFRQWUROWRDKHDWFKDOOHQJHDWG &K 70WKHUPDOPDQLSXODWHGGXULQJHPEU\RJHQHVLV
DQG70&K70WRDKHDWFKDOOHQJHDWG'DWDZHUHDQDO\]HGFRQVLGHULQJLQFXEDWLRQDQGKHDW
FKDOOHQJHZLWKLQLQFXEDWLRQLH&K ,QFXEDWLRQ DVPDLQHIIHFWV7KHGLIIHUHQWOHWWHUVLQGLFDWH
VLJQL¿FDQWGLIIHUHQFHVEHWZHHQWUHDWPHQWV DEP< 'DWDDUHSUHVHQWHGDVPHDQV“6(0'
'HLRGLQDVH''HLRGLQDVH3*&Į33$5ȖFRDFWLYDWRUĮ+.+H[RNLQDVH&6&LWUDWH
V\QWKDVH8&38QFRXSOLQJ3URWHLQ

7KHSKRVSKRU\ODWLRQRINLQDVHVUHJXODWLQJSURWHLQV\QWKHVLV 6ULERVRPDOSURWHLQDQG6.LQDVH 
ZDVVLJQL¿FDQWO\LQKLELWHGE\KHDWFKDOOHQJHZLWKLQLQFXEDWLRQFRQGLWLRQVZKHUHDV30LWRJHQ
DFWLYDWHGSURWHLQNLQDVHLQYROYHGLQVWUHVVDQGLQÀDPPDWRU\UHVSRQVHVVKRZHGDQLQFUHDVHG
phosphorylation state in TM conditions.

In conclusion, several genes and proteins controlling energy metabolism and stress response in the
Pectoralis majorPXVFOHZHUHDIIHFWHGE\HPEU\RWKHUPDOPDQLSXODWLRQUHÀHFWLQJDSRVVLEOHORQJ
lasting adaptation, which may modify peripheral metabolism.

Acknowledgements
Funding: Agence Nationale de la Recherche, Project ANR-09-JCJC-0015-01 THERMOCHICK.

References
Boussaid-Om Ezzine, S., N. Everaert, S. Metayer-Coustard, N. Rideau, C. Berri, R. Joubert, S. Temim, A. Collin, and
67HVVHUDXG(IIHFWVRIKHDWH[SRVXUHRQ$NW6.VLJQDOLQJDQGH[SUHVVLRQRIJHQHVUHODWHGWRSURWHLQ
and energy metabolism in chicken (Gallus gallus) pectoralis major muscle. Comp. Biochem. Physiol. B-Biochem.
Mol. Biol. 157: 281-287.
0XMDKLG$.6DWR<$NLEDDQG07R\RPL]X$FXWHKHDWVWUHVVVWLPXODWHVPLWRFKRQGULDOVXSHUR[LGHSURGXFWLRQ
LQEURLOHUVNHOHWDOPXVFOHSRVVLEO\YLDGRZQUHJXODWLRQRIXQFRXSOLQJSURWHLQFRQWHQW3RXOW6FL  
Piestun Y., D. Shinder, M. Ruzal, O. Halevy, J. Brake, and S. Yahav, 2008. Thermal manipulations during broiler
HPEU\RJHQHVLVHIIHFWRQWKHDFTXLVLWLRQRIWKHUPRWROHUDQFH3RXOW6FL
Walter I. and F. Seebacher, 2007. Molecular mechanisms underlying the development (Gallus gallus): a new role of
PGC-1a? Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, 2315-2322.

266 Energy and protein metabolism and nutrition in sustainable animal production
Heat stress-induced overproduction of mitochondrial reactive oxygen
species is down-regulated in laying-type chickens
M. Kikusato and M. Toyomizu
Division of Life Sciences, Graduate School of Agricultural Science, Tohoku University, 1-1
7VXWVXPLGRUL$PDPL\DPDFKL$REDNX6HQGDL-DSDQ[email protected]

Introduction
Heat stress is an environmental factor that gives rise to oxidative stress. We have previously shown
that acute heat stress-induced oxidative damage resulted from the overproduction of mitochondrial
reactive oxygen species (mitROS) in the skeletal muscle of meat-type chickens. Recently, we further
FODUL¿HGWKDWWKHRYHUSURGXFWLRQRIPLW526UHVXOWHGIURPDQLQFUHDVHRIWKHPLWRFKRQGULDOPHPEUDQH
SRWHQWLDO ǻȌ DQGWKDWWKLVZDVDFFRPSDQLHGE\DGHFUHDVHLQSURWRQOHDNGXHWRWKHUHGXFHG
expression of avian uncoupling protein (avUCP) (Kikusato et al., 2013). Given that avUCP exerts
XQFRXSOLQJDFWLYLW\WRGLVVLSDWHǻȌDQGWKHUHE\UHGXFHPLW526SURGXFWLRQLWFRXOGEHSRVWXODWHG
that down-regulation of avUCP expression might be a factor responsible for the overproduction of
mitROS under heat stress conditions. On this basis, we hypothesized that the overproduction of
mitROS could be down-regulated when avUCP is highly expressed, even under heat stress conditions.
We attempt to verify this hypothesis using two different types of chickens (meat-type and laying-type)
whose muscle avUCP content is quantitatively different: avUCP expression is higher in the skeletal
muscle tissue of laying-type chickens compared with meat-type chickens (Toyomizu et al., 2011).

Material and methods

Meat-type (Ross) and laying-type (WLH) chickens (n=8) at 0 day of age were obtained from
commercial hatcheries and fed a standard diet for 3 and 10 wks, respectively. At the end of the
respective periods the chickens were transferred to a diet containing 19% crude protein and 2,900
kcal/kg metabolizable energy until body weights reached 1.2 kg. Thereafter, they were divided into
two sub-groups for each type, with one of the two groups exposed to 34 °C for 12 h and the other
JURXSPDLQWDLQHGDWƒ&)ROORZLQJWKLVWUHDWPHQWWKHFKLFNHQVZHUHVDFUL¿FHGE\GHFDSLWDWLRQ
3HFWRUDOLVVXSHU¿FLDOLV muscles were removed, and mitochondria isolated and incubated at 38 °C
LQDSRWDVVLXPEDVHGDVVD\PHGLXP %6$S+ FRQWDLQLQJȝ0FDUER[\DWUDFW\ORVLGH
which inhibits the uncoupling activity of adenine nucleotide translocator. Succinate was added to
initiate mitochondrial respiration, and the O2FRQVXPSWLRQUDWHDQGPHPEUDQHSRWHQWLDO ǻȌ ZHUH
simultaneously measured by using electrodes sensitive to O2 and to the potential-dependent probe
TPMP+, respectively. To measure proton leak kinetics, state 4-respiration was titrated with sequential
DGGLWLRQVRIPDORQDWH XSWRP0 ,QDGGLWLRQDUDFKLGLRQLFDFLG ȝ0IUHVKO\SUHSDUHG 
ZDVXVHGWRDFWLYDWHWKHXQFRXSOLQJDFWLYLW\RIDY8&30LW526SURGXFWLRQZDVÀXRURPHWULFDOO\
determined by assay with Amplex Red (Ex./Em. = 544/590 nm).

Results and discussion

Body weights of meat- and laying-type chickens decreased in response to the heat exposure, but the
extent of the decrease (expressed as a ratio of the weights of heat-stressed versus control chickens) was
VLJQL¿FDQWO\OHVVLQOD\LQJW\SHFKLFNHQVWKDQLQPHDWW\SHFKLFNHQV)HHGLQWDNHZDVDOVRGHFUHDVHG
by the heat treatment for both types, but no difference between them was observed. As shown in
Figure 1, no difference in basal proton leak between the thermoneutral and heat-stressed groups was
REVHUYHGIRUPHDWRUOD\LQJW\SHFKLFNHQV,QFRQWUDVWKRZHYHUǻȌDWVWDWH IXUWKHVWULJKWKDQG
SRLQWLQWKHNLQHWLFFXUYH ZDVVLJQL¿FDQWO\LQFUHDVHGE\KHDWH[SRVXUHLQPHDWW\SHFKLFNHQVEXW
was unchanged in laying-type chickens. Mitochondrial proton leak increased markedly for both
W\SHVRIFKLFNHQVIROORZLQJWKHDGGLWLRQRIDUDFKLGRQLFDFLGEXWWKHLQFUHDVHZDVQRWDVVLJQL¿FDQW

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 267
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_87, © Wageningen Academic Publishers 2013
)LJXUH%DVDO FLUFOHV DQGDUDFKLGRQLFDFLGLQGXFHGDY8&3DFWLYDWHG GLDPRQGV SURWRQOHDN
LQVNHOHWDOPXVFOHPLWRFKRQGULDIURPPHDWDQGOD\LQJW\SHFKLFNHQVXQGHUWKHUPRQHXWUDO RSHQ
V\PEROV DQGKHDWVWUHVVHG FORVHGV\PEROV FRQGLWLRQV9DOXHVDUHPHDQV“6(Q 

for the heat-stressed group compared with the thermoneutral control group in meat-type chickens.
No difference in arachidonic acid-induced proton leak was observed between the thermoneutral and
heat-stressed groups in laying-type chickens, which suggests that avUCP might be highly expressed
even under heat stress conditions.

0LW526SURGXFWLRQLQWKHSUHVHQFHRIDUDFKLGRQLFDFLGZDVVLJQL¿FDQWO\LQFUHDVHGE\WKHKHDW
exposure in meat-type chickens, but was unchanged in laying-type chickens, probably because
WKHDY8&3PHGLDWHGSURWRQOHDNZDVQRWLQÀXHQFHGE\WKHKHDWVWUHVVLQWKHOD\LQJW\SH,QD
similar fashion, lipid peroxidation (expressed in terms of malondialdehyde content) in muscle
ZDVVLJQL¿FDQWO\LQFUHDVHGE\WKHKHDWWUHDWPHQWLQPHDWW\SHFKLFNHQVEXWRQO\PDUJLQDOO\LQWKH
laying-type. Taken together, these results suggest that avUCP plays an important role in regulating
the acute heat stress-induced overproduction of mitochondrial ROS, probably via a decrease in the
ǻȌRIVNHOHWDOPXVFOHPLWRFKRQGULDLQELUGV

References
Kikusato, M. and M. Toyomizu, 2013. Crucial role of membrane potential in heat stress-induced overproduction of
reactive oxygen species in avian skeletal muscle mitochondria. PloS One, in press.
Toyomizu, M., M. Kikusato, Y. Kawabata, M. A. K. Azad, E. Inui and T. Amo, 2011. Meat-type chickens have a higher
HI¿FLHQF\RIPLWRFKRQGULDOR[LGDWLYHSKRVSKRU\ODWLRQWKDQOD\LQJW\SHFKLFNHQV&RPS%LRFKHP3K\VLRO$
159, 75-81.

268 Energy and protein metabolism and nutrition in sustainable animal production
)XQFWLRQDOUROHVRIDTXDSRULQVDQGXUHDWUDQVSRUWHUVLQXUHDÀX[DFURVV
the ruminal epithelium
M.E. Walpole1, B.L. Schurmann1, P. Górka1,2, G.B. Penner1, M.E. Loewen and T. Mutsvangwa1
1'HSDUWPHQWRI$QLPDODQG3RXOWU\6FLHQFH8QLYHUVLW\RI6DVNDWFKHZDQ6DVNDWRRQ6.61$
Canada; [email protected]
2'HSDUWPHQWRI$QLPDO1XWULWLRQ8QLYHUVLW\RI$JULFXOWXUH.UDNRZ3RODQG
'HSDUWPHQWRI%LRPHGLFDO6FLHQFHV8QLYHUVLW\RI6DVNDWFKHZDQ6DVNDWRRQ6.61$&DQDGD

Introduction
In ruminants, urea that is recycled to the rumen is an important source of N for microbial growth.
Urea transport (UT-B) proteins facilitate urea movement across the ruminal epithelium, although other
mechanisms must be involved as inhibiting UT-B does not completely abolish urea transport (Stewart
et al., 2005). Of the aquaporins (AQP), a family of membrane-spanning proteins predominantly
involved in water transport, AQP-3, -7, and -10 are also permeable to urea (Litman et al., 2009);
however, it is not clear if AQP contribute to urea transport across the ruminal epithelium. Røjen et al.
(2011) observed that mRNA abundance for AQP-3, -7, and -10 in ruminal epithelium was altered by
dietary N content, suggesting that AQP might be involved in trans-epithelial urea transport. Increasing
ruminal carbohydrate digestion improves urea transfer to the rumen (Reynolds and Kristensen,
2008); however, the mechanisms responsible for this response remain obscure. Our objectives were
to determine: (1) the relative functional roles of AQP and UT-B in ruminal urea transport; and (2) if
functional adaptation of AQP and UT-B occurred in response to increased diet digestion.

Material and methods

7ZHQW\¿YHZHDQHG+ROVWHLQEXOOFDOYHV Q  ZHUHDVVLJQHGWRDFRQWUROGLHW &21KD\
and 8.5% vitamin-mineral premix) or a medium grain diet (MGD; 41.5% barley grain, 50% hay,
and 8.5% vitamin-mineral premix) that was fed for 3 (G3), 7 (G7), 14 (G14), or 21 (G21) d. All
calves were fed at 2.25% BW at 0800 h. Calves were killed at 1000 h and ruminal epithelium was
collected for mounting in Ussing chambers under short-circuit conditions and for analysis of mRNA
abundance of UT-B and AQP-3, -7, and -10. To mimic physiologic conditions, the mucosal buffer
S+ FRQWDLQHGQRXUHDZKLOHWKHVHURVDOEXIIHU S+ FRQWDLQHGPM urea. The serosal-to-
PXFRVDOÀX[HVRI14C-urea (Jsm-ureaN%TPO DQG3H-mannitol (Jsm-mannitol; 37 kBq/10 ml) were
measured, with Jsm-mannitol being used as an indicator of hydrophilic movement. Serosal addition of
phloretin (1 mM) was used to inhibit UT-B-mediated urea transport, while NiCl2 (1 mM) was used
to inhibit AQP-mediated urea transport. Gene transcript abundance was measured using real-time
qPCR, with GAPDH as a control. Data were analyzed using Proc Mixed as a randomized complete
block design, with polynomial contrasts used to test for linear, quadratic, and cubic effects of the
duration of adaptation to the MGD. Correlation analysis was performed to examine the relationships
between Jsm-urea and mRNA abundance.

Results and discussion

The Jsm-urea tended (3 0.075) to increase linearly as the duration of adaptation to MGD increased
(Table 1). Phloretin- (3 0.54) and NiCl2-sensitive (3  -sm-urea were not affected by diet.
Phloretin-insensitive Jsm-urea tended to increase linearly (3 0.075) as the duration of adaptation
to MGD increased; however, NiCl2-insensitive Jsm-urea was unaffected by diet (3 0.11; data not
shown). Across treatments, the addition of phloretin or NiCl2 reduced (P<0.001) the Jsm-urea and,
when both inhibitors were added simultaneously, Jsm-urea was further reduced (P<0.001; Figure 1).
Gene transcript abundance for AQP-3 (3 0.001) and UT-B (3 0.007) increased linearly as the
duration of MGD adaptation increased (Table 1). Gene transcript abundance for AQP-3 (3 0.011)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 269
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_88, © Wageningen Academic Publishers 2013
Table 1. Total Jsm-urea QPROFP2K DQGP51$DEXQGDQFH IROGFKDQJHUHODWLYHWRFRQWURO IRU87%
and AQP in bovine ruminal epithelium from animals adapted to grain.

Days on grain SEM Contrasts

Item 0 3 7 14 21 0 vs. all Linear Quad Cubic

Jsm-urea 107.7  112.8 122.8 144.2 14.5  0.075  0.85
Relative mRNA abundance, arbitrary units
AQP-3 1.00 1.55 2.40 4.47  0.72 0.014 0.001 0.45 0.47
AQP-7 1.00 0.03 0.04 0.13 0.15 0.29 0.015 0.20 0.12 0.18
AQP-10 1.00 0.13 0.11 -0.02 0.01 0.32 0.019 0.090 0.17 0.38
UT-B 1.00 1.38 1.35 2.44 3.02 0.52  0.007 0.85 0.78

150
a
QPROÂ FP2)-1ÂK-1)

b
100
c
Jsm-urea

50 d

Control NiCl2 3KORUHWLQ NiCl23KORUHWLQ

Figure 1. Total, NiCl2-sensitive, and phloretin-sensitive Jsm-urea across bovine ruminal epithelium.

and UT-B (3 0.019) was correlated with Jsm-urea. For AQP-7 (3 0.015) and AQP-10 (3 0.019),
gene transcript abundance in animals fed MGD was lower relative to those fed CON (Table 1). These
results demonstrate that both AQP and UT-B have functional roles in urea transport across the ruminal
epithelium, but more work is needed to understand the regulation of these urea transport mechanisms.

References
Litmann, T., R. Søgaard, and T. Zeuthen, 2009. Ammonia and urea permeability of mammalian aquaporins. Handb.
Exp. Pharmacol. 190, 327-358.
Reynolds, C. K., and N. B. Kristensen, 2008. Nitrogen recycling through the gut and the nitrogen economy of ruminants:
DQDV\QFKURQRXVV\PELRVLV-$QLP6FL((
Røjen, B., A. S. B. Poulsen, P. K. Theil, R. A. Fenton, and N. B. Kristensen, 2011. Effects of dietary nitrogen
concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in
ruminal papillae from lactating Holstein cows. J. Dairy Sci. 94, 2587-2591.
Stewart, G. S., C. Graham, S. Cattell, T. P. L. Smith, and C. P. Smith, 2005. UT-B is expressed in bovine rumen: potential
UROHLQUXPLQDOXUHDWUDQVSRUW$P-3K\VLRO5HJXO,QWHJU&RPS3K\VLRO55

270 Energy and protein metabolism and nutrition in sustainable animal production
Modulation of insulin signaling by n-3 PUFA in chicken liver
S. Tesseraud1, S. Métayer-Coustard1, P. Chartrin1, D. Hermier2, N. Simon, C. Peyronnet, M.
Lessire1 and E. Baéza1
1,15$855HFKHUFKHV$YLFROHV1RX]LOO\)UDQFH[email protected]
285$JUR3DULV7HFK,15$31&$3DULV)UDQFH
21,'2/3DULV)UDQFH

Introduction
N-3 polyunsaturated fatty acids (PUFA) are crucial for normal development and organ functioning
in vertebrates. Their pleiotropic effects are well documented as regulators of lipid and glucose
metabolism, but less known with regard to protein metabolism. Recent studies indicate that the
consumption of long-chain PUFA (LC-PUFA) promotes muscle protein anabolism both in farm
animals (Gingras et al., 2007) or humans (Smith et al. 2011a,b). These studies suggest that n-3 PUFA
may act by increasing insulin sensitivity rather than by regulating gene transcription. It should be
QRWHGWKDWWKHVHVWXGLHVZHUHSHUIRUPHGXVLQJQ/&38)$DEXQGDQWLQ¿VKRLOV DQGFDUERQV 
DQGHIIHFWVRIWKHYHJHWDOSUHFXUVRUĮOLQROHQLFDFLG $/$&Q KDYHQHYHUEHHQH[SORUHG
Moreover, the consequences on other tissues than muscle are unknown. Our aim was to investigate
the role of n-3 PUFA (ALA or LC-PUFA) on liver protein metabolism in chickens, by focusing on
their potential function as co-regulators of the insulin signaling cascade.

Material and methods

Ross male broilers were divided into 3 dietary treatments. Diets were isoproteic (22% CP), isoenergetic
NFDO0(NJ DQGKDGVLPLODUOLSLGVXSSO\  ZLWKGLIIHUHQWOLSLGVRXUFHVROHLFVXQÀRZHURLO
ULFKLQ&Q FRQWUROJURXS ¿VKRLOULFKLQ/&38)$DQGUDSHVHHGDQGOLQVHHGRLOVSURYLGLQJ
ALA. At 3 weeks of age, we studied insulin signaling cascade in the liver compared to the Pectoralis
major (PM) muscle in chickens submitted to an i.v. injection of insulin or saline. Liver and PM muscle
lysates were prepared as previously described (Duchene et al., 2008) and subjected to SDS-PAGE gel
HOHFWURSKRUHVLVDQGZHVWHUQEORWWLQJXVLQJWKHDSSURSULDWHSKRVSKRVSHFL¿FDQWLERG\$IWHUZDVKLQJ
membranes were incubated with an Alexa Fluor secondary antibody. Bands were visualized and
TXDQWL¿HGE\WKH2G\VVH\® infrared Imaging System. Values are presented as means ±SEM. Data
ZHUHVXEMHFWHGWR$129$WRGHWHFWVLJQL¿FDQWLQWHUJURXSGLIIHUHQFHV7KHPHDQVZHUHFRPSDUHG
E\)LVKHU¶VOHDVWVLJQL¿FDQWGLIIHUHQFHWHVWLQWKHFDVHRIDVLJQL¿FDQWHIIHFW

Results and discussion

Providing diets enriched in n-3 PUFA, i.e. rich in LC-PUFA as in ALA, for 3 weeks improved chicken
ERG\ZHLJKW 7DEOH /LYHUZHLJKWVLQWKH/&38)$JURXSZHUHVLJQL¿FDQWO\KLJKHUWKDQWKRVHRI
the other two groups (P<0.001). The PM muscle was heavier in chickens fed the ALA diet compared
to the other two diets (P<0.05). Our data indicated that n-3 PUFA led to different activation patterns
of insulin signaling in the liver and the muscle (Figure 1). In the PM muscle, ALA-enriched diet
PD\LPSURYHLQVXOLQVHQVLWLYLW\ZLWKDJUHDWHUDFWLYDWLRQRIWKHLQVXOLQLQGXFHG6.6SDWKZD\
involved in mRNA translation into proteins, thereby potentially increasing muscle protein synthesis
and growth. In the liver, conversely to PMPXVFOHLQVXOLQLQGXFHGSKRVSKRU\ODWLRQOHYHOVRI6
ZHUHORZHULQWKH$/$DQG/&38)$JURXSVFRPSDUHGWRWKH2OHLFJURXS7KHVH¿QGLQJVVXJJHVW
WKDWQ38)$KDGDWLVVXHVSHFL¿FHIIHFWRQLQVXOLQVLJQDOLQJ7KHEDVLFPHFKDQLVPVVXVWDLQLQJ
WKHWLVVXHVSHFL¿FHIIHFWVRIQ38)$ $/$DQGRU/&38)$ UHPDLQXQFOHDU2QHK\SRWKHVLVLV
that these differential effects may be due to some differences in membrane fatty acid composition
between the liver and muscle, which still requires investigation.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 271
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_89, © Wageningen Academic Publishers 2013
7DEOH%RG\DQGWLVVXHZHLJKWVRIZNROGFKLFNHQVIHG2OHLF$/$DQG/&38)$H[SHULPHQWDO
diets and injected intravenously with either insulin or saline.

Diet (D)1 Oleic ALA LC-PUFA SEM P

Treatment (T)2 C I C I C I D T D×T

BW, g  975 1,010 1,009 1,010  9.1 <0.01 0.51 0.85
Liver weight, g   21.57 22.72 24.84 25.25  <0.001  
PM weight, g       1.83 <0.05 0.34 0.89

12OHLFGLHWFRQWDLQLQJROHLFVXQÀRZHURLOULFKLQ$/$GLHWULFKLQ$/$SURYLGHGE\UDSHVHHGDQGOLQVHHGRLOV

/&38)$GLHWULFKLQ/&Q38)$SURYLGHGE\¿VKRLO
2 Treatment, saline (C) and insulin (I).

Muscle Control (C) Liver

Insulin (I)
3 3 a
p-S6/Vinculin (a.u.)

pS6/Vinculin (a.u.)
a
b
2 b b 2 b

1 1
c c c
0 0
Oleic ALA LC-PUFA Oleic ALA PUFA
LC-PUFA
)LJXUH6SKRVSKRU\ODWLRQOHYHOVLQWKH30PXVFOHDQGOLYHURIZNROGFKLFNHQVIHG2OHLF$/$
DQG/&38)$GLHWVDQGLQMHFWHGLQWUDYHQRXVO\ZLWKHLWKHULQVXOLQRUVDOLQH FRQWURO 7KHUHZDVD
'LHWî7UHDWPHQWLQWHUDFWLRQLQWKHOLYHU OHWWHUVLQGLFDWHGLIIHUHQFHVEHWZHHQWKHJURXSV EXWQRW
LQWKH30PXVFOH OHWWHUVLQGLFDWHGLIIHUHQFHVEHWZHHQGLHWV 

In conclusion, our results suggest that n-3 PUFA can act as co-regulators of insulin anabolic signaling
FDVFDGHLQJURZLQJFKLFNHQV)XUWKHUVWXGLHVPRUHVSHFL¿FDOO\IRFXVHGRQWKHXQGHUO\LQJPHFKDQLVPV
and their consequences on anabolic processes are likely to have new nutritional applications in the future.

References
Duchene, S., S. Métayer, E. Audouin, K. Bigot, J. Dupont, and S. Tesseraud, 2008. Refeeding and insulin activate the
$.7S6NLQDVHSDWKZD\ZLWKRXWDIIHFWLQJ,56,W\URVLQHSKRVSKRU\ODWLRQLQFKLFNHQPXVFOH'RPHVW$QLP
Endocrinol. 34, 1-13.
Gingras, A.A., P.J. White, P.Y. Chouinard, P. Julien, T.A. Davis, L. Dombrowski, Y. Couture, P. Dubreuil, A. Myre,
K. Bergeron, A. Marette, and M.C. Thivierge, 2007. Long-chain omega-3 fatty acids regulate bovine whole-body
SURWHLQPHWDEROLVPE\SURPRWLQJPXVFOHLQVXOLQVLJQDOOLQJWRWKH$NWP7256.SDWKZD\DQGLQVXOLQVHQVLWLYLW\
-3K\VLRO
Smith, G.I., P. Atherton, D.N. Reeds, B.S. Mohammed, D. Rankin, M.J. Rennie, and B. Mittendorfer, 2011a.
Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-
K\SHUDPLQRDFLGDHPLDLQKHDOWK\\RXQJDQGPLGGOHDJHGPHQDQGZRPHQ&OLQ6FL
Smith, G.I., P. Atherton, D.N. Reeds, B.S. Mohammed, D. Rankin, M.J. Rennie, and B. Mittendorfer, 2011b. Dietary
omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized
controlled trial. Am. J. Clin. Nutrition 93, 402-12.

272 Energy and protein metabolism and nutrition in sustainable animal production
+RUPRQHDQGPHWDEROLWHOHYHOVGLIIHUEHWZHHQFHUHEURVSLQDOÀXLGDQG
plasma of periparturient dairy cows
T. Laeger1, H. Sauerwein2, C.C. Metges1 and B. Kuhla1
1/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 ,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶
'XPPHUVWRUI*HUPDQ\[email protected]
28QLYHUVLW\RI%RQQ,QVWLWXWHRI$QLPDO6FLHQFH3K\VLRORJ\ +\JLHQH8QLW%RQQ*HUPDQ\

Introduction
Hormones and metabolites act as satiety signals in the brain and play an important role in control
of feed intake. These signals can reach the hypothalamus and brainstem, two major centers of feed
LQWDNHUHJXODWLRQYLDWKHFHUHEURVSLQDOÀXLG &6) 7KHFRQWULEXWLRQRISXWDWLYHDQRUH[LFRURUH[LF
&6)VLJQDOVSRVVLEO\OHDGLQJWRWKHLQVXI¿FLHQWIHHGLQWDNHE\KLJK\LHOGLQJGDLU\FRZVGXULQJHDUO\
lactation has not been studied so far. Therefore, the aim of this study was to elucidate associations
existing between both plasma and CSF hormones and metabolites during the periparturient period.

Material and methods

Ten multiparous German Holstein cows were fed ad libitum a total mixed ration offered 2-times
daily. Energy intake, milk yield, and energy balance (EB) were determined. CSF from the spinal cord
was obtained by lumbar puncture after local anesthesia, and blood was withdrawn from the jugular
vein before morning feeding on d -20, -10, +1, +10, +20 and +40 relative to calving. Concentrations
RIȕK\GUR[\EXW\ULFDFLG %+%$ JOXFRVHODFWDWHDQGQRQHVWHUL¿HGIDWW\DFLGV 1()$ ZHUH
determined photometrically in blood plasma and CSF. Amino acid concentrations were analyzed by
+3/& &FROXPQZLWKDXWRPDWHGSUHFROXPQGHULYDWL]DWLRQDQGÀXRUHVFHQFHGHWHFWLRQ .XKOD
et al., 2011). The concentration of leptin was measured by a double antibody enzyme immunoassay
(Sauerwein et al., 2004). Concentrations of acylated ghrelin were determined by RIA (Börner et al.,
2013). All data are presented as means and were analyzed by the residual option in the GLIMMIX
procedure of SAS (version 9.3) to construct the block diagonal structure of the residual covariance
matrix for each animal. The one-way ANOVA was used to analyze zootechnical variables and two-
ZD\$129$IRUWKHDQDO\VLVRIPHWDEROLWHVDQGKRUPRQHV ¿[HGHIIHFWVERG\ÀXLGDQGWLPH $
PRGHOZLWKWKH¿[HGHIIHFWERG\ÀXLGDQGWKHFRYDULDWHGD\ZDVXVHGWRHVWLPDWHWKHVORSHVIRU
plasma and CSF variables for evaluating their parallel course.

Results and discussion

Energy intake declined starting 5 days before parturition (>80 MJ NEL/d) until calving (51 MJ
NELG DQGLQFUHDVHGWKHUHDIWHUWRUHDFK0-1(L/d until the 40th day after parturition (P<0.05).
(QHUJ\FRUUHFWHGPLONLQFUHDVHGUDSLGO\ZLWKLQWKH¿UVWZHHNDIWHUSDUWXULWLRQDQGUHDFKHGDSODWHDX
at around 42 kg/d. All cows were in positive EB prepartum and in negative EB postcalving until the
end of the sampling period. While ghrelin did not change during the periparturient period, leptin
concentrations decreased after calving and remained low in early lactation in both CSF and plasma
(Table 1). Considering the well-known anorexic effect of leptin, diminished leptin concentrations do
QRWH[SODLQLQVXI¿FLHQWIHHGLQWDNHLQHDUO\ODFWDWLRQ1H[WWROHSWLQWKHPHWDEROLWHV%+%$ODFWDWH
and NEFA showed different responses during the periparturient period, either in plasma or CSF
(Table 1). This is presumably due to a different permeability of the blood-brain barrier bordering the
periphery from brain and due to an altered brain energy metabolism. In addition, Gln concentrations
were increased in CSF on d 1 postpartum. Gln may be utilized by the brain for ATP production, the
latter signals for satiety (Kola, 2008). Therefore, Gln could act as central anorexigenic signal around
parturition. Furthermore, BHBA (Sun et al., 1997) and branch-chained amino acids such as Leu
(Morrison et al., 2007) have been shown to exert anorexic responses when administered into the

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 273
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_90, © Wageningen Academic Publishers 2013
brain. The increased CSF BHBA and Leu concentration in early lactation (Table 1) can be suggested
WRFRQWULEXWHWRWKHLQVXI¿FLHQWHQHUJ\LQWDNHDIWHUFDOYLQJ

7DEOH3HULSDUWXULHQWSODVPD 3 DQG&6)FRQFHQWUDWLRQVRIKRUPRQHVDQGPHWDEROLWHV

Fluid1 -20 -10 +1 +10 +20 +40

Ghrelin (pg/ml) P 209  127 185 184 125

CSF  3.9 4.8 3.5 4.1 3.2
Leptin (ng/ml)2 P* 8.5 8.1  4.3 4.7 5.0
CSF*   0.5 0.4 0.4 0.4
BHBA (mM)2 P* 0.3 0.3 0.8 0.9 1.1 1.0
CSF* 0.1 0.2 0.2 0.2 0.3 0.2
Glucose (mM) P* 4.3 4.3 4.2   
CSF* 2.7 2.7  2.0 2.1 2.0
Lactate (mM)2 P* 1.5 1.7 1.0 0.7  0.7
CSF* 2.2 2.1 2.0 1.8 1.8 1.7
NEFA (μM)2 P* 257 402 1,232 1,890  1,437
CSF 22.0 14.3 15.1 11.4  24.1
Gln (μM) P* 340 355 338 288  241
CSF* 495 505 581 497 450 451
Ile (μM) P 102   105 117 112
CSF* 0.8 1.3 0.9 1.2 1.5 
Leu (μM) P 134 139 83 121 132 117
&6) 2.1 3.3 2.2 2.8 3.3 3.0
Val (μM) P 244 255 142 212 253 240
CSF* 2.5 3.8  3.1 4.4 

1 LQGLFDWHVDVLJQL¿FDQWVORSH P” RIWKHWUHQGOLQHZKHUHDVLQGLFDWHVP<0.1.

2,QGLFDWHVVLJQL¿FDQWO\GLIIHUHQWVORSHVRISODVPDYHUVXV&6)WUHQGOLQHV

References
Börner, S., M. Derno, S. Hacke, U. Kautzsch, C. Schäff, S. Thanthan, H. Kuwayama, H. M. Hammon, M. Röntgen,
R. Weikard, C. Kühn, A. Tuchscherer and B. Kuhla, 2013. Plasma ghrelin is positively associated with body fat,
OLYHUIDWDQGPLONIDWFRQWHQWEXWQRWZLWKIHHGLQWDNHRIGDLU\FRZVDIWHUSDUWXULWLRQ-(QGRFULQRO
Kola, B. 2008. Role of AMP-activated protein kinase in the control of appetite. J. Neuroendocrinol. 20, 942-951.
Kuhla B., G. Nürnberg, D. Albrecht, S. Görs, H. M. Hammon and C. C. Metges, 2011. Involvement of skeletal muscle
protein, glycogen and fat metabolism in the adaptation on early lactation of dairy cows. J. Proteome Res. 10,

Morrison, C. D., X. Xi, C. L. White, J. Ye and R. J. Martin, 2007. Amino acids inhibit Agrp gene expression via an
P725GHSHQGHQWPHFKDQLVP$P-3K\VLRO(QGRFULQRO0HWDE((
Sauerwein, H., U. Heintges, A. Hennies, T. Selhorst and A. Daxenberger, 2004. Growth hormone induced alterations
of leptin serum concentrations in dairy cows as measured by a novel enzyme immunoassay. Livest. Prod. Sci.
87, 189-195.
Sun, M., R. J. Martin and G. L. Edwards, 1997. ICV beta-hydroxybutyrate: effects on food intake, body composition,
DQGERG\ZHLJKWLQUDWV3K\VLRO%HKDY

274 Energy and protein metabolism and nutrition in sustainable animal production
+HSDWLFĮ1DQGȕ2-adrenergic receptors in dairy cows with different fat
mobilization during early lactation
H.M. Hammon, U. Kautzsch, C. Reiko, B. Kuhla, M. Röntgen, M. Derno and C.C. Metges
1XWULWLRQDO 3K\VLRORJ\ µ2VNDU .HOOQHU¶ /HLEQL] ,QVWLWXWH IRU )DUP $QLPDO %LRORJ\ )%1 
Dummerstorf, Germany; [email protected]

Introduction
Catecholamines increase around parturition in dairy cows and the hepatic adrenergic system is
involved in metabolic adaptation during early lactation, e. g., by stimulation of hepatic glucose
production (glycogenolysis and gluconeogenesis) after parturition to cover glucose demands
(McDowell, 1983; Nonogaki, 2000; Weber et al., 2013). Effects of adrenaline and noradrenaline
DUHPHGLDWHGE\KHSDWLFĮ1DQGȕ2-adrenergic receptors (AR), which are the dominant subtypes
in bovine liver (Carron et al., 2004; Ontsouka et al. 7KHREMHFWLYHRIWKHSUHVHQWVWXG\ZDV
WRLQYHVWLJDWHĮ1DQGȕ2-adrenergic receptors (AR) in liver of dairy cows varying in fuel selection
during the transition from pregnancy to lactation. Cows differed in their extent of whole body fat
mobilization and varied in their respiration quotient (RQ) before and after parturition (Börner et al.,
 7KHK\SRWKHVLVZDVWHVWHGWKDWKHSDWLFĮ1DQGȕ2-AR are involved in regulation of hepatic
HQHUJ\PHWDEROLVPWKDWWKHGHQVLW\RIKHSDWLFĮ1DQGȕ2-AR depend on the metabolic type of the
cow (high versus low fat mobilization post partum [pp]) and that the number of hepatic AR are
related to RQ and carbohydrate (COX) and fat oxidation (FOX) pp in dairy cows.

Material and methods

1LQHWHHQ*HUPDQ+ROVWHLQFRZV !NJPLONG•ndODFWDWLRQ ZHUHFODVVL¿HGE\OLYHU
fat concentration (LFC) pp in low (L; <240 mg total fat/g dry matter [DM]; n=9) and high (H; >240
mg total fat/g DM; n=10) fat mobilization around parturition. Cows were studied from dry off up
to 40 d pp and were fed total mixed rations ad libitum. Milk yield increased pp to 44.1 kg/d by the
5th wk of lactation and did not differ between groups. Liver biopsies were taken on d 3, 18, and
30 pp to measure LFC. During the 2nd wk pp, respiration quotient (RQ) and COX and FOX were
determined from gaseous exchange measurements in respiration chambers (Derno et al., 2013).
'DWDIRU54IURPFRZV RXWRIFRZV ZHUHUHFHQWO\SUHVHQWHGE\%|UQHUet al. (2013). In
addition, [U-13C]-glucose was infused to measure endogenous glucose production (eGP) and glucose
oxidation (GOx). Cows were slaughtered at d 40 pp and liver samples were snap frozen. For AR
measurements, membrane suspensions (2 mg protein/ml) were prepared. Saturation binding assays
with increasing concentrations of (3H)-prazosin and (3H)-CGP-12177 were performed for the
GHWHUPLQDWLRQRIĮ1DQGȕ2-AR, respectively (Carron et al., 2005; Ontsouka et al. 0D[LPDO
binding capacity (Bmax DQGELQGLQJDI¿QLW\ .D) were calculated using unlabeled phentolamine
Į1$5 DQGSURSUDQRORO ȕ2-AR) as competitors. Data are presented as LSMeans ± SE and were
DQDO\]HGE\*HQHUDO/LQHDU0RGHORI6$6ZLWK/)&DV¿[HGHIIHFW&RUUHODWLRQVZHUHFDOFXODWHG
between Bmax and KD of AR and eGP, GOx, RQ, COX, and FOX, respectively.

Results and discussion

Bmax and KDIRUKHSDWLFĮ1DQGȕ2-AR were not affected by LFC, respectively. LSMeans for H
DQG/FRZVZHUH“IPROPJSURWHLQ %max DQG“Q0 .D IRUĮ1$5DQG“
fmol/mg protein (Bmax DQG“Q0 .D IRUȕ2-AR. In addition, body fat mobilization did not
LQÀXHQFHH*3DQG*2[ /60HDQVIRU+DQG/FRZVZHUH“PPRO>NJ%:îK@IRUH*3
and 0.11±0.01 mmol/[kg BW × h] for GOx). However, there was a trend for a positive correlation
between BmaxRIĮ1$5DQGH*3 U 3 0.1) in H cows, but a strong negative correlation between
BmaxRIĮ1-AR and eGP (r=-0.93; P< LQ/FRZV$OWKRXJKKHSDWLFĮ1$5ZDVQRWLQÀXHQFHG

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 275
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_91, © Wageningen Academic Publishers 2013
E\/)&WKHUHODWLRQVKLSEHWZHHQĮ1-AR and eGP depended on the degree of fat mobilization after
calving. Interestingly, Bmax and KDRIKHSDWLFȕ2-AR were not related to eGP in our cows, but hepatic
ȕ2-AR is involved in stimulation of eGP in human (Rizza et al., 1980). We have recently found a
FORVHUHODWLRQVKLSEHWZHHQKHSDWLFĮ1$5EXWQRWȕ2-AR, and eGP in neonatal calves, which may
VXSSRUWWKHSUHVHQW¿QGLQJVWKDWKHSDWLFĮ1$5PRUHWKDQȕ2-AR may be involved in regulation of
eGP in cattle (Rohrbeck et al., 2012).

Independent of LFC, BmaxRIĮ1$5FRUUHODWHGSRVLWLYHO\ZLWK*2[ U P<0.05) and COX

U P< EXWQHJDWLYHO\ZLWK)2; U P< 7KHUHZHUHQRVLJQL¿FDQWFRUUHODWLRQV
EHWZHHQKHSDWLFȕ2-AR (Bmax) and data for substrate oxidation. These data again point to the
LPSRUWDQFHRIKHSDWLFĮ1-AR expression for regulation of fuel utilization in dairy cows, whereas
DGUHQHUJLFHIIHFWVPHGLDWHGE\KHSDWLFȕ2-AR may be not regulated at the receptor density level.

In conclusion, our data indicate that the hepatic adrenergic system is involved in regulation of fuel
VHOHFWLRQLQGDLU\FRZVGXULQJHDUO\ODFWDWLRQ,QWHUHVWLQJO\Į1$5EXWQRWȕ2-AR, is the dominant
adrenergic receptor type that takes part in regulation of glucose supply and substrate oxidation.

Acknowledgements
The research was funded by German Research Foundation (DFG).

References
Börner, S., M. Derno, S. Hacke, U. Kautzsch, C. Schäff, S. ThanThan, H. Kuwayama, H. M. Hammon, M. Röntgen,
R. Weikard, C. Kühn, A. Tuchscherer, and B. Kuhla, 2013. Plasma ghrelin is positively associated with body fat,
OLYHUIDWDQGPLONIDWFRQWHQWEXWQRWZLWKIHHGLQWDNHRIGDLU\FRZVDIWHUSDUWXULWLRQ-(QGRFULQRO
Carron, J., C. Morel, H. M. Hammon, and J. W. Blum, 2005. Ontogenetic development of mRNA levels and binding
VLWHVRIKHSDWLFȕDGUHQHUJLFUHFHSWRUVLQFDWWOH'RPHVW$QLP(QGRFULQRO
Derno, M., G. Nürnberg, P. Schön, A. Schwarm, M. Röntgen, H. M. Hammon, C. C. Metges, R. M. Bruckmaier, and
B. Kuhla, 2013. Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows. J.
'DLU\6FL
0F'RZHOO*++RUPRQDOFRQWURORIJOXFRVHKRPRHRVWDVLVLQUXPLQDQWV3URF1XWU6RF
Nonogaki, K., 2000. New insights into sympathetic regulation of glucose and fat metabolism. Diabetologia 43, 533-549.
2QWVRXND(&<=ELQGHQ+0+DPPRQDQG-:%OXP2QWRJHQHVLVRIP51$OHYHOVDQGELQGLQJVLWHV
RIKHSDWLFȕDGUHQRFHSWRUVLQ\RXQJFDWWOH'RPHVW$QLP(QGRFULQRO
Rizza, R. A., P. E. Cryer, M. W. Haymond, and J. E. Gerich, 1980. Adrenergic mechanisms of catecholamine action
RQJOXFRVHKRPHRVWDVLVLQPDQ0HWDEROLVP
Rohrbeck, D., J. Steinhoff-Wagner, E. Kanitz, and H. M. Hammon, 2012. Ontogenic changes of hepatic glucocorticoid
and alpha1- and beta2DGUHQHUJLFUHFHSWRUVLQQHRQDWDOFDOYHV-'DLU\6FL 6XSSO 
Weber, C., C. Hametner, A. Tuchscherer, B. Losand, E. Kanitz, W. Otten, S. P. Singh, R. M. Bruckmaier, F. Becker,
W. Kanitz, and H. M. Hammon, 2013. Variation in fat mobilization during early lactation differently affects feed
LQWDNHERG\FRQGLWLRQDQGOLSLGDQGJOXFRVHPHWDEROLVPLQKLJK\LHOGLQJGDLU\FRZV-'DLU\6FL

276 Energy and protein metabolism and nutrition in sustainable animal production
Insulin signaling of glucose uptake in skeletal muscle of lactating dairy cows
S.K. Spachmann, U. Schönhusen, B. Kuhla, M. Röntgen and H.M. Hammon
,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 
Dummerstorf, Germany; [email protected]

Introduction
Insulin response in skeletal muscles is thought to be impaired in dairy cows during early lactation to favor
nutrient supply, especially glucose, towards the mammary gland. However, the molecular mechanisms of
insulin action on glucose metabolism in cows and in other ruminants are still not completely understood,
particularly during early lactation. Previous studies provide evidence that peripartal insulin resistance in
skeletal muscle of ruminants is caused by post-receptor changes of the insulin signaling pathway. The
protein content of glucose transporter 4 (GLUT4) is reduced in skeletal muscle of goats and cows at
begin of lactation (Balage et al., 1997; Kuhla et al., 2011). On the other hand, lactation does not have
DQHIIHFWRQWKHQXPEHUWKHDI¿QLW\RUWKHDFWLYLW\RIWKHW\URVLQHNLQDVHRIWKHLQVXOLQUHFHSWRU ,QV5 
in sheep (Wilson et al. DQGJRDWV %DODJHet al., 1992). Furthermore, Duhlmeier et al. (2005)
showed differences in protein amount of GLUT 1 and 4 in oxidative and glycolytic muscles of lactating
cows. We have tested the hypothesis that insulin signaling in skeletal muscle of dairy cows is related
to the stage of lactation and differs among various muscle types. Furthermore, we have investigated
WKHLQÀXHQFHRIGLIIHUHQWSRVWFDOYLQJPHWDEROLFVWDWXVRIGDLU\FRZVDVUHÀHFWHGE\GLYHUJHQWIDW
mobilization and liver fat concentration (LFC) on insulin signaling in muscle tissue.

Material and methods

*HUPDQ+ROVWHLQFRZV Q !NJPLONG•nd lactation) were fed twice daily a total mixed
ration according to their physiological state. Liver biopsies were taken on d 3, 18, and 30 post partum
(pp) to determine LFC. Depending on their mean LFC pp, cows were retrospectively grouped in either
low LFC (LLFC) (n=9) or high LFC (HLFC) (n=10). Muscle biopsies of M. semitendinosus (ST) were
taken on d 17 ante partum (ap), and d 3 and 30 pp to perform relative quantitative real time RT-PCR
using the LightCycler 2.0 (Roche Molecular Biochemicals, Mannheim, Germany) and SYBR Green I
DVWKHÀXRUHVFHQWG\H +DPPRQet al., 2009) to analyze parameters involved in insulin signaling: insulin
receptor (InsR), insulin receptor substrate 1 (IRS1), regulatory subunit (p85) and catalytic subunit (p110)
of phosphatidylinositol-3-kinase (PI3K), insulin sensitive glucose transporter (GLUT4) and insulin-
independent glucose transporter (GLUT1). Hydroxymethylbilane synthase (HMBS) and splicing factor
3 subunit 1 (SF3A1) were used as reference genes (Pérez et al., 2008). Corresponding blood samples
for measurement of plasma insulin concentrations were taken before tissue sampling on d 17 ap and d
3 and 30 pp, respectively. Cows were slaughtered on d 40 pp and samples of ST, M. longissimus dorsi
(LD), and M. masseter (MA) were collected to investigate the insulin signaling pathway on mRNA level
in different muscle types. Additionally in all three muscle types the protein abundance of GLUT1 and
GLUT4 was determined by Western Blot. Data are presented as LSmeans ± SE and were analyzed by
WKH0L[HG0RGHORI6$6ZLWKJURXSDQGPXVFOHW\SHRUWLPHDV¿[HGHIIHFWV

Results and discussion

3ODVPDLQVXOLQFRQFHQWUDWLRQVGHFUHDVHGVLJQL¿FDQWO\IURPDSWRSS P<0.001) but were not affected
by LFC (pooled LSmean ± SE: 0.75±0.1 μg/l, 0.25±0.1 μg/l and 0.35±0.1 μg/l on d 17 ap, and d
3 and d 30 pp). In ST muscle biopsies, mRNA expression of all parameters except for GLUT4
changed with time, with GLUT1 increasing after calving (P<0.01), InsR, p85, and p110 being
highest around calving (P<0.01), and IRS1 tending to decrease after calving (P< )XUWKHUPRUH
InsR showed a group × time interaction (P<0.05) and p85 was higher expressed in LLFC than in
HLFC (P< GXULQJWKHWUDQVLWLRQIURPSUHJQDQF\WRODFWDWLRQ7KHVHUHVXOWVFRQ¿UPGHFUHDVHG

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 277
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_92, © Wageningen Academic Publishers 2013
plasma insulin concentrations pp and the hypothesis that there are insulin signaling post-receptor
changes during lactation in bovine muscle tissue, but the gene expression of GLUT4 is not involved
in the pp insulin resistance in muscle tissue of cows (Komatsu et al. +RZHYHUWKHUHPLJKW
be post-transcriptional regulation of GLUT4 in skeletal muscle, because the protein expression of
GLUT4 is reduced with onset of lactation (Kuhla et al., 2011). This assumption is supported by
the observed increase of p85 abundance around calving which may lead to a shift from p85-p110
heterodimers to p85 monomers, resulting in a decline of PI3K activity and consequently reduced
insulin signaling on GLUT4 translocation (Ueki et al., 2002). Additionally, the tendency of IRS1
WRGHFUHDVHDIWHUFDOYLQJVXJJHVWVUHGXFHGLQVXOLQVLJQDOLQJEHFDXVH,56LVWKH¿UVWGRZQVWUHDP
signal of the intracellular insulin signaling cascade and hence superior for the further signaling steps
regulating GLUT4 translocation. The increased GLUT1 mRNA expression after calving in this study
supports the idea of an insulin-independent glucose transport for maintaining a basic glucose supply
for glycolytic muscle cells during lactation (Duhlmeier et al., 2005). The increased expression of
InsR on d 3 pp, mainly in HLFC cows, may indicate an inverse response to the reduced plasma
insulin concentrations around parturition (Hammon et al., 2009).

At slaughter on d 40 pp, mRNA expression of all parameters except p110 depend on the muscle type
with GLUT4 and InsR being highest and p85 and IRS1 being lowest in MA (P<0.01). GLUT1 and
GLUT4 were higher (P<0.05; P<0.01), but IRS1 and p85 were lower (P<0.01) expressed in ST
than in LD. LFC affected p85 with higher (P<0.05) mRNA abundance in LLFC than HLFC. The
protein concentrations of GLUT4 corresponded with its mRNA concentrations in all three muscle
W\SHVDQGWKHSURWHLQDEXQGDQFHRI*/87ZHUHVLJQL¿FDQWO\ORZHU P<0.01) in MA than in ST
DQG/'2XUUHVXOWVDUHLQFRQVLVWHQFHZLWK¿QGLQJVLQURGHQWVGHVFULELQJWKDWLQVXOLQVLJQDOLQJLV
related to the type of muscle (Song et al., 1999). Results on GLUT1 and GLUT4 protein abundance
were supported by those of Duhlmeier et al. (2005).

In conclusion our results indicate that insulin signaling in bovine skeletal muscle depends on skeletal
muscle type and stage of lactation, but is barely affected by post-calving fat mobilization.

References
Balage, M., C. Sornet, and J. Grizard. 1992. Insulin receptor binding and kinase activity in liver and skeletal muscles
RIODFWDWLQJJRDWV$P-3K\VLRO 3W (
Balage, M., J.F. Hoquette, B. Graulet, P. Ferré, and J. Grizard. 1997. Skeletal muscle glucose transporter (GLUT-4)
SURWHLQLVGHFUHDVHGLQODFWDWLQJJRDWV$QLP6FL
Duhlmeier, R., A. Hacker, A. Widdel, W. von Engelhardt, and H. P. Sallmann. 2005. Mechanisms of insulin-dependent
glucose transport into porcine and bovine skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 289:R187-R197.
Hammon, H. M., G. Stürmer, F. Schneider, A. Tuchscherer, H. Blum, T. Engelhard, A. Genzel, R. Staufenbiel, and W.
Kanitz. 2009. Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-
\LHOGLQJGDLU\FRZVZLWKKLJKDQGORZIDWFRQWHQWLQOLYHUDIWHUFDOYLQJ-'DLU\6FL
Komatsu, T., F. Itoh, S. Kushibiki, and K. Hodate. 2005. Changes in gene expression of glucose transporters in lactating
DQGQRQODFWDWLQJFRZV-$QLP6FL
Kuhla B., G. Nürnberg, D. Albrecht, S. Görs, H.M. Hammon, and C.C. Metges, 2011. Involvement of skeletal muscle
SURWHLQJO\FRJHQDQGIDWPHWDEROLVPLQWKHDGDSWDWLRQRQHDUO\ODFWDWLRQRIGDLU\FRZV-3URWHRPH5HV
Pérez, R., I. Tupac-Yupanqui, and S. Dunner. 2008. Evaluation of suitable reference genes for gene expression studies
in bovine muscular tissue. BMC Mol Biol. 9:79.
6RQJ;0-:5\GHU<.DZDQR$9&KLEDOLQ$.URRNDQG-5=LHUDWK0XVFOH¿EUHW\SHVSHFL¿FLW\LQ
LQVXOLQVLJQDOWUDQVGXFWLRQ$P-3K\VLRO 3W 5
Ueki, K., D.A. Fruman, S.M. Brachmann, Y. Tseng, L.C. Cantley, and C.R. Kahn. 2002. Molecular balance between
the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol. Cell.
Biol.   .
:LOVRQ/$6(0LOOV()LQOH\(.LOJRXU3-%XWWHU\DQG5*9HUQRQ(IIHFWRIODFWDWLRQRQLQVXOLQVLJQDO
WUDQVGXFWLRQLQVKHHSDGLSRVHWLVVXHDQGVNHOHWDOPXVFOH-(QGRFULQRO  

278 Energy and protein metabolism and nutrition in sustainable animal production
,QÀXHQFHRIDGAT1 polymorphism on response of dairy cows to ruminal
supplementation of linseed oil
A.M. van Vuuren1, L. Kruijt1, H.C.A. Widjaja1, A. Klop1, A.J. Cnossen1, D. Anjema2 and J. van Baal
1:DJHQLQJHQ 85 /LYHVWRFN 5HVHDUFK 32 %R[   $% /HO\VWDG WKH 1HWKHUODQGV
[email protected]
2&HQWUDO9HWHULQDU\,QVWLWXWH32%R[$%/HO\VWDGWKH1HWKHUODQGV
$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands

Introduction
The DGAT1JHQHHQFRGHVIRUWKHGLDF\OJO\FHURODF\OWUDQVIHUDVHHQ]\PHZKLFKFDWDO\]HVWKH¿QDO
step in triacylglycerol synthesis. Various polymorphisms have been reported for DGAT1. One of
them, the K232A DGAT1 polymorphism in which lysine as the 232nd amino acid is replaces by
alanine, is associated with increased milk yield, lower fat and protein concentrations (Grisart et al.,
2001) and altered milk fatty acid (FA) composition (Schennink et al.,2007). Milk FA composition
is, on the other hand, also affected by nutritional factors including dietary FA composition (Sterk
et al., 2011). Until now, no literature data could be retrieved to establish whether the response in
PLON)$FRPSRVLWLRQRIGDLU\FRZVWRFKDQJHVLQGLHWDU\)$FRPSRVLWLRQLVLQÀXHQFHGE\DGAT1
polymorphism. Therefore, a study was performed with 8 rumen-cannulated dairy cows in mid-
lactation with different DGAT1 polymorphism, receiving either palm fat or linseed oil.

Material and Methods

Eight rumen-cannulated cows were selected and blocked depending on DGAT1 polymorphism: 4
cows were genotyped as AA and 4 as KK mutant. Cows were allocated to treatments in a change-
over designed experiment with 2 periods. Cows were fed a partial mixed ration (49% grass silage,
33% corn silage, 11% soy bean meal, 5% and rapeseed meal and 2% premix on dry matter basis) ad
libitum and 1 kg of concentrates during milking. Dietary treatments were palm fat and linseed oil
supplementation at 500 g/d, dosed intra-ruminal in 2 equal portions after milking. Each treatment
was given for two weeks. During the last 4 milkings of each period, milk yield was recorded and
milk samples were taken to determine fat, protein and lactose content and milk FA composition.
Data were statistically analyzed by ANOVA with cow and period as random, and genotype and
WUHDWPHQWDV¿[HGHIIHFWV

Results and discussion

&RZVFRQVXPHGRQDYHUDJHNJGU\PDWWHUGD\LQFOXGLQJNJRIFUXGHSURWHLQGNJ
RI1')GDQGNJFUXGHIDWG&RPSDUHGWR..JHQRW\SHFRZVZLWK$$JHQRW\SHSURGXFHG
numerically less milk and had a lower milk fat and protein content (Table 1). The lower milk yield
for cows with the AA genotype is in contrast with other observations (Grisart et al., 2001; Schennink
et al., 2007), but was attributed to the difference in stage of lactation between cows. Compared to
KK genotype, milk fat of cows with AA genotype had higher proportions of oleic and linoleic acids,
which was compensated by lower proportions of caproic, caprylic and capric acid (Table 2), the
latter in agreement with observations by Schennink et al. (2007).

As expected, compared to palm fat, feeding linseed oil resulted in a lower milk fat content. Compared
to palm fat, feeding linseed oil resulted in higher proportions of C:18 FA in milk fat, which was
FRPSHQVDWHGE\ORZHUSURSRUWLRQVRI&DQG&)$*HQHUDOO\QRJHQRW\SHîIDWVRXUFH
interactions could be observed, except for stearic acid and cetoleic acid (C20:1, n11).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 279
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_93, © Wageningen Academic Publishers 2013
Table 1. Effect of lipid supplementation and DGAT1 polymorphism on milk yield and milk composition
of dairy cows.

Parameter Treatment (lipid source) SED P-value

Linseed Palm

DGAT1 genotype G L L×G

AA KK AA KK

Milk production
Milk, kg /d 18.0 21.4 18.8 22.0 0.7 0.50  0.94
Fat, kg/d 0.59 0.81  1.14 0.07 0.04 0.01 0.05
Protein, kg/d  0.89  0.90 0.03 0.18 0.32 0.85
Milk composition
Fat, g/kg 33.0 40.7 37.3 51.9 2.4 0.02 0.01 0.10
Protein, g/kg  42.3  41.5 0.97 0.03  0.54

1 L: lipid source; G: DGAT1 mutation; L×G: interaction.

Table 2. Effect of lipid supplementation and DGAT1SRO\PRUSKLVPRQPLONIDWW\DFLGSUR¿OH J

J)$ 

Fatty acid Treatment (lipid source) SED P-value

Palm Linseed

DGAT1 genotype G L L×G

AA KK AA KK

C4:0 3.9 4.2 4.0 4.2 0.07 0.43 0.18 0.35

& 1.9 2.3 1.9 2.3 0.05 0.03 1.00 0.51
C8:0 0.8 1.1 0.9 1.1 0.04 0.04  
C10:0 1.7 2.2 1.8 2.2 0.12 0.05 0.43 0.79
C12:0 2.3  2.4 2.8 0.13  0.12 
C14:0 8.9 9.2 9.5 9.7   0.03 0.95
& 32.3 34.1 22.9 25.5 0.92  <0.001 0.57
&Q 2.2 2.2 1.4  0.08 0.90 <0.001 0.15
C18:0  9.4 10.0 10.0 0.25 0.59 0.01 
C18:1, n9 24.3 20.7 25.8 21.8 0.45 0.03 0.01 
C18:1, trans   4.2 4.3 0.49 0.91 0.01 0.84
C 18:1 other 1.3 1.2 2.3 2.1 0.14 0.27 <0.001 0.81
&Q 1.3 1.2 1.5 1.4 0.04 0.01 0.001 1.00
C18:2, c9,t11 0.8 0.7 1.7  0.20 0.49 0.01 0.93
C18:2, trans 1.2 1.0 3.3 3.0  0.40 <0.001 0.70
C18:3, n3 0.4 0.4 0.5 0.4 0.03 0.28 0.03 
C 20:1, n11 0.1 0.1 0.2 0.2 0.02  <0.001 0.03

1 L: lipid source; G: DGAT1 mutation; L×G: interaction.

280 Energy and protein metabolism and nutrition in sustainable animal production
Conclusion
Although K232A DGAT1SRO\PRUSKLVPDOWHUHGPLONFRPSRVLWLRQDQGPLON)$SUR¿OHWKHUHVSRQVH
LQPLONFRPSRVLWLRQDQGPLON)$SUR¿OHWRVXSSOHPHQWLQJOLQVHHGRLOZDVVLPLODUIRU$$DQG..
genotype cows.

References
Grisart, B., Coppieters, W., Farnir, F., Karim, L., Ford, C., Berzi, P., Cambisano, N., Mni, M., Reid, S., Simon, P.,
6SHOPDQ5*HRUJHV06QHO53RVLWLRQDOFDQGLGDWHFORQLQJRID47/LQGDLU\FDWWOH,GHQWL¿FDWLRQRI
a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition. Genome Res.
12, 222-231.
Schennink, A., Stoop, W.M., Visker, M.H.P.W., Heck, J.M.L., Bovenhuis, H., van der Poel, J.J., van Valenberg, H.J.F.
and van Arendonk J.A.M., 2007. DGAT1 underlies large genetic variation in milk-fat composition. Anim. Genet.


Energy and protein metabolism and nutrition in sustainable animal production 281
Correlations between plasma ghrelin and parameters of fat metabolism
in early lactating dairy cows
S. Börner1, M. Derno1, H.M. Hammon1, M. Röntgen1, S. Thanthan2, H. Kuwayama2 and B. Kuhla1
1Institute of Nutritional Physiology ‘Oskar Kellner’, Leibniz Institute for Farm Animal Biology
)%1 'XPPHUVWRUI*HUPDQ\[email protected]
2Agriculture and Veterinary Medicine Inada-cho, Department of Life Science and Agriculture,
2ELKLUR8QLYHUVLW\RI2ELKLUR&LW\1LVKL+RNNDLGR-DSDQ

Introduction
The peptide hormone ghrelin is produced in the ruminal and proximal duodenal wall with a small
SRUWLRQRIJKUHOLQEHLQJSRVWWUDQVODWLRQDOO\PRGL¿HGE\IDWW\DFLGVDW6HU%RWKIRUPV GHVDF\O
and acyl ghrelin) are released into the blood stream and have been initially assigned a role in the
control of feed intake. While acyl ghrelin increases feed intake, desacyl ghrelin may exert opposing
effects. However, there is accumulating evidence for the involvement of ghrelin in fat allocation
and utilization involving liver, muscle and adipose tissue (Barazzoni et al., 2005; Jennings et al.,
2011; Rodríguez et al., 2009). Therefore, it seems possible that ghrelin may be involved in body
fat metabolism of dairy cows during the transition period. Here we examined the hypothesis that
ghrelin could play a role in the extent of body fat mobilization during early lactation in dairy cows.

Material and methods

Sixteen Holstein cows (2nd-4th lactation) were kept in respiratory chambers to determine the
UHVSLUDWRU\TXRWLHQW 54 GXULQJWZRH[SHULPHQWDOZHHNV ZNDQGUHODWLYHWRSDUWXULWLRQ 
Prior to each experimental period, body weight and back fat thickness (BFT) were measured. On
the 1st day of each experimental period animals received ad libitum a total mixed ration (TMR)
offered twice daily (07:00 and 15:00 h). Dry matter intake (DMI) was recorded daily and milk yield
and milk composition was analyzed for each milking. At the beginning of the 2nd day, animals were
feed-deprived for 10 h to study the preprandial ghrelin surge. Afterwards, animals were re-fed ad
libitum to determine 14 h-compensatory feed intake. On the 1st and 2nd day, serial blood samples
ZHUHWDNHQLQPLQLQWHUYDOVDQGDQDO\]HGIRUSODVPDDF\ODQGWRWDO DF\OGHVDF\O JKUHOLQQRQ
HVWHUL¿HGIDWW\DFLGV 1()$ DQGWULJO\FHULGHV2QWKHth day, liver biopsies were taken. Based on
the liver fat content (LFC) in week 2 post partum (pp), cows were retrospectively assigned to a high
(H, n=8, LFC>32% fat/g dry matter (DM)) and low (L, n=8, LFC<31% fat/g DM) fat mobilizing
group. Data were evaluated by the MIXED procedure of SAS. Linear correlations were calculated
using the CORR procedure of Base SAS. The concentration of the plasma ghrelin peak (after 9 h
feed-deprivation) was correlated with the subsequent 14 h-compensatory feed/energy intake or with
SDUDPHWHUVUHÀHFWLQJIDWPHWDEROLVP

Results and discussion

Ad libitum and 14-h compensatory feed intake did not differ between groups, neither ante partum (ap)
nor pp. BFT was higher (3 0.01) but RQ was lower in H cows (P<0.05), both ap and pp. During
ad libitum feeding, plasma NEFA concentrations were higher in H cows pp (P<0.01), while group
differences were not evident during the feed-deprivation period (3 0.2). Preprandial triglycerides
were lower pp (P<0.01), whereas preprandial total ghrelin concentration were lower ap (3 0.03)
in H cows. Preprandial acyl ghrelin concentration (P<0.01) and the preprandial acyl:total ghrelin
ratio (P<0.02) were higher in H cows, both ap and pp. The pp preprandial acyl ghrelin concentration
correlated positively with plasma NEFA concentrations (Table 1). Correlations between pp preprandial
WRWDOJKUHOLQDQGSDUDPHWHUVUHÀHFWLQJIDWPHWDEROLVPRUIHHGLQWDNHZHUHQRWHYLGHQW (Table 1). On
the contrary, the preprandial acyl:total ghrelin ratio pp correlated positively with LFC, BFT, and milk

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 283
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_94, © Wageningen Academic Publishers 2013
fat content, but negatively with RQ, plasma NEFA and TG (Table 1). There were no correlations
between acyl ghrelin, total ghrelin or the acyl:total ghrelin ratio with DMI or NEL intake. These
results imply that endogenous ghrelin forms are not major players in controlling feed intake in dairy
cows. The acyl:total ghrelin ratio is rather suggested to play a prominent role in determining fat
allocation, fatty liver, and fat utilization during the periparturient period.

7DEOH&RUUHODWLRQFRHI¿FLHQWVEHWZHHQSSSUHSUDQGLDODF\OJKUHOLQWRWDOJKUHOLQWKHDF\OWRWDO
JKUHOLQUDWLRDQGSDUDPHWHUVUHÀHFWLQJIDWPHWDEROLVPRUIHHGLQWDNHZLWKP<

acyl ghrelin total ghrelin acyl:total ghrelin ratio

Plasma NEFA  ns ns

Plasma TG ns ns ns
Milk fat content ns ns 
LFC ns ns 
BFT ns ns 0.75
RQ ns ns -0.78
DMI ns ns ns
NEL intake ns ns ns

QV QRVLJQL¿FDQFH

Acknowledgements
6XSSRUWHGE\')* .8 *HUPDQ\

References
Barazzoni R., A. Bosutti, M. Stebel, M.R. Cattin, E. Roder, L. Visintin, L. Cattin, G. Biolo, M. Zanetti, and G. Guarnieri.
2005. Ghrelin regulates mitochondrial-lipid metabolism gene expression and tissue fat distribution in liver and
skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 288, E228-235.
Jennings J.S., A.E. Wertz-Lutz, R.H. Pritchard, A.D. Weaver, D.H. Keisler, and K. Bruns 2011. Circulating ghrelin and
leptin concentrations and growth hormone secretagogue receptor abundance in liver, muscle, and adipose tissue
of beef cattle exhibiting differences in composition of gain. J. Anim. Sci. 89, 3954-3972.
Rodríguez A., J. Gómez-Ambrosi, V. Catalán, M.J. Gil, S. Becerril, N. Sáinz, C. Silva, J. Salvador, I. Colina, and G.
Frühbeck,. 2009. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Int. J.
Obes. (Lond). 33, 541-552.

284 Energy and protein metabolism and nutrition in sustainable animal production
Effects of realimentation after nutrient restriction during early to mid-
gestation on pancreatic digestive enzymes in beef cattle
F.E. Doscher1, E.A. Nere1, R.D. Yunusova1, L.E. Camacho1, C.O. Lemley2, L.D. Prezotto1, K.A.
Vonnahme1, J.S. Caton1 and K.C. Swanson1
1'HSWRI$QLPDO6FLHQFH1RUWK'DNRWD6WDWH8QLYHUVLW\$OEUHFKW%RXOHYDUG)DUJR1'
86$[email protected]
2'HSWRI$QLPDODQG'DLU\6FLHQFH0LVVLVVLSSL6WDWH8QLYHUVLW\:LVH&HQWHU'U0LVVLVVLSSL
6WDWH0686$

Introduction
Maternal nutrition has been shown to impact fetal pancreatic endocrine development (Dahri et
al., 1995). However, less is known about its effects on exocrine function. The objectives were to
determine the effect of nutrient restriction and realimentation during gestation on maternal and fetal
pancreatic mass and digestive enzymes.

Material and methods

2QGD\RISUHJQDQF\PXOWLSDURXVEHHIFRZV Q “NJ ZHUHUDQGRPO\DVVLJQHGWR
GLHWDU\WUHDWPHQWVFRQWURO &15&Q  DQGQXWULHQWUHVWULFWLRQ 515&Q  2Q
GD\RIJHVWDWLRQFRZVZHUHHLWKHUVODXJKWHUHG &Q DQG5Q  RUUHPDLQHGRQFRQWURO &&
n=12), restricted (RR; n=12) or were realimented to control (RC; n=11). On day 140 cows were
HLWKHUVODXJKWHUHG &&Q 55Q 5&Q  RUUHPDLQHGRQFRQWURO &&&Q 5&&Q  RU
UHDOLPHQWHGWRFRQWURO 55&Q  2QGD\DOOUHPDLQLQJFRZVZHUHVODXJKWHUHG0DWHUQDODQG
fetal pancreas were weighed and a subsample collected and snap-frozen in liquid nitrogen and stored
DWƒ&XQWLODQDO\VLVIRUĮDP\ODVHDQGWU\SVLQDFWLYLWLHV'DWDZHUHDQDO\]HGDVDFRPSOHWHO\
randomized block (breeding group) design within slaughter date using the mixed procedure of SAS.
For fetal measurements, sex was also included in the model. Fetal pancreas was not collected at day 85.

Pancreatic tissue (0.25 g) was homogenized in 0.9% NaCl (2.25 ml) using a polytron (Brinkmann
,QVWUXPHQWV,QF:HVWEXU\1<86$ $FWLYLW\RIĮDP\ODVHZDVGHWHUPLQHGXWLOL]LQJDNLWIURP
Teco Diagnostics (Anaheim, CA, USA). Trypsin activity was assayed using the methods described
E\*HLJHUDQG)ULW]  DIWHUDFWLYDWLRQZLWK8OHQWHURNLQDVH *OD]HUDQG6WHHU 2QH
unit (U) of enzyme activity equals 1 μmole product produced per min. Enzyme activity data are
expressed as U/g wet tissue, kU/pancreas, and U/kg BW.

Results and discussion

No effects (P>0.05) were observed for maternal pancreas measurements on day 85. Absolute weight
of maternal pancreas (g) on day 140 was greater (P<0.04) in CC cows compared to RC and RR cows.
Relative weight (% of BW) also varied with CC and RR being greater (P<0.01) than RC. Maternal
ĮDP\ODVHDFWLYLW\RQGD\ZDVQRWDIIHFWHG P> E\WUHDWPHQW 7DEOH 0RUHRYHUQRHIIHFWV
(P>0.33) on fetal pancreatic weight or enzyme activity were observed on day 140 among treatments.
Plane of nutrition affects maternal pancreatic weights which could impact pancreatic function.

On day 254, no effects (P>0.10) on maternal pancreatic weight or enzyme activity were observed.
)HWDOĮDP\ODVHDFWLYLW\ 8NJRI%: ZDVJUHDWHU P<0.04) in RRC compared to CCC and RCC
7DEOH 1XWULHQWUHVWULFWLRQDQGUHDOLPHQWDWLRQGXULQJHDUO\JHVWDWLRQLQÀXHQFHGWKHDFWLYLW\RI
ĮDP\ODVHLQIHWDOSDQFUHDVGXULQJODWHJHVWDWLRQVXJJHVWLQJWKDWPDWHUQDOQXWULWLRQPD\LQÀXHQFH
the development of the fetal exocrine pancreas.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 285
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_95, © Wageningen Academic Publishers 2013
7DEOH,QÀXHQFHRIQXWULHQWUHVWULFWLRQDQGUHDOLPHQWDWLRQRQSDQFUHDWLFĮDP\ODVHDFWLYLW\LQ
SUHJQDQWFRZVDWGD\RIJHVWDWLRQ

Item Treatment SEM P-value

CC1 RC2 RR3

BW (kg)   542 32.4 0.22

Pancreas weight
g 474 354 434 33.2 0.048
% of BW 0.078  0.079 0.004 0.011
Į$P\ODVH
U/g  90.4 174 52.3 0.44
kU/pancreas 71.5 33.7 71.9 21.84 0.32
U/kg of BW   140 40.34 

1 Fed at 100% of NRC recommendations from day 30 to 140.

2)HGDWRI15&UHFRPPHQGDWLRQVIURPGD\WR
3)HGDWRI15&UHFRPPHQGDWLRQVIURPGD\WRDQGRI15&UHFRPPHQGDWLRQVIURPGD\WR

7DEOH,QÀXHQFHRIUHDOLPHQWDWLRQRQSDQFUHDWLFĮDP\ODVHDFWLYLW\LQRYLQHIHWXVHVDWGD\
of gestation.

Item Treatment SEM P-value

CCC1 RCC2 RRC3

BW (kg)  35.4 35.8 2.30 0.77

Pancreas weight
g 24.4 23.3   0.35
% of BW   0.073 0.0048 0.24
Į$P\ODVH
U/g 1.84 5.55  2.020 0.09
kU/pancreas  0.131  0.0505 0.10
U/kg of BW 1.13a 3.45b 4.23b 1.118 0.049

1 Fed at 100% of NRC recommendations from day 30 to 254.

2)HGDWRI15&UHFRPPHQGDWLRQVIURPGD\WRDQGRI15&UHFRPPHQGDWLRQVIURPGD\WR
3)HGDWRI15&UHFRPPHQGDWLRQVIURPGD\WRDQGRI15&UHFRPPHQGDWLRQVIURPGD\WRWR
ab Means with uncommon superscripts differ (P<0.05).

References
'DKUL6%5HXVHQ&5HPDFOH--+RHW1XWULWLRQDOLQÀXHQFHVRQSDQFUHDWLFGHYHORSPHQWDQGSRWHQWLDOOLQNV
ZLWKQRQLQVXOLQGHSHQGHQWGLDEHWHV3URF1XWU6RF  
*HLJHU5DQG+)ULW]7U\SVLQ3DJHVLQ0HWKRGVRI(Q]\PDWLF$QDO\VLV+%HUJPH\HUHG$FDG
Press, New York, NY.
Glazer, G. and M.L. Steer, 1977. Requirements for activation of trypsinogen and chymotrypsinogen in rabbit pancreatic
juice. Anal. Biochem. 77:130-140.

286 Energy and protein metabolism and nutrition in sustainable animal production
,QKLELWRU\HIIHFWRIDPPRQLDRQXUHDÀX[DFURVVUXPHQHSLWKHOLXP
depends on level of serosal urea
M.E. Walpole, G.B. Penner and T. Mutsvangwa
'HSDUWPHQWRI$QLPDODQG3RXOWU\6FLHQFH8QLYHUVLW\RI6DVNDWFKHZDQ6DVNDWRRQ6.61$
Canada; [email protected]

Introduction
Ruminants perpetually depend on their ability to transfer urea from blood to the rumen in order to
maintain a positive N balance. Estimates indicate that 40 to 80% of endogenously-produced urea can
be recycled to the rumen where it provides ruminally-available N for microbial growth (Lapierre and
Lobley, 2001), so it is important to understand the regulatory mechanisms that control trans-epithelial
urea transfer into the rumen. Increasing ruminal ammonia (NH3-N) concentration decreases trans-
epithelial urea transfer from blood into the rumen (Abdoun et al., 2009); however, the mechanisms
that are responsible for this response are uncertain. Facilitative urea transporter proteins (UT-B) are
expressed in ruminal epithelium and the addition of phloretin, an inhibitor of UT-B function, reduces
WUDQVHSLWKHOLDOÀX[RIXUHDLQLVRODWHGUXPLQDOHSLWKHOLXP 6WHZDUWet al., 2005), thus providing proof
that these UT-B might have a functional role in ruminal urea transfer. However, the functional role of
UT-B in mediating the effects of ruminal NH31RQWUDQVHSLWKHOLDOÀX[RIXUHDUHPDLQVREVFXUH:H
evaluated the effects of mucosal NH3 and serosal urea concentrations on total and UT-B-dependent
LHSKORUHWLQVHQVLWLYH XUHDÀX[DFURVVWKHLVRODWHGERYLQHUXPLQDOHSLWKHOLXP

Material and methods

Six Holstein male calves fed a common diet for ad libitum intake at 0700 h were used. The diet
was comprised of 38% alfalfa hay, 31% barley silage, 20% rolled barley and 11% protein-mineral
supplement, with a dietary crude protein (CP) content of 14.2% (DM basis). Calves were slaughtered
(1000 h) and the ruminal epithelium was collected and mounted in Ussing chambers under short-
circuit conditions. To mimic physiological conditions, the serosal buffer (pH 7.4) contained 1.78
(LU) or 7.14 (HU) mMXUHDZKHUHDVWKHPXFRVDOEXIIHU S+ KDGQRXUHDEXWFRQWDLQHG /$ 
or 8.8 (HA) mM (NH4)2CO37KHVHURVDOWRPXFRVDOÀX[RI14C-urea (Jsm-urea; 42 kBq/15 ml) and
3H-mannitol (J
sm-mannitol; 37 kBq/15 ml) were measured, with Jsm-mannitol being used as an indicator
of hydrophilic movement. Serosal addition of phloretin (1 mM) was used to inhibit UT-B-mediated
urea transport. Data were analyzed using the Proc Mixed procedure (SAS, 2001) as a factorial
design, with regression analysis used to determine the relationship between Jsm-urea and Jsm-mannitol.

Results and discussion

Ruminal NH3DQGEORRGXUHD1FRQFHQWUDWLRQVDWVODXJKWHUDYHUDJHGDQGPM, respectively.
Flux measurements are presented in Table 1. High NH3 tended to inhibit total Jsm-urea with HU, but
there was no effect of NH3 on total Jsm-urea with LU (interaction, 3 0.055). The phloretin-sensitive
Jsm-urea was not affected by serosal urea (3 0.34) or mucosal NH3 (3  KRZHYHUSKORUHWLQ
insensitive Jsm-urea tended to be higher with LA than HA when incubated with HU but was not
different with LU (interaction, 3  7KH-sm-mannitol was not affected by urea (3  RU1+3
(3 0.22) concentration. The Jsm-urea and Jsm-mannitol tended to be positively correlated for HA but
not LA in combination with LU (Figure 1). The same pattern was observed with HU treatments
where Jsm-urea and Jsm-mannitol tended to be positively correlated for HA but not LA (Figure 1). We
conclude that luminal NH3 has an inhibitory effect on Jsm-urea when serosal urea concentration is
high, and that Jsm-urea is mediated via hydrophilic pathways at a low serosal urea concentration but
is mediated through non UT-B-dependent pathways when serosal urea concentration is high. Future
studies should determine other potential transporters involved in urea movement across the GIT.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 287
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_96, © Wageningen Academic Publishers 2013
7DEOH8UHDÀX[UDWHV>QPROFP2/h] across ruminal epithelial tissue incubated in Ussing chambers
ZLWKFRPELQDWLRQVRIKLJK +8 RUORZ /8 VHURVDOXUHD 8 DQGKLJK +$ RUORZ /$ PXFRVDO
DPPRQLD $ ZLWKRUZLWKRXWSKORUHWLQ 3 

Treatment SE P-value

HU LU U A U ×A

Item LA HA LA HA

Total Jsm-urea 771.8    30.4 <0.001  
P-insensitive Jsm-urea 781.7 717.5 181.7  34.4 <0.001 0.10 
Total Jsm-mannitol   51.1 44.9 7.3  0.22 0.38

70 70
Jsm-mannitol (nmolÂ(cm2)-1ÂK-1)

Jsm-mannitol (nmolÂ(cm2)-1ÂK-1)
R² = 0.301 R² = 0.324
60 60
P = 0.08 P = 0.08
50 50
40 40
30 30
20 R² = 0.027 20 R² = 0.111
10 P = 0.70 10 P = 0.32
0 0
0 100 200 300 0 500 1000
Jsm-urea (nmolÂ(cm2)-1ÂK-1) Jsm-urea (nmolÂ(cm2)-1ÂK-1)
Figure 1. Regression analysis between Jsm-urea and Jsm-mannitol across the ruminal epithelium in
8VVLQJFKDPEHULQFXEDWLRQVFRQWDLQLQJP0VHURVDOXUHDZLWK Ƈ RU Ŷ P0DPPRQLD
OHIWSDQHO RU8VVLQJFKDPEHULQFXEDWLRQVFRQWDLQLQJP0VHURVDOXUHDZLWK Ƈ RU
Ŷ P0DPPRQLD ULJKWSDQHO 

References
Abdoun, K., F. Stumpff, I. Rabbani, and H. Martens, 2010. Modulation of urea transport across sheep rumen epithelium
in vitro by SCFA and CO2. Am. J. Physiol. Gastrointest. Liver Physiol. 298, G190-G202
Lapierre, H., and G. E. Lobley, 2001. Nitrogen recycling in the ruminant: a review. J. Dairy Sci. 84(E. Suppl.),
((
Stewart, G. S., C. Graham, S. Cattell, T. P. L. Smith, and C. P. Smith, 2005. UT-B is expressed in bovine rumen: potential
UROHLQUXPLQDOXUHDWUDQVSRUW$P-3K\VLRO5HJXO,QWHJU&RPS3K\VLRO55

288 Energy and protein metabolism and nutrition in sustainable animal production
Effects of starch derived substrates on pancreatic and mucosal enzyme
activities in milk-fed calves
A.J. Pantophlet1, M.S. Gilbert2, J.J.G.C. van den Borne2, R.J. Vonk1, W.J.J. Gerrits2, A. Pluschke,
H.A. Schols, M.G. Priebe1 and J. Roelofsen1
1&HQWUHIRU0HGLFDO%LRPLFV8QLYHUVLW\0HGLFDO&HQWHU*URQLQJHQ32%R[55
Groningen, the Netherlands
2$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands; [email protected]
&HQWUHIRU1XWULWLRQDQG)RRG6FLHQFHV8QLYHUVLW\RI4XHHQVODQG6W/XFLD$XVWUDOLD
/DERUDWRU\RI)RRG&KHPLVWU\:DJHQLQJHQ8QLYHUVLW\32%R[(9:DJHQLQJHQWKH
Netherlands

Introduction
Į$P\ODVH DQGWKHPXFRVDOHQ]\PHVPDOWDVHDQGLVRPDOWDVHSOD\DQLPSRUWDQWUROHLQWKH
GLJHVWLRQRIVWDUFK3DQFUHDWLFĮDP\ODVHLVUHVSRQVLEOHIRUWKHEUHDNGRZQRISRO\VDFFKDULGHVLQWR
oligosaccharides such as maltose, maltotriose and dextrin. These oligosaccharides are subsequently
converted to monosaccharides by small intestinal mucosal enzymes. Veal calves are fed with milk
replacers (MR) which commonly contain 40-50% of lactose, originating from whey. The price of
ZKH\KRZHYHULVVXEMHFWHGWRH[WUHPHO\ODUJHÀXFWXDWLRQVDQGSURGXFHUVRI05DUHLQVHDUFKRI
alternatives such as starch-based substrates (SBSs).

The high lactase activity of calves enables rapid digestion of large amounts of lactose. If lactose is
UHSODFHGE\6%6VRWKHUHQ]\PHVVXFKDVĮDP\ODVHPDOWDVHDQGRULVRPDOWDVHDUHUHTXLUHGIRUWKH
complete breakdown of these products. However, the capacity and inducibility for enzymatic starch
digestion in milk-fed calves is unclear. Therefore, a study was designed to evaluate the effect of SBSs
RQSDQFUHDWLFĮDP\ODVHDQGPXFRVDOHQ]\PHDFWLYLWLHVZKHQJUDGXDOO\LQWURGXFHGLQPLONIHGFODYHV

Material and methods

)RUW\PDOH+ROVWHLQ)ULHVLDQFDOYHV “NJ DWWKHDJHRIZHHNVZHUHDOORWWHGWRRI
GLHWDU\WUHDWPHQWV'XULQJWKH¿UVWZHHNWKHFDOYHVUHFHLYHGDPLONUHSODFHUFRQWDLQLQJODFWRVHDV
exclusive source of carbohydrates. Thereafter, one group (n=8) remained on this control diet. In
the other groups (n=8 each) lactose was partly replaced by one of four SBSs. Each SBS required
GLIIHUHQWFRPELQDWLRQVRIHQ]\PHVIRUFRPSOHWHEUHDNGRZQ  JHODWLQL]HGVWDUFK ĮDP\ODVH
DQGPDOWDVH   PDOWRGH[WULQ ĮDP\ODVHDQGPDOWDVH   PDOWRGH[WULQZLWKĮEUDQFKLQJ
ĮDP\ODVHPDOWDVHDQGLVRPDOWDVH   PDOWRVH PDOWDVH 

$OOGLHWVLQFOXGHG&R('7$DVDQLQGLJHVWLEOHPDUNHU$IWHUDQDGDSWDWLRQSHULRGRIZHHNVLQ
which the percentage of SBSs was gradually increased to 18% at the expense of lactose, the calves
ZHUHVDFUL¿FHGDQGGLJHVWDIURPWKHDERPDVXP $% WKH¿UVWDQGVHFRQGKDOIRIWKHVPDOOLQWHVWLQH
60DQG60UHVSHFWLYHO\ ZHUHFROOHFWHGWRPHDVXUHWKHĮDP\ODVHDFWLYLW\ %LR9LVLRQ0LOSLWDV
USA). Also, mucosal scrapings were collected from three parts of the small intestine (SM1, SM2 and
the terminal ileum), to measure lactase, maltase and iso-maltase activity by the Dahlqvist method
'DKOTYLVW 

Results and discussion

Preliminary results of the mucosal enzyme activity of 4 calves from the control group and the
gelatinized starch group and 2 calves from the other groups are depicted in Figure 1A-C. Lactase
activity did not differ between dietary treatments, but differed between segments (P<0.01) and was

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 289
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_97, © Wageningen Academic Publishers 2013
 240 240

Maltase activity
Lactase activity 200 200
160 160
120 A 120 B
80 80
40 40
0 0
SM 1 SM 2 IL SM 1 SM 2 IL
240
3.50
Iso-maltase activity

Į-amylase activity
200 3.00
160 2.50
120 2.00
C 1.50 D
80
1.00
40 0.50
0 0.00
SM 1 SM 2 IL AB SM1 SM2
Control Gel starch Dextrin Dextrin with branching Maltose
)LJXUH(Q]\PHDFWLYLWLHVRI$ /DFWDVH LQ8PJSURWHLQ % 0DOWDVH LQ8PJSURWHLQ & 
,VRPDOWDVH LQ8PJSURWHLQ ' Į$P\ODVH LQ8PJ&R IRUHDFKRIWKH¿YHGLHWDU\WUHDWPHQWV
60 ¿UVWKDOIRIWKHVPDOOLQWHVWLQH60 VHFRQGKDOIRIWKHVPDOOLQWHVWLQHDQG,/ ,OHXP

KLJKHULQ60 “8PJSURWHLQ WKDQLQ60 8PJSURWHLQ /DFWDVHDFWLYLW\ZDV

DOPRVWXQGHWHFWDEOHLQWKHWHUPLQDOLOHXP “8PJSURWHLQ 0DOWDVHDQGLVRPDOWDVHDFWLYLWLHV
were low in SM1 (4.8±1.1 and 2.5±0.3 U/mg protein, respectively) and higher (P<0.01) in SM2
“DQG“8PJSURWHLQUHVSHFWLYHO\ DQGWHUPLQDOLOHXP “DQG“8
PJSURWHLQUHVSHFWLYHO\ +RZHYHUWKHUHZDVQRVLJQL¿FDQWGLIIHUHQFHDPRQJGLHWDU\WUHDWPHQWV

/XPLQDOĮDP\ODVHDFWLYLW\ZDVPHDVXUHGLQDOOFDOYHVH[SUHVVHGUHODWLYHWRWKHLQGLJHVWLEOHPDUNHU
&R DQGWKHUHVXOWVDUHGHSLFWHGLQ)LJXUH'7KHĮDP\ODVHDFWLYLW\LQWKHGLJHVWDRIWKHFRQWURO
calves was almost undetectable (0 for AB, 0.3±0.3 and 0 U/g Co for SM1 and SM2, respectively),
but increased by feeding of SBSs, particularly in SM2 (1.7±0.3 U/g Co). This was probably due to
WKHLQGXFWLRQRIPLFURELDOĮDP\ODVHDFWLYLW\LQ60+RZHYHUZLWKWKHDP\ODVHDFWLYLW\NLWLWZDV
not possible to distinguish between pancreatic and bacterial amylase activity.

Based on these preliminary results we conclude that the activity of mucosal enzymes required for
the hydrolysis of starch cannot be induced by feeding SBSs in milk-fed calves. We speculate that a
considerable part of the starch disappearance at the terminal ileum in milk-fed calves is caused by
degradation by microbial enzymes.

References
'DKOTYLVW$0HWKRGIRUDVVD\RILQWHVWLQDOGLVDFFKDULGHV$QDO%LRFKHP

290 Energy and protein metabolism and nutrition in sustainable animal production
Effect of heat stress on intake and metabolism of Bos taurus (Angus) and
Bos indicus (Nellore)
E.E.L. Valente1, M.L. Chizzotti1, C.V.R. Oliveira1, M.C. Galvão1, S.S. Domingues1, A.C. Rodrigues1
M.M. Ladeira1 R.A. Ferreira1 and F.H.M. Chizzotti2
1Animal Science Departament, Universidade Federal de Lavras, Lavras, Brazil
2 Animal Science Departament, Universidade Federal de Viçosa, Viçosa, Brazil;
PDULRFKL]]RWWL#G]RXÀDEU

Introduction
During heat stress, ruminants like other homeothermic animals, use pathways to increase heat
loss and reduce heat production to maintain euthermia (Bernabucci et al., 2010). Environmentally
LQGXFHGK\SHUWKHUPLDGHFUHDVHVHI¿FLHQF\DQGSURGXFWLRQ 2¶%ULHQet al. 2QHRIWKH¿UVW
noticeable signs of heat stress (HS) during production is reduced nutrient intake, which is presumably
an evolutionary strategy to reduce the ‘heat increment’ of feeding (O’Brien et al., 2010). The aim
of this study was to evaluate the effect of different air temperatures on diurnal, nocturnal, and daily
feed intake as well as water intake, heart rate, respiratory rate and body temperature.

Material and methods

Sixteen young bulls (8 Angus, Bos taurus, and 8 Nellore, Bos indicus ZLWK“NJDQG
months of age were used. The experimental period was divided in two 10 days periods, after 7 days
of adaptation. Two climate-controlled rooms, with a 12×12 h photoperiod, were used. In period 1 all
animals were housed in thermoneutral ambient temperature (TN, 25.4±0.4 °C). In period 2, 8 bulls (4
$QJXVDQG1HOORUH ZHUHVXEMHFWHGWRORZKHDWVWUHVV /+6“ƒ&LQGD\WLPHDQGDPELHQW
temperature at night, 25±0.2 °C) and 8 bulls (4 Angus and 4 Nellore) were subjected to high heat
stress (HHS, 31.4±0.4 °C during the daytime and ambient temperature at night, 25±0.2 °C). The diet
ZDVFRPSRVHGE\RIFRUQVLODJHDQGRIFRQFHQWUDWH &3 DQGRIIHUHGWZLFHSHUGD\
DWDPDQGSP2UWVZHUHZLWKGUDZQDQGVDPSOHG DWDSURSRUWLRQRI EHIRUHHDFKIHHGLQJ

We measured diurnal, nocturnal and daily feed intake as well as daily water intake. Heart rate was
obtained using heart rate monitors (Polar RS800, Finland) on days 9 and 10 of each period, only
in Angus bulls at an interval of 1-min for 48 h. Respiratory rate was measured in days 9 and 10 of
HDFKSHULRGE\FRXQWLQJÀDQNPRYHPHQWRYHUDPLQLQWHUYDOLQGLIIHUHQWWLPHVSHUGD\(\H
and skin temperatures were measured by thermal imaging (Fluke, USA) on day 10 of each period.
Eye temperature was considered as the maximum value in the eyeball and skin temperature was the
average of temperature of a similar area under the eyes.

The experiment was conducted under a randomized design using 2×2 factorial arrangement with
two genetic groups and two heat stresses. The variables were evaluated according to a completely
randomized design with repeated measures over time using the mixed models.

Results and discussion

Diurnal and daily intake of Nellore bulls was not affected (P>0.05) by heat stress, but an increase
(P<0.05) was observed in nocturnal intake with high heat stress. However, Angus bulls decreased
their diurnal intake by 24% and decreased nocturnal intake by 5% with a total decrease of 15% in
daily intake during high heat stress (Table 1). At TN, Angus bulls had higher (P<0.05) daily intake
JNJRI%: WKDQ1HOORUHEXOOV JNJRI%: EXWGXULQJ++6WKH\KDGVLPLODU P>0.05)
LQWDNHV DQGJNJRI%:UHVSHFWLYHO\ 7KHUHZDVDQREVHUYHGLQFUHDVH P<0.05) in BR,
but eye and skin temperatures and water intake were not affected (P>0.05) with temperature increase.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 291
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_98, © Wageningen Academic Publishers 2013
The HR decreased (P<0.05) with heat stress suggesting that occurred reduction in metabolic activity
to decrease metabolic heat production. Different anatomical and morphological characteristics
partially explain differences in heat tolerance among species and breeds (Gaughan et al., 2010).
Thus, Nellore bulls showed more adapted to high temperatures than Angus bulls.

We concluded that Angus feed intake is decreases more than that of Nellore on high air temperature.
Angus cattle present lower diurnal intake in high heat stress than on thermoneutral conditions and
partially compensates this reduction by increasing the intake in nocturnal period. However, total
daily intake of Angus is decreased on high heat stress.

7DEOH(IIHFWRIKHDWVWUHVV PHDQVDQGVWDQGDUGHUURU6( RQGDLO\LQWDNHKHDUWEHDWEUHDWKUDWH

and skin temperature according of genetic group.

Genetic group Treatment1 SE P-value

Thermoneutral Low stress High stress

Daily intake (g/kg of BW)

Angus $D 35.4Aa E 0.71 0.003
Nellore 29.1B 29.8B 30.2 0.71 0.504
SE  0.95 0.95
P-value <0.001 0.003 0.439
Heart beat (beats/min)
Angus 93.0a 89.9ab 87.0b 1.21 
Breath rate (breaths/min)
Angus $E $D 104.0Aa 1.83 <0.001
Nellore 29.5Bb 42.0Ba 45.2Ba 1.95 0.003
SE 1.73 2.45 
P-value <0.001 <0.001 <0.001
Skin temperature (°C)
Angus 34.3b 35.9a D 0.12 <0.001
Nellore 34.1b 35.9a D 0.12 <0.001
SE 0.11  
P-value 0.278  0.072

16XEVFULSWORZHUFDVHOHWWHUVZLWKLQDURZDQGVXEVFULSWFDSLWDOOHWWHUVZLWKLQDFROXPQGHQRWHVLJQL¿FDQWGLIIHUHQFH

(P<0.05).

Reference
Bernabucci U, Lacetera N, Baumgard LH, Rhoads RP, Ronchi B and Nardone A 2010. Metabolic and hormonal
DFFOLPDWLRQWRKHDWVWUHVVLQGRPHVWLFDWHGUXPLQDQWV$QLPDO
Gaughan JB, Mader TL, Holt SM, Sullivan ML and Hahn GL 2010. Assessing the heat tolerance of 17 beef cattle
JHQRW\SHV,QW-%LRPHWHRURO
O’Brien, M.D., Rhoads, R.P.,Sanders, S.R., Duff, G.C., Baumgard, L.H. 2010. Metabolic adaptations to heat stress in
JURZLQJFDWWOH'RPHVW$QLP(QGRFULQ

292 Energy and protein metabolism and nutrition in sustainable animal production
Growth hormone releasing factor and secretion of growth hormone in
Iberian and Landrace gilts
J.M. Rodríguez-López, L. González-Valero, M. Lachica and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQL¿JDUHV#HH]FVLFHV

Introduction
Compared to modern breeds, Iberian pigs have lower growth rate and protein deposition (Nieto et
al., 2002). However, factors that limit growth performance of Iberian pigs are still under debate. In a
previous study, greater basal serum insulin, leptin and IGF-1 with no difference in growth hormone
(GH) compared to Landrace pigs was reported (Fernández-Fígares et al., 2007). The objective of
the present work was to explore the possibility that Iberian have lower capacity of GH secretion
than Landrace pigs, which could explain their lower growth capacity.

Material and methods

,EHULDQ Q  DQG/DQGUDFH Q  JLOWV “NJ%: ZHUH¿WWHGZLWKSHUPDQHQWFDURWLGDUWHU\
catheters. Following surgery recovery and after 18 hours fasting pigs were injected intravenously 3.32
μg growth hormone releasing hormone (GHRH)/kg BW and blood samples collected at -15, 0, 15,
DQGPLQ3ODVPDVDPSOHVZHUHIUR]HQ ƒ& LQDOLTXRWVXQWLODQDO\VLV
of GH, insulin and amino acids (AA). GH was analysed using a porcine GH RIA kit. Insulin was
determined with a porcine insulin RIA kit using human insulin as standard. Plasma free AA were
analysed by HPLC using the Waters Pico Tag method which involves pre-column derivatization with
phenylisothiocianate and a reverse phase column. Data were evaluated using the mixed procedure
of SAS with repeated measures with breed, time of sampling and their interaction in the model
statement. Concentration at time zero of the analyte was included as a covariate in the statistical
DQDO\VLV6LJQL¿FDQWGLIIHUHQFHVDPRQJWUHDWPHQWVZHUHDVVHVVHGXVLQJ7XNH\¶VPXOWLSOHUDQJHWHVW

Results and discussion

Landrace had greater GHRH stimulated GH release than Iberian pigs (50%, P<0.001). Previous
studies with growing Iberian pigs (15 to 50 kg BW) demonstrated their low genetic potential for
lean tissue deposition (Nieto et al., 2002) compared with conventional breeds. Metabolic differences
among pigs with distinct genotypes can be expected in blood concentration of metabolites and
hormones. We have reported increased serum insulin, leptin and IGF-1, decreased glucose and no
change in GH in Iberian compared to Landrace gilts (Fernández-Fígares et al., 2007), although a single
SRLQWGHWHUPLQDWLRQZDVXVHGZKLFKPD\QRWEHVXI¿FLHQWIRUGHWHUPLQDWLRQRISXOVDWLOHVHFUHWLRQ
hormones such as GH. Similarly to the present study, GH clearly differed when extreme breeds
were compared and was lower when a reduced growth rate coincides with high daily deposition of
fat (Kasser et al., 1981; Wangness et al., 1981).

Increased GH paralleled decreased insulin in the plasma of pigs subjected to a GHRH challenge,
so that Iberian showed increased serum insulin compared to Landrace gilts (50%, P<0.01). An
increase in plasma insulin and glucose after GH injection has been repeatedly reported (Wray-Cahen
et al., 1991; Caperna et al., 1993). Although total AA did not change (P>0.10), branched chain
AA, sulphur AA and aromatic AA increased (P<0.05) in Iberian compared to Landrace pigs after
D*+5+FKDOOHQJH DQGUHVSHFWLYHO\ $GPLQLVWUDWLRQRI*+LVDVVRFLDWHGZLWKUDSLG
changes in protein and energy metabolism. (Dunshea et al., 1992; Caperna et al., 1993). Moreover, a
VLJQL¿FDQWGHFUHDVHLQV\VWHPLFHVVHQWLDOQRQHVVHQWLDOEUDQFKHGFKDLQDQGWRWDO$$FRQFHQWUDWLRQV
was found in GH treated pigs (Vann et al., 2000; Bush et al., 2002) found. In conclusion, Landrace

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 293
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_99, © Wageningen Academic Publishers 2013
had greater capacity of GHRH stimulated GH secretion than Iberian pigs, which may explain the
lower growth capacity of Iberian pigs.

7DEOH3ODVPDJURZWKKRUPRQH *+QJPO LQVXOLQ —8PO DQGDPLQRDFLG $$P0 FRQFHQWUDWLRQ

LQ,EHULDQDQG/DQGUDFHJLOWV Q  DIWHUi.v. injection of porcine growth hormone releasing hormone.

Landrace Iberian

GH “a 8.2±0.77b
Insulin 5.5±0.59a 8.3±0.54b
Total AA 3.58±0.085a 3.72±0.085a
Branched chain AA 0.53±0.009a “b
Sulphur AA 0.15±0.014a 0.33±0.014b
Aromatic AA 0.093±0.0040a 0.138±0.0040b
a,b9DOXHVLQWKHVDPHURZZLWKGLIIHUHQWVXSHUVFULSWVDUHVLJQL¿FDQWO\GLIIHUHQW P<0.05).

References
Bush J.A., G. Wu, A. Suryawan, H.V. Nguyen and T.A. Davis, 2002. Somatotropin-induced amino acid conservation
LQSLJVLQYROYHVGLIIHUHQWLDOUHJXODWLRQRIOLYHUDQGJXWXUHDF\FOHHQ]\PHDFWLYLW\-1XWU
Caperna T.J., N.C. Steele, C.M. Evock-Clover, D. Brocht, J.P. McMurtry and R.W. Rosebrough, 1993. Acute effects
of administration of porcine growth hormone on circulating levels of hormones and metabolites in 20-, 40-, and
NLORJUDPJLOWV-$QLP6FL
Dunshea, F.R., D.E. Bauman, R.D. Boyd, and A.W. Bel, 1992. Temporal response of circulating metabolites and
hormones during somatotropin treatment of growing pigs. J. Anim. Sci. 70, 123-131.
)HUQiQGH])tJDUHV,0/DFKLFD51LHWR0*5LYHUD)HUUHDQG-)$JXLOHUD6HUXPSUR¿OHRIPHWDEROLWHV
DQGKRUPRQHVLQREHVH ,EHULDQ DQGOHDQ /DQGUDFH JURZLQJJLOWVIHGEDODQFHGRUO\VLQHGH¿FLHQWGLHWV/LYHVW
Sci. 110, 73-81.
Kasser, T.R., R.J. Martin, J.H. Gahagan and P.J. Wangsness, 1981. Fasting plasma hormones and metabolites in feral
and domestic newborn pigs. J. Anim. Sci. 53, 420-425.
Nieto, R., A. Miranda, M.A. García and J.F. Aguilera, 2002. The effect of dietary protein content and feeding level
on the rate of protein deposition and energy utilization in growing Iberian pigs from 15 to 50 kg body weight.
Br. J. Nutr. 88, 39-49.
Vann, R.V., H.V. Nguyen, P.J. Reeds, D.G. Burrin, M.L. Fiorotto, N.C. Steele, D.R. Deaver, and T.A. Davis, 2000.
Somatotropin increases protein balance by lowering body protein degradation in fed, growing pigs. Am. J. Physiol.
278, E477-E483.
Wangness, P.J., W.A. Acker, J.H. Burdette, L.F. Krabill and R. Vasilatos, 1981. Effect of fasting on hormones and
PHWDEROLWHVLQSODVPDRIIDVWJURZLQJOHDQDQGVORZJURZLQJREHVHSLJV-$QLP6FL
Wray-Cahen, D., D.A. Ross, D.E. Bauman and R.D. Boyd. 1991. Metabolic effects of porcine somatotropin: Nitrogen
and energy balance and characterization of the temporal pattern of blood metabolites and hormones. J. Anim.
6FL

294 Energy and protein metabolism and nutrition in sustainable animal production
Effects of a simple or a complex starter microbiota on the gastric
WUDQVFULSWRPHSUR¿OHRIFDHVDUHDQGHULYHGSLJOHWV
D. Priori1, M. Colombo1, S.J. Koopmans2, A.J.M. Jansman, G. Schiavo1, P. Trevisi1 and P. Bosi1
1'HSDUWPHQWRI$JULFXOWXUDODQG)RRG6FLHQFHV8QLYHUVLW\RI%RORJQD%RORJQD,WDO\
[email protected]
2Department of Animal Sciences, Adaptation Physiology Group of Wageningen University,
Wageningen, the Netherlands
Wageningen UR Livestock Research, Lelystad, the Netherlands

Introduction
Recent research suggests that early exposure of piglets to a diverse microbiota can shape the skills
of gut-associated lymphoid tissue to respond to exogenous molecules (Lewis et al., 2012). In pig, a
VKRUWHQFRXQWHUZLWKDFRPSOH[PLFURELRWDLQWKHHDUO\OLIHFDQEHVXI¿FLHQWWRLQÀXHQFHWKHLQWHVWLQDO
microbiota in the following weeks (Jansman et al., 2012). The priming effect of the complexity of
the microbiota that enter the digestive tract, is not fully characterized. The gastric barrier of newborn
piglets is not fully active and early contact with microbes can affect functional maturation of the
stomach. Thus we aimed at evaluating the effect of complexity of starter microbiota on the gastric
WUDQVFULSWRPHSUR¿OHRI\RXQJSLJV

Material and methods

Twelve caesarean derived piglets were assigned to two treatment groups by litter and body weight
on day of birth (d 0), housed in two separate clean laboratory rooms with a hygiene barrier, received
serum from sow blood from d 0 to d 2, and were orally dosed a ‘simple’ microbiota consisting of
10-107 cfu from each Lactobacillus amylovorus, Clostridium glycolium and Parabacteroides sp
from d 1 to d 3. In addition, on d 3 and d 4, the groups received a fecal inoculant obtained from a
conventional adult sow (Complex Association, CA), or a placebo inoculant (Simple Association, SA).
$OOSLJOHWVZHUHIHGDPLONUHSODFHUGLHWGXULQJGD\WRDQGDPRLVWGLHWIURPGD\RQZDUGV3LJV
were euthanized on d 14 and samples of oxyntic tissue in the stomach were obtained and deep frozen.

On quality-tested mRNA, the analysis of whole transcript expression per each pig was done by
$II\PHWUL[‹3RUFLQH*HQH67DUUD\VWULSV$5REXVW0XOWLFKLS$QDO\VLVZDVGRQHRQ&(/¿OHV
by Affymetrix Expression Console. Transcripts ID’s, in general characterized by several exonic
VHTXHQFHVZHUHDVVRFLDWHGWR+XPDQJHQHQDPHVEDVHGRQ6XVVFURID(QVHPEOH2QJHQH
expression values, exploratory functional analysis was done with Gene Set Enrichment Analysis
(C2.BP catalog of gene sets, Molecular Signatures Database v3.0, http://www.broadinstitute.org/
gsea/msigdb/Index.jsp), comparing the CA and SA treatment. Normalized enrichment score (NES)
ZDVFDOFXODWHGIRUHDFKJHQHVHWDQGVWDWLVWLFDOVLJQL¿FDQFHZDVGH¿QHGZKHQ)DOVH'LVFRYHU\5DWH
% <25 and P-values of NES <0.05.

Results and discussion

3KHQRW\SHV6$VLJQL¿FDQWO\HQULFKHGJHQHVHWVFRPSDUHGWR&$ 7DEOH 0RVWRIWKH
pathways with the highest NES were related to cell replication and mitosis, and also to the interplay
with xenobiota (Toll like receptors signaling), immune response (chemokine receptors binding
FKHPRNLQHV DQGKRVWLQWHUDFWLRQZLWK+XPDQ,PPXQRGH¿FLHQF\9LUXVIDFWRUV6HYHUDOFKHPRNLQHV
characterized by the CXC motif were represented at the top of the core genes characterizing enriched
JHQHVHWV2QO\IRXUVHWVRIJHQHVZHUHVLJQL¿FDQWO\HQULFKHGLQSKHQRW\SHV&$FRPSDUHGWR6$
They relate to the expression of epidermal growth factor (EGF), hormone biosynthesis, signaling
of peroxisome proliferator-activated receptors and keratinocyte pathways. Among the core genes

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 295
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_100, © Wageningen Academic Publishers 2013
within these sets of genes were genes encoding for EGF, deiodinase 1 and adiponectin. The results
suggest that early association of newborn pigs with a complex microbiota favorably prevents the
DFWLYDWLRQRISDWKZD\VUHODWHGWRFHOOGLYLVLRQDQGLQÀDPPDWRU\GHYHORSPHQWLQWKHJDVWULFPXFRVD
at an age of two weeks compared to the association with a simple microbiota.

7DEOH)LUVWJHQHVHWVHQULFKHGLQWKHJDVWULFR[\QWLFWLVVXHZLWKVLPSOL¿HGPLFURELRWDDVVRFLDWLRQ
compared to complex microbiota association.

Name Size NES FDR q-val

Mitotic M G1 phases 122  0.0000

Mitotic prometaphase 70 -2.55 0.0000
Chemokine receptors bind chemokines 42 -2.45 0.0000
Cell cycle mitotic  -2.45 0.0000
DNA replication pre-initation 58 -2.23 0.0005
M G1 transition 48 -2.18 0.0017
Regulation of APC activators between G1 S and early anaphase 57 -2.17 0.0020
CDC20 phospho-APC-mediated degradation of cyclin A 52 -2.14 0.0021
Spliceosome  -2.14 0.0020
Toll Like Receptor signaling pathway 79 -2.14 0.0018
&'7DVVRFLDWLRQZLWK&'&25&RULJLQFRPSOH[ 43 -2.12 0.0024
HIV_infection 140 -2.11 0.0027
6&)ȕ75&3PHGLDWHGGHJUDGDWLRQRI(0, 40 -2.08 0.0031
Processing of capped intron containing pre-mRNA 93 -2.08 0.0029
S phase 82 -2.05 0.0041
Regulation of ornithine decarboxylase 39 -2.05 0.0039
G1 S transition 78 -2.05 0.0037
G2 M_checkpoints 34 -2.03 0.0052
Cell cycle checkpoints 89 -2.02 
Host interactions of HIV factors 93 -2.00 
,QÀXHQ]DOLIHVF\FOH 100 -2.00 
HIV life cycle 79 -2.00 
G alpha I signaling events 130 -2.00 
Synthesis of DNA 71 -1.99 
Peptide ligand binding receptors 130 -1.97 0.0075

Acknowledgements
Research funded by the European Union (Interplay project, Grant agreement no. 227549).

References
Jansman, A. J. M., J. Zhang, S. J. Koopmans, R. A. Dekker and H. Smidt, 2012. Effects of a simple or a complex starter
microbiota on intestinal microbiota composition in caesarean derived piglets. J. Anim. Sci. 90 (Suppl.4): 433-435.
Lewis M. C., C. F. Inman, D. Patel, B. Schmidt., I. Mulder, B. Miller, B. P. Gill, J. Pluske, D. Kelly, C. R. Stokes and
0%DLOH\'LUHFWH[SHULPHQWDOHYLGHQFHWKDWHDUO\OLIHIDUPHQYLURQPHQWLQÀXHQFHVUHJXODWLRQRILPPXQH
UHVSRQVHV3HGLDWU$OOHUJ\,PPXQRO

296 Energy and protein metabolism and nutrition in sustainable animal production
%LWWHUWDVWHUHFHSWRUJHQHVLQSLJV613LGHQWL¿FDWLRQE\XVLQJQH[W
generation semiconductor sequencing
L. Fontanesi1, F. Bertolini1, E. Scotti1, G. Schiavo1, P. Trevisi1, M. Colombo1, P.L. Martelli2, R.
Casadio2 and P. Bosi1
1'HSDUWPHQWRI$JULFXOWXUDODQG)RRG6FLHQFHV ',67$/ 'LYLVLRQRI$QLPDO3URGXFWLRQ8QLYHUVLW\
RI%RORJQD9LDOH)DQLQ%RORJQD,WDO\[email protected]
2%LRFRPSXWLQJ*URXS %L*H$'HSW 8QLYHUVLW\RI%RORJQD9LD6DQ*LDFRPR%RORJQD,WDO\

Introduction
Diet selection in animals is directly affected by their taste perception. It is commonly accepted that
WKHWDVWHFKHPRVHQVRU\V\VWHPLQPDPPDOVFDQGHWHFW¿YHEDVLFWDVWHVVZHHWVDOWVRXUXPDPLDQG
bitter (Behrens and Meyerhof, 2009). In particular, bitter sensing evolved as a central warning signal
to protect against ingesting potentially toxic bitter-tasting substances. It is sensed by a family of bitter
taste receptors (referred as TAS2Rs) that in mammalian genomes are encoded by approximately
10-40 functional TAS2R genes (Bachmanov and Beauchamp, 2007). Variability in these genes
could play an important role in feed preferences and feed intake in livestock species. Therefore, it
could be also possible to apply information about animals with different variants giving different
VHQVLWLYLW\WRELWWHUWDVWDQWVIRUGLHWIRUPXODWLRQDQGIHHGLQJSUDFWLVHV7RZDUGWKLVDLPWKH¿UVWVWHS
LVWKHLGHQWL¿FDWLRQRISRO\PRUSKLVPVDWWKH'1$OHYHODQDO\VLQJOLYHVWRFNJHQRPHV

In this study we resequenced ten TAS2R genes in different pig breeds using a next generation
sequencing platform (Ion Torrent PGM technology; Rothberg et al., DQGLGHQWL¿HGDTXLWH
large number of single nucleotide polymorphisms (SNPs), some of which might potentially have
a functional effect.

Material and methods

7$65JHQHVHTXHQFHVZHUHLGHQWL¿HGPLQLQJWKH6VFURIDJHQRPHYHUVLRQDQG*HQ%DQN
database. PCR primers were designed to amplify fragments of 800-1,200 bp covering all coding
UHJLRQVDQGSDUWRI¶DQG¶ÀDQNLQJUHJLRQVLQFOXGLQJ¶DQG¶XQWUDQVODWHGUHJLRQVRIWKHVHOHFWHG
genes. PCR was carried out on DNA pools constructed using equimolar quantities of genomic DNA
of 10-50 pigs of the same breed. Different pools were prepared for pigs of different breeds: 2 pools
for Italian Large White, one for Italian Duroc, one for Italian Landrace, one for Casertana and one
for Pietrain. The pooling strategy was designed to exploit as much as possible the throughput of
the Ion Torrent technology reducing the time and cost of the preparatory works needed to obtain
material to be sequenced with this new machine.

6HTXHQFLQJRIWKHDPSOL¿HGSURGXFWVZHUHVHTXHQFHGXVLQJWKH,RQ7RUUHQW3*0VHTXHQFHU /LIH
7HFKQRORJLHV %ULHÀ\3&5SURGXFWVREWDLQHGIURP'1$SRROVRIWKHVDPHEUHHGZHUHSRROHG
IUDJPHQWHGIRUOLEUDU\SUHSDUDWLRQEDUFRGHGDQGWKHQSRROHGDJDLQIRUVHTXHQFLQJUHDFWLRQRQD
Ion Torrent chip. Obtained ionograms were automatically selected using barcoding information and
compared with reference sequences. Average coverage on the obtained sequences was approximately
20,000×.

Single nucleotide polymorphisms were called using appropriate statistics based on number of animals
LQWKHGLIIHUHQW'1$SRROVEUHHGVFRPELQDWLRQ613VLGHQWL¿HGLQWKHTAS2R1 gene using the Ion
7RUUHQWSODWIRUPZHUHFRQ¿UPHGE\6DQJHUVHTXHQFLQJRIDPSOL¿HGSURGXFWVREWDLQHGE\JHQRPLF
DNA from single animals.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 297
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_101, © Wageningen Academic Publishers 2013
Results and discussion
Mining the pig genome and other sequences available in DNA databases (GenBank/EMBL) made it
possible to select 10 genes of the TAS2 family with homologous in the human and/or mouse genome.
These genes were: 7$657$657$657$657$657$657$657$65
7$65and 7$655HVHTXHQFLQJGDWDREWDLQHGZLWKWKH,RQ7RUUHQWSODWIRUPLGHQWL¿HGDWRWDO
RI613V7KHODUJHVWQXPEHURI613VZDVLGHQWL¿HGLQWKHTAS2R1 gene (n=12), the lowest was
LGHQWL¿HGLQWKH7$65 sequences (n=2). Most of the polymorphisms located in coding regions were
V\QRQ\PRXVVXEVWLWXWLRQVHLJKWZHUHPLVVHQVHPXWDWLRQV)RXUPLVVHQVHPXWDWLRQVZHUHLQGHQWL¿HG
in the TAS2R1 gene. Difference in allele frequencies between breeds were observed (presence or
absence of the two allelic forms in one or another breed) or inferred (based on the number of reads
ZLWKWKHDOWHUQDWLYHDOOHOHV IURPWKHVHTXHQFLQJLQIRUPDWLRQ$OO613VLGHQWL¿HGXVLQJWKH6DQJHU
VHTXHQFLQJDSSURDFKZHUHDOVRLGHQWL¿HGZLWKWKH,RQ7RUUHQWVHTXHQFHULQGLFDWLQJWKDWWKLVSODWIRUP
can produce reliable sequence information, improving throughput and speed. To our knowledge,
WKLVLVWKH¿UVWVWXG\WKDWDSSOLHGWKLVVHTXHQFLQJWHFKQRORJ\WRLGHQWLI\613VLQDIDUPDQLPDOV

7KHVHUHVXOWVUHSUHVHQWWKH¿UVWVWHSWRLQYHVWLJDWHSRWHQWLDOHIIHFWVRIWKHYDULDELOLW\LQWDVWHUHFHSWRU
genes with differences of individual sensitivity to feedstuffs including bitter but not harmful
substances. Additional studies are under way to further characterize the targeted genes, including gene
expression data (Colombo et al., 2012), and to evaluate association between these polymorphisms
and feed intake and related production traits in pigs.

Acknowledgment
Research funded by the European Union (Interplay project, Grant agreement no. 227549) and Italian
MiPAAF (Innovagen project).

References
Bachmanov A.A. and G.K. Beauchamp, 2007. Taste receptor genes. Annu. Rev. Nutr. 27, 389-414.
Behrens M. and W. Meyerhof, 2009. Mammalian bitter taste perception. Results Probl. Cell Differ. 47, 203-220.
&RORPER037UHYLVL**DQGRO¿DQG3%RVL$VVHVVPHQWRIWKHSUHVHQFHRIFKHPRVHQVLQJUHFHSWRUVEDVHG
on bitter and fat taste in the gastrointestinal tract of young pig. J. Anim. Sci. 90, Suppl 4, 128-130.
Rothberg J.M., W. Hinz, T.M. Rearick, J. Schultz, W. Mileski, M. Davey, J.H. Leamon, K. Johnson, M.J. Milgrew,
M. Edwards, J. Hoon, J.F. Simons, D. Marran, J.W. Myers, J.F. Davidson, A. Branting, J.R. Nobile, B.P. Puc, D.
Light, T.A. Clark, M. Huber, J.T. Branciforte, I.B. Stoner, S.E. Cawley, M. Lyons, Y. Fu, N. Homer, M. Sedova,
X. Miao, B. Reed, J. Sabina, E. Feierstein, M. Schorn, M. Alanjary, E. Dimalanta, D. Dressman, R. Kasinskas, T.
Sokolsky, J.A. Fidanza, E. Namsaraev, K.J. McKernan, A. Williams, G.T. Roth and J. Bustillo, 2011. An integrated
semiconductor device enabling non-optical genome sequencing. Nature 475, 348-452.

298 Energy and protein metabolism and nutrition in sustainable animal production
Post weaning growth and metabolism in F2-offspring of protein
restricted mink dams
C.F. Matthiesen and A.-H. Tauson
Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences,
University of Copenhagen, Denmark; [email protected]

Introduction
Malnutrition during fetal life may lead to structural and functional metabolic changes in the offspring,
adaptations aiming to maximize the utilization of available nutrients in order to optimize fetal
development and survival. Low protein provision during pregnancy in mink has resulted in lower
birth weight of F1- but higher birth weight of F2-generation offspring (Matthiesen et al., 2010a,b).
Furthermore, the relative mRNA abundance of some enzymes in glucose metabolism was affected
by fetal life protein provision (Matthiesen et al., 2010a,b). The objectives of the present study were
to investigate post-weaning growth and quantitative metabolism in male F2-generation mink born by
dams exposed to either low (L1) or adequate (A1) protein provision during fetal life (maternal fetal
treatment) and fed either a low (L2) or adequate (A2) protein diet during gestation (male kit fetal life
treatment), and the male kits’ response to a low (L) or an adequate (A) protein diet.

Material and methods

Sixteen F2 male offspring, four from each maternal and own fetal life treatment (A1A2, A1L2, L1A2,
L1L2) were used to study post-weaning growth, energy metabolism and substrate oxidation. They
were measured in three balance periods including respiration experiments (indirect calorimetry) at
DQGZHHNVRIDJH'LHWVZHUHDGHTXDWH $±RIPHWDEROL]DEOHHQHUJ\ 0( 
from protein; P, fat; F and carbohydrates; CHO) or low (L-19 P:48 F:33 CHO % of ME) in protein
and fed during the balance and respiration experiments.

The data were analysed using the mixed procedure in SAS, version 9.2. Fixed effects of maternal-,
and kit fetal life protein provision and post weaning diet and their interactions were analysed.
'LIIHUHQFHVZHUHGHQRWHGDVVLJQL¿FDQWLIP<0.05.

Results and discussion

7KHPDLQUHVXOWVDUHSUHVHQWHGLQ7DEOH3RVWZHDQLQJJURZWKVKRZHGVLJQL¿FDQW P<0.001)
interactions between maternal fetal life and own fetal life treatment resulting in A1A2 and L1L2 having
the highest body weights at 50 weeks of age and A1L2 and L1A2 the lowest. The heat production
+( ZDVVLJQL¿FDQWO\ 3 0.04) lower in males born by L1 than A1 dams in contrast to F1 males
born by L dams which tended to have higher HE than A kits (Matthiesen et al., 2012). The energy
UHWHQWLRQ 5( ZDVVOLJKWO\QHJDWLYHLQDOOJURXSVDQGDVLJQL¿FDQWLQWHUDFWLRQEHWZHHQPDWHUQDO
fetal life and male kit fetal life treatment (3 0.04) resulted in the most negative RE in L1A2 males.
7KHRQO\VLJQL¿FDQWHIIHFWVRISRVWZHDQLQJGLHWFRQFHUQHGR[LGDWLRQRISURWHLQDQGFDUERK\GUDWH
ZKLFKUHÀHFWHGWKHGLHWFRPSRVLWLRQ6LJQL¿FDQWLQWHUDFWLRQVEHWZHHQPDWHUQDODQGNLWIHWDOOLIH
WUHDWPHQWUHVXOWHGLQVLJQL¿FDQWO\KLJKHUR[LGDWLRQRISURWHLQ 2;3 DQGORZHUR[LGDWLRQRIIDW
(OXF) for A1A2 and L1L2 than A1L2 and L1A2 males, the lower OXP of L1A2PDOHVFRQ¿UPLQJ
SUHYLRXV¿QGLQJVLQ)1 males (Matthiesen et al., 2012).

In conclusion, both maternal and own fetal life protein provision affected post-weaning growth and
metabolism of F2JHQHUDWLRQPDOHRIIVSULQJ7KH¿QGLQJWKDWPDWHUQDOIHWDOOLIHORZSURWHLQSURYLVLRQ
(L1) resulted in lower HE in their offspring indicates an adaptation towards saving energy. Protein
oxidation data were, however, inconclusive due to a lower OXP in L1A2 animals indicating a strive

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 299
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_102, © Wageningen Academic Publishers 2013
Table 1. The intake of ME, HE, RE, substrate oxidation and body weight of male offspring born by
GDPVH[SRVHGWRHLWKHUORZ /1 RUDGHTXDWH $1 IHWDOOLIHSURWHLQSURYLVLRQDQGEHLQJH[SRVHGWR
HLWKHUORZ /2 RUDGHTXDWH $2 SURWHLQSURYLVLRQGXULQJWKHLURZQIHWDOOLIHDQGWKHQIHGDORZRU
DQDGHTXDWHSURWHLQGLHWDWDQGZHHNVRIDJH

Treatment P-value

A1A2 A1L2 L1A2 L1L2 W D M F M×F

Body weight, g <0.001 NS NS NS <0.001

10 weeks 1,455 1,335 1,424 1,304
ZHHNV 3,275ab 2,785a ab 3,479b
50 weeks 3,189a 2,541b bc 3,083ac

ME kJ/kg0.75 575a 553ab 431b 572a 0.01 NS NS NS 0.08

HE kJ/kg0.75 a a ab 599b <0.001 NS 0.04 NS NS
RE kJ/kg 0.75 -58ab -73ab -188a -10b <0.001 NS NS 0.08 0.04
Substrate oxidation in % of HE
OXP 27a 22ab 20b 27a NS <0.001 NS NS 0.004
OXF a b b 58a 0.01 NS NS NS 0.005
OXCHO 17 11 11 15 <0.001 <0.001 NS NS 

: ZHHNRU' GLHW±ORZRUDGHTXDWH0 PDWHUQDOIHWDOOLIHWUHDWPHQW) PDOHV¶RZQIHWDOOLIHWUHDWPHQW

0HDQYDOXHVZLWKLQDURZZLWKXQOLNHORZHUFDVHVXSHUVFULSWOHWWHUV DEF ZHUHVLJQL¿FDQWO\GLIIHUHQWZLWKLQWUHDWPHQW
combination.

to save protein but not in A1L2 and L1L2 animals where OXP was similar to controls. Effects of
ORZSURWHLQSURYLVLRQWRSUHJQDQWPLQNGDPVPD\DVGHPRQVWUDWHGKHUHQRWRQO\EHOLPLWHGWR¿UVW
generation offspring but also be evident in the following generation.

References
Matthiesen C.F., Blache D., Thomsen P.D., Hansen N.E. and Tauson A.-H. 2010a. Effect of late gestation low protein
supply to mink (Mustela vison) dams on reproductive performance and metabolism of dam and offspring. Arch.
$QLP1XWU
Matthiesen C.F., Blache D., Thomsen P.D. and Tauson A.-H. 2010b. Feeding mink (Neovison vison) a protein-restricted
diet during pregnancy induces higher birth weight and altered hepatic gene expression in the F2 offspring. Br. J.
Nutr. 104: 544-553.
Matthiesen C.F., Blache D., Thomsen P.D. and Tauson A.-H. 2012. Foetal life protein restriction in male mink (Neovison
vison NLWVORZHUVSRVWZHDQLQJSURWHLQR[LGDWLRQDQGWKHUHODWLYHDEXQGDQFHRIKHSDWLFIUXFWRVHELVSKRVSKDWDVH
P51$$QLPDO

300 Energy and protein metabolism and nutrition in sustainable animal production
Investigation of the glucose metabolism of the embryonic and neonatal
broiler chicks by injection of insulin
L. Franssens1, J. Lesuisse1, A. Koppenol1,2, Y. Wang1, E. Willems1, J. Buyse1, E. Decuypere1 and
N. Everaert1
1Division of Animal-Nutrition-Quality, Lab of Livestock Physiology, KU Leuven, Belgium;
[email protected]
2Division of Animal, ILVO, Belgium

Introduction
In contrast to most mammals, chickens have blood glucose concentrations that are twice as high as
these in mammals. In addition, they develop a resistance to insulin early in life (Mc Cormick et al.,
1978; Seki et al., 2001) So far, the response of insulin to elevated glucose levels in embryonic and
neonatal chicks is not well documented and the aim is to explore the glucose metabolism by the
injection of insulin in the embryonic and neonatal chick.

Material and methods

828 Ross broiler eggs were incubated under standard conditions. Eggs (12 eggs/group) were injected
RQHPEU\RQLFGD\ (' RU('LQEORRGFDSLOODULHVRIWKHFKRULRDOODQWRLFPHPEUDQHRUQHRQDWDO
chicks were injected in the blood on postnatal day (D) 2 with 200 μl of insulin (1 μg/g embryo or
chick BW) dissolved in a saline solution. An injection of 200 μl saline was used as a sham group.
Another group served as a control group and did not receive any treatment. Blood and liver samples
were taken before and every two hours after injections (until 23 hours post-injection (PI)). Plasma
glucose concentrations were determined. mRNA expression of glucose transporters (GLUT) 1, 2,
DQGLQWKHOLYHUDWKRXUV3,ZDVGHWHUPLQHGXVLQJ()ĮDVDKRXVHNHHSLQJJHQH*OXFRVH
concentrations and mRNA expression levels were analyzed by using the 2-way ANOVA model in
SAS 9.2., using treatment and time of injection (glucose concentrations) and treatment and day of
LQMHFWLRQ P51$H[SUHVVLRQOHYHOV ZLWKWKHLULQWHUDFWLRQDVYDULDEOHV:KHQWKHUHZDVDVLJQL¿FDQW
GLIIHUHQFHD7XNH\¶VWHVWZDVSHUIRUPHG$FRQ¿GHQFHLQWHUYDOWKUHVKROG P<0.05) was set.
'DWDDUHVKRZQDVPHDQ“6(07KHFRPSDULVRQEHWZHHQWKHFRQWURODQGVDOLQHJURXSZDV¿UVW
SHUIRUPHGEXWGLGQRWUHYHDODQ\VLJQL¿FDQWGLIIHUHQFHV

Results
For every injection day, injection of saline did not affect plasma glucose concentrations compared
to non-injected embryos or chicks. On all time points for every injection day, except 21 hours PI on
D2, the insulin-injected group had lower plasma glucose concentrations than their sham counterparts.
On every injection day, plasma glucose concentrations decreased to a minimum level 7 hours PI.
7KHQD¿UVWVLJQL¿FDQWULVHLQJOXFRVHOHYHOVZDVVHHQDWDQGKRXUV3,RQ('DQG('
respectively. For embryonic insulin injections, glucose levels did not reach the basal level after 21
KRXUV3, ¿JXUH$% 2Q'SODVPDJOXFRVHFRQFHQWUDWLRQV¿UVWUHPDLQHGORZDIWHUK3,WKHQ
LQFUHDVHGVLJQL¿FDQWO\DWKRXUV3,UHDFKLQJFRQWUROOHYHOVDWKRXUVSRVWLQMHFWLRQDQGWKHQ
GHFUHDVHGDJDLQ ¿JXUH& 7KHP51$OHYHOVRI*/87VLQQRQLQMHFWHGHPEU\RVRUFKLFNVZHUH
QRWVLJQL¿FDQWO\GLIIHUHQWIURPWKHVHRIWKHVDOLQHJURXSUHJDUGOHVVRIGD\RILQMHFWLRQ7KHLQVXOLQ
LQMHFWLRQRQ('('RU'GLGQRWDOWHUWKHP51$OHYHOVRI*/87DQG GDWDQRWVKRZQ 
As shown in table 1, the mRNA levels of GLUT2 in the liver of insulin-injected embryos/chicks on
('DQG'ZDVVLJQL¿FDQWO\ORZHUFRPSDUHGWROHYHOVRIWKHVDOLQHDQGFRQWUROJURXS2Q('
the mRNA expression levels of this gene did not differ between treatments.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 301
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_103, © Wageningen Academic Publishers 2013
 A B C

Glucose concentration (mg/dL)

200

Glucose concentration (mg/dL)

Glucose concentration (mg/dL)
200 200
a
150 150 150

100 100 100 b

b
b,c
a
50 a a,b 50 a,b a,b a a 50
a,b a,b,c c,d c,d
a,b,c a,b,d a,b,c c,d c,d
a,b,d c,d c,d
a,b,d b,d b,c,d d
0 c,d 0 c,d d 0
d d d

0 3 5 7 9 11 13 15 17 19 21 23 0 3 5 7 9 11 13 15 17 19 21 23 0 3 5 7 9 11 13 15 17 19 21 23

Time after treatment (h) Time after treatment (h) Time after treatment (h)

)LJXUH3ODVPDJOXFRVHFRQFHQWUDWLRQ PJGO RIWKHVKDPJURXS ZKLWHFLUFOHV RULQVXOLQJURXS

EODFNFLUFOHV DWGLIIHUHQWWLPHSRLQWV KRXUVK EHIRUHDQGDIWHULQVXOLQLQMHFWLRQRQ(' $ ('
% DQG' & 9DOXHVDUHPHDQV“6(0Q DEFGHPHDQYDOXHVDUHVLJQL¿FDQWO\GLIIHUHQW
between time points of the insulin-injected group.

7DEOH)ROGFKDQJH DUELWUDU\XQLWV RI*/87DWKRXUV3,9DOXHVDUHPHDQV“6(0Q D

DQGEPHDQYDOXHVDUHVLJQL¿FDQWO\GLIIHUHQWEHWZHHQWKHJURXSVDQGWKHLQMHFWLRQGD\V()ĮZDV
used as a reference gene.

Treatment Injection day

(' ED 18 D2

Control -7.99E-11a±0.40 -3.55E-11a±0.29 -4.44E-11a±0.40

Sham 0.08a±0.32 -0.71a±0.21 -0.02a±0.38
Insulin -2.08b±0.42 -0.47a“ -2.70b±0.27

Discussion
On every injection day, the injection of insulin clearly caused a decrease in the plasma glucose
concentrations. Interestingly, the recovery pattern back to pre-injection level was different in respect
WRWKHLQMHFWLRQGD\V7KH3,LQFUHDVHLQSODVPDJOXFRVHFRQFHQWUDWLRQVZDVPRUHVORZO\RQ('
and ED18 when compared with that on D2. Therefore, it seems that embryos are more sensitive
to insulin than neonatal chicks. GLUT 2, the major GLUT in liver, is present on the sinusoidal
membrane of hepatocytes and allows for the bi-directional transport of glucose, which is adjusted
by dietary and hormonal control (Wood and Trayhurn, 2003). In rats, insulin suppresses hepatic
glucose production, and this suppression is associated with a decrease in the hepatocyte plasma
membrane-bound quantity of the facilitative glucose transport protein GLUT2 (Nathan et al., 2001).
Indeed, our results showed that the insulin-injected group had a decrease in GLUT2 mRNA levels
RQ('DQG',QDGGLWLRQ7KRUQet al. (2012) also demonstrated that GLUT2 mRNA levels were
diminished after insulin supplementation in fetal sheep with hypoglycemia. However, reasons of the
absence of changes in GLUT2 mRNA level in the insulin injected group on ED18 are unknown and
WKLV¿QGLQJZLOOEHIXUWKHULQYHVWLJDWHGE\DQDO\]LQJDGGLWLRQDO3,WLPHSRLQWV

In conclusion, the sensitivity to exogenous insulin seems to diminish towards the end of the embryonic
period and continues during neonatal stage, and a down-regulation of GLUT2 gene expression
might be involved.

References
Mc Cormick, K.L., J.A. Widness, J.B. Susa and R. Schwarz, 1978. Effects of chronic hyperinsulinemia on hepatic
enzymes involved in lipogenesis and carbohydrate metabolism in the young rat. Biochem. J. 172, 327-331.

302 Energy and protein metabolism and nutrition in sustainable animal production
Nathan, J.D., P.D. Zdankiewicz, J. Wang, S.A. Spector, G. Aspelund, B.P. Jena, N.E. Seymour, J.P. Geibel and D.K.
Andersen, 2001. Impaired hepatocyte glucose transport protein (GLUT2) internalization in chronic pancreatitis.
Pancreas 22, 172-178.
Seki, Y., K. Sato, H. Ohtsu and Y. Akiba, 2001. Persistent hypoglycemia is induced by tolbutamide administration in
broiler chickens fed a low-carbohydrate diet. Domest. Anim. Endocrinol. 20, 109-122.
Thorn, S.R., S.M. Sekar, J.R. Lavezzi, M.C. O’Meara, L.D. Brown, W.W. Jr. Hay and P.J. Rozance, 2012. A physiological
increase in insulin suppresses gluconeogenic gene activation in fetal sheep with sustained hypoglycemia. Am. J.
3K\VLRO5HJXO,QWHJU&RPS3K\VLRO55
Wood, I.S. and P. Trayhurn, 2003. Glucose transporters (GLUT and SGLT): expanded families of sugar transport
proteins. Br. J. Nutr. 89, 3-9.

Energy and protein metabolism and nutrition in sustainable animal production 303
Stage of egg production regulates protein turnover and lysine
partitioning for broiler breeders
R.D. Ekmay, K. Vignale, C. Salas, J. England, S. Cerrate and C.N. Coon
University of Arkansas, Poultry Science Department, Center of Excellence for Poultry Science, John
7\VRQ%XLOGLQJ2)D\HWWHYLOOH$586$[email protected]

Introduction
Among the regulators of protein turnover, amino acids play a critical role. Lysine, leucine and
isoleucine were found to alter protein synthesis, degradation and its regulation (Yoshizawa, 2004;
Nakashima et al., 2005; Tesseraud et al., 2008). Furthermore, Tesseraud et al. (2000) reported that
differences in the fractional degradation rates (FDR) between fast-growing and slow-growing birds
are observed in young birds but not older ones, suggesting a possible age effect.

7KHFXUUHQWEURLOHUEUHHGHULQGXVWU\SUDFWLFHWDUJHWVEHWZHHQWRNFDO0(GIRUSHDNHJJ
SURGXFWLRQZLWKGLHWVFRQWDLQLQJD¿[HGGLHWDU\SURWHLQ&UXGHSURWHLQDQGO\VLQHLQWDNHV
often exceed 28 g/d and 1000 mg/day, respectively. A series of studies were conducted to determine
if additional protein and energy intake impact protein turnover in broiler breeders during different
stages of egg production.

Methods
,QWKH¿UVWVWXG\&REEEURLOHUEUHHGHUKHQVZHUHUDQGRPO\DVVLJQHGWRRQHRIVL[GLHWDU\
treatments in a 2×3 fashion. Two levels of energy (390 and 450 kcal/d) and three levels of protein
DQGJ&3G ZHUHXWLOL]HG7KLUW\VL[KHQVZHUHDGPLQLVWHUHGDGDLO\RUDOGRVHRIPJ
of 15N-Lys for a period of two weeks. After this enrichment period, hens were allowed a period of
two days in which no isotopes were given. After two days, a daily oral dose of 15mg of 2D4-Lys
was administered at which point, breast muscle was excised and snap frozen between the 2nd and
4th egg. Muscle and egg were analyzed via GC-MS for the ratio of labeled to unlabeled lysine.
Three time periods were assessed: 25, 29 and 45 wk. Data was analyzed as two-way ANOVA and
contrasts were performed to determine line shape.

,QWKHVHFRQGVWXG\WHQKHQVSHUWUHDWPHQWZHUHJLYHQDQLQWUDYHQRXVÀRRGLQJGRVHRI13KHDW
10 ml/kg. After 10 min, birds were slaughtered and breast muscle excised and snap-frozen in liquid
QLWURJHQ)UHHSKHQ\ODODQLQHZDVTXDQWL¿HGYLD*&06DQGWKHSURWHLQERXQGIRUPZDVTXDQWL¿HG
via GC-C-IRMS. 3-methylhistidine was utilized to determine protein degradation rates. Protein
WXUQRYHUZDVGHWHUPLQHGDWZNDQG5HODWLYHH[SUHVVLRQRIFDOSDLQSURWHDVRPH&
subunit, and F box protein 32 were determined via RT-PCR at wk 20, 25, and 44.

Results and discussion

Results show that over 78% of all labeled lysine was found in breast muscle. In the egg, the portion
of total labeled lysine that was deposited varied by age. At wk 25 and 45, the amount of lysine that
RULJLQDWHGIURPVNHOHWDOPXVFOHDQGZDVGHSRVLWHGLQWR\RONZDVVLJQL¿FDQWO\KLJKHUWKDQWKHDPRXQW
deposited from the diet (P<0.05). At wk 29, diet became the major source of lysine deposited into
yolk. Diet was the major source of lysine for albumen formation at wk 25 and 29, and an even split
with skeletal muscle at wk 45. No difference in the partitioning of lysine was determined by energy
or protein intake. Contrasts indicate fractional synthesis rate and FBXO32 increase to a maximum
DWZNDQGDGHFUHDVHWKHUHDIWHU7KHVHUHVXOWVVXJJHVWWKDWWKHUHLVDQXSUHJXODWLRQRISURWHLQ
GHJUDGDWLRQGXULQJWKHSHULRGVRIORZHUHJJSURGXFWLRQ$VLJQL¿FDQWGURS P<0.05) in PSMA1
and FBXO32 was observed between 25 and 44 wk and matched the decrease observed in FBR.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 305
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_104, © Wageningen Academic Publishers 2013
)LJXUH&KDQJHVWRIUDFWLRQDOV\QWKHVLV )65 DQGEUHDNGRZQUDWHV )%5 RIPectoralis muscle
GXULQJWKHWUDQVLWLRQLQWRVH[XDOPDWXULW\DQGSDVWSHDNHJJSURGXFWLRQLQ&REEEURLOHUEUHHGHU
hens.

However, no differences in the expression of calpain were observed. Furthermore, no differences

were detected in the levels of fractional synthesis and degradation rates, or the expression of CAPN2,
PSMA1, and FBXO32, due to protein or energy intake. In summary, skeletal muscle appears to be
a major source of lysine for egg formation during the transition into sexual maturity but not at peak
production. Fractional degradation rates are upregulated coming into production but decrease with
higher rates of egg production. Results suggest that stage of egg production may play an important
role in the regulation of protein turnover. The observed changes in degradation also appear to be
mediated by the ubiquitin-proteasome pathway.

References
1DNDVKLPD.,VKLGD$<DPD]DNL0DQG$+LUR\XNL/HXFLQHVXSSUHVVHVP\R¿EULOODUSURWHRO\VLVE\
down-regulating ubiquitin-proteasome pathway in chick skeletal muscles. Biochem. Biophys. Res. Commun.

Tesseraud, S., Bouvarel, I., Collin, A., Audouin, E., Crochet, S., Seiliez, I. and C. Letterier. 2008. Dily variations in
dietary lysine content alter the expression of genes related to proteolysis in chicken pectoralis major muscle. J.
Nutr. 139:38-43.
Tesseraud, S., Chagneau, A.M., and J. Grizard. 2000. Muscle protein turnover during early development in chickens
GLYHUJHQWO\VHOHFWHGIRUJURZWKUDWH3RXOW6FL
Yoshizawa F. 2004. Regulation of protein synthesis by branched-chain amino acids in vivo. Biochem Biophys Res
Commun. J313: 417-22.

306 Energy and protein metabolism and nutrition in sustainable animal production
Lipid utilization for egg formation in broiler breeders
C. Salas, R. Ekmay, J. England, S. Cerrate and C.N. Coon
University of Arkansas, Poultry Science Department, Center of Excellence for Poultry Science,
)D\HWWHYLOOH$586$[email protected]

Introduction
Energy utilization is a key component of animal production. The study of the mechanisms by which
lipids and carbohydrates are mobilized and deposited in the egg, can give us clues about how breeders
utilize energy during the production period. Is the dietary energy directly utilized during yolk
formation or is there a dynamic metabolic system that consistently provides nutrients to the egg at
the expense of body tissue? Are the fatty acids in the yolk derived from direct intestinal absorption,
endogenous synthesis, or maternal body stores? Do the systems change as the breeder ages and egg
production decreases? The aim of the present studies was to better understand the mechanisms of
energy partitioning in broiler breeder hens.

Material and methods

The contribution of dietary fatty acids (FA), tissue FA or newly synthesized FA utilized for the
formation of egg yolk during different periods of egg production was determined in broiler breeders
using stable isotopes. In Exp 1, 20 broiler breeder pullets (BB) were fed a 25 mg meal of U-13C-
OLQROHLFDFLG /$ RQWKH¿UVWGRIRYLSRVLWLRQ7ZRELUGVZHUHVDFUL¿FHGHDFKGIRUWKHQH[WGDQG
DOOODUJH\HOORZIROOLFOHVZHUHFROOHFWHGIURPWKHRYDU\LGHQWL¿HGDQGVWRUHGDWƒ&IRUDQDO\VLV
The accumulation of labeled LA acid in the follicles was determined using a GC-MS. The objective
was to evaluate the pattern of incorporation of the labeled essential FA into eggs of sexually maturing
BB hens. In Exp 2, a group of pullets were fed a daily 15 mg dose of U-13C-glucose (G) 10 d prior
WRVH[XDOPDWXULW\FRQWLQXLQJWKURXJKGIROORZLQJ¿UVWHJJ$PJPHDORI8&/$ZDV
DOVRRUDOO\DGPLQLVWHUHGGSULRUWRWKHLUHVWLPDWHG¿UVWHJJ2QWKHGRIWKHLU¿UVWHJJHDFKELUG
received a 25 mg meal of D31-LA. All eggs were collected for the next 10 d. The incorporation
of U-13C-LA, D31-LA and labeled palmitic acid (PA) from U-13C-G metabolism in the egg was
determined using a GC-MS. The enrichment with three different labeled FA was repeated at peak
egg production (30 wk) and 45 wk of age. In Exp 3, broiler breeders were randomly assigned to three
different levels of feed allocation to achieve 3 different growth curves during rearing period: 20%
heavier (HBW), standard (SBW) and 20% lighter (LBW) than the breeder target. Exp 3 was conducted
to determine the effect of body weight and body composition during the egg production cycle on
gene expression for lipoprotein lipase (LPL), fatty acid binding protein (FABP), apolipoprotein B
(ApoB) and apolipoprotein VLDL-II (ApoVLDL-II). Body composition was determined for each
hen using DEXA technology and levels of estradiol in blood were also measured.

Results and discussion

7KHUHVXOWVRI([SVKRZWKDW¿UVWDQGVHFRQGGDIWHUGRVLQJWKHELUGVDW¿UVWHJJWKHVPDOOHU)
F7 follicles received a higher relative dose of the labeled LA. This is consistent with other reports,
where the peak of incorporation of labeled LA into the yolk was on day 4 or 5 after forced feeding
(Burghelle-Mayeur et al., 1990).

7KHUHVXOWVIRU([SLQGLFDWHWKDWWKHUHFRYHU\RIWKHODEHOHGLVRWRSHVLVDOWHUHGDVWKHÀRFNDJHV
$W¿UVWHJJWKHGHSRVLWLRQRIODEHOHG3$LVKLJKHUZKHQFRPSDUHGWRLWVGHSRVLWLRQDWZNDQG
ZNRIDJH7KHGHSRVLWLRQRIWKHWZR/$ODEHOVZDVORZIRU¿UVWRYLSRVLWLRQDQGLQFUHDVHGDW
peak production and 45 wk. In older birds, the rate of glucose oxidation has been shown to increase

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 307
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_105, © Wageningen Academic Publishers 2013
relative to the nonoxidative route of glucose utilization (Buyse et al., 2004). As the hens aged in Exp
2, their incorporation of U-13C-G (as acetate) in the formation of PA is decreased.

7KHKHSDWLFH[SUHVVLRQRIWKHVHOHFWHGJHQHVZDVVLJQL¿FDQWO\LQFUHDVHGDIWHUVWDUWRIOD\DQGUHDFKHG
DSODWHDXDIWHU¿UVWRYLSRVLWLRQ )LJXUH 7KHH[SUHVVLRQRI/3/VLJQL¿FDQWO\GHFUHDVHGWKURXJKRXW
the production period for all groups. The levels of expression of all the genes were strongly correlated
WRWKHOHYHOVRIHVWUDGLROLQHDFKVDPSOLQJSHULRG7KHERG\FRPSRVLWLRQRIWKHELUGVZDVVLJQL¿FDQWO\
different between growth curves at 22 wk and at peak egg production, but these differences did not
seem to directly affect the expression of the selected genes. Changes throughout the production
period in the mechanisms of lipid formation and mobilization are not only a matter of an increased
production of lipoproteins, but also of the increasing inhibition of LPL.

)LJXUH*HQHWLFH[SUHVVLRQRIJHQHVFRGLQJIRU$ $SR% OLYHU % IDWW\DFLGELQGLQJSURWHLQ OLYHU 

& $SR9/'/,, OLYHU ' /3/ DGLSRVHWLVVXH GHWHUPLQHE\573&5IRUEURLOHUEUHHGHUVUHDUHG
LQGLIIHUHQWJURZWKFXUYHV9DOXHVDUHPHDQVRIǻ&WUHODWLYHWR%DFWLQ

References
%XUJKHOOH0D\HXU&07L[LHU%LRFKDUG30HUDWDQG<'HPDUQH,QÀXHQFHRIWKHVH[OLQNHGGZDU¿QJJHQH
GZ RQWKHFRQWULEXWLRQRIGLHWDU\OLSLGWR\RONOLSLGV\QWKHVLV%U3RXOW6FL
Buyse J., B. Geypens, R.D. Malheiros,V.M. Moraes,Q. Swennen, and Y. Demarne. 2004. Assessment of age-related
glucose oxidation rates of broiler chickens by using stable isotopes. Life Sciences 75:2245-55.

308 Energy and protein metabolism and nutrition in sustainable animal production
The heat-induced production of reactive oxygen species regulates protein
content in cultured chick skeletal muscle cells
H. Yoshida, M. Kikusato, K. Souma and M. Toyomizu
Animal Nutrition, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-
$PDPL\DPDFKL$REDNX6HQGDL-DSDQ[email protected]

Introduction
Heat stress is a major factor affecting meat production in the poultry industry, especially in hotter
regions of the world. A previous in vivo study by our group highlighted that acute heat stress stimulates
mitochondrial reactive oxygen species (ROS) production in the skeletal muscle of broiler chickens.
5HFHQWO\ZHIXUWKHUFODUL¿HGWKDWWKHRYHUSURGXFWLRQRIPLWRFKRQGULDO526UHVXOWHGIURPDQ
increase in the mitochondrial membrane potential, probably due to an increase in substrate oxidation
(Kikusato et al., 2010) and a decrease in avian uncoupling protein (avUCP) content (Mujahid et
al. )XUWKHUPRUHLWZDVUHSRUWHGWKDWKHDWVWUHVVLQFUHDVHVSURWHLQR[LGDWLRQDQGP\R¿EULOODU
proteolysis in chick cultured skeletal muscle cells (Nakashima et al., 2004). Considering the fact
that ROS play a pivotal role in muscle proteolysis in sepsis (Supinski and Callahan, 2007), it could
be postulated that heat treatment stimulates mitochondrial ROS production, thereby leading to an
increase in skeletal muscle proteolysis. To verify this possibility, we have investigated the effect of
heat stress on ROS production, protein content, and the ROS-related expression of genes for avUCP,
heme oxygenase-1 (HO-1) and NADPH oxidase 4 (NOX4) in chick skeletal muscle cells in primary
culture. We also examine the reducing effects of 4-hydroxy-TEMPO (Tempol), which is a strong
antioxidant, on these parameters.

Material and methods

Skeletal muscle cells were isolated from the SHFWRUDOLVVXSHU¿FLDOLV muscles of chicks (Ross
VWUDLQ DWGD\RIDJHDQGVXVSHQGHGLQEDVDOPHGLXP 'XOEHFFR0RGL¿HG(DJOH¶V0HGLXP
and 20% Medium 199) supplemented with 10% fetal bovine serum, 1.5×105 U/l penicillin and
0.15 g/l streptomycin. The cell suspension was transferred to uncoated culture dishes to allow
¿EUREODVWDWWDFKPHQW7KHVXSHUQDWDQWZDVFROOHFWHGDQGP\REODVWVZHUHFRXQWHGDQGVHHGHGRQWR
collagen-coated plates at a density of 4.5×104 cells/cm2. Incubation was carried out at 37 °C in a
5% CO2HQULFKHGDWPRVSKHUHRIKXPLGL¿HGDLUXQWLOVXEFRQÀXHQFH,QDVXEVHTXHQWVWHSWKHFHOOV
ZHUHIXUWKHULQFXEDWHGDWQRUPDO ƒ& RUKLJK ƒ& WHPSHUDWXUHIRUKLQDVHUXPIUHHEDVDO
PHGLXP:KHUHUHOHYDQW7HPSRO ¿QDOFRQFHQWUDWLRQPPROO ZDVDGGHGWRFHOOFXOWXUHPHGLD
1 h before the heat treatment.

&HOOXODU526SURGXFWLRQZDVGHWHUPLQHGÀXRURPHWULFDOO\XVLQJ&0+2'&)'$ ȜH[ȜHP 

QP DFHOOSHUPHDQWPROHFXOHWKDWIRUPVDÀXRUHVFHQWDGGXFWXSRQUHDFWLQJZLWK5267KHSURWHLQ
content of muscle cells was measured by the micro-BCA protein assay. Expression levels of genes
for avUCP, HO-1 and NOX4 were measured by real-time RT-PCR, with expression referenced to
that of 18S ribosomal RNA as an internal control.

Results and discussion

Cellular ROS production in the cultured chick skeletal muscle cells was increased in response to
the heat treatment. As expected, the cellular protein content was also decreased by this treatment,
FRQ¿UPLQJWKDWDGHFUHDVHLQSURWHLQFRQWHQWRFFXUVFRQFRPLWDQWO\ZLWKDQLQFUHDVHLQ526SURGXFWLRQ
under heat stress conditions. Also in agreement with our previous in vivo study, the expression of
DY8&3P51$ZDVVLJQL¿FDQWO\GHFUHDVHGE\WKHKHDWWUHDWPHQWZKLFKVXJJHVWVWKDWWKHGRZQ
regulation of avUCP might result in the overproduction of mitochondrial ROS in chick muscle cells.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 309
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_106, © Wageningen Academic Publishers 2013
Furthermore, the expression of HO-1 mRNA (HO-1 being a heme-catabolizing enzyme that produces
VXEVWUDWHVH[HUWLQJDSURWHFWLYHHIIHFWDJDLQVWR[LGDWLYHVWUHVV ZDVVLJQL¿FDQWO\GHFUHDVHGE\WKH
heat treatment, while the expression of NOX4 mRNA (NOX4 being an enzyme related to superoxide
production) was increased by heat treatment. These results suggest that changes in the levels of
these enzymes could be partly responsible for the overproduction of ROS in response to heat stress.

The addition of Tempol to culture media attenuated the heat-induced increase in cellular ROS
production and the decrease in protein content (Figure 1A), indicating that ROS scavenging by
7HPSROFDQVXSSUHVVWKHQHJDWLYHHIIHFWVFDXVHGE\WKHKHDWVWUHVVFRQGLWLRQWKLV¿QGLQJDOVR
suggests that the heat-induced ROS production might have led to a decrease in protein content.
Also, Tempol reduced the heat-induced decrease in HO-1 expression and the heat-induced increase
in NOX4 expression, but did not affect avUCP expression (Figure 1B). These results indicate that
Tempol scavenges mitochondrial ROS produced by the down-regulation of avUCP, thereby causing
HO-1 and NOX4 gene expression levels to be up- and down-regulated, respectively.

It can be concluded that heat stress induces the overproduction of cellular ROS, which causes a
GHFUHDVHLQWKHSURWHLQFRQWHQWRIFKLFNPXVFOHFHOOV)XUWKHUPRUHWKLVVWXG\SURYLGHVWKH¿UVW
evidence that HO-1, NOX4 and avUCP are factors involved in the heat-induced production of ROS.

A B
2 a 1.5
Relative amount

Relative amount

a a
b a a b b
b 37 °C
b a a 1
1 b b 41 °C
b
0.5
41 °C + Tmp
0 0
ROS Protein avUCP HO -1 NOX4
production content
mRNA expression (/18S ribosomal RNA)
)LJXUH(IIHFWRI7HPSRORQ526SURGXFWLRQDQGSURWHLQFRQWHQW $ DQGRQWKHH[SUHVVLRQRI
DY8&3+2DQG12;P51$ % LQFXOWXUHGFKLFNPXVFOHFHOOVH[SRVHGWRKHDWVWUHVV9DOXHV
DUHH[SUHVVHGUHODWLYHWRWKHDYHUDJHDWƒ&DQGH[SUHVVHGDVWKHPHDQ“6( Q  6WDWLVWLFDO
analysis was performed with one-way ANOVA followed by the Tukey Kramer multiple comparison
WHVW a,bP< 

References
Kikusato, M., A.J.J. Ramsey, T. Amo, and M. Toyomizu, 2010. Application of modular kinetic analysis to mitochondrial
oxidative phosphorylation in skeletal muscle of birds exposed to acute heat stress. FEBS Lett. 584, 3143-3148.
0XMDKLG$.6DWR<$NLEDDQG07R\RPL]X$FXWHKHDWVWUHVVVWLPXODWHVPLWRFKRQGULDOVXSHUR[LGHSURGXFWLRQ
LQEURLOHUVNHOHWDOPXVFOHSRVVLEO\YLDGRZQUHJXODWLRQRIXQFRXSOLQJSURWHLQFRQWHQW3RXOW6FL
1DNDVKLPD.,1RQDND60DVDNL0<DPD]DNLDQG+$EH0\R¿EULOODUSURWHRO\VLVLQFKLFNPXVFOHFHOO
FXOWXUHVGXULQJKHDWVWUHVV$QLPDO6FLHQFH-RXUQDO
6XSLQVNL*6DQG&DOODKDQ/$)UHHUDGLFDOPHGLDWHGVNHOHWDOPXVFOHG\VIXQFWLRQLQLQÀDPPDWRU\FRQGLWLRQV
-$SSO3K\VLRO

310 Energy and protein metabolism and nutrition in sustainable animal production
Part 5. Modeling / systems biology
Fasting heat production and metabolic body size in non-ruminant
growing farm animals
J. Noblet1, E. Labussière1, S. Dubois1, C.F.M. de Lange2, R. Barea2, J. Lasnier1, V. Rivera1, M.
Warpechowski and J. van Milgen1
1,15$6DLQW*LOOHV)UDQFH[email protected]
2University of Guelph, Canada
Federal University of Parana, Curitiba, Brazil

Introduction
Fasting heat production (FHP) of animals is indicative of their basal metabolic rate and is used to estimate
the maintenance energy requirements and for calculating the net energy value of feeds. However,
estimates of FHP vary with experimental conditions, as well as with measurement and calculation
methods. In addition, FHP is often related to metabolic body weight (MBW) with the idea that FHP
per unit of MBW is constant for a given group of animals over a large BW range. For comparing FHP
in adult animals of different species, MBW is frequently expressed as BW0.75 but validity of the 0.75
exponent has been questioned for growing animals (Noblet et al., 1999; Labussière et al., 2008). The
objective of this study is to combine FHP measurements obtained according to a common methodology
in four species of growing monogastric farm animals (veal calves, pigs, turkeys and broilers).

Material and methods

In each trial, single housed (calves, pigs) or group-housed (broilers, turkeys) animals were kept for
GD\VLQDUHVSLUDWLRQFKDPEHUDQGDQHQHUJ\DQGSURWHLQEDODQFHZDVFDUULHGRXWGXULQJWKH¿UVW
days. On the 7th day, no feed was provided. The continuous recording of gas exchange in the chamber
and of physical activity (with force sensors) of the animals on the fasting day allowed calculating,
according to modelling techniques, the asymptotic value of HP at zero activity considered to be FHP
(Labussière et al., 2011). The total number of FHP observations was 18 in 25-100 kg BW castrated
pigs (fed ad libitum or restricted; de Lange et al. LQNJ%:EURLOHUV WULDOVIHG
ad libitum; unpublished), 19 in 0.4-24.0 kg BW male turkeys (fed ad libitum; Rivera-Torres et al.,
 DQGLQNJ%:PDOHPLONIHGFDOYHV IHGad libitum or restricted; Labussière et al.,
2009). For each species, BW range was divided into 3 intervals to determine the effect of BW on ME
intake and FHP (Table 1). The exponent for calculating MBW was estimated from the relationship
between FHP and MBW and ME intake during the previous days (log basis) to account for residual
KHWHURVFHGDVWLFLW\DQGHIIHFWRIIHHGLQJOHYHORQ)+3WKHGLIIHUHQFHZLWKXVXDOFRHI¿FLHQWVZDV
assessed with the extra sum of square reduction test (Labussière et al., 2008).

Results and discussion

7KHEHVWH[SRQHQWRI0%:GXULQJWKHJURZLQJSHULRGZDVDQGLQJURZLQJSLJV
broilers and calves, respectively which all differed (P<0.05) from 0.75. In turkeys, it equalled
0.70 and did not differ from 0.75. Differences in the exponent among species can be attributed to
differences in the allometric growth of visceral organs relative to the whole body (from 0.7 in pigs
WRPRUHWKDQLQFDOYHV5REHOLQ1REOHW et al., 1999; Rivera-Torres et al., 2011). With
increasing BW, FHP decreased in turkeys and calves and tended to decrease in pigs (3 0.11) because
of the correlated variations in ME intake (Table 1; Labussière et al., 2011). In close to ad-libitum
fed animals, daily FHP averaged 740 kJ/kg BW in pigs, 438 kJ/kg BW0.70 in broilers, 413 kJ/
kg BW0.70 in turkeys and 317 kJ/kg BW0.85 in calves over the growing period. The rate of change
of FHP per additional ME intake (kJ/kJ) was 0.13 in turkeys, 0.14 in pigs and 0.23 in calves (Table
 7RFRQFOXGHVSHFL¿FSDUDPHWHUVPXVWEHXVHGIRU0%:RIJURZLQJQRQUXPLQDQWDQLPDOVDQG
FHP and maintenance ME requirements must be adjusted for differences in ME intake.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 313
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_107, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIERG\ZHLJKW %: DQGSUHYLRXV0(LQWDNHRQIDVWLQJKHDWSURGXFWLRQ )+3 RI
growing pigs, broilers, turkeys and calves.

Species MBW1 BW, ME intake (range), FHP2, ¨)+3¨0(1

kg kJ/kg MBW/day kJ/kg MBW/day

Pigs BW 55.4 2,084a (1,578-2,598)   0.14

n=18 70.4 2,082a   
 2,003b (1,485-2,528)  
Broilers BW0.70 1.0 1,558a 439 -
WULDOV 1.8 1,472b 431
Q  2.5 1,398c 435
Turkeys BW0.70  1,479a 458a (431) 0.13
n=19  1,275b 453a (422)
21.1 1,025c 391b 
Calves BW0.85 72.8 a  298a (312) 0.23
n=47  b  301a (322)
 554c  280b 

abc LS-means within each species with different superscripts are different (P<0.05).
10%:PHWDEROLF%:¨)+3¨0(VORSHRIWKHUHODWLRQVKLSEHWZHHQ)+3DQG0(LQWDNH
2 In brackets, FHP in close to ad libitum ME intake (2580 kJ/kg BWGD\LQSLJVN-NJ%:0.70/day in turkeys

and 710 kJ/kg BW0.85/day in calves).

References
'H/DQJH&-YDQ0LOJHQ-1REOHW6'XERLVDQG6%LUNHWW3UHYLRXVIHHGLQJOHYHOLQÀXHQFHVSODWHDXKHDW
production following a 24 h fast in growing pigs. Br. J. Nutr. 95, 1082-1087.
Labussière E., S. Dubois, J. van Milgen, G. Bertrand and J. Noblet, 2008. Fasting heat production and energy cost of
standing activity in veal calves. Br. J. Nutr. 100, 1315-1324.
Labussière E., G. Maxin, S. Dubois, J. van Milgen, G. Bertrand and J. Noblet, 2009. Effect of feed intake on heat
SURGXFWLRQDQGSURWHLQDQGIDWGHSRVLWLRQLQPLONIHGYHDOFDOYHV$QLPDO
Labussière E., J. van Milgen, C.F.M. de Lange and J. Noblet, 2011. Maintenance energy requirements of growing pigs
DQGFDOYHVDUHLQÀXHQFHGE\IHHGLQJOHYHO-1XWU
Noblet J., C. Karege, S. Dubois and J. van Milgen, 1999. Metabolic utilization of energy and maintenance requirements
LQJURZLQJSLJVHIIHFWVRIVH[DQGJHQRW\SH-$QLP6FL
Rivera-Torres V., J. Noblet, S. Dubois and J. van Milgen, 2010. Dynamics of energy utilization in male and female
turkeys during growth. Animal 5, 202-210.
Rivera-Torres V., J. Noblet and J. van Milgen, 2011. Changes in chemical composition in male turkeys during growth.
3RXOW6FL
5REHOLQ-&RPSRVLWLRQFRUSRUHOOHGHVERYLQVpYROXWLRQDXFRXUVGXGpYHORSSHPHQWHWGLIIpUHQFHVHQWUHUDFHV
Thesis, Université de Clermont-Ferrand, France.

314 Energy and protein metabolism and nutrition in sustainable animal production
The evolution of INRA feeding systems for ruminants based on absorbed
nutrients and animal responses
P. Nozière1, D. Sauvant2, J.L. Peyraud and co-workers
1UMR Herbivores, Theix, France; [email protected]
2UMR Mosar, Paris, France
UMR Pegase, St Gilles, France
UMR Selmet, Montpellier, France
UR Zootechniques, Petit-Bourg; INRA, France

Introduction
The current feed evaluation systems for ruminants still have some limits regarding emerging
FKDOOHQJHVLQUXPLQDQWQXWULWLRQSDUWLFXODUO\IRUSUHGLFWLRQRIIHHGFRQYHUVLRQHI¿FLHQF\SURGXFW
quality, animal health, and emissions to the environment. Further, they do not appropriately cover
the case of either low quality or very intensive diets. A working group of INRA’s researchers has
been constituted for 3 years (the ‘Systali’ project) to address these issues. Continuously accumulated
new published data on digestive, metabolic and productive responses of ruminant to diets have thus
EHHQLQWHJUDWHGZLWKLQDµQHZJHQHUDWLRQ¶RIIHHGXQLWV\VWHPV7KLVLVEDVHGRQÀRZVRIQXWULHQWV
DQGPXOWLSOHDQLPDOUHVSRQVHVWRWKHVHÀRZVDQGDOORZVWKHXSGDWHGV\VWHPWREHXVHGRQHQODUJHG
¿HOGVRIDSSOLFDWLRQ

Material and methods

Large data bases of digestion, metabolic balance and animal performances, representative of the
major feeding practices, have been built and interpreted by meta-analysis, allowing dissociation
of intra vs. inter experiment variability (Sauvant et al. 7RHQVXUHHQODUJHPHQWRI¿HOGVRI
application, a particular attention has been devoted to low digestibility (including tropical forages)
and high concentrate diets. An integrated mechanistic model of digestion, that ensures consistency
across empirical relationships issued from the various meta-analyses, has been built (Sauvant
and Nozière, 2013). In contrast with the gut model included in the current INRA systems (INRA,
2007), this new model integrates the effects of the main digestive interactions related to feeding
level (FL), proportion of concentrate (PCO), and rumen protein balance (RPB), on transit, substrate
degradation, microbial synthesis and ruminal fermentation. There also are improvement in the
TXDQWL¿FDWLRQRIHQGRJHQRXVORVVHVDQGWKHLULPSOLFDWLRQRQSURWHLQUHTXLUHPHQWVIRUPDLQWHQDQFH
The major equations of this model permit calculation of new nutritive values of feeds and diets, as
ZHOODVDEVRUEDEOHQXWULHQWÀRZVDQGVXEVHTXHQWO\UHYLVLWLQJWKHUHTXLUHPHQWVDQGUHVSRQVHVWR
GLHWV0XOWLSOHPDUJLQDOUHVSRQVHVRIDQLPDOV LHIHHGHI¿FLHQF\PLON\LHOGJURZWKUDWHPLONDQG
carcass composition, risk of acidosis, N and CH4 emissions...) to feeding practices and subsequent
nutrient supply are also being provided. These features are currently undergoing extensive validation.
A simulation tool, which also integrates models of intake updated at pasture (Delagarde et al., 2011)
is being developed, and allows simulation of hundreds of dietary and animal situations, and thereby
global checking of the consistency of the values of supply, requirements and responses. Coupled
to external data, this allows evaluation and validation of the whole renewed system, across a large
range of feeding practices and animal characteristics.

Results and discussion

7KHJOREDOVWUXFWXUHRIWKHFXUUHQW,15$  V\VWHPVKDVQRWFKDQJHGEXWVLJQL¿FDQWPRGL¿FDWLRQV
KDYHEHHQDGRSWHG2QO\WKHPDMRUPRGL¿FDWLRQVDUHSUHVHQWHGLQWKHSUHVHQWSDSHU'LHWDU\SURWHLQ
supply remains a calculation from in saccoGHJUDGDWLRQKRZHYHUSDVVDJHUDWHVDUHQRORQJHU¿[HG
but modulated according to FL and PCO. The estimation of energy available in the rumen is improved

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 315
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_108, © Wageningen Academic Publishers 2013
E\WDNLQJLQWRDFFRXQWGLJHVWLYHLQWHUDFWLRQVDQGLQWHVWLQDOGLJHVWLELOLW\RIVXEVWUDWHVLWVHI¿FLHQF\
IRUPLFURELDOJURZWKLVQRORQJHU¿[HGEXWUHJXODWHGE\UXPHQSDUDPHWHUV7KH20GLJHVWLELOLW\RI
IHHGVZLWKLQDGLHWLVQRORQJHU¿[HGEXWUHJXODWHGE\)/3&2DQG53%/RVVHVRIHQHUJ\WKURXJK
CH4 and urines, that partly compensate digestive interactions, are more clearly dissociated and
TXDQWL¿HG$PDMRUDGYDQFHUHODWHVWRWKHIDFWWKDWHQHUJ\DQGSURWHLQDUHPRUHFOHDUO\LQWHUDFWLQJ
through an iterative effect of the RPB on energy digestion and consequently microbial protein
synthesis that itself affects RPB. The protein maintenance requirements, based on fecal endogenous
and minimal urinary N losses, have been reconsidered to account for FL. Subsequent recalculation
of requirements and responses, and their validation, are still in progress.

Conclusion
The renewed INRA feeding systems for ruminants will be based on a more precise representation of
GLJHVWLYHSURFHVVHVDQGÀRZVRIQXWULHQWVDQGRIPXOWLSOHDQLPDOUHVSRQVHVWRWKHVHVÀRZV7KH\
are built from integration of a large amount of data, carefully interpreted, incorporated and validated
DFFRUGLQJWRDZLGHUDQJHRIIHHGLQJSUDFWLFHV:KLOHPDLQWDLQLQJWKHIXQFWLRQDOLW\DQGVSHFL¿FLW\
RIWKHFXUUHQWV\VWHPVWKHVHPRGL¿FDWLRQVH[WHQGWKHLU¿HOGVRIDSSOLFDWLRQDQGWKHVSHFWUXPRI
predicted responses.

References
INRA, 2007. Alimentation des bovins, ovins et caprins – Besoins des animaux – Valeurs des aliments – Tables INRA
2007, Versailles, 307 pp.
Sauvant, D. and P. Nozière, 2013. Integrative model of the digestive tract including the interactions involved in
energy and protein digestion in Ruminants. In: J.W. Oltjen, E. Kebreab, H. Lapierre (editors), Energy and protein
metabolism and nutrition in sustainable animal production. Wageningen Academic Publishers, Wageningen, the
Netherlands, EAAP Series 134:317-318.
Sauvant, D., P. Schmidely, J.J. Daudin and N.R. St-Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2, 1203-1214.
Delagarde, R., P. Faverdin, C. Baratte and J.L. Peyraud, 2011. GrazeIn: a model of herbage intake and milk production
for grazing dairy cows. 2. Prediction of intake under rotational and continuously stocked grazing management.
*UDVVDQG)RUDJH6FLHQFH

316 Energy and protein metabolism and nutrition in sustainable animal production
Integrative model of the digestive tract including the interactions
involved in energy and protein digestion in ruminants
D. Sauvant1 and P. Nozière2
1UMR INRA-AgroParisTech Mosar, Paris, France; [email protected]
2UMR INRA Herbivores, Theix, France

Introduction
In ruminants, a major limitation of feeding systems in predicting the responses to diets is the
LQVXI¿FLHQWLQWHJUDWLRQRIWKHYDULRXVLQWHUDFWLRQVLQYROYHGLQHQHUJ\DQGSURWHLQGLJHVWLRQDQG
metabolism. To bridge this gap, in the new feeding systems built at INRA (Nozière et al., 2013),
a focus was made on digestive interactions by combining data issued from meta-analysis and a
mechanistic model of digestion, as already proposed (Sauvant and Mertens, 2008).

Material and methods

Three large data bases of results of published digestion studies were built: on cattle digestion (800
H[SHULPHQWV([SWUHDWPHQWV7U RQFDORULPHWULFVWXGLHVRQFDWWOHVKHHSDQGJRDWV ([S
7U DQGRQVKHHSGLJHVWLRQ ([S7U 7KHVHEDVHVKDYHEHHQVWXGLHGE\PHWDDQDO\VLVXVLQJ
encoding of data allowing careful study of the meta-designs and dissociation of intra vs. inter experiments
variability (Sauvant et al., 2008). This phase provided numerous empirical equations quantifying the
digestive and some metabolic responses to changes in the diet composition or in feeding practices.

A number of these empirical equations were directly used in the new feeding system to calculate
the new versions of net energy (NE) and metabolisable protein (PDI). The other equations related to
digestive phenomena that lead to the production of absorbable nutrients, (volatile fatty acids, VFA,
glucose and long chain fatty). Equations were also derived that relate to chewing activities, saliva
UHF\FOLQJUXPHQS+DQGYROXPHSURGXFWLRQRI&+DQGWUDQVLWÀRZUDWHVDVZHOODVWKHVL]HVDQG
DGMDFHQWÀRZVRIPDMRUFRPSDUWPHQWVLQWKHUXPHQ1') GHJUDGDEOH'RUQRWLQWKHODUJHRU
small particles), protein (soluble-S, or D and undegradable), starch (S or D), water, VFA and microbes.

7REXLOGWKHQHZIHHGXQLWVWKUHHPDMRUIDFWRUVDIIHFWLQJGLJHVWLYHLQWHUDFWLRQVZHUHLGHQWL¿HGWKH
dry matter intake per LW (DMI%LW), the proportion of concentrate (PCO) and the rumen protein
EDODQFH 53% LQJHVWHG&3±GXRGHQDO&3  >1+DEVRUSWLRQ±8UHD1UHF\FOLQJLQWKH
rumen]). These 3 factors affect transit of particles and liquids and digestible organic matter (DOM)
in the whole tract. They also alter the proportions of gross energy dissipated in CH4 and urine. Feed
degradation and by-pass predictions of protein and starch were based on in sacco measurements
and transits; they were validated on in vivoGXRGHQDOÀRZV7KHFDOFXODWLRQRIIHUPHQWDEOH20LQ
WKHUXPHQ )20 IURP'20ZDVPRGL¿HGWREHFORVHUWRWKHDFWXDO)20LQWKHUXPHQWKDQWKH
previous expressions proposed in the literature (and in the former INRA system). Microbial protein
ÀRZDWWKHGXRGHQXPZDVDOVRUHGH¿QHGIURP)203&2DQG53%

To solve the challenge of insuring global consistency among all these equations, an integrated
mechanistic model of gut has been built. Its rumen part, the kernel of the model, is a minimal model
based on the 12 above mentioned compartments. In contrast to other mechanistic models of rumen, the
current one was built through a top-down procedure using a framework of key structuring relationships
issued from the meta-analyses. By this procedure, the most important equations above were used
to calibrate the model and to check mutual consistency and for any unexpected interactions. This
procedure of optimization was performed with Modelmaker III through virtual experimentations
using plausible ranges of values of the most important dietary inputs: DMI%LW, PCO, dietary NDF,
CP and starch, effective degradability in sacco of N and starch.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 317
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_109, © Wageningen Academic Publishers 2013
Results and discussion
Major evaluations of the model were performed by comparing the responses of the model with
YDOXHVSUHGLFWHGE\WKHVWUXFWXULQJHTXDWLRQV7KHVLPXODWHGGXRGHQDOÀRZVRISURWHLQDQGVWDUFK
were very close to the predicted ones and, globally, the equations used to predict the new feed units
DUHPXWXDOO\FRQVLVWHQW,QFRQWUDVWSUHGLFWLRQVRIFRPSDUWPHQWVDQGÀRZVRI1')DQG+2ZHUH
a bit less accurate. For some characteristics, as chewing time, the framework provided more than
one equation to calculate that entity according to various factors, which gives fairly different results
according to the equation used.

The model has been incorporated within two calculation and simulation tools to ensure its application
to small ruminants, evaluate its animal’s requirements and responses, integrate with the INRA’s
¿OOXQLWV\VWHPRILQWDNHDQGYDOLGDWHWKHZKROHUHQHZHGV\VWHPDFURVVVLPXODWLRQRIKXQGUHGVRI
dietary and animal situations.

Conclusion
A new feeding system model has been constructed from numerous equations extracted from meta-
analysis of large datasets of published results ensuring a basis in real digestive phenomena. It
includes a better integration of digestive interactions and allows the calculation of not only the new
YHUVLRQRIWKHHQHUJ\DQGSURWHLQXQLWVEXWDOVRWKHÀRZVRIDEVRUEDEOHQXWULHQWVDQGVHYHUDORWKHU
key aspects of digestion in ruminants.

References
Nozière P. and D. Sauvant, 2013. The evolution of INRA feeding systems for Ruminants based on absorbed nutrients
and animal responses. In: J.W. Oltjen (editor), Energy and protein metabolism and nutrition. Wageningen Academic
Publishers, Wageningen, the Netherlands, EAAP Series XXX:xx-xx.
Sauvant, D. and D.R. Mertens, 2008. Use of metaanalysis to build a mechanistic model of responses of rumen digestion
WRGLHWDU\¿EUHLQFDWWOH&DQ-$QLP6FL
Sauvant D, P. Schmidely, J.J. Daudin and N.R. St-Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2, 1203-1214.

318 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWVRIGLHWFRPSRVLWLRQGXULQJWKH¿QLVKLQJSHULRGRQSURWHLQDQG
lipid deposit in young bulls
J. Agabriel, M. Al-Jammas, I. Ortigues-Marty, C. Villetelle, P. Nozière and F. Garcia-Launay
,15$805+HUELYRUHV6DLQW*HQqV&KDPSDQHOOH)UDQFH
[email protected]

Introduction
The INRA feeding system aims at simulating young bull’s growth and body composition in response
WRGLHWDU\FKDQJHVGXULQJWKH¿QLVKLQJSHULRG3UHVHQWO\WLVVXHGHSRVLWLRQLVPRGL¿HGE\QHWHQHUJ\
intake in relation to age and breed, without considering the relative proportion of energy nutrients. But
DW¿[HG1(GHQVLW\UDWLRQVEDVHGRQFRQFHQWUDWHRUPDL]HVLODJHVXSSRUWIDWWHUJDLQV *DUFLDet al.,
 ZKLFKMXVWL¿HVEHWWHUFKDUDFWHUL]DWLRQRIGLHWFRPSRVLWLRQLQSUHGLFWLRQPRGHOV2XUREMHFWLYH
LVWRVLPXODWHWKHDEVRUEHGHQHUJ\QXWULHQWÀX[HVWKHLUWLPHYDULDWLRQDQGUHODWLYHSURSRUWLRQVDV
ZHOODVWKHLUHIIHFWVRQOLSLGVDQGSURWHLQVRIFDUFDVVDQGYLVFHUDOGHSRWVRI¿QLVKLQJEXOOV$G\QDPLF
model of growing cattle (Hoch and Agabriel, 2004) provides the framework and helps target variables
of interest to reveal these effects. The present work aims to quantify diet effects on energy nutrient
ÀX[HVRI\RXQJEXOOVGXULQJIDWWHQLQJDQGWROLQNWKHPWRERG\FDUFDVVRUYLVFHUDOOLSLGFRQWHQW

Material and methods

We used two complementary approaches: (A1) an analysis of publications chosen from the
LQWHUQDWLRQDOOLWHUDWXUHWKDWDOORZHGHVWLPDWLRQRIGDLO\QXWULHQWÀX[HVIURPSXEOLVKHGGDWD Q  
DQGOLQNLQJWKHPZLWKIDWGHSRVLWLRQLQFDUFDVVDQGYLVFHUD Q  DQG $ DQDQDO\VLVRIWZRSUHYLRXV
INRA experiments where young bulls, Blond d’Aquitaine – late maturing breed (Micol et al., 2007),
and Salers – medium maturing breed (Geay et al., ZHUHLQGLYLGXDOO\IHGGLHWV FRUQVLODJHYV
hay and concentrate; grass silage vs. concentrate). Individual weights, gains, daily intake of each
feed, and detailed slaughter data were available.

Diets were estimated from INRA Feed Tables (OMD, Fermentable OM, protein digestible in the
intestine PDI, digestible lipids DLi, DE, ME) and from Loncke et al. (2009) predictions of net
portal appearance (mmol/j/kg LW) of VFA, Glucose, BOH, Lactate and alpha-amino-N. Net portal
DSSHDUDQFHRIHDFKQXWULHQWZDVH[SUHVVHGRQDQHQHUJ\EDVLV'DLO\ÀX[HVZHUHFDOFXODWHGGXULQJ
WKH¿QLVKLQJSHULRGIRUJURXSVLQH[SHULPHQWV$RUDYHUDJHGDFURVVHDFKLQGLYLGXDOLQ$7KH
HIIHFWVRIGLHWVRQUHFDOFXODWHGQXWULHQWVÀX[HVDQGERG\FRPSRVLWLRQZHUHWHVWHG6$6*/0ZDV
used for statistical analysis between individual values.

Results and discussion

The calculated patterns between absorbed energy and nitrogen type nutrients differed between diets
within experiments and helped to describe the observed differences in fat depots. Using A1 results,
we found that total LW gain and carcass weight were greater on groups fed ME rich diets, while
the quantity of body lipids at slaughter depended on the composition of ME, especially its content
in starch and DLi.

A linear combination of energy from nutrients (R) including DLi and portal amino acids and acetate
ZDVVLJQL¿FDQWO\DVVRFLDWHGZLWKWKH¿QDOTXDQWLWLHVRIFDUFDVVOLSLGV )LJXUH 8VLQJLQGLYLGXDOV
$ VLJQL¿FDQWGLIIHUHQFHVLQSRUWDOHQHUJ\ÀX[HVRI9)$DQG'/LZHUHQRWLFHG3UHYLRXVOLQHDU
combination R was individually calculated and compared to carcass lipids (%) or body lipids (%) for
WKHWZRH[SHULPHQWV$VLJQL¿FDQWHIIHFWRIGLHWDQGEUHHGLVREVHUYHGZKLOHLQGLYLGXDOV¶GLIIHUHQFHV
remains poorly predicted within group (Table 1).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 319
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_110, © Wageningen Academic Publishers 2013
)LJXUH5HODWLRQVKLSVEHWZHHQDFRPELQDWLRQRIDYHUDJHGDLO\HQHUJ\IURPQXWULHQWV5 HQHUJ\RI
GLJHVWLEOHOLSLGV HQHUJ\ SRUWDODPLQRDFLGVDQGDFHWDWH DQGFDUFDVVFRPSRVLWLRQ FDUFDVVZHLJKW
&&: LQFKRVHQH[SHULPHQW $ 

7DEOH  $YHUDJH YDOXHV RI 5 DQG IDWQHVV /LSLGV FDUFDVV&DUFDVV :HLJKW &&:  /LSLGV
YLVFHUD&&: LQH[SHULPHQW$

Exp Salers(Geay et al., 1997) Blond d’Aquitaine(Micol et al., 2007)

Diets Hay Grass Corn P-value Concentrate Corn Hay P-value

silage silage silage

R D 0.259 b 0.285 c <0.001 F 0.257 b 0.223 a <0.001

±0.002 “ ±0.001 ±0.007 ±0.0003 ±0.005
LipC%CCW D E 19.74 b 0.004 9.29 ab 10.87 b 7.99 a 0.002
Lip Visera %CCW 4.58 a 5.58 b E  4.04 ab 4.73 b 3.47 a 0.002

In the future and after validation, evolution of the growth model could incorporate a modulation of
the Gompertz function for lipid carcass synthesis by the R combination of energy nutrients.

Acknowledgements
The authors wish to thank the two companies INZO and Limagrain for their support of this research.

References
Garcia F., R.D. Sainz, J. Agabriel, L.G. Barioniand J.W. Oltjen, 2008. Comparative analysis of two dynamic mechanistic
models of beef cattle growth. Animal Feed Science and Technology 143, 220-241.
Geay Y., B. Picard, R.D Jailler, R.T. Jailler, A. Listrat, C. Jurie, M.C. Bayle and C. Touraille, 1997. Effets de la nature
de la ration sur les performances, les caractéristiques musculaires et la qualité de la viande de taurillons Salers.
Renc. Rum. 4, 307-310.
Hoch T. and J. Agabriel, 2004. A mechanistic dynamic model to estimate beef cattle growth and body composition: 1.
Model description. Agric. Syst. 81, 1-15.
/RQFNH&0RGpOLVDWLRQGHVUHODWLRQVHQWUHO¶DOLPHQWDWLRQHWOHVÀX[VSODQFKQLTXHVGHQXWULPHQWVpQHUJpWLTXHV
chez le ruminant. Thèse de Doctorat de l’Institut des Sciences et Industries du Vivant et de l’Environnement :
$JUR3DULV7HFKS
Micol D., H. Dubroeucq, C. Martin, F. Garcia, M.M. Mialon and J. Agabriel, 2007. Utilisation de rations de valeurs
FRQWUDVWpHVSRXUOD¿QLWLRQGHMHXQHVERYLQVGHUDFH%ORQGG¶$TXLWDLQH5HQF5HFK5XP

320 Energy and protein metabolism and nutrition in sustainable animal production
Integrating nutritional and reproductive models to improve reproductive
HI¿FLHQF\LQGDLU\FDWWOH
S.L. Shields1, H. Woelders2, M. Boer, C. Stötzel, S. Röeblitz, J. Plöntzke and J.P. McNamara1
1Department of Animal Sciences, Washington State University, Pullman, WA, USA;
[email protected]
2Animal Breeding and Genomics Centre, Wageningen UR Livestock Research, Lelystad, the
Netherlands
Adaptation Physiology Group, Department of Animal Sciences, Wageningen University, Wageningen,
the Netherlands
Computational Systems Biology Group, Zuse Institute Berlin, Berlin, Germany

Successful reproduction requires coordination among neural, endocrine and nutritional systems
leading to ovulation, insemination and a uterine environment that allows embryonic growth and
attachment. These processes are a function of genetics, dietary nutrient composition and intake,
DQGKRXVLQJDQGFOLPDWH:HODFNDV\VWHPVELRORJ\DSSURDFKWRVWXG\DQGGH¿QHWKHFRQWURORI
reproduction in the dairy cow. Pregnancy rates in the best managed herds only approach 25 to
30%, and may be due to the multifactorial nature of reproductive processes (McNamara, 2010).
Lower fertility increases the cost for insemination because of low reproduction performance and
UHPDLQVDSULPDU\UHDVRQIRUFXOOLQJFRZVLQWKH¿UVWWKUHHZHHNVRIODFWDWLRQ &KDJDVet al., 2007).
Additional days the cow is not pregnant beyond the optimal time post-calving are costly to the dairy
producer. Low fertility has often been blamed on high rates of milk production, but this is not the
only factor affecting reproduction (Sangsritavong et al., 2002). There are three major systems of
reproduction that can be affected by genetics, nutrition and management: the hypothalamic-pituitary
axis controlling initialization of cycling, the follicular development in the ovary leading to ovulation;
and the successful fertilization and growth of an embryo in the uterine environment. Thus, the goal
LVWRLPSURYHUHSURGXFWLRQHI¿FLHQF\ZKLOHGHFUHDVLQJDQ\HQYLURQPHQWDOLPSDFWV%HWWHUQXWULWLRQDO
management, genetic selection for fertility, and more attention to success can improve reproductive
HI¿FLHQF\,QRUGHUWRGRDOOWKLVHI¿FLHQWO\ELRV\VWHPVPRGHOVFDQEHRIJUHDWHI¿FDF\

Our hypothesis is that a dynamic, mechanistic, deterministic metabolic systems model of nutrition
and reproduction can be used to describe the control of reproductive processes in the dairy cow.
Therefore, our objective was to integrate two existing mechanistic, dynamic models of nutritional
and reproductive processes in the dairy cow. The objective of this research model is to be suitable
for evaluation of data, concepts, and hypotheses regarding underlying genetic, nutritional, and
physiological control of reproduction. A model of metabolism (Molly, UC Davis); which describes
metabolism of glucose, VFA, and amino acids for fat and protein synthesis and degradation and
milk component production, as well as tracking energy transactions (ADP/ATP) (Baldwin, 1995)
was integrated with a model of reproductive processes (Stötzel et al., 2011), which describes growth
and decay of the follicles and corpus luteum, gonadotropin releasing hormone, follicle stimulating
KRUPRQHOXWHLQL]LQJKRUPRQHSURJHVWHURQHHVWURJHQR[\WRFLQDQGSURVWDJODQGLQ)ĮRYHUWLPH
7KHWZRPRGHOVDUHLQWHJUDWHGDWVSHFL¿FSRLQWVLQFUHDVHGERG\IDWDQGJOXFRVHDYDLODELOLW\GHFUHDVH
the post-partum anovulatory interval; glucose and IGF-I increase rates of follicle stimulating hormone,
luteinizing hormone, and follicular growth; and increased degradation of estrogen and progesterone
in the liver affect follicular development, maintenance of the corpus luteum and embryonic survival.
7KHHQHUJ\EDODQFHRIWKHFRZDQGUHVXOWDQWÀX[WKURXJKWKHDGLSRVHWLVVXHDQGOLYHUDOWHUWKHSDWWHUQ
RIF\FOLFLW\DQGDIIHFWVWKHRQVHWRIIROOLFXODUZDYHV,QDGGLWLRQJOXFRVHÀX[SUHFDOYLQJDQGLQ
HDUO\ODFWDWLRQDIIHFWV,*)FRQFHQWUDWLRQVOLYHUGHULYHG,*)UHJXODWHV¿QDOPDWXUDWLRQRIWKH
dominant follicle (Kawashima et al. )LQDOO\IDVWHUPHWDEROLFÀX[WKURXJKWKHOLYHULQFUHDVHV
degradation of estrogen and progesterone (Sangsritavong et al., 2002), with a resultant decrease in
the signs of visible estrus, reducing the number of inseminations. The reduction in progesterone

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 321
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_111, © Wageningen Academic Publishers 2013
concentration, prior to insemination and during the luteal phase of the cycle after insemination, is
associated with low embryo survival (Diskin et al. 

The model behavior is such that it describes the pattern and direction of response in reproductive
processes consistent with literature values. That is: lower energy balance and glucose availability
reduce adipose tissue amount and delay the onset of cycling; lower IGF1 (as a function of glucose
and energy balance) reduces follicular growth; and increased metabolic rate (conversion of ATP
to ADP in the total viscera) as a function of feed intake or milk production increases reduces the
peak and average concentrations of estrogen and progesterone. A reduction in body fat of 1 kg
ZLOOLQFUHDVH',0DW¿UVWF\FOLQJE\G WKXVNJOHVVERG\IDWGHOD\V¿UVWHVWUXVE\G 
Increasing circulating concentration of IGF1 by 20 ng/ml will increase follicular growth by 1 mm
in 18 days (versus an average of 12 mm pre-ovulation). Increasing AtAdv (total ATP conversion to
ADP) from 372 to 1488 moles/d decreases peak estrogen from 0.342 to 0.281 (arbitrary units; peak
E = 153e(0.0004(AtAdv)))and degrades the pattern of peaks; progesterone at peak is decreased from
1.023 to 0.913 (peak P = 274e(8X10-4*AtAdv)) and time between peaks is increased.

7KHPRGHOZLOOEHWHVWHGDJDLQVWRWKHUUHVHDUFKDQG¿HOGGDWDLQRUGHUWRLGHQWLI\ZHDNQHVVHVLQ
understanding, to use as a basis for further research. The model is to be used in design and testing
RIVSHFL¿FUHVHDUFKK\SRWKHVHVLQWHJUDWHGRYHUJHQHWLFVQXWULWLRQDQGUHSURGXFWLRQWRFRQQHFW
the basic biology with animal level function. It can be used in reproductive biology, nutrition and
animal management research, and can be used in classes and extension efforts to assist in integrating
complex functions toward better management decisions.

References
%DOGZLQ5/0RGHOOLQJUXPLQDQWGLJHVWLRQDQGPHWDEROLVP&KDSPDQ +DOO1HZ<RUN86$SS
Chagas, L. M., J. J. Bass, D. Blache, C. R. Burke, J. K. Kay, D. R. Lindsay, M. C. Lucy, G. B. Martin, S. Meier, F.
M. Rhodes, J. R. Roche, W. W. Thatcher, and R. Webb, 2007. Invited Review: New perspectives on the roles of
nutrition and metabolic priorities in the subfertility of high-producing dairy cows. J. Dairy Sci. 90, 4022-4032
Diskin, M. G. and D. G. Morris, 2008. Embryonic and early foetal losses in cattle and other ruminants. Reprod. Dom.
$QLP 6XSSO 
Kawashima, C., S. Fukihar, M. Maeda, E. Kaneko, C. A. Montoya, M. Matsui, T. Shimizu, N. Matsunaga, K. Kida,
Y. I. Miyake, D. Schams, and A. Miyamoto, 2007. Relationship between metabolic homones and ovualtion of
GRPLQDQWIROOLFOHGXULQJWKH¿UVWIROOLFXODUZDYHSRVWSDUWXPLQKLJKSURGXFLQJGDLU\FRZV5HSUR
McNamara, J.P., 2010. Nutrigenomics for improved reproduction, Chapter 19 in Reproductive Genomics in Domestic
Animals by Jiang, Z. and T. Ott, eds. 27.
Sangsritavong, S., D. K. Combs, R. Sartori, L. E. Armentano, and M. C. Wiltbank, 2002. High feed intake increases
OLYHUEORRGÀRZDQGPHWDEROLVPRISURJHVWHURQHDQG(VWUDGLROȕLQGDLU\FDWWOH-'DLU\6FL
Stötzel, C., J. Plöntzke, and S. Röblitz, 2011. Advances in modeling of the bovine estrous cycle: Administration of
3*)Į=,%UHSRUW=XVH,QVWLWXWH%HUOLQ

322 Energy and protein metabolism and nutrition in sustainable animal production
Use of thermodynamic equations to improve predictions of volatile fatty
acid production by the Molly cow model
S. Ghimire1, R.A. Kohn2, P. Gregorini and M.D. Hanigan1
19LUJLQLD3RO\WHFKQLF,QVWLWXWHDQG6WDWH8QLYHUVLW\%ODFNVEXUJ9$86$VDQG#YWHGX
2$QLPDODQG$YLDQ6FLHQFHV8QLYHUVLW\RI0DU\ODQG&ROOHJH3DUN0'86$
'DLU\1=/WG3ULYDWH%DJ+DPLOWRQ1HZ=HDODQG

Introduction
Volatile fatty acids are important products of fermentation in ruminant animals contributing about 72%
of the total energy supply (Bergman, 1990). They also affect ruminal hydrogen supply, which dictates
methane production. Therefore it is imperative to be able to accurately predict VFA production. The
0ROO\FRZPRGHOLVDG\QDPLFPHFKDQLVWLFPRGHOZKLFKXVHVVWRLFKLRPHWU\FRHI¿FLHQWVGHULYHG
by Murphy et al.  WRSUHGLFW9)$SURGXFWLRQ7KHFRHI¿FLHQWVDUHEDVHGRQWKHDVVXPSWLRQ
that substrate supply is a primary determinant of VFA production rates, and interconversions are
assumed to either not occur or be constant across diets. Our recent work demonstrated that the model
poorly predicted VFA production rates and that accounting for variable interconversion rates may
improve prediction accuracy. The objectives of this study were to incorporate VFA interconversion
equations and evaluate them in the Molly cow model.

Material and methods

7KHWKHUPRG\QDPLFHTXDWLRQVGHVFULEHGE\8QJHUIHOGDQG.RKQ  ZHUHXVHGWRUHSUHVHQW
interconversions among VFA in the model with ruminal concentrations of VFA and pH the main
driving variables. Rate constants for each equation were algebraically calculated from several literature
observations, mean estimates were used in the model. Global sensitivity of VFA concentrations and
V\QWKHVLVWRWKH0XUSK\FRHI¿FLHQWVDQGLQWHUFRQYHUVLRQUDWHFRQVWDQWVZDVFRQGXFWHGXVLQJWKH
Fourier amplitude sensitivity test as described by Saltelli et al. (1999) with sampling boundaries of
75 and 125% of initial parameter estimates. For evaluation and parameter estimation purposes, a VFA
production dataset containing 8 studies was combined with a data set containing VFA concentration
GDWDIURPVWXGLHV7KHODWWHUZDVDVXEVHWRIWKRVHXVHGE\WKH15&FRPPLWWHHWRIRUPXODWH
the 2001 Nutrient Requirement model. The model was set to simulate for 10 d to ensure steady
state, and predictions from the last day were compared to observed values. Parameters that were
HVWLPDWHGLQFOXGHGWKHGHQRYR9)$SURGXFWLRQFRHI¿FLHQWVIRUDFHWDWHSURSLRQDWHDQGEXW\UDWHGH
novo production from starch, soluble carbohydrate, cellulose, and protein for mixed diets plus the
absorption rate constants for each. Residual errors of prediction were used to calculate root means
square prediction errors (RMSPE), which were expressed as a percentage of the observed mean,
and RMSPE were partitioned into mean bias, slope bias, and dispersion.

Results and discussion

7KHUHGHULYHGFRHI¿FLHQWVZHUHGLIIHUHQWWKDQWKHRULJLQDOVHW7KHSDUDPHWHUVWKDWZHUHPRVWDIIHFWHG
were de novo acetate, propionate, and butyrate synthesis from cellulose, and acetate and butyrate
synthesis from hemicelluloses with more than 90% decrease followed by de novo synthesis of
SURSLRQDWHIURPVWDUFKDQGVWUXFWXUDOFDUERK\GUDWH DQGGHFUHDVHUHVSHFWLYHO\ 3DUDPHWHUV
for denovo butyrate synthesis from starch, and acetate and propionate absorption were least affected
(7, 10 and 9%, respectively). he results of residual error analyses are presented in Table 1. The revised
model predicted net synthesis rates of propionate and acetate substantially better than without the
thermodynamic equations primarily due to a reduction in the previously unexplained dispersion
error. Predictions of butyrate synthesis were not improved. Ruminal acetate synthesis rates were
sensitive to parameters for acetate production from starch, soluble carbohydrate and cellulose and

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 323
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_112, © Wageningen Academic Publishers 2013
DFHWDWHFRQYHUVLRQWREXW\UDWH VHQVLWLYLW\FRHI¿FLHQWVRIDQGUHVSHFWLYHO\ 
Similarly, net propionate synthesis was sensitive to changes in parameters for production from starch,
VROXEOHFDUERK\GUDWHDQGEXW\UDWH VHQVLWLYLW\FRHI¿FLHQWVRIDQGUHVSHFWLYHO\ 1HW
production of butyrate was primarily affected by changes in parameters for interconversion from
acetate to butyrate and from butyrate to propionate (0.27 and 0.34, respectively) with little sensitivity
WRWKH0XUSK\FRHI¿FLHQWV

This preliminary work indicted that a fully exchanging three pool VFA production model that
FRQVLGHUVWKHUPRG\QDPLFLQÀXHQFHVEHWWHUUHSUHVHQWHGREVHUYHGUXPLQDOSURGXFWLRQUDWHVIRU
propionate in the Molly cow model. Based on the sensitivity analyses, additional improvements in
prediction accuracy, particularly for butyrate, may be possible if interconversion rate constants are
SDUDPHWHUL]HGLQFRQFHUWZLWKWKHGHQRYR9)$V\QWKHVLVFRHI¿FLHQWV7KHSUHVHQFHRIPHDQELDVLQ
predictions of acetate and butyrate concentrations suggests that interconversions with those pools
PD\DOVRQHHGSDUDPHWHUL]DWLRQRUWKDWIXUWKHUSURJUHVVLQGH¿QLQJWKHVHFRHI¿FLHQWVPD\EHSRVVLEOH

Table 1. Residual error analyses for predictions by the Molly cow model with the original Murphy et
al  FRHI¿FLHQWV ,QLWLDO DQGDOWHUHGWRUHSUHVHQWWKHUPRG\QDPLFDOO\GULYHQLQWHUFRQYHUVLRQV
DPRQJ9)$DQGDQHZO\GHULYHGFRHI¿FLHQWV )LQDO 

Variable N Observed mean1 RMSPE Mean bias Slope bias

Initial Final Initial Final Initial Final

% of Mean % of MSPE

Net synthesis
Acetate 23 23.1  58 4 10 39 13
Propionate 23 10.9 59 38 20 3 1 22
Butyrate 21  48  1 21 54 30
Concentration
Acetate 193  27 25 41 13 8 25
Propionate 193 25.3 44 40 45 35 10 11
Butyrate 193  39 38 41 31 19 21

1 Values for net synthesis are in mol/d and for concentration are in mmol/l.

Acknowledgements
Research funded by the New Zealand Agricultural Greenhouse Gas Research Centre.

References
Bergman, E.N., 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species.
3K\VLRORJLFDO5HYLHZV
Murphy, M.R., Baldwin, R.L., Koong, L.J., 1982. Estimation of stoichiometric parameters for rumen fermentation of
roughage and concentrate diets. J. Anim. Sci. 55, 411-421.
Saltelli, A., Tarantola, S., Chan, K.P.S., 1999. A Quantitative Model-Independent Method for Global Sensitivity Analysis
RI0RGHO2XWSXW7HFKQRPHWULFV
8QJHUIHOG(0.RKQ5$7KHUROHRIWKHUPRG\QDPLFVLQWKHFRQWURORIUXPLQDOIHUPHQWDWLRQ,Q6HMUVHQ
K., Hvelplund, T., Nielsen, M.O. (editors), Ruminant physiology: digestion, metabolism and impact of nutrition
RQJHQHH[SUHVVLRQLPPXQRORJ\DQGVWUHVV:DJHQLQJHQ$FDGHPLF3XEOLVKHUV:DJHQLQJHQ

324 Energy and protein metabolism and nutrition in sustainable animal production
BR-CORTE 1.0: online software for diet optimization on tropical
conditions
S.C. Valadares Filho1,2, M.L. Chizzotti, P.A.S. Machado1,2, H.F. Amaral1 and T. Furtado1
18QLYHUVLGDGH)HGHUDOGH9LoRVD'HSDUWPHQWRI$QLPDO6FLHQFH9LoRVD0*%UD]LO
2,QVWLWXWR1DFLRQDOGH&LrQFLDH7HFQRORJLDHP&LrQFLD$QLPDO'=28)99LoRVD0*
Brazil; PDULRFKL]]RWWL#G]RXÀDEU
8QLYHUVLGDGH)HGHUDOGH/DYUDV'HSDUWPHQWRI$QLPDO6FLHQFH/DYUDV0*%UD]LO

3UHFLVLRQIHHGLQJLVHVVHQWLDOWRLPSURYHWKHHFRQRPLFDODQGHQYLURQPHQWDOHI¿FLHQF\RIEHHIFDWWOH
production. BR-CORTE is a free-access, on-line software (www.brcorte.ufv.br) that integrates
the nutrient requirement model for Zebu (Valadares Filho et al., 2010a) with the Brazilian feed
composition tables (CQBAL 3.0, Valadares Filho et al., 2010b) to optimize diets for beef cattle.

BR-CORTE 2010 (Valadares Filho et al., 2010a) is a mechanistic static model that uses animal and
growing system information to estimate the nutritional requirements of Bos indicus and their crosses.
The model is based on meta-analysis of a dataset of 25 comparative slaughter studies uses as input the
SK\VLRORJLFDOVWDJHEUHHG 1HOORUHRUFURVVHVZLWK%RVWDXUXV ¿QLVKLQJV\VWHP SDVWXUHRUIHHGORW 
sex (bull, steer and heifer), weight and average weight gain to predict the requirements of digestible
energy, rumen degradable protein, rumen non-degradable protein and minerals. A summary of the
prediction equations for the requirements of energy and protein is reported in Table 1.

The CQBAL 3.0 (www.ufv.br/cqbal) is an on-line feed composition database containing more than
UHIHUHQFHVIURPWKHVLVDQGGLVVHUWDWLRQVUHSRUWLQJIHHGFRPSRVLWLRQLQ%UD]LO 9DODGDUHV)LOKR
et al.E 7KHVRIWZDUHXVHVIHHGFRPSRVLWLRQ UHTXLUHVQHXWUDOGHWHUJHQW¿EHUFRUUHFWIRUDVKDQG
SURWHLQOLJQLQFUXGHSURWHLQQHXWUDODQGDFLGGHWHUJHQW¿EHUSURWHLQVQRQ¿EURXVFDUERK\GUDWHV
ether extract, in situ water soluble fraction and in situ potentially degradable fraction and its rate of
degradation) and feeding conditions to predict feed digestible fractions and protein fractions based
on equations of Dettman et al. (2008) and exports that information for the BR-CORTE 1.0 software.

BR-CORTE 1.0 then uses animal information to predict the requirements for a target weight and feeds
VHOHFWHGIURPWKH&4%$/GDWDVHWDQGWKHLUFRVWVSHUNJDVIHGWR¿QGWKHRSWLPDOVROXWLRQRI
SURSRUWLRQRIVHOHFWHGIHHGVEDVHGRQWKHPLQLPXPFRVWRIWKH¿QDOGLHWXVLQJWKHVLPSOH[PHWKRG
and returns the best solution (Murty, 1983). The software allows bounds to limit the proportion of
ingredients as well as the proportion of roughage to concentrate. The output reports the proportion of
ingredients, in as fed and dry matter basis, and the amount of each nutrient supplied by each ingredient.

The BR-CORTE 1.0 is a useful tool for modeling nutritional requirement of Zebu cattle as well as
for teaching and diet formulation.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 325
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_113, © Wageningen Academic Publishers 2013
7DEOH6XPPDU\RIWKH%5&257(SUHGLFWLRQHTXDWLRQVIRUWKHHQHUJ\DQGSURWHLQUHTXLUHPHQWV
of beef cattle.

DMI Nellore: -2.7878 + (0.08789×SBW0.75  î$'* ± î$'*2);

&URVVEUHG î6%:0.75  î$'* ± î$'*2)
EBW Feedlot: 0.895×SBW;
3DVWXUHî6%:
EBG )HHGORW1HOORUHî$'*)HHGORW&URVVEUHGî$'*3DVWXUHî$'*
EQEBW Nellore: (EBW/430)×440; Crossbred:(EBW/455)×440
NEm Feedlot:0.0742×EBW0.75; Pasture 0.0717×EBW0.75
NEg Bulls:0.053×EQEBW0.75 ×EBG1.0956WHHUVî(4(%:0.75×EBG1.095; Heifers:
0.072×EQEBW0.75×EBG1.095; Pasture: 0.052×EQEBW0.75×EBG
%REp 1.1404×(RE/EBG) -1.137
Kg 0.327/[0.539 + (%REp/100)]
Km Nellore: 0.513+(0.173×Kg)+(0.100×EBG);
Crossbred: 0.513+(0.173×Kg)+(0.073×EBG)
ME (NEm/km) + (NEg/Kg)
TDN ME/0.82/4.409
NPg 1HOORUH%XOOV î(%* ± î1(J 1HOORUH6WHHUVDQG+HLIHUV
î(%* ± î1(J &URVVEUHG%XOOV î(%* ± î1(J 
&URVVEUHG6WHHUVDQG+HLIHUV î(%* ± î1(J
K ,I6%:”NJ± î(4(%: LI6%:!NJ
MPm Feedlot: 4.0×BW0.75; Pasture: 4.5×BW0.75
MP MPm + (NPg/K)
MCP 120×TDN
CP î0&3 ^> 03± 0&3î @`

DMI = DM intake, kg/day; SBW = shrunk BW, kg; ADG = average daily gain, kg/day; EBW = empty BW, kg; EBG =
empty body gain, kg/day; EQEBW = equivalent empty BW, kg; NEm = NE for maintenance, Mcal/day; NEg = NE for
growth, Mcal/day; %Rep = percentage of energy retained as protein, %; KJ HI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROL]DEOH
energy for growth, %; KP HI¿FLHQF\RIXWLOL]DWLRQRIPHWDEROL]DEOHHQHUJ\IRUPDLQWHQDQFH0( PHWDEROL]DEOH
energy, Mcal/day; TDN = total digestible nutrients, kg/day; NPg = Net protein for growth, g/day; K HI¿FLHQF\RI
utilization of metabolizable protein for growth, %; MPm = metabolizable protein for maintenance, g/day; MP = total
metabolizable protein, g/day; MCP = microbial crude protein, g/day; CP = total dietary crude protein, g/day.

References
Detmann, E., S. C. Valadares Filho, D. S. Pina, L. Henriques, M. F. Paulino, K. A. Magalhaes, P.A. Silva and M. L.
Chizzotti, 2008. Prediction of the energy value of cattle diets based on the chemical composition of the feeds
under tropical conditions. Anim. Feed Sci. Tech. 143, 127-147.
Murty, K. G, 1983. Linear programming. John Wiley & Sons Inc.: New York, 482p.
Valadares Filho, S. C., M. I. Marcondes, M. L. Chizzotti and P. V. R. Paulino, 2010a. Nutrient Requirements of Zebu
Beef Cattle: BR-CORTE. 2. ed. UFV: Viçosa, 185p.
Valadares Filho, S. C., P. A. Silva, M. L. Chizzotti, H. F. Amaral, K. A. Magalhães, V. R. Rocha Junior and E. R. Capelle,
2010b. Tabelas Brasileiras de composição de alimentos para bovinos. 2. ed. UFV: Viçosa, 502p.

326 Energy and protein metabolism and nutrition in sustainable animal production
A structural equation model to analyze energy utilization in lactating
dairy cows
L.E. Moraes1, A.B. Strathe2, E. Kebreab1, D.P. Casper, J. Dijkstra, J. France and J.G. Fadel1
1 'HSDUWPHQW RI $QLPDO 6FLHQFH 8QLYHUVLW\ RI &DOLIRUQLD 'DYLV &$  86$
[email protected]
2Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Denmark
'DLU\6FLHQFH'HSDUWPHQW6RXWK'DNRWD6WDWH8QLYHUVLW\%URRNLQJV6'86$
$QLPDO1XWULWLRQ*URXS:DJHQLQJHQ8QLYHUVLW\$+:DJHQLQJHQWKH1HWKHUODQGV
Department of Animal and Poultry Science, University of Guelph, ON, N1G 2W1, Canada

Introduction
Energy balance trials from lactating cows have traditionally been analyzed using the regression
approach. However, univariate analysis of mutually interacting animal traits can provide biased
parameter estimates if one trait is used as an explanatory variable to model a second trait and the
VXEPRGHORIWKH¿UVWWUDLWLVLJQRUHG *LDQRODDQG6RUHQVHQ 0RUHRYHUPXOWLYDULDWHPHWKRGV
should be preferred because animal responses are correlated and relationships among responses can
EHTXDQWL¿HGWKURXJKWKHXVHRIVWUXFWXUDOHTXDWLRQV)XUWKHUPRUHWKH86FXUUHQWHQHUJ\HYDOXDWLRQ
V\VWHPRIGDLU\FDWWOH 15& UHOLHVRQHQHUJHWLFSDUDPHWHUVIURPWKH¶VEXWRYHUWKH
past decades genetic improvement of dairy cattle has substantially increased the ability of cows to
produce milk. The objective of the present study was to analyze energy utilization in lactating cows
using structural equations.

Material and methods

A structural equation model was developed to analyze energy utilization in lactating cows using
HQHUJ\EDODQFHUHFRUGVIURPVWXGLHVFRQGXFWHGIURPWR5HFRUGVUHSUHVHQWDW
least 4 consecutive days of lactating cows in respiration chambers and were collected at the former
USDA Energy Metabolism Unit at Beltsville, Maryland. The model was developed under a Bayesian
framework, in which minimally informative priors were assigned to all parameters and statistical
inference was based on Markov Chain Monte Carlo methods. The response vector, comprised by
metabolizable energy (ME) intake, milk energy and tissue energy balance, was modeled through a
multivariate normal distribution.

The structural parameters represent the causal relationships among responses and, under our model
VWUXFWXUHGHWHUPLQHGVWUXFWXUDOSDUDPHWHUVFDQEHLQWHUSUHWHGDVWKHHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQ
(DFKUHVSRQVHZDVPRGHOHGZLWKDVHWRIH[RJHQRXVFRYDULDWHVDQGFURVVFODVVL¿HGUDQGRPHIIHFWV
of animal and study. The structural equation model can be represented by: yijk ȁdyijk + Xijkȕd +
ai + sj + eijk, where yijk is the (r × 1) response vector of the kth record on animal i and study jȁd is
the (r × r) matrix of structural parameters describing the causal structure among responses on the
dth decade, Xijk is the (r × p PDWUL[RIH[RJHQRXVFRYDULDWHVȕd is the (p × 1) vector of regression
FRHI¿FLHQWVRQWKHGth decade, ai and sj are (r × 1) vectors of animal and study random effects and
eijk is the (r × 1) vector of errors. In this notation, r represents the number of responses and p the
QXPEHURIH[RJHQRXVFRYDULDWHV6WUXFWXUDOSDUDPHWHUVDQGUHJUHVVLRQFRHI¿FLHQWVZHUHDOORZHGWR
GHSHQGRQWKHGHFDGHWKHH[SHULPHQWZDVFRQGXFWHG LHDQG 
HVWLPDWLQJGHFDGHVSHFL¿FSDUDPHWHUVDQGLQYHVWLJDWLQJWKHFKDQJHVRQWKHVHHQHUJHWLFSDUDPHWHUV
RYHUWKH\HDUV0DLQWHQDQFHUHTXLUHPHQWVDQGHQHUJHWLFHI¿FLHQFLHVZHUHHVWLPDWHGXVLQJWKH
functional form described by Strathe et al. (2011). Parameters’ 95% credible intervals and Bayesian
P-values, computed as the proportion of posterior samples which are different from zero, were used
to compare the differences between energetic parameters from the second and third decades with
HVWLPDWHVIURP

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 327
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_114, © Wageningen Academic Publishers 2013
Results and discussion
Energetic parameters and their 95% credible intervals are reported in Table 1. The metabolizability
(q), estimated as the slope of the regression of ME intake on gross energy (GE) intake, was similar
between decades (3 0.94 and 3  DVZHOODVWKHHI¿FLHQF\ kT) of utilizing tissue energy to
produce milk (3 0.81 and 3 0.31). However, net energy requirements for maintenance (NEM)
increased substantially over the years (3 0.02 and P< DVZHOODVWKHHI¿FLHQF\ kL) of utilizing
ME intake for milk production (3 DQGP< 6LPLODUO\WKHHI¿FLHQF\ kG) of utilizing ME
intake for growth increased substantially over the three decades (3 0.005 and P<0.001). Therefore,
PRGHUQODFWDWLQJFRZVKDYHKLJKHUPDLQWHQDQFHHQHUJ\UHTXLUHPHQWVWKDQFRZVIURPWKH¶VDQG
DOVRKDYHDKLJKHUHI¿FLHQF\IRUXWLOL]LQJ0(LQWDNHIRUPLONSURGXFWLRQDQGWLVVXHJDLQ

7DEOH(QHUJHWLFSDUDPHWHUVHVWLPDWHVDQGWKHLUFUHGLEOHLQWHUYDOV

Parameter Decade Estimate 95% CI

q MJ/MJ  0.52 (0.50, 0.53)

q MJ/MJ 1973-1983 0.52 (0.50, 0.54)
q MJ/MJ 1983-1995 0.53 (0.51, 0.55)
NEM MJ/kg0.75/day  0.25 (0.22, 0.29)
NEM MJ/kg0.75/day 1973-1983 0.32 (0.27, 0.37)
NEM MJ/kg0.75/day 1983-1995 0.41 
kL MJ/MJ  0.57 (0.55, 0.59)
kL MJ/MJ 1973-1983  
kL MJ/MJ 1983-1995  
kT MJ/MJ  0.89 (0.83, 0.94)
kT MJ/MJ 1973-1983 0.89 (0.83, 0.97)
kT MJ/MJ 1983-1995 0.84 (0.74, 0.93)
kG MJ/MJ  0.57 
kG MJ/MJ 1973-1983  
kG MJ/MJ 1983-1995 0.72 

References
Gianola, D. and D. Sorensen, 2004. Quantitative genetic models for describing simultaneous and recursive relationships
EHWZHHQSKHQRW\SHV*HQHWLFV
NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th ed. National Academy Press, Washington, DC.
Strathe, A.B., J. Dijkstra, J. France, S. Lopez, T. Yan and E. Kebreab, 2011. A Bayesian approach to analyze energy
balance data from lactating dairy cows. J. Dairy Sci. 94, 2520-2531.

328 Energy and protein metabolism and nutrition in sustainable animal production
Protein deposition potential and modeling of methionine requirements
in homozygous (Na/Na) and heterozygous (Na/na) naked neck meat type
chicken
D.R. Khan, C. Wecke and F. Liebert
Division Animal Nutrition Physiology, Department of Animal Sciences, Georg-August-University,
.HOOQHUZHJ*RHWWLQJHQ*HUPDQ\[email protected]

Introduction
+LJKDPELHQWWHPSHUDWXUH $7 GHFUHDVHVWKHJURZWKRIIDVWJURZLQJFKLFNHQEHFDXVHRIGLI¿FXOW\
in dissipating heat through the feather coverage. Consequently, they become more sensitive to high
AT (Yunis and Cahaner, 1999). Introduction of the naked neck gene (Na) in modern meat type
genotypes could be helpful in tolerating high AT by providing more surface area for heat dissipation
compared to their normally feathered (na/na) counterparts. The aims of this study were to determine
the (1) protein deposition potential and (2) modeling the methionine requirement for these genetics.

Material and methods

Birds of three genotypes (Na/Na; Na/na; na/na) were obtained by mating their heterozygous (Na/
na) parents. In total 144 average weight birds (72 Na/Na male and female; 72 Na/na male and
female) were selected for two N-balance experiments involving both starter and grower periods.
Each experimental period was divided into a 5 d adaptation period and two 5d consecutive excreta
FROOHFWLRQSHULRGV(DFKEDODQFHVWXG\XWLOL]HGELUGV Na/NaNa/na) involving both genders,
UDQGRPO\DOORWWHGWR¿YHGLHWVZLWKJUDGHGGLHWDU\SURWHLQVXSSO\'XHWRSULQFLSOHVRIWKHGLHW
dilution technique, the dietary amino acid ratio was kept constant. Methionine (Met:Cys = 1:1.01)
ZDVLGHQWL¿HGDVWKH¿UVWOLPLWLQJDPLQRDFLGLQHDFKGLHW0HWKLRQLQHUHTXLUHPHQWVGHSHQGLQJRQ
age and growth rate (protein deposition) were derived according to Samadi and Liebert (2008).

The daily N maintenance requirement (NMR) was determined using the exponential regression
between N intake (NI, mg/BWkg) and total daily N excretion (NEX; mg/BWkg). The threshold
value of the function (Equation 1 and 2) was estimated by application of the Levenberg-Marquardt
algorithm within the statistical program package SPSS (Vers. 19).

NR = NRmax7 íHíE1,) (1)

ND = NRmax7 íHíE1,) – NMR (2)

In the equations ND is daily N-deposition, NR is daily N-retention (ND + NMR; mg/BWkg);

NRmaxT is theoretical maximum for daily NR (mg/BWkg); and b is the slope of the N-retention
curve (indicating the feed protein quality independent of N intake).

Results and discussion

Results of both NMR and NDmaxT estimation are summarized in Table 1. Daily NMR did not reveal
DQ\VWDWLVWLFDOGLIIHUHQFHEHWZHHQJHQHWLFVDQGDJHSHULRGV+RZHYHUWKHUHZDVDVLJQL¿FDQWGLIIHUHQFH
between male and female birds. NMR data as observed were applied for further estimation of NDmaxT.

Making use of the yielded model parameters, as an example Methionine requirement data were
derived for targeted CP deposition depending on genotype and age (Table 2).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 329
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_115, © Wageningen Academic Publishers 2013
7DEOH(VWLPDWHVRIGDLO\PDLQWHQDQFHUHTXLUHPHQW 105 DQGRIWKHRUHWLFDOSRWHQWLDOIRUGDLO\
1GHSRVLWLRQ 1'max7 

Starter period Grower period

Na/Na Na/na Na/Na Na/na

NMR NDmaxT NMR NDmaxT NMR NDmaxT NMR NDmaxT

mgN/BWkg/d

Male 259 3,849 229 4,053 298  319 3,094

Female 339  385 3,890 351 2,489  

Table 2. Example for modeling the Met-requirement of male naked neck chicken with dietary Met-
HI¿FLHQF\DVREVHUYHGDQGDLPHG&3GHSRVLWLRQ PHDQ%:VWDUWHUJJURZHUJ 

Na/Na Na/na

Item Starter Grower Starter Grower

CP deposition (g/d) 9 11 13 15 9 11 13 15
0HWHI¿FLHQF\ EF-1) 182 182 252 252    
Met-requirement (mg/BWkg/d) 209 298 327 409 198 274 324 394

Predicted feed intake (g/d) Met-content needed in the diet (%) depending on feed intake

Starter Grower

 120 0.34 0.49 0.27 0.34 0.33  0.27 0.33

70 130 0.30 0.42 0.25 0.31 0.28 0.39 0.25 0.30

Methionine requirements for Na/Na were slightly higher as compared to Na/na in starter period.
However, yielded Met-requirement data were in good agreement with previous studies with normally
feathered chicken. In conclusion, for a dietary ratio of Met:Cys near 1:1, reduced feathering did not
interfere with Methionine requirements.

References
6DPDGLDQG)/LHEHUW(VWLPDWLRQRIQLWURJHQPDLQWHQDQFHUHTXLUHPHQWVDQGSRWHQWLDOIRUQLWURJHQGHSRVLWLRQ
in fast-growing chickens depending on age and sex. Poult. Sci. 85, 1421-1429.
Samadi and F. Liebert, 2008. Modelling the optimal lysine to threonine ratio in growing chickens depending on age
DQGHI¿FLHQF\RIGLHWDU\DPLQRDFLGXWLOLVDWLRQ%ULW3RXOW6FL
Yunis, R. and A. Cahaner, 1999. The effect of naked neck (Na) and frizzle (F) genes on growth and meat yield of broiler
and their interactions with ambient temperatures and potential growth rate. Poult. Sci. 78, 1347-1352.

330 Energy and protein metabolism and nutrition in sustainable animal production
/LQHDUDQGQRQOLQHDUHVWLPDWHVRIWKHHI¿FLHQF\ZLWKZKLFK
metabolizable energy is used for maintenance or gain
C.A. Old1 and H.A. Rossow2
1$QWHORSH5DQFK/H*UDQG&$86$DQWHORSHUDQFK#JPDLOFRP
26FKRRORI9HWHULQDU\0HGLFLQH8QLYHUVLW\RI&DOLIRUQLD'DYLV&$86$

Introduction
/LQHDUHVWLPDWHVRIWKHHI¿FLHQF\RIPHWDEROL]DEOHHQHUJ\ 0( IRUPDLQWHQDQFH Nm) or gain (kg),
FDOFXODWHGE\/RIJUHHQDQG*DUUHWW  WKHEDVLVIRUWKH&DOLIRUQLD1HW(QHUJ\6\VWHP &1(6 
differ from estimates calculated from metabolic pathways; observations in the former are consistent
with the statement that km is greater than kg and the converse for the latter. In their analysis these
authors describe metabolizable energy intake as both response and predictor variable. A further
assumption is that the relationship between retained energy (RE) and ME intake is rectilinear; this
DVVXPSWLRQLVEDVHGRQDVWDWLFPDLQWHQDQFHUHTXLUHPHQW$QDQDO\VLVE\%OD[WHU  DQGRWKHUV
showed that maintenance is dynamic. This study was undertaken to evaluate equations representing
either linear or nonlinear relationships between ME intake and (RE) to compare estimates of km and kg.

Material and methods

'DWDIURP/RIJUHHQDQG*DUUHWW  IRUJURZLQJEHHIVWHHUVDQGKHLIHUVZHUHDQDO\]HGXVLQJHLWKHU
the linear models employed by those investigators, log heat production (HP) = a + b*ME intake
(MEI) (1) and MEI = c + RE/kg (2); a represents log fasting HP (FHP) and c is MEm (ME intake at
5(  WKHSRLQWLQFRPPRQ7KHWKLUGHTXDWLRQFRQWDLQHGERWK¿UVWRUGHUDQGOLQHDUHOHPHQWVDQG
was MEI = (a1ek1RE + a2ek2RE)/km + RE/kg (3); an are parameter estimates, kn are rate constants. All
HQHUJ\WHUPVXVHGE\/RIJUHHQDQG*DUUHWW  ZHUHVFDOHGE\:0.75 (W = empty body weight,
kg); no such convention was employed in the exponential model. Parameter estimates for the linear
analysis are least squares estimators and reliability of parameter estimates was evaluated using a
Markov Chain Monte Carlo (MCMC) simulation (1000 simulations) utilizing the method described
by Martin et al. (2011) through the public domain language R (R, 2011). Parameter estimates for the
exponential model were calculated using a non-linear optimization technique (R, 2011). Reliability
of parameter estimates k1, k2, km and kg was also evaluated using the same technique as for the
linear model.

Results and discussion

)RUWKHOLQHDUPRGHO  DFRQ¿GHQFHLQWHUYDODERXWWKHLQWHUFHSW :0.75) ranged from
0.114 to 0.148. Fasting heat production was calculated to be 0.077/W0.75; km is 0.077/0.133, or 0.579.
For kg  DFRQ¿GHQFHLQWHUYDOZDVIURPWR52, while adequate (0.784),
suggests that not all the assignable variability in the response variable was captured by variability
LQWKHSUHGLFWRUYDULDEOH7HVWVIRUJRRGQHVVRI¿WLQGLFDWHWKDWHTXDWLRQ  XVHGE\/RIJUHHQDQG
*DUUHWW  LVPLVVSHFL¿HG 3  UHLQIRUFLQJWKHSUHYLRXVVWDWHPHQW3DUDPHWHUHVWLPDWHV
IRUPRGHOZHUHVWDEOHFRQ¿GHQFHLQWHUYDOVZHUH“DERXWWKHPHDQ0RGHOSDUDPHWHU
estimates were unstable; MCMC simulation results were largely outside the range of biologically
SRVVLEOHUDQJHIRUPRGHOVLPXODWLRQUHVXOWVZHUHELRORJLFDOO\LQIHDVLEOHRIWKH
simulations indicated either MEm was less than 0 or kg greater than 1.

For the second model R2ZDVLPSURYHG  WKHHVWLPDWHRINm was 0.278±0.001; kg was
“WKHVHDUHVLPLODUWRWKHRUHWLFDOPD[LPDIRUZKLFKNm=0.279 (8 ATP per mole of acetate
oxidized) and kg=0.723. Estimates of km for 1000 simulations ranged from 0.250 to 0.305 and for
kg the range was from 0.599 to 0.732; all simulations gave estimates that were not different from

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 331
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_116, © Wageningen Academic Publishers 2013
those that are biologically possible. Considering that the model is overparametized, this stability is
notable. Koong et al. (1983) and more recently, Labussierre et al. (2011) noted that maintenance
requirement is not a constant function of body weight, but rather increases with energy intake. It is
possible that the results observed in the second model are simply due to shifting HP from synthesis
to maintenance; if this were the case it is expected that km and kg would be correlated. Parameter
estimates km and kg from the MCMC simulation were not correlated (r=0.008). It should be noted
that for model 3 solutions for km and kg are simultaneous; this is not the case for the linear solutions
of km and kg.

7KLVDQDO\VLVLQGLFDWHVWKDWWKHSDUDPHWHUHVWLPDWHVUHJDUGLQJHI¿FLHQFLHVRI0(XVHIRUPDLQWHQDQFH
and gain obtained from linear equations scaled by W0.75DUHKLJKO\XQVWDEOHDQGPD\QRWUHÀHFWWKH
true parameters. While adequate for the intended purposes, that is, prediction of gain in growing and
¿QLVKLQJEHHIFDWWOHRXUDQDO\VLVLQGLFDWHVWKDWWKHVHHVWLPDWHVVKRXOGQRWEHXVHGDVWKHEDVLVIRUD
PHFKDQLVWLFH[SODQDWLRQUHJDUGLQJHI¿FLHQFLHVRI0(XWLOL]DWLRQ1RQOLQHDUHVWLPDWHVRIHI¿FLHQFLHV
are similar to theoretical estimates obtained from biochemical pathways and more of the variability
in ME intake was described by this equation. Incorporation of these concepts into models used to
SUHGLFWLQSXWRXWSXWUHODWLRQVKLSVIRUJURZLQJDQG¿QLVKLQJEHHIFDWWOHPD\DOORZIRUPRUHDFFXUDWH
prediction of performance.

References
%OD[WHU./7KHQXWULWLYHYDOXHRIIHHGVDVDVRXUFHRIHQHUJ\DUHYLHZ-'DLU\6FL
Koong, L.J., J.A. Nienaber and H.J. Mersmann. 1983. Effects of plane of nutrition on organ size and fasting heat
SURGXFWLRQLQJHQHWLFDOO\REHVHDQGOHDQSLJV-1XWU
Labussierre, E., J. van Milgen, C.F.M. de Lange and J. Noblet. 2011. Maintenance energy requirements of growing
SLJVDQGFDOYHVDUHLQÀXHQFHGE\IHHGLQJOHYHO-1XWU
/RIJUHHQ*3DQG:1*DUUHWW$V\VWHPIRUH[SUHVVLQJQHWHQHUJ\UHTXLUHPHQWVDQGIHHGYDOXHVIRUJURZLQJ
DQG¿QLVKLQJEHHIFDWWOH-$QLP6FL
Martin, A.D., K.M. Quinn and J.H. Park. 2011. MCMC pack: Markov Chain Monte Carlo in R. J. Stat. Soft. 42: 1-5.
R, D.C.T., 2011. R: A language and environment for statistical computing. R Foundation for Statistical Computing.

332 Energy and protein metabolism and nutrition in sustainable animal production
Responses of laying hens to methionine and cystine intake
H.C.P. Bendezu, N.K. Sakomura, L. Hauschild, J. Sato, J.C.P. Dorigam and E.P. Silva
Department of Animal Science, University of Agrarian and Veterinary Sciences of UNESP, Jaboticabal,
63%UD]LO[email protected]

Introduction
The knowledge of the importance of amino acid requirements is related to precise amino acid balance
to prevent loss of energy due to an unbalanced diet and maximize economic results. Many studies
have been conducted to determine the sum of methionine and cystine (Met+Cys) requirements and
WKHUHVXOWVGLIIHUEHFDXVHWKHUHDUHPDQ\LQÀXHQWLDOIDFWRUVVXFKDVJHQHWLFVPDQDJHPHQWDJHGLHWV
environmental conditions and methodology used. In addition, requirements have been estimated based
on the response of the birds over a long period and does not consider the dynamics of body changes
in the birds. For this reason, the focus of this experiment was to determine the optimal Met+Cys
intake for Dekalb White laying hens in four periods (32 to 48 weeks) according to dilution technique.

Material and methods

Two hundred and eighty-eight Dekalb White laying hens were used during the period from 32 to 48
weeks of age. The hens were distributed in a completely randomized design into eight treatments,
six replicates and six hens per experimental unit. Diets were isocaloric (2,850 kcal/kg) with different
OHYHOVRI0HW&\V DQG DQGZHUHREWDLQHGE\
dilution technique (Fisher and Morris, 1970) with the eighth treatment as a control treatment used to
verify bird response for Met+Cys limitation. Egg production, mass, feed intake and feed conversion
per egg mass were evaluated. Data were analyzed using PROC GLM SAS 9.0. LSMEAN was used
WRFRPSDUHWUHDWPHQWVPHDQVE\WLPH)RUWKRVHWKDWZHUHVLJQL¿FDQWE\SHULRGWKH\ZHUHVXEMHFWHG
to regression analysis according the broken line (BL), quadratic (Q) and BL + Q model for each
SHULRG ZHHNV DQGWKRVHWKDWZHUHQRWVLJQL¿FDQWE\SHULRGZHUHDQDO\]HGE\DPRGHOIRUWRWDO
H[SHULPHQWWLPH ZHHNV 

Results and discussion

7KHFRQWUROHLJKWKWUHDWPHQWFRQ¿UPHGWKDW0HW&\VZHUHWKHOLPLWLQJDPLQRDFLGVLQWKLVH[SHULPHQW
Egg mass was affected by Met+Cys level (P<0.0001) and by the interaction between level and period
(3  )RUWKLVUHDVRQWKHGDWDZHUHDQDO\]HGE\SHULRG(JJPDVVLQFUHDVHVZLWKDJHRIWKH
hen (Johnston and Gous, 2007). Values of egg mass were adjusted by the BL, Q and BL + Q models
for each period. All models had high R2, however, the BL + Q model was chosen because it has a
biological interpretation. The following equations and the results of optimal Met+Cys intake by BL
+ Q, expressed per day by each period, are numbered sequentially in chronological order:

 0HW&\V±0HW&\V2PJ 

 0HW&\V±0HW&\V2; 728 mg (2)

 0HW&\V±0HW&\VW2; 743 mg (3)

 0HW&\V±0HW&\V2; 770 mg (4)

The feed conversion per egg mass (FC) was affected by level (P< EXWWKHUHZDVQRVLJQL¿FDQW
interaction between level and period (3 0.313). Feed conversion per egg mass covered a wide
range, with the value of the lower level treatment much different from the others. For this reason,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 333
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_117, © Wageningen Academic Publishers 2013
the Q model did not have any problem in estimating the optimal Met + Cys intake and the result is
in agreement others. Equation 5 presented the optimal daily feed intake.

FC = 13.17104 – 0.03711 Met+Cys + 0.0000279 Met+Cys 2PJ 

References
Fisher C. and T.R. Morris, 1970. The determination of the methionine requirement of laying pullets by a diet dilution
WHFKQLTXH%ULWLVK3RXOWU\6FLHQFH
Johnston, S.A. and R.M. Gous, 2007. Modelling the changes in the proportions of the egg components during a laying
cycle. British Poultry Science, 48: 347-353.

334 Energy and protein metabolism and nutrition in sustainable animal production
Assessment of ideal dietary amino acid ratios between branched-chain
amino acids for growing chicken
A. Pastor, C. Wecke and F. Liebert
Division Animal Nutrition Physiology, Department of Animal Sciences, Georg-August-University,
.HOOQHUZHJ*RHWWLQJHQ*HUPDQ\[email protected]

Introduction
Improvement of amino acid (AA) recommendations are a precondition for sustainable conversion of
feed to food proteins and have to be based on physiological acceptable and validated procedures to
derive both quantitative daily AA requirements and optimal dietary AA ratios. According to earlier
DSSOLFDWLRQVRIDQH[SRQHQWLDOQLWURJHQ 1 XWLOL]DWLRQPRGHO 6DPDGLDQG/LHEHUW
Liebert, 2008), two experiments were conducted to obtain new data about ideal amino acid ratio
(IAAR) between branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ile), valine (Val)
and lysine (Lys) in meat type chicken diets.

Material and methods

Experiment 1

7KH1EDODQFHVWXG\XWLOL]HGPDOHFKLFNHQV 5RVVDJHSHULRG WRSURYLGHWKHPRGHO

parameters (NMR: N-maintenance requirement; NDmaxT: theoretical maximum for daily
1GHSRVLWLRQ ,QWRWDOGLHWVZLWKJUDGHGSURWHLQVXSSO\ &3 ZHUHIHG Q  RYHUD
period of 15 days (5 days adaptation, 2×5 days collection period) for both starter (day 10-20) and
grower period (day 25-35). Excreta were collected every 12 hours and stored at -20 °C until further
analysis. Samples were analyzed according to the German VDLUFA standards (Naumann and
%DVVOHU 105DQG1'maxT were estimated following iteration steps by the Levenberg-
Marquardt-Algorithm within the SPSS statistical package (version 19). Observed NMR and NDmaxT
were applied for model parameter b (protein quality parameter, independent on NI).

Experiment 2

7KH1EDODQFHVWXG\H[DPLQHG¿YHGLHWV Q  YDU\LQJLQLQGLYLGXDO$$VXSSO\E\$$GLOXWLRQ
>EDODQFHGFRQWUROGLHW%'GLHWVOLPLWLQJLQ/\V  /HX  ,OH  DQG9DO RIWKH
assumed requirement)]. Protein quality parameter (b) was applied to evaluate the individual AA
HI¿FLHQF\ EF-1), taking into account the dietary concentration (c) of the limiting AA. According to
6DPDGLDQG/LHEHUW  REVHUYHG$$HI¿FLHQFLHVZHUHXWLOL]HGIRUFRQFOXVLRQRI,$$5>,$$5
EFLys-1: bcindividual BCAA-1]. However, this proportion is only valid if the AA under study is really
LQWKHOLPLWLQJSRVLWLRQLQWKHH[SHULPHQWDOGLHWDVLQGLFDWHGE\DVLJQL¿FDQWGHFOLQHRIEYDOXH
between balanced control diet and the AA diluted diet under study.

Results and discussion

Experiment 1 yielded daily NMR (113mg and 215mg N/BWkg for the starter and grower period,
respectively) and daily NDmaxT (4593mg and 4302mg N/BWkg for starter and grower period,
respectively). Observed model parameters were utilized for further model applications. Experiment
\LHOGHGPRVWHI¿FLHQW1XWLOL]DWLRQGXHWRWKHEDODQFHGGLHW%' 7DEOH $VLJQL¿FDQWGHFOLQH
of protein quality (parameter b) was observed following each of the AA diluted diets. However, in
WKHVWDUWHUSHULRGDQLQFUHDVHGVWDWLVWLFDOULVNKDGWREHDFFHSWHGWRSURYHVLJQL¿FDQFHIRUIXUWKHU
conclusion of Leu requirement data. Consequently, for this age period a further validation of Leu
related observations is needed.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 335
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_118, © Wageningen Academic Publishers 2013
7DEOH5HVXOWVRI1EDODQFHVWXG\ ([SHULPHQW DQGGHULYHGLGHDODPLQRDFLGUDWLRIRUWKH
EUDQFKHGFKDLQDPLQRDFLGV /\VLQH  'LIIHUHQWVXSHUVFULSWOHWWHUVUHYHDOVLJQL¿FDQWGLIIHUHQFHV
among the experimental diets.

BD1 Leu1 Ile1 Val1 Lys1

Starter period (10-20d)

Average BW3 (g) a±155 398a±150 a±142 343a±142 b±92
NI4 (mg/d)7 3,555a±341 3,010bc±493 3,209ab±500 2,977bc±572 2,597c±531
NEX5 (mg/d)7 a±177 895a“ 1,085a± 1,114a±288 887a±248
ND (mg/d)7 2,439a“ 2,115b±275 2,124b±313 bc±300 1,710c±297
IAAR
b-value 221a±12 215b±142 199b±10 185c±10 191bc±9
bcAA-1experimental diet 42±2 73±4 “ 40±2
IAAR (%)8 94 55  100
Recommendation NRC (1994) 109 73 82 100

Grower period (25-35d)

AverageBW3(g) 1,445a±280 1,152b±207 1,141b± 1,129b±214 1,234ab±204
NI4 (mg/d)7 3,322a±341 2,299c±414 2,430bc±573 2,392bF“ 2,843b±298
NEX5 (mg/d)7 947ab±150 749b±214 813ab± a±258 990a±174
ND (mg/d)7 2,375a±231 1,550c±232 bc±372 1,424c± 1,853b“E
IAAR
b-value 258a±15 218b±13 217b±15 191c±15 b±12
bcAA-1experimental diet 42±3 “ “ 45±2
IAAR (%)8   72 100
Recommendation NRC (1994) 109 73 82 100

1 P<0.05.
2 P<0.2 (Duncan test or Games-Howell test, according to Levene test).
3 BW = body weight.
4 NI = N intake.
5 NEX = N excretion.
 ND = N deposition.
7 Related to metabolic BW (BW ).
kg
8%DVHGRQFDOFXODWLRQZLWKVKDUS QRWURXQGHG ¿JXUHV

The yielded IAAR for BCAA in meat type chicken was lower than data reported earlier. Breeding
SURJUHVVFDQEHH[SHFWHGDVRQHIDFWRUOHDGLQJWRHQKDQFHG1XWLOL]DWLRQDQGPRGL¿HGUHTXLUHPHQWV
Accordingly, in the starter period we expected a more distinctive limitation of Leu by its dilution
down to 80% of the expected requirement. This observation indicates that previously reported
Leu recommendations could overestimate the physiological requirement for actual broiler strains.
2I¿FLDO$$UHFRPPHQGDWLRQV HJ15& DUHROGHUWKDQ\HDUVDQGFRXOGEHLQDGHTXDWHIRU
current genotypes. For Leu and Val, a clear differentiation between starter and grower period became
obvious. Consequently, we expect different ratios for both of the BCAA between age periods. For
Val, we have support for this expectation from ongoing studies.

In conclusion, the derived IAAR for the BCAA was lower than earlier reports, indicating an
RYHUHVWLPDWLRQRIWKH%&$$ZLWKRI¿FLDOUHFRPPHQGDWLRQV,QDGGLWLRQFXUUHQWUHVXOWVJLYHVXSSRUW
for an increased relative importance of Leu and Val with increasing age of the birds.

336 Energy and protein metabolism and nutrition in sustainable animal production
References
1DXPDQQ.DQG5%DVVOHU9'/8)$0HWKRGHQEXFK,,,'LHFKHPLVFKH8QWHUVXFKXQJYRQ)XWWHUPLWWHOQ
Mit Ergänzungslieferungen 1983, 1988, 1993, 1997. Darmstadt: VDLUFA-Verlag.
NRC (National Research Council), 1994. Nutrient Requirements of Poultry. 9th Rev. ed., National Academy Press,
Washington, D.C.
Liebert, F., 2008. Modelling of protein metabolism yields amino acid requirements dependent on dietary amino acid
HI¿FLHQF\JURZWKUHVSRQVHJHQRW\SHDQGDJHRIJURZLQJFKLFNHQ$Y%LRO5HV
6DPDGLDQG)/LHEHUW(VWLPDWLRQRIQLWURJHQPDLQWHQDQFHUHTXLUHPHQWVDQGSRWHQWLDOIRUQLWURJHQGHSRVLWLRQ
in fast-growing chickens depending on age and sex. Poult. Sci. 85, 1421-1429.
Samadi and F. Liebert, 2008. Modelling the optimal lysine to threonine ratio in growing chickens depending on age
DQGHI¿FLHQF\RIGLHWDU\DPLQRDFLGXWLOL]DWLRQ%ULW3RXOW6FL

Energy and protein metabolism and nutrition in sustainable animal production 337
Meta-analysis of the response of growing pigs to valine content of the diet
J. van Milgen1, M. Gloaguen1, N. Le Floc’h1, L. Brossard1, Y. Primot and E. Corrent2
1 ,15$$JURFDPSXV 2XHVW 805 3(*$6(  6DLQW*LOOHV )UDQFH
[email protected]
2$MLQRPRWR(XURO\VLQHVDV3DULV&HGH[)UDQFH

Introduction
The use of crystalline amino acids allows reducing the CP in the diet without compromising
performance. The extent to which the CP content in the diet can be reduced under practical conditions
depends on accurate knowledge of the requirements of amino acids that may become limiting when
pigs are offered low-CP diets. Valine is now considered the next-limiting amino acid after Lys, Met
(+Cys), Thr, and Trp in cereal-soybean meal based diets. Nevertheless, experimental studies on the
response of pigs to the Val supply are scarce. Our objective was to analyze studies for growing pigs
fed diets with increasing levels of L-Val.

Data collection and methods of analyses

Twenty-eight dose-response experiments were collected, originating from 20 publications, 9 of
which were peer-reviewed. Within an experiment, diets only differed in the supply of L-Val with
a minimum of 4 levels of Val. To account for differences in design (e.g. due to differences in
body weight or differences in expressing the Val requirement) data were standardized within each
experiment. Average daily feed intake (ADFI) and daily gain (ADG) were used as response criteria,
and responses were expressed relative to that observed at the highest level of Val-supplementation.
The Val content was reported or recalculated from feed ingredients using table values (Sauvant et
al., 2004). The standardized ileal digestible (SID) Val content was expressed relative to the Val
requirement estimate of the NRC (1998) as a percentage in the diet (using the average body weight
GXULQJWKHH[SHULPHQW RUUHODWLYHWR/\V 6,'9DO/\V GHSHQGLQJRQWKHH[SHULPHQWDOGHVLJQ
A quadratic regression analysis was carried out for each experiment to assess if the increase in Val
content resulted in an increase in ADFI or ADG. Experiments for which there was no indication for
a response (P>0.25) or where the magnitude of the response was less than 10% were not considered
further. A bent-stick model was then used to analyze the response to the Val supply (Van Milgen
et al., 2012). This model is composed of an ascending line and a plateau, connected by quadratic
transition phase. The transition accounts for the fact that the Val requirement declines during the
experiment while the experimental diets do not change. The model was parameterized to include
a Val requirement (midpoint of the transition phase) and plateau for each experiment, whereas the
slope of the ascending line was shared across experiments. The Val supply required to maximize
ADFI or ADG was also determined.

Results and discussion

0RVWH[SHULPHQWVZHUHFDUULHGRXWLQ\RXQJSLJVDQGWKH¿QDOERG\ZHLJKWZDVOHVVWKDQNJLQ
25 of the 28 experiments. In 9 experiments, there was no indication for a response to the increasing
Val supply. In 4 experiments, the lack of response could be explained because the lowest level of Val
was only slightly lower (or even greater) than the NRC requirement estimate. In 3 other experiments,
the Val requirement was expressed relative to Lys, but Lys may not have been second-limiting for
growth, with Lys levels exceeding the NRC requirement estimate by 20%. For 2 experiments, the
Met+Cys and Thr contents of the diet could have been second-limiting for performance (before
Lys), thereby limiting the response of the pigs to the increasing Val supply. In 19 experiments (18 in
young pigs, 1 in grower pigs), a response to the increasing Val supply was observed (P<0.25). The
estimated Val requirements (midpoints of the transition phase) were similar for ADFI or ADG, and

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 339
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_119, © Wageningen Academic Publishers 2013
both estimates were highly correlated (r=0.97). A very low Val requirement was estimated for an
experiment published in the 1950s (53% of the NRC requirement estimate), which was not included
LQWKHIXUWKHUDQDO\VLV7KHHVWLPDWHG9DOUHTXLUHPHQWVUDQJHGIURPWRRIWKH15&  
requirement estimate for ADFI and from 84 to 114% for ADG (94% on average for both). A Val
GH¿FLHQF\UHVXOWHGLQDYHU\VWURQJUHGXFWLRQLQSHUIRUPDQFHZLWKVORSHVRIWKHDVFHQGLQJOLQHVRI
DQGIRU$'),DQG$'*UHVSHFWLYHO\$OWKRXJKWKHPLGSRLQWRIWKHWUDQVLWLRQSKDVHZDV
considered as ‘the requirement’, a greater Val supply is required to maximize performance. Between
DQGRIWKH15&UHTXLUHPHQWHVWLPDWHZRXOGEHUHTXLUHGWRPD[LPL]H$'* DYHUDJH
 ([SUHVVHGUHODWLYHWR/\V6,'9DO/\VZRXOGEHUHTXLUHGWRDWWDLQWKHPLGSRLQWRIWKH
WUDQVLWLRQSKDVHZKLOH6,'9DO/\VZRXOGEHUHTXLUHGWRPD[LPL]H$'*7KLVLQFUHDVHLQ9DO
supply would increase ADG by 5%.

To further analyze the variation in Val requirement estimates, correlations were calculated between
the Val requirement estimates and the supply of other essential amino acids (expressed relative
to the requirement estimates of the NRC). These correlations were positive for all amino acids,
suggesting that the protein supply has an impact on the Val requirement. An increased protein intake
PD\LQFUHDVHSURWHLQWXUQRYHUUHVXOWLQJLQDORZHUHI¿FLHQF\RI9DOXWLOL]DWLRQDQGDQLQFUHDVHG
9DOUHTXLUHPHQW7KHJUHDWHVWFRUUHODWLRQFRHI¿FLHQWVEHWZHHQWKHDPLQRDFLGFRQWHQWDQGWKH9DO
UHTXLUHPHQWZHUHREVHUYHGIRU3KH  3KH7\U  +LV  7KU  DQG/\V  
7KHFRUUHODWLRQFRHI¿FLHQWVZLWK/HXDQG,OHZHUHORZHU DQGUHVSHFWLYHO\ VXJJHVWLQJ
that the supply of branched-chain amino acids has no impact on the Val requirement. This contrasts
with Ile requirement, where the supply of other branched-chain amino acids or large neutral amino
acids other appears to affect the Ile requirement. Although there are indications that an excess
VXSSO\RI/HXDJJUDYDWHVWKHHIIHFWRID9DOGH¿FLHQF\ *ORDJXHQet al., 2011), this effect could not
EHTXDQWL¿HGEHFDXVHUHVSRQVHWRD9DOGH¿FLHQF\ZDVDVVXPHGWREHVLPLODUIRUDOOGRVHUHVSRQVH
studies in this meta-analysis.

,QFRQFOXVLRQ\RXQJSLJVUHVSRQGWRDGLHWDU\9DOGH¿FLHQF\E\UHGXFLQJ$'),ZKLFKLQWXUQUHVXOWV
LQDUHGXFWLRQLQ$'*7RPD[LPL]HSHUIRUPDQFH6,'9DO/\VLVUHTXLUHG:KHWKHUWKHVH
UHVXOWVREWDLQHGPRVWO\LQ\RXQJSLJVFDQEHH[WUDSRODWHGWRJURZHUDQG¿QLVKHUSLJVUHPDLQVWREH
FRQ¿UPHG7KHHIIHFWRIWKHSURWHLQVXSSO\RQDPLQRDFLGUHTXLUHPHQWVPD\UHTXLUHPRUHDWWHQWLRQ

References
Gloaguen M., N. Le Floc’h, L. Brossard, R. Barea, Y. Primot, E. Corrent and J. van Milgen, 2011. Response of piglets to
the valine content in diet in combination with the supply of other branched-chain amino acids. Animal 5, 1734-1742.
NRC, 1998. Nutrient requirements of swine, 10th revised edition. National Academy Press, 189 pp.
Sauvant D., J.M. Perez and G. Tran, 2004. Tables of composition and nutritional value of feed materials. Pigs, poultry,
FDWWOHVKHHSJRDWVUDEELWVKRUVHV¿VK,15$3DULVSS
Van Milgen J., M. Gloaguen, N. Le Floc’h, L. Brossard, Y. Primot and E. Corrent, 2012. Meta-analysis of the response
RIJURZLQJSLJVWRWKHLVROHXFLQHFRQFHQWUDWLRQLQWKHGLHW$QLPDO

340 Energy and protein metabolism and nutrition in sustainable animal production
Prediction of dry matter intake in dairy calves
A.L. Silva1, M.I. Marcondes1, M.M. Campos2, F.S. Machado2, M.M.D. Castro1 and A.S. Trece1
1Department of Animal Science, Federal University of Viçosa, Brazil; [email protected]
2Brazilian Agricultural Research Corporation, Embrapa Dairy Cattle, Brazil

Introduction
Dry matter intake (DMI) directly affects animal performance; it is the main determinant of nutrients
used to meet the requirements for maintenance and production. Therefore, accurate models of dry
PDWWHULQWDNHLQFDOYHVLVIXQGDPHQWDOWRIRUPXODWLQJGLHWVWRPHHWUHTXLUHPHQWVDQGWKHHI¿FLHQWXVH
of the nutrients by the animals (NRC, 2001; Chizzotti et al., 2007). The objective was to establish a
model to determine dry matter intake (DMI) and free water intake (WI) for crossbred Holstein-Zebu
FDOYHVDJHGEHWZHHQDQGGD\V

Material and methods

The experiment was conducted in the Department of Animal Science, Universidade Federal de
Viçosa, Brazil. Eighteen male calves, crossbreed Holstein-Zebu, with an initial body weight of
“NJZHUHXVHG7KHDQLPDOVZHUHGLVWULEXWHGDFFRUGLQJWRDFRPSOHWHO\UDQGRPL]HGGHVLJQ
LQWRWUHDWPHQWVZLWKUHSOLFDWLRQVDQGWKHVHWUHDWPHQWVFRQVLVWHGRIWKUHHGLIIHUHQWOHYHOVRIPLON
intake, which were 2 (2L), 4 (4L) and 8 (8L) liters per day. The animals had free access to water
and concentrate (starter), which was formulated in accordance with the requirements presented in
15&  7KHDQLPDOVZHUHIHGWZLFHDGD\DWDQGDQGZDWHUDQGVWDUWHULQWDNHZHUH
PHDVXUHGHYHU\GD\DWK7KHUHZHUHSHUIRUPHGGLJHVWLELOLW\DVVD\VDWDQGGD\VRIOLIH
During those periods, there were collected samples of feeds offered. Milk samples were dried by
lyophilization (Method INCT-CA G-002/1). All samples were analyzed for dry matter, according
to the method (INCT-CA G-003/1), describe by Detmann et al., (2012). Environmental variables
were also considered: relative humidity (RH), temperature and humidity index (THI) and black
globe temperature and humidity index (BGTHI). These estimates were made from daily uptakes of
black globe temperature, dry bulb temperature, wet bulb temperature, and maximum and minimum
temperatures. Environmental effects, milk intake and age were used in a multiple regression model,
considering both linear and quadratic effects, to estimate calves DMI and WI. The test was conducted
XVLQJWKH0,;('SURFHGXUHRIWKH6$6DWWKHOHYHORIVLJQL¿FDQFHRI

Results and discussion

The DMI was affected by milk intake (P<0.001), THI (3 0.0453) and age of the animals (P<0.001).
+RZHYHUQRVLJQL¿FDQWTXDGUDWLFHIIHFWVZHUHREVHUYHG P> WKXVWKH¿QDOUHJUHVVLRQFDQEH
expressed:

'0, î0±î7+,îGD\ 

where DMI= dry matter intake (kg/day), M= milk intake (kg/day of DM), THI= temperature and
humidity index (without dimension), Day= Age of animal (days). The DMI observed between
treatments varied and increased with increasing the milk intake, as expected. On the other hand,
starter intake (SI), followed the opposite behavior, and decreased as the milk intake was increased
(Table 1). The reduction in solids intake by calves drinking more milk was due to the fact that these
animals have reached satiety by chemical-physiological mechanisms (higher blood glucose) and by
SK\VLFDOIDFWRUV FRQWLQXRXVJXW¿OOLQJEHFDXVHRIFXUGIRUPDWLRQ.KDQet al., 2011). The SI can be
estimated by the difference between the total DMI and the DMI from milk.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 341
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_120, © Wageningen Academic Publishers 2013
7DEOH'U\PDWWHU NJGD\ VWDUWHU NJGD\'0 DQGZDWHULQWDNHV OGD\ DQGSUHGLFWLRQHTXDWLRQV
for dry matter and water intakes.

L 21 L4 L8 Regression

DMI  0.795 1.114 '0, î0±î7+,î'D\

SI 0.348 0.249 0.123 SI = DM Itotal – DMI milk
WI 2.274 1.979 1.273 :, î7dbî'D\î6,

1 L2: 2 l/day; L4: 4 l/day; L8: 8 l/day; DMI: dry matter intake, SI: starter intake, WI: free water intake.

All the environmental variables (dry bulb temperature, THI, BGHI) affected water intake (P<0.0001).
However, as the dry bulb temperature had the greater correlation with WI (r2=0.185), the other
YDULDEOHVZHUHQRWLQFOXGHGLQWKHPRGHOWRHVWLPDWH:,,WZDVYHUL¿HGVLJQL¿FDQWHIIHFWVRIGU\
bulb temperature (P<0.0001), age in days (P<0.0001), and starter intake (3 0.0028) on WI. The
same way as occurred to DMI, it wasn’t observed quadratic effects for any of the variables tested
(P>0.05) and the multiple linear regression to estimate WI in calves can be expressed:

:, î7dbîGD\î6, 

where WI = free water intake (liters /day), Tdb = dry bulb temperature (°C), Day= age of animal
(days), SI = starter intake (kg/day). The water intake presented a direct relationship with the starter
intake (Table 1), and with the percentage of dry matter on the diet, and these were 25.2; 17.7 and
13.9% of the natural matter for the treatments 2L, 4L and 8L, respectively. NRC (2001) mention
that among the many factors that can change the free water intake, the percentage of the dry matter
on the diet is the main factor. Therefore, higher solid feed intake tends to be followed of the higher
water intake, as observed in this study.

It can be concluded that the dry matter intake in calves can be estimated using parameters as milk
intake, THI and age of animals. Furthermore, the free water intake can be estimated using parameters
as dry bulb temperature, age of animals and starter intake.

References
Chizzotti, M. L., S.C. Valadares Filho, R. F. Diniz Valadares, F. H. M. Chizzotti, M. I. Marcondes, and M. A. Fonseca,
2007. ‘Intake, digestibility and nitrogen metabolism in Holstein cows with different milk production levels.’ Braz.
-$QLP6FL
Detmann, E., M.A. Souza, and S.C. Valadares Filho, 2012. Métodos Para Análises De Alimentos. ed. E. Detmann,
M.A. Souza, and S.C. Valadares Filho. 1st ed. Visconde do Rio Branco: Suprema.
Khan, M A, D. M. Weary, and M. A. G. Von Keyserlingk, 2011. ‘Invited Review : Effects of Milk Ration on Solid
Feed Intake, Weaning, and Performance in Dairy Heifers.’ J. Dai. Sci. 94, 1071-1081.
National Research Council – NRC, 2001. Nutrient Requirements of Dairy Cattle. 7th ed. Washington D.C.: National
Academic Press.

342 Energy and protein metabolism and nutrition in sustainable animal production
Re-evaluation of lysine requirement of broilers based on protein
GHSRVLWLRQDQGO\VLQHHI¿FLHQF\
J.C.P. Dorigam, N.K. Sakomura, L. Hauschild and E.P. Silva
Department of Animal Science, University of Agrarian and Veterinary Sciences of UNESP, Jaboticabal,
63%UD]LO[email protected]

Introduction
The quality of protein is related to concentration and availability of amino acids in feed and differs
between geographic regions because of the composition of feedstuffs. Differences in environment,
PDQDJHPHQWJHQRW\SHDQGSK\VLRORJLFDOVWDWHFDQDOVRLQÀXHQFHWKHUHVSRQVHRIELUGVWRDPLQR
acid intake. Due to these factors a re-evaluation of amino acid requirements is necessary. The lysine
requirement is constantly studied because of its importance as a reference amino acid in the ideal
SURWHLQFRQFHSW$OVRWKHDPRXQWRIO\VLQHLQJHVWHGKDVDGLUHFWLQÀXHQFHRQJURZWKSHUIRUPDQFH
because it is used for body protein deposition. Thus a growth model for broilers (Samadi and Liebert,
2007) that takes into account nonlinear functions integrated in a factorial approach considering lysine
HI¿FLHQF\SURWHLQGHSRVLWLRQVH[DQGDJHZDVXVHGWRUHHYDOXDWHWKHRSWLPXPGLHWDU\O\VLQHOHYHO
for cobb genotype.

Material and methods

1LWURJHQ 1 EDODQFHVWXGLHVZHUHFRQGXFWHGIRUWKUHHDJHSHULRGV DQGG DQG
eighty four broilers of cobb500 genotype were used (42 males and 42 females) per period. The birds
were distributed in a completely randomized design with seven treatments (six levels of crude protein
&3 1 1 1 1 1 DQG1 JNJLQGU\PDWWHU '0 DQGVL[
replicates for each sex. In the seventh treatment 4 g of L-Lysine HCl (78%) /kg in the diet N1 was
DGGHGDVDFRQWUROWRFKHFNLIO\VLQHZDVWKH¿UVWOLPLWLQJDPLQRDFLG7KHGLHWVZHUHREWDLQHGE\
GLOXWLQJDKLJKSURWHLQGLHWZLWKD1IUHHGLHWFRPSRVHGRIFRUQVWDUFKWRPDNHO\VLQHWKH¿UVWOLPLWLQJ
DPLQRDFLG J/\VJ&3 (DFK1EDODQFHVWXG\ODVWHGGD\V ¿YHGD\VRIDGDSWDWLRQ
DQGWZRSHULRGVRIH[FUHWDFROOHFWLRQRI¿YHGD\V $WWKHHQGRIWKHFROOHFWLRQSHULRGWKHH[FUHWD
were homogenized and freeze dried for DM and N analysis. The exponential regression between N
intake (NI) and N excretion (NEX) allowed estimation of N maintenance requirements (NMR) when
NI=0, according to Samadi and Liebert (2007). Based on the exponential function between NI and
N retention (NR), the theoretical maximum for NR (NRmaxT) was estimated using the equation: NR
= NRmaxT (1 – e-b*NI), where b = slope of NR curve. NRmaxT minus NMR results in the theoretical
maximum for N deposition (NDmaxT). For modeling the requirement of limiting amino acid (LAA)
an equation was applied: LAAI = [lnNRmax7íOQ 15max7í15 @EFí; where LAAI is the intake
of LAA and bcí is the slope of LAA concentration (c) on feed protein quality (b).

Results and discussion

The responses of the control treatment were intermediate to those obtained in the treatments N1
DQG1FRQ¿UPLQJWKDWO\VLQHZDVWKHOLPLWLQJDPLQRDFLGLQGLHWV7KHUHVXOWVRIWKH1EDODQFH
studies are summarized in Table 1. The estimated NMR values in each age period were used to
calculate NDmaxT and are in accordance to results of Samadi and Liebert (2007). The NDmaxT
HVWLPDWHGZHUHDQGPJ%:kgGIRUPDOHDQGDQGPJ
BWkg/d for female in the periods I, II, and III, respectively. This continuous decrease with age
RFFXUVEHFDXVHWKHHI¿FLHQF\RIDPLQRDFLGGHSRVLWLRQLQFDUFDVVGHFUHDVHVZLWKDJH 6NODQDQG
1R\ $OVRVH[DIIHFWVWKHHI¿FLHQF\RIXWLOL]DWLRQRIWKHDPLQRDFLG '¶0HOOR OHDGLQJ
to different NDmaxT observed.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 343
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_121, © Wageningen Academic Publishers 2013
Table 1. Applied model parameter depending on age and sex for NMR and NRmaxT.

Sex Age Mean BW NMR NRmaxT NDmaxT R2

(days) (g) (mg/BWkg/d) (mg/BWkg/d) (mg/BWkg/d)

Male  370 220   0.98

22-37 1,438  3,401 3,137 0.98
38-53   2,480 2,204 0.98
Female  338 225 3,845  0.98
22-37  277 3,315 3,038 0.98
38-53 2,485 271 2,319 2,048 0.98

7KHGDWDIRUPRGHOLQJO\VLQHUHTXLUHPHQWVDUHSUHVHQWHGLQ7DEOH%DVHGRQHI¿FLHQF\RIO\VLQH
utilization the required dietary digestible lysine for males and females broilers were 1.07 and 0.9%
G DQG G DQG G FRUUHVSRQGLQJWRDQGJRI
WKHGDLO\IHHGLQWDNHUHVSHFWLYHO\7KHHVWLPDWHVIRUDJHGDQGGZDVORZHUWKDQDFWXDO
digestible lysine recommendations (Bernal et al., 2012) of 1.22 for males and 1.24% for females at
GDQGIRUERWKJHQGHUVDWG+RZHYHUDWGWKHHVWLPDWLYHZHUHLGHQWLFDO
to the requirement of 0.90 and 0.72% for males and females, estimated by Samadi and Liebert
(2007) after 50 days with a daily feed intake of 180g. The result indicates that broilers have become
more demanding and require more nutrients for maximum performance, primarily at younger ages.

7DEOH&DOFXODWLRQRIWKHO\VLQHUHTXLUHPHQWVIRU&REEDFFRUGLQJWRHI¿FLHQF\RIO\VLQH
utilization and CP deposition.

Age GD\V 22-37 days 38-53 days

Male Female Male Female Male Female

CP deposition (g/d)2 7.2  15.0 14.2 17.7 14.1

/\VHI¿FLHQF\ EF-1) 58×10 î î 71×10 î 104×10
Lys requirement (mg/d) 535.5 482.3 1,208.5  1,447.4 1,145.7

1&RQVLGHUHGRI1'
maxT for simulate practical performance condition.

Acknowledgements
7KHDXWKRUVDUHJUDWHIXOWR)$3(63IRUWKH¿QDQFLDOVXSSRUW

References
Bernal L.E.P., F.C. Tavernari, G.R. Lelis, H.S. Rostagno, 2012. Nutritional requirement of digestible lysine for cobb
500 broiler chickens. In: World´s Poultry Congress, 2012, Salvador. Abstract. Salvador: WSPA, 2012. J. Poult.
6FLYVXSO
D’Mello J. P. F. 2003. Adverse effects of amino acid. In: D’mello JPF Amino acids in animal nutrition. 2ed. Edinburgh:
CABI Publishing, pp. 187-202.
Samadi, and F. Liebert, 2007. Lysine requirement of fast growing chickens – effects of age, sex, level of protein
GHSRVLWLRQDQGGLHWDU\O\VLQHHI¿FLHQF\-3RXOW6FL
Sklan, D. and Y. Noy, 2004. Catabolism and deposition of amino acids in growing chicks: effect of dietary supply.
3RXOW6FL

344 Energy and protein metabolism and nutrition in sustainable animal production
Ideal ratio (relative to lysine) of methionine + cystine and threonine for
broiler breeders
A.R. Troni1, N.K. Sakomura1, C.F.S. Oliveira2, E.P. Silva1, F.G.P. Costa2 and D.C.Z. Donato1
1StateUniversity of São Paulo, UNESP, Jaboticabal, SP, Brazil; [email protected]
2Federal University of Paraíba, UFPB, Areia, PB, Brazil

Introduction
To establish the amino acid requirements for broilers, the ideal ratio of lysine to the amino acids is
used. This criterion is based on the small variation in the proportion of amino acids with lysine. Few
studies deal with amino acid requirements for broiler breeders. A factorial method for calculating the
amino acid requirement was proposed by Fisher et al. (1973) for egg production and body weight
maintenance, including the standard deviation of requirements and an economic factor. Based on
WKLVPHWKRGWKHREMHFWLYHRIWKLVVWXG\ZDVWRGHWHUPLQHWKHHI¿FLHQF\RIXWLOL]DWLRQRIPHWKLRQLQH
and cystine (Met+Cys) and threonine (Thr) and their ideal ratio to lysine (Lys).

Material and methods

Three ten-week (six week adaptation, four week data collection) experiments with 147 broiler
breeders housed in individual cages were based on a completely randomized design with seven
OHYHOVRIDPLQRDFLGDQGVHYHQUHSOLFDWHV$FRQWUROWRFRQ¿UPWKDWWKHVWXGLHGDPLQRDFLGZDV¿UVW
limiting was included. Diets were based on corn, soybean meal and synthetic amino acids. Egg
mass was used in the model: AAI=(a×Emax)+(b×BW)+y, where AAI is amino acid intake, mg/
day; a is requirement for egg mass production, mg/g; Emax is egg mass maximum response, g/day;
b is body weight maintenance requirement, mg/kg, BW is body weight, kg; and y is the standard
GHYLDWLRQ ı RIWKHUHTXLUHPHQW Dîı(PD[  Eîı%: &RHI¿FLHQWVZHUHDGMXVWHGE\WKH()*
VRIWZDUHPRGXOHDPLQRDFLGRSWLPL]HU(I¿FLHQF\RIXWLOL]DWLRQ (II\ ZDVREWDLQHGE\GLYLGLQJWKH
deposition of amino acid in the egg (d; mg/g) by the amino acid requirement for egg mass production
DPJJ (II\ GD7KHLGHDOUDWLRZDVREWDLQHGIURPWKHFRHI¿FLHQWVUDWHDRI7KU D7KUD/\V DQG
Met+Cys (aMet+Cys/aLys).

Results and discussion

,QFUHDVLQJOHYHOVRI/\V7KUDQG0HW&\VLQFUHDVHGEURLOHUEUHHGHUHJJPDVVDQG
123.88%, respectively (Table 1). The Reading Model partitions amino acid requirements into
maintenance and egg production. Based on model parameters, daily requirements of Lys, Thr
DQG0HW&\VIRUDELUGSURGXFLQJDQDYHUDJHJHJJZHUHFDOFXODWHGDVDQGPJ
UHVSHFWLYHO\ 7DEOH 7KHUHTXLUHPHQWIRUPDLQWHQDQFHRI/\V7KUDQG0HW&\VUHSUHVHQWV
0.01 and 7.49% of amino acid intake, respectively. This maintenance requirement is not in accordance
to Bonato et al. (2011) and Siqueira et al. (2011); however, they are similar to those found by Fisher
et al. (2001). The ratio of lysine to amino acids may vary depending on the maintenance requirement,
thus any change in this parameter changes the values (Fisher, 1998). Therefore, we determined the
LGHDOUDWLRXVLQJWKHSDUDPHWHUDZKLFKUHSUHVHQWVWKHHJJPDVVUHTXLUHPHQWDQGGRHVQRWLQÀXHQFH
WKHPDLQWHQDQFHUHTXLUHPHQW7KHLGHDOUDWLR7KU/\VZDV%\FRQVLGHULQJWKHPDLQWHQDQFH
UHTXLUHPHQWRI/\V PJNJ DQG7KU PJNJ IRXQGE\1RQLVDQG*RXV  WKHLGHDOUDWLRLV
EHFDXVHRIWKHLQFUHDVHRIPDLQWHQDQFHRYHUWKHWRWDOLQWDNH7KLVUHVXOWVXSSRUWVWKHK\SRWKHVLV
of an effect of maintenance on the ideal ratio of amino acids. The technique is a good alternative
WR¿QGUHODWLRQVKLSVRIDQGHI¿FLHQF\RIXWLOL]DWLRQRIDPLQRDFLGVEXWWKHYDOXHVIRUPDLQWHQDQFH
in the literature are discrepant and it is important to rely on the requirements of production of egg
mass a to determine the responses.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 345
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_122, © Wageningen Academic Publishers 2013
7DEOH5HVSRQVHVRIHJJPDVV (0JGD\ RIEURLOHUEUHHGHUVWRGLIIHUHQWLQWDNHVRIDPLQRDFLGV
PJGD\ 

Treatment Lysine (220-733) Threonine (178-594) 0HW&\V 

Intake EM Intake EM Intake EM

1 270.3 22.2 237.0  208.2 20.1

2 384.3 33.9 342.0 40.0 292.7 29.2
3 489.0 39.8 441.0 42.0  
4 572.1 42.1 528.0 47.9  44.0
5  42.2  50.5  47.0
 847.0 42.7  50.1 750.0 45.0
7 952.5 45.5   - -

7DEOH3DUDPHWHUVRIWKHPRGHOYDOXHVRIHI¿FLHQF\ (II\ DQGDPLQRDFLG $$ UDWHWRO\VLQH $$

/\V IRUEURLOHUVEUHHGHUV

Amino acid Emax1 BW ı(PD[ ı%: a b d Eff AA/Lys

Lysine 43.8 4.12 10 0.412 11.39 0.80 8.3 73 100

Threonine  4.30 12  7.42 0.01 5.7 77 
Met + Cys 45.5 4.41 5 0.441 8.82 7.37  78 77

1(PD[ HJJPDVVPD[LPXPUHVSRQVHJGD\%: ERG\ZHLJKWNJı VWDQGDUGGHYLDWLRQD UHTXLUHPHQWIRUHJJ

mass production, mg/g; b = requirement for BW maintenance, mg/kg; d = amino acid deposition in the egg (mg/g).

Acknowledgements
:HWKDQN)$3(63IRU¿QDQFLDOVXSSRUWDQG&13TIRUDVFKRODUVKLS

References
Bonato, M. A., N.K. Sakomura, J.C. Siqueira, J.B.K. Fernandes and R.M. Gous, 2011. Maintenance requirements
for methionine and cysteine, and threonine for poultry. South African Journal of Animal Science 41, 209-222.
Bowmaker, J. E. and R.M. Gous, 1991. The response of broiler breeder hens to dietary lysine and methionine. British
3RXOWU\6FLHQFH
Fisher, C., 1998. Amino acid requirements of broiler breeders. Poultry Science 77, 124-133.
Fisher, C., R.M. Gous, L. Goddard, 2001. Lysine requirements of broiler breeders. In: Benalve D, (ed.), Australian
3RXOWU\6FLHQFH6\PSRVLXPY3RXOWU\5HVHDUFK)RXQGDWLRQ6\GQH\SS
Fisher, C., T.R. Morris and R.C. Jennings, 1973. A model for the description and prediction of the response of laying
KHQVWRDPLQRDFLGLQWDNH%ULWLVK3RXOWU\6FLHQFH
Nonis, M. K. and R.M. Gous, 2008. Threonine and lysine requirements for maintenance in chickens. South African
Journal of Animal Science 38, 75-82.
Siqueira, J. C., N.K. Sakomura, H.S. Rostagno, M.A. Bonato, S.R.F. Pinheiro and D.C.N. Nascimento, 2011. Exigência
de lisina para mantença determinada com galos de diferentes genótipos. Revista Brasileira de Zootecnia 40, 812-820.

346 Energy and protein metabolism and nutrition in sustainable animal production
Prediction of net hepatic release of glucose using a ‘hybrid’ mechanistic
model in ruminants applied to positive energy balance
L. Bahloul1, I. Ortigues-Marty1, J. Vernet1, H. Lapierre, P. Nozière1 and D. Sauvant2
1,15$8QLWp0L[WHGH5HFKHUFKHVVXUOHV+HUELYRUHV6W*HQqV&KDPSDQHOOH)UDQFH
28050RGpOLVDWLRQ6\VWpPLTXH$SSOLTXpHDX[5XPLQDQWV$JUR3DULV7HFK3DULV)UDQFH
[email protected]
$JULFXOWXUHDQG$JUL)RRG&DQDGD6KHUEURRNH4&-,0&&DQDGD

Introduction
Ruminants depend on hepatic gluconeogenesis to meet most of their metabolic demand for glucose
which relies on availability of precursors from diet supply and animal requirements (Loncke et al.,
2010 Several mechanistic models of the metabolic fate of nutrients across the liver exist that have
been parameterized for dairy cows. They cannot be directly used to predict hepatic gluconeogenesis
in all types of ruminants in different physiological status. A hybrid mechanistic model of nutrient
ÀX[HVDFURVVWKHOLYHULVSUHVHQWO\EHLQJGHYHORSHG %DKORXOet al., 2012), that is calibrated empirically
based on meta-analysis (Sauvant and Mertens, 2008) to be applicable to all types of ruminants in
different physiological status and to usual nutritional practices. The objectives of the present work
were to test the hybrid liver model in its present state of development to simulate the net hepatic
UHOHDVHRIJOXFRVHZKHQWKHJOXFRJHQLFNHWRJHQLFQLWURJHQRXVQXWULHQWSUR¿OHHQWHULQJWKHOLYHUYDULHV
7KLV¿UVWDSSOLFDWLRQRIWKHPRGHOZDVOLPLWHGWRUXPLQDQWVLQSRVLWLYHFDOFXODWHGHQHUJ\EDODQFH
in which the nutrient fate across the liver is mostly directed by mass action laws.

Material and methods

1HWYHQRDUWHULDOÀX[HVRIQXWULHQWVFRUUHVSRQGWRWKHLQSXWV QHWSRUWDODSSHDUDQFH13$ DQG
outputs (net splanchnic release, NSR) of the mechanistic model. Nutrients considered are: acetate
& SURSLRQDWH & EXW\UDWH & JOXFRVHODFWDWHȕK\GUR[\EXW\UDWH %+% WRWDODPLQRDFLGV
(TAA), urea, NH3, O2, CO2DQGWKHDEVRUEHGIDWW\DFLGV7KHLUÀRZVDUHH[SUHVVHGLQPPRORI
C/h/BW except for NH3, expressed in mmol of N/h/BW. Ten hepatic compartments are included:
C2, C3, C4, BHB, glycogen, protein, glucose, lactate, TAA and CO2&RQYHUVLRQÀX[HVEHWZHHQ
compartments, including metabolic compartments summarizing the Krebs cycle, represent the
main hepatic metabolic pathways. The model based on mass action laws is adjusted on empirical
relationships of the net hepatic transfer of nutrients derived by meta-analysis (Loncke, 2009) from
FLORA database, allowing estimation of NSR of nutrients. The empirical equation used to predict
glucose NSR was based on the sum of the NPA of precursors (Loncke et al., PRGL¿HGWRWDNH
into account all AA potentially convertible to glucose. Indeed, if only the NPA of glucogenic AA
LVFRQVLGHUHGDGH¿FLWRIFDUERQLVFDOFXODWHGWRPHHWJOXFRVHRXWSXW6HWVRIH[SHULPHQWDOGDWD
representative of nutritional practices representing different combinations of ketogenic, glucogenic
DQGQLWURJHQQXWULHQWLQSXWVZHUHXVHGWRDGMXVWSDUDPHWHUVRIWKHFRQYHUVLRQÀX[HVLQFDWWOHDQG
sheep, only in calculated positive energy balance. Nutritional status ranged from 1 to 5 × ME and
WRîPHWDEROL]DEOHSURWHLQPDLQWHQDQFHUHTXLUHPHQWV3DUDPHWHUVZHUHDGMXVWHGVRWKDWWKH
simulated model outputs converge at best with the values predicted by empirical relationships. The
model was evaluated by: (1) calculating the full C balance across the liver; and (2) by comparing
outputs (NSR) of glucose simulated by the model with values predicted by the empirical relationships.

Results and discussion

The full C balance across the liver simulated by the model (Figure 1) indicates a tight agreement
EHWZHHQ&RXWSXWVDQGLQSXWVZLWKDVPDOOGH¿FLWDYHUDJLQJPRO&G&RPSDULVRQEHWZHHQ
the model simulated and the empirically predicted NSR of glucose (Figure 2) shows a good (93%)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 347
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_123, © Wageningen Academic Publishers 2013
 25

20

Car bon out put

15

10
Y= -0.007± 0.029(NS) + 0.994* * * ± 0.001 X
n= 80, RMSE= 0.065, adj R² = 99.9

5
5 10 15 20 25
Car bon input

)LJXUH5HODWLRQVKLSEHWZHHQWKHFDUERQRXWSXW < VLPXODWHGE\WKHPRGHODQGWKHFDUERQLQSXW

; LQPPRORI&K%: P<

6
NSR of glucose ( simulat ed)

2
Y= 0.255± 0.055* * * + 0.933* * * ± 0.013 X
1
n= 80, RMSE= 0.23, adj R² = 98.3
0

0 1 2 3 4 5 6 7 8
NSR of glucose ( pr edict ed)

)LJXUH5HODWLRQVKLSEHWZHHQWKH165RIJOXFRVH < VLPXODWHGE\WKHPRGHODQGSUHGLFWHGE\

VWUXFWXUDOUHODWLRQVKLS ; LQPPRORI&K%: P<

simulation. For C3 and TAA, the major glucogenic nutrients, the simulated outputs compare well
with the values predicted by the structural relationship. Parameters of mass action laws obtained by
model adjustment show that 93%/h and 173%/h of intrahepatic pool of TAA and C3 is metabolized
to oxaloacetate (OA) respectively; 124% of intrahepatic pool of OA is converted to glucose. Almost,
all TAA up taken by the liver (73%) appeared used for neoglucogenesis. The hepatic uptake of C3
(91%) seems to be converted completely to glucose, only 19%/h and 0.8%/h of intrahepatic pool
of C3 are released and converted to pyruvate respectively. A strength of the approach is to identify
situations responsible for poor adjustments: an underestimation (0.34 mmol of C/h/BW) of the
simulated NSR glucose is observed at elevated TAA NPA (>3 mmol of C/h/BW).

The hybrid model based strictly on mass action laws simulates well the prediction of hepatic release
of glucose for ruminants in positive energy balance. The model was found to be stable in term of C
EDODQFHZLWKDVPDOOQHJOLJLEOHGH¿FLWLQ&7KHQH[WVWHSFRQVLVWVRQWKHDSSOLFDWLRQRIWKHPRGHO
to ruminants in negative energy balance. If necessary, regulations representing hepatic metabolism
will be introduced to take account interaction between nutrients and tissues (nutrient requirements).

Acknowledgements
Financial support from INZO and Limagrain is acknowledged.

348 Energy and protein metabolism and nutrition in sustainable animal production
References
Bahloul, L., D. Sauvant, J. Vernet, H. Lapierre, P. Nozière and I. Ortigues-Marty, 2012. Mechanistic modeling based on
PHWDDQDO\VLVSUHGLFWLRQRIWKHQHWKHSDWLFÀX[HVRIHQHUJHWLFQXWULHQWVLQUXPLQDQW&DQ-$QLP6FL
/RQFNH&0RGpOLVDWLRQGHVUHODWLRQVHQWUHO¶DOLPHQWDWLRQHWOHVÀX[VSODQFKQLTXHVGHQXWULPHQWVpQHUJHWLTXHV
chez le ruminant. Thèse de doctorat, AgroParisTech. France.
Loncke, C., P. Noziere, S. Lemosquet, J. Vernet, H. Lapierre, D. Sauvant and I. Ortigues-Marty, 2010. Splanchnic
release of glucose in relation to dietary supply and animal performances. Renc. Rech. Ruminants 17, 317.
Sauvant, D. and D. Mertens, 2008. Use of meta-analysis to build a mechanistic model of responses of rumen digestion
WRGLHWDU\¿EUHLQFDWWOH&DQ-$QLP6FL

Energy and protein metabolism and nutrition in sustainable animal production 349
Part 6. Products and health
Recent advances in understanding the interactions between nutrients
and immunity in farm animals
K.C. Klasing and V.J. Iseri
Department of Animal Science, University of California, Davis, CA, USA; [email protected]

Introduction
1XWULWLRQLVDQLPSRUWDQWGHWHUPLQDQWRIWKHGHYHORSPHQWDQGHI¿FDF\RIWKHLPPXQHV\VWHP
Nutrition is also sometimes used as a management tool to affect changes in the type or magnitude
RIDQLPPXQHUHVSRQVH7KHVHGHYHORSPHQWVKDYHEHHQWKHUHVXOWRIGLI¿FXOWUHVHDUFKWKDWUHOLHV
on accepted principles of nutrition to inform the somewhat unknown frontiers of immunology and
disease resistance. Historically, plethoric levels of nutrients were thought to boost, improve, or
stimulate immunity and these claims were a tool of nutrient marketing because of the intellectual
vacuum. These claims illustrate sloppiness in thinking, which fortunately has diminished due to our
new appreciation for the role of nutrients in the immune system.

In the case of nutritionally required nutrients, both the absolute amounts and the balance of individual
QXWULHQWVLQÀXHQFHLPPXQLW\7KHUHLVOLWWOHGRXEWWKDWVHYHUHQXWULWLRQDOGH¿FLHQFLHVLPSDLUWKH
LPPXQHV\VWHPDQGLQFUHDVHVXVFHSWLELOLW\WRLQIHFWLRXVGLVHDVHV+RZHYHUVHYHUHGH¿FLHQFLHVWKDW
UHVXOWLQFODVVLFDOQXWULWLRQDOSDWKRORJ\DUHYHU\UDUHZKHQOLYHVWRFNDUHIHGVFLHQWL¿FDOO\IRUPXODWHG
FRPPHUFLDOGLHWV0DUJLQDOGH¿FLHQFLHVWKDWGRQRWPDUNHGO\LPSDFWJURZWKRUUHSURGXFWLYHRXWSXW
DUHPXFKPRUHFRPPRQDQGWKHLPPXQHV\VWHPLVVHQVLWLYHWRPRGHUDWHGH¿FLHQFLHVRIVRPH
nutrients. However, the immune system is essential for life and appears to have a very high priority for
PDQ\QXWULHQWVUHODWLYHWRPXVFOHDFFUHWLRQRUUHSURGXFWLRQDQGLVVXUSULVLQJO\UHVLOLHQWWRGH¿FLHQFLHV
RIPDQ\QXWULHQWV8QGHUVWDQGLQJZKLFKQXWULHQWVZKHQPDUJLQDOO\GH¿FLHQWLPSDLULPPXQLW\LV
of great practical importance. Considerable recent progress has been made toward elucidating the
mechanisms by which some nutrients are prioritized between leukocytes and other cells types.

Additionally many nutrients, including some that are not essential for growth or reproduction,
regulate and modify immune responses. Progress has also been made towards understanding the
mechanisms by which nutrients impose their regulatory effects. These developments have permitted
the use of surfeit amounts of select nutrients to induce predictable immunoregulatory outcomes for
WZRSULPDU\SXUSRVHVWRGHFUHDVHWKHLQFLGHQFHRIVSHFL¿FLQIHFWLRXVGLVHDVHVDQGWRPLQLPL]HWKH
untoward effects of immune responses on growth, reproduction, and the incidence of metabolic
diseases (Athanasiadou and Huntley, 2008; Cook, 1991; Ingvartsen and Moyes, 2013; Klasing,
2007; Sordillo et al., 2009; Zebeli and Metzler-Zebeli, 2012). The following review examines recent
advances in our understanding of the nutritional needs of the immune system as well as the regulatory
DFWLRQVRIQXWULHQWVRQWKHLPPXQHV\VWHP1XWULWLRQDOVRLQÀXHQFHVWKHJURZWKRIFRPPHQVDODQG
SDWKRJHQLFPLFURÀRUDLQWKHJDVWURLQWHVWLQDOWUDFWDOWKRXJKWKLVDUHDRIUHVHDUFKLVKLJKO\UHOHYDQW
and exciting, it is beyond the scope of this review.

The majority of the research on nutrition and immunity has utilized rodents and these studies inform
much of what we know about nutrient-leukocyte interactions at a mechanistic level. Chickens have
historically been used as a basic research model because of their utility in studying the development of
B cells in their bursa. Swine have recently become a favored model for understanding the development
of mucosal immunity and are increasingly being used for studies on systemic immunity due to their
similarities with humans (Fairbairn et al., 2011). Consequently, basic research using chickens and
pigs supplements and extends the lessons we have gained from mouse models.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 353
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_124, © Wageningen Academic Publishers 2013
Meeting the needs for development, maintenance and responses of
leukocytes
For most nutrients the most important nutritional strategy for optimizing immunity is meeting the
HVWDEOLVKHGUHTXLUHPHQWVIRUPD[LPL]LQJJURZWKIHHGHI¿FLHQF\DQGSURGXFWLYLW\)RUPDQ\RI
WKHHVVHQWLDOQXWULHQWVGH¿FLHQFLHVWKDWDUHVXI¿FLHQWO\VHYHUHWRVORZJURZWKRUUHSURGXFWLRQDUH
also deleterious to the adaptive immune system and markedly increase susceptibility to pathogens
(for reviews, see Kidd, 2004; Korver, 2012; Latshaw, 1991). For some nutrients, leukocytes are
DPRQJWKHPRVWVHQVLWLYHWRPDUJLQDOGH¿FLHQFLHVRIDQ\FHOOW\SHZKLOHIRURWKHUQXWULHQWVWKH
LPPXQHV\VWHPLVODUJHO\XQDIIHFWHGE\GH¿FLHQFLHV7KLVGLFKRWRP\FDQEHH[SODLQHGE\VHYHUDO
mechanisms. First, with the exception of primary immune tissues of the neonate (thymus, bursa,
etc.), most leukocytes in healthy animals are exceptionally inactive and have very low nutrient
needs. Second, many lineages of leukocytes have an excellent capacity to compete with other cells
when circulating levels of nutrients are low. This high priority for nutrients is greatly enhanced in
those cells that respond to an infectious challenge. Third, an immune response is accompanied by
the mobilization of nutrients from muscle and other tissues, which supplies adequate amounts of
some, but not all, nutrients to leukocytes.

Size and nutrient content of the immune system

There has been considerable conjecture but very little investigation on the size and nutrient content
of the immune system. Nutritionists, despite rigorous application of quantitative theory and modeling
to nutrient needs for growth or reproduction, have been remiss in applying this fundamental concept
WRLPPXQLW\,VHULDQG.ODVLQJ  UHFHQWO\TXDQWL¿HGWKHDPRXQWRIO\VLQHWKDWLVFRQWDLQHGLQ
the cells and protective proteins of the systemic (non-mucosal) immune system of an adult healthy
chicken. Lysine was chosen because it is the reference amino acid in the ideal protein system used
commonly in non-ruminant nutrition. The cells and proteins of the systemic immune system at
PDLQWHQDQFHKDYHWKHVDPHO\VLQHFRQWHQWDVRIDPHGLXPHJJRURIDSHFWRUDOLVPXVFOH
'XULQJWKHDFXWHSKDVHDQLYLQMHFWLRQRI¿[HGE. coli, the additional lysine accreted into the cells and
protective proteins of the immune system is equal to 17% of an egg or 5.4% of a pectoralis muscle.
Liver hypertrophies increase markedly during the acute phase of the immune response for the rapid
SURGXFWLRQRIDFXWHSKDVHSURWHLQV+HSDWLFK\SHUWURSK\UHTXLUHVWKHO\VLQHFRQWDLQHGLQRID
PHGLXPHJJGXULQJWKH¿UVWKUSRVWE. coli challenge. Because the liver is recruited to become
part of the immune defenses during the acute phase response, it is the most expensive part of the
response. The amount of lysine needed for the adaptive phase of the response (antibody production
and new lymphocytes) is much less than that needed for the acute phase of the response. In fact with
the E-coli challenge model system described above, the entire lysine needs for the adaptive response
can be provisioned by the decline of the acute phase response and degradation of the acute phase
SURWHLQV,QWHUHVWLQJO\WKHDGGLWLRQDOO\VLQHQHHGVIRUWKHV\QWKHVLVRIVSHFL¿FLPPXQRJOREXOLQV 
mg lysine) is very small compared to the other components of the adaptive phase of the response.
$VDUHVXOWWKHO\VLQHFRQWHQWRIDQDQWLERG\UHVSRQVH VSHFL¿F,JSOXVDGGLWLRQDOFHOOV LVHTXDOWR
only 1.3% of an egg.

The study by Iseri et al. (2013) supports the concept that the cost of an immune response is mostly
due to protective processes unrelated to the needs of leukocytes. Even when the hypertrophy of
the liver and the massive production of acute phase proteins are included, the amount of nutrients
diverted to protective processes accounts for very little of the depression in growth or reproduction
that occurs during the response. Clearly other factors such as decreased appetite, increased metabolic
UDWHDQGPHWDEROLFLQHI¿FLHQFLHVGRPLQDWHWKHQXWULWLRQDOFRVWVRIDQLPPXQHUHVSRQVH

354 Energy and protein metabolism and nutrition in sustainable animal production
Priority of the immune system for nutrients

The transport of the essential nutrients across the cell membrane has been examined in many cell
types, including leukocytes. For example, the essential amino acids lysine and arginine are transported
by the cationic amino acid transporter (CAT), of which there are several isoforms and each isoform
KDVGLIIHUHQWWUDQVSRUWNLQHWLFV&KLFNHQPXVFOHVH[SUHVVUHODWLYHO\ORZOHYHOVRIWKHKLJKDI¿QLW\
CAT isoform, whereas bursal tissue expresses high levels (Humphrey et al. 7KLV
GLIIHUHQFHLQFUHDVHVGUDPDWLFDOO\GXULQJDO\VLQHGH¿FLHQF\FDXVHGE\IHHGLQJDQLQDGHTXDWHGLHWRU
E\UHVWULFWLQJIHHGLQWDNH7KHGLVSDULW\LQH[SUHVVLRQRIKLJKDI¿QLW\WUDQVSRUWHUVDOORZVWKHEXUVD
WRRXWFRPSHWHPXVFOHIRUO\VLQHGXULQJSHULRGVRIGLHWDU\O\VLQHGH¿FLHQF\DQGPDLQWDLQQRUPDO
numbers of differentiating bursa cells even when muscle growth is severely impaired. Interestingly
the thymus has a priority for lysine that is lower than skeletal muscle and this tissue fails to maintain
populations of developing thymocytes during periods of lysine restriction. Thus, T-lymphocyte
PHGLDWHLPPXQLW\EXWQRWDQWLERG\UHVSRQVHVDUHLPSDLUHGE\PDUJLQDOO\VLQHGH¿FLHQFLHV

Changes in the amount and type of glucose transporter expression is coordinated to allow chicken
lymphocytes to develop and respond appropriately as neonatal chicks transition from the lipid energy
dominant environment of the egg to the carbohydrate energy supplied by the diet (Rudrappa and
Humphrey, 2007). The energy consumption of a naïve lymphocyte is relatively small and energy is
acquired from the breakdown of glucose, lipids and amino acids (Fox et al., 2005). After antigenic
VWLPXODWLRQJOXFRVHFRQVXPSWLRQLQFUHDVHVIROGZLWKLQWKH¿UVWKRXUZKLFKLVIDFLOLWDWHGE\
increases in glucose transporters (Greiner et al., 1994; Humphrey and Rudrappa, 2008). The
VLJQL¿FDQWLQFUHDVHLQJOXFRVHXSWDNHLVWRQRWRQO\WRSURYLGHHQRXJKHQHUJ\IRUELRFKHPLFDO
reactions, but to generate cellular components for the proliferating cell. Glucose is diverted to
precursors such as acetyl CoA for fatty acid synthesis, glycolytic intermediates for non-essential
amino acids production, and ribose for nucleotides synthesis (Van der Heiden et al., 2009). In
FKLFNHQVERWK7DQG%O\PSKRF\WHVH[SUHVVKLJKDI¿QLW\UHFHSWRUVWKDWDOORZWKHPWRFRPSHWH
successfully for glucose when concentrations are low (Humphrey and Rudrappa, 2008; Rudrappa
and Humphrey, 2007). This, observation helps to explain why moderate dietary energy restriction
does not impinge on immune responses.

Another example of nutrient prioritization occurs in chicken macrophages, which express very
high levels of metallothionein when activated. This permits macrophages to sequester high levels
of zinc (Laurin et al., 1990) to supply their anabolic needs and endows them with a higher priority
for limiting levels of this trace nutrient relative to many other cell types. This is important because
the need for zinc markedly increases when cells are activated and become highly anabolic. In
summary, it appears that evolution has provisioned some leukocyte lineages with a high priority
IRUPDQ\FULWLFDOQXWULHQWVOLNHO\WKRVHWKDWZHUHFRPPRQO\GH¿FLHQWLQQDWXUDOGLHWVDVDQLPDOV
evolved. Nutrients that appear to fall into this category include zinc (Mohanna and Nys, 1999), most
of the amino acids (Kidd, 2004) except possibly methionine (Cook, 1991; Rama Rao et al., 2003)
and energy supplying compounds such as glucose (Humphrey et al., 2004). Conversely, for some
nutrients there are not mechanisms that permit leukocytes to establish priority over other cell types
DQGWKH\DUHDIIHFWHGGXULQJGH¿FLHQFLHVLQDPDQQHUVLPLODUWRDOORWKHUWLVVXHV HJFRSSHU.RK
et al. )RUVRPHQXWULHQWVWKHUHODWLYHSULRULW\DQGUHTXLUHPHQWIRUGHYHORSPHQWRUUHVSRQVH
of the immune system has not been examined and compared to the requirement for other processes
such as growth or reproduction. This is particularly true for many vitamins and trace minerals and
¿OOLQJWKLVLQIRUPDWLRQJDSVKRXOGEHDKLJKSULRULW\

Appropriation of nutrients when the immune system responds

7KHSURLQÀDPPDWRU\F\WRNLQHVUHOHDVHGE\SKDJRF\WHVLQUHVSRQVHWRSDWKRJHQVLQGXFHPHWDEROLF
changes, including increased protein degradation and insulin resistance, which divert nutrients from

Energy and protein metabolism and nutrition in sustainable animal production 355
skeletal muscle and other tissues. These appropriated nutrients become available for the increased
demands of the liver and responding leukocytes (Sirimongkolkasem, 2007). These same cytokines
also mediate decreased food consumption. An important question is if the nutrients appropriated
GXULQJWKHDFXWHSKDVHUHVSRQVHMX[WDSRVLWLRQHGZLWKGLPLQLVKHGIRRGLQWDNHDUHVXI¿FLHQWLQ
amount and balance for the immune response. Research in rodent models and humans suggest a
mismatch between supply and demand (Obled et al., 2002). It has been suggested that the main
contributor to the increased degradation of skeletal muscle is due to the mismatch in amino acid
SUR¿OHVEHWZHHQSURWHFWLYHSURWHLQV LHDFXWHSKDVHSURWHLQV DQGVNHOHWDOPXVFOH 5HHGVDQG-DKRRU
2001). Furthermore, amino acids are not only used for the production of acute phase proteins, but
for increasing liver mass, synthesizing immunoglobulins, and the clonal expansion of leukocytes.
Recent work indicate that cysteine is the most limiting amino acid during the acute phase response
in chickens (Iseri and Klasing, 2013; Sirimongkolkasem, 2007) and also in rats (Breuille et al.
Breuille and Obled, 2000). This is due to a mismatch between muscle cysteine release and hepatic
demand for production of acute phase proteins and glutathione.

Implications for nutritionists

In general, developing T lymphocytes are the most susceptible leukocyte population to nutrient
GH¿FLHQFLHVGXHWRWKHLULQDELOLW\WRFRPSHWHZLWKRWKHUWLVVXHV6HYHUHGH¿FLHQFLHVRIDPLQRDFLGV
like lysine (Humphrey et al. RUEUDQFKHGFKDLQDPLQRDFLGV .RQDVKL et al., 2000) that
markedly impair growth of chicks cause involution of the thymus but not the bursa. The thymus
LVPRUHVHQVLWLYHWRGH¿FLHQFLHVRIWKHEUDQFKHGFKDLQDPLQRDFLGVWKDQDQ\RWKHUJURXSRIDPLQR
DFLGV+RZHYHUWKHGH¿FLHQF\PXVWEHVHYHUHDQGWKHWK\PXVDQGSHULSKHUDO7FHOOSRSXODWLRQVDUH
QRWVHQVLWLYHWRPDUJLQDOGH¿FLHQFLHVRIWKHEUDQFKHGFKDLQDPLQRDFLGV .LGG.RQDVKL et
al., 2000). With diminished lymphocyte-mediated immunity, exaggerated responses by the innate
LPPXQHV\VWHPRIWHQRFFXUGXULQJLQIHFWLRQZKLFKUHVXOWLQUREXVWLQÀDPPDWRU\UHVSRQVHVDQG
accompanying immunopathology.

Feed restriction is commonly used in the management of animals, especially in breeding females.
$ZLGHYDULHW\RIVWXGLHVKDYHH[DPLQHGWKHLQÀXHQFHRIIHHGUHVWULFWLRQRQLPPXQRFRPSHWHQFH
(Hangalapura et al., 2005; Nir et al.3UDKDUDM et al. DQGWKHJHQHUDOFRQFOXVLRQLVWKDW
mild to moderate feed restriction enhances many measures of immunity. Very severe feed restriction
impairs cellular immunity but may enhance innate (Hangalapura et al., 2005) or antibody responses
(Khajavi et al., 2003). The endocrine and metabolite changes that accompany feed restriction likely
mediate many of the effects on immunocompetence in chickens (Nir et al. ,QSHULSDUWXPGDLU\
cows in negative energy balance, derangements in metabolites accompanied by endocrine changes
impair immunity and increase susceptibility to infections such as mastitis (Ingvartsen and Moyes,
2013). Preventing these metabolic changes is a key management strategy for optimizing immunity.

Immunoregulatory nutrients
A variety of nutrients modulate the immune system by direct actions on regulatory mechanisms
of leukocytes (Ballou et al.*RII/LOOHKRMDQG/HH/LSSROLV:DOODFH et
al., 2010). Required nutrients with indisputable immunoregulatory actions in rodents and livestock
include the long-chain polyunsaturated fatty acids (PUFA) and vitamins A, D, and E. Additionally,
some nutrients that are not normally considered as being dietary essential also modulate immunity,
including carotenoids, phytonutrients (e.g. conjugated linoleic acids, curcumin, capsicum, genistein,
essential oils, etc) and vitamin C. In general those nutrients that are not structural components or
co-factors for enzymes are most likely to be immunomodulatory. Unlike increases in nutrients from
GH¿FLHQWWRVXI¿FLHQWOHYHOVZKHUHPDQ\LQGLFHVRILPPXQRFRPSHWHQFHDUHHOHYDWHGVXSSOHPHQWDWLRQ
of immunomodulatory nutrients causes some components of immunity to be elevated and others to
be diminished; in other words the type and intensities of responses have been changed.

356 Energy and protein metabolism and nutrition in sustainable animal production
0DQ\LPPXQRPRGXODWRU\QXWULHQWVLQÀXHQFHWKHEDODQFHRIF\WRNLQHVDQGHLFRVDQRLGVUHOHDVHGE\
UHJXODWRU\FHOOV)RUH[DPSOH38)$VRIWKHQVHULHVHQKDQFHWKHUHOHDVHRIOHVVLQÀDPPDWRU\
HLFRVDQRLGVFRPSDUHGWRQIDWW\DFLGV )ULWVFKH 6HYHUDOQXWULHQWVFKDQJHWKHDFWLYLW\RI
QXFOHDUIDFWRUNDSSD% 1)ț% DQGFRQVHTXHQWO\PRGXODWHF\WRNLQHH[SUHVVLRQ,QURGHQWVDQG
FKLFNVQXWULHQWVWKDWGHFUHDVH1)ț%DFWLYLW\DQGGDPSHQWKHLQÀDPPDWRU\UHVSRQVHLQFOXGHD
YDULHW\RIQ38)$VDQWLR[LGDQWVYLWDPLQ$DQGOXWHLQ,QDGGLWLRQWRGDPSHQLQJWKHLQÀDPPDWRU\
UHVSRQVHE\GHFUHDVLQJWKHUHOHDVHRISURLQÀDPPDWRU\F\WRNLQHVWKHVHQXWULHQWVRIWHQVKLIW
lymphocyte responses from T-cytotoxic (Th1, cell-mediated) towards antibody responses (Th2).
This polarization of adaptive immunity has implications for the susceptibility of experimental
DQLPDOVWRDXWKHQWLFLQIHFWLRQV )ULWVFKH.ODVLQJDQG/HVKFKLQVN\ %HFDXVHRIWKHLU
LPPXQRPRGXODWRU\HIIHFWVRQWKH7K7KEDODQFHWKHEHQH¿WRIWKHVHQXWULHQWVGHSHQGVRQWKH
types of pathogens to which animals are exposed. A diet that modulates the immune system towards a
7KUHVSRQVHPD\EHEHQH¿FLDOZLWKVRPHSDWKRJHQVEXWEHGHOHWHULRXVZLWKRWKHUV WKRVHSURWHFWHG
by Th1 response).

,QWKHIHZVWXGLHVWKDWKDYHORRNHGWKHVSHFL¿FHIIHFWVRILPPXQRPRGXODWRU\QXWULHQWVDUHKLJKO\
dose dependent. This has been clearly shown for PUFAs, vitamin E and vitamin A, where the
immunomodulatory actions follow a bell-shaped curve and high levels may not be as useful as
PRGHUDWHOHYHOV /HVKFKLQVN\DQG.ODVLQJ/LQDQG&KDQJ6NODQ et al., 1994).

)XUWKHUFRPSOLFDWLQJWKHSLFWXUHWKHQHWLPPXQRPRGXODWRU\LQÀXHQFHRIDGLHWLVQRWDVLPSOHVXPRI
the actions of individual nutrients because there are robust and sometimes unpredictable interactions
EHWZHHQGLIIHUHQWLPPXQRPRGXODWRU\QXWULHQWV)RUH[DPSOHWKHDQWLLQÀDPPDWRU\HIIHFWRIGLHWDU\
lutein on chicken macrophages depends on the amount of PUFAs in the diet (Selvaraj and Klasing,
6HOYDUDM et al. 7KHFRQYHUVHLVDOVRWUXHLQWKDWWKHDQWLLQÀDPPDWRU\HIIHFWVRIQ
PUFAs are dependent upon the amount of lutein in the diet. This interaction is mediated by nuclear
hormone receptors that respond to these two nutrients (RXR for lutein and PPAR for PUFAs), which
LQWXUQDIIHFWWKHH[SUHVVLRQRI1)ț%6LPLODUO\GLIIHUHQWGLHWDU\IDWW\DFLGVRIWKHQDQGWKHQ
series have separate and interactive effects when supplemented to diets of chicks (Parmentier et al.,
2002; Sijben et al., 2001), as does vitamin E and selenium (Singh et al.6ZDLQ et al., 2000).

Implications for nutritionists

%RWKH[SHULPHQWDODQGFOLQLFDOUHVXOWVFOHDUO\LQGLFDWHWKDWWKHVSHFL¿FLPPXQRPRGXODWRU\DFWLRQV
of a nutrient and its interactions with other dietary nutrients must be understood before application
WRDQLPDOSRSXODWLRQVEHFDXVHLWVHI¿FDF\LVGHSHQGHQWRQWKHPLOLHXRILQIHFWLRXVDQGPHWDEROLF
GLVHDVHVWKDWDUHSUHVHQW,QVLWXDWLRQVZKHUHDVSHFL¿FGLVHDVHGRPLQDWHVWKHSURGXFWLRQORVVHVDQG
where it is clearly known what type of immune response optimally protects against that disease,
supplementation of a nutrient that modulates the immune system towards that optimal response is
indicated. At this time there are few examples that meet these criteria.

References
Athanasiadou, S. and Huntley, J. F. 2008. Emerging technologies and their applications in interactions between nutrition
and immunity to gastrointestinal parasites in sheep. Parasite immunology, 30, 101-111.
Ballou, M. A., Cruz, G. D., Pittroff, W., Keisler, D. H. and DePeters, E. J. 2008. Modifying the acute phase response of
-HUVH\FDOYHVE\VXSSOHPHQWLQJPLONUHSODFHUZLWKRPHJDIDWW\DFLGVIURP¿VKRLO-'DLU\6FL
%UHXLOOH'%HFKHUHDX)%XI¿HUH&'HQLV33RX\HW&DQG2EOHG&%HQH¿FLDOHIIHFWRIDPLQRDFLG
VXSSOHPHQWDWLRQHVSHFLDOO\F\VWHLQHRQERG\QLWURJHQHFRQRP\LQVHSWLFUDWV&OLQ1XWU
Breuille, D. and Obled, C. 2000. Cysteine and glutathione in catabolic states. Nestle Nutrition Workshop Series, 3,
173-191.
&RRN0(1XWULWLRQDQGWKHLPPXQHUHVSRQVHRIWKHGRPHVWLFIRZO&ULW5HY3RXOW%LRO

Energy and protein metabolism and nutrition in sustainable animal production 357
Fairbairn, L., Kapetanovic, R., Sester, D. P. and Hume, D. A. 2011. The mononuclear phagocyte system of the pig as
a model for understanding human innate immunity and disease. J Luek Biol, 89, 855-871.
Fox, C. J., Hammerman, P. S. and Thompson, C. B. 2005. Fuel feeds function: energy metabolism and the T-cell
response. Nat Rev Immunol, 5, 844-852.
)ULWVFKH.)DWW\DFLGVDVPRGXODWRUVRIWKHLPPXQHUHVSRQVH$Q5HY1XWU
*RII-30DMRUDGYDQFHVLQRXUXQGHUVWDQGLQJRIQXWULWLRQDOLQÀXHQFHVRQERYLQHKHDOWK-'DLU\6FL
1292-1301.
Greiner, E. F., Guppy, M. and Brand, K. 1994. Glucose is essential for proliferation and the glycolytic enzyme induction
WKDWSURYRNHVDWUDQVLWLRQWRJO\FRO\WLFHQHUJ\SURGXFWLRQ-%LRO&KHP
Hangalapura, B. N., Nieuwland, M. G., De Vries Reilingh, G., Buyse, J., Van Den Brand, H., Kemp, B. and Parmentier,
H. K. 2005. Severe feed restriction enhances innate immunity but suppresses cellular immunity in chicken lines
divergently selected for antibody responses. Poult Sci, 84, 1520-1529.
Humphrey, B. D. and Rudrappa, S. G. 2008. Increased glucose availability activates chicken thymocyte metabolism
and survival. J. Nutr., 138, 1153-1157.
Humphrey, B. D., Stephensen, C. B., Calvert, C. C. and Klasing, K. C. 2004. Glucose and cationic amino acid
transporter expression in growing chickens (Gallus gallus domesticus). Comp Biochem Physiol A Mol Integr
Physiol, 138, 515-525.
+XPSKUH\%'6WHSKHQVHQ&%&DOYHUW&&DQG.ODVLQJ.&/\VLQHGH¿FLHQF\DQGIHHGUHVWULFWLRQ
independently alter cationic amino acid transporter expression in chickens (Gallus gallus domesticus). Comparative
biochemistry and physiology. Part A, Molecular & integrative physiology, 143, 218-227.
Ingvartsen, K. L. and Moyes, K. 2013. Nutrition, immune function and health of dairy cattle. Animal, 7 Suppl 1, 112-122.
Iseri, V. J. and Klasing, K. C. 2013. Dynamics of the systemic components of the chicken (Gallus gallus domesticus)
immune system following activation by Escherichia coli; implications for the costs of immunity. Dev. Comp.
Immuno., In Press.
Khajavi, M., Rahimi, S., Hassan, Z. M., Kamali, M. A. and Mousavi, T. 2003. Effect of feed restriction early in life
on humoral and cellular immunity of two commercial broiler strains under heat stress conditions. Br Poult Sci,
44, 490-497.
.LGG071XWULWLRQDOPRGXODWLRQRILPPXQHIXQFWLRQLQEURLOHUV3RXOW6FL
Klasing, K. C. 2007. Nutrition and the immune system. Br Poult Sci, 48, 525-537.
Klasing, K. C. and Leshchinsky, T. V. 1999. Interactions between nutrition and immunity: Lessons from animal
DJULFXOWXUH,Q0(*HUVKZLQ HG +DQGERRNRI1XWULWLRQDQG,PPXQRORJ\ SS 1HZ<RUN+XPDQD
Press.
.RK763HQJ5.DQG.ODVLQJ.&'LHWDU\FRSSHUOHYHODIIHFWVFRSSHUPHWDEROLVPGXULQJOLSRSRO\VDFFKDULGH
LQGXFHGLPPXQRORJLFDOVWUHVVLQFKLFNV3RXOW6FL
.RQDVKL67DNDKDVKL.DQG$NLED<(IIHFWVRIGLHWDU\HVVHQWLDODPLQRDFLGGH¿FLHQFLHVRQLPPXQRORJLFDO
YDULDEOHVLQEURLOHUFKLFNHQV%U-1XWU
Korver, D. R. 2012. Implications of changing immune function through nutrition in poultry. Anim. Feed Sci. Technol.,

Latshaw, J. D. 1991. Nutrition--mechanisms of immunosuppression. Vet Immunol Immunopathol, 30, 111-120.
Laurin, D. E., Barnes, D. M. and Klasing, K. C. 1990. Rates of metallothionein synthesis, degradation and accretion
LQDFKLFNHQPDFURSKDJHFHOOOLQH3URF6RF([S%LRO0HG
Leshchinsky, T. V. and Klasing, K. C. 2001. Relationship between the level of dietary vitamin E and the immune
response of broiler chickens. Poult Sci, 80, 1590-1599.
Lillehoj, H. S. and Lee, K. W. 2012. Immune modulation of innate immunity as alternatives-to-antibiotics strategies
WRPLWLJDWHWKHXVHRIGUXJVLQSRXOWU\SURGXFWLRQ3RXOW6FL
/LQ<)DQG&KDQJ6-(IIHFWRIGLHWDU\YLWDPLQ(RQJURZWKSHUIRUPDQFHDQGLPPXQHUHVSRQVHRIEUHHGHU
chickens. Asain-Aust. J. Anim. Sci., 19, 884-891.
/LSSROLV-',PPXQRORJLFDOVLJQDOLQJQHWZRUNVLQWHJUDWLQJWKHERG\¶VLPPXQHUHVSRQVH-$QLP6FL
(
Mohanna, C. and Nys, Y. 1999. Effect of dietary zinc content and sources on the growth, body zinc deposition and
retention, zinc excretion and immune response in chickens. Br Poult Sci, 40, 108-114.

358 Energy and protein metabolism and nutrition in sustainable animal production
1LU,1LWVDQ='XQQLQJWRQ($DQG6LHJHO3%$VSHFWVRIIRRGLQWDNHUHVWULFWLRQLQ\RXQJGRPHVWLFIRZO
0HWDEROLFDQGJHQHWLFFRQVLGHUDWLRQV:RUOG3RXOW6FL-
Obled, C., Papet, I. and Breuille, D. 2002. Metabolic bases of amino acid requirements in acute diseases. Curr Opin
Clin Nutr Metab Care, 5, 189-197.
Parmentier, H. K., Awati, A., Nieuwland, M. G., Schrama, J. W. and Sijben, J. W. 2002. Different sources of dietary
QSRO\XQVDWXUDWHGIDWW\DFLGVDQGWKHLUHIIHFWVRQDQWLERG\UHVSRQVHVLQFKLFNHQV%U3RXOW6FL
3UDKDUDM1.*URVV:%'XQQLQJWRQ($1LU,DQG6LHJHO3%,PPXQRUHVSRQVLYHQHVVRIIDVWJURZLQJ
FKLFNHQVDVLQÀXHQFHGE\IHHGLQJUHJLPHQ%U3RXOW6FL
Rama Rao, S. V., Praharaj, N. K., Ramasubba Reddy, V. and Panda, A. K. 2003. Interaction between genotype and
dietary concentrations of methionine for immune function in commercial broilers. Br Poult Sci, 44, 104-112.
Reeds, P. J. and Jahoor, F. 2001. The amino acid requirements of disease. Clin. Nutr., Supplement 1, 15-22.
Rudrappa, S. G. and Humphrey, B. D. 2007. Energy metabolism in developing chicken lymphocytes is altered during
the embryonic to posthatch transition. J. Nutr., 137, 427-432.
6HOYDUDM5.DQG.ODVLQJ.&/XWHLQDQGHLFRVDSHQWDHQRLFDFLGLQWHUDFWWRPRGLI\L126P51$OHYHOV
WKURXJKWKH33$5JDPPD5;5SDWKZD\LQFKLFNHQVDQG+'FHOOOLQHV-1XWU
Selvaraj, R. K., Shanmugasundaram, R. and Klasing, K. C. 2011. Effects of dietary lutein and PUFA on PPAR and
5;5LVRPHUH[SUHVVLRQLQFKLFNHQVGXULQJDQLQÀDPPDWRU\UHVSRQVH&RPS%LRFKHP3K\VLRO$0RO,QWHJU
Physiol, 157, 198-203.
Sijben, J. W., Nieuwland, M. G., Kemp, B., Parmentier, H. K. and Schrama, J. W. 2001. Interactions and antigen
GHSHQGHQFHRIGLHWDU\QDQGQSRO\XQVDWXUDWHGIDWW\DFLGVRQDQWLERG\UHVSRQVLYHQHVVLQJURZLQJOD\HUKHQV
Poult Sci, 80, 885-893.
6LQJK+6RGKL6DQG.DXU5(IIHFWVRIGLHWDU\VXSSOHPHQWVRIVHOHQLXPYLWDPLQ(RUFRPELQDWLRQVRIWKH
two on antibody responses of broilers. Br Poult Sci, 47, 714-719.
Sirimongkolkasem, P. 2007. Amino acid partitioning during the acute phase response. Ph.D Thesis, UC Davis, Davis, CA.
Sklan, D., Melamed, D. and Friedman, A. 1994. The effect of varying levels of dietary vitamin A on immune response
in the chick. Poult Sci, 73, 843-847.
6RUGLOOR/0&RQWUHUDV*$DQG$LWNHQ6/0HWDEROLFIDFWRUVDIIHFWLQJWKHLQÀDPPDWRU\UHVSRQVHRI
SHULSDUWXULHQWGDLU\FRZV$QLPDOKHDOWKUHVHDUFKUHYLHZV
Swain, B. K., Johri, T. S. and Majumdar, S. 2000. Effect of supplementation of vitamin E, selenium and their different
combinations on the performance and immune response of broilers. Br Poult Sci, 41, 287-292.
Vander Heiden, M. G., Cantley, L. C. and Thompson, C. B. 2009. Understanding the Warburg effect: the metabolic
requirements of cell proliferation. Science, 324, 1029-1033.
Wallace, R. J., Oleszek, W., Franze, C., Hahn, I., Baser, K. H. C., Mathe, A. and Reichmann, K. 2010. Dietary plant
ELRDFWLYHVIRUSRXOWU\KHDOWKDQGSURGXFWLYLW\%U3RXOW6FL
=HEHOL4DQG0HW]OHU=HEHOL%8,QWHUSOD\EHWZHHQUXPHQGLJHVWLYHGLVRUGHUVDQGGLHWLQGXFHGLQÀDPPDWLRQ
in dairy cattle. Res. Vet. Sci., 93, 1099-1108.

Energy and protein metabolism and nutrition in sustainable animal production 359
0LONIDWW\DFLGSUR¿OHLQGDLU\FRZVGXULQJDQHJDWLYHHQHUJ\EDODQFHLQ
early lactation and feed-restriction in mid-lactation
J.J. Gross1, H.A. van Dorland1, R.M. Bruckmaier1 and F.J. Schwarz2
19HWHULQDU\3K\VLRORJ\9HWVXLVVH)DFXOW\8QLYHUVLW\RI%HUQ%UHPJDUWHQVWUDVVHD%HUQ
Switzerland; [email protected]
27HFKQLVFKH8QLYHUVLWlW0QFKHQ&KDLURI$QLPDO1XWULWLRQ/LHVHO%HFNPDQQ6WUDVVH
Freising-Weihenstephan, Germany

Introduction
Besides diet, mainly lactational stage along with energy balance in dairy cows have an impact on fatty
DFLG )$ SUR¿OHLQFRZCVPLON'XULQJWKH¿UVWFRXSOHRIZHHNVDIWHUSDUWXULWLRQWKHRFFXUUHQFHRI
DQHJDWLYHHQHUJ\EDODQFH 1(% LVDFRPPRQSKHQRPHQRQREVHUYHGLQGDLU\FRZV7KHGH¿FLHQF\
of nutrients and energy is compensated by mobilization of body reserves, predominantly adipose
WLVVXHDVVRFLDWHGZLWKWKHUHOHDVHRI)$'XULQJLQVXI¿FLHQWVXSSO\DQGTXDOLW\RIIHHGD1(%PD\
also occur later in lactation.

7KHIRFXVRIWKHSUHVHQWVWXG\LVVHWRQWKHLQWHUDFWLRQVEHWZHHQHQHUJ\EDODQFHDQGPLON)$SUR¿OH
in dairy cows. Contrary to earlier studies, the present study investigates effects of both, the NEB in
early lactation and a deliberately induced NEB by feed-restriction at around 100 days in milk on
milk FA composition.

Material and methods

The animal trial and diets were described previously by Gross et al. (2011). According to their
energy balance (EB) up to wk 12 p.p. (period 1), 40 multiparous Holstein cows were allocated
equably to a control (C, n=20) or a restriction group (R, n=20, -30% of energy requirements) for 3
wks at 100 days in milk (period 2). Thereafter, R-cows were (re)fed similarly as C-cows for 8 wks
(period 3). EB of each cow was calculated from daily feed intake, maintenance requirement and
milk yield. Blood samples were taken once a week and analyzed for glucose, NEFA and BHBA.
0LONVDPSOHVZHUHDQDO\]HGIRU)$FRPSRVLWLRQE\JDVFKURPDWRJUDSK\LQZNDQGSS
RISHULRGZHHNO\GXULQJSHULRGDQGLQZNDQGRISHULRG&KDQJHVLQPLON)$SUR¿OH
over time during lactation and feed-restriction with subsequent realimentation were evaluated by
DPL[HGPRGHOLQ6$6ZLWKJURXSZNDQGWKHJURXS[ZNLQWHUDFWLRQDV¿[HGHIIHFW'LIIHUHQFHV
over time and within groups during feed-restriction (period 2) and realimentation (period 3) were
GHWHFWHGE\WKH%RQIHUURQLCVWWHVW P<0.05).

Results and discussion

Lactational stage clearly affected daily milk yield and milk composition. Daily milk yield started
DW“NJGLQZNSSSHDNHGLQZNSS “NJG DQGGHFUHDVHGWR“NJG
in wk 12 p.p. During period 2 restricted cows (27.4 kg/d) had a lower milk yield than control cows
(30.5 kg/d; P<0.05). Milk yield of restricted cows increased already in wk 1 of realimentation period
IURPWRNJGDQGZDVWKHUHDIWHUHYHQEHWZHHQDQGNJGDERYHWKHFRQWUROJURXS
(wks 2-8 in phase 3; P>0.05).

Energy balance in dairy cows was most negative in wk 1 p.p. and improved with increasing feed
LQWDNHEXWZDVVWLOOQHJDWLYHLQZNSS,QRUGHUWRFRPSHQVDWHIRUWKHLQVXI¿FLHQWHQHUJ\LQWDNH
p.p., considerable amounts of body fat were mobilized resulting in elevated plasma concentrations
of NEFA and BHBA. During the NEB in early lactation, plasma glucose concentration in the present
study showed a nadir of 3.30±0.04 mmol/l in wk 2 p.p., whereas plasma concentrations were highest

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 361
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_125, © Wageningen Academic Publishers 2013
IRU1()$LQZNSS “PPROO DQGIRU%+%$LQZNSS “PPROO $IWHUWKH
observed peak of lactation, energy requirements could be met by consumed feed resulting in a positive
energy balance. With progressing lactation and improvement of energy balance through increasing
IHHGLQWDNHHVSHFLDOO\PLON)$SUR¿OHPDUNHGO\FKDQJHG0RVWFKDQJHVLQPLON)$SUR¿OHWRRNSODFH
GXULQJWKHREVHUYHG1(%IURPZNWRSSZKLOH)$FRPSRVLWLRQZDVUHODWLYHO\FRQVWDQWEHWZHHQ
ZNWRSS)$XSWR&VKRZHGORZHVWSURSRUWLRQVLQZNSSWKDWLQFUHDVHGWRUHODWLYHO\
FRQVWDQWSURSRUWLRQVIURPZNSSRQZDUGV6DWXUDWHG)$ 6)$ HVSHFLDOO\&LQFUHDVHGIURP
wk 1 to 12 p.p., while monounsaturated FA (MUFA), mainly represented by C18:1,9c decreased
until wk 12 p.p. with improving energy balance. The proportion of polyunsaturated FA (PUFA) was
UHODWLYHO\FRQVWDQWIURPZNXSWRSS0LON)$SUR¿OHRI&FRZVZDVVWDEOHGXULQJWKHZKROH
WLPHRISHULRGDQG)RU5FRZVWKHSURSRUWLRQRIPRVW)$”& HJ&&&
&&& ZDVGHFUHDVHGGXULQJWKH1(%LQGXFHGE\IHHGUHVWULFWLRQFRPSDUHGWRWKH
respective initial values, whereas preformed FA, especially C17:1,9c, C18:0 and C18:1,9c arising
from body fat mobilization increased markedly during feed-restriction. These changes occurred
UDSLGO\ZLWKLQWKH¿UVWZNRISHULRGDQGGLVDSSHDUHGFRPSOHWHO\ZLWKLQRQHZNRIUHDOLPHQWDWLRQ
(4 d on average). During period 2, SFA decreased, while MUFA (especially C18:1,c9) increased for
R-cows compared to C-cows. PUFA were stable during period 2 and 3 in R-cows. Although less in
their extent, milk FA clearly showed a similar pattern during the NEB in period 1 and 2.

6KRUWDQGPHGLXPFKDLQ)$XSWR&LQFUHDVHGZLWKWKHGHFUHDVLQJ1(%SSZKLOHORQJFKDLQ
FA, especially C18:1,9c decreased as mobilization of body fat reserves declined. The responses of
)$SUR¿OHVRIFRZVCPLONGXHWRD1(%DWWZRODFWDWLRQDOVWDJHVLQWKHSUHVHQWVWXG\±WKH1(%LQ
early lactation and the deliberately induced NEB by feed-restriction – was similarly directed. Despite
WKHPDLQWHQDQFHRIDKLJK1(%GXULQJWKHIHHGUHVWULFWLRQSHULRGFKDQJHVLQPLON)$SUR¿OHZHUH
less pronounced compared to changes during the NEB in early lactation and tended to adjust to
WKHLQLWLDOFRPSRVLWLRQ+RZHYHUPLON)$SUR¿OHFKDQJHGZLWKLQDIHZGD\VDIWHULQLWLDWLRQRIWKH
GHOLEHUDWHO\LQGXFHG1(%DQGVKRZHGQRPRUHGLIIHUHQFHVZLWKLQWKH¿UVWZNRIUHDOLPHQWDWLRQ
compared to control cows. For the dietary composition and feeding regimen in the present study,
the close relationship with energy balance makes changes in C18:1,9c as well as in groups of FA
(SFA, MUFA, de novo synthesized and preformed FA) suitable indicators of the energy balance in
dairy cows.

References
*URVV-+$YDQ'RUODQG50%UXFNPDLHUDQG)-6FKZDU]3HUIRUPDQFHDQGPHWDEROLFSUR¿OHRIGDLU\
cows during a lactational and deliberately induced negative energy balance by feed restriction with subsequent
realimentation. J. Dairy Sci., 94, 1820-1830.

362 Energy and protein metabolism and nutrition in sustainable animal production
The effect of long-term butyrate supplementation to high producing
periparturient dairy cows
L. Vandaele1, J. Van Eys2, B. D’Heer2, B. Ampe1 and S. De Campeneere1
1,/92$QLPDO6FLHQFHV8QLW6FKHOGHZHJ0HOOH%HOJLXP[email protected]
21XWULDG,QWHUQDWLRQDO19+RRJYHOG'HQGHUPRQGH%HOJLXP

Introduction
Black et al.  LQGLFDWHGWKDWLQFUHDVLQJUXPLQDOFRQFHQWUDWLRQVRIEXW\UDWHPLJKWEHEHQH¿FLDOWR
dairy cows in early lactation, by preventing the oxidation of pyruvate and by increasing the pyruvate
to oxaloacetate conversion. Secondly, extra-mammary tissues can spare glucose by using the increased
OHYHOVRIEHWDK\GUR[\EXW\UDWHIURPEXW\UDWH +ROWHQLXVDQG+ROWHQLXV %RWKPLJKWOHDGWR
better performance in transition cows (DeFrain et al., 2004). This study aimed to explore the effect
of daily butyrate supplementation around calving on milk production and composition, and blood
glucose level in dairy cattle.

Material and methods

Sixteen Holstein-Friesian multiparous cows were divided in two homogenous groups. Cows in
WKHEXW\UDWHJURXS %87 UHFHLYHGGDLO\JVRGLXPEXW\UDWH ZHSUHFDOYLQJXQWLOZHSRVW
calving). Diets of control (CTRL) and BUT were kept iso-nitrogenous and iso-energetic through
the inclusion of rumen inert fat into the CTRL diet. Roughage (offered ad libitum) was the same
IRUERWKJURXSVPDL]HVLODJHDQGVWUDZGXULQJWKHIDURIISHULRG WKH¿UVWZHHNRIWKHWULDO DQG
pre-wilted grass silage, maize silage and pressed beet pulp during close-up and after calving. Cows
were offered a dry cow concentrate before calving and increasing amounts of a protein corrector and
FRQFHQWUDWHVDIWHUFDOYLQJ IURPNJXSWRNJLQGD\V 'DLO\PLONSURGXFWLRQDQGZHHNO\
bodyweight were recorded. Milk and blood samples were taken weekly. Roughages and concentrates
were analyzed to calculate individual energy and protein intake (Dutch protein evaluation system).
Data on dietary composition were compared using student’s T-test. Post calving data were compared
with a longitudinal model with correction for repeated measures within cow (SAS 9.3 for Windows).

Results and discussion

The pre calving CTRL diet contained higher NDF (only far off) and higher NEL than the BUT diet
(far off and close-up), but both diets were similar for DPI and RDPB (Table 1). During the post
calving period the dietary composition was similar for CTRL and BUT (Table 1). Daily intake of
NEl (149±5 and 147±5 MJ/d), DPI (2,207±45 and 2,178±55 g/d), RPDB (-128±75 and -139±102
g/d) and DM (20.1±0.3 and 20.4±0.3 kg/day) did not differ between groups post-calving (Table 2).

7KHPHDQPLON\LHOGRI%87FRZVZDVVLJQL¿FDQWO\ORZHUWKDQRI&75/FRZVEXWIDWDQGSURWHLQ
FRQWHQWZHUHVLJQL¿FDQWO\KLJKHULQ%87JURXS 7DEOH 7KHIDFWWKDW%87FRZVVHHPHGWREH
more sensitive to metabolic problems post calving (two cows with displaced abomasum in BUT and
none in CTRL) might partially explain the lower milk yield in the BUT group.

,QWHUHVWLQJO\EORRGJOXFRVHOHYHOVDQGKDIWHUIHHGLQJZHUHVLJQL¿FDQWO\ORZHUIRU%87WKHQIRU
CTRL. This observation might be related to higher insulin levels as direct response to butyrate as
shown by Mineo et al. (1990) in sheep. Higher insulin levels might depress glucose levels despite
the higher butyric acid concentration.

In conclusion, long term supplementation with butyrate leads to a lower milk yield and lower post
calving blood glucose levels. However this did not lead to lower daily fat nor protein production.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 363
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_126, © Wageningen Academic Publishers 2013
7DEOH'LHWDU\FRPSRVLWLRQLQ&75/DQG%87LQJNJ'0 PHDQ“6(0 

Far off Close-up Postpartum

CTRL BUT CTRL BUT CTRL BUT

NDF 375±2.2 “* “ 312±4.9 298±3.4 290±4.0

Starch 259±1.8 252±1.9 “ 211±3.9 “ 203±8.7
NEL (MJ/kg) “ “ 7.2±0.03 7.0±0.01* 7.4±0.04 7.3±0.04
DPI 53±0.4 52±0.3 74±0.7 72±0.9 110±1.4 109±1.5
RDPB 0±2.5 “ 14±2.7 15±3.2 “ -7±5.2

NEl = net energy lactation; DPI = true protein digested in the small intestine; RDPB = rumen degradable protein balance.
5HVXOWVDUHVLJQL¿FDQWO\GLIIHUHQWEHWZHHQJURXSVZLWKLQWKHVDPHSHULRG

7DEOH/HDVWVTXDUHPHDQV “6(0 RISRVWFDOYLQJ'0LQWDNHDQGSHUIRUPDQFHVLQERWKWUHDWPHQW

groups, including P-value.

CTRL BUT P-value

Milk yield (kg/day) “ 32.9±0.70 <0.05

Fat (%) 4.90±0.077 5.27±0.087 <0.01
Protein (%) 3.30±0.029 3.50±0.033 <0.01
Lactose (%) “ “ <0.05
Fat prod. (kg/day) “ “ 
Protein prod. (kg/day) “ “ 0.50
Glucose 2 h (mg/dl) 43±1.5 35±1.7 <0.01
Glucose 4 h (mg/dl) “ “ <0.01

References
%ODFN$/-/XLFN)0ROOHU56$QDQG3\UXYDWHDQGSURSLRQDWHPHWDEROLVPLQODFWDWLQJFRZV(IIHFWRI
butyrate on pyruvate metabolism. J. Biol. Chem. 241, 5233-5237.
DeFrain J.M., A.R. Hippen, K.F. Kalscheur, D.J. Schingoethe, 2004. Feeding lactose increases ruminal butyrate and
SODVPDEHWDK\GUR[\EXW\UDWHLQODFWDWLQJGDLU\FRZV-'DLU\6FL
+ROWHQLXV3DQG.+ROWHQLXV1HZDVSHFWVRINHWRQHERGLHVLQHQHUJ\PHWDEROLVPRIGDLU\FRZV$UHYLHZ
Zbl. Vet. Med. A. 43, 579-587.
Mineo H., M. Kanai, S. Kato, J.I. Ushijima, 1990. Effects of intravenous injection of butyrate, valerate and their isomers
RQHQGRFULQHSDQFUHDWLFUHVSRQVHVLQFRQVFLRXVVKHHS&RPS%LRFKHP3K\VLRO

364 Energy and protein metabolism and nutrition in sustainable animal production
Peripartal energy balance and peripheral blood mononuclear cell
activation in normal and high mobilizing dairy cows
A. Schwarm1,2, T. Viergutz, B. Kuhla1, H.M. Hammon1 and M. Schweigel-Röntgen1
1Institute of Nutritional Physiology „Oskar Kellner’, Leibniz Institute for Farm Animal Biology
)%1 'XPPHUVWRUI*HUPDQ\[email protected]
2Animal Nutrition Group, Institute of Agricultural Sciences, Swiss Federal Institute of Technology
(7+ =XULFK6ZLW]HUODQG
,QVWLWXWHRI5HSURGXFWLYH%LRORJ\/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 'XPPHUVWRUI
Germany

Introduction
In mammals, the time of pregnancy and lactation is highly energy demanding and shifts the priority
of energy partitioning from the immune system and body reserves towards the conceptus and the
mammary gland (Nelson et al., 2002). The resulting peripartal breakdown of (acquired) immunity has
been determined most of all by diminished blood leukocyte DNA synthesis in response to mitogens
(e.g. humans: Redwine et al., 2001, cow: reviewed by Mallard et al., 1998). The cell cycle phase
preceding the DNA synthetic (S) phase, i.e. immune cell activation (G1 phase), has received little
attention, but the few available data suggest that leukocyte activation may also be impaired by the
limited energy/nutrient availability.

Dairy cows with a high liver fat content postpartum, i.e. mobilizing body reserves to an above
average extent, have a more severe negative energy balance (EB) and a higher risk for metabolic
and infectious diseases than cows with a normal liver fat content.

7KHUHIRUHWKHDLPRIWKLVVWXG\ZDVWR¿QGRXWZKHWKHUWKHHQHUJ\FRQVXPLQJSURFHVVRISHULSKHUDO
blood mononuclear cells (PBMC) activation – in terms of oxygen consumed over basal levels after
in vitro stimulation – is differentially altered by EB in normal and high mobilizing cows.

Material and methods

Twelve multiparous high-yielding Holstein cows were retrospectively grouped according to normal
1“RIGU\PDWWHU'0Q PHDQ“6' DQGKLJK +“RI'0Q  OLYHUIDWFRQWHQW
(calculated from C, N glycogen and glucose content) at day 18 postpartum. Oxygen consumption rate
(OCR, as a measure of ATP production) was determined in phytohaemagglutinin (PHA)-stimulated
3%0&DWZHHNDQGUHODWLYHWRSDUWXULWLRQ2&5RI3%0&ZDVDVVHVVHGÀXRURPHWULFDOO\
KDIWHUWUHDWPHQWZLWK3+$ —JPO RUZLWK530,PHGLXP FRQWURO  6FKZDUPet al. 2013).
The activation index was calculated as the PHA-induced 24 h-increase of OCR above baseline. In
addition, we determined cellular lactate production, DNA+RNA synthesis and cell size.

The animals’ EB was estimated antepartum from metabolizable energy intake and postpartum from
netto energy intake for lactation and energy-corrected milk yield. Furthermore, we assessed body
weight, body condition score, backfat thickness, and selected plasma parameters, e.g. glucose, insulin,
QRQHVWHUL¿HGIDWW\DFLGDQGȕK\GUR[EXW\ULFDFLGFRQFHQWUDWLRQ

The data were analysed by Student´s t-test, Mann-Whitney-U-test, one-way repeated measures
DQDO\VLVRIYDULDQFH 50$129$ DQG6SHDUPDQFRUUHODWLRQFRHI¿FLHQW 6&& XVLQJ6\VWDW
(UNUDWK*HUPDQ\ 7KHVLJQL¿FDQFHOHYHOZDVVHWWRĮ DQG3YDOXHVEHWZHHQDQG
were considered as trends.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 365
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_127, © Wageningen Academic Publishers 2013
Results and discussion
H cows started antepartum with a higher (week -5, t-test, 3 0.020) backfat thickness than N cows
but had a similar (week -5, U-test, 3  (%Postpartum EB was more negative (t-test, 3 0.001)
in H than N cows at its nadir in the second week in milk, when the energy-corrected milk yield had
its maximum.

Basal OCR of PBMC did not change during the studied period of the reproduction cycle [N cows:
1.2±0.4 nmol/min/(107FHOOV Q ) 3 0.791; H cows: 1.1±0.4 nmol/min/(107 cells), n=5-
) 3 0.159], which is in accordance to previous results (Schwarm et al., 2013). However,
peripartal PBMC activation was differentially altered in N and H cows. Antepartum, activation
LQGLFHVRI3%0&WHQGHGWREHSRVLWLYHO\FRUUHODWHGZLWK(%LQ1FRZV Q 6&& 3 0.071),
EXWQRVXFKUHODWLRQVKLSZDVREVHUYHGLQ+FRZV Q 6&& 3 0.433). It is tempting to
speculate, that H cows have a superior nutritional and energetic state antepartum because less
energy is partitioned to immune function. In contrast, N cows directly channel the surplus energy
(of feeding the more energy dense close-up diet) towards immune function. Correspondingly,
high levels of plasma glucose coincided in time with high in vitro PBMC activation indices in N
FRZV Q 6&& 3  EXWQRWLQ+FRZV Q 6&& 3 0.480). The pattern is
reversed postpartum: a positive relationship between EB and activation indices of PBMC has been
observed only in H cows (n=11, SCC=0.73, 3 1FRZVQ 6&& 3 0.123). This
energy partitioning towards the immune system during early lactation may additionally deteriorate
the poor nutritional and energetic state of H cows, without reaching host defense.

We conclude that differences in disease susceptibility between N and H cows may be related to the
observed differences in energy partitioning towards immune function. Our results corroborate the
assumption that immune modulation during reproduction is a facultative (resource-driven) response
rather than an obligatory side effect of going through reproduction per se (French et al., 2007).

References
French, S.S., DeNardo, D.F., Moore, M.C., 2007. Trade-offs between the reproductive and immune systems: facultative
responses to resources or obligate responses to reproduction? Am. Nat. 170, 79-89.
Mallard, B.A., Dekkers, J.C., Ireland, M.J., Leslie, K.E., Sharif, S., Vankampen, C.L., Wagter, L., Wilkie, B.N., 1998.
$OWHUDWLRQLQLPPXQHUHVSRQVLYHQHVVGXULQJWKHSHULSDUWXPSHULRGDQGLWVUDPL¿FDWLRQRQGDLU\FRZDQGFDOI
health. J. Dairy Sci. 81, 585-595.
Nelson, R., Demas, G., Klein, S., Kriegsfeld, L., 2002. Energetics and immune function. In: Seasonal Patterns of Stress,
Immune Function, and Disease. Cambridge University Press, Cambridge, pp. 151-189.
Redwine, L.S., Altemus, M., Leong, Y.M., Carter, C.S., 2001. Lymphocyte responses to stress in postpartum women:
UHODWLRQVKLSWRYDJDOWRQH3V\FKRQHXURHQGRFULQRORJ\
Schwarm, A., Viergutz, T., Kuhla, B., Hammon, H. M. and M. Schweigel-Röntgen, 2013. Fuel feeds function: Energy
EDODQFHDQGERYLQHSHULSKHUDOEORRGPRQRQXFOHDUFHOODFWLYDWLRQ&RPS%LRFKHP3K\VLRO$

366 Energy and protein metabolism and nutrition in sustainable animal production
Impact of CFA and dietary protein supply on acute phase responses and
nitrogen retention in pigs
E. Kampman-van de Hoek1,2, P. Sakkas2, J.J.G.C. van den Borne2, W.J.J. Gerrits2, C.M.C. van der
Peet-Schwering1, H. van Beers and A.J.M. Jansman1
1'HSDUWPHQWRI$QLPDO1XWULWLRQ:DJHQLQJHQ85/LYHVWRFN5HVHDUFK32%R[$%
Lelystad, the Netherlands; [email protected]
2$QLPDO 1XWULWLRQ *URXS :DJHQLQJHQ 8QLYHUVLW\ 32 %R[  $+ :DJHQLQJHQ WKH
Netherlands
6'D7KH1HWKHUODQGV9HWHULQDU\0HGLFLQHV$XWKRULW\<DOHODDQ&08WUHFKWWKH
Netherlands.

Introduction
During immune system activation, an increased competition occurs between amino acids (AA) for
body protein deposition and for immune system functioning (Klasing and Johnstone, 1991; Sandberg
et al., 2007). The production of acute phase proteins (APP) has been suggested to increase the
demand for especially aromatic AA (Reeds et al., 1994). When muscle protein is mobilized to supply
AA for APP production, this leads to an imbalance in AA available for body protein deposition, as
the AA composition of APP differs largely from the composition of muscle protein (Reeds et al.,
1994). As a consequence, increased AA oxidation and N loss via urine occur. It is hypothesized that
the competition between AA increases when dietary protein supply is reduced. In addition, there is
LQFUHDVLQJHYLGHQFHWKDWWKHGLHWDU\SURWHLQRU$$VXSSO\FDQPRGXODWHWKHLQÀDPPDWRU\UHVSRQVH
during immune system activation (Grimble, 2001; Calder and Yaqoob, 2012). The aim of the present
study was to quantify the interaction between acute phase protein (APP) responses, induced by
immune system activation, and dietary protein supply on nitrogen (N) metabolism.

Material and methods

To quantify the interaction between acute phase protein (APP) responses and dietary protein supply
RQ1PHWDEROLVPZHFKDOOHQJHGSLJV NJ%: ZLWK&RPSOHWH)UHXQG¶V$GMXYDQW &)$ DW
WZROHYHOVRIGLHWDU\SURWHLQVXSSO\$OOSLJVZHUHVXUJLFDOO\¿WWHGZLWKFDWKHWHUVLQWKHMXJXODUYHLQ
housed in metabolic cages and assigned to a diet inducing either an adequate (A) or restricted (R,
70% of A) dietary protein supply, relative to the supply of digestible energy, fed at 2.7× the energy
requirements for maintenance. Two consecutive 5 d N-balance measurements were performed using
funnels underneath the cages for urine collection under addition of sulphuric acid to prevent NH3
losses, one before and one 2-7 d after intravenous administration of CFA (0.15-0.20 ml CFA/kg
BW, divided over 3 or 4 equal portions in 2 days). Blood samples were collected daily throughout
the experimental period up to 8 d after the CFA challenge and analysed for concentrations of acute
phase proteins (APP).

Results and discussion

Serum concentrations of haptoglobin, C-Reactive Protein (CRP) and pig Major Acute Phase
3URWHLQ SLJ0$3 ZHUHSURIRXQGO\LQFUHDVHGEHWZHHQGDQGSRVW&)$FKDOOHQJH P<0.01),
and decreased afterwards on d 7 and 8 post challenge. Serum albumin concentrations were reduced
post challenge (P<0.01). Prior to the CFA challenge, a restricted dietary protein supply reduced
the concentrations of CRP (P<0.05) and albumin (P<0.01), but did not affect the concentrations of
KDSWRJORELQDQGSLJ0$33RVW&)$FKDOOHQJHWKHVHUXPDOEXPLQFRQFHQWUDWLRQZDVVLJQL¿FDQWO\
reduced by dietary protein restriction (P<0.01), while the concentrations of haptoglobin, pig-MAP
and CRP were not affected by dietary protein supply. Houdijk et al. (2007) also found lower plasma
CRP concentrations in pigs with sub-clinical colibacillosis fed a low protein diet, than in pigs fed

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 367
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_128, © Wageningen Academic Publishers 2013
a high protein diet. In line, plasma albumin concentrations decreased in pigs (Jahoor et al., 1999)
and rats (Lunn et al. LQFDVHRIDGH¿FLHQWGLHWDU\SURWHLQVXSSO\2XUUHVXOWVLQGLFDWHWKDWWKH
concentrations of CRP and albumin are affected by the quantitative dietary protein and AA supply.
Due to the challenge, N-retention tended to be reduced in A-fed, but not in R-fed pigs (3 0.07).
This reduction was mainly caused by increased urinary N losses during the post-challenge period.
In line, a reduced N retention due to immune system activation was found by Williams et al. (1997)
and Daiwen et al.  7KHVH¿QGLQJVVXJJHVWWKDWOLPLWDWLRQVLQGLHWDU\SURWHLQVXSSO\UHVWULFW
the APP responses to a systemic CFA challenge in growing pigs. Furthermore, our results suggest
that the priority of partitioning dietary protein over immune system functioning and body protein
deposition depends on the dietary protein supply.

7DEOH(IIHFWRILPPXQHV\VWHPDFWLYDWLRQLQGXFHGE\&)$DGPLQLVWUDWLRQRQ1PHWDEROLVP DOO
data expressed in g/ BWG 

Dietary protein Adequate (A) Restricted (R) Pooled P-values

Challenge Pre Post Pre Post SEM Diet Chall. D×C

N intake 2.32 2.32 1.77 1.75 

Faecal N excretion 0.12 0.12 0.13 0.12 0.003 0.41  0.34
Urinary N excretion  0.73 0.42 0.43 0.031 <0.001 0.04 0.14
Total N excretion  0.85 0.55 0.55 0.031 <0.001 0.09 0.11
N retention 1.57 1.47 1.22 1.20 0.038 <0.001 0.07 0.28

References
Calder, P.C., and P. Yaqoob, 2012. Nutrient regulation of the immune response present knowledge in nutrition. In:
Erdman, J.W., Macdonald, I.A., and S.H. Zeisel (editors), Present Knowledge in Nutrition. John Wiley & Sons,
,QF:LOH\%ODFNZHOO7HQWKHGLWLRQ
'DLZHQ&=.H\LQJDQG:&KXQ\DQ,QÀXHQFHVRIOLSRSRO\VDFFKDULGHLQGXFHGLPPXQHFKDOOHQJHRQ
performance and whole-body protein turnover in weanling pigs. Livest. Sci. 113, 291-295.
*ULPEOH5)1XWULWLRQDOPRGXODWLRQRILPPXQHIXQFWLRQ3URF1XWU6RF
Houdijk, J.G.M., F.M. Campbell, P.D. Fortomaris, P.D. Eckersall, and I. Kyriazakis. 2007. Effects of sub-clinical
post-weaning colibacillosis and dietary protein on acute phase proteins in weaner pigs. Livest. Sci. 108, 182-185.
Jahoor, F., L. Wykes, M. Del Rosario, M. Frazer, and P.J. Reeds. 1999. Chronic protein undernutrition and an acute
LQÀDPPDWRU\VWLPXOXVHOLFLWGLIIHUHQWSURWHLQNLQHWLFUHVSRQVHVLQSODVPDEXWQRWLQPXVFOHRISLJOHWV-1XWU

Klasing, K.C., and B.J. Johnstone. 1991. Monokines in growth and development. Poult. Sci. 70, 1781-1789.
/XQQ3*DQG6$XVWLQ'LIIHUHQFHVLQQLWURJHQPHWDEROLVPEHWZHHQSURWHLQGH¿FLHQWDQGHQHUJ\GH¿FLHQW
rats with similarly restricted growth rates. Ann. Nutr. Metab. 27, 242-251.
Reeds, P.J., C.R. Fjeld, and F. Jahoor. 1994. Do the differences between the amino acid compositions of acute-phase
DQGPXVFOHSURWHLQVKDYHDEHDULQJRQQLWURJHQORVVLQWUDXPDWLFVWDWHV"-1XWU
Sandberg, F.B., G.C. Emmans, and I. Kyriazakis. 2007. The effects of pathogen challenges on the performance of naïve
DQGLPPXQHDQLPDOVWKHSUREOHPRISUHGLFWLRQ$QLPDO
Williams, N.H., T.S. Stahly, and D.R. Zimmerman. 1997. Effect of chronic immune system activation on body nitrogen
UHWHQWLRQSDUWLDOHI¿FLHQF\RIO\VLQHXWLOL]DWLRQDQGO\VLQHQHHGVRISLJV-$QLP6FL

368 Energy and protein metabolism and nutrition in sustainable animal production
Nano-nutrition as a method of anticancer therapy
E. Sawosz1, M. Grodzik1, S. Jaworski1, M. Wierzbicki1, M. Prasek1 and A. Chwalibog2
1Warsaw University of Life Science, Division of Biotechnology and Biochemistry of Nutrition,
&LV]HZVNLHJR:DUVDZ3RODQG[email protected]
2University of Copenhagen, Department of Veterinary Clinical and Animal Sciences, Groennegaardsvej
)UHGHULNVEHUJ'HQPDUN

Introduction
Nano-nutrition is a method of providing cells with nutritional factors, disintegrated to nanoparticles
or transported by nanoparticles. Bioactive substances attached to nanoparticles may penetrate
organism, bypass blood system and through the membranes go directly to the cells. Consequently,
the transported nutritive substances may be delivered to cells, also to cancer cells switching on the
mechanism of programmed cell death, leading to decreased tumour growth.

Graphene is one of carbon allotropes, regarded as the thinnest material in the world (Geim and
Novoselov, 2007). It is made of one layer of carbon atoms, forming a hexagonal structure connected
by sp2 type bonds. Moreover, graphene as a carbon structure is not very toxic and used at an optimal
level may form drug delivery platform. Graphene may be easy functionalised with amino acids and
GHOLYHUWKHPWRWKHFHOOVWRPRGLI\VSHFL¿FLW\RIERQGV$WWDFKHGWRJUDSKHQHDPLQRDFLGV DUJLQLQH
and hyroxyproline) may block p53 – MDM2 binding by enriching hydroxyproline or arginine in
proline-rich domain of p53 protein, leading to apoptosis of cancer cells.

The objective of the experiments was to use graphene as a platform to deliver arginine and
hydroxyproline to glioblastoma multiforme (GM) cancer cells, activate p53 protein and induce
apoptosis.

Material and methods

Graphene nanoparticles were obtained from ITE (Warsaw, Poland) and from SkySpring Nanomaterials
(Houston, USA). The size of graphene sheets varied around 450 nm.

The experiments were carried out with in vitro and in ovo methods. Human glioblastoma U87
and U118 cell lines were obtained from the American Type Culture Collection and maintained in
DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C
LQDKXPLGL¿HGDWPRVSKHUHRI&22/95% air.

To maintain tumour of GM the fertilized eggs Ross 308 were incubated at 37 °C and 70% humidity.
After 7 days the silicone ring with the deposited 3-4×10 U87 or U118 glioma cells suspended in 30
ȝORIFXOWXUHPHGLXPZDVSODFHGRQWKHFKRULRDOODQWRLFPHPEUDQH &$0 LQWKHDUHDRIIRUPHGEORRG
vessels (Grodzik et al., 2011). The eggs were incubated for 5 days and then experimental solution
(control vs. graphene or graphene with attached arginine and hydroxyproline) were injected directly
into the tumour. After 5 days the eggs were opened and tumours were resected for further analysis.

Morphology, histology, mortality, expression of protein involved in stress (p-53) as well as and
apoptosis and necrosis were measured in vitro with GM cells and using tumour model maintained
in ovo.

Data were analyzed using one-way analysis of variance using STATGRAPHICS® Plus 4.1.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 369
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_129, © Wageningen Academic Publishers 2013
Results and discussion
Graphene, but also graphene with attached amino acids affected morphology of the GM cells but this
HIIHFWZDVGLIIHUHQWEHWZHHQWKH8DQG8OLQHV,QERWKFDVHVJUDSKHQHVKRZHGDI¿QLW\WR*0
cells, locating and adhering to the body of cells. However, because of the size (450 μm) the graphene
ÀDNHVGLGQRWHQWHUWKHFHOOHV7KHPHDVXUHPHQWVRIFHOOPRUWDOLW\XVLQJWKHXVHRI7U\SDQEOXHG\H
demonstrated the dose depended toxic effect of graphene nanoparticles on glioma cells, as previously
observed by Chang et al., 2011. Graphene had the highest toxicity at the concentration of 100 mg/
kg but was not toxic at the levels less than 20 mg/kg. Toxic effect was observed by evaluating cell
membrane functionality and integrity. Analyzing the integrity of membranes between two glioma
FHOOOLQHVVLJQL¿FDQWGLIIHUHQFHVZHUHVHHQ7KHPHPEUDQHLQWHJULW\LQ8FHOOVZDVPXFKPRUH
disrupted than in U118 even at the low graphene concentrations.

Graphene and also graphene with attached amino acids induced apoptosis in U118 GM and apoptosis
and necrosis in U87 GM. Protein p53 is the main activator of apoptosis, but U118 GM showed
reduced possibilities to activate apoptosis via p53 induction. It can be supposed that grapheme can
activate apoptosis by an alternative way, being in a direct contact with cell membranes and membrane
UHFHSWRUV$PLQRDFLGVDWWDFKHGWRJUDSKHQHGLGQRWVLJQL¿FDQWO\DIIHFWWKHREVHUYHGPHFKDQLVP

In experiments with GM tumour graphene injected into the tumour decreased size of tumour and
increased number of apoptotic cells. Morphology of the tumour indicated less number of disrupted
mitochondria. Expression of p53 and MDM2 protein also changed; however, tendency to increase
apoptosis by graphene and graphene attached with amino acids was not correlated with p53, which
also may suggest an alternative way of apoptosis.

7KHUHVXOWVGHPRQVWUDWHGWKDWJUDSKHQHÀDNHVUHJDUGOHVVRIDWWDFKHGDUJLQLQHDQGK\GUR[\SUROLQH
at a level less than 20 mg/kg seem to be not toxic, however, they activate mechanisms of apoptosis
in U118 GM cell line and this may indicate anticancer characteristics of graphene.

References
Chang, Y., S. T. Yang, J. H. Liu, E. Dong, Y. Wang, Y. Liu, H. Wang, 2011. In vitro toxicity evaluation of graphene
oxide on A549 cells. Toxicology Lett. 200, 201-210.
*HLP$.DQG.61RYRVHORY7KHULVHRIJUDSKHQH1DW0DWHU
Grodzik, M., E. Sawosz, M. Wierzbicki,P. Orlowski, A. Hotowy, T. Niemiec, M. Szmidt, K. Miture, A. Chwalibog.
2011. Nanoparticles of carbon allotropes inhibit glioblastoma multiforme angiogenesis in ovo. Int. J. Nanomedicine.


370 Energy and protein metabolism and nutrition in sustainable animal production
Differential expression of innate immune system genes in liver of beef
cattle with divergent phenotypes for RFI
K.A. Johnson1, J.J. Michal1, G.E. Carstens2, $1+DÀD2 and T.D.A. Forbes2
1'HSDUWPHQW RI $QLPDO 6FLHQFHV :DVKLQJWRQ 6WDWH 8QLYHUVLW\ 3XOOPDQ :$  86$
[email protected]
2'HSDUWPHQWRI$QLPDO6FLHQFH7H[DV$ 08QLYHUVLW\&ROOHJH6WDWLRQ7;86$

Introduction
5HVLGXDOIHHGLQWDNH 5), TXDQWL¿HVLQWHUDQLPDOYDULDQFHVLQGU\PDWWHULQWDNH '0, LQGHSHQGHQW
of variation in body size and productivity (e.g. growth, milk). In beef cattle, Herd and Arthur (2009)
estimated that approximately one-third of the biological variation in RFI was due to inter-animal
differences in digestion, heat increment, body composition, and activity, with the remaining variation
associated with energy expenditure of metabolic processes. Given that RFI is independent of BW
and level of production, it is an ideal trait for discovery of genomic regions (Rolf et al., 2011) and
functional genes (Chen et al. DVVRFLDWHGZLWKHI¿FLHQF\RIIHHGXWLOL]DWLRQ6RPHYDULDWLRQ
in RFI may be caused by individual differences in immune system function. The immune system
is an energetically expensive system. A previous study showed that metabolic heat production and
oxygen consumption increased 20-30% in mice that were immunized with a relatively benign antigen
(Demas et al., 1997). The objective of this work was to examine whether the genes associated with
the immune system were differentially expressed by beef cattle with divergent RFI phenotypes.

Material and methods

7UDQVFULSWDEXQGDQFHVLQOLYHUIURP%RQVPDUDKHLIHUV Q 7ULDO DQG¿QLVKLQJFURVVEUHGVWHHUV
(n=12; Trial 2) with divergent phenotypes for RFI were initially screened with custom-designed
NimbleGen bovine gene expression arrays. Several genes involved with innate immune functions
DQGLQÀDPPDWRU\UHVSRQVHVZHUHLGHQWL¿HGDVELRORJLFDOO\VLJQL¿FDQWEHWZHHQWKH5),JURXSV
Genes investigated included serpin peptidase inhibitor, clade I (pancipin), member 2 (SERPINI2),
tetraspanin 13 (TSPAN13), caspase 3 (CASP3), interleukin 1, beta (IL1B), interleukin 18 (1L18),
LQWHUIHURQDOSKDLQGXFLEOHSURWHLQ ,), LQWHUIHURQLQGXFHGWUDQVPHPEUDQHSURWHLQ ,),70 
interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), interleukin 20 receptor, alpha
(IL20RA), and folliculin interacting protein 1 (FNIP1).

7RIXUWKHUH[DPLQHWKHELRORJLFDOVLJQL¿FDQFHRIWKHVHJHQHVDGGLWLRQDOOLYHUVDPSOHVZHUHFROOHFWHG
IURPFDWWOHZLWKGLYHUJHQW5),7ULDOLQFOXGHG6DQWD*HUWUXGLVKHLIHUV Q  DQGEXOOV Q  WULDO
FRPSULVHGFURVVEUHGVWHHUV Q  DQGWULDOLQFOXGHG%RQVPDUDKHLIHUV Q  &DWWOHLQWULDOV
3 and 5 were fed a low-energy diet, whereas, a high-energy diet was fed in trials 2 and 4. Total RNA
was extracted from liver, and reverse-transcribed to cDNA. Primers for real-time PCR were designed
ZLWK4XDQW3ULPHDQGWUDQVFULSWDEXQGDQFHVLQDOOFDWWOHZHUHGHWHUPLQHGXVLQJ6<%5JUHHQEDVHG
detection and a BioRad iCycler iQ detection system. Beta-actin was used as a housekeeping gene
for data normalization, and transcript abundance expressed relative to a reference sample (pooled
F'1$IURPVWHHUV 7KH352&0,;('SURFHGXUHRI6$6ZDVXVHGWRGHWHUPLQHWKH¿[HG
effect of RFI group (high or low) on mRNA expression for each trial separately.

Results and discussion

The expression of immune function genes by heifers and bulls consuming a low energy diet did
not vary by RFI phenotype. However, there was a tendency (P<0.10) for IFITM2 to be down-
UHJXODWHGDQGIRU,/5$WREHXSUHJXODWHGLQDQLPDOVZLWKORZ5),SKHQRW\SHV PRUHHI¿FLHQW 
7KLVLVLQFRQWUDVWWRHI¿FLHQWVWHHUVIHGDKLJKJUDLQGLHWLQZKLFK,/%763$1DQG6(53,1,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 371
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_130, © Wageningen Academic Publishers 2013
ZHUHVLJQL¿FDQWO\GRZQUHJXODWHG2WKHUVKDYHDOVRUHSRUWHGWKDW6(53,1,DQG763$1ZHUH
down regulated in liver of cattle with high and low RFI (Chen et al., 2011). Serpins are involved
LQUHJXODWLRQRIGHYHORSPHQWFRDJXODWLRQ¿EULQRO\VLVDQGLQÀDPPDWRU\SURFHVVHV 5DJJ 
ZKLOHWHWUDVSDQLQVDUHLQYROYHGLQFHOODGKHVLRQIXVLRQPHPEUDQHWUDI¿FNLQJ5HFHQWHYLGHQFH
suggests that tetraspanins play a key role in the course of pathogenic infection (Monk and Partridge,
2012). Expression of IL1B, TSPAN13 and SERPIN12 were positively correlated (P<0.01) to DMI
UHVSHFWLYHO\ IHHGFRQYHUVLRQUDWLR UHVSHFWLYHO\ DQG5), 
UHVSHFWLYHO\ LQVWHHUVIURPWULDO7KHRWKHUJHQHVH[DPLQHGVXFKDV,),7 GRZQ
regulated), IL20RA (up regulated) tended to be altered in the steers fed a high grain diet in trials 2 &
,QWHUOHXNLQEHWD,/&$63,),7,),70,),DQG,/5$DUHJHQHVWKDWDUHLQYROYHG
in the innate immune response. Examination of the locus in Angus cattle that explained 10% of
the variation in DMI revealed that FNIP1 may be important in the regulation of feed intake (US
Consortium et al. ,QWKLVVWXG\)1,3H[SUHVVLRQZDVSRVLWLYHO\FRUUHODWHG P<0.05)
to DMI in one heifer trial.

The genes involved in the innate immune system may be affected by diet, sex and the RFI phenotype
in growing cattle. Pathway analysis of additional genes involved in this system may provide a clearer
YLHZRIKRZLQÀDPPDWLRQDQGWKHLQQDWHLPPXQHV\VWHPFRQWULEXWHWRHQHUJ\PHWDEROLVP

References
&KHQ<&*RQGUR.4XLQQ50+HUG3)3DUQHOODQG%9DQVHORZ*OREDOJHQHH[SUHVVLRQSUR¿OLQJ
reveals genes expressed differentially in cattle with high and low residual feed intake. Anim. Genet. 42, 475-490.
Demas, G.E., V. Chefer, M.I. Talan and R.J. Nelson, 1997. Metabolic costs of mounting an antigen-stimulated immune
UHVSRQVHLQDGXOWDQGDJHG&%/-PLFH$P-3K\VLRO,QWHJU&RPS3K\VLRO55
+HUG50DQG3)$UWKXU3K\VLRORJLFDOEDVLVIRUUHVLGXDOIHHGLQWDNH-$QLP6FL((
Monk, P.N. and L.J. Partridge, 2012. Tetraspanins – Gateways for infection. Infect. Disord. Drug Targets. 12, 4-17.
5DJJ+7KHUROHRIVHUSLQVLQWKHVXUYHLOODQFHRIWKHVHFUHWRU\SDWKZD\&HOO0RO/LIH6FL
Rolf, M.M., J.F. Taylor, R.D. Schnabel, S.D. McKay, M.C. McClure, S.L. Northcutt, M.S. Kerley and R.L. Weaber,
*HQRPHZLGHDVVRFLDWLRQDQDO\VLVIRUIHHGHI¿FLHQF\LQ$QJXVFDWWOH$QLP*HQHW
86&RQVRUWLXPIRUWKH*HQHWLF,PSURYHPHQWRI)HHG(I¿FLHQF\LQ%HHI&DWWOH3ODQWDQG$QLPDO*HQRPLFV
Meeting. P0521.

372 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWRIKLJKIDWE\SURGXFWVSHOOHWVLQ¿QLVKLQJGLHWVIRUVWHHUV
P. Górka1,2, J.J. McKinnon1 and G.B. Penner1
1'HSDUWPHQWRI$QLPDODQG3RXOWU\6FLHQFH8QLYHUVLW\RI6DVNDWFKHZDQ61$6DVNDWRRQ
Saskatchewan, Canada; [email protected]
2Department of Animal Nutrition and Feed Management, University of Agriculture in Krakow,
.UDNRZ3RODQG

Introduction
Cereal grain prices have increased in recent years resulting in increased feed costs for feedlot
operators. To offset these costs, investigation into alternative energy sources, relative to cereal
grains, has been initiated (Marx et al., 2000). Applying single ingredient substitution strategies may
OLPLWWKHXVHRIVRPHE\SURGXFWVIRU¿QLVKLQJFDWWOHVXFKDVJUDLQVFUHHQLQJVSHDVFUHHQLQJVRURDW
KXOOVGXHWRLQVXI¿FLHQWHQHUJ\FRQWHQW 0DU[et al., 2000) or alternatively may limit the amount
of cereal grain that can be replaced. One strategy to eliminate overfeeding of nutrients from high
byproduct inclusion rates is to utilize strategic combinations of various byproducts to optimize
ruminal and postruminal energy and protein availability (Zenobi et al., 2012). The aim of this study
was to determine the effect of including strategically blended high-fat by-product pellets as a partial
UHSODFHPHQWIRUEDUOH\JUDLQDQGFDQRODPHDOLQ¿QLVKLQJGLHWVIRUVWHHUV

Material and methods

Two hundred and sixty four crossbreed steers (average BW of 375±0.72 kg) were randomly allocated
to one of 12 outdoor pens (22 steers/pen) with each pen randomly allocated to 1 of 3 treatments (4
pens/treatment). The control (CON) diet consisted of 89% concentrate (barley and canola meal),
EDUOH\VLODJHDQGYLWDPLQPLQHUDOSHOOHWV '0EDVLV +LJKIDWSHOOHWV FUXGHIDW
29.8% NDF, 5.23 MJ NEg/kg on DM basis) were included in diets by replacing 30% (HFP30)
RU +)3 RIWKHEDUOH\DQGFDQRODPHDOUHODWLYHWR&217KH+)3FRQWDLQHG '0EDVLV 
JURXQGZKHDWZKHDWVFUHHQLQJVZKROHFDQRODSHDVFUHHQLQJVDQG
11.9% oat hulls. Diets were formulated to be isonitrogenous and isoenergetic with CP and NEg
contents of 13.4% and 5.02 MJ NEg/kg (DM basis), respectively, and were offered ad libitum once
daily. Steers were weighed on 2 consecutive days at the start and end, and monthly weights were
taken throughout the study. Feed bunks were cleaned every 2 wk and DMI was calculated based on
the amount of feed offered and refused. After completing 155 d on feed, steers were weighed and
shipped to commercial slaughter plant and carcasses were evaluated using the Computer Vision
Grading System (VBG 2000 e+v Technology GmbH, Oranienburg, Germany). Data were analyzed
as a completely randomized design using the mixed model of SAS (Cary, NC, USA) except for
carcass grade, which were analyzed using Proc GLIMMIX. Contrasts were used to determine the
effect of HFP inclusion and whether the level of inclusion affected the response.

Results and discussion

,QLWLDODQG¿QDO%:DQGFXPXODWLYH$'*ZHUHQRWGLIIHUHQWDPRQJWUHDWPHQWV P>0.05; Table 1).
Dry matter intake was higher and the gain:feed ratio was lower (P< IRU+)3DQG+)3DV
FRPSDUHGWR&21ZLWKQRGLIIHUHQFHVREVHUYHGEHWZHHQ+)3DQG+)3+RWFDUFDVVZHLJKW
rib eye area, and marbling score were not affected by treatment (P>0.05). Quality grade and carcass
yield were not different among treatments (P>0.05). Back fat thickness was higher (3 0.02) for
+)3DQG+)3DVFRPSDUHGWR&21ZLWKQRGLIIHUHQFHVEHWZHHQ+)3DQG+)3

Partially replacing barley grain with strategically blended HFP allowed for high inclusion rates of
E\SURGXFWV DQGIRU+)3DQG+)3UHVSHFWLYHO\ LQ¿QLVKLQJGLHWVIRUEHHIVWHHUV

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 373
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_131, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRISDUWLDOO\UHSODFLQJEDUOH\JUDLQDQGFDQRODPHDOZLWKKLJKIDWSHOOHW +)3 RQ
BW, ADG, DMI, and carcass yield and carcass grade.

Treatment SEM Contrasts

CON HLP30 +/3 CON vs. HLP +/3YV+/3

Initial BW (kg) 374 374 375 0.2 0.18 

Final BW (kg)    1.8 0.27 0.38
ADG (kg/d) 1.90 1.88  0.010 0.27 0.38
DMI (kg/d) 10.7 11.3 11.4 0.10 <0.01 0.17
Gain:feed 0.18 0.17  0.01 <0.01 0.10
Hot carcass (kg) 379 387 382 2.5 0.31 0.39
Rib eye area (cm2) 94.8 93.9   0.78 0.13
Back fat (cm) 0.77 0.93 0.88 0.28 0.02 0.38

DQGWKXVWKHSRWHQWLDOWRUHSODFHDVLJQL¿FDQWDPRXQWRIKLJKSULFHGFHUHDOJUDLQV$OWKRXJK+)3
LQFOXVLRQUHGXFHGIHHGHI¿FLHQF\WKHUHZHUHQRGLIIHUHQFHVLQJURZWKSHUIRUPDQFHRUFDUFDVV\LHOG
and quality among treatments suggesting that higher DMI compensated for the potential negative
effects feeding diets high in NDF with a small particle size and high in lipid on passage rate and
digestibility. Thus, when NEm and NEg were calculated based on intake and performance, the
NEm and NEg 0-NJ ZHUHDQGDQGDQGDQGIRU&21+)3DQG
+)3UHVSHFWLYHO\

7KHUHVXOWVLQGLFDWHWKDWXSWRRIEDUOH\DQGFDQRODPHDOLQ¿QLVKLQJGLHWVFDQEHUHSODFHGZLWK
strategically blended HFP without negative effects on ADG of steers and carcass quality. However,
gain:feed ratio may be compromised.

References
Marx, T.A., J.J. McKinnon, A.F. Mustafa, D.A. Christensen, and V.J. Racz, 2000. The feeding value of grain screenings
IRUUXPLQDQWV&KHPLFDOFRPSRVLWLRQDQGQXWULHQWXWLOL]DWLRQ&DQ-$QLP6FL
Zenobi, M.G., P. Yu, D.A. Christensen, P.G. Jefferson, H.A. Lardner, and J.J. McKinnon, 2012. Performance of cattle
fed diets based on blended by-product pellets varying in rumen avaliale energy and protein content. J. Anim. Sci.
90 (Suppl. 3): 711.

374 Energy and protein metabolism and nutrition in sustainable animal production
Microbial activity in the large intestine of chickens fed diets containing
different sources of inulin-type fructans
M. Taciak, M. Barszcz, $7XĞQLR(ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá
Department of Monogastric Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition,
3ROLVK$FDGHP\RI6FLHQFHV-DEáRQQD3RODQG[email protected]

Introduction
Inulin-type fructans are water-soluble compounds of plant origin with varying length of polysaccharide
FKDLQVZLWKȕIUXFWRV\OIUXFWRVHJO\FRVLGLFERQGV7KLVW\SHRIERQGPDNHVLWUHVLVWDQWWR
HQ]\PDWLFK\GURO\VLVE\VPDOOLQWHVWLQDOGLJHVWLYHHQ]\PHV VSHFL¿FIRUĮJO\FRVLGLFERQGV $VD
result, inulin fructans are indigestible by enzymes of animal origin and are fermented in the large
intestine. The inulin fructans can be found in many plants, but by the industry is mainly extracted
from chicory and Jerusalem artichoke. Maintaining animal welfare through the modulation of large
intestine function may play an important role, therefore the aim of the study was to determine the
effect of diets’ supplementation with different sources of inulin-type fructans on microbial activity
in the large intestine of chickens.

Material and methods

The experiment was performed on 40 male chickens (Ross 308) divided into 5 groups (n=8). Birds
ZHUHIHGIURPWKH¿UVWGD\RIOLIHFHUHDOGLHWVVXSSOHPHQWHGZLWKRILQXOLQH[WUDFWHGIURPFKLFRU\
URRWZLWKGHJUHHRISRO\PHUL]DWLRQ '3 •RILQXOLQZLWK'3•RIGULHG-HUXVDOHP
artichoke and 0.8% of dried chicory, corresponding with 0.4% of pure inulin. Control group was
JLYHQGLHWZLWKRXWVXSSOHPHQWDWLRQ%LUGVZHUHVDFUL¿FHGDWWKHDJHRIGD\VDQGGLJHVWDVDPSOHV
were taken from the caecum and colon for analysis of microbial activity indices. Digesta pH was
measured using pH-meter, SCFA concentration was analyzed by gas chromatography (Barszcz et al.,
 &DHFDOȕJOXFRVLGDVHDFWLYLW\ZDVGHWHUPLQHGVSHFWURSKRWRPHWULFDOO\ -XĞNLHZLF]et al., 2011).
Statistical analysis was performed by one-way ANOVA procedure and post hoc Tukey HSD test.

Results and discussion

1HLWKHUFDHFDOQRUFRORQLFGLJHVWDS+ZDVVLJQL¿FDQWO\DIIHFWHGE\GLHWV 7DEOH KRZHYHUELUGV
fed diet with dried chicory tended to have lower ceacal pH compared to other groups. Bacterial
HQ]\PHDFWLYLW\DQG6&)$FRQFHQWUDWLRQLQFDHFDOGLJHVWDDOVRGLGQRWGLIIHUVLJQL¿FDQWO\EHWZHHQ
experimental groups. Concentration of SCFA in colonic digesta was affected by experimental
GLHWV)HHGLQJ-HUXVDOHPDUWLFKRNHVLJQL¿FDQWO\LQFUHDVHGDFHWLFDQGSURSLRQLFDFLGFRQFHQWUDWLRQV
FRPSDULQJWRFKLFRU\DQGLVREXW\ULFDFLGFRPSDULQJWRFKLFRU\DQGLQXOLQZLWK'3•,QFUHDVHG
concentration of isobutyric acid in the colon may be a result of more intensive bacterial catabolism
of endogenous or dietary protein (Hughes et al., 2000). Concentration of valeric acid in the caecum
and valeric and isovaleric acids in the colon were under detection level.

&RQWUDU\WRWKHH[SHFWDWLRQVPLFURELDODFWLYLW\LQWKHODUJHLQWHVWLQHRIFKLFNHQLVVOLJKWO\PRGL¿HG
by feeding diets with different sources of inulin-type fructans, at the level of 0.4%, since the only
difference was found in SCFA concentration in the colon, except for butyric acid concentration.
Neither inulin preparations nor dried plants rich in inulin decreased digesta pH and activity of
detrimental bacterial enzyme.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 375
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_132, © Wageningen Academic Publishers 2013
7DEOHS+VKRUWFKDLQIDWW\DFLGVFRQFHQWUDWLRQV —0JRIGLJHVWD DQGDFWLYLW\RIȕJOXFRVLGDVH
8JRIGLJHVWD LQWKHFDHFXPDQGFRORQRIFKLFNHQ

pH Acetic Propionic Isobutyric Butyric Isovaleric ȕJOXFRVLGDVH

Caecum
Control  31.91 0.830 0.351 7.92 0.223 273.8
IN10  27.28 0.875 0.321  0.211 
IN23 5.90 33.08 0.892 0.327 7.95 0.248 297.9
JA  31.81    0.259 285.2
CH 5.43 29.14 0.801  9.14  
SEM  0.959 0.039 0.017 0.425 0.023 8.098
P-value 0.053 0.331  0.139 0.192  
Colon
Control  18.23ab ab ab 4.34 bd na
IN10  19.31ab ab 0.287a 4.01 bd na
IN23  ab 0.521ab 0.315ab 3.81 bd na
JA  28.19b 0.721b 0.520b 3.44 bd na
CH  7.75a 0.183a a 0.97 bd na
SEM  1.950 0.053 0.030 0.512 bd na
P-value  0.020 0.019 0.002 0.251 bd na

IN10 LQXOLQH[WUDFWHGIURPFKLFRU\URRWVZLWKGHJUHHRISRO\PHUL]DWLRQ•,123 = inulin extracted from chicory

URRWVZLWKGHJUHHRISRO\PHUL]DWLRQ•-$ GULHG-HUXVDOHPDUWLFKRNH&+ GULHGFKLFRU\EG EHORZGHWHFWLRQ
na = not analyzed.
a,bPHDQYDOXHVZLWKLQDFROXPQZLWKGLIIHUHQWOHWWHUVGLIIHUVLJQL¿FDQWO\3”

Acknowledgements
)LQDQFLDOVXSSRUWRI7KH1DWLRQDO&HQWHUIRU5HVHDUFKDQG'HYHORSPHQW3URMHFW1R15

References
%DUV]F]007DFLDNDQG-6NRPLDá$GRVHUHVSRQVHHIIHFWVRIWDQQLFDFLGDQGSURWHLQRQJURZWKSHUIRUPDQFH
FDHFDOIHUPHQWDWLRQFRORQPRUSKRORJ\DQGȕJOXFXURQLGDVHDFWLYLW\RIUDWV-$QLP)HHG6FL
Hughes, R., E.A.M. Magee and S. Bingham, 2000. Protein degradation in the large intestine: relevance to colorectal
cancer. Curr. Issues Intest. Microbiol. 1(2), 51-58.
-XĞNLHZLF]-==GXĔF]\N(ĩDU\6LNRUVND%.UyO-0LODODDQG$-XUJRĔVNL(IIHFWRIWKHGLHWDU\
polyphenolic fraction of chicory root, peel, seed and leaf extracts on caecal fermentation and blood parameters in
rats fed diets containing prebiotic fructans. Brit. J. Nutr. 105, 710-720.

376 Energy and protein metabolism and nutrition in sustainable animal production
Effect of dietary protein and carbohydrates on phenolic compounds
formation in the large intestine of pigs
M. Taciak, $7XĞQLRM. Barszcz, (ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá
Department of Monogastric Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition,
3ROLVK$FDGHP\RI6FLHQFHV-DEáRQQD3RODQG[email protected]

Introduction
Phenolic and indolic compounds, potentially toxic substances, are formed in the gut in bacterial
degradation of the aromatic amino acids e.g. phenol, p-cresol and phenylpropionate originate from
tyrosine, phenylacetate from phenylalanine and indole, indole propionate and indole acetate from
tryptophan (Hughes et al. 7KLVSURFHVVPD\EHPRGL¿HGE\IHHGLQJGLHWFRQWDLQLQJGLIIHUHQW
FDUERK\GUDWHVDQGSURWHLQVRXUFHV7KHDLPRIWKHSUHVHQWVWXG\ZDVWRDVVHVVWKHLQÀXHQFHRISURWHLQ
RIDQLPDORUSODQWRULJLQDQGWKUHHW\SHVRIFDUERK\GUDWHVXVHGDVDQHQHUJ\VRXUFHIRUPLFURÀRUD
on the processes of aromatic amino acid degradation in the large intestine of pigs.

Material and methods

$WZRIDFWRULDOH[SHULPHQWZDVFRQGXFWHGRQFDVWUDWHGPDOHSLJVRIDERXWNJLQLWLDOERG\ZHLJKW
$QLPDOVZHUHGLYLGHGLQWRJURXSVUHFHLYLQJFHUHDOIHHGVGLIIHULQJLQWKHW\SHRIFDUERK\GUDWHVDGGHG
to diets (cellulose, potato starch, pectin) and the type of protein (potato protein concentrate or casein).
After two weeks of experiment pigs were slaughtered and samples of caecal and proximal, middle
and distal colon digesta were collected and analyzed for phenol, p-cresol and indole concentration
using the gas chromatography method.

Results and discussion

Neither type of carbohydrate nor type of protein in the diet affected concentration of phenol in all
segments of the large intestine of pigs. In the caecum, proximal and distal colon concentration of
p-cresol and indole was affected by the protein source in the diet. Potato protein concentrate increased
concentration of these products, may be as a result of substantial amount of protein passing to the
large intestine from ileum because of the apparent resistance of potato protein to enzymatic digestion
7XĞQLRet al., 2011). Type of carbohydrate in the diet affected quantity of indole in the caecum
and proximal colon, and p-cresol in proximal and distal colon. Both products concentration in the
proximal colon, as well as indole in the ceacum were higher in pigs fed diets supplemented with
cellulose compared to pectin. Potato starch in the diet resulted in increased concentration of p-cresol
in the distal colon also in comparison with pectin. Microbial growth is stimulated by the presence of
fermentable carbohydrate. This leads to an increased demand for amino acids by microorganisms, as
may by suggested by reduced protein catabolism in the gut. Therefore, the production of potentially
harmful compounds, such as phenolic compounds, would be less intensive in the presence of a
carbohydrate source (Williams et al., 2001).

Increased concentration of p-cresol and indole, resulting from microbial metabolism of protein,
and enhanced by the presence of cellulose, a carbohydrate of low fermentation capability, may be
reduced by the addition of highly fermentable pectin.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 377
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_133, © Wageningen Academic Publishers 2013
7DEOH3KHQROFUHVRODQGLQGROFRQFHQWUDWLRQV —0JRIGLJHVWD LQWKHFDHFXP 6 SUR[LPDO
& PLGGOH & DQGGLVWDO & FRORQRISLJV1

PPC CAS SEM P-values

CEL PEC PS CEL PEC PS Protein Carbohydrate P×C

S phenol  1.30 0.73 0.70  0.70 0.28 0.373 0.445 0.435
cresol 1.30  1.22 0.90 0.88 0.73 0.28 0.013  
indole 0.08 0.02 0.04 0.04 0.01 0.02 0.01 0.007 0.000 
C25 phenol      0.73 0.05 0.370  0.887
cresol 2.32 1.23 1.95 0.95 0.88 1.11 0.22 0.000 0.022 
indole 0.09 0.02 0.05 0.03 0.01 0.03 0.01 0.001 0.001 0.042
C50 phenol     0.59 0.71  0.823 0.303 0.703
cresol 2.08 1.85 2.03 1.10 0.89 1.39  0.000  0.732
indole 0.07 0.05 0.04 0.03 0.01 0.02 0.01 0.000  0.331
C75 phenol 0.58 0.57     0.04 0.458  
cresol 3.20  4.43 2.94 2.14 3.47  0.307 0.039 0.821
indole 0.09  0.04 0.05 0.04 0.05 0.01 0.223 0.130 0.233

1 PPC = potato protein concentrate; CAS = casein; CEL = cellulose; PEC = pectin; PS = potato starch.

Acknowledgements
)LQDQFLDOVXSSRUW3URMHFW1R11

References
Hughes, R., E.A.M. Magee and S. Bingham, 2000. Protein degradation in the large intestine: relevance to colorectal
cancer. Curr. Issues Intest. Microbiol. 1(2), 51-58.
7XĞQLR$%3DVWXV]HZVND(ĝZLĊFKDQG07DFLDN5HVSRQVHRI\RXQJSLJVWRIHHGLQJSRWDWRSURWHLQDQG
SRWDWR¿EUH±QXWULWLRQDOSK\VLRORJLFDODQGELRFKHPLFDOSDUDPHWHUV-$QLP)HHG6FL
Williams B.A., M.W.A. Verstegen and S. Tamminga, 2001. Fermentation in the large intestine of single-stomached
animals and its relationship to animal health. Nutr. Res. Rev. 14, 207-227.

378 Energy and protein metabolism and nutrition in sustainable animal production
Microbial activity in the large intestine of piglets fed diets with different
sources of inulin
M. Barszcz, M. Taciak, $7XĞQLR(ĝZLĊFKà6WDĞNLHZLF]DQG-6NRPLDá
Department of Monogastric Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition,
3ROLVK$FDGHP\RI6FLHQFHV-DEáRQQD3RODQG[email protected]

Introduction
Inulin-type fructans are natural food ingredients present as storage carbohydrates in a number of
plants. Currently only two species, both from Compositae family, i.e. chicory and Jerusalem artichoke
are used by the industry for extraction of these compounds (Kaur and Gupta, 2002). Inulin-type
fructans resist digestion by small intestinal enzymes of monogastric animals but are fermented in
the large intestine (Roberfroid, 2005). Modulation of large intestine function plays an important
role in maintaining animal welfare, therefore the aim of this study was to determine the effect of
diets’ supplementation with different sources of inulin-type fructans on microbial activity in the
large intestine of piglets.

Material and methods

The experiment was performed on 40 castrated male piglets (PIC) divided into 5 groups (n=8).
Piglets were fed from the 2nd week of life cereal diets supplemented with 2% of inulin extracted from
FKLFRU\URRWVZLWKGHJUHHRISRO\PHUL]DWLRQ '3 • ,110 RILQXOLQZLWK'3• ,123);
4% of dried Jerusalem artichoke (JA) and 4% of dried chicory (CH) corresponding to 2% of inulin
PHQWLRQHGEHIRUH&RQWUROJURXSZDVJLYHQGLHWZLWKRXWVXSSOHPHQWDWLRQ3LJOHWVZHUHVDFUL¿FHGDW
the age of 50 days and digesta samples were taken from the caecum and proximal, middle and distal
colon for analysis of microbial activity indices. Digesta pH was measured using pH-meter, short-
FKDLQIDWW\DFLGV 6&)$ FRQFHQWUDWLRQZDVDQDO\]HGE\JDVFKURPDWRJUDSK\ȕJOXFXURQLGDVHDQG
ȕJOXFRVLGDVHDFWLYLW\ZDVGHWHUPLQHGVSHFWURSKRWRPHWULFDOO\DQGVWDWLVWLFDODQDO\VLVZDVSHUIRUPHG
by one-way ANOVA and Tukey test.

Results and discussion

)HHGLQJH[SHULPHQWDOGLHWVGLGQRWDIIHFWVLJQL¿FDQWO\S+RIGLJHVWDLQWKHSLJOHWODUJHLQWHVWLQH
but it affected SCFA concentration (Table 1). Acetic, propionic and butyric acids were the major
metabolites in the large intestine, which agrees with previous studies (Loh et al.+DODVet al.,
 ,QWKHFDHFDOGLJHVWDYDOHULFDFLGFRQFHQWUDWLRQZDVVLJQL¿FDQWO\KLJKHULQSLJOHWVIHGGLHW
with JA than in piglets fed diets with IN10 and IN23 (P<0.01). Similarly, in the proximal colon valeric
DFLGFRQFHQWUDWLRQLQSLJOHWVIHG-$GLHWGLIIHUHGVLJQL¿FDQWO\IURPSLJOHWVIHG,110 diet (P<0.05).
Valeric acid is considered as an isoacid resulted from protein degradation in the intestine and its
concentration increases in piglet fed diets supplemented with inulin, especially that with greater DP
(Passlack et al. 7KHUHZDVDOVRDVLJQL¿FDQWHIIHFWRQDFHWLFDFLGFRQFHQWUDWLRQZKLFKZDV
smaller in piglets fed IN10 diet than in piglets fed IN23 and CH diet (P<0.01). This may indicate
that inulin with greater DP affects microbial activity more pronounced than inulin with smaller DP,
which was suggested by Passlack et al. (2012). There were no differences in SCFA concentration
LQWKHPLGGOHDQGGLVWDOFRORQ%DFWHULDOHQ]\PHVDFWLYLW\DOVRGLGQRWGLIIHUVLJQL¿FDQWO\EHWZHHQ
H[SHULPHQWDOJURXSVH[FHSWFDHFDOȕJOXFRVLGDVHDFWLYLW\ZKLFKZDVKLJKHULQSLJOHWVIHG-$GLHW
compared to animals from other groups (P<0.05).

Contrary to the expectations, microbial activity in the large intestine of piglets is only slightly
PRGL¿HGE\IHHGLQJGLHWVZLWKGLIIHUHQWVRXUFHVRILQXOLQW\SHIUXFWDQV1HLWKHULQXOLQSUHSDUDWLRQV
nor dried plants rich in inulin decreased digesta pH and activity of detrimental bacterial enzymes.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 379
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_134, © Wageningen Academic Publishers 2013
7KH\DOVRGLGQRWFKDQJHIHUPHQWDWLRQSUR¿OHWRZDUGVLQFUHDVHGSURGXFWLRQRIEXW\ULFDFLGZKLFK
is the most important SCFA for colonic epithelium.

7DEOH6KRUWFKDLQIDWW\DFLGVFRQFHQWUDWLRQV —0JRIGLJHVWD LQWKHVHJPHQWVRISLJOHWODUJH

intestine.

Acetic Propionic Isobutyric Butyric Isovaleric Valeric

Caecum
Control 35.8 19.0 0.30 10.5 0.19 1.82ab
IN10 33.4 17.7 0.30 9.3 0.22 b
IN23 37.7  0.35 10.1 0.28 b
JA  18.2 0.30 9.9 0.27 2.72a
CH  19.5 0.30 10.3 0.18 ab
SEM 0.98 0.49  0.41 0.024 0.124
P-value   0.878 0.888 0.589 0.010
Proximal colon
Control 35.3ab 17.9   0.42 2.18ab
IN10 30.8b 15.8  9.1 0.57 b
IN23 39.8a 17.9  11.8 0.73 2.59ab
JA 34.8ab 18.3 0.47 10.9 0.49 3.17a
CH 39.3a 19.2 0.42 11.5  2.22ab
SEM 0.92 0.51 0.032 0.41 0.052 0.151
P-value 0.008 0.285 0.094 0.278 0.174 0.015

IN10 LQXOLQH[WUDFWHGIURPFKLFRU\URRWVZLWKGHJUHHRISRO\PHUL]DWLRQ•,123 = inulin extracted from chicory roots

ZLWKGHJUHHRISRO\PHUL]DWLRQ•-$ GULHG-HUXVDOHPDUWLFKRNH&+ GULHGFKLFRU\
a,bPHDQVZLWKLQDFROXPQZLWKGLIIHUHQWOHWWHUVGLIIHUVLJQL¿FDQWO\ P” 

Acknowledement
)LQDQFLDOVXSSRUWRI7KH1DWLRQDO&HQWUHIRU5HVHDUFKDQG'HYHORSPHQW3URMHFW1R15

References
Halas, D., C.F. Hansen, D.J. Hampson, B.P. Mullan, J.C. Kim, R.H. Wilson and J.R. Pluske, 2010. Dietary supplementation
with benzoic acid improves apparent ileal digestibility of total nitrogen and increases villous height and caecal
PLFURELDOGLYHUVLW\LQZHDQHUSLJV$QLP)HHG6FL7HFK
Kaur, N. and A.K. Gupta, 2002. Applications of inulin and oligofructose in health and nutrition. J. Biosci. 27, 703-714.
/RK*0(EHUKDUG50%UXQQHU8+HQQLJ6.XKOD%.OHHVVHQDQG&&0HWJHV,QXOLQDOWHUVWKH
intestinal microbiota and short-chain fatty acid concentrations in growing pigs regardless of their basal diet. J.
1XWU
Passlack, N., M. Al-Samman, W. Vahjen, K. Männer and J. Zentek, 2012. Chain length of inulin affects its degradation
and the microbiota in the gastrointestinal tract of weaned piglets after a short-term dietary application. Livest.
6FL
Roberfroid, M.B., 2005. Introducing inulin-type fructans. Brit. J. Nutr. 93, Suppl. 1, S13-S25.

380 Energy and protein metabolism and nutrition in sustainable animal production
7UDQVJHQLFÀD[LQKLJKIDWGLHWLQKLELWVLQÀDPPDWRU\VWDWHGHYHORSPHQW
in mice liver
M. Matusiewicz1, I. Kosieradzka1, 0ĩXN2 and J. Szopa2
1Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Warsaw University of
/LIH6FLHQFH±6**:&LV]HZVNLHJR:DUVDZ3RODQG[email protected]
2)DFXOW\RI%LRWHFKQRORJ\:URFáDZ8QLYHUVLW\3U]\E\V]HZVNLHJR:URFáDZ3RODQG

Introduction
Polyphenols are well known as antioxidants very important for health. Polyphenol-rich plants of
ÀD[ Linum usitatissimum L.) were obtained using two strategies: simultaneous overexpression of
WKUHHJHQHVFRGLQJIRUHQ]\PHVRIÀDYRQRLGV\QWKHVLV FKDOFRQHV\QWKDVHFKDOFRQHLVRPHUDVHDQG
GLK\GURÀDYRQROUHGXFWDVH ±OLQH:RURIJHQHFRGLQJIRUWKHHQ]\PHWKDWUHJXODWHVVWDELOLW\
RIWKHVHFRPSRXQGV±JOXFRVHWUDQVIHUDVH±OLQH*7 ĩXNet al., 2011). In comparison with the
QRQWUDQVJHQLFOLQHWKHVHHGFDNHH[WUDFWVRIERWKWUDQVJHQLFÀD[OLQHVZHUHFKDUDFWHUL]HGE\DQ
HQULFKHGFRQWHQWRIÀDYRQRLGV NDHPSIHUROTXHUFHWLQ DQWKRF\DQLQVOLJQDQVDQGSKHQROLFDFLGV
important for antioxidant potential. The objective of the experiment was to determine the effect of
GLHWULFKLQÀD[VHHGFDNHRIWUDQVJHQLFOLQHVRQVHUXPUHGR[VWDWHDVZHOODVRQWKHGHYHORSPHQWRI
WKHLQÀDPPDWRU\VWDWHLQPLFHOLYHU

Material and methods

Research was conducted on male mice that were derived from outbred stock by crossing four inbred
VWUDLQV$6W%1D%$/%FDQG&%/-Q)LYHJURXSVRIPLFHZHUHIHG ad libitum IRU
days one of the isoprotein diets: (1) standard diet; (2) high-fat diet; (3) diet supplemented with 30%
RIVHHGFDNHRIWKHQRQWUDQVJHQLFÀD[OLQH /LQROD   RIVHHGFDNHRIWKHWUDQVJHQLFÀD[OLQH
:RU  RIVHHGFDNHRIWKHWUDQVJHQLFÀD[OLQH*7 7DEOH 7KHGDWDZHUHH[DPLQHGE\
one-way analysis of variance, a PYDOXHZDVFRQVLGHUHGVLJQL¿FDQW

Table 1. Composition of experimental diets and their nutritional value.

Component Group diet

Standard High-fat Linola W92 GT

Flaxseed cake (%) - - 30 30 30

Nutritional value (% DM)
Total protein 20.0 20.1 19.7 19.7 19.5
Crude fat 2.8 12.1  11.2 11.9
GE (kcal/100g)  515 512 510 514
GE from crude fat (kcal/100g) 27 115 110  113

Results and discussion

0LFHIHGWKHGLHWFRQWDLQLQJVHHGFDNHRI*7OLQHGHPRQVWUDWHGVLJQL¿FDQWO\KLJKHUOHYHORIVHUXP
total antioxidant status (TAS), detected by spectrophotometric method, comparing to animals fed
high-fat diet and standard diet. Detected spectrophotometrically, concentration of serum thiobarbituric
DFLGUHDFWLYHVXEVWDQFHV±7%$56WKHSURGXFWVRIR[LGDWLYHGHJUDGDWLRQRIOLSLGVZHUHVLJQL¿FDQWO\
ORZHULQDOORIWKHÀD[JURXSVFRPSDUHGWRKLJKIDWGLHW )LJXUH 

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 381
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_135, © Wageningen Academic Publishers 2013
)LJXUH6HUXPWRWDODQWLR[LGDQWVWDWXV 7$6  D DQGWLREDUELWXULFDFLGUHDFWLYHVXEVWDQFHV 7%$56 
E RIPLFHIHGGLIIHUHQWGLHWV'DWDDUHH[SUHVVHGDVPHDQ“6(0P<

([SHULPHQWVXVLQJ(/,6$WHVWV )LJXUH VKRZHGWKDWWKHOHYHORISURLQÀDPPDWRU\F\WRNLQH71)Į

ZDVWKHVDPHLQÀD[JURXSVWKHOHYHORIWKLVF\WRNLQHZDVGHFUHDVHGLQWKHOLYHURIPLFHIHGOLQH
GT and non-transgenic line compared to high-fat diet but these differences were not statistically
VLJQL¿FDQW7KHFRQFHQWUDWLRQRIWKHRWKHUSURLQÀDPPDWRU\F\WRNLQH,)1ȖZDVWKHVDPHLQÀD[
JURXSVUHJDUGOHVVRIJHQHWLFIRUP6WXGLHVGLGQRWVKRZDQ\GLIIHUHQFHLQFRQFHQWUDWLRQRI,/
and adiponectin between the groups. The level of liver C-reactive protein was the same in mice fed
VHHGFDNHRIDOORIWKHÀD[OLQHVDQGFRQFHQWUDWLRQRIWKLVSURWHLQZDVVLJQL¿FDQWO\VPDOOHULQPLFH
IHGÀD[VHHGFDNHRIOLQH*7WKDQPLFHIHGKLJKIDWGLHW

7KHHIIHFWRIGLHWVXSSOHPHQWHGZLWKRIVHHGFDNHRIH[DPLQHGÀD[WUDQVJHQLFOLQHVRQVHOHFWHG
SDUDPHWHUVRIVHUXPUHGR[VWDWHDQGLQÀDPPDWRU\VWDWHLQPLFHOLYHUPD\UHVXOWIURPWKHFKDQJHV
of biologically-active substances concentration.

)LJXUH&RQFHQWUDWLRQRILQÀDPPDWRU\PDUNHUVLQPLFHOLYHULQWHUIHURQJDPPD ,)1Ȗ  D 
&UHDFWLYHSURWHLQ &53  E 'DWDDUHH[SUHVVHGDVPHDQ“6(0P<

References
ĩXN0$.XOPD/'\PLĔVND.6]RáW\VHN$3UHVFKD-+DQX]D-6]RSD)ODYRQRLGHQJLQHHULQJRIÀD[
potentiate its biotechnological application. BMC Biotechnology 2011 11:10.

382 Energy and protein metabolism and nutrition in sustainable animal production
Part 7. Tissue metabolism
0HFKDQLVPVXQGHUO\LQJYDULDWLRQLQEHHIFDWWOHIHHGHI¿FLHQF\UROHVRI
muscle and adipose tissues.
R.A. Hill1,2, C.M. Welch1, M. McGee1, S. Acharya1, S. Ji1, C.S. Schneider1 and J.K. Ahola
1Department of Animal and Veterinary Science, University of Idaho, ID, USA; [email protected]
286'$5HJLRQDO3URMHFW:,QWHJUDWHG$SSURDFKWR(QKDQFH(I¿FLHQF\RI)HHG8WLOL]DWLRQLQ
Beef Production Systems, USA
Department of Animal Sciences, Colorado State University, CO, USA

Introduction
7KHSRWHQWLDOIRUWKHFRQWULEXWLRQVRIPXVFOHDQGDGLSRVHWLVVXHWRYDULDWLRQLQIHHGHI¿FLHQF\ )( LV
ODUJH%RWKHQHUJ\VWRUDJHDQGHQHUJ\WXUQRYHUPHFKDQLVPVPD\FRQWULEXWHWRYDULDWLRQLQHI¿FLHQW
use of feed energy inputs (Hill and Ahola, 2012). This paper provides a brief review of some of the
PHFKDQLVPVWKDWFRQWULEXWHWRYDULDWLRQLQHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQ

Role of the insulin-like growth factor axis

Due to the relationship between insulin-like growth factor-1 (IGF-I) and linear growth, and the
actions of the IGF axis directly in muscle, the IGF axis has been suggested as a source of potential
SK\VLRORJLFDOLQGLFDWRUVRI)(*HQHH[SUHVVLRQSUR¿OLQJRIGLIIHUHQWLDOO\H[SUHVVHGJHQHVEHWZHHQ
animals divergent for FE may serve to identify potential candidate genes associated with FE. Red
Angus sires divergent for maintenance energy (MEM) expected progeny difference (EPD) and cross-
bred dams were used (AI) to breed progeny across three cohorts (n=222). Progeny plasma IGF-I
(at weaning) was negatively correlated (r=-0.22, P<0.01) with MEM EPD (Welch et al., 2011). In
a sub-set of animals, (n=37) biopsies of biceps femoris muscle were sampled and studied for gene
expression using real time PCR. Expression of IGFBP3 was marginally different (P<0.10) between
the high and low MEM EPD groups, with gene expression levels in the high MEM EPD group
showing marginally greater IGFBP3 expression (1.5 fold) when compared to the low MEM EPD
group. A difference in IGFBP2 (P<0.05) expression was detected between MEM EPD groups, with
gene expression levels in the high MEM EPD group being greater (approximately 1.8 fold) when
compared to the low MEM(3'JURXS6NHOHWDOPXVFOHH[SUHVVHV,*)%3VDQG )ORULQL
et al 7KHPDMRUIXQFWLRQVRIWKHVHELQGLQJSURWHLQVDUHWRDVVLVWLQWKHWUDQVSRUWRI,*)V
both locally and systemically, to prolong the half-life of IGFs and regulate plasma clearance, and to
modulate the interactions of IGFs with their receptors (reviewed in Kokta et al. (2004)). These data
VXJJHVWWKDWPRGXODWLRQRI,*),DWPXVFOHWLVVXHOHYHOPD\DIIHFW,*)VLJQDOLQJDVLQLQHI¿FLHQW
animals, it appears that IGF-binding protein levels are higher and this may result in lower availability
of local IGF-1. Thus, the IGF axis is implicated in variation in energy utilization in muscle.

Role of redox pathways

In addition, we have studied the potential for variation in oxidative pathways to affect myogenic
GLIIHUHQWLDWLRQDQGWKXVWKHHQHUJHWLFHI¿FLHQF\RIPXVFOHJURZWKDQGGHYHORSPHQW5HDFWLYH
oxygen species (ROS) are important regulators of several intracellular signaling pathways. Real
time PCR analysis demonstrated that NADPH oxidase-4 (Nox4) isoform is primarily expressed
in differentiating myogenic cells and contributes to the generation of ROS during differentiation.
Both silencing and over-expression of Nox4 during myoblast differentiation was accompanied by
reduced intracellular ROS concentrations and inhibition of differentiation (P<0.05). Thus, optimum
cellular ROS concentrations appear to be necessary for optimum differentiation and suggests that
physiological regulation of ROS concentrations provides a potential mechanism that underpins
variation in myogenic energy utilization (Acharya et al., 2013).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 385
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_136, © Wageningen Academic Publishers 2013
Leptin pathway
A polymorphism in the leptin gene (Nkrumah et al., 2004) appears to be related to gain in backfat
thickness (3 0.02), ultrasound backfat thickness (3 0.07), and lean meat yield (3 0.007). Further
studies by this research group have indicated that polymorphisms in the leptin promoter may have
potential to predict intake and possibly FE (Nkrumah et al., 2005). Animals having one of the
KRPR]\JRXVJHQRW\SHVVKRZHGVLJQL¿FDQWO\KLJKHUIHHGLQWDNH P<0.001), growth rate, metabolic
BW (P<0.05), and live weight at slaughter (P<0.10). While at another locus, animals with an
homozygous genotype also showed higher feed intake (3 0.001), growth rate (P<0.10), and BW
(P< 7KHUHZHUHVLJQL¿FDQWGLIIHUHQFHVDPRQJWKHGLIIHUHQWJHQRW\SHFRPELQDWLRQVLQ$'*
(3 0.04) and feed conversion ratio (3 0.019) (Nkrumah et al ,QDGGLWLRQZHKDYHVKRZQ
that bovine muscle responds to leptin and insulin in a manner that suggests its ability for differential
substrate utilization consistent with physiology in which there is low circulating glucose (Strat et al.,
2005). Thus, there appear to be mechanisms that link muscle and adipose tissue that have potential
to modulate partitioning of energy substrate utilization, and that genetic variation within the leptin
axis may affect individual animal physiology, resulting in differences in FE.

Conclusion
7KHVHSDWKZD\VSURYLGHWDUJHWVIRUIXUWKHUVWXG\RIHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQLQEHHIFDWWOH
Evidence presented here suggests that there are interacting genetic and physiological bases that may
account for variation in FE, and that these differences manifest in both muscle and adipose and their
functional interactions.

References
Acharya, S., A.M. Peters, A.S. Norton, G.K. Murdoch, and R.A. Hill. 2013. Change in Nox4 expression is accompanied
E\FKDQJHVLQP\RJHQLFPDUNHUH[SUHVVLRQLQGLIIHUHQWLDWLQJ&&P\REODVWV3ÀJHUV$UFKLY(XURS-3K\VLRO
(in press). doi: 10.1007/s00424-013-1241-0.
)ORULQL-5'=(ZWRQDQG6$&RROLFDQ*URZWKKRUPRQHDQGWKHLQVXOLQOLNHJURZWKIDFWRUV\VWHPLQ
myogenesis. Endocrin. Rev. 17: 481-517.
+LOO5$DQG-.$KROD)HHGHI¿FLHQF\LQWHUDFWLRQVZLWKRWKHUWUDLWV*URZWKDQGSURGXFWTXDOLW\,Q5$
+LOO HG )HHGHI¿FLHQF\LQWKHEHHILQGXVWU\S:LOH\%ODFNZHOO
Kokta, T.A., M.V. Dodson, A. Gertler, and R.A. Hill. 2004. Intercellular signaling between adipose tissue and muscle
tissue. Domest. Anim. Endocrinol. 27: 303-331.
Nkrumah, J.D., J.A. Basarab, M.A. Price, E.K. Okine, A. Ammoura, S. Guercio, C. Hansen, C. Li, B. Benkel, B.
0XUGRFKDQG660RRUH'LIIHUHQWPHDVXUHVRIHQHUJHWLFHI¿FLHQF\DQGWKHLUSKHQRW\SLFUHODWLRQVKLSV
with growth, feed intake, and ultrasound and carcass merit in hybrid cattle. J. Anim. Sci. 82: 2451-2459.
1NUXPDK-'&/L'+.HLVOHU(/6KHUPDQ=:DQJ%00XUGRFKDQG660RRUH3RO\PRUSKLVPV
LQWKHOHSWLQJHQHDQGWKHLUDVVRFLDWLRQVZLWKSHUIRUPDQFHIHHGHI¿FLHQF\DQGFDUFDVVPHULWRIEHHIFDWWOHWK
:RUOG&RQJUHVVRQ*HQHWLFV$SSOLHGWR/LYHVWRFN3URGXFWLRQ$XJXVW%HOR+RUL]RQWH0*%UD]LO
Nkrumah, J.D., C. Li, J. Yu, C. Hansen, D.H. Keisler, and S.S. Moore. 2005. Polymorphisms in the bovine leptin
promoter associated with serum leptin concentration, growth, feed intake, feeding behavior, and measures of
carcass merit. J Anim Sci 83: 20-28.
Strat, A.L., T.A. Kokta, M.V. Dodson, A. Gertler, Z. Wu, and R.A. Hill. 2005. Early signaling interactions between the
LQVXOLQDQGOHSWLQSDWKZD\VLQERYLQHP\RJHQLFFHOOV%LRFKLP%LRSK\V$FWD
Welch, C. M., G.K. Murdoch, C.S. Schneider, K. Chapalamadugu, K.J. Thornton, J.K. Ahola, J.B. Hall, and R.A. Hill.
2011. Gene expression of Red Angus sired steers and heifers evaluated for residual feed intake. J. Anim. Sci. 89
(E Suppl. 2): 718.

386 Energy and protein metabolism and nutrition in sustainable animal production
$GLSRVHWLVVXHSUHIHUHQFHVIRUDFHWDWHDQGJOXFRVHE\¿QLVKLQJVWHHUV
W.A.D. Nayananjalie1, T.R. Wiles1, D.E. Gerrard2, M.A. McCann2 and M.D. Hanigan1
1'HSWRI'DLU\6FLHQFH9LUJLQLD3RO\WHFKQLF,QVWLWXWHDQG6WDWH8QLYHUVLW\%ODFNVEXUJ9$
USA; [email protected]
2Dept. of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg,
9$86$

Introduction
Increased marbling enhances the quality grade of beef, and as such, high starch diets are often used
by feedlots to stimulate this type of fat deposition. In vitro studies suggest that adipocytes from
intramuscular fat (IMF) and subcutaneous fat (SCF) may have different preferences for acetate
and glucose (Smith and Crouse, 1984). If such differential precursor preference exists, it should be
connected to differential fat synthesis rates from precursors by different depots. The objectives of
this study were to assess the acetate and glucose turnover rates, palmitate synthesis rate, and acetate
and glucose preference by subcutaneous (SCF), intramuscular (IMF) and visceral adipocytes (VF)
LQ¿QLVKLQJVWHHUV

Material and methods

Experiment was conducted as a complete randomized design. Sixteen, Angus x Simmental steers
ZHUHIHGDFRUQVLODJHEDVHG¿QLVKLQJUDWLRQIRUG(LJKWDQLPDOV “GRIDJHDQG“NJ
BW) were infused continuously with [2H3@DFHWDWH PPROPLQ DQGWKHUHPDLQGHU “GRI
age and 549±20 kg BW) were infused with [U-13C] glucose (0.07 mmol/min) for 12 h immediately
prior to animal harvest. Blood samples were collected before and at the end of infusions and adipose
tissue samples from SCF, IMF and VF depots were collected at slaughter. Lipids were extracted from
tissue samples and palmitate enrichment in lipids, and acetate and glucose enrichment in blood were
determined by GC-MS. Concentrations of DNA in tissue samples were also determined. Plasma
enrichments of acetate and glucose, and palmitate enrichment in each depot were used to calculate
fractional palmitate synthesis rates (FSRs, %/h). Data were analyzed using the GLIMMIX procedure
of SAS and means were separated using Turkey’s test. Further, acetate to glucose preference across
depots was compared using contrast statements. Data were presented with least square means ± SEM.

Results and discussion

&RQFHQWUDWLRQVRI'1$ZHUHVLJQL¿FDQWO\GLIIHUHQWE\GHSRWZKHUH,0)KDGWKHJUHDWHVW'1$
concentrations (143±4.9 ng/mg of tissue) and VF the least (79±4.9 ng/mg of tissue). Acetate turnover
ZDVVLJQL¿FDQWO\JUHDWHUWKDQWKDWRIJOXFRVHWXUQRYHU “PPROPLQPHWDEROLFERG\ZHLJKW
(MBW) vs. 0.04±0.01 mmol/min/MBW). Palmitate FSR from acetate were greater than FSR from
glucose (0.27±0.04%/h vs. 0.02±0.04; P< +RZHYHU)65ZDVQRWVLJQL¿FDQWO\GLIIHUHQWDPRQJ
the depots for either acetate or glucose (P>0.05), and the acetate to glucose preference ratio was
QRWVLJQL¿FDQWO\GLIIHUHQWDFURVVGHSRWV P>0.05). In conclusion, 13 times more acetate was used
for lipid synthesis than glucose, and there were no differences in preference for acetate and glucose
among the major fat depots. Thus, diets leading to high glucose supply will not preferentially direct
energy storage to intramuscular stores.

References
Smith, S.B. and J.D. Crouse, 1984. Relative contributions of acetate, lactate and glucose to lipogenesis in bovine
intramuscular and subcutaneous adipose tissue. J. Nutr. 114: 792-800.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 387
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_137, © Wageningen Academic Publishers 2013
Growth rate in beef cattle affects adipose gene expression and skeletal
PXVFOH¿EHUW\SH
P.A. Lancaster, M.A. Vaughn, J.D. Starkey, E.D. Sharman, C.R. Krehbiel and G.W. Horn
2NODKRPD6WDWH8QLYHUVLW\6WLOOZDWHU2.86$SDODQFDVWHU#XÀHGX
7H[DV7HFK8QLYHUVLW\/XEERFN7;86$

Introduction
*URZWKUDWHLQEHHIFDWWOHDIIHFWVIDWGHSRVLWLRQDQGVNHOHWDOPXVFOH¿EHUFKDUDFWHULVWLFV <DPED\DPED
and Price, 1991), but individual fat depots appear to be regulated by different mechanisms (Ortiz-
Colon et al., 2009). Calkins et al  IRXQGWKDWVNHOHWDOPXVFOH¿EHUW\SHFRPSRVLWLRQH[SODLQHG
RIWKHYDULDWLRQLQPDUEOLQJVFRUHZLWKPRUHR[LGDWLYH¿EHUW\SHVKDYLQJDSRVLWLYHFRUUHODWLRQ
ZLWKPDUEOLQJVFRUH7KXVPDQDJHPHQWVWUDWHJLHVFRXOGEHXVHGWRLQÀXHQFHVNHOHWDOPXVFOH
FKDUDFWHULVWLFVZKLFKFRXOGHQKDQFHLQWUDPXVFXODUIDWGHSRVLWLRQLQWKH¿QLVKLQJSKDVH7KHSXUSRVH
of this study was to evaluate growth rate during the stocker phase on adipose tissue development
DQGVNHOHWDOPXVFOHFKDUDFWHULVWLFVRIJURZLQJ¿QLVKLQJEHHIFDWWOH

Material and methods

7ZRH[SHULPHQWVZHUHFRQGXFWHGXVLQJ$QJXVVWHHUV ([SQ ([SQ  DVVLJQHGWRRI
4 winter grazing treatments in a completely randomized design: (1) control, grazing dormant native
range (DNR) and fed 1.0 kg/d of a 40% CP cottonseed meal-based supplement (CON); (2) grazing
'15DQGIHGDJURXQGFRUQVR\EHDQPHDOEDVHG&3VXSSOHPHQWDWRI%: aNJG 
(CORN); (3) grazing winter wheat pasture (WP) at a high stocking rate (3.21 steers/ha) to achieve a
low rate of BW gain (LGWP); and (4) grazing WP at a low stocking rate (0.99 steers/ha) to achieve
a high rate of BW gain (HGWP). At the end of the stocker phase, a subset of steers was slaughtered
at similar age (Exp. 1; 3 steers per treatment; 138 d) or hot carcass weight (Exp. 2; 4 steers per
WUHDWPHQWDQGGIRUWUHDWPHQWV DQGWKHUHPDLQLQJVWHHUVZHUHIHGDFRPPRQ
¿QLVKLQJGLHWWRDQXOWUDVRXQGSUHGLFWHGULEIDWWKLFNQHVVRIFP$WHDFKKDUYHVWVDPSOHVRI
ORQJLVVLPXVPXVFOHZHUHFROOHFWHGIRU¿EHUW\SLQJE\LPPXQRÀXRUHVFHQFHPLFURVFRS\ ([S
only), and samples of intramuscular (IM) and subcutaneous (SC) adipose tissue were collected for
P51$H[SUHVVLRQRIJHQHVLQYROYHGLQDGLSRJHQHVLV 33$5Ȗ65(%)&(%3ȕDQG3UHI DQG
OLSRJHQHVLV *3'+)$61DQG'*$7 *URZWKFDUFDVVDQG¿EHUW\SLQJGDWDZHUHDQDO\]HG
using a general linear model that included treatment. Gene expression data were analyzed using
MANOVA with a general linear model that included treatment, adipose tissue and the interaction
WHUPZKHQVLJQL¿FDQW/6PHDQVZHUHVHSDUDWHGXVLQJ)LVKHU¶VSURWHFWHG/6' Į  

Results and discussion

At the end of the stocker phase in Exp. 1, marbling score increased linearly with ADG (180, 217, 280,
340 ± 18 for CON, CORN, LGWP, and HGWP, respectively), whereas rib fat thickness increased
DWDQLQFUHDVLQJUDWH DQG“FP 7KHUHZDVQRWUHDWPHQW[DGLSRVHWLVVXH
interaction for adipogenic or lipogenic gene expression indicating that adipose tissues responded
similarly to the treatments (Figure 1). Adipogenic gene expression was lesser (P<0.05) for CON
steers than the other steers and lipogenic gene expression was greater (P<0.05) for LGWP and
HGWP steers than CON and CORN steers. Subcutaneous adipose tissue had greater (P<0.05)
expression of adipogenic and lipogenic genes than IM. In Exp. 2, LGWP and HGWP steers had
greater (P<0.05) marbling score than CON and CORN steers (158, 143, 315, 228 ± 22), but there was
no relationship with ADG. In contrast to Exp. 1, there was a treatment x adipose tissue interaction
(P<0.05) for both lipogenic and adipogenic gene expression. For adipogenic and lipogenic genes,
WUHDWPHQWLQÀXHQFHGH[SUHVVLRQLQ6&EXWQRWLQ,0,Q([SWKH&21VWHHUVWHQGHG P<0.15)

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 389
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_138, © Wageningen Academic Publishers 2013
WRKDYHJUHDWHUSHUFHQWDJHRI7\SH R[LGDWLYH PXVFOH¿EHUVDWLQWHUPHGLDWHKDUYHVW 
DQG“ FRPSDUHGZLWKWKHRWKHUVWHHUVDQGWKHJUHDWHVWFDSLOODU\GHQVLW\ 
DQG“ EXWWKLVZDVQRWVLJQL¿FDQWO\GLIIHUHQWIURPRWKHUVWHHUV$W¿QDOKDUYHVWWKHUH
was no difference (P>0.10) in marbling score among steers in Exp. 1 or 2, and there was no effect
of treatment on adipogenic or lipogenic gene expression in Exp. 2 (data not shown). In Exp. 2,
WKHUHZDVQRGLIIHUHQFHLQPXVFOH¿EHUW\SHVDPRQJVWHHUVDW¿QDOKDUYHVWEXW&21VWHHUVWHQGHG
(P<0.15) to have greater FDSLOODU\GHQVLW\WKDQWKHRWKHUVWHHUV “ 7KHVH
data indicate that rate of gain to similar BW affects metabolic pathways in SC and IM differently
than rate of gain to similar age, and that low rates of gain may result in skeletal muscle characteristics
that are more favorable for IM development.

)LJXUH&DQRQLFDOYDULDWHIURPPXOWLYDULDWHDQDO\VLVRIYDULDQFHRIDGLSRJHQLF $DQG& DQG

OLSRJHQLF %DQG' JHQHH[SUHVVLRQRILQWUDPXVFXODU ,0 DQGVXEFXWDQHRXV 6& DGLSRVHWLVVXH
RIVWHHUVIURPLQWHUPHGLDWHKDUYHVWLQ([S $DQG% DQG &DQG' 
abc0HDQVZLWKLQDWUHDWPHQWRUWLVVXHZLWKRXWDFRPPRQVXSHUVFULSWGLIIHU P< 

References
&DONLQV&575'XWVRQ*&6PLWK=/&DUSHQWHUDQG*:'DYLV5HODWLRQVKLSRI¿EHUW\SHFRPSRVLWLRQ
WRPDUEOLQJDQGWHQGHUQHVVRIERYLQHPXVFOH-)RRG6FL
Ortiz-Colon, G., A.C. Grant, M.E. Doumi and D.D. Buskirk, 2009. Bovine intramuscular, subcutaneous, and perirenal
stromal-vascular cells express similar glucocorticoid receptor isoforms, but exhibit different adipogenic capacity.
J. Anim. Sci. 87:1913-1920.
Yambayamba, E. and M.A. Price, 1991. Fiber-type proportions and diameters in longissimus muscle of beef heifers
undergoing catch-up (compensatory) growth. Can. J. Anim. Sci. 71:1031-1035.

390 Energy and protein metabolism and nutrition in sustainable animal production
First evidence of an insulin-sensitive glucose transporter in chicken:
GLUT-12
E. Coudert1, J. Dupont2, J. Simon1, E. Cailleau-Audouin1, S. Crochet1, M.J. Duclos1, S. Tesseraud1
and S. Métayer-Coustard1
1855HFKHUFKHV$YLFROHV1RX]LOO\)UDQFH[email protected]
280535&1RX]LOO\)UDQFH

Introduction
Facilitated transport of glucose into cells is mediated by a family of facilitative-diffusion glucose
transporter (GLUT) proteins. In mammals, mostly in adipose and muscle tissues, some GLUTs, called
‘insulin-sensitive GLUTs’, are recruited at the plasma membrane in response to insulin. Facilitative-
diffusion glucose transporter-4 is the best characterized (Bryant et al., 2002). So far, no functional ‘insulin-
sensitive GLUTs’ has been characterized in chicken tissues. This species exhibits some peculiarities
for glucose metabolism: a high glycaemia despite the presence of insulin circulating at ‘normal’
concentrations, and a low sensitivity to exogenous insulin, reminiscent of mammalian type-2 diabetes.

The chicken genome database contains several sequences that are suggested as encoding glucose
transporter-like proteins, but none encoding a GLUT-4 homolog. A sequence is predicted as encoding
a chicken GLUT-12 homolog (ENSGALG00000013980); it is located on chromosome 3, comprises
H[RQVDQGHQFRGHVDDPLQRDFLGSURWHLQH[KLELWLQJLGHQWLW\ZLWKKXPDQ*/87
Interestingly, in mammals, GLUT-12 appears to act as an ‘insulin-sensitive GLUT’ in a way
qualitatively similar to GLUT-4 (Stuart et al., 2009).

Material and methods

Tissue distribution of chicken GLUT-12

5RVV0DOHEURLOHUFKLFNHQV Q  ZHUHVODXJKWHUHGDW¿YHZHHNVRIDJHDQGGLIIHUHQWWLVVXHVZHUH
removed and snap frozen into liquid nitrogen.

GLUT-12 mRNA was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR)

LQYDULRXVFKLFNHQWLVVXHVXVLQJVSHFL¿FSULPHUVIRUFKLFNHQ*/87VHTXHQFH

Protein distribution was analyzed by western blot. Tissues lysates were prepared as previously
described and subjected to SDS-PAGE gel electrophoresis and western blotting using a commercial
GLUT-12 antibody directed against a highly conserved region of the corresponding human protein
(87% of identity between human and predicted chicken sequences). After washing, the membranes
were incubated with an Alexa Fluor labeled secondary antibody, and the signals were visualized
using the Odyssey® infrared Imaging System.

GLUT-12 regulation

In order to evaluate the insulin sensitivity of GLUT-12, we used a previously described model of
LQVXOLQLPPXQRQHXWUDOL]DWLRQLQFKLFNHQVDJHGRUGD\V 'XSRQWet al., 2008). Fed chickens
were injected with 3 i.v. injections of anti-insulin serum (5 hr-insulin-immunoneutralization) or
normal serum (Fed state) at 2 h intervals and compared to chickens fasted for 5 hours and injected
with 3 i.v. injections of normal serum (Fasted) (n=7 per experimental group). Muscle samples (leg
and Pectoralis major) were removed and snap frozen. The expression of GLUT-12 was analyzed by
T573&5DQGQRUPDOL]HGZLWKȕDFWLQRUF\WRFKURPHE F\WE 7KHGDWDZHUHVXEMHFWHGWR$129$
WRGHWHFWVLJQL¿FDQWGLIIHUHQFHVDQGWKHPHDQVIXUWKHUFRPSDUHGE\D7XNH\.UDPHUWHVW

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 391
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_139, © Wageningen Academic Publishers 2013
In preliminary studies, GLUT-12 translocation was assessed. Membrane proteins were extracted using
the Promokine kit (Promocell) according to the manufacturer’s recommendations, from muscles of
animal fasted, fed and/ or injected with insulin. The enrichment of GLUT-12 in the plasma membrane
was measured by western blot.

Results and discussion

GLUT-12 mRNA was expressed in chicken skeletal and heart muscles, i.e. ‘insulin sensitive
tissues’ (Figure 1A, lanes 1-4), where an immunoreactive band, showing the expected size, was also
detected (Figure 1B, lanes a-d). No signal was detected in an insulin insensitive tissue, the liver. The
LPPXQRUHDFWLYH*/87ZDVDOVRGHWHFWHGLQSXUL¿HGOHJPXVFOHPHPEUDQHVZKLFKLQFUHDVHG
following insulin administration (data not shown), suggesting translocation of the GLUT-12 protein
to the plasma membrane.

GLUT-12 mRNA expression was further characterized in vivo in chicken muscle using the insulin
LPPXQRQHXWUDOL]DWLRQPRGHO*/87P51$H[SUHVVLRQZDVVLJQL¿FDQWO\ORZHULQIDVWHGDQG
insulin-immunoneutralized conditions compared to the fed condition (Figure 2).

In conclusion, the facilitative-diffusion glucose transporter protein GLUT-12 is present in chicken

muscle and could act as an ‘insulin-sensitive’ transporter in a way qualitatively similar to GLUT-4
in mammals. An extensive characterization of the role of GLUT-12 in chicken is now required.

A B Mouse Chicken
100 pb -
75 kDa -
1 2 3 4 5 6 7 b c d e f g
)LJXUH7LVVXHGLVWULEXWLRQRI*/87 $ P51$H[SUHVVLRQRI*/87LQFKLFNHQWLVVXHV 
3HFWRUDOLVPDMRUPXVFOH6DUWRULXVPXVFOH3RVWHULRU/DWLVVLPXV'RUVLPXVFOHKHDUW
GXRGHQXPEUDLQDQGOLYHU  % 3URWHLQGLVWULEXWLRQRI*/87LQPRXVHPXVFOH D DQGLQ
FKLFNHQ E3HFWRUDOLVPDMRUPXVFOHFOHJPXVFOHGKHDUWHEUDLQIWHVWLVDQGJOLYHU 

)LJXUH*/87P51$H[SUHVVLRQLQOHJPXVFOH $ DQGLQ3HFWRUDOLVPDMRUPXVFOH % RI

FKLFNHQVLQWKHIHGVWDWH LQEODFN IDVWHG LQZKLWH RUDIWHUKULQVXOLQLPPXQRQHXWUDOL]DWLRQ LQ
JUH\ 9DOXHVDUHSUHVHQWHGDVPHDQV“6(07KHPHDQVZHUHFRPSDUHGE\D7XNH\.UDPHUWHVW

References
Bryant, N.J., R. Govers and D.E. James, 2002. Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol.
&HOO%LRO
Dupont, J., S. Tesseraud, M. Derouet, A. Collin, N. Rideau, S. Crochet, E. Godet, E. Cailleau-Audouin, S. Métayer-
Coustard, M.J. Duclos, C. Gespach, T.E. Porter, L.A. Cogburn and J. Simon, 2008. Insulin immuno-neutralization
in chicken: effects on insulin signaling and gene expression in liver and muscle. J Endocrinol. 197(3):531-42.
Stuart C.A., M.A. Howell, Y. Zhang and D. Yin, 2009. Insulin-stimulated translocation of glucose transporter (GLUT)
12 parallels that of GLUT4 in normal muscle. J. Clin. Endocrinol. Metab. 94, 3535-3542.

392 Energy and protein metabolism and nutrition in sustainable animal production
,QÀXHQFHRIPLWRFKRQGULDOIXQFWLRQRQIHHGHI¿FLHQF\RIEURLOHUVZLWK
and without growth enhancing levels of minerals supplementation
during a coccidiosis challenge
G. Acetoze1, R. Kurzbard2, J.J. Ramsey1, K.C. Klasing2 and H.A. Rossow1
1School of Veterinary Medicine, University of California, Davis, CA, USA; [email protected]
2Department of Animal Science, University of California, Davis, CA, USA

Introduction
Mitochondrial conversion of energy as NADH and FADH to ATP is an important contributor to
energy supply accounting for approximately 20-30% of resting energy requirements (Owen et al.,
1978; Zurlo et al 7KHUHIRUHFKDQJHVLQPLWRFKRQGULDOHI¿FLHQF\ZLOOKDYHODUJHLPSDFWV
RQHQHUJHWLFDQGIHHGHI¿FLHQF\ %RWWMHDQG&DUVWHQV %URLOHUFKLFNHQVKDYHPRUHHI¿FLHQW
PXVFOHPLWRFKRQGULDWKDQOD\LQJFKLFNHQVDQGWKLVLVFRUUHODWHGZLWKWKHLUKLJKHUIHHGHI¿FLHQF\
and increased growth rates (Bottje et al., 2002). But, it is unknown how diet (mineral levels) affects
PLWRFKRQGULDOHI¿FLHQF\LQFKLFNHQV,Q)DULVVet al. showed protection from oxidative stress
by administration of antioxidants such as vitamin E and ubiquinone. Copper and Zinc are known as
minerals that have antibiotic and antioxidant properties by reducing the effects of secondary bacterial
infection and macromolecular damage by free radicals through the action of superoxide dismutase
(Nonn et al 8QGHUVWDQGLQJWKHUROHRIPLWRFKRQGULDOHI¿FLHQF\LQIHHGHI¿FLHQF\ZLOODLGLQ
IHHGLQJGHFLVLRQVDQGVHOHFWLRQRIEURLOHUVWKDWDUHPRUHHQHUJHWLFDOO\HI¿FLHQWWRRSWLPL]HQXWULHQW
XVH0RUHHQHUJHWLFDOO\HI¿FLHQWEURLOHUVZLOOUHGXFHIHHGLQWDNHDQGFRVWVRISURGXFWLRQOHDGLQJ
WRLPSURYHGIHHGHI¿FLHQF\DQGVXVWDLQDELOLW\7KLVUHVHDUFKZLOODVVHVVWKHLQÀXHQFHRIIHHGLQJ
LQFUHDVLQJOHYHOVRIDQWLR[LGDQWVXSSOHPHQWV FRSSHURU]LQF RQPLWRFKRQGULDOHI¿FLHQF\DQGIHHG
HI¿FLHQF\LQGD\ROGEURLOHUFKLFNVGXULQJDFRFFLGLRVLVFKDOOHQJH

Material and methods

Sixteen 7 days-old broilers (4 birds/treatment; 2 birds/pen) were randomly assigned to 4 treatments:
DQLQIHFWHGFRQWUROGLHW &XPJNJDQG=QPJNJ Eimeria maxima; 245 mg/kg Cu from
Tribasic Copper Chloride + Eimeria maxima (TBCC); QHJDWLYHFRQWURO &XPJNJDQG=QPJ
kg) – Eimeria maxima; and 2,000 mg/kg Zn from ZnO + Eimeria maxima. The diet was composed of
FRUQVR\EHDQPHDOYHJHWDEOHRLO '0 &3 21.37%, Fat=7.7%, ME=2.89
0FDOGD\ DQGZDVIHGIRUGD\V,QLWLDODQG¿QDOERG\ZHLJKWVDQGGU\PDWWHULQWDNHZHUHFDOFXODWHG
XQWLOWKHFXOOLQJGD\$WGD\RIDJHVWKHELUGVZHUHVDFUL¿FHGE\&2 and immediately dissected.
Approximately 1 gram of liver tissue was used for isolated mitochondria analysis of respiratory control
ratio (RCR), proton leak kinetics and membrane damage. Mitochondria were isolated from liver using
VOLJKWPRGL¿FDWLRQVRIWKHSURFHGXUHVRI&KDSSHOODQG+DQVIRUG  DQG5LFNZRRGet al. (1987) as
previously described (Ramsey et al., 2004). Mitochondrial oxygen consumption was measured using
methods described by Harper et al. (1998) and Lal et al. (2001). All of the mitochondria isolation
procedures are well established and have been sited repeatedly in work measuring mitochondrial
respiration and/or membrane potential. Integrity of the mitochondrial preparations was determined
by measuring RCR, with accepted values greater than 2.0 (State3/State4; Chappell and Hansford,
1972). All measurements were completed in duplicate using mitochondrial protein (1.0 mg/ml)
in incubation medium with 5μM rotenone, 0.4 μg nigericin and 8.0 μg of oligomycin. Data were
analyzed using R software using ANOVA and a Tukey-Kramer test for treatment mean differences.
7KHVWDWLVWLFDOPRGHOXVHGZHUH<LM —;LMȕIJLİLMLQZKLFK<LLVWKHGHSHQGHQWYDULDEOH—LV
WKHRYHUDOOPHDQ;LMDUHWKHFRYDULDWHVIRUWUHDWPHQWLDQGDQLPDOMIJLLVWKH¿[HGHIIHFWRIWUHDWPHQW
ȕLVWKHYHFWRURI¿[HGHIIHFWVDQGİLMDUHWKHUDQGRPHUURUV

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 393
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_140, © Wageningen Academic Publishers 2013
Results and discussion
Average gain/feed values per pen were 1.15 and 1.20 (SEM=0.42) kg feed/kg gain for the treatment
containing 245 mg/kg of Cu from TBCC and treatment containing 2,000 mg/kg ZnO, respectively.
1RVWDWLVWLFDOVLJQL¿FDQFHZDVIRXQGIRUWKHHIIHFWRIWUHDWPHQWRQJDLQIHHGHI¿FLHQF\ 3 0.83).
Oxygen consumption (nmolO2/mg protein/min) in State 3 and State 4 were 28.2 (SD=4.2) and 9.4
6'  IRUWKHQHJDWLYHFRQWUROJURXS 6'  DQG 6'  IRUWKHSRVLWLYHFRQWURO
group, 23.2 (SD=1.4) and 9.4 (SD=0.7) for 245 mg/kg of Cu from TBCC and 28.9 (SD=3.8) and
 6'  IRUPJNJ=Q27KH5&5YDOXHVZHUHDQG 6(0  
for the negative control group, the positive control group, 245 mg/kg of Cu from TBCC and 2,000
mg/kg ZnO, respectively and were different (3 0.02). Levels of minerals (245 mg/kg Cu as TBCC
and 2,000 mg/kg Zn as ZnO) could possibly be decreasing the activity of Eimeria maxima by acting
DVDQWLFRFFLGLDOVSURWHFWLQJWKHFHOOVIURPLQÀDPPDWLRQZKLFKLVDQLPSRUWDQWSURFHVVRQWKH
production of reactive oxygen species production (ROS) which is related to macromolecular damage,
mitochondrial uncoupling, loss of membrane integrity and decrease in oxidative phosphorylation.

References
Bottje, W., Z.X. Tang, M.D. Iqbal, D. Cawthon, R. Okimoto, T. Wang and M. Cooper, 2002. Association of mitochondrial
IXQFWLRQZLWKIHHGHI¿FLHQF\ZLWKLQDVLQJOHJHQHWLFOLQHRIPDOHEURLOHUV3RXOW6FL
%RWWMH:*DQG*(&DUVWHQV$VVRFLDWLRQRIPLWRFKRQGULDOIXQFWLRQDQGIHHGHI¿FLHQF\LQSRXOWU\DQGOLYHVWRFN
VSHFLHV-$QLP6FL((
Chappell, J.B. and R.G. Hansford, 1972. Preparation of mitochondria from animal tissues and yeasts. In: Subcellular
Components: Preparation and Fractionation (ed. Birnie GD). University Park Press, Baltimore. 77-90.
Fariss, M.W., C.B. Chan, M. Patel, B. van Houten and S. Orrenius, 2005. Role of mitochondria in toxic oxidative
stress. Mol. Interven. 5, 98-114.
Harper, M.E., S. Monemdgou, J.J. Ramsey and R. Weindruch, 1998. Age-related increase in mitochondrial proton leak
DQGGHFUHDVHLQ$73WXUQRYHUUHDFWLRQVLQPRXVHKHSDWRF\WHV$P-3K\VLRO((
Lal, S.B., J.J. Ramsey, S. Monemdjou, R. Weindruch and M.E. Harper, 2001. Evidence that caloric restriction lowers
PLWRFKRQGULDOSURWRQOHDNLQVNHOHWDOPXVFOHIURPROGUDWV-*HURQWRO$%%
Nonn, L., R.R. Willians, R.P. Erickson and G. Powis, 2003. The absence of mitochondrial thioredoxin 2 causes massive
DSRSWRVLVH[HQFHSKDO\DQGHDUO\HPEU\RQLFOHWKDOLW\LQKRPR]\JRXVPLFH0RO&HOO%LRO
Owen, O.E., G.A. Reichard, G. Boden, M.S. Parel and V.E. Trapp, 1978. Inter-relationships among key tissues in the
utrilization of metabolic substrate. Adv. Mod. Nutr. 2,517-550.
Ramsey, J.J., K. Hagopian, T.M. Kenny, E.K. Koomson, L. Bevilacqua, R. Weindruch and M.E. Harper, 2004. Proton leak
DQGK\GURJHQSHUR[LGHSURGXFWLRQLQOLYHUPLWRFKRQGULDIURPHQHUJ\UHVWULFWHGUDWV$P-3K\VLRO((
Rickwood, D., M.T. Wilson and V.M. Darley-Usmar, 1987. Isolation and characteristics of intact mitochondria. In:
Mitochondria: A Practical Approach (eds. Darley-Usmar VM, Rickwood D, Wilson MT). IRL Press, Washington,
'&
Zurlo, F., K. Larson, C. Bogardus and E. Ravussin, 1990. Skeletal muscle metabolism is a major determinant of resting
HQHUJ\H[SHQGLWXUH-&OLQ,QYHVW

394 Energy and protein metabolism and nutrition in sustainable animal production
Expression of amino acid transporter in porcine skeletal muscles during
postnatal development
A. Ishida, A. Ashihara, K. Nakashima and M. Katsumata
1,/*67VXNXED-DSDQDLNR#DIIUFJRMS

Introduction
Amino acids are transported across the plasma membrane by the transporters that often overlap in their
VXEVWUDWHVSHFL¿FLWLHV%HFDXVHTXDOLW\RIPHDWWDVWHLQSDUWLFXODULVGHSHQGHQWRQWKHFRQFHQWUDWLRQV
and the composition of amino acids in skeletal muscle (Nishimura and Kato, 1988), we aim to change
the concentrations or the proportion of amino acids in skeletal muscle by regulating amino acid
transporter (AAT) expression in pigs. However, our knowledge on regulation of expression of the
AATs in porcine skeletal muscle is limited (Ai-Min et al., 2010, García-Villalobos et al., 2012, Shi-
Geng et al. 6NHOHWDOPXVFOHLVFRPSULVHGRIP\R¿EUHVXESRSXODWLRQVZKLFKKDYHGLIIHUHQW
PHWDEROLFSURSHUWLHV0RUHRYHUSURSHUWLHVRISRUFLQHP\R¿EUHVFRQWLQXHWRGHYHORSSRVWQDWDOO\
'DYLHV 7KLVOHGXVWRK\SRWKHVL]HWKDWWKHH[SUHVVLRQRIWKH$$7VLVPXVFOHVSHFL¿FDOO\
regulated and its levels change during postnatal development. Therefore, we determined mRNA
expression of amino acid transporters in three distinct skeletal muscles at 5 time points during the
¿UVWZHHNVSRVWQDWDOO\

Material and methods

$WRWDORIPDOHSLJVZHUHVHOHFWHGIURPOLWWHUV SLJVOLWWHUELUWKZHLJKW“NJ 7KHSLJV
were housed under a common practical condition and were fed commercial diets i.e. starting creep
IHHGLQJRQWKHGD\ZHDQLQJRQWKHGD\DQGVWDUWLQJJURZHUPHDORQWKHGD\Longissimus
dorsi, rhomboideus, and biceps femoris muscles were taken from six pigs from each litter at 5
VODXJKWHUWLPHSRLQWVZLWKLQKDIWHUELUWK GD\ROG DQGRQDQGGD\VROG7KH
longissimus dorsi muscle has large proportion of a fast-twitch glycolytic muscle. The rhomboideus
and the biceps femoris muscles are mixed slow- and fast-twitch oxido-glycolytic muscles, and the
rhomboideus muscle has more slow-oxidative muscle than the biceps femoris muscle. Therefore,
WKHVHP\R¿EHUW\SHKDYHGLIIHUHQWFKDUDFWHULVWLFVLQPHWDEROLVPGXHWRGLIIHUHQFHLQP\R¿EHUW\SH
The levels of mRNA expression of the AAT genes (cationic amino acid transporter (Cat)-1; Cat-2,
Cat-3, system y + L-amino acid transporter-1 (y+LAT-1, neutral and cationic amino acid transporter),
sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) (ATB0,+), B0, + type
amino acid transporter (B0,+AT)-1, excitatory amino acid carrier 1 (EAAC1, cationic amino acids
and cysteine transporter), system ASC neutral amino acid transporter (ASCT) 1, system N2 (SN2,
glutamate transporter)) were determined by a real-time RT-PCR method. In order to examine the
effects of growth stage, data normalization was carried out using RPL4, HPRT1 and HMBS as the
reference genes. The Tukey-Kramer multiple comparisons test was used to determine differences
(P<0.05) between the means of the different ages in days.

Results and discussion

Although the mRNA expression of Cat-1 and SN2 on the day 1 was higher than that of the other
time points in all the muscle examined (P<0.05), the mRNA expression of Cat-2 and ASCT1 was
the lowest on the day 1 and increased with the pigs grew (P<0.05). Although the expression of
EAAC 1 was detected in the skeletal muscles, the growth stage did not affect it. The expression of
Cat-3, B0,+AT and ATB0,+ZDVQRWGHWHFWHGRUEHORZWKHOLPLWRIDFFXUDWHTXDQWL¿FDWLRQLQDOOWKH
muscles examined at any time points. Contrary to our hypothesis, the patterns of the change of mRNA
expression of the AATs as the development of the pigs occurred did not differ among the muscles.
Consistent with our hypothesis, the expression of mRNAs of the AATs changed as the pigs grew.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 395
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_141, © Wageningen Academic Publishers 2013
Table 1. The expression pattern of porcine AAT genes in different developmental stages of porcine
skeletal muscles1.

Day 1 12  45 75 Pooled SEM

BW(kg) 1.5  9.2 18.8 39.1

Gene Muscle
Cat-1 LD 1.15a 0.32b 0.30b 0.21b 0.27b 
RH 1.74a 0.85b 0.73b b b 0.04
BF a 0.92b 0.72b 0.59b b 0.13
Cat-2 LD 0.75c 3.22b 3.78ab 3.99ab a 0.13
RH 0.35b 0.83a a 1.05a a 0.03
BF 0.21d 0.89c 1.02bc 1.34ab a 0.04
SN2 LD 1.05a 0.47b 0.37b 0.38b 0.30b 0.04
RH a b b 0.71b 0.54b 0.03
BF 2.23a 0.93b 0.77b b b 0.08
ASCT1 LD 1.55 2.87  2.40 3.17 0.17
RH 0.45b 0.88a 0.85a 0.80ab a 0.05
BF 0.53b 1.21a 1.01ab 0.99ab 1.03a 0.05

1'D\DQGQ GD\Q /' Longissimus dorsi; RH = Rhomboideus; BF = Biceps femoris. Data were
normalized by 3 stably expressed housekeeping genes as described in material and methods.
a,b,c,d9DOXHVZLWKLQDURZZLWKGLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\ P<0.05).

7RRXUNQRZOHGJHWKLVLVWKH¿UVWREVHUYDWLRQRIFKDQJHRIWKHH[SUHVVLRQRIWKHFHUWDLQ$$7VLQ
porcine skeletal muscle during postnatal development. The role of changes in mRNA expression of
the AATs in porcine skeletal muscle during postnatal development need to be established.

References
Ai-Min, Z., Z. Xiang-Yan, Z. Jian-Jun, Z. Shi-Geng, H. Zhi-Yi, W. Xiao-Lan, L. TAO and F. Ding-Yuan, 2010.
Molecular Cloning, Tissue Distribution and Expression of Porcine y[+]L Amino Acid Transporter-1. Asian-Aust.
J. Anim. Sci. 23, 272-278.
'DYLHV$63RVWQDWDOFKDQJHVLQWKHKLVWRFKHPLFDO¿EUHW\SHVRISURFLQHVNHOHWDOPXVFOH-$QW
García-Villalobos, H., A. Morales-Trejo, B. A. Araiza-Piña, J. K. Htoo and M. Cervantes-Ramírez, 2012. Effects of
dietary protein and amino acid levels on the expression of selected cationic amino acid transporters and serum
DPLQRDFLGFRQFHQWUDWLRQLQJURZLQJSLJV$UFK$QLP1XWU
Nishimura, T. and H. Kato, 1988. Taste of free amino acids and peptides. Food Rev. Int. 4, 175-194.
Shi-Geng, Z., Z. Ai-Nlin, Z. Xiang-Yan, Z. Jian-Jun, Y. ZHANG, H. Zhi-Yi, X. Ping-Wen and F. Ding-Yuan, 2009.
Molecular Cloning, Segmental Distribution and Ontogenetic Regulation of Cationic Amino Acid Transporter 2
in Pigs. Asian-Aust. J. Anim. Sci. 22, 712-720.

396 Energy and protein metabolism and nutrition in sustainable animal production
Rate of rumen epithelial adaptation for sodium and short chain fatty
acid absorption
B.L. Schurmann1, M.E. Walpole1, P. Górka1,2 and G.B. Penner1
1'HSDUWPHQWRI$QLPDODQG3RXOWU\6FLHQFH8QLYHUVLW\RI6DVNDWFKHZDQ6DVNDWRRQ6.61$
Canada; EOV#PDLOXVDVNFD
2Department of Animal Nutrition and Feed Management, University of Agriculture in Krakow,
.UDNRZ3RODQG

Introduction
Past studies evaluating ruminal adaptation have largely focused on changes in papillae surface area
and epithelial histology as indicators for adaptation. However, abruptly increasing the proportion of
GLHWDU\FRQFHQWUDWHLQFUHDVHVWKHQHWÀX[RI1D+ (Jnet-Na) with marked changes occurring within one
week of the dietary change (Etschmann et al., 2009). Given that Jnet-Na is driven via an ATP-dependent
electrochemical gradient, the increase in Jnet-Na indicates that there must also be corresponding
increases in energy substrate transport to supply cellular ATP. Presumably, the primary energy
substrates would be from apical absorption of short-chain fatty acids (SCFA; Bergman, 1990).
The objectives of this study were to establish the timeline for SCFA and Na+ absorption across the
ruminal epithelia following an abrupt increase in diet fermentability.

Material and methods

7ZHQW\¿YHZHDQHG+ROVWHLQEXOOFDOYHV “NJ ZHUHUDQGRPO\DVVLJQHGWRRIWUHDWPHQWV
the control (CON; 91.5% hay and 8.5% vitamin and mineral supplement) or G3, G7, G14, and G21
which received the same diet (41.5% barley grain, 50% hay and 8.5% vitamin and mineral) but for
different durations (3, 7, 14 or 21 d, respectively). All calves were provided feed at 2.25% BW at
K5HWLFXODUS+ZDVUHFRUGHGHYHU\PLQIRUKSULRUWRNLOOLQJDWK5XPLQDOÀXLGZDV
collected to determine the concentration of acetate, butyrate, and propionate, and ruminal tissue from
the caudal dorsal blind sac was collected and prepared for mounting in Ussing chambers. Tissues
ZHUHLQFXEDWHGXQGHUVKRUWFLUFXLWFRQGLWLRQVZLWKDPXFRVDODQGVHURVDOEXIIHUS+RIDQG
UHVSHFWLYHO\7KHPXFRVDOWRVHURVDOÀX[RI3H-acetate (JMS-acetate; 100 kBq/15 ml) and 14C-butyrate
(JMS-butyrate; 72 kBq/15 ml) were measured in parallel. To evaluate the pathway of SCFA absorption
(Aschenbach et al., 2009), incubation buffer solutions contained HCO3-, were free of HCO3-, or
were free of HCO3- and contained nitrate (40 mM) in order to determine the bicarbonate-dependent,
bicarbonate-independent, bicarbonate-independent nitrate-sensitive, and bicarbonate-independent
QLWUDWHLQVHQVLWLYHÀX[7KH-net-Na was measured by difference using the JNa-MS and JNa-SM (80
kBq 22Na+/15 ml) for tissues paired only when tissue conductance differed by <20%. Data were
analyzed as a randomized complete block design using the Mixed Procedure of SAS (version 9.2,
Cary, NC, USA) using polynomial contrasts to test whether linear, quadratic, or cubic responses
occurred over time.

Results and discussion

Reticular pH decreased (quadratic P< IURP&21  WR*  WKHQLQFUHDVHGWR*
6(0  7RWDO6&)$FRQFHQWUDWLRQDQGWKHPRODUSURSRUWLRQRISURSLRQDWHZHUHQRW
affected with means of 84.1 mM (SEM=1.704) and 20.4 mol/100 mol (SEM=0.402), respectively.
The molar proportion (mol/100 mol) of acetate decreased (cubic 3 0.008 and butyrate increased
(quadratic 3 0.008) from CON to G21.

The JMS-Na and JSM-Na were not affected, but JNet-Na increased cubically (P< IURPIRU&21
WR—PRO FP2 × h) for G21, with the highest JNa-Net (2.41 μmol/(cm2 × h)) for G7, supportive

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 397
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_142, © Wageningen Academic Publishers 2013
of previous research (Etschmann et al., 2009; Table 1). Total and bicarbonate-independent JMS-acetate
increased from CON to G7 but decreased thereafter (cubic, P<0.05). A quadratic increase (P<0.05)
in the bicarbonate-independent nitrate-insensitive JMS-acetateZDVREVHUYHGZLWKÀX[UDWHVRIIRU
CON, 0.59 for G7 and 0.52 μmol/(cm2 × h) for G21. Total JMS-butyrate increased linearly (P<0.05)
from CON to G21. The bicarbonate-independent nitrate-insensitive JMS-butyrate increased quadratically
(P<0.05) from 0.74 for CON to 1.12 μmol/(cm2 × h) for G21, with maximum uptake at 1.17 for G14.

These results indicate that the ruminal epithelium rapidly adapts to changes in diet fermentability
and that increases in SCFA absorption occur in concert with increases in Na+ absorption. It appears
that much of the increase for SCFA absorption is via lipophilic absorption.

Table 1. SCFA concentrations and Na, acetate, and butyrate absorption rates.

Item Treatment1 SEM Contrasts1

CON G3 G7 G14 G21

Na uptake, μmol/(cm2 × h)
Net4   2.41 1.58   0.042C
Mucosal-serosal 1.89 2.81 3.40  2.90 0.141 NSA
Serosal-mucosal 0.73 1.13 0.99 1.03 1.24  NS
Acetate uptake, μmol/(cm2 × h)
Total 1.07 1.12 1.31 1.02 1.14 0.078 0.045C
Bicarbonate-dependent  0.47  0.48 0.52 0.099 NS
Bicarbonate-independent   0.71 0.55   C
Nitrate sensitive 0.09  0.12 0.04 0.10  NS
Nitrate insensitive 0.37 0.49 0.59 0.51 0.52 0.052 0.043Q
Butyrate uptake, μmol/(cm2 × h)
Total 1.25 1.35 1.53 1.53 1.72  0.002L
Bicarbonate-dependent 0.57 0.47 0.38 0.55 0.70  NS
Bicarbonate-independent  0.88 1.15 0.98 1.02 0.133 NS
Nitrate sensitive  0.04  -0.19 0.09 0.132 NS
Nitrate insensitive 0.74 0.84 1.09 1.17 1.12  0.005Q

1 L = linear; Q = quadratic; C = cubic; NS = all P-YDOXHVZHUHQRWVLJQL¿FDQW P>0.05).

References
Aschenbach, J.R., S. Bilk, G. Tadesse, F. Stumpff and G. Gabel, 2009. Bicarbonate-dependent and bicarbonate-
independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep. Am. J.
3K\VLRO*DVWURLQWHVW/LYHU3K\VLRO**
Bergman, E.N. 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species.
3K\VLRO5HY
Etschmann, B., A. Suplie and H. Martens, 2009. Change of ruminal sodium transport in sheep during dietary adaptation.
$UFKLYHVRI$QLP1XWU

398 Energy and protein metabolism and nutrition in sustainable animal production
5XPLQDQWVSHFL¿FPROHFXODUDQGV\VWHPLFDGDSWDWLRQRIUHQDOHOHFWURO\WH
handling to low N intake
S. Starke1, C. Cox2, K.-H. Südekum2 and K. Huber1
1'HSDUWPHQWRI3K\VLRORJ\8QLYHUVLW\RI9HWHULQDU\0HGLFLQH+DQQRYHU%LVFKRIVKROHU'DPP
+DQQRYHU*HUPDQ\[email protected]
2,QVWLWXWHRI$QLPDO6FLHQFH8QLYHUVLW\RI%RQQ(QGHQLFKHU$OOHH%RQQ*HUPDQ\

Introduction
For economical and ecological reasons precise protein rationing is essential in the nutrition of
ruminant livestock. This dietary adjustment should consider the endogenous capacity to recycle
urea-N for maintaining rumen microbial protein synthesis. However, results of our previous study
showed that despite urea recycling, dietary intake of a low N diet affected not only N metabolism
but also systemic and renal molecular electrolyte handling in growing male Saanen-type goats
(Starke et alXQSXEOLVKHGGDWD :HK\SRWKHVL]HGWKDWWKHVHFKDQJHVZHUHDOVRUHÀHFWHGE\FKDQJHV
in electrolyte excretion in vivo. Therefore, the main objective of the present study was to evaluate
the impact of adaptive changes due to low crude protein (LCP) intake on electrolyte handling and
excretion in vivo LQJURZLQJJRDWV7RH[DPLQHZKHWKHUWKLVDGDSWDWLRQLVUXPLQDQWVSHFL¿FDVLPLODU
H[SHULPHQWZDVFRQGXFWHGRQJURZLQJUDWVFRQVXPLQJD/&3GLHWLQFRQVLGHUDWLRQRIWKHVSHFL¿F
differences in nutrition, digestive physiology and metabolism of rats and goats.

Material and methods

7ZHOYHPDOH6DDQHQW\SHJRDWV DQLPDOVSHUJURXS DQGPDOH:LVWDUUDWV DQLPDOVSHUJURXS 
were fed diets either high or low in CP content (as-fed basis: HCP: 20%, LCP: 8%) for 5 weeks.
During the 5thZHHN13DQG&DEDODQFHWULDOVZHUHSHUIRUPHGRYHUGD\VLQJRDWVDQGGD\V
LQUDWV'LUHFWO\DWVDFUL¿FLQJEORRGVDPSOHVZHUHFROOHFWHGWRPHDVXUHXUHD&DSKRVSKDWH 3L 
insulin like growth factor-1 (IGF-1), 1,25-dihydroxyvitamin D3 (calcitriol), CrossLaps (CTX) and
parathyroid hormone (PTH) concentrations. Furthermore, renal expression of the sodium-dependent
Pi transporter types IIa and IIc (NaPi IIa and IIc) and the PTH receptor (PTHR) was determined
VHPLTXDQWLWDWLYHO\E\:HVWHUQ%ORWWLQJ$VVRFLDWLRQVEHWZHHQWKHPHDVXUHGYDULDEOHVZHUHLGHQWL¿HG
by linear regression analyses. Differences between the feeding groups within one species were tested
by unpaired Student’s t-test, data comparing the two species were analysed for interaction between
species and feeding by Two-Way ANOVA.

Results
3ODVPDXUHDFRQFHQWUDWLRQ PPROOJRDWV+&3/&36(0P<0.001, rats: HCP
/&36(0P<0.01) and urinary N excretion (g/d; goats: HCP 7.17, LCP 1.38, SEM
0.24, P<0.001, rats: HCP 0.47, LCP 0.13, SEM 0.02, P<0.001) decreased in both species due to
LCP intake, whilst daily amounts of retained N were unaffected. In goats, plasma IGF-1 (P<0.01)
and, by trend, calcitriol (P<0.1) concentration decreased (Figure 1A), but Ca and P excretion and
retention (g/d) were unchanged. Furthermore, renal PTHR protein expression declined (P<0.05).
Changes of the modulators of systemic electrolyte handling, IGF-1 and calcitriol, were correlated
with changes of other modulators of systemic and also of renal molecular electrolyte handling, like
e.g. PTH (calcitriol negatively correlated with PTH; r2=0.38, P<0.1) and NaPi IIc (IGF-1 negatively
correlated with NaPi IIc; r2=0.44, P<0.05). In rats, contrary to goats, plasma IGF-1 and calcitriol
concentrations did not change due to LCP intake (Figure 1B). However, daily urine excretion (g/d;
HCP 14.41, LCP 9.28, SEM 1.22, P<0.001) and daily urinary P excretion (g/d; HCP 0.037, LCP
0.024, SEM 0.003, P<0.01) decreased. Furthermore, urinary Ca concentration increased (g/kg; HCP
0.05, LCP 0.08, SEM 0.008, P<0.001). Despite these changes, the renal expression of the detected

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 399
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_143, © Wageningen Academic Publishers 2013
)LJXUH3ODVPD,*)DQGFDOFLWULROFRQFHQWUDWLRQVLQ $ JRDWVDQG % UDWVGXULQJ/&3LQWDNH
FRPSDUHGWRWKHUHVSHFWLYH+&3JURXS P< P<

proteins remained unchanged. In rats mainly renal molecular modulators of electrolyte homeostasis,
like PTHR and NaPi IIc, were correlated with each other.

By direct comparison, in goats with LCP intake plasma urea concentrations decreased stronger than
in rats (feeding: P<0.001, species: P<0.05, interaction: P<0.05), accompanied by a greater reduction
of urinary and fecal N excretion than in rats, when related to metabolic body size. Furthermore,
urinary Ca excretion during LCP intake was more decreased in goats than in rats (feeding: P<0.05,
species: P<0.001, interaction: P<0.05; Figure 2).

)LJXUH $ &RPSDULVRQRIXULQDU\&DH[FUHWLRQUHODWHGWRPHWDEROLFERG\VL]HLQUDWVDQGJRDWV
GXULQJ/&3LQWDNH % 3HUFHQWDJHGLIIHUHQFHRIXULQDU\&DH[FUHWLRQ H[FU RIWKH/&3JURXSVWR
WKHLUUHVSHFWLYH+&3JURXSLQUDWVDQGJRDWV P<

Summary and conclusion

7KHPDLQ¿QGLQJVRIRXUVWXG\ZHUH
1. Both rats and goats exhibited adaptive changes of N metabolism and electrolyte homeostasis due
to LCP intake. Particularly the regulation of Ca homeostasis appeared to underlie these adaptive
changes.
2. Only in goats, LCP intake decreased concentration of the regulatory hormone IGF-1.
3. Only in goats the observed adaptations resulted in changes in the daily in vivo Ca excretion
related to metabolic body size.

7KHVHUHVXOWVFRQ¿UPWKHH[LVWHQFHRIDJRDWVSHFL¿FDGDSWDWLRQRI1PHWDEROLVPDQGV\VWHPLFDQG
renal electrolyte handling to low N intake.

400 Energy and protein metabolism and nutrition in sustainable animal production
1XWULHQWXWLOL]DWLRQGXULQJLQÀDPPDWLRQGLIIHUVEHWZHHQSLJVVHOHFWHG
IRUGLIIHUHQFHVLQIHHGHI¿FLHQF\
E. Labussière, E. Merlot, J.-N. Thibault, J. Noblet, N. Le Floc’h and J. van Milgen
,15$8053HJDVH6DLQW*LOOHV)UDQFH[email protected]
$JURFDPSXV2XHVW8053HJDVH5HQQHV)UDQFH

Introduction
When based on productive traits, genetic selection does not account for ‘non-productive functions’
such as the animal’s defense systems. Two lines of pigs were divergently selected with a low (RFI-)
or a high (RFI+) residual feed intake (RFI), which is calculated as the difference between the actual
and the theoretical feed intake required for maintenance and growth (Gilbert et al., 2007). In healthy
growing animals, the immune system has low nutritional requirements, which can dramatically
LQFUHDVHGXULQJLQÀDPPDWLRQ:KHWKHUVHOHFWLRQIRU5),DIIHFWVWKHDELOLW\WRSDUWLWLRQQXWULHQWV
between growth and immune response is not known. The objective of this study is to compare results
obtained in 2 experiments, where the effect of RFI on energy and protein utilization was evaluated
LQSLJVVXEMHFWHGWRDQLQÀDPPDWRU\FKDOOHQJH

Material and methods

$FKURQLFOXQJLQÀDPPDWLRQZDVLQGXFHGWKURXJKDQLQWUDYHQRXVLQMHFWLRQRI&RPSOHWH)UHXQG¶V
Adjuvant (CFA; Melchior et al. ,Q([SWKHHIIHFWVRILQÀDPPDWLRQRQUHFWDOWHPSHUDWXUH
SODVPDJOXFRVHKDSWRJORELQDQG,)1ȖFRQFHQWUDWLRQVKDIWHUWKHPHDOZHUHPHDVXUHGLQSLJVIURP
both lines (n=20; mean body weight – BW: 15-20 kg) from the day before to the 7th day following
CFA. On the 8thGD\IROORZLQJ&)$LQMHFWLRQSURWHLQV\QWKHVLV XVLQJDÀRRGLQJGRVHRI>15N]valine)
and proteolytic enzyme activities in the liver and longissimus dorsi ZHUHTXDQWL¿HGDQGLQÀDPPDWRU\
cytokines mRNA in the pulmonary lymph nodes were measured. In Exp. 2 (from 3 day before to
GD\DIWHU&)$LQMHFWLRQ WKHHIIHFWVRIJHQRW\SHDQGLQÀDPPDWLRQRQKHDWSURGXFWLRQ +3 
respiratory quotient (RQ), N and energy balance and oxidation of dietary [U-13C]glucose (measured
as 24 h-13CO2 production on the day before and on the 3rd day after CFA) were measured in pigs
from both lines (n=13; BW: 15-30 kg) that were housed individually in a respiration chamber. In
both experiments, pigs were offered a commercial starter diet that contained 20.9% CP and 14.9 MJ
ME/kg. In Exp. 1, pigs were offered feed ad libitum whereas they were restricted at 1.72 MJ ME/
kg BW/d in Exp. 2 (0.34 on the day of CFA). Data were analyzed (PROC MIXED, SAS, 2004)
for the effects of genotype, day and interaction in Exp. 1 and for genotype, period (before or after
CFA) and interaction in Exp. 2 according to a mixed linear model. In Exp. 2, RQ was analyzed for
the effects of genotype, day as repeated measurement and interaction.

Results and discussion

In both experiments, feed intake did not differ between genetic lines. Plasma haptoglobin concentration
was higher (P<0.05) in both lines up to 7 days after CFA but did not differ between RFI lines (Exp.
1). Rectal temperature increased during 3 days following CFA and was higher (P<0.05) on the
GD\RI&)$LQ5),WKDQLQ5),SLJV YV“ƒ& $WVODXJKWHU,/,/DQG,/
P51$H[SUHVVLRQLQSXOPRQDU\O\PSKQRGHVDQGSODVPD,)1ȖOHYHOVZHUHORZHU P<0.10) in
5),WKDQLQ5),SLJVVXJJHVWLQJWKDWWKHLQÀDPPDWLRQZDVOHVVVHYHUHLQ5),SLJVRUWKDWWKH\
recovered faster, in agreement with the lower proteolytic enzyme activities in the muscle. Differences
in utilization of nutrients were observed between the two lines before CFA: HP and oxidation of
dietary glucose was lower in RFI- than in RFI+ pigs although glucose was oxidized at a faster rate
in RFI- than in RFI+ pigs (Exp.2, Table 1). In contrast to RFI+ pigs, the RQ in RFI- pigs was lower
on the day following CFA (1.03) than that measured before or on the 2nd or the 3rd days after CFA

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 401
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_144, © Wageningen Academic Publishers 2013
injection (1.08 on average). Accordingly, plasma glucose concentration (Exp. 1, Figure 1) and
dynamic patterns of glucose oxidation on the 3rd day following CFA injection were not affected by
WKHLQÀDPPDWLRQLQ5),SLJVZKHUHDVWKHVHSDUDPHWHUVZHUHPRGL¿HGLQ5),SLJV ([S7DEOH
 ,QFRQFOXVLRQWKHVHUHVXOWVLQGLFDWHWKDWWKHHIIHFWVRILQÀDPPDWLRQRQQXWULHQWXWLOL]DWLRQDQG
metabolism were expressed to a greater extent in RFI- pigs during the 1st day, whereas the effects
lasted longer in RFI+ pigs.

7DEOH(IIHFWRIJHQRW\SH * SHULRG 3 DQGLQWHUDFWLRQ 3î* RQKHDWSURGXFWLRQDQGNLQHWLFV

RIR[LGDWLRQRIGLHWDU\>8C]glucose.

Genotype RFI- RFI+ Rsd 6LJQL¿FDQFH

Period Before After Before After

CFA CFA CFA CFA

Heat production (% of ME intake)     2.1 G*

Respiratory quotient 1.08 1.07  1.08 0.01 P×G*
13C recovery as 13CO (% of intake) 48.0 50.2  49.3 2.4 P×G*
2
Time to recover 50% of 13CO2 (h) y 3.7y 4.8x 3.9y 0.2 P**,G*,P×G**

*P<0.05; **P<0.01.
xy LS-means with different superscript letters within the same row differ P<0.05.

1,300
concentration (mg/L)
Plasma glucose

1,100

900
*
700 *
*
500
-1 1 3 5 7
Day (relative to induction of
inflammation)
)LJXUH(IIHFWRIJHQHWLFVHOHFWLRQ 5),LQEODFNDQG5),LQJUH\ RQSODVPDJOXFRVHFRQFHQWUDWLRQ
PHDVXUHGWZRKRXUVDIWHUIHHGLQJEHIRUHRUDIWHULQGXFWLRQRILQÀDPPDWLRQ ([SLQWHUDFWLRQ
EHWZHHQJHQRW\SHDQGGD\ZDVVLJQL¿FDQWP= P<IRU5), 

Acknowledgements
The study was funded by the French National Research Agency (L’Agence Nationale de la Recherche,
ANR, ANR-08-GENM038 PIG_FEED Project).

References
Gilbert H., J.P. Bidanel, J. Gruand, J.C. Caritez, Y. Billon, P. Guillouet, H. Lagant, J. Noblet and P. Sellier, 2007. Genetic
parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat
quality traits. J. Anim. Sci 85, 3182-3188.
0HOFKLRU'%6HYHDQG1/H)ORF¶K&KURQLFOXQJLQÀDPPDWLRQDIIHFWVSODVPDDPLQRDFLGFRQFHQWUDWLRQVLQ
pigs. J. Anim. Sci 82, 1091-1099.
SAS 2004. SAS/STAT® 9.1 User’s Guide.

402 Energy and protein metabolism and nutrition in sustainable animal production
Differential protein deposition in tissues of growing Iberian and
Landrace × Large White pigs under identical nutritional management
R. Nieto, R. Barea, L. Lara, R.A. Márquez and J.F. Aguilera
,QVWLWXWHRI$QLPDO1XWULWLRQ(VWDFLyQ([SHULPHQWDOGHO=DLGtQ6SDQLVK&RXQFLOIRU6FLHQWL¿F
5HVHDUFK &6,& &DPLQRGHO-XHYHVVQ$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
The present work is part of an experimental program aimed at explaining the biological basis for the
ORZHUPHWDEROLFHI¿FLHQF\RIWKH,EHULDQSLJDVORZJURZLQJQDWLYHREHVHSLJZKHQFRPSDUHGWR
conventional lean pig types (Barea et al., 2011).

As a key process for growth, we have focused on protein deposition (PD). Previous work in the
literature suggests that pig genotypes with different potential for lean growth might differ not only in
WKHUDWHRI3'EXWDOVRLQWKHHI¿FLHQF\RIXVHRIGLHWDU\SURWHLQIRU3' )XOOHU et al., 1995), although
there are contradictory results regarding this issue (Kyriazakis and Emmans, 1995). Additionally,
WKHGLVWULEXWLRQRI3'DPRQJWLVVXHVZLWKGLIIHUHQWPHWDEROLFUROHVPD\LQÀXHQFHWKHHI¿FLHQF\RI
whole body (WB) PD. To investigate further these aspects, we performed a comparative study with
Iberian and Landrace × Large White (LRW) pigs to assess genotype differences in PD at the WB,
carcass and viscera (with clearly different body functions) level during two stages of growth and
under identical nutritional management.

Material and methods

The experiment started with 19 Iberian and 19 LRW pigs. Three pigs per genotype were slaughtered
DWWKHEHJLQQLQJRIWKHVWXG\ “NJ%: WRDVVHVVLQLWLDOERG\FRPSRVLWLRQ7KHUHPDLQLQJ
32 pigs were fed isoenergetic diets (14.4 MJ ME/kg DM) containing either 130 or 170 g CP/kg
DM to match protein requirements for each breed. These CP contents were maintained throughout
the experiment for simplicity. Eight pigs per genotype were allocated to each CP level. Pigs were
individually housed in an environmentally controlled room. Daily feed allowance was adjusted
RQDZHHNO\EDVLVDFFRUGLQJWR%:DQG¿[HGDWîad libitum intake of the Iberian pig breed.
$W“NJ%:SLJV JHQRW\SH&3OHYHO ZHUHVODXJKWHUHGZKHUHDVWKHUHVWRISLJV  
FRQWLQXHGWKHVWXG\XSWRVODXJKWHUDW“NJ%:$IWHUVODXJKWHUWKHJDVWURLQWHVWLQDOWUDFW
was emptied and weighed as well as the rest of visceral organs. Four components were obtained for
each pig (carcass, head and feet, viscera, and blood) which were kept at -20 °C until analyses. The
right half of the carcass and the rest of body components were separately ground, homogenized and
sub-samples were freeze-dried and analysed for nutrient composition. The results were subjected
to ANOVA for a factorial arrangement of treatments, with included genotype (G), growth stage (P:
15 to 50 and 50 to 115 kg BW), dietary CP content and their interactions using the GLM procedure
of SAS. The experimental protocol was approved by the CSIC Bioethical Committee.

Results and discussion

Growth rate was higher for LRW pigs during both growing periods (Table 1; PG<0.001). The effect
RIGLHWDU\&3LVQRWVKRZQDVLWZDVQRWVLJQL¿FDQWIRUPRVWRIWKHSDUDPHWHUVDQDO\VHG2QDYHUDJH
WB and carcass PD in LRW pigs was from 0.8 to >2 fold higher than in Iberian pigs (PG<0.001).
No effect of growth stage on WB and carcass PD was detected for Iberian pigs, whereas in LRW
both parameters increased (PG×P<0.001) from 19 to 50% in the heavier pigs (P<0.05). Average PD
in carcass relative to WB was higher in LWR pigs (PG<0.001) and increased in the heavier pigs of
both pig genotypes. On the contrary, PD in visceral tissues relative to total PD was always greater
in Iberian pigs (0.117 vs. 0.08 for Iberian vs. LWR pigs, PG 7KHHI¿FLHQF\RI3' :%3'

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 403
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_145, © Wageningen Academic Publishers 2013
7DEOH3URWHLQGHSRVLWLRQ 3' DWZKROHERG\ :% FDUFDVVDQGYLVFHUDOHYHORI,EHULDQDQG
/DQGUDFHî/DUJH:KLWH /5: SLJVJURZLQJIURPWRNJ%: 3 DQGIURPWRNJ%:
3 

Iberian LRW SEM P-value1

P1 P2 P1 P2 G P G×P

ME intake, MJ/d 21.1 38.0 20.9 37.4 0.3 0.458 0.001 0.744
Protein intake, g/d 235 499  500 3 0.491 0.001 0.447
Growth rate, g/d 445  594 908 7 0.001 0.001 0.001
WB PD, g/d 53.8 48.2 91.5 118.5 2.2 0.001 0.022 0.001
Carcass PD, g/d 38.3 35.7 71.3 98.3 1.9 0.001 0.004 0.001
Viscera PD, g/d  5.59 8.30 8.31 0.31 0.001  
Carcass PD/WB PD 0.711 0.741 0.778 0.829  0.001 0.002 0.385
Viscera PD/WB PD 0.117 0.117 0.094  0.003 0.001  0.073
WB PD/digestible protein 0.314 0.127 0.527 0.307 0.008 0.001 0.001 0.329
intake

1 G = genotype.

to protein ingested or to digestible protein) was considerably lower for Iberian pigs at both stages
of growth (PG<0.001).

Our results show relevant genotype differences in PD pattern during growth that can contribute to
H[SODLQWKHORZHUPHWDEROLFHI¿FLHQF\RIWKH,EHULDQSLJZKHQFRPSDUHGWRFRQYHQWLRQDOSLJW\SHV

Acknowledgements
)XQGLQJE\WKH6SDQLVK0,1(&2 JUDQW$*/ LVJUDWHIXOO\DFNQRZOHGJHG

References
Barea, R., R. Nieto, F. Vitari, C. Domeneghini and J.F. Aguilera, 2011. Effects of pig genotype (Iberian vs.
Landrace×Large White) on nutrient digestibility, relative organ weight and small intestine structure at two stages
of growth. Animal 5, 547-557.
Fuller, M.F., M.F. Franklin, R. McWilliam and K. Pennie, 1995. The response of growing pigs, of different sex and
JHQRW\SHWRGLHWDU\HQHUJ\DQGSURWHLQ$QLP6FL
.\ULD]DNLV,DQG*&(PPDQV'REUHHGRISLJGLIIHULQWKHHI¿FLHQF\ZLWKZKLFKWKH\XVHDOLPLWLQJSURWHLQ
supply? Br. J. Nut. 74, 183-195.

404 Energy and protein metabolism and nutrition in sustainable animal production
Intravenous administration of arginine to twin-bearing ewes enhances
birth weight and peri-renal fat stores of female offspring in sheep
S. McCoard1, F. Sales1, N. Wards1, Q. Sciascia1, M. Oliver2, J. Koolaard1 and D. van der Linden1
1$J5HVHDUFK /LPLWHG 7HQQHQW 'ULYH 3ULYDWH %DJ  3DOPHUVWRQ 1RUWK 1HZ =HDODQG
[email protected]
2The Liggins Institute, University of Auckland, Auckland, New Zealand

Introduction
$GYDQFHVLQJHQHWLFVHOHFWLRQDQGEUHHGLQJKDYHVLJQL¿FDQWO\LQFUHDVHGWKHSURSRUWLRQRIPXOWLSOH
bearing pregnancies in sheep. However, competition between littermates in mid to late gestation
leads to lower birth weight and increased mortality compared to their singleton counterparts, even
when ewes are fed on a high plane of nutrition (McCoard et al., 2000; Wu et al., ,QWHUYHQWLRQ
strategies are not currently available to ameliorate the effect of fetal growth restriction in utero that
results from multiple-bearing pregnancies. Recent research in sheep has indicated that maternal
supplementation with L-arginine in mid-late gestation can relieve the restriction on fetal growth
induced by under-feeding or increased litter number (quadruplets; Lassala et al., 2011, 2012). The
objective of this study was to evaluate the effects of parenteral administration of L-arginine to well-
fed twin-bearing ewes from 100 days gestation to birth on lamb birth weight and body composition.

Material and methods

Fifty synchronized Romney ewes were naturally mated to one of two sires to minimize paternal
JHQHWLFHIIHFWVRQVL]HDQGZHLJKWRIIHWXVHV7ZLQEHDULQJHZHVZHUHLGHQWL¿HGIROORZLQJSUHJQDQF\
GLDJQRVLVDWGD\VSRVWPDWLQJ(ZHVZHUHDFFOLPDWL]HGWRLQGRRUKRXVLQJDWGD\VRIJHVWDWLRQ
(P80) and fed a lucerne-based pellet diet to meet 100% of NRC feeding recommendations. Between
P100 and birth, ewes received an i.v. bolus of either L-arginine-mono-hydrochloride (Arg; 345 μmol/
kg BW) or sterile saline solution (control) 3 times a day via a catheter placed in the tarsal vein. At
P140, 20 ewes (Arg group n=9; control group n=11) were euthanized and fetal mass and organ mass
evaluated. The remaining ewes (Arg group n=13; control group n=12) were allowed to lamb and
lamb birth weight was recorded. Blood samples were collected from the ewes and fetuses 1 hour
post-bolus administration at P140 and from ewes and lambs 2 hours post-birth. Plasma amino acids
were determined by ion-exchange chromatography as previously described (van der Linden et al.,
2012). Amino acid data were analysed using the nlme package in R. All other data were analyzed
XVLQJWKH0,;('SURFHGXUHLQ6$6/LQHDUPRGHOVZHUH¿WWHGDQGLQFOXGHGWKH¿[HGHIIHFWVRI
treatment group (arginine vs. control) for maternal data. The linear model for lamb/fetal data included
WKH¿[HGHIIHFWRIWUHDWPHQWJURXSVH[RIODPE IHPDOHYVPDOH WUHDWPHQWE\VH[LQWHUDFWLRQDQG
the random effect of ewe to adjust for the twinning effect.

Results and discussion

Ewe live weight and average daily feed intake did not differ (P>0.05) between groups during the
treatment period (data not shown). At P140, fetal weight (5.3±0.1 vs. 5.3±0.1 kg) and the weight of
the internal organs did not differ (P>0.05) between control and Arg-supplemented groups (data not
VKRZQ 7KHQRWDEOHH[FHSWLRQZDVSHULUHQDOIDWVWRUHVZKLFKZHUHLQFUHDVHGE\LQIHWXVHVIURP
Arg-supplemented ewes relative to controls (8.7±0.4 vs. 7.7±0.4 g, P< 7KLV¿QGLQJLVFRQVLVWHQW
ZLWKPDWHUQDO$UJVXSSOHPHQWDWLRQWRHLWKHUXQGHUIHGVKHHS 6DWWHU¿HOGet al., 2011) whereby fetal
peri-renal fat stores at 125 days of gestation are increased indicative of Arg stimulation of brown
adipose tissue development. At birth, there was a trend for an interaction (3  EHWZHHQ$UJ
supplementation and sex for birth weight of the lamb. The ewe lambs from Arg-supplemented ewes

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 405
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_146, © Wageningen Academic Publishers 2013
ZHUHKHDYLHUDWELUWKFRPSDUHGZLWKFRQWUROV “YV“NJ3 0.03) whereas birth
weight of male lambs did not differ between Arg and control groups (5.4±0.2 vs. 5.4±0.1, 3 0.80).

At P140, ewes treated with Arg had increased (P<0.05) plasma concentrations (μM; LSM ± SEM) of
arginine (992 vs. 109±84), ornithine (394 vs. 137±38), and decreased (P<0.05) plasma concentrations
RIJO\FLQH YV“ VHULQH YV“ DQGPHWKLRQLQH YV“ UHODWLYHWRFRQWUROV
Fetuses from ewes supplemented with Arg had increased (P<0.05) plasma concentrations of ornithine
YV“ DQGGHFUHDVHG P<0.05) plasma concentrations of taurine (47 vs. 84±13), threonine
YV“ JO\FLQH YV“ PHWKLRQLQH YV“ W\URVLQH YV“ 
DQGKLVWLGLQH YV“ UHODWLYHWRFRQWUROIHWXVHVDW3

Two hours post-birth, ewes treated with Arg had elevated (P<0.05) concentrations of ornithine (113
YV“ ZKHUHDVPHWKLRQLQHUHPDLQHGGHFUHDVHG YV“P<0.01). Lambs born to ewes
treated with Arg had increased (P<0.05) concentrations of isoleucine (181 vs. 80±29) and leucine
YV“ FRPSDUHGWRODPEVERUQWRFRQWUROHZHVDWELUWK7KHGLIIHUHQFHVLQWKHDPLQRDFLG
concentrations of ewes and their offspring at P140 and post-birth are likely due to Arg administration
being stopped 2-12 hours prior to birth.

7KHVH[VSHFL¿FHIIHFWRI$UJVXSSOHPHQWDWLRQRQODPEELUWKZHLJKWLVLQWULJXLQJ2QDYHUDJH
female lambs are 10-15% lighter at birth compared to males, an effect ameliorated in this study. Sex
VSHFL¿FHIIHFWVRIQXWULWLRQDOLQWHUYHQWLRQVKDYHEHHQUHSRUWHGSUHYLRXVO\ -DTXLHU\et al., 2012).
+RZHYHULWLVSRVVLEOHWKDWWKHUHZHUHLQVXI¿FLHQWPDOHVLQHDFKJURXSWRGHWHFWDWUHDWPHQWHIIHFW
)XUWKHUYDOLGDWLRQRI¿QGLQJVLVUHTXLUHG7KH¿QGLQJVRIWKLVVWXG\LQGLFDWHWKDWPDWHUQDO$UJ
supplementation ameliorates the effects of fetal growth restriction in female twin lambs. Increased
birth weight coupled with peri-renal fat stores may have important implications for the survival of
the newborn with importance for both agriculture and medicine.

References
-DTXLHU\$/ 0+ 2OLYHU 0 +RQH\¿HOG5RVV -( +DUGLQJ DQG )+ %ORRP¿HOG  3HULFRQFHSWLRQDO
XQGHUQXWULWLRQLQVKHHSDIIHFWVDGXOWSKHQRW\SHRQO\LQPDOHV-1XWU0HWDEGRL
/DVVDOD$):%D]HU7$&XGG6'DWWD'+.HLVOHU0&6DWWHU¿HOG7(6SHQFHUDQG*3DUHQWHUDO
administration of L-arginine prevents fetal growth restriction in undernourished ewes. J. Nutr. 140, 1242-1248.
/DVVDOD$):%D]HU7$&XGG6'DWWD'+.HLVOHU0&6DWWHU¿HOG7(6SHQFHUDQG*:X3DUHQWHUDO
administration of L-arginine enhances fetal survival and growth in sheep carrying multiple fetuses. J. Nutr. 141,
849-855.
McCoard, S.A., W.C. McNabb, S.N. McCutcheon, P.M. Harris and S.W. Peterson, 2000. Muscle growth, cell number,
type and morphometry in single and twin fetal lambs during mid to late gestation. Reprod. Fert. Dev. 12, 319-327.
6DWWHU¿HOG0&.$'XQODS'+.HLVOHU):%D]HUDQG*:X$UJLQLQHQXWULWLRQDQGIHWDOEURZWQDGLSRVH
WLVVXHGHYHORSPHQWLQQXWULHQWUHVWULFWHGVKHHS$PLQR$FLGV'2,V
:X*):%D]HU-0:DOODFH7(6SHQFHU%RDUGLQYLWHGUHYLHZLQWUDXWHULQHJURZWKUHWDUGDWLRQLPSOLFDWLRQV
IRUWKHDQLPDOVFLHQFHV-$QLP6FL

406 Energy and protein metabolism and nutrition in sustainable animal production
Effect of dietary protein concentration and forage type on nitrogen
PHWDEROLVPDQGQXWULHQWÀX[DFURVVWKHSRUWDOGUDLQHGYLVFHUDDQGWKH
liver in lactating dairy cows
C.E.S. Barratt, L.A. Crompton, C. Green, D.J. Humphries, R.D. Pilgrim and C.K. Reynolds
6FKRRORI$JULFXOWXUH3ROLF\DQG'HYHORSPHQW8QLYHUVLW\RI5HDGLQJ32%R[(DUOH\*DWH
5HDGLQJ5*$58QLWHG.LQJGRP[email protected]

Introduction
With regard to dairy cow nutrition, past efforts have focused on maximising milk output, rather
WKDQIHHGFRQYHUVLRQHI¿FLHQF\0RUHUHFHQWO\DWWHQWLRQKDVUHYHUWHGWRWKHHI¿FLHQF\RIPLON
production, and in particular dietary nitrogen (N) utilisation due to increasing concerns regarding
nitrogenous emissions from dairy cattle. The contribution of dairy production to environmental
pollutants, particularly nitrous oxide and ammonia, is an issue of increasing concern. Current feed
SUHGLFWLRQVFKHPHVFRQFHUQLQJPHWDEROLVDEOHSURWHLQDUHOLPLWHGGXHWRDQRYHUVLPSOL¿FDWLRQRI
SRVWDEVRUSWLYHHYHQWV7KHUHDUHIHZUHFHQWUHOLDEOHGDWDRQWKHHI¿FLHQF\RIFRQYHUWLQJGLHWDU\1
into milk protein and there is still much variation seen between different studies (Hristov and Ropp,
2003; Lee et al., 2009). The present results are from a larger study concerned with describing the
absorption and metabolism of nutrients and metabolites by the splanchnic tissues in lactating dairy
cows in response to varying amounts of absorbable protein and two forage types.

Material and methods

6L[+ROVWHLQGDLU\FRZVLQPLGODWHODFWDWLRQVXUJLFDOO\SUHSDUHGZLWKDUXPHQ¿VWXODDQGVSODQFKQLF
blood sampling catheters were fed ad libitum in equal meals provided hourly, total mixed rations
consisting of a 50:50 mixture (dry matter (DM) basis) of forage:concentrate, with the forage
comprised of either 25:75 or 75:25 grass:maize silage (DM basis). Rations were formulated to contain
crude protein levels of 12.5, 15 and 17.5% of diet DM giving a 2×3 factorial design. The experiment
was conducted as repeated 3×3 Latin squares, with the effect of protein level tested within squares
and forage source as the square effect. Within each square treatment periods were 3 weeks long,
with blood and digesta sampling taking place in the last week of each treatment period. A total of
KRXUO\EORRGVDPSOHVVHWVZHUHREWDLQHGDQGDQDO\]HGLQGLYLGXDOO\1HWÀX[RIQXWULHQWVDFURVV
the portal drained viscera (PDV) and liver (LIV) including glucose, lactate andȕ-hydroxybutyrate
%2+% ZHUHFDOFXODWHGDVYHQRXVDUWHULDOFRQFHQWUDWLRQGLIIHUHQFHWLPHVSODVPDÀRZUDWHPHDVXUHG
E\ȡDPLQRKLSSXUDWHGLOXWLRQ'DWDZHUHDQDO\]HGXVLQJPL[HGPRGHOVSURFHGXUHVDFFRXQWLQJIRU
¿[HGHIIHFWVRIVTXDUHSHULRGZLWKLQVTXDUHIRUDJHSURWHLQDQGIRUDJHE\SURWHLQLQWHUDFWLRQDQG
random effects of animal.

Results and discussion

Dry matter intake (DMI), N intake (NI), milk yield (MY) and milk N yield increased (P” ZLWK
LQFUHDVLQJGLHWSURWHLQFRQFHQWUDWLRQEXWZHUHQRWVLJQL¿FDQWO\DIIHFWHGE\IRUDJHW\SH5XPHQ
ammonia concentrations, and net PDV release of ammonia (NH3; Table 1) increased linearly
(P<0.001) as diet protein concentration increased and were higher when the grass-based diet was
fed, as NI was numerically higher for the grass-based diet. Arterial urea concentration and LIV urea
production (Table 1) increased (P<0.001 and P<UHVSHFWLYHO\ ZLWKLQFUHDVLQJGLHWSURWHLQ
concentration and arterial urea concentration was greater (3  ZKHQWKHJUDVVEDVHGGLHWZDVIHG
compared to the maize-based diet. Higher dietary protein levels are associated with the degradation
of protein in the rumen, causing increased rumen ammonia concentration (Broderick, 2003) and the
capture of ammonia for microbial protein synthesis is energy dependent; therefore the amount and
GHJUDGDELOLW\RIGLHWDU\FDUERK\GUDWHFDQLQÀXHQFHDPPRQLDDEVRUSWLRQ7KHLQFUHPHQWLQUXPHQ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 407
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_147, © Wageningen Academic Publishers 2013
7DEOH'U\PDWWHULQWDNHPLON\LHOG1SURGXFWLRQHI¿FLHQF\DQGVSODQFKQLFQXWULHQWÀX[

Maize Grass SEM P-values1

12.5 15.0 17.5 12.5 15.0 17.5 For Pro Int

DMI2 20.5 22.3 22.4 19.7 20.3 21.1 0.7 0.185 0.014 0.431
MY2 24.8 27.7 29.7 25.2 27.2 31.1 2.5 0.903 0.001 0.582
NI3 402 500 583  540   0.128 0.001 
Milk N3 137 150  139 149  13 0.935 0.054 0.938
1(I¿FLHQF\ 0.35 0.30 0.29 0.31 0.28 0.25 0.03 0.332 0.097 0.982
Rumen NH34 3.13 3.52 4.09 4.19 4.77 5.58 0.38 0.024 0.010 0.750
Arterial urea4   9.0  7.4 10.4  0.058 0.001 0.849
PDV NH35 301  484  453 529 23 0.039 0.001 0.580
LIV Urea5    403  725 101 0.392 0.057 0.422
LIV Glucose5 733 747 857 740 742 773   0.037 
LIV BOHB5 284 301 312 189 253 411 25  0.003 0.010

1 Probability of effects of forage type (For), protein level (Pro) and interaction (Int).
2 kg/day; 3 g/day; 4 mM; 5 mmol/h.

DPPRQLDZDVDFFRPSDQLHGE\DQLQFUHDVHLQDUWHULDOXUHDFRQFHQWUDWLRQ1HI¿FLHQF\ PLON1
NI; Table 1) tended to be affected by an increase in protein level (3 0.10), with N capture in milk
protein relative to NI decreasing as NI increased with protein level within each diet. Net LIV release
of glucose and BOHB both increased linearly (P<0.05) as diet protein concentration, DMI and milk
yield increased (Table 1), but the increase in LIV BOHB release was greater for the grass-based diet
compared to the maize-based diet (Pint=0.01).

These data demonstrate the extent to which forage type (grass versus maize silage) and level of
protein supply affect post-absorptive metabolism of N and other metabolites. Results show that there
is a strong relationship between NI, net PDV ammonia absorption, net LIV urea production and N
XVHHI¿FLHQF\DVSURWHLQOHYHOVDUHLQFUHDVHGEXWWKLVLVQRWDIIHFWHGE\IRUDJHW\SH

Acknowledgments
Funded by the Commission of the European Communities (FP7 KBBE-2007-1; REDNEX).

References
Broderick, G.A., 2003. Effects of varying dietary protein and energy levels on the production
RIODFWDWLQJGDLU\FRZV-'DLU\6FL
Hristov, A.N. and J.K. Ropp, 2003. Effect of dietary carbohydrate composition and availability on utilization of ruminal
DPPRQLDQLWURJHQIRUPLONSURWHLQV\QWKHVLVLQGDLU\FRZV-'DLU\6FL
Lee, M.R.F. Theobald, V.J. Tweed, J.K.S. Winters, A.L. and N.D. Scollan, 2009. Effect of feeding fresh or conditioned
UHGFORYHURQPLONIDWW\DFLGVDQGQLWURJHQXWLOL]DWLRQLQODFWDWLQJGDLU\FRZV-'DLU\6FL

408 Energy and protein metabolism and nutrition in sustainable animal production
Effect of abomasal amino acid infusion on splanchnic metabolism in
postpartum transition dairy cows
M. Larsen1, C. Galindo2, D.R. Ouellet, G. Maxin, N.B. Kristensen1 and H. Lapierre
1'HSDUWPHQW RI $QLPDO 6FLHQFH $DUKXV 8QLYHUVLW\ )RXOXP  7MHOH 'HQPDUN
[email protected]
2'pSDUWHPHQWGHV6FLHQFHV$QLPDOHV8QLYHUVLWp/DYDO4XpEHF4&*9$&DQDGD
$JULFXOWXUHDQG$JUL)RRG&DQDGD6KHUEURRNH4&-0&&DQDGD

Introduction
Postpartum transition cows are in negative energy and protein balance since the high nutrient demand
FUHDWHGE\LQLWLDWLRQRIODFWDWLRQLVQRWPHWE\VXI¿FLHQWIHHGLQWDNH,QFRPSDULVRQWRHQHUJ\WKHUHLV
a paucity of data on metabolic and production effects of dietary attempts to alleviate the postpartum
SURWHLQGH¿FLHQF\7KHDLPRIWKHVWXG\ZDVWRLQYHVWLJDWHWKHHIIHFWRILQFUHDVLQJSURWHLQVXSSO\RQ
tissue amino acid (AA) metabolism in postpartum cows.

Material and methods

Nine multiparous Holstein cows implanted with rumen cannula and permanent indwelling catheters
in major splanchnic vessels were used in a generalised randomised incomplete block design with
repeated measurements. Cows, blocked according to parity (2nd and 3rd+), were assigned to one
of two treatments: continuous abomasal infusion of water (CTRL; n=4) or of a mixture of free AA
$$&1FDVHLQSUR¿OHQ  LQLWLDWHGDWWKHGD\RISDUWXULWLRQ st day in milk, DIM). Infusion of
$$&1ZDVDGPLQLVWHUHGZLWKKDOIRIIXOOGRVHDW',0IXOOGRVH “JG IURPWR',0
followed by a linear daily decrease (571±2 g/d at 15 DIM and 227±1 g/d at 29 DIM). All cows
UHFHLYHGWKHVDPHWRWDOPL[HGUDWLRQ J&3NJ'0 DOORFDWHGDGOLELWXP6L[VHWVRIDUWHULDO
venous portal, hepatic and mammary venous samples were collected at 45 min intervals at 5, 15,
DQG',0'DWDZHUHDQDO\VHGZLWKDPL[HGPRGHOLQFOXGLQJWKH¿[HGHIIHFWVRIEORFNWUHDWPHQW
(Trt), DIM, and Trt × DIM; DIM was tested as a repeated measurement using the autoregressive
order 1 covariance structure.

Results and discussion

Increasing the early postpartum AA supply induced a substantial increase in both milk (7.8±1.3
kg/d), and milk protein yield (Table 1), but milk protein content was unaffected (3 0.31). Arterial
concentrations of essential AA (EAA) tended to be greater for AA-CN compared with CTRL (3 0.08;
1,175 vs. 908±84 μM). The increased milk yield was not supported by a greater feed intake as dry
PDWWHULQWDNHGHFUHDVHGE\“NJGZLWK$$&1,QVWHDGWKHLQFUHDVHGPLON\LHOGVHHPHGWREH
SDUWLDOO\VXSSRUWHGE\JUHDWHUPRELOLVDWLRQRIERG\IDWDVWKHDUWHULDOFRQFHQWUDWLRQRIQRQHVWHUL¿HG
fatty acids were higher at 5 DIM for AA-CN as compared with CTRL (723 vs. 382±78 μM), but
did not differ by 29 DIM (299±52 μM; interaction: 3 0.05). Furthermore, arterial concentrations
of urea-N were greater for AA-CN compared with CTRL (3 YV“P0 LQGLFDWLQJ
that the increased AA supply was partially utilised for energetic purposes.

Net portal release of group 1 EAA (His+Met+Phe+Trp+Tyr; Table 1) was greater with AA-CN. Their
liver removal, however, increased equivalently resulting in an unaltered net release by splanchnic
WLVVXHV0RUHRYHUOLYHUUHPRYDOUHODWLYHWRWRWDOLQÀX[RI+LV0HW3KHDQG7USZDVJUHDWHUZLWK
$$&1 ”P” LQGLFDWLQJDQXSUHJXODWLRQRIKHSDWLFSDWKZD\VXWLOLVLQJWKHVH($$2YHUDOO
WKHGH¿FLWEHWZHHQWKHQHWVSODQFKQLFUHOHDVHDQGPLONSURWHLQVHFUHWLRQRIJURXS($$ZDVQRW
reduced in AA-CN cows.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 409
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_148, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIDERPDVDODPLQRDFLG $$ LQIXVLRQLQpostpartum dairy cows on production and
WLVVXHPHWDEROLVPRIHVVHQWLDO$$ ($$ DQGQRQ($$

Item Postpartum Trt SEM P-values

CTRL AA-CN Trt DIM Trt × DIM

Dry matter intake, kg/d1 20.4 18.8 0.4 0.02 ‫ޒ‬ 0.32
Milk, kg/d1 38.2  0.8 <0.01 ‫ޒ‬ 0.29
Milk protein, g/d2 1301 1521 43 0.01 0.03 0.50
Group 1 EAA2 PDV3, mmol/h 78 90 4 0.05  0.44
Liver, mmol/h -33 -49 4 0.03 0.53 0.37
TSP3, mmol/h 43 42  0.89  0.70
MG3, mmol/h   2 0.02 0.10 0.52
Milk, mmol/h 57  2 0.01 0.03 
PDV:milk ratio 1.39 1.38 0.05 >0.99 0.04 0.51
TSP:milk ratio 0.75  0.07 0.15  
MG:milk ratio 0.99 1.01 0.01  0.49 0.95
Group 2 EAA2 PDV, mmol/h 121 144 9 0.13 0.53 0.34
Liver, mmol/h -4.4  11 0.53  0.91
TSP, mmol/h 118 132 12  0.53 0.27
MG, mmol/h -137  5 <0.01  0.49
Milk, mmol/h   4 0.01 0.03 
PDV:milk ratio 0.98 1.00 0.07 0.88 0.10 0.83
TSP:milk ratio 0.94 0.91 0.07 0.79 0.13 0.49
MG:milk ratio 1.09 1.15 0.02 0.02 0.18 0.82
Non-EAA2 PDV, mmol/h 217 284 21 0.12 0.25 0.82
Liver, mmol/h -140 -242 27  <0.01 
TSP, mmol/h 72 50 28  0.70 0.32
MG, mmol/h -150 -174 11 0.22 0.03 0.71
Milk, mmol/h 244 284 8 0.01 0.03 
PDV:milk ratio 0.89 1.01 0.08 0.43 0.07 0.78
TSP:milk ratio 0.29 0.17 0.09 0.47  
MG:milk ratio   0.04 0.85 0.33 

1 Averageof daily observations from 1 to 29 DIM.

2 Averageof 3 sampling days; Group 1 EAA = His+Met+Phe+Tyr+Trp; Group 2 EAA = Ile+Leu+Val+Lys.
3 PDV = portal-drained viscera; TSP = total splanchnic tissue; MG = mammary gland.

The net portal release of group 2 EAA (Ile+Leu+Val+Lys) was numerically greater with AA-CN,
whereas their small liver removal was unaffected by the increased supply. Consequently, net portal and
VSODQFKQLFUHOHDVHVRIWKHEUDQFKHGFKDLQ$$DQG/\VZHUHFORVHWREHVXI¿FLHQWWRPHHWPLONSURWHLQ
secretion and that was not affected by AA-CN. However, mammary uptake to output ratio increased
with AA-CN. The net portal release of non-EAA was numerically greater with AA-CN, but liver
removal increased to a greater extent, resulting in an unaltered net splanchnic release. Moreover, as
the liver removed a large fraction of non-EAA portal release (except for Glu), there was a substantial
GH¿FLHQF\RIQRQ($$UHODWLYHWRPLONVHFUHWLRQZLWKERWKWUHDWPHQWV

In conclusion, the attempt to alleviate the early postpartumSURWHLQGH¿FLHQF\E\DERPDVDO$$

infusion induced a profound increment in milk and milk protein yield. Moreover, the greater AA
supply allowed more AA for liver anabolism and catabolism, though the partitioning could not be
assessed. Thus, the early postpartumSURWHLQGH¿FLHQF\SHUVLVWHGDVWKHFRZHI¿FLHQWO\XWLOLVHGWKH
additional AA for anabolic processes intended for the new born calf.

410 Energy and protein metabolism and nutrition in sustainable animal production
Net portal appearance of amino acids in Iberian compared to Landrace pigs
L. González-Valero, J.M. Rodríguez-López, M. Lachica and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQL¿JDUHV#HH]FVLFHV

Introduction
Compared to modern breeds, Iberian pigs have lower rates of muscle protein deposition and
greater viscera weight. Factors that limit growth performance of Iberian pigs are still unknown. We
hypothesized that differences in net portal appearance (NPA) of amino acids (AA) might partially
explain the lower growth rate reported in Iberian pigs compared to modern breeds (Nieto et al., 2002).

Material and methods

1HWSRUWDODEVRUSWLRQRI$$ZDVPHDVXUHGLQ,EHULDQ ,E DQG/DQGUDFH /G JLOWV NJ%: 
¿WWHGZLWKFKURQLFFDWKHWHUVLQSRUWDOYHLQ 39 PHVHQWHULFYHLQ 09 DQGFDURWLGDUWHU\ &$ %ORRG
VDPSOHVIURP39DQG&$ZHUHWDNHQPLQDQGDQGKDIWHUIHHGLQJ
25% of the daily intake of 2 isoenergetic diets (14-14.5 MJ ME/kg DM) with different CP (13 vs.
/&3DQG+&3UHVSHFWLYHO\ FRQWHQWLQDFURVVRYHUGHVLJQZLWKDQDGDSWDWLRQSHULRGWRWKH
diet of 1 week. Plasma free AA were analysed by HPLC using the Waters Pico Tag method which
involves pre-column derivatization with phenylisothiocianate and a reverse phase column. Net portal
AA absorption was calculated by multiplying portal-arterial plasma AA concentration difference
E\39SODVPDÀRZHVWLPDWHGE\DQLQGLFDWRUGLOXWLRQWHFKQLTXHLQIXVLQJSDUDDPLQRKLSSXULFDFLG
into one MV. Data were subjected to ANOVA using the MIXED procedure. The statistical model
DSSOLHGLQFOXGHGWKH¿[HGHIIHFWRIEORFNGLHWEUHHGVDPSOLQJWLPHDQGFRUUHVSRQGLQJLQWHUDFWLRQV
Time of sampling within pig was considered as a repeated measure. Concentration at time zero of
WKHDQDO\WHZDVLQFOXGHGDVDFRYDULDWHLQWKHVWDWLVWLFDODQDO\VLV6LJQL¿FDQWGLIIHUHQFHVDPRQJ
treatments were tested according to the factorial design.

Results and discussion

Portal and arterial plasma concentration and NPA of AA are presented in Table 1. Feeding increased
(P<0.001) concentrations of plasma AA in both portal and arterial samples and net portal appearance
of AA throughout the postprandial period (data not shown). No interactions of breed or diet with
time was found (P>0.10). The postprandial increase in portal and arterial concentrations of AA and
the subsequent gradual decrease are in agreement with results in the literature (Yen et al., 2004).

Portal and arterial non-essential (NEAA) and total AA were lower (P<0.05) in Ib compared to
Ld pigs with no difference (P>0.10) in portal and arterial essential AA (EAA) concentrations.
Furthermore, NPA was lower in Ib compared to Ld for total AA, EAA and NEAA (52, 45 and 50%,
respectively; P< 'LIIHUHQFHVLQ($$13$DUHWKHUHIRUHGXHWRWKHORZHUSRUWDOEORRGÀRZ
in Ib compared to Ld pigs (Rodríguez-López et al., 2010). Interestingly, no differences in Lys and
Met absorption were found between Ib and Ld pigs fed barley-soybean diets of different protein
concentration (González-Valero et al., 2012).

Regarding CP content of the diet, portal EAA concentration was greater (P<0.05) when pigs were
given the HCP, with no differences in portal total AA and NEAA concentrations. Pigs fed the
LCP diet had, however, greater arterial total AA and NEAA (P<0.05) concentrations although no
difference in arterial EAA concentration was found (P>0.10). Consequently, NPA of total AA, EAA
DQG1($$GXULQJWKHKSRVWSUDQGLDOSHULRGZDVORZHU P<0.01) when pigs were fed the LCP
diet (41, 52 and 35%, respectively) than the HCP diet. Lys and Met NPA were reported to be lower

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 411
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_149, © Wageningen Academic Publishers 2013
7DEOH$UWHULDODQGSRUWDOFRQFHQWUDWLRQV >P0@ DQGQHWSRUWDODSSHDUDQFH 13$PPROPLQ RI
WRWDO 7 HVVHQWLDO ( DQGQRQHVVHQWLDO 1( DPLQRDFLGV $$ LQ,EHULDQ ,E DQG/DQGUDFH /G 
SLJV Q SLJVEUHHG UHFHLYLQJHLWKHU / RU + &3GLHWV

Ib Ld SE P-value

LCP HCP LCP HCP Breed Diet Breed×Diet

Portal [TAA]   4.8 4.9 0.12 0.008 NS NS

Arterial [TAA] 4.3 4.0 4.4 4.3 0.10 0.040 0.052 NS
TAA NPA 0.15 0.33 0.40  0.041 <0.001 <0.001 NS
Portal [EAA]  1.8  1.7  NS 0.031 NS
Arterial [EAA] 1.5 1.5 1.5 1.4 0.05 NS NS NS
EAA NPA 0.07 0.15 0.14 0.28 0.018 <0.001 <0.001 NS
Portal [NEAA] 2.7 2.5 3.0 3.0 0.08 <0.001 NS NS
Arterial [NEAA]  2.2  2.7  <0.001 0.005 <0.001
NEAA NPA 0.07 0.18 0.23 0.28 0.025 <0.001 0.002 NS

when pigs consumed LCP compared to HCP diet (González-Valero et al., 2012). Simoes Nunes et
al. (1991) reported decreased AA absorption when pigs fed 12% casein were compared to pigs fed
24% casein diets.

Differences in AA NPA absorption may partially explain the disparate growth capacity of Ib pigs
compared to modern genotypes. This may be the result of the lower digestibility of protein (Rivera-
Ferre et al.,%DUHDet al., 2011) or higher utilization of AA by the portal-drained viscera in
the Ib pigs compare to the Ld pigs or both. Further research on individual AA may modulate the
overall conclusion.

References
Barea, R., R. Nieto, F. Vitari, C. Domeneghini and J.F. Aguilera, 2011. Effects of pig genotype (Iberian vs. Landrace
× Large White) on nutrient digestibility, relative organ weights and small intestine architecture at two stages of
growth. Animal 5, 547-557.
González-Valero, L., J.M. Rodríguez-López, M. Lachica and I. Fernández-Fígares, 2012. Differences in portal
appearance of lysine and methionine in Iberian and Landrace pigs. J. Anim. Sci. 90, 110-112.
Nieto, R., A. Miranda, M.A. García and J.F. Aguilera, 2002. The effect of dietary protein content and feeding level
on the rate of protein deposition and energy utilization in growing Iberian pigs from 15 to 50 kg body weight.
Br. J. Nutr. 88, 39-49.
5LYHUD)HUUH0*-)$JXLOHUDDQG51LHWR'LIIHUHQFHVLQZKROHERG\SURWHLQWXUQRYHUEHWZHHQ,EHULDQDQG
/DQGUDFHSLJVIHGDGHTXDWHRUO\VLQHGH¿FLHQWGLHWV-$QLP6FL
Rodríguez-López, J.M., M. Lachica, L. González-Valero and I. Fernández-Fígares, 2010. Energy expenditure of
VSODQFKQLFWLVVXHVLQ,EHULDQDQG/DQGUDFHJURZLQJJLOWV/LYHVW6FL
Simoes Nunes, C., I. Galibois, A. Rérat, L. Savoie and P. Vaugelade, 1991. Hepatic and portal-drained viscera balances
of amino acids, insulin, glucagon and gastrin in the pig after ingestion of casein or rapeseed proteins. Reprod.
Nutr. Dev. 31, 217-231.
Yen, J.T., B.J. Kerr, R.E. Easter and A.M. Parkhurst, 2004. Difference in rates of net portal absorption between
crystalline and protein-bound lysine and threonine in growing pigs fed once daily. J. Anim. Sci. 82, 1079-1090.

412 Energy and protein metabolism and nutrition in sustainable animal production
8UHDQLWURJHQDEVRUEHGIURPWKHKLQGJXWLVXVHGHI¿FLHQWO\IRUERG\
SURWHLQGHSRVLWLRQLQSLJVIHGDGLHWGH¿FLHQWLQQRQHVVHQWLDODPLQRDFLG
nitrogen
W. Mansilla1, D. Columbus1, J.K. Htoo2 and C.F.M. de Lange1
1Department of Animal and Poultry Science, University Guelph, Guelph, ON, N1G 2W1, Canada;
[email protected]
2(YRQLN,QGXVWULHV$*5RGHQEDFKHU&KDXVVHH+DQDX*HUPDQ\

Introduction
The absorption of nitrogen (N) from the hindgut of monogastric animals is thought to be of little
value for supporting body protein synthesis. However, N that is absorbed from the lower gut,
largely in the form of ammonia, can be used for synthesis of non-essential amino acids (NEAA) or
converted to urea. The latter can be excreted in urine or recycled into the upper gut and contribute
WRPLFURELDOSURGXFHGDPLQRDFLGVWKDWFDQEHRIEHQH¿WWRWKHKRVW )XOOHU 7KHREMHFWLYHRI
WKHSUHVHQWH[SHULPHQWZDVWRH[SORUHWKHHI¿FLHQF\RIXVLQJ1DEVRUEHGIURPWKHKLQGJXWIRUERG\
SURWHLQGHSRVLWLRQLQJURZLQJSLJVIHGDGLHWGH¿FLHQWLQ1($$1

Material and methods

1LQHEDUURZ<RUNVKLUHSLJV LQLWLDO%:“NJ ZHUH¿WWHGZLWKDVLPSOH7FDQQXODLQWKH
caecum. All pigs were fed 3 equal meals daily (8:00, 13:00 and 17:30 h) of a cornstarch, casein and
crystalline amino acids-based diet limiting in NEAA-N. Ratios among essential amino acids (EAA)
were based on the ideal protein concept (NRC, 1998), while the EAA-N to total N ratio was high
(0.75) and above the required ratio (Heger, 1998). Water was supplied with feed in a 3 to 1 ratio.

Pigs were randomly assigned to 1 of 3 treatments, representing a control and 2 different urea-N
infusion rates into the caecum (1.5, 3.0 g/d), according to two different (one repeated) 3x3 Latin
Square designs and during 3 successive experimental periods. Experimental periods consisted of 5
d adaptation followed by a 4 d N-balance period.

During each N-balance period, urine was collected quantitatively at 24 h intervals in containers
with sulphuric acid to keep urine pH below 2. Fecal samples were collected continuously, frozen
immediately, pooled per pig and N-balance period, and freeze dried. Urine and feces were analyzed
for total N content. Nitrogen retention was calculated as the difference between dietary N intake
(corrected for feed wastage) plus infused N and urinary plus fecal N excretion. Data were analyzed
using the PROC MIXED procedure of SAS (v. 9.2; SAS Institute Inc., Cary, NC) with pig, period
and urea-N infusion rate as sources of variation.

Results and discussion

'XHWRLQVXI¿FLHQWIHHGLQWDNHRULQFRPSOHWHXULQDU\1FROOHFWLRQVRPHREVHUYDWLRQVZHUHUHPRYHG
SULRUWRDQDO\VLV:KROHERG\1UHWHQWLRQGLIIHUHGDPRQJWUHDWPHQWV DQGJG
P<0.001) and increased with urea infusion rate (Table 1). Moreover, BW gain increased with urea
LQIXVLRQUDWH DQGJGP<0.05) and there was a difference between the control and the
highest urea infusion rate (P< 7KHVHUHVXOWVDUHLQDJUHHPHQWZLWK5RVHDQG'HNNHU  ZKR
VKRZHGWKDWVXSSO\LQJUDWVIHGDGLHWGH¿FLHQWLQ1($$ZLWKXUHDLPSURYHGJURZWKSHUIRUPDQFH

Fecal and urinary N excretion did not differ among treatments (P>0.10), indicating that all N that
ZDVLQIXVHGLQWRWKHFDHFXPZDVDEVRUEHGDQGUHWDLQHGLQWKHERG\7KHPDUJLQDOHI¿FLHQF\RIXVLQJ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 413
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_150, © Wageningen Academic Publishers 2013
Table 1. Growth performance and aspects on whole body N utilization in growing pigs fed a diet
GH¿FLHQWLQ1($$1DQGLQIXVHGZLWKYDU\LQJDPRXQWVRIXUHDLQWRWKHFDHFXP

Urea-N infused, g/d P-value

0 (control) 1.5 3.0

(n=7) Q  (n=8)

Mean BW, kg 32.1±0.70 31.7±0.70 32.3±1.00 0.41

Whole body N retention, g/d “a “b 7.75±0.19c <0.001
Difference vs. control, g/d - +1.54±0.21a +2.89±0.19b <0.001
BW gain, g/d “a 313±17ab “b <0.05
Difference vs. control, g/d - +47±17 +93±15 0.18
Dietary dry matter intake, g/d “ “ “ 0.13
Dietary N intake, g/d “ “ 8.72±0.07 0.13
N infused into caecum, g/d - 1.49±0.05a 3.02±0.05b <0.001
Fecal N excretion, g/d 1.54±0.11 1.57±0.14 1.33±0.10 0.25
Urinary N excretion, g/d 2.38±0.20 2.21±0.22 “ 0.39
(VWLPDWHGLOHDO1ÀRZJG1 0.59±0.01 0.59±0.01 0.59±0.01 
Lower gut N disappearance, g/d2 “a 0.49±0.11b “c <0.001
Difference vs. control, g/d - +1.45±0.11a +3.22±0.10b <0.001
Marginal N retention3 - 104.1±9.7 87.4±7.9 0.20

1 Calculated based on an ileal N digestibility of 93.3% (NRC, 2012).

2&DOFXODWHGDVORZHUJXW1GLVDSSHDUDQFH  LOHDO1ÀRZ1LQIXVLRQ ±IHFDO1ÀRZ
3 Calculated as: (Increase in N retention vs. control/N infused into lower gut) × 100.
a,b,c9DOXHV OHDVWVTXDUHPHDQV“VWDQGDUGHUURU LQWKHVDPHURZIROORZHGE\GLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\

(P<0.05).

N infused into the caecum for whole body N retention was not different for the two urea infusion
treatments (104.1 and 87.4% for 1.5 and 3.0 g/d of urea-N infused, respectively; 3 0.20).

,QFRQFOXVLRQWKHVHUHVXOWVVKRZWKDW1DEVRUEHGIURPWKHKLQGJXWFDQEHXVHGHI¿FLHQWO\IRUZKROH
ERG\1UHWHQWLRQLQSLJVIHGGLHWVWKDWDUHGH¿FLHQWLQ1($$1ZLWKQRH[FHVVRI($$UHÀHFWLQJWKH
XVHRIDEVRUEHGQRQSURWHLQ1IRUHQGRJHQRXVV\QWKHVLVRI1($$ZKHQWRWDO1VXSSO\LVGH¿FLHQW

References
Fuller, M.F., 2012. Determination of protein and amino acid digestibility in foods including implications of gut microbial
DPLQRDFLGV\QWKHVLV%U-1XWU66
Heger, J., 1998. Effect of essential: total nitrogen ratio on protein utilization in the growing pig. Br. J. Nutr. 80, 537-544
National Research Council (NRC), 2012. Nutrient requirements of swine. The national academies press, Washington, DC.
5RVH:DQG(('HNNHU8UHDDVDVRXUFHRIQLWURJHQIRUWKHELRV\QWKHVLVRIDPLQRDFLGV-%LRO&KHP
223, 107-121.

414 Energy and protein metabolism and nutrition in sustainable animal production
Supplementation with a leucine pulse during continuous feeding
stimulates protein synthesis and suppresses protein degradation
pathways in skeletal muscle of neonatal pigs
C. Boutry1, S.W. El-Kadi1,2, A. Suryawan1, S.M. Wheatley1, R.A. Orellana1, H.V. Nguyen1 and T.A. Davis1
1USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of
Medicine, Houston, TX, USA; [email protected]
2Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA

Introduction
Orogastric tube feeding, using either continuous or intermittent bolus delivery, is a common clinical
practice in pediatric patients who are unable to feed normally. We have shown, using the neonatal pig
as a model for the human neonate, that intermittent bolus feeding (BOL) has a greater stimulatory
effect on muscle protein synthesis (PS) than continuous orogastric infusion (CON) (Gazzaneo et
al., 2011; El-Kadi et al., 2012). This effect is largely due to the rise in insulin and amino acid (AA)
concentrations after BOL feeding which increases the activation of mammalian target of rapamycin
(mTOR) and its downstream signaling proteins leading to translation initiation in muscle (Suryawan
et al., 2007).

The branched-chain AA, leucine (Leu), acts as a nutrient signal to stimulate PS in skeletal muscle
(Anthony et al., 2000). Previously, our laboratory demonstrated that a 1 h parenteral infusion of Leu
increases PS in skeletal muscle of neonatal pigs (Escobar et al., 2005). However, the response to Leu
was not sustained, likely due to a Leu-induced decrease in the circulating levels of other AA, and
particularly the branched-chain AA. Recently, we demonstrated that the Leu-induced stimulation
of PS can be sustained up to 24 h if the Leu-induced reduction in essential AA is prevented by
infusion (Wilson et al., 2010). Limited evidence suggests that Leu may also suppress muscle protein
degradation.

The aim of this study was to determine if administration of a Leu pulse during CON feeding can
enhance PS and reduce protein degradation in muscle of the neonate.

Material and methods

Twenty 8-d-old piglets were fed by orogastric tube, for 24 h, equivalent amounts of a milk replacement
IRUPXODHLWKHUFRQWLQXRXVO\ &21Q  RUE\EROXVLHHYHU\KIRUPLQ %2/Q  $WKLUG
group (CON+Leu, n=7) was fed continuously and received an additional parenteral Leu infusion
ȝPRONJK IRUKHYHU\K$IWHUKRILQIXVLRQEORRGVDPSOHVZHUHFROOHFWHGHYHU\
PLQIRUKDQGHYHU\KXQWLOKRILQIXVLRQ$IWHUKDÀRRGLQJGRVHRI3H-phenylalanine was
injected to determine PS rates. Protein immunoblot analysis by western blot was used to identify the
intracellular mechanism involved. Piglets were euthanized and muscle samples were taken at 25.25 h.
Data were analyzed using one-way ANOVA and using mixed models for repeated-measures analysis.

Results and discussion

Insulin and glucose concentrations increased 15 min after the bolus meal (P<IURP“
WR“—8PODQG“WR“PJGOUHVSHFWLYHO\ DQGUHWXUQHGWREDVHOLQHE\
2 h; they were unchanged in the CON and CON+Leu groups where they averaged 10.8±3.3 μU/
ml and 188.3±4.7 mg/dl, respectively. In the CON+Leu pigs, plasma Leu was higher after the Leu
SXOVH KDIWHULQIXVLRQIURP“WR“—0P<0.0005). BOL feeding
DOVRVLJQL¿FDQWO\LQFUHDVHGSODVPD/HXFRQFHQWUDWLRQ KDIWHULQIXVLRQIURP“
WR“—0 ZLWKQRFKDQJHLQWKH&21JURXS “—0LQDYHUDJH /HXLQIXVLRQ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 415
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_151, © Wageningen Academic Publishers 2013
during CON feeding decreased essential AA concentrations (P<“YV“
μM) compared to CON feeding. Protein synthesis rates in the longissimus dorsi muscle were greater
(P< LQWKH&21/HX “G DQG%2/ “G JURXSVWKDQ
in the CON group (11.7±0.9%/d). The gastrocnemius (P<0.003) and soleus (P<0.0001) muscles
UHVSRQGHGVLPLODUO\7KHUHZDVDVLJQL¿FDQWLQFUHDVHRI36DIWHU%2/IHHGLQJLQWKHVPDOOLQWHVWLQH
compared to CON and CON+Leu groups (P<“YV“DQG“G
respectively). No difference between groups was found on PS of the heart and liver. Phosphorylation
RIULERVRPDOSURWHLQ6NLQDVHDQG(ELQGLQJSURWHLQDQGIRUPDWLRQRIWKHDFWLYHHXNDU\RWLF
LQLWLDWLRQIDFWRU H,) H,)(‡H,)*FRPSOH[ZHUHDOVRKLJKHULQPXVFOHRI&21/HXDQG%2/WKDQ
CON suggesting increased translation initiation (P<0.05). There was no effect on AMP-activated
SURWHLQNLQDVHĮHXNDU\RWLFLQLWLDWLRQIDFWRUĮDQGHXNDU\RWLFHORQJDWLRQIDFWRUSKRVSKRU\ODWLRQ
The ratio of LC3-II to total LC3 in muscle was lower in CON+Leu (0.27±0.1 arbitrary units A.U.)
and BOL (0.32±0.1 A.U.) compared to CON pigs (0.70±0.1 A.U.) suggesting reduced autophagy-
lysosome system activation (P<0.05). There were no differences between groups in indices of the
ubiquitin-proteasome pathway, i.e. Atrogin-1 and MURF-1 abundance and FoxO3 phosphorylation.

In conclusion, administration of a leucine pulse during continuous orogastric feeding increases skeletal
muscle protein synthesis in neonatal pigs by stimulating translation initiation. The leucine pulse
also suppresses the autophagy-lysosome, but not the ubiquitin-proteasome, degradation pathways
in skeletal muscle of neonatal pigs.

Acknowledgements
6XSSRUWHGE\1,+$5DQG86'$$56

References
Anthony, J.C., Anthony, T.G., Kimball, S.R., Vary, T.C. and Jefferson, L.S. 2000. Orally administered leucine stimulates
protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J. Nutr.
130, 139-145.
El-Kadi, S.W., Suryawan, A., Gazzaneo, M.C., Srivastava, N., Orellana, R.A., Nguyen, H.V., Lobley, G.E. and Davis,
T.A. 2012. Anabolic signaling and protein deposition are enhanced by intermittent as compared with continuous
IHHGLQJLQVNHOHWDOPXVFOHRIQHRQDWHV$P-3K\VLRO(QGRFULQRO0HWDE(
Escobar, J., Frank, J.W., Suryawan, A., Nguyen, H.V., Kimball,S.R., Jefferson, L.S. and Davis, T.A. 2005. Physiological
rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor
activation. Am. J. Physiol. Endocrinol. Metab. 288, E914-921.
Gazzaneo, M.C., Suryawan, A., Orellana,R.A., Torrazza, R.M., El-Kadi, S.W., Wilson, F.A., Kimball, S.R., Srivastava,
N., Nguyen, H.V., Fiorotto, M.L. and Davis, T.A. 2011. Intermittent bolus feeding has a greater stimulatory effect
on protein synthesis in skeletal muscle than continuous feeding in neonatal pigs. J. Nutr. 141, 2152-2158.
Suryawan, A., Orellana, R.A., Nguyen, H.V., Jeyapalan, A.S., Fleming, J.R. and Davis, T.A. 2007. Activation by insulin
and amino acids of signaling components leading to translation initiation in skeletal muscle of neonatal pigs is
GHYHORSPHQWDOO\UHJXODWHG$P3K\VLRO(QGRFULQRO0HWDE(
Wilson, F.A., Suryawan, A., Gazzaneo, M.C., Orellana, R.A., Nguyen, H.V. and Davis. T.A. 2010. Stimulation of
muscle protein synthesis by prolonged parenteral infusion of leucine is dependent on amino acid availability in
QHRQDWDOSLJV-1XWU

416 Energy and protein metabolism and nutrition in sustainable animal production
Effects of copper nanoparticles on metabolic rate and development of
layer embryos
L. Pineda, E. Sawosz, K.P. Vadalasetty and A. Chwalibog
University of Copenhagen, Department Veterinary Clinical and Animal Sciences, Groennegaardsvej
)UHGHULNVEHUJ'HQPDUN[email protected]

Introduction
The poor bioavailability of Copper (Cu) has resulted in high excretion rate of Cu in the faeces of
animals. Regardless of inclusion level, the major part of Cu (70-90%) is excreted and its effect on
soil microorganisms, plants and aquatic species is today one of the crucial environmental concerns
(Gonzales-Eguia, 2009; Zhao et al., 2010).

&RSSHULVDQHI¿FLHQWKHDOWKDQGJURZWKSURPRWHUXVHGDVDIHHGDGGLWLYHIRUSRXOWU\$WSUHVHQW
there are no effective alternatives having similar growth promoting effects as Cu and the withdrawal
of Cu from animal diets will cause severe health, performance and economic drawbacks in intensive
poultry production.

It was hypothesized that nanoparticles of Cu (CuNano), because of high physical reactivity, may affect
O2 consumption and stimulate growth and development; and thereby can be used as an alternative
health and growth promoter for animals. The objective of the study was to investigate the effects
of in ovo injection of CuNano and timing of injection on metabolic rate (O2 consumption and heat
production, HP) and development of layer hatchlings.

Material and methods

On day 1 of incubation, 192 fertile eggs from 29 week old Lohmann breeder strain were distributed
into 4 groups according to the following treatment descriptions: Group 1 – CuNanoD1, Group 2 –
CuNanoD10, Group 3 – CuNanoD1+D10 and Group 4 – Control. Colloidal CuNano with 50 mg/
kg concentration was introduced via in ovo injection. Eggs from Group 1 were injected at day 1
(D1), while eggs in Group 2 at day 10 (D10). In Group 3 eggs were injected twice at D1 and then at
D10 of incubation. Eggs in Group 4 did not receive any injections, and served as the non-injected
negative control. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP
ZDVFDOFXODWHGIRUDQGGD\ROGHPEU\RV$WKRXUDIWHUKDWFKLQJFKLFNVZHUHHXWKDQL]HG
and blood samples were collected to evaluate the effects of the in ovo CuNano injection on plasma
concentration of immunoglobulins (IgG and IgM). Gene expression of nuclear factor kappa-light-
FKDLQHQKDQFHURIDFWLYDWHG%FHOOV 1)N% DQGWXPRUQHFURVLVIDFWRUDOSKD 71)Į RQWKHP51$
level was measured using quantitative polymerase chain reaction method. Yolk free body weight
(YFBW) and the relative organ weights were used as a measure of hatchling development.

Data were analysed using GLM procedure (one-way analysis of variance) of SAS (SAS Institute
,QF 7KH7XNH\.UDPHUKRQHVWO\VLJQL¿FDQWGLIIHUHQFHWHVWZDVXVHGWRWHVWWKHVHSDUDWLRQ
RIWKHPHDQVDWDVLJQL¿FDQFHOHYHORIP<0.05.

Results and discussion

The O2 consumption and HP, hence, the metabolic rate was affected by the treatments. In ovo injection
of CuNano at different days during incubation decreased metabolic rate compared with the control
group (P<0.05). The residual yolk sac weight in the treated groups was higher than the control
group (P<0.01), indicating that CuNano injection did not enhance oxidation of fat, which could
be associated with the rates of O2 consumption of embryos in these groups (P<0.01). Accordingly,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 417
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_152, © Wageningen Academic Publishers 2013
the organ weights relative to YFBW were also lower in embryos injected with CuNano (P<0.05).
Interestingly, the difference in metabolic rate and organ development between treatments was not
UHÀHFWHGLQ<)%:7KHUHVXOWLVQRWFRQVLVWHQWZLWKWKHUHSRUWHGLPSURYHPHQWLQJURZWKSHUIRUPDQFH
of piglets supplemented with 50 mg CuNano/kg in the diet (Gonzales-Eguia et al., 2009), which can
be attributed to the differences in species and to the form of nanoparticles used.

&X1DQRLQMHFWLRQGLGQRWLQÀXHQFHWKHSODVPDFRQFHQWUDWLRQRI,J0DQG,J* ERWKP>0.05; data

not shown), suggesting that it did not interact with humoral system. Furthermore, it did not affect
P51$H[SUHVVLRQRI1)N%DQG71)Į ERWKP>0.05; data not shown), indicating the absence of
LQÀDPPDWRU\UHVSRQVHRULPPXQHVXSSUHVVLRQSURSHUWLHVRI&X1DQR

The results demonstrated that in ovo CuNano injection, regardless of day of injection, altered
metabolic rate of embryos and depressed the development of organs, however, did not affect YFBW
of hatchling, concentration of immunoglobulins and expression of immune-related genes.

7DEOH2[\JHQFRQVXPSWLRQ 22 KHDWSURGXFWLRQ +3 \RONIUHHERG\ZHLJKW <)%: \RONVDF

ZHLJKW <6 ZHLJKWVUHODWLYHWR<)%:RILQWHVWLQHOLYHUDQGKHDUWRIOD\HUHPEU\RVLQRYRLQMHFWHG
ZLWKFRSSHUQDQRSDUWLFOHV &X1DQR DWGLIIHUHQWVWDJHVRIHPEU\RQLFGHYHORSPHQW1

Parameter Day of injection of CuNano SEM P-value

Control D1 D10 D1+10

O2, ml/h 31.1a 28.5b 27.2b 28.7b 0.044 0.001

HP, J/h a b 543.1b b 17.49 0.002
YFBW, g 34.9 34.7 35  0.08 0.18
YS, g 3.5a 5.7b b 5.3b 0.01 <0.0001
Intestine weight, % YFBW c 3.9b 4.0b b 0.004 <0.0001
Liver weight, % YFBW 2.9a b 2.5b 2.4b 0.001 0.0002
Heart weight, % YFBW 0.7a b b b 0.004 0.002

1 Numbers in a row with a different superscript differ, P”

References
Gonzales-Eguia, A., C.-M. Fu, F-Y. Lu and T.F. Lien, 2009. Effects of nanocopper on copper availability and nutrients
GLJHVWLELOLW\JURZWKSHUIRUPDQFHDQGVHUXPWUDLWVRISLJOHWV/LYHVWRFN6FL
SAS Institute Inc. 2009. SAS1 Procedure Guide. Version 9.2. Cary (NC): SAS Institute Inc.
Zhao, J., R.B. Shirley, M. Vazquez-Anon, J.J. Dibner, J.D. Richards, P. Fisher, T. Hampton, K.D. Christensen, J.P.
Allard and A.F. Giesen, 2010. Effects of chelated trace minerals on growth performance, breast meat yield, and
IRRWSDGKHDOWKLQFRPPHUFLDOPHDWEURLOHUV-$SSOLHG3RXOWU\5HV

418 Energy and protein metabolism and nutrition in sustainable animal production
$WURJLQDPXVFOHVSHFL¿FXELTXLWLQOLJDVHLVKLJKO\H[SUHVVHGLQWKH
smooth muscle of the chicken gizzard
K. Nakashima, A. Ishida and M. Katsumata
1$52,QVWLWXWHRI/LYHVWRFNDQG*UDVVODQG6FLHQFH7VXNXED-DSDQ[email protected]

Introduction
Muscle proteolysis in catabolic conditions is due primarily to activation of the ubiquitin-proteasome
proteolytic pathway, whereby the proteins destined to be degraded are linked to a chain of ubiquitin
molecules, which targets them of rapid breakdown by the proteasome. Evidence suggests that
atrogin-1, an E3 ubiquitin ligase also referred to as MAFbx (muscle atrophy F-box), plays a pivotal
role in muscle atrophy (Gomes et al., 2001). Atrogin-1 plays a critical role in development of muscle
proteolysis and its gene expression is a reliable index of muscle proteolysis (Ohtsuka et al., 2011).

Atrogin-1 mRNA is expressed in smooth muscle, and its gene expression is increased in the smooth
muscle of uterine of the postpartum period and in the smooth muscle of intestine of feed deprivation.
In chicken, the gizzard is a characteristic avian smooth muscle sac functioning to crush the feed and
begin in digestion of the proteins. However, whether atrogin-1 is expressed in the smooth muscle
of the chicken gizzard has yet to be investigated.

We previously reported that the expression of atrogin-1 mRNA was increased by fasting, and
decreased by refeeding in the skeletal muscle of chicken (Nakashima et al +RZHYHU
regulation of atrogin-1 expression in the smooth muscle of the chicken gizzard by food deprivation and
QXWULWLRQDOVXSSO\KDV\HWWREHGH¿QHG7KXVLQWKHSUHVHQWVWXG\WKHHIIHFWVRIIDVWLQJDQGUHIHHGLQJ
on the mRNA level of atrogin-1 in the smooth muscle of the chicken gizzard were investigated.

Material and methods

At 11 days of age, four chicks with body weights of 114±1.31 (mean ± SD) were subjected to
tissue sampling (skeletal muscle, heart, gizzard, brain and liver) to established tissue distribution
of atrogin-1 mRNA.

At 11 days of age, another eighteen chicks of similar body weight were selected and housed in wire-
bottomed aluminum cages. They were given free access to a commercial starter diet and water for 3
G$WWKHVWDUWRIWKHH[SHULPHQWGD\ROGFKLFNVZHUHGLYLGHGLQWRWKUHHJURXSV Q JURXS IHG
group, food-deprived group and refed group. Fed chicks were continuously given free access to the
diet for 3 days before being killed. Food-deprived chicks subjected to food deprivation for 24 h before
they were killed on day 15. Refed chicks subjected to food deprivation for 24 h and then refed for 2
KEHIRUHEHLQJVDFUL¿FHGRQGD\7KHWLVVXHVDPSOHVZHUHIUR]HQZLWKOLTXLGQLWURJHQDQGVWRUHG
at -80 °C until analysis. The mRNA level of atrogin-1 was measured by a real-time RT-PCR method.

Data were analyzed by Paired Student’s t-test to evaluate tissue distribution of atrogin-1 mRNA or
ANOVA and Tukey’s multiple comparison test to determine the effect of feeding regime on atrogin-1
mRNA level in smooth muscle. A PRIYDOXHZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQL¿FDQW(DFK
result is expressed as the mean ± standard deviation (SD). Gene expression levels were estimated on
WKHEDVLVRI3&5HI¿FLHQF\DQGWKUHVKROGF\FOHGHYLDWLRQRIDQXQNQRZQVDPSOHYHUVXVDFRQWURO
18S ribosomal RNA was chosen as reference gene.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 419
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_153, © Wageningen Academic Publishers 2013
Results and discussion
As shown in Table 1, the expression of atrogin-1/MAFbx mRNA in the smooth muscle of the gizzard
is higher than that of skeletal muscle and heart in chicken.

$OWKRXJKJL]]DUGZHLJKW J ZDVVLJQL¿FDQWO\GHFUHDVHGE\IDVWLQJ IHG“JYHUVXVIDVWHG

5.99±0.37 g, P<0.05), refeeding failed to restore these tissues weight to the fed level within 2 h (refed,
“J ,QWKHVPRRWKPXVFOHRIWKHJL]]DUGH[SUHVVLRQRIDWURJLQP51$ZDVVLJQL¿FDQWO\
LQFUHDVHGE\IDVWLQJZKHUHDVUHIHHGLQJVLJQL¿FDQWO\VXSSUHVVHGLW 7DEOH 

In conclusion, we demonstrated here that the smooth muscle of the chicken gizzard expresses high
levels of atrogin-1 gene and its expression is associated with the muscle atrophy that occurs during
food deprivation.

Table 1. Tissue distribution of chicken atrogin-1 mRNA in 11-day-old chicken.

Tissue Relative expression

Skeletal muscle 1.00±0.49

Heart 1.22±0.43
Gizzard 13.0±2.91**
Brain “
Liver 0.12±0.03**

Results are expressed as ratios to relative to the 18S rRNA levels in skeletal muscle, whose expression level was taken
to be equal to 1. Value are means±SD (n=4). Symbols: *P<0.05; **P<0.01.

Table 2. Effects of fasting and refeeding on the mRNA levels of atrogin-1 in the smooth muscle
JL]]DUG RIFKLFNHQV

Treatment Relative expression

Fed 1.00±0.28b
Fasted “a
Refed “b

5HVXOWVRI51$TXDQWL¿FDWLRQDUHH[SUHVVHGDVUDWLRVWRUHODWLYHWRWKH6U51$OHYHOVLQIHGFKLFNHQVZKRVHH[SUHVVLRQ
OHYHOZDVWDNHQWREHHTXDOWR9DOXHVDUHPHDQV“6' Q  
a,b P<0.05.

References
*RPHV0'6+/HFNHU57-DJRH$1DYRQDQG$/*ROGEHUJ$WURJLQDPXVFOHVSHFL¿F)ER[SURWHLQ
highly expressed during muscle atrophy. Proc. Natl. Acad. Sci. USA, 98, 14440-14445.
1DNDVKLPD.<<DNDEH0<DPD]DNLDQG+$EH(IIHFWVRIIDVWLQJDQGUHIHHGLQJRQH[SUHVVLRQRIDWURJLQ
and Akt/FOXO signaling pathway in skeletal muscle of chicks. Biosci. Biotechnol. Biochem., 70, 2775-2778.
2KWVXND$1.DZDWRP.1DNDVKLPD7$UDNLDQG.+D\DVKL*HQHH[SUHVVLRQRIPXVFOHVSHFL¿FXELTXLWLQ
ligase, atrogin-1/MAFbx, possitively correlates with skeletal muscle protelysis in food-deprivated broiler chickens.
-3RXOW6FL

420 Energy and protein metabolism and nutrition in sustainable animal production
An in ovo 13C-tracer approach to explore liver intermediary metabolism
in developing chicken embryos
Q. Hu, U. Agarwal and B.J. Bequette
$QLPDODQG$YLDQ6FLHQFHV'HSDUWPHQW8QLYHUVLW\RI0DU\ODQG&ROOHJH3DUN0'86$
[email protected]

Introduction
7KHQXWULHQWFRPSRQHQWVRIWKHHJJDUH¿QLWHWKXVWKHFKLFNHQHPEU\RPXVWSURSHUO\FRRUGLQDWH
the utilization of these substrates to support developmental requirements. However, the pathways
that the macronutrients are allocated towards and the relative contributions the substrates make to
WKHYDULRXVSDWKZD\ÀX[HVDWWKHWLVVXHDQGZKROHERG\OHYHOVRIWKHHPEU\RDUH\HWWREHGH¿QHG
The commercial availability of stable isotope tracers enables researchers to explore the intricacies of
individual as well as connecting metabolic pathways networks. The objective of the present study was
to employ a constant infusion protocol for the in ovo delivery of [13C]glucose to attain steady-state
labeling and high rates of incorporation of 13C into intermediates of gluconeogenesis-glycolysis and
the Krebs cycle for accurate measurement of 13&SRVLWLRQDOLVRWRSRPHUVIRUÀX[DQDO\VLV

Material and methods

A sterile solution of [13C]glucose (7 mg/50 mg saline) was constantly infused (45 mg solution/h)
over 8 h into the chorio-allantoic compartment of chicken eggs on embryonic (e) day 14 and 19 (n=4).
At the end of infusion, blood was withdrawn from a vitelline vessel, and the whole liver rinsed with
ice-cold saline and frozen for analysis. Glucose was extracted from blood with ice-cold acetone and
converted to the isopropylidene pentaacetate derivative prior to selected ion monitoring (SIM) by
gas chromatography-mass spectrometry (GC-MS) under electron impact mode (Sunny and Bequette,
2010). Liver was homogenized in ice-cold sulfosalicylic acid (10% w/v), amino acids isolated by
cation-exchange chromatography and converted to the t-butyldimethylsilyl derivitive prior to SIM
by GC-MS under electron impact mode. Mass fragmentology analysis of aspartate and glutamate
was conducted because these amino acids are in metabolic equilibrium with their corresponding
LQWHUPHGLDU\PHWDEROLWHR[DORDFHWDWHDQGĮNHWRJOXWDUDWHUHVSHFWLYHO\*OXFRQHRJHQHVLVZDV
FDOFXODWHGDFFRUGLQJWR+D\PRQGDQG6XQHKDJ  ZLWKPRGL¿FDWLRQVDQG.UHEVF\FOHDQG
QRQHVVHQWLDODPLQRDFLGV 1($$ ÀX[HVDFFRUGLQJWR%HWKROGet al. (1994). Results were analyzed
by using student’s t-test of SAS 9.2 (SAS Institute, Cary, NC, USA). Probability of P<0.05 was
FRQVLGHUHGWREHVWDWLVWLFDOO\VLJQL¿FDQW

Results and discussion

Employing the constant infusion approach, Cori cycling, i.e. glucose carbon recycling, tended to be
higher in e14 embryos (Table 1) which is similar to earlier work from our lab using a daily injection
approach (Sunny and Bequette, 2010). Glucose entry, gluconeogenesis and glycogen turnover were all
greater in e14 compared to e19 embryos, which suggests that embryos at later stages of development
conserve more glycogen for post-hatch energy requirements.

7KHUHZDVVLJQL¿FDQWLQFRUSRUDWLRQRI13C glucose carbon into many NEAA (aspartate, glutamate,

glutamine, serine, glycine and alanine) in the liver indicating the capacity of the embryo to synthesize
these amino acids. Importantly, by e11, there is only ~125 mg of preformed carbohydrates remaining
in the residual yolk and albumen (Hu et al., 2011), suggesting that non-glucose sources such as
JO\FHURO \RON DQGDPLQRDFLGV DOEXPHQ PXVWEHVLJQL¿FDQWFRQWULEXWRUVWR1($$V\QWKHVLV*LYHQ
the high degree of labeling of aspartate and glutamate, it was possible to calculate the proportion
of the Krebs cycle oxaloacetate and acetyl-CoA pools in the liver derived from the 3-carbon pool

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 421
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_154, © Wageningen Academic Publishers 2013
7DEOH%ORRGJOXFRVHÀX[HVLQHDQGHFKLFNHQHPEU\RV

e14 e19 SEM P-value

Cori cycling (%) 51.1 29.2  0.09

Fractional gluconeogenesis 0.49 0.32  NS1
Glucose entry (mg/g BW/d) 21.1 7.2 3.4 <0.05
Gluconeogenesis (mg/g BW/d) 10.75 2.53 2.15 <0.05
Glycogen turnover (mg/g BW/d) 10.35 4.70 1.27 <0.01

116QRWVLJQL¿FDQW P>0.10).

(eg. pyruvate). For the oxaloacetate pool, which is a major anaplerotic reaction to supply carbon
VNHOHWRQVIRU1($$V\QWKHVLVDQGWRPDLQWDLQ.UHEVF\FOHÀX[HVWKHFRQWULEXWLRQIURPWKHFDUERQ
pool ranged from 7.5 to 10% (SEM 1.8%) and did not differ between e14 and e19. Thus, substrates
WKDWDUHPHWDEROL]HGYLDRWKHUURXWHVLQWRWKH.UHEVF\FOHDUHPRUHVLJQL¿FDQWSUHFXUVRUVIRU1($$
synthesis. For the acetyl-CoA pool, which is the major oxidation route and precursor for fatty acid
synthesis, the contribution from the 3-carbon pool ranged from 8.8 to 15% (SEM 3.4%) and did not
differ between e14 and e19. Hence, fatty acids are the major oxidative substrates.

In conclusion, this tracer approach employing [13C]glucose allow for the measurements of Krebs
F\FOHÀX[HVDQGDFWLYLW\JOXFRQHRJHQHVLVJO\FRO\VLVJO\FRJHQWXUQRYHUDQGLGHQWL¿FDWLRQRISRWHQWLDO
precursors derived from yolk and albumen that contribute to oxidation and NEAA synthesis. The
results generated from this study will also lead to a better understanding of the nutritional adequacy
SURYLGHGE\WKHHJJFRQWHQWVDQGWKHLULQÀXHQFHRQHPEU\RJURZWKDQGVXUYLYDO

References
Bethold, H. K., L. J. Wykes, F. Jahoor, P. D. Klein, and P. J. Reeds, 1994. The use of uniformly labeled substrates and
mass isotopomer analysis to study intermediary metabolism. Proc. Nutr. Soc. 53, 345-354.
Haymond, M.W. and A. L. Sunehag, 2000. The reciprocal pool model for the measurement of gluconeogenesis by use
of [U-13C] glucose. Am. J. physiol. Endocrinol. Metab. 278: E140-E145.
Hu, Q., U. Agarwal, K.R. Somers, K.M. Bailey and B. J. Bequette, 2011. Energy balance regulation and carbohydrate
utilization in developing chicken embryos. FASEB J. 25: 774.11.
Sunny, N. E. and B. J. Bequette, 2010. Gluconeogenesis differs in developing chick embryos derived from small
compared with typical size broilder breeder eggs. J. Anim. Sci. 88:912-921.

422 Energy and protein metabolism and nutrition in sustainable animal production
7KHKLJKIDWGLHWDQGÀD[VHHGFDNHLQÀXHQFHGRQOLSLGPHWDEROLVPLQ
mice selected for body weight
M. Matusiewicz1, S. Fiedorowicz1, K. Fiszdon2, I. Kosieradzka1 and W. Bielecki
1Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Warsaw University of
/LIH6FLHQFH±6**:&LV]HZVNLHJR:DUVDZ3RODQG[email protected]
2Department of Genetics and Animal Breeding, Faculty of Animal Science, Warsaw University of
/LIH6FLHQFH±6**:&LV]HZVNLHJR:DUVDZ3RODQG
Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw
8QLYHUVLW\RI/LIH6FLHQFH±6**:1RZRXUV\QRZVNDF:DUVDZ3RODQG

Introduction
It is becoming increasingly evident that consumption of high-fat diet rich in saturated fatty acids
UHVXOWVLQQHJDWLYHHIIHFWRQOLSLGPHWDEROLVPDQGUHGR[VWDWH2SSRVLWHHIIHFWZDVFRQ¿UPHGIRU
ÀD[VHHGVWKDWKDYHDQXPEHURIKHDOWKEHQH¿WVUHVXOWIURPWKHFRQWHQWRIQXWULHQWVLQFOXGLQJRLO
ULFKLQSRO\XQVDWXUDWHGIDWW\DFLGVGLHWDU\¿EHUDQGSRO\SKHQROLFFRPSRXQGV/LQRODFXOWLYDUVHHGV
ZHUHFKDUDFWHUL]HGE\UHGXFHGFRQWHQWRIĮOLQROHQLFDFLGJUHDWHUVWDELOLW\DQGR[LGDWLRQUHVLVWDQFH
7KHREMHFWLYHRIWKHVWXG\ZDVWRHYDOXDWHWKHLQÀXHQFHRIKLJKIDWGLHWULFKLQODUGDQGDGGLWLRQRI
ÀD[VHHGFDNHRQVHUXPUHGR[VWDWHDQGOLSLGSUR¿OHFRQFHQWUDWLRQRIOLYHUDGLSRQHFWLQ±LPSRUWDQW
cytokine secreted by fat tissue cells as well as effect on the development of the perirenal adipose
tissue in mice selected for body weight. The selection had resulted in differences in serum lipid
SUR¿OHDQGDQWLR[LGDQWVWDWH

Material and methods

Experiment was carried out on male mice that were divergently selected over 130 generations for
high (line C) and low (line L) body weight at the postnatal day 21. These lines of animals were
GHULYHGIURPRXWEUHGVWRFNE\FURVVLQJIRXULQEUHGVWUDLQV$6W%1D%$/%FDQG&%/-Q

7KHH[SHULPHQWDODQLPDOVZHUHGLYLGHGLQWRJURXSVZLWKLQGLYLGXDOVLQHDFKDQGIHG ad
libitum IRUGD\VRQHRIWKHLVRSURWHLQGLHWV  OLQH&DQG  OLQH/ZLWKVWDQGDUGGLHW  OLQH
&DQG  OLQH/ZLWKKLJKIDWGLHWULFKLQSRUNODUG  OLQH&DQG  OLQH/ZLWKGLHWVXSSOHPHQWHG
ZLWKRIÀD[VHHGFDNH 7DEOH 6HUXPWRWDODQWLR[LGDQWVWDWXV 7$6 OLSLGSHUR[LGHVLQVHUXP
measured as thiobarbituric acid reactive substances (TBARS), total cholesterol (TC) and triglycerides
(TG) were detected by the spectrophotometric method. Concentration of adiponectin in liver was
determined by enzyme-linked immunosorbent assay.

Results and discussion

0LFHIHGGLHWWKDWFRQWDLQVÀD[VHHGFDNHGHPRQVWUDWHGVLJQL¿FDQWO\KLJKHUOHYHORIVHUXP7$6
FRPSDULQJWRDQLPDOVIHGVWDQGDUGGLHW 7DEOH 7KH7$6ZDVDOVRVLJQL¿FDQWO\KLJKHULQOLQH/
RIPLFHWKDQLQOLQH&&RQFHQWUDWLRQRIVHUXP7%$56ZDVVLJQL¿FDQWO\ORZHULQÀD[VHHGFDNH
JURXSWKDQLQKLJKIDWGLHW)HHGLQJÀD[VHHGFDNHUHVXOWHGLQGHFUHDVHGFRQFHQWUDWLRQRIVHUXP7&
DQG7*FRPSDUHGWRKLJKIDWGLHW/LQH/RIPLFHZDVFKDUDFWHUL]HGE\VLJQL¿FDQWO\ORZHU7&DQG
TG compared to line C. Two-way ANOVA revealed the interaction in liver adiponectin between
diet and mice line. Interaction between diet and mice line demonstrated that feeding SD to line L
LQFUHDVHG$'3ZKLOHIHHGLQJ+)'RUÀD[VHHGFDNHGLHWWROLQH/UHGXFHG$'39LVXDOL]DWLRQRI
perirenal adipose tissue seemed to appear similar in all groups of mice.

$GGLWLRQRIÀD[VHHGFDNHWRWKHKLJKIDWGLHWLQÀXHQFHGSUR¿WDEO\RQVHUXPUHGR[VWDWHDQG
examined parameters of serum lipid metabolism in mice. Line L was characterized by better values

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 423
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_155, © Wageningen Academic Publishers 2013
RIWKDWSDUDPHWHUVWKDQOLQH&7KHEHQH¿FLDOHIIHFWRIGLHWVRQOLYHUDGLSRQHFWLQFRQFHQWUDWLRQLV
greater in line L.

7DEOH&RPSRVLWLRQRIH[SHULPHQWDOGLHWVDQGWKHLUQXWULWLRQDOYDOXH '0 1

Component Group diet

Standard High-fat Flaxseed cake

Flaxseed cake (%) - - 30

Total protein 20.0 20.1 19.7
Crude fat 2.8 12.1 
GE (kcal/100 g)  515 512
GE from crude fat (kcal/100 g) 27 115 110

1 Perirenal adipose tissue was visualized by scanning electron microscopy (FEI Quanta 200). The results were analyzed

by two-way ANOVA with means compared using Tukey’s correction, a difference of P<0.05 between means was
FRQVLGHUHGWREHVLJQL¿FDQW

7DEOH7ZRZD\$129$ GLHWPLFHOLQH IRUH[DPLQHGSDUDPHWHUVRIPLFH PHDQ“6(0 1

Factor Red-ox state /LSLGSUR¿OH Adiponectin

(ng/mg protein)
TAS TBARS TC TG
(mmol/l) (nmol/ml) (mmol/l) (mmol/l)

Diet * * * * *
SD 1.91±0.13a 0.28±0.03ab 1.54±0.11a 1.34±0.18a “
HFD 2.12±0.13ab 0.38±0.03b “b 1.90±0.18b 390.98±47.88
Flaxseed cake 2.51±0.13b 0.27±0.03a 1.91±0.11a 1.29±0.18a 323.77±47.88
Mice line * NS * * NS
C 2.00±0.10 0.32±0.02 2.29±0.09 1.77±0.15 352.52±39.09
L “ 0.30±0.02 1.78±0.09 1.25±0.15 259.74±39.09
Interaction NS NS NS NS *
SD C “ 0.29±0.04 “ 1.27±0.25 “
SD L 2.12±0.18 0.27±0.04 “ 1.40±0.25 “
HFD C 1.80±0.18 0.38±0.04 “ 2.37±0.25 “
HFD L 2.45±0.18 0.39±0.04 “ 1.43±0.25 “
Flaxseed cake C 2.50±0.18 0.30±0.04 “ “ “
Flaxseed cake L 2.52±0.18 0.25±0.04 “ 0.91±0.25 “

1 VLJQL¿FDQWHIIHFW P< 16 QRQVLJQL¿FDQW a,b,c values within a factors with different superscripts differ
VLJQL¿FDQWO\ P<0.05).

424 Energy and protein metabolism and nutrition in sustainable animal production
%UDQFKHGFKDLQĮNHWRDFLGVLQSODVPDRIJURZLQJFKLFNHQZKHQLVWKH
time for blood sampling?
A. Pastor, A. Sünder and F. Liebert
Division Animal Nutrition Physiology, Department of Animal Sciences, Georg-August-University,
.HOOQHUZHJ*RHWWLQJHQ*HUPDQ\[email protected]

Introduction
Complementary to quantitative requirement studies for the branched-chain amino acids (BCAA)
OHXFLQHLVROHXFLQHDQGYDOLQHVWXGLHVRQG\QDPLFVRIĮNHWRDFLGV\LHOGHGE\GHJUDGDWLRQRIWKH
%&$$FRXOGSURYLGHLPSRUWDQWDGGLWLRQDOPHWDEROLFLQVLJKWV*HQHUDOO\WKH¿UVWVWHSLQFDWDEROLVP
of the BCAAs is undertaken by the same enzyme (branched-chain amino acid transferase; Harper
et al DQG\LHOGVWKHFRUUHVSRQGLQJEUDQFKHGFKDLQĮNHWRDFLGV %&.$ ĮNHWRLVRFDSURLF
DFLG .,& ĮNHWRPHWK\OYDOHULFDFLG .09 DQGĮNHWRLVRYDOHULFDFLG .,9 IRUOHXFLQHLVROHXFLQH
DQGYDOLQHUHVSHFWLYHO\,QKXPDQEHLQJVDGH¿FLHQF\RIWKLVHQ]\PHPD\FUHDWHDEXLOGXSRIWKH
BCAA and their corresponding BCKA, leading to the maple syrup urine disease (Langenbeck et al.,
1978). Therefore, determination of the BCKA concentration in blood plasma is a standard procedure
in humans. However, for growing chicken, guidelines are not available to indicate the optimal time
schedule for sampling. The current study was conducted both to establish a method to detect BCKA
in chicken plasma and to yield information about the time course of BCKA in blood from the jugular
vein for an improved understanding of the BCAA metabolism in growing chicken.

Material and methods

Eight groups (n=5) of 37-day old male meat type chicken (Ross 308) were fasted overnight (12
hours), subsequently getting access to feed for half an hour. Afterwards, diets were removed and
blood samples were taken from the vena jugularis dextra at 1, 3, 5, 9, 11, 12 and 23 hours after feed
removal. The diet fed met the National Research Council (1994) requirements. Blood samples were
centrifuged to yield blood plasma, which was stored at -80 °C until further analysis.

Analysis of BCKA was carried out by high performance liquid chromatography (HPLC), adapted
from Kandár et al. (2009) for human beings.

Statistical analyses (P” GHSHQGLQJRQWLPHZHUHFDUULHGRXWXVLQJWKH6366VWDWLVWLFDOVRIWZDUH

package (version 19.0 for Windows, IBM SPSS Statistics, Inc. Chicago, IL). Data were subjected to
DYHUL¿FDWLRQRIYDULDQFHKRPRJHQHLW\DFFRUGLQJWR/HYHQHWHVWIROORZLQJ7XNH\RU*DPHV+RZHOO
post-hoc tests.

Results and discussion

The method of Kandár et al. (2009) was successfully adapted for determination of BCKA in chicken
SODVPDPDNLQJXVHRIVHYHUDOPRGL¿FDWLRQV HJFRQFHQWUDWLRQRILQWHUQDOVWDQGDUGLQMHFWLRQ
volume, calibration line). The observed time course of the BCKA related to food deprivation (h) is
demonstrated in Figure 1.

7KUHHKDIWHUIHHGUHPRYDOWKH.,&DQG.,9FRQFHQWUDWLRQLQEORRGSODVPDGHFUHDVHGVLJQL¿FDQWO\
(P<0.05) compared to a feed deprivation of 1 h, but remained relatively stable until 11 h after feed
UHPRYDO7ZHOYHDQGKDIWHUIHHGGHSULYDWLRQRQO\.,&FRQFHQWUDWLRQLQFUHDVHGVLJQL¿FDQWO\
compared to 3 h after feed removal, suggesting enhanced catabolism of Leu. For KMV concentration,
DVLJQL¿FDQWHIIHFW P<0.05) was observed only between 3 h and 12 h of fasting. Accordingly,
Leslie and Saunderson (1985) reported the highest BCKA concentration in blood 1 h after feeding.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 425
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_156, © Wageningen Academic Publishers 2013
 30
D
25
&RQWHQWRIĮNHWRDFLGV
DE D

DE
D DE
20
—PROOSODVPD

DE

15 b D
KIC
DE
DE DE
DE KMV
10 b
DE DE
DE
D KIV
DE DE DE
5 b DE
DE

0
1 3 5 7 9 11 12 23
)HHGGHSULYDWLRQ K
)LJXUH7LPHFRXUVHRIWKHEUDQFKHGFKDLQĮNHWRDFLGVGHSHQGLQJRQWLPHRIIHHGGHSULYDWLRQ 
K YDOXHVZLWKDGLIIHUHQWVXSHUVFULSWGLIIHUZLWKLQDVDPHOLQH P<

It is concluded, that 5 h to 11 h after feeding is the preferable time for blood sampling in meat type
chicken for further analysis of BCKA. Within this timeframe, concentration of BCKA demonstrated
low variation. Ongoing experiments study effects of varying dietary BCAA supply on BCKA
concentration in blood plasma to improve the understanding of BCAA related metabolic processes
DQGLQWHUUHODWLRQVKLSVWRWKHPRGHOLQJRI$$HI¿FLHQF\EDVHGUHTXLUHPHQWVLQJURZLQJFKLFNHQ

References
Harper, A., R. Miller and K. Block, 1984. Branched-chain amino acid metabolism. Ann. Rev. Nutr. 4, 409-454
.DQGiU53äiNRYi--LURãRYiDQG06ODGNi'HWHPLQDWLRQRIEUDQFKHGFKDLQDPLQRDFLGVPHWKLRQLQH
SKHQ\ODODQLQHW\URVLQHDQGĮNHWRDFLGVLQSODVPDDQGGULHGEORRGVDPSOHVXVLQJ+3/&ZLWKÀRXUHVFHQFH
GHWHFWLRQ&OLQ&KHP/DE0HG
Langenbeck, U., U. Wendel, A. Mench-Hoinowski, D. Kuschel, K. Becker, H. Przyrembel and H. J. Bremer, 1978.
&RUUHODWLRQVEHWZHHQEUDQFKHGFKDLQDPLQRDFLGVDQGEUDQFKHGFKDLQĮNHWRDFLGVLQEORRGLQPDSOHV\UXSXULQH
disease. Clin. Chim. Acta. 88, 283-291.
/HVOLH6DQG&/6DXQGHUVRQ0HDVXUHPHQWRIEUDQFKHGFKDLQĮNHWRDFLGVLQWKHEORRGSODVPDSIWKHGRPHVWLF
fowl and poultry. Comp. Biochem. Physiol. 80B, 99-102.
National Research Council, 1994. Nutrient Requirements of Poultry. 9th Rev. ed. Natl. Acad. Press, Washington, D.C.

426 Energy and protein metabolism and nutrition in sustainable animal production
Lean accretion and protein turnover are enhanced by intermittent bolus
feeding in neonatal pigs
S.W. El-Kadi1,2, C. Boutry1, A. Suryawan1, M.C. Gazzaneo1, R.A. Orellana1, N. Srivastava1, H.V.
Nguyen1, M.L. Fiorotto1 and T.A. Davis1
1USDA/ARS Children Nutrition Research Center, Pediatrics, Baylor College of Medicine, Houston,
TX, USA; [email protected]
2Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA

Introduction
Orogastric tube feeding is indicated in neonates with impaired ability to ingest food normally and
can be administered by intermittent bolus (INT) or continuous (CON) infusion. Insulin and amino
acids play important roles in the regulation of protein synthesis in the neonate. While the sensitivity
to insulin is developmentally regulated (Davis et al., 1998) and greatly diminishes with age (Wray-
Cahen et al., 1997), amino acids exert a positive effect on muscle protein synthesis throughout life
(Denne et al., 1991; Davis et al., 1998; Volpi et al., 1998). In addition to providing amino acids to
RWKHURUJDQVZKHQGLHWDU\VXSSOLHVDUHLQVXI¿FLHQWKLJKUDWHVRISURWHRO\VLVDUHQHFHVVDU\WRSURYLGH
amino acids for ongoing tissue modeling and rapid growth. The two most important proteolytic
pathways in skeletal muscle are thought to be the ubiquitin-proteasome and autophagy-lysosome
V\VWHPV 9HQWDGRXUDQG$WWDL[ 7KHDLPRIWKLVVWXG\ZDVWRGHWHUPLQHLIWKHVHIHHGLQJ
modalities affect growth and lean tissue accretion, and the mechanisms for this response.

Material and methods

1HRQDWDOSLJV Q GROG“NJ%: ZHUHVXUJLFDOO\¿WWHGXQGHUJHQHUDODQHVWKHVLDXVLQJ
sterile techniques with catheters in the carotid artery, jugular vein, and gastrostomy tube. Pigs were
assigned to treatment using completely randomized design and fed the same sow milk replacer at
240 ml/kg/d in equivalent amounts either continuously (CON; 10 ml/kg/h) or intermittently (INT;
meal given over 15 min at 4 h intervals; 40 ml/kg/bolus) for 21d. Diets supplied 12.8 g/kg/d crude
protein and 175 kcal/kg/d. Dual X-ray absorptiometry was used before initiation of the study and 18
d after feeding to determine body composition and linear growth. Feed conversion was calculated at
the end of the study based on feed intake and protein and energy retention. Plasma samples, taken
over 4 h during the last day of feeding were analyzed for glucose and insulin concentrations as
described previously (El-Kadi et al., 2012). Fractional protein synthesis was determined using the
ÀRRGLQJGRVHPHWKRG 'DYLV et al., 1999). Pigs were euthanized 1 h following the last meal for the
INT group, and at the same time in the CON group. Muscle samples were removed and frozen at
-70 °C until analysis. Translation initiation and protein degradation signaling were determined by
polyacrylamide gel electrophoresis (El-Kadi et al., 2012). Data were analyzed using the MIXED
procedure of SAS (version 8.0). To investigate temporal effects, all samples were considered in a
repeated measures analysis. Values presented are means ± SEM.

Results and discussion

We recently demonstrated that 24 h after the initiation of feeding, protein synthesis was enhanced
by intermittent bolus feeding compared to continuous feeding with no change in protein degradation
(El-Kadi et al., 2012). Whether this acute response is maintained over long-term feeding periods
has not been determined.

Weight gain was greater for INT than for CON pigs and resulted in heavier body weights from 9
XQWLOGRIIHHGLQJ YV“NJ  P< 7KHHI¿FLHQF\RISURWHLQDQGHQHUJ\UHWHQWLRQV
were enhanced by 30 and 44% in INT compared to CON fed pigs (P<0.05). Lean tissue mass (4.75

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 427
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_157, © Wageningen Academic Publishers 2013
vs. 3.53±0.12 kg) and spine length (28 vs. 30±0.4 cm) were greater in INT than CON pigs (P<0.05).
Glucose and arterial insulin levels measured on the last day of feeding were greater for INT after the
meal than for CON pigs (P<0.05). Muscle protein synthesis increased by 40% in longissimus dorsi
YV“G DQGLQJDVWURFQHPLXV YV“ DQGVROHXV YV“ 
of the INT as compared to the CON group (P<0.05).

To establish the mechanism for this response, translation initiation and protein degradation signaling
were studied in longissimus dorsi muscle. Insulin receptor and insulin receptor substrate-1 expression
and protein kinase B phosphorylation in skeletal muscle were unaffected by feeding modality.
Formation of the active eukaryotic initiation factor (eIF) 4E-eIF4G complex and phosphorylation of
ULERVRPDOSURWHLQ6NLQDVHZHUHKLJKHU P< DQGSKRVSKRU\ODWLRQRIH,)ĮZDVORZHU P<0.05)
in INT compared to CON fed pigs indicating increased translation initiation. Sodium-coupled neutral
amino acid transporter 2 expression (P<0.05), but not L-type amino acid transporter 1, was higher in
,17FRPSDUHGWR&21SLJVVXJJHVWLQJHQKDQFHGJOXWDPLQHWUDQVSRUW$EXQGDQFHRIPXVFOHVSHFL¿F
XELTXLWLQOLJDVHVPXVFOH5,1*¿QJHUDQG)ER[SURWHLQDWURJLQ0$)E[ZDVKLJKHUIRU,17
compared to CON pigs (P<0.05).

These results suggest that intermittent feeding enhances lean tissue accretion as compared to
continuous feeding, and that this difference is apparent by 9 d and persists for the duration of feeding.
The increased rate of lean tissue accretion in pigs fed intermittently occurs in response to an increased
activation of translation initiation. In addition, intermittent feeding enhances lean tissue accretion
by increasing amino acid transport and protein turnover.

Acknowledgements
6XSSRUWHGE\1,+$5DQG86'$$56

References
Davis, T.A., D.G. Burrin, M.L. Fiorotto, P.J. Reeds and F. Jahoor, 1998. Roles of insulin and amino acids in the
regulation of protein synthesis in the neonate. J. Nutr. 128, 347S-350S.
Davis, T.A., M.L. Fiorotto, H.V. Nguyen and D.G. Burrin, 1999. Aminoacyl-tRNA and tissue free amino acid pools are
HTXLOLEUDWHGDIWHUDÀRRGLQJGRVHRISKHQ\ODODQLQH$P-3K\VLRO(QGRFULQRO0HWDE(
Denne, S.C., E.M. Rossi and S.C. Kalhan, 1991. Leucine kinetics during feeding in normal newborns. Ped. Res. 30,
23-27.
El-Kadi, S.W., A. Suryawan, M.C. Gazzaneo, N. Srivastava, R.A. Orellana, H.V. Nguyen, G.E. Lobley and T.A. Davis,
2012. Anabolic signaling and protein deposition are enhanced by intermittent compared with continuous feeding
LQVNHOHWDOPXVFOHRIQHRQDWHV$P-3K\VLRO(QGRFULQRO0HWDE(
9HQWDGRXU6DQG'$WWDL[0HFKDQLVPVRIVNHOHWDOPXVFOHDWURSK\&XUU2SLQ5KHXPDWRO
Volpi, E., A.A. Ferrando, C.W. Yeckel, K.D. Tipton and R.R. Wolfe, 1998. Exogenous amino acids stimulate net muscle
protein synthesis in the elderly. J. Clin. Invest. 101, 2000-2007.
Wray-Cahen, D., P.R. Beckett, H.V. Nguyen and T.A. Davis, 1997. Insulin-stimulated amino acid utilization during
glucose and amino acid clamps decreases with development. Am. J. Physiol. Endocrinol. Metab. 273, E305-E314.

428 Energy and protein metabolism and nutrition in sustainable animal production
Changes in tissue amino acid composition and protein metabolism in
piglets due to a limiting supply of total sulphur amino acids
J.A. Conde-Aguilera1,2, C. Cobo-Ortega1,2, N. Le Floc’h1,2, Y. Mercier and J. van Milgen1,2
1,15$8053(*$6(6DLQW*LOOHV)UDQFH[email protected]
2$JURFDPSXV2XHVW8053(*$6(5HQQHV)UDQFH
$GLVVHR)UDQFH6$6$QWRQ\)UDQFH

Introduction
In growing pigs, amino acid (AA) requirements are usually estimated using body weight gain or N
retention as response criteria which imply that the AA composition of total body protein should be
constant. However, there are indications that the AA composition of body protein is affected by the
SURWHLQHQHUJ\RU$$VXSSO\$GH¿FLHQWWRWDOVXOSKXU$$ 76$$0HW&\V VXSSO\KDVEHHQVKRZQ
WRPRGLI\WKH$$SUR¿OHRIWLVVXHSURWHLQV &RQGH$JXLOHUDet al., 2010). Protein retention results
from the rate of protein synthesis exceeding that of protein breakdown. Whether one or both of these
processes are affected by a limiting AA supply to preserve the metabolic needs of the animal remains
to be explored. The objective of this study was to evaluate the response of protein metabolism to a
GLHWGH¿FLHQW 76$$ RUVXI¿FLHQW 76$$ LQ76$$VXSSO\E\DVVHVVLQJWKHSURWHLQV\QWKHVLV
proteasome enzyme activity, and the AA composition of different tissues and organs in piglets.

Material and methods

Twelve Piétrain × (Large White × Landrace) piglets were used 14 d after weaning (at 42 d of age)
DQGZHUH¿WWHGZLWKDMXJXODUFDWKHWHU7KH\ZHUHGLYLGHGLQJURXSVDQGUHFHLYHGHLWKHU76$$RU
TSAA+ for 10 d. Diets were formulated to meet the nutritional recommendations except for the Met
and TSAA supplies in TSAA-, which were 31 and 28% below requirements, respectively. The daily
feed allowance was about 25% below the ad libitum intake capacity. To measure protein synthesis,
SLJOHWVZHUHLQMHFWHGZLWKDÀRRGLQJGRVHRI>13C] l-Val and were slaughtered 14 min thereafter.
Samples of the liver, kidneys, longissimus dorsi (LM), rhomboideus (RM) and semitendinous (SM)
muscles, intestines, kidneys, and skin were collected for analysis.

Results and discussion

7KHGH¿FLHQW76$$VXSSO\GHFUHDVHGZHLJKWJDLQ P<0.001), the protein synthesis rate in LM by
32% (P<0.01) and in SM by 17% (3 7DEOH 7KH51$HI¿FLHQF\ZDVUHGXFHGE\DQG
22% for LM and SM, respectively (P” 0RUHRYHU60SURWHDVRPHDFWLYLW\ZDVJUHDWHU  
in piglets receiving diet TSAA- (P<0.05). Compared with pigs receiving diet TSAA+, the relative
weights of LM and distal jejunum were, respectively lower (3 0.09) and higher (P<0.01), the Met
content of tissue protein decreased in both SM (3 0.09) and LM (P<0.05), and was greater in the
liver (P<0.05) in piglets offered diet TSAA- (Table 2). Also, the Cys content was lower in the liver
(P<0.05) but higher in the distal jejunum and ileum (P< LQ76$$SLJVZKLFKPD\UHÀHFW
an adaptation of TSAA metabolism to maintain glutathione and tissue redox status (Richie et al.,
2004). Regarding the contents of other AA in tissue protein, the responses were minimal for the
proximal jejunum, ileum, kidneys and skin, but more important for LM, SM, RM, distal jejunum,
DQGOLYHU&KDQJHVLQWKH$$SUR¿OHRISURWHLQVPD\EHDVVRFLDWHGZLWKFKDQJHVLQWKHQDWXUHRI
protein deposited (Conde-Aguilera et al. ,QFRQFOXVLRQWKHDQLPDOVKRZVFHUWDLQÀH[LELOLW\
LQUHVSRQVHWRDOLPLWLQJ76$$VXSSO\TXHVWLRQLQJWKHXVHRIDFRQVWDQW$$SUR¿OHWRHVWDEOLVK$$
requirements. The functional consequences of these changes remain to be studied.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 429
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_158, © Wageningen Academic Publishers 2013
7DEOH3HUIRUPDQFHDQGSURWHLQPHWDEROLVPLQSLJOHWVRIIHUHGGLHW76$$RU76$$

TSAA- TSAA+ RSD P-value

Final BW (kg) 12.3 13.0 1.2 0.24

Weight gain (g/d) 292 375  <0.001
Feed conversion (g gain/g feed) 0.78 0.99 0.10 <0.01
Protein synthesis (%/d) LM 4.33  0.90 <0.01
RM 4.20 3.85  0.39
SM 4.11 4.95 0.72 0.10
(I¿FLHQF\RISURWHLQV\QWKHVLV1 LM 2.95 4.91  <0.001
RM 4.38   0.43
SM 2.98 3.80 0.50 0.03
Proteasome activity2 LM 2,170 1,959 384 0.39
RM 2,741 2,788 513 0.88
SM 2,284 1,914  0.05

1 Expressed as g of protein synthesized per day per mg of RNA.

2([SUHVVHGDVUHODWLYHÀXRUHVFHQFHXQLWVSHUPLQSHUPJRISURWHLQ

7DEOH:HLJKWDQGFRPSRVLWLRQRIZHLJKWLQSLJOHWVRIIHUHGGLHW76$$RU76$$1.

Tissue Diet Weight Weight Protein JJ1

(g) (g/kg (g/kg
BW) BW) Lys Met Cys Thr Val Ile Leu His

SM TSAA- 43.3 3.45    1.25 4.57  4.77 8.18 4.11**
TSAA+ 44.5 3.43  8.79 3.02 1.23 4.52 5.21 4.80 8.37 3.45
LM TSAA- 194   8.90  1.17 4.44  4.57  3.99**
TSAA+ 222 17.1   2.91 1.15 4.58 5.18 4.72 8.00 3.52
RM TSAA- 5.22 0.42   2.50  4.37 4.88* 4.33* 7.75 2.97**
TSAA+ 5.45 0.42 154 8.27  1.25 4.34 5.07 4.51 7.82 2.82
Liver TSAA- 308  174 7.29 2.50* 1.85* 4.55* 5.72** 4.48**  2.85*
TSAA+ 313 24.1  7.04 2.28 2.00 4.10 5.41 4.24 8.14 
Proximal TSAA- 148 11.7 135  2.14 1.44 4.43 4.89 3.93  2.27
jejunum TSAA+  11.2 131  2.20 1.49 4.24 4.85 3.93  2.34
Distal TSAA-  13.2**  7.58 2.40 1.58*  5.13 4.20 7.95 2.52
jejunum TSAA+ 141 10.8 122 7.45 2.37 1.40 4.41 5.07 4.17 7.85 
Ileum TSAA-  13.5  7.33 2.21 1.40* 4.19 4.73 3.89  2.19
TSAA+  12.4 119  2.17 1.35 4.10 4.71 3.92 7.17 2.27
Kidneys TSAA-  5.55  7.03 2.27  4.31 5.33 4.11 8.14 2.58
TSAA+ 70.4 5.45 135  2.21  4.34 5.38 4.19 8.15 
Skin TSAA- 35.1  194 4.45 1.47  2.71 3.45 2.18 4.54 2.01
TSAA+ 34.1 2.50  4.40 1.53 1.19 2.75 3.55 2.13 4.55 2.38

16LJQL¿FDQWOHYHOZLWKLQDFROXPQDQGWLVVXHE\ P” P” P”P”

References
&RQGH$JXLOHUD-$5%DUHD1/H)ORF¶K//HIDXFKHXUDQG-YDQ0LOJHQ$VXOIXUDPLQRDFLGGH¿FLHQF\
changes the amino acid composition of body protein in piglets. Animal 4, 1349-1358.
Richie, J. P., D. Komninou, Y. Leutzinger, W. Kleinman, N. Orentreich, V. Malloy and J. A. Zimmerman, 2004. Tissue
glutathione and cysteine levels in methionine-restricted rats Nutrition 20, 800-805.

430 Energy and protein metabolism and nutrition in sustainable animal production
Changes in fatty acid composition of intramuscular fat in growing
Iberian and Landrace × Large White pigs under identical nutritional
management
I. Seiquer, A. Haro, J.F. Aguilera and R. Nieto
,QVWLWXWHRI$QLPDO1XWULWLRQ(VWDFLyQ([SHULPHQWDOGHO=DLGtQ6SDQLVK&RXQFLOIRU6FLHQWL¿F
5HVHDUFK$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
*HQRW\SHLVRQHRIWKHPDLQIDFWRUVDIIHFWLQJOLSLGSUR¿OHRIPHDWLQSLJVDOWKRXJKRWKHUDVSHFWV
LQFOXGLQJGLHWFRPSRVLWLRQDQGVODXJKWHUERG\ZHLJKW %: PD\DOVRKDYHDGHHSLQÀXHQFHRQWKLV
variable (Wood et al., 2008).

The Iberian pig grows at a slower rate than leaner pig genotypes; it has lower potential for lean tissue
GHSRVLWLRQKLJKHUZKROHERG\IDWFRQWHQWDQGFRPSDUDWLYHO\ORZHUHI¿FLHQF\IRUQXWULHQWXWLOL]DWLRQ
than pigs from conventional breeds (Nieto et al., 2012). The high quality and consumer acceptability
of Iberian meat products are related to its high intramuscular fat content and particular fatty acid (FA)
SUR¿OHFRPSDUHGZLWKFRQYHQWLRQDOSLJV7KHLQLWLDOK\SRWKHVLVRIWKLVVWXG\LVWKDWWKHSDWWHUQRI)$
XQVDWXUDWLRQGXULQJJURZWKPLJKWFRQWULEXWHWRH[SODLQWKHRYHUDOOORZHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQ
previously observed in this native obese breed. This study is part of an experimental program aiming
DWH[SODLQLQJWKHORZHUPHWDEROLFHI¿FLHQF\RIWKH,EHULDQSLJFRPSDUHGWROHDQSLJW\SHV

Material and methods

The FA composition of intramuscular fat of longissimum dorsi (LD) and biceps femoris (BF) muscles
from Iberian and Landrace × Large White (LRW) pigs at different stages of growth, under identical
nutritional management, was investigated. A total of 38 pigs (19/genotype) were involved in the
VWXG\7KUHHSLJVJHQRW\SHZHUHVODXJKWHUHGDWWKHEHJLQQLQJRIWKHH[SHULPHQW “NJ%: 
the remainder 32 pigs were housed in individual pens and fed isoenergetic diets (14.4 MJ/kg DM)
containing either 130 or 170 g CP/kg DM, to match protein requirements of each breed (8 pigs/
genotype/CP level). Feeding level was restricted to 0.8 × ad libitum intake of the Iberian breed. At
“NJ%:SLJV JHQRW\SH&3OHYHO ZHUHVODXJKWHUHGE\H[VDQJXLQDWLRQDIWHUHOHFWULFDO
VWXQQLQJZKHUHDVWKHUHVWRIWKHSLJV  ZHUHVODXJKWHUHGDW“NJ%::LWKLQPLQ
after slaughter, samples of LD and BF were removed, trimmed of visible intermuscular fat, vacuum
packed and stored at -80 °C until analyses. Total intramuscular fat was extracted with chloroform/
PHWKDQRODQG)$ZHUHWUDQVPHWK\ODWHGDFFRUGLQJWR0RUULVRQDQG6PLWK  )$SUR¿OHZDV
REWDLQHGE\JDVFKURPDWRJUDSK\XVLQJD7KHUPR6FLHQWL¿FHTXLSPHQW )RFXV*&5RGDQR,WDO\ 
FA were expressed as % of total FA methyl esters. Statistical analysis revealed no effect of dietary
CP level on the FA analysed; therefore, results for each muscle type were re-analysed by a two-way
$129$UDQGRPL]HGGHVLJQLQFOXGLQJJHQRW\SH * DQG%:DV¿[HGIDFWRUVDQGWKHLULQWHUDFWLRQV
The experimental protocol was approved by the CSIC Bioethical Committee.

Results and discussion

6LJQL¿FDQWHIIHFWVRIJHQRW\SHDQG%:RQWKH)$FRPSRVLWLRQRILQWUDPXVFXODUIDWIRUERWKPXVFOHV
was found, except for total saturated FA (SFA), which was not affected by pig genotype (Table 1).
Monounsaturated FA (MUFA) increased (PBW<0.001) progressively with BW and were always
higher (PG=0.009) in intramuscular fat from Iberian pig´s muscles, mainly due to the more elevated
content of oleic acid (PG<0.001) compared with LRW pigs. On the contrary, total polyunsaturated
FA (PUFA) decreased (PBW<0.001) with increasing BW with percentage values always lower in

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 431
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_159, © Wageningen Academic Publishers 2013
7DEOH0DMRUIDWW\DFLGFRPSRVLWLRQ WRWDO)$PHWK\OHVWHUV RILQWUDPXVFXODUIDWLQ/'DQG%)
muscles from LRW and Iberian pigs slaughtered at different BW.

LRW (kg) Iberian (kg) SEM P-value

15 50 115 15 50 115 G1 BW G×BW

LD
& 25.5  27.0 25.8 28.1  0.40  0.341 
C18:1n9 31.5 35.5 41.3  40.7   0.021 <0.001 
&Q 18.3 10.5 7.4   3.9 0.31 <0.001 <0.001 
SFA 40.0 45.3 41.9 40.3 44.8  0.81 0.795  0.937
MUFA  41.4 48.9 40.2 47.5 54.3 0.73 0.009 <0.001 0.742
PUFA 22.4 13.3 9.2 19.5 7.7 5.0 0.43 <0.001 <0.001 0.547
QQ 11.9 10.3 12.4  12.8 12.7 0.21 <0.001 0.002 0.008
BF
& 23.4 24.5 24.7 25.2 27.2 23.9 0.22 0.015 0.005 0.003
18:1n9   39.4 32.4 39.3 45.0 0.42 <0.001 <0.001 0.547
Q 21.4  10.9 17.3 8.4 7.8 0.34 <0.001 <0.001 
SFA  40.2 38.7 38.8  35.8 0.33 0.759 <0.001 0.003
MUFA 32.8 41.7  39.1   0.49 <0.001 <0.001 0.583
PUFA  18.0 14.4 22.1 11.0  0.48 <0.001 <0.001 0.245
QQ 12.0 10.4 13.3 19.1 13.4 10.7 0.22 <0.001 <0.001 <0.001

1 G = genotype.

,EHULDQWKDQLQ/5:PXVFOHV$WWKH¿QLVKLQJVWDJHQQUDWLRLQ%)ZDVORZHULQ,EHULDQWKDQLQ
LRW pigs, and no differences were found for LD (PG×BW=0.008).

The results show relevant differences in the FA composition of intramuscular fat between genotypes,
and a clear evolution associated to increasing BW. The composition of the de novo synthesized FA
ZRXOGKDYHDGHHSHIIHFWRQ)$SUR¿OHRILQWUDPXVFXODUIDWDQGPLJKWDOVRLQÀXHQFHWKHHI¿FLHQF\
of energy utilization.

Acknowledgements
)XQGLQJE\WKH6SDQLVK0,1(&2 JUDQW$*/ LVJUDWHIXOO\DFNQRZOHGJHG

References
0RUULVRQ:5DQG/06PLWK3UHSDUDWLRQRIIDWW\DFLGPHWK\OHVWHUVDQGGLPHWK\ODFHWDOVIURPOLSLGVZLWK
ERURQÀXRULGHPHWKDQRO-/LSLG5HV
Nieto, R., L. Lara, R. Barea, R. García-Valverde, M.A. Aguinaga, J.A. Conde-Aguilera and J.F. Aguilera, 2012.
Response analysis of the Iberian pig growing from birth to 150 kg body weight to changes in protein and energy
supply. J. Anim. Sci. 90, 3809-3820.
Wood, J.D., M. Enser, A.V. Fisher, G.R. Nute, P.R. Sheard, R.I. Richardson, S.I. Hughes and F.M. Whittington, 2008.
Fat deposition, fatty acid composition and meat quality: A review. Meat Sci. 78, 343-358.

432 Energy and protein metabolism and nutrition in sustainable animal production
Portal-drained viscera heat production in pigs fed betaine and
conjugated linoleic acid (CLA) supplemented diets
M.L. Rojas-Cano, M. Lachica, L. Lara, A. Haro and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQL¿JDUHV#HH]FVLFHV

Introduction
Betaine and CLA have the potential to alter growth and body composition in swine (Fernández-
Fígares et al LPSURYLQJIHHGHI¿FLHQF\SURWHLQDFFUHWLRQUDWHDQGUHGXFLQJERG\IDW
Although their mode of growth promotion is not well understood. We hypothesised that differences
in portal-drained viscera (PDV) heat production might partially explain differences in growth in
pigs fed betaine and CLA (Fernández-Fígares et al., 2008).

Material and methods

Sixteen barrows (Sus scrofa mediterraneus; 19±0.28 kg BW) were individually penned and fed
barley-soybean meal-based diets containing no betaine or CLA (Control), 0.5% betaine, 1% CLA, or
0.5% betaine + 1% CLA (Clabet), at 70% of ad libitum energy intake. At 30 kg BW three permanent
catheters were placed in each pig: in carotid artery and portal vein for blood sampling, and in ileal
YHLQIRUSDUDDPLQRKLSSXULFDFLG 3$+ LQIXVLRQWRPHDVXUHSRUWDOEORRGÀRZ3LJVUHFRYHUHGIURP
surgery on metabolic cages and resumed their regular daily intake within three days after surgery. The
WULDOEHJDQEHWZHHQGDIWHUVXUJHU\)RUW\¿YHPLQSULRUEORRGVDPSOLQJDPOSXOVHGRVHRI
PAH (2% w/v) was infused into ileal vein, followed by continuous infusion of 0.8 ml/min until the
end of the sampling period. Blood samples were anaerobically taken simultaneously from carotid
DUWHU\DQGSRUWDOYHLQHYHU\PLQIRUKDQGKRXUO\XQWLOKDIWHUIHHGLQJJRIGLHWLQWRDQ
heparinized tube for instantaneous measurement of blood parameters and haematocrit. Plasma was
VWRUHGDWƒ&XQWLO3$+DQDO\VLV:KROHEORRGÀRZDQG22 consumption rates were based on
Fick principle. The assumed energy equivalent for O2 was 20.4 kJ/l. Data were subjected to ANOVA
XVLQJWKH0,;('SURFHGXUH7KHVWDWLVWLFDOPRGHOLQFOXGHGWKH¿[HGHIIHFWRIEORFNGLHWVDPSOLQJ
time and corresponding interactions. Time of sampling within pig was considered as a repeated
measure. Concentration at time zero of the analyte was included as a covariate in the statistical
DQDO\VLV6LJQL¿FDQWGLIIHUHQFHVDPRQJWUHDWPHQWVZHUHDVVHVVHGXVLQJ7XNH\¶VPXOWLSOHUDQJHWHVW

Results and discussion

Haematocrit and haemoglobin concentration were lower (P<0.001) in pigs fed CLA and Clabet
diets compared to Control (Table 1). Arterial-portal difference in O2FRQFHQWUDWLRQZDVVLJQL¿FDQWO\
lower (P<0.001, 14.3%) in pigs fed Clabet diets compared to Control. Furthermore, O2 consumption
was greater in Control compared to betaine, CLA and Clabet pigs (P<0.01, 21-35%). Interestingly,
betaine tended towards reduced PDV heat production (3 0.08) while CLA and Clabet had decreased
PDV heat production compared to Control pigs (P<0.01; 27 and 58%, respectively), indicating a
potential synergistic effect of betaine and CLA reducing PDV heat production. Visceral tissues have
a disproportionate high metabolism with respect to their masses and under certain circumstances may
compromise nutrient availability to the periphery (Yen et al., 1989). Splanchnic tissues contribution
to total body weight is greater in Iberian than in Landrace pigs (Rivera-Ferre et al., 2005), although
heat production (HP) associated to PDV was greater in Landrace than in Iberian pigs (Rodríguez-
López et al., 2010). Oxygen concentration difference between arterial and portal venous blood
and O2 consumption by PDV in the present experiment were within the range of published data
(Yen and Nienaber, 1992; Rodríguez López et al., 2010). The decreased O2 consumption and heat
production by PDV in pigs fed CLA and Clabet diets would indicate a reduction in energy and nutrient

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 433
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_160, © Wageningen Academic Publishers 2013
requirement of PDV, with a consequent increase in the portion of absorbed energy and nutrients to
be utilized for growth of muscle and other non PDV tissues, which probably accounts for some of
the improvement in the rate of weight gain in growing pigs fed Betaine + CLA supplemented diets
(Fernández-Fígares et al., 2008).

7DEOH(IIHFWVRIGLHWDU\EHWDLQH  DQG&/$  RQEORRGSDUDPHWHUVDQGSRUWDOGUDLQHG

YLVFHUDKHDWSURGXFWLRQRISLJV Q WUHDWPHQW 

Control Betaine CLA Betaine+CLA SEM

Haematocrit, % a 31.7a b 29.7c 0.29

Haemoglobin, g/l 102a 103a 99b c 0.95
Blood O2 concentration, mmol/l
Arterial 5.98a 5.95a 5.81ab b 0.08
Portal 3.98ab 4.12a 3.89b 3.89b 
CCA O2 – CPV O21, mmol/l 2.05a 1.95a 1.97a 1.75b 0.05
2
PDV O2 consumption , mmol/min a 2.1b 2.1b 1.7b 0.14
3 0.75
PDVHP , kJ/kg /day 123a 102ab 90bc 78c 

abc:LWKLQDURZYDOXHVZLWKGLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\
P<0.001).
1 O2 concentration difference between arterial and portal vein blood.
2 PDV = portal-drained viscera; 3 PDVHP = portal-drained viscera heat production.

Acknowledgements
7KLVVWXG\ZDV¿QDQFHGE\JUDQW$*/0LQLVWU\RI6FLHQFHDQG,QQRYDWLRQ

References
Fernández-Fígares, I., J.A. Conde-Aguilera, R. Nieto, M. Lachica and J.F. Aguilera, 2008. Synergistic effects of betaine
DQGFRQMXJDWHGOLQROHLFDFLGRQJURZWKDQGFDUFDVVFRPSRVLWLRQRIJURZLQJ,EHULDQSLJV-$QLP6FL
Fernández-Fígares, I., D. Wray-Cahen, N.C. Steele, R.G. Campbell, D.D. Hall, E. Virtanen and T.J. Caperna, 2002.
Effect of dietary betaine on energy utilization and partitioning in the young growing feed restricted pig. J. Anim.
Sci. 80, 421-428.
Rivera-Ferre, M.G., J.F. Aguilera and R. Nieto, 2005. Muscle fractional protein synthesis is higher in Iberian than in
/DQGUDFHJURZLQJSLJVIHGDGHTXDWHRUO\VLQHGH¿FLHQWGLHWV-1XWU
Rodríguez-López, J.M., M. Lachica, L. González-Valero and I. Fernández-Fígares, 2010. Energy expenditure of
VSODQFKQLFWLVVXHVLQ,EHULDQDQG/DQGUDFHJURZLQJJLOWV/LYHVW6FL
<HQ-7DQG-$1LHQDEHU,QÀXHQFHRIFDUEDGR[RQIDVWLQJR[\JHQFRQVXPSWLRQE\SRUWDOYHLQGUDLQHGRUJDQV
and by the whole animal in growing pigs. J. Anim. Sci. 70, 478-483.
Yen, J.T., D.A.H. Nienaber and W.G. Pond, 1989. Oxygen consumption by portal vein-drained organs and by whole
animal in consciuos growing swine. Proc. Soc. Exp. Biol. Med. 190, 2393-2395.

434 Energy and protein metabolism and nutrition in sustainable animal production
The effect of dietary carbohydrate composition on net portal appearance
of nutrients and AA liver uptake in dairy cows fed low protein diets
G. Cantalapiedra-Hijar, J.M. Rodriguez-Lopez, A. Illovies, F. Messad and I. Ortigues-Marty
,QVWLWXW1DWLRQDOGHOD5HFKHUFKH$JURQRPLTXH8058QLWp0L[WHGH5HFKHUFKHVVXUOHV
+HUELYRUHV7KHL[6W*HQqV&KDPSDQHOOH)UDQFH[email protected]

Introduction
7KHHI¿FLHQF\E\ZKLFKUXPLQDQWVWUDQVIRUPWKHIHHG1LQWRPLONSURWHLQKDVEHHQVKRZQ +XKWDQHQ
DQG+ULVWRY WREHDIIHFWHGE\WKHGLHWDU\FDUERK\GUDWHFRPSRVLWLRQ &+2VWDUFKYV¿EHU 
However, no evidences exist on the metabolic adaptations that may be involved. A sparing effect
of glucogenic nutrients on amino acids (AA) has been traditionally evoked, but this has not been
found at the splanchnic level in a recent study using high protein diets (18-19% CP; Larsen and
Kristensen, 2012). The aim of this study was to test whether the absorbed nutrients could impact
WKHOLYHUXSWDNHRIHVVHQWLDO$$ ($$ ZKHQVWDUFKUHSODFHG¿EHUDVWKHPDLQGLHWDU\FDUERK\GUDWH
in low CP dairy diets.

Material and methods

)LYHPXOWLSDURXV-HUVH\FRZV “NJRI%: LQPLGODFWDWLRQZHUHXVHGLQD[/DWLQVTXDUH
GHVLJQZLWKWKH¿IWKFRZXVHGDVDQH[WUDREVHUYDWLRQ&KURQLFLQGZHOOLQJFDWKHWHUVZHUHVXUJLFDOO\
implanted into the mesenteric artery, portal and hepatic veins for blood sampling and mesenteric and
ruminal veins for para-aminohippuric acid (PAH) infusions. Four iso-NEL diets were formulated
WRSURYLGHGLIIHUHQW&3FRQWHQWV DQG DQGGLIIHUHQW&+2 JVWDUFKDQG
J1')NJ'0IRUWKHVWDUFKGLHWVDQGJVWDUFKDQGJ1')NJ'0IRUWKH¿EHUGLHWV
respectively). Each experimental period lasted 28 d and feed was offered in equal quantities every
hour. On d 27 and 1 hour after the onset of a continuous PAH infusion (38 mmol/h), hourly blood
VDPSOHVZHUHWDNHQRYHUKRXUVIURPHDFKVDPSOLQJYHVVHORIHDFKFRZ&RQFHQWUDWLRQVRIDFHWDWH
& SURSLRQDWH & EXW\UDWH & JOXFRVHODFWDWHȕK\GUR[\EXW\UDWH %+%$ DPPRQLD1
XUHD1($$DQGGHDFHW\ODWHG3$+ZHUHREWDLQHGDQGDYHUDJHG1HWÀX[HVRIQXWULHQWVDFURVVWKH
portal drained viscera (NPA) and liver (NHF) were determined. Dry matter intake was measured
on the last 3 days of each period. The effects of CP, CHO and CP×CHO on variables were analyzed
by GLM procedure while residuals from the regression of EAA-NHF on EAA-NPA were analyzed
for the effects of CP and CHO using one-way ANOVA (Minitab14).

Results and discussion

Dry matter intake was similar (P>0.05) across dietary treatments (Table 1). Diets rich in starch vs.
¿EHUGHFUHDVHGWKH13$RIYRODWLOHIDWW\DFLGV 3 0.01), BHBA (3 0.02) and molar proportion of
C2 (3 0.04), while increased the NPA of glucose (3 0.01) and molar proportion of C4 (P<0.01).
Low CP diets decreased (P<0.01) the NPA of EAA, ammonia-N and urea-N compared to Normal
CP diets. At low protein levels, diets rich in starch promoted a 20% higher EAA-NPA compared to
GLHWVULFKLQ¿EHUEXWWKLVHIIHFWZDVQRWVLJQL¿FDQW P>0.05). A residuals analysis from the regression
of EAA-NHF on EAA-NPA was carried out to evaluate whether the CHO itself could impact the
EAA liver uptake (Figure 1). The EAA liver uptake was affected by neither of the studied effects
(P>0.05) although a tendency for diets rich in starch (3 0.10) to have higher EAA-NHF (lower
XSWDNH ZDVIRXQG7KLVVWXG\FRQ¿UPHGWKHODFNRIDVLJQL¿FDQWHIIHFWRIJOXFRJHQLFGLHWVRQ($$
liver uptake (Larsen and Kristensen, 2012). However, the low number of animals used in this type
of studies along with some tendencies found in our experiment warrant further research.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 435
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_161, © Wageningen Academic Publishers 2013
Table 1. Dry matter intake and net portal appearance of nutrients promoted by treatments.

12.0%CP &3 SEM P-values1

Starch Fiber Starch Fiber

DM intake, kg/d  14.0 14.7 15.4 0.398 NS

NPA, mmol/min
VFA  31.3 24.7 32.9 1.91 CHO*
C2, %     0.023 CHO*
C3, %   27.8 25.7 0.021 NS
C4, % 10.3 5.49 10.0   CHO***
Glucose 1.01 -1.14 0.77 -0.91 0.574 CHO*
Lactate 1.79  1.70  0.187 NS
BHBA 2.89 3.37 2.80 3.70 0.217 CHO*
Essential AA 3.21  4.07 4.04 0.153 CP***
Ammonia-N 4.29  7.49   CP**
Urea-N -1.73 -1.91 -3.93   CP**

116 QRQVLJQL¿FDQW&+2 FDUERK\GUDWHFRPSRVLWLRQ&3 SURWHLQFRQWHQW P<0.05; **P<0.01 and *** P<0.001.

200
Starch Fiber Starch Fiber
150
100
50
Residuals

0
-50
-100
-150
-200 12.0%CP 16.5%CP

)LJXUH5HVLGXDOVDQDO\VLVIURPWKHUHJUHVVLRQRIHVVHQWLDO$$OLYHUXSWDNH < RQHVVHQWLDO$$

QHWSRUWDODSSHDUDQFH ; DFFRUGLQJWRWKHGLHWDU\FDUERK\GUDWHFRPSRVLWLRQ P= DQGSURWHLQ
FRQWHQW P= <  “P=  “P< ; U2  

Acknowledgment
This study was granted by the Commission of the European Communities; project FP7-BBE-2007-1
µ5HGQH[¶:HDOVRWKDQN$GLVVHRIRU¿QDQFLDOVXSSRUWWRFRQGXFWWKLVH[SHULPHQW

References
Huhtanen P., and A.N. Hristov. 2009. A meta-analysis of the effects of dietary protein concentration and degradability
RQPLONSURWHLQ\LHOGDQGPLON1HI¿FLHQF\LQGDLU\FRZV-'DLU\6FL
Larsen M. and N.B. Kristensen. 2012. Effects of glucogenic and ketogenic feeding strategies on splanchnic glucose
and amino acid metabolism in postpartumWUDQVLWLRQ+ROVWHLQFRZ-'DLU\6FL

436 Energy and protein metabolism and nutrition in sustainable animal production
Uptake of arterial amino acids by ruminal tissue in Holstein cows under
washed rumen conditions
A.C. Storm1, M. Aguilar2, M.D. Hanigan2, N.B. Kristensen1 and M. Larsen1
1'HSDUWPHQW RI $QLPDO 6FLHQFH $DUKXV 8QLYHUVLW\ )RXOXP  7MHOH 'HQPDUN
[email protected]
2Department of Dairy Science, VirginiaTech, Blacksburg, VA, USA

Introduction
Non mesenteric-drained viscera (MDV) metabolism of amino acids (AA) has been proposed to
represent as much as 40% of the MDV release of AA (MacRae et al., 1997). There is limited
knowledge of the ruminal vein-drained visceral (RDV) use of AA which might be dependent on
arterial AA concentration, epithelial metabolism, or both. Ruminal absorption of AA might occur,
so ruminal tissue may metabolize AA of both arterial and ruminal origin. The objective of the study
was to quantify the effect of dietary CP and ruminal absorption of fermentation products on RDV
use of arterial AA when ruminal AA absorption is excluded.

Material and methods

Six Holstein cows catheterized in the rumen vein (RV) and an artery (A) were used in a washed
rumen study (Kristensen et al., 2010; Storm et al., 2011). In brief, cows were subjected to two dietary
levels of CP (17% and 13% CP, n=3) for 3 wk prior to the washed rumen trial. Further, 3 ruminal
buffers (Ammonia, Butyric, and Control) were administrated in a split-plot design with CP as whole
plot and buffer as subplot factor arranged in a Latin square. Blood were collected at 9, 20, and 30
PLQDIWHUDGPLQLVWUDWLRQRIWKHEXIIHUV7KH59SODVPDÀRZZDVFDOFXODWHGIURPUXPLQDOFOHDUDQFH
and A-RV concentration difference of D2O. Plasma was analyzed for AA by GC-MS by isotope
GLOXWLRQ'DWDZHUHDQDO\]HGXVLQJDPRGHOLQFOXGLQJWKH¿[HGHIIHFWRI&3EXIIHUVDPSOLQJWLPH
period and interactions. Sampling time within cow and buffer was considered as a repeated measure
(spatial Gaussian) and cow within CP level as random. Data are presented as LS means ± SEM.

Results and discussion

Overall, net RDV uptake of arterial essential AA (EAA) and total AA was decreased 37 and 33%,
respectively (Table 1), when CP was reduced. The A–RV concentration differences of AA were in
general not affected by dietary CP (0.10<P<0.87), except for Phe, Val, Asp, and Gln, (3 
0.08, 0.05, respectively). Nor were the A–RV concentration differences and the RDV uptake of AA
affected by buffer (0.10<P<0.95), except for A-RV of Asp and Asn (P<0.01) and RDV uptake for
Asn (P<0.01). Thus, low CP induced a decrease in RDV uptake of EAA and total AA that might
EHUHODWHGWR59EORRGÀRZ7KH59EORRGÀRZZDVQRWLQFUHDVHGE\&3 3 0.10) but by butyrate
buffer (P< WKHUHE\UHMHFWLQJDVWULFW59EORRGÀRZUHODWHGH[SODQDWLRQ7KLVLQGLFDWHVWKDW
WKHUHGXFHGIHHGLQWDNHDQGQXPHULFDOO\ORZHU59EORRGÀRZZLWKWKHORZ&3PD\LQWHUOLQNLQWKH
regulation of RDV AA uptake. However the RDV extraction ratio ([A–RV]/A) were not affected by
CP, but ratios of Ala, Asp, Gly, Pro, Ile, Leu, Thr, and Val decreased at low CP and butyrate buffer
(PCP×Buffer UHVSHFWLYHO\ 5HGXFHG5'9H[WUDFWLRQ
of AA with low CP and high butyrate absorption indicate that RDV down-regulates branched-chain
AA uptake when AA availability is low and metabolism in the ruminal wall is high. Hence, RDV
uptake of AA is not only controlled by mass action.

Relating the current RDV uptake of EAA with 17% CP to net PDV release of EAA observed with
the same cows at 29 days after caling fed diets at similar intake and CP level (Larsen and Kristensen,
2012) show that RDV uptake of EAA was equal to 5% of net PDV release. Thus, RDV uptake of

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 437
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_162, © Wageningen Academic Publishers 2013
Table 1. Ruminal tissue uptake of arterial AA in dairy cows under washed rumen conditions.

Item Dietary CP SEM P-value

17% 13% CP Buffer

Dry matter intake, kg/d 20.5 18.3 0.5 <0.01 -

5XPLQDOYHLQEORRGÀRZOK 280 209 37 0.24 <0.01
1HW5'9ÀX[PPROK
EAA 12.3 7.8 1.5 0.10 0.47
His 1 0.9 0.3 0.79 0.29
Ile 1.7 1 0.2 0.04 0.32
Leu 2.8  0.3 0.05 
Lys 1.7 1.1 0.2 0.09 0.34
Met 0.4 0.2 0.1 0.08 
Phe 1  0.1 <0.01 0.51
Thr  1.2 0.2 0.28 
Trp 0.2 0.1 0.1 0.24 0.99
Val 2.1 1.1 0.2 <0.01 0.85
Non-EAA  15.1 1.5 <0.01 0.27
Ala 4.8 2.9 0.8 0.17 0.87
Asp 0.4 0.1¥ 0.1 0.08 <0.01
Asn 0.5 0.4 0.1 0.22 0.28
Cys 0.5 0.2 0.1  0.95
Gln   0.8  0.55
Glu 0.3¥ -0.2¥ 0.4 0.43 0.83
Gly 7.4 3.8 1.0  0.18
Pro 2.2 1.3 0.3 0.08 
Ser -0.3¥ -0.4¥ 0.3  0.82
Tyr 0.7 0.5 0.1 0.07 0.17
Total AA 34.9 22.9 2.5 0.03 0.43

¥ Estimate not different from zero (t-test).

EAA could only account for 7.5% of the non-MDV uptake of EAA, assuming that 40% of the MDV
release is equals non-MDV uptake. Overall, the data shows that the majority of non-MDV use of
arterial AA is related to tissue other than RDV.

References
Kristensen, N.B., A.C. Storm and M. Larsen, 2010. Effect of dietary nitrogen content and intravenous urea infusion on
UXPLQDODQGSRUWDOGUDLQHGYLVFHUDOH[WUDFWLRQRIDUWHULDOXUHDLQODFWDWLQJ+ROVWHLQFRZV-'DLU\6FL
Larsen, M. and N.B. Kristensen, 2012. Effects of glucogenic and ketogenic feeding strategies on splanchnic glucose
and amino acid metabolism in postpartumWUDQVLWLRQ+ROVWHLQFRZV-'DLU\6FL
MacRae J.C., L.A. Bruce, D.S. Brown, D.A.H. Farningham and M. Franklin, 1997. Absorption of amino acids from the
LQWHVWLQHDQGWKHLUQHWÀX[DFURVVWKHPHVHQWHULFDQGSRUWDOGUDLQHGYLVFHUDRIODPEV-$QLP6FL
Storm, A.C., M.D. Hanigan and N.B. Kristensen, 2011. Effects of ruminal ammonia and butyrate concentrations
RQUHWLFXORUXPLQDOHSLWKHOLDOEORRGÀRZDQGYRODWLOHIDWW\DFLGDEVRUSWLRQNLQHWLFVXQGHUZDVKHGUHWLFXORUXPHQ
conditions in lactating dairy cows. J. Dairy Sci. 94, 3980-3994.

438 Energy and protein metabolism and nutrition in sustainable animal production
Uptake of arterial amino acids by ruminal tissue in periparturient
Holstein cows
M. Larsen, A.C. Storm and N.B. Kristensen
'HSDUWPHQW RI $QLPDO 6FLHQFH $DUKXV 8QLYHUVLW\ )RXOXP  7MHOH 'HQPDUN
[email protected]

Introduction
Ruminal tissue mass increases with increasing feed intake in the periparturient dairy cow requiring
amino acids (AA) for protein synthesis. Thus, arterial-ruminal venous (A-V) concentration differences
of AA were hypothesised to increase after parturition in response to increasing feed intake. Further,
the aim was to assess the ruminal-drained visceral (RDV) uptake of arterial AA in relation to
mesenteric-drained visceral (MDV) release of AA.

Material and methods

Nine periparturient Holstein cows catheterised in major splanchnic vessels were used in a complete
randomised design with repeated measurements to investigate effects of postpartum feeding strategy
on splanchnic glucose and AA metabolism. Cows were assigned to 1 of 3 feeding strategies at
calving: a glucogenic, a ketogenic, or an alfalfa-glucogenic strategy. Eight sample sets of arterial,
ruminal venous, and portal venous blood were collected at -14, +4, +15, and +29 days relative to
FDOYLQJ '57& 3RUWDOGUDLQHGYLVFHUDO 3'9 SODVPDÀRZZDVPHDVXUHGE\GRZQVWUHDPGLOXWLRQ
of paraDPLQRKLSSXULFDFLGDQG5'9SODVPDÀRZZDVDVVXPHGWRFRQWULEXWHWRWKHSRUWDO
SODVPDÀRZ *LUDUGDQG'HVURFKHUV6WRUPet al., 2011). Data were analysed with a model
LQFOXGLQJWKH¿[HGHIIHFWRIWUHDWPHQW'57&DQGWKHLQWHUDFWLRQ'57&ZLWKLQFRZZDVFRQVLGHUHG
as a repeated measurement (autoregressive order 1). Data is presented as means ± SEM for DRTC
as no feeding strategy effects were observed (P>0.11, not Ala), for ruminal A-V differences.

Results and discussion

Ruminal A-V differences were positive for most AA (Table 1), except for Glu and Ser that were
negative, and for His, Asn, Asp, Cys, and Gln that did not differ from zero (P<0.05, thus not
presented). The general positive ruminal A-V differences indicate an overall RDV uptake of arterial
AA, and consequently, no net absorption of AA from the ruminal lumen. Ruminal A-V differences
did not change with DRTC either linearly or quadratically (Table 1), except for Ala, Pro, and Ser.
However, net RDV uptake of Leu, Lys, Thr, Trp, and Val increased linearly with increasing DRTC
ZKHQDSSO\LQJWKHVLPXOWDQHRXVLQFUHDVHLQ5'9SODVPDÀRZ 7DEOH EXWGLGQRWFKDQJHIRURWKHU
essential AA (EAA). Loss of AA in sloughed epithelia, ruminal tissue proliferation, and catabolism
are the primary fates of the AA taken up in the RDV, however, catabolism would likely be limited
GXULQJSHULSDUWXULHQWSURWHLQGH¿FLHQF\

7KHQHW3'9UHOHDVHRI($$KDVEHHQIRXQGWRFRPSULVHEHWZHHQDQGRIWKHQHW0'9
release of EAA in sheep and cattle, implying that the amount of EAA net utilised in non-MDV
tissues is equivalent to the amount of EAA unaccounted for in PDV release. On average in the
periparturient period, the non-MDV utilisation of EAA could be estimated to be between 39±1 and
“PPROKDVVXPLQJHLWKHURU3'9UHFRYHU\RI0'9UHOHDVHUHVSHFWLYHO\+HQFHWKH
RDV uptake of EAA could account for between 11 and 29% of the EAA utilised by the non-MDV
tissues. In sheep, Rémond et al. (2003) observed the RDV uptake of arterial EAA to account for
30% of non-MDV uptake of EAA. Thus, other non-MDV tissues than the RDV seem to utilise the
major part of EAA utilised in non-MDV, likely the abomasum and pancreas synthesising digestive
enzymes and hormones.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 439
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_163, © Wageningen Academic Publishers 2013
Table 1. Ruminal tissue uptake of arterial amino acids in periparturient Holstein cows.

Item Day relative to calving SEM P-contrasts

-14 +4 +15 +29 Linear Quadratic

Dry matter intake, kg/d 10.0 12.9   1.0 <0.01 0.77
3RUWDOSODVPDÀRZOK 759 980 1312  47 <0.01 0.55
5XPLQDO$9GLIIHUHQFHVȝ0 Ile 7.8 5.4 5.9 5.3 1.3 0.21 0.54
Leu 9.7 10.8 10.3 11.1 1.9  0.94
Lys 5.5 8.2 7.3 7.7 1.3 0.29 0.41
Met 1.7   1.5 0.4  
Phe 3.0 3.5 3.3   0.70 0.30
Thr  4.3 5.4 5.5 0.9 0.72 0.25
Trp 0.9 0.8 1.2 1.4 0.2 0.12 0.47
Val  7.2 9.2  1.9 0.75 0.49
Ala 17.4  15.5 22.0 1.4  0.01
Glu -2.8 -1.5 -2.5 -1.5 0.8 0.35 0.88
Gly 24.9  28.0 29.7 2.3  0.97
Pro   5.4 7.0 0.9  0.37
Ser -4.7 -8.7 -5.7  1.3 0.84 0.05
Tyr 2.7 2.4 2.4 1.8   0.72
Net RDV uptake, mmol/h EAA 7.8 8.9  12.8 1.7  
Ile 1.2 1.0 1.4  0.3  0.49
Leu 1.5 2.1 2.1 3.2 0.5 0.04 0.52
Lys 0.8 1.7 1.7 2.1 0.3 0.02 0.54
Met 0.3 0.3 0.4 0.4 0.1 0.20 0.82
Phe 0.5 0.7 0.8 0.7 0.1 0.18 0.37
Thr 1.0 0.8 1.2 1.5 0.2 0.08 0.30
Trp 0.1 0.2 0.3 0.4 0.1 <0.01 0.12
Val 1.0 1.5 1.9 2.0 0.3  
Ala  3.3 3.9  0.4 <0.01 0.05
Glu -0.3 -0.3 -0.7 -0.4 0.2 0.53 
Gly  5.3  8.1 0.5 <0.01 
Pro 0.7 0.9 1.2 2.2 0.2 <0.01 0.08
Ser -0.7 -1.9 -1.7  0.3 0.04 0.02
Tyr 0.5 0.5   0.1 0.53 >0.99
Total AA  18 24 27 4.4 0.08 0.81
Net PDV release, mmol/h EAA  144 178 245 13 <0.01 0.98
Total AA 128 303 414   <0.01 0.48

References
*LUDUG&/DQG$'HVURFKHUV1HWÀX[RIQXWULHQWVDFURVVVSODQFKQLFWLVVXHVRIODFWDWLQJGDLU\FRZVDVLQÀXHQFHG
by dietary supplements of biotin and vitamin B12-'DLU\6FL
5pPRQG'/%HUQDUG%&KDYHDX31R]LqUHDQG&3RQFHW'LJHVWLRQDQGQXWULHQWQHWÀX[HVDFURVVWKH
rumen, and the mesenteric- and portal-drained viscera in sheep fed with fresh forage twice daily: net balance and
G\QDPLFDVSHFWV%U-1XWU
Storm, A.C., M.D. Hanigan and N.B. Kristensen, 2011. Effects of ruminal ammonia and butyrate concentrations
RQUHWLFXORUXPLQDOHSLWKHOLDOEORRGÀRZDQGYRODWLOHIDWW\DFLGDEVRUSWLRQNLQHWLFVXQGHUZDVKHGUHWLFXORUXPHQ
conditions in lactating dairy cows. J. Dairy Sci. 94, 3980-3994.

440 Energy and protein metabolism and nutrition in sustainable animal production
Effects of ethanol on splanchnic nutrient metabolism in sheep at
different intake levels
T. Obitsu, K. Nishimura, Y. Udaka, T. Sugino and K. Taniguchi
*UDGXDWH6FKRRORI%LRVSKHUH6FLHQFH+LURVKLPD8QLYHUVLW\.DJDPL\DPD+LJDVKLKLURVKLPD
VKL-DSDQ[email protected]

Introduction
Ruminants fed silage-based diets are likely to ingest alcohols, due to alcohol production in fermented
IHHGV 1LVKLQRDQG6KLQGH $OWKRXJKDEVRUEHGDOFRKROVDUHSDUWO\GHWR[L¿HGPDLQO\LQWKH
liver, this process may affect nutrient utilization by the tissues. In human, absorption and elimination
of ingested alcohol are affected by food consumption (Jones et al., 1997). Thus, we investigated the
effects of ruminal infusion of ethanol (EtOH) on splanchnic nutrient metabolism in sheep given a
diet at different levels.

Material and methods

6L[VKHHS DYHUDJHERG\ZHLJKW“NJ ¿WWHGZLWKDUXPLQDOFDQQXODDQGYHVVHOFDWKHWHUVLQ
the splanchnic region were used in a split-plot crossover design. The experiment consisted four 7
day periods. After adaptation period, three sheep were assigned to a low (0.7 times maintenance
energy) intake level (L) for 2 weeks (period 1 and 2), then the feeding levels were switched to a
high (1.7 times maintenance energy) level (H) for 2 weeks (period 3 and 4). Other 3 sheep were
assigned to the reverse sequence of the feeding levels (H to L). At each period, sheep were assigned
the treatments with or without administration of EtOH. Sheep were given 4 equal portions of a diet
&3'0 FRQVLVWLQJRIFRQFHQWUDWHDQGKD\DWKLQWHUYDOV)RU(W2+WUHDWPHQWV
EtOH (1.5 g/kg BW/d, 20% solution) was dosed into the rumen immediately after each feeding for
GD\V5XPHQÀXLGVDPSOHVZHUHWDNHQDWKDIWHUIHHGLQJRQGD\RIHDFKSHULRGWRPHDVXUH
FRQFHQWUDWLRQRI(W2+YRODWLOHIDWW\DFLGVDQGHVWHUV2QGD\EORRGVDPSOHVZHUHFROOHFWHGIRU
KDQGSODVPDÀRZUDWHVZHUHHVWLPDWHGE\SDUDDPLQRKLSSXULFDFLGGLOXWLRQ5XPLQDOFKDUDFWHULVWLFV
Q RU DUWHULDOFRQFHQWUDWLRQV Q RU QHWSRUWDOÀX[HV Q RU DQGQHWWRWDOVSODQFKQLF
763 ÀX[HV Q RU RISODVPDPHWDEROLWHVZHUHFRPSDUHGZLWKIHHGLQJOHYHOVHWKDQROLQIXVLRQ
and their interactions as main effects, using the MIXED procedure of SAS. For treatments with
(W2+SODVPDFRQFHQWUDWLRQDQGÀX[HVRI(W2+DQGFOHDUDQFHUDWHRIDUWHULDO(W2+FDOFXODWHGE\
a two-compartment model were compared between L and H by paired t-test.

Results and discussion

'U\PDWWHULQWDNHZDVKLJKHUIRU+WKDQ/LQWDNHOHYHO YVNJG6(0 P<0.01),
but not affected by EtOH infusion. Ruminal EtOH concentration in sheep infused with EtOH was
higher for L compared with H intake level (5.9 vs. 2.9 mmol/l, SEM=0.77, P<0.10) at 2 h after
feeding. Arterial EtOH concentration peaked (4 mM) at 1.0 (H) and 1.5 h (L) after EtOH dose. The
SRUWDOUHFRYHU\RILQIXVHG(W2+GLGQRWGLIIHUEHWZHHQ/ “ DQG+ “ +RZHYHU
clearance rates of arterial EtOH were higher (1.40±0.27 vs. 1.13±0.30 /h, 3 0.07) for H than L intake.
3ODVPDÀRZVRISRUWDODQGKHSDWLFYHLQZHUHKLJKHUIRU+WKDQ/LQWDNH P<0.01), but not affected
by EtOH infusion (Table 1). Arterial acetate concentration increased (P<0.01) with EtOH infusion.
However, net portal absorption of acetate decreased (P<0.05) with EtOH infusion. Interaction of
feeding levels and EtOH was observed (P<0.10) for net acetate releases by TSP. Arterial concentration
DQGQHWSRUWDODQG763ÀX[HVRIJOXFRVHZHUHKLJKHUIRU+WKDQ/LQWDNHEXWQRWDIIHFWHGE\(W2+
Arterial lactate concentration increased (P<0.05) with EtOH infusion, but not affected by feeding
levels. Net portal releases of lactate decreased (P<0.05) but net TSP releases numerically increased
ZLWK(W2+1HLWKHUDUWHULDOFRQFHQWUDWLRQVQRUQHWÀX[HVRIK\GUR[\EXW\UDWHDQGQRQHVWHUL¿HG

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 441
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_164, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIUXPLQDOHWKDQRO (W2+ LQIXVLRQRQQXWULHQWQHWÀX[E\VSODQFKQLFWLVVXHVLQVKHHS
at different intake levels.

High intake3 Low intake3 SEM Probability

-EtOH +EtOH -EtOH +EtOH Intake EtOH Interaction

3ODVPDÀRZUDWHl/hr
Portal1  110  72 7.9 <0.001 0.798 0.413
Hepatic2 133 120  75 13.3 0.020 0.439 0.941
Arterial concentration1, mmol/l
Acetate 0.93  1.11 1.57 0.19 0.310 <0.001 0.353
Glucose 3.90 3.84  3.47 0.11 <0.001  0.315
Lactate   1.01  0.23 0.294 0.012 0.583
Alanine 0.15 0.15 0.21  0.01 0.018 0.077 0.103
1HWSRUWDOÀX[1, mmol/h
Acetate 121.7 80.0 53.9  11.29 0.001 0.030 
Glucose 2.3 3.2 -1.3 -1.8 2.00 0.059 0.912 0.730
Lactate 8.2 3.8   1.29 0.238 0.010 0.814
Alanine 3.2 4.7 1.0  0.48 <0.001 0.041 
1HWWRWDOVSODQFKQLFÀX[2, mmol/h
Acetate 183.9 112.8  97.7 29.70 0.045 0.447 0.097
Glucose 57.9 35.0 14.9   0.012 0.251 0.180
Lactate 13.0 20.3 1.7 3.9 4.41 0.031 0.354 
Alanine    -2.3 1.27 0.819 0.031 

1Q RUIRUKLJKLQWDNHDQGORZLQWDNHUHVSHFWLYHO\
2 n=3 except for low intake without EtOH (n=2).
3 High intake: 1.7 times maintenance energy. Low intake: 0.7 times maintenance energy.

fatty acids were affected by EtOH infusion. Arterial concentration (3  DQGQHW763ÀX[
(3 0.053) of triglyceride tended to increase with EtOH infusion but not affected by feeding levels.
$UWHULDOFRQFHQWUDWLRQVDQGQHWSRUWDOÀX[HVRIPRVWHVVHQWLDODPLQRDFLGVZHUHQRWDIIHFWHGE\
EtOH. However, arterial concentration (3  DQGQHW763ÀX[ P<0.05) of alanine decreased
but net portal alanine absorption increased (P<0.05) with EtOH infusion.

In summary, effects of EtOH load on net splanchnic metabolism of most nutrients, except for acetate,
ZHUHQRWPRGL¿HGE\IHHGLQJOHYHOV$OWHUQDWLRQRIODFWDWHDQGDODQLQHÀX[HVZLWKLQWKHVSODQFKQLF
WLVVXHVPD\UHÀHFWWKHHQKDQFHGFRQYHUVLRQEHWZHHQWKHVHVXEVWUDWHVXQGHU(W2+ORDGLQVKHHS

References
Jones A.W., K.A. Jonsson and S. Kechagias, 1997. Effect of high-fat, high-protein, and high-carbohydrate meals on
WKHSKDUPDFRNLQHWLFVRIDVPDOOGRVHRIHWKDQRO%U-&OLQ3KDUPDFRO
Nishino, N. and S. Shinde. 2007. Ethanol and 2,3-butanediol production in whole-crop rice silage. Grassl. Sci. 53,


442 Energy and protein metabolism and nutrition in sustainable animal production
Contribution of amino acids to glucose and lactose synthesis in lactating
dairy cows
G. Maxin, D.R. Ouellet and H. Lapierre
$JULFXOWXUH DQG $JUL)RRG &DQDGD 6KHUEURRNH 4XpEHF -0 & &DQDGD
[email protected]

Introduction
Despite a nutritional approach splitting protein and energy inputs in the models used to balance
dairy rations, many studies indicated important interactions between energy and protein metabolism.
For example, in the dairy cow, increases in milk protein and lactose yields are both induced with
increased protein supply. This increase in lactose yield is not always paralleled by an incremental
increase in mammary glucose uptake but is associated with an increased uptake of branched-chain
amino acids (AA). Although AA are used for gluconeogenesis, Bequette et al. (2005) demonstrated
in vitro that some essential AA could contribute to lactose synthesis by mammary cells supporting
earlier observations that arterial glucose would not be the only precursor of milk lactose (Bickerstaffe
et al., 1974). Therefore, the objective of this work was to study the interactions between AA and
glucose metabolism in dairy cows receiving different AA mixtures and to determine if AA could
contribute to lactose synthesis within the mammary gland.

Material and methods

6L[GDLU\FRZVDYHUDJLQJ 6' NJ%:DQG 6' ',0HTXLSSHGZLWKUXPHQ
¿VWXODDQGFKURQLFFDWKHWHUVLQPHVHQWHULFDUWHU\DQGSRUWDOKHSDWLFDQGPHVHQWHULFYHLQVZHUH
used in a replicated 3×3 Latin Square with 14-d periods. Cows were fed a corn-grass silage based
GLHWSURYLGLQJDQGRIWKHLUQHWHQHUJ\DQGPHWDEROL]DEOHSURWHLQ 03 UHTXLUHPHQWV
respectively. Treatments were abomasal infusions of water (CTL, 10 l/d) or a mixture of total AA
JGFDVHLQSUR¿OH HVWLPDWHGWRVXSSO\ZLWKWKHGLHWRI03UHTXLUHPHQWV 7$$ RURQO\
WKHHVVHQWLDO$$RIWKH7$$PL[WXUH JG($$ 0LON\LHOGDQGFRPSRVLWLRQZDVGHWHUPLQHG
on the last 3 d of each period. On d 14, D-[U-13C@JOXFRVH “PPROK ZDVLQIXVHGIRUK
into a jugular vein. Starting at 1 h after the initiation of the infusion, six hourly blood samples were
taken simultaneously, from arterial, portal, and hepatic sources to determine the WB and tissue net
DQGWRWDOUHOHDVHRIJOXFRVH3RUWDODQGVSODQFKQLFSODVPDÀRZVZHUHGHWHUPLQHGE\GRZQVWUHDP
dilution of para-aminohippurate after deacetylation. In addition, cows were milked right after
the last blood samples, the milking being performed after oxytocin injection to remove residual
milk. Concentrations and isotopic enrichment (IE) of plasma glucose (on all blood samples) and
HQULFKPHQWRIJDODFWRVHDQGJOXFRVHIURPHQ]\PDWLFDOO\K\GURO\]HGODFWRVH IURPPLONLQJDWK 
were determined by gas-chromatography-mass spectrometry. Data (average for each cow × period)
were analyzed according to the replicated 3×3 Latin square using the mixed procedure of SAS (2001)
ZLWKSHULRGDQGWUHDWPHQWDV¿[HGHIIHFWDQGFRZDVUDQGRPHIIHFW0XOWLSOHFRPSDULVRQVRIPHDQV
were performed with an adjusted Tukey-Kramer test.

Results and discussion

'U\PDWWHULQWDNHZDVQRWPRGL¿HGE\WUHDWPHQWDQGDYHUDJHGNJ'0G&RPSDUHGWR&7/
treatment, the infusion of TAA increased milk yield (P<0.01) as previously observed by Doepel and
Lapierre (2010). Milk protein yield increased more with TAA treatment compared with EAA treatment
(Table 1). The infusion of TAA increased the WB Ra of glucose compared to the other treatments,
whereas WB Ra of glucose was not different between CTL and EAA treatments (Table 1). Although
ERWKSRUWDODQGKHSDWLFWUXHJOXFRVHUHOHDVHZHUHQRWDIIHFWHGE\WUHDWPHQWWKHVSODQFKQLFWUXHÀX[
of glucose observed with TAA was numerically higher than for CTL treatment: the increment of WB

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 443
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_165, © Wageningen Academic Publishers 2013
Table 1. Effect of different AA mixture supply on milk yield and glucose kinetics in dairy cows.

Treatments1 SEM P-value

Trt
CTL EAA TAA

Milk, kg/d 33.1b 35.0ab 37.3a 2.0 <0.01

Milk protein, kg/d 0.97c 1.11b 1.21a 0.72 <0.01
Glucose kinetics, mmol/h
Whole body rate of appearance 798b 811b 902a 39 <0.01
Net glucose release
Portal 43a -51b 9ab 21 0.03
Hepatic 725 807 780  0.53
Splanchnic  734 799  0.79
Glucose utilization
Portal a -192b ab 27 
Hepatic -17 -7 -31 32 0.83
Splanchnic -113 -152 -171 33 0.39
True glucose release
Portal 139 141   
Hepatic 742 815 811 77 0.38
Splanchnic 882 902 957 81 0.50
Isotopic enrichment (IE) in milk lactose2
Glucose, atom percent excess 1.08 1.11 1.02 0.04 0.19
Galactose, atom percent excess a 0.57ab 0.51b 0.03 0.03
Galactose IE /Glucose IE a 0.52ab 0.50b 0.01 <0.01

1$ERPDVDOLQIXVLRQRI&7/ZDWHU Q  7$$PL[WXUHRIWRWDO$$ Q  ($$RQO\HVVHQWLDO$$RI7$$ Q  

22QVDPSOHFROOHFWHGDIWHUKRI'>813C ]glucose infusion.

a,b,c Values with different superscript in the same row are different: P<0.05.

Ra of glucose could therefore be mainly related to increased hepatic glucose production, probably
by conversion of nonessential AA to glucose.

Six h after the initiation of the D-[U-13C]glucose infusion, IE of glucose in milk lactose averaged
0.94 (SEM: 0.04) of arterial IE and this ratio was not altered by treatment. This ratio not different
from unity (P>0.10) suggests that arterial glucose is the major, if not the sole, contributor of carbons
to glucose in milk lactose. The IE of galactose in milk lactose was, however, lower than the IE of
glucose in milk lactose (Table 1) indicating that other sources of carbon than arterial glucose have
been used for galactose synthesis in the mammary gland. Moreover, the ratio of the IE of galactose on
IE of glucose in milk lactose decreased with TAA treatment (P<0.05) suggesting that the contribution
of carbons other than arterial glucose was altered by AA supply.

,QFRQFOXVLRQWKLVVWXG\FRQ¿UPVHDUOLHUREVHUYDWLRQVWKDWDUWHULDOJOXFRVHZRXOGQRWEHWKHRQO\
precursor of milk lactose, especially as other sources of carbon would contribute to galactose synthesis
and suggests that AA could be involved in the milk lactose synthesis.

References
Bequette B.J., S.L. Owens, S.W. El-Kadi, N.E. Sunny and A. Shamay, 2005. Use of 13C-mass isotope distribution
DQDO\VLV 0,'$ WRGH¿QHSUHFXUVRUVIRUODFWRVHDQGDPLQRDFLGV\QWKHVLVE\ERYLQHPDPPDU\H[SODQWV-'DLU\
Sci. 88 Supp. 1, 289.

444 Energy and protein metabolism and nutrition in sustainable animal production
Bickerstaffe R. and E.F. Annison, 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy
cows J. Agri. Sci. 82, 71-85.
Doepel, L. and H. Lapierre, 2010. Changes in production and mammary metabolism of dairy cows in response to
HVVHQWLDODQGQRQHVVHQWLDODPLQRDFLGLQIXVLRQV-'DLU\6FL

Energy and protein metabolism and nutrition in sustainable animal production 445
Contribution of essential amino acids to glucose metabolism and lactose
secretion in late lactation dairy cows
H. Lapierre1, S. Lemosquet2 and D.R. Ouellet1
1$JULFXOWXUHDQG$JUL)RRG&DQDGD6KHUEURRNH4&-0&&DQDGD[email protected]
2,15$805'DLU\3URGXFWLRQ6DLQW*LOOHV)UDQFH

Introduction
Increased protein supply is known to increase both milk and milk lactose yields as well as whole
body rate of appearance (Ra) of glucose (Lapierre et al., 2010). However, despite earlier observations
reporting that arterial glucose would not be the only precursor of milk lactose (Bickerstaffe et al.,
1974), little attention has been paid to the potential contribution of amino acids (AA) to mammary
lactose synthesis. Although, Bequette et al.  UHFHQWO\GHPRQVWUDWHGin vitro that some essential
AA could contribute to mammary lactose synthesis, no in vivo study has examined the contribution
of essential AA to milk lactose secretion and if that contribution would be altered by AA provision.
Therefore, the objective of this study was to examine if increased supply of essential AA, in excess
of requirement, would affect the contribution of glucose to lactose synthesis in lactating dairy cows.

Material and methods

)RXUUXPHQ¿VWXODWHGGDLU\FRZVLQODWHODFWDWLRQDYHUDJLQJ 6' NJZHUHXVHGLQFURVV
RYHUGHVLJQZLWKWZRGSHULRGV7KURXJKRXWWKHVWXG\FRZVZHUHIHGD¿[HGDPRXQWUHVWULFWHG
at 98% of ad libitum intake, of a grass hay-based diet providing 100% of net energy (28.8 Mcal/d)
and metabolizable protein (1,907 g/d) requirements (NRC, 2001). Treatments were abomasal
infusions of water (CTL) or a mixture of essential AA (EAAinfJGFDVHLQSUR¿OH HVWLPDWHG
to provide 140% of the essential AA requirements (diet + infusion). Milk yield was measured daily
DQGPLONFRPSRVLWLRQZDVGHWHUPLQHGRQWKHODVWGRIHDFKSHULRG2QG'>2H2]glucose
(24.4±0.5 mmol/h) was infused into jugular vein preceded by a priming dose (20.3±1.8 mmol).
Blood samples were taken 1:15, 1:45, 2:15, 2:45, 3:15 and 4:15 h after the initiation of the glucose
infusion. Furthermore, cows were milked right after the last two blood samples, the milking at
3:15 h being performed after oxytocin injection to remove residual milk. Concentrations and
isotopic enrichment (IE) of plasma glucose (on all blood samples) and enrichment of galactose and
glucose from enzymatically hydrolyzed lactose (from milking at 4:15 h) were determined by gas-
chromatography-mass spectrometry. Whole body glucose Ra was estimated using a non-steady-state
model, as proposed by Brockman (1984). Data were analyzed using GLM procedures of Minitab
YHUVLRQ ZLWKWUHDWPHQWSHULRGDQGFRZDV¿[HGHIIHFW

Results and discussion

Dry matter intake was not affected by treatment (as there was no orts) and averaged 18.0±0.0 kg/d
(LSM ± SEM for CTL vs. EAAinf). Milk, protein, fat and lactose yields were not affected (P>0.20)
by EAA infusion (Table 1). Milk protein concentration, however, increased (3 0.009) with EAAinf
(38.5 vs. 39.9±0.08 g/kg). Plasma glucose concentration was not affected (3 0.77) by treatment
(3.77 vs. 3.77±0.004 mM), nor was whole body Ra of glucose (Table 1). The proportion of whole
body glucose Ra accounted for by milk lactose secretion, assuming glucose as the sole precursor of
ODFWRVHDYHUDJHGDQGZDVQRWDIIHFWHGE\WUHDWPHQW 3 0.94).

In milk lactose, IE of glucose (3 0.02) and galactose (3  GHFUHDVHGZLWKWKH($$inf. In addition,

galactose IE was lower (P<0.001) than glucoselactose IE, but EAAinf did not alter this ratio, the IE
of galactose being on average 82% that of glucose. After 4:15 h of labeled glucose infusion, the IE

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 447
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_166, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRILQFUHDVLQJLQWHVWLQDOVXSSO\RIHVVHQWLDO$$ ($$inf LQH[FHVVRIUHTXLUHPHQWRQ
PLON\LHOGJOXFRVHZKROHERG\UDWHRIDSSHDUDQFH :%5D DQGLVRWRSLFHQULFKPHQW ,( RIJOXFRVH
and galactose during an infusion of labeled glucose.

Parameter Treatment SEM P-value

CTL EAAinf

Milk yield, kg/d 22.2 22.8 0.52 

Milk protein yield, g/d 857 905 20.1 0.23
Milk fat yield, g/d 999  24.0 
Milk lactose yield, g/d 989  27.3 0.88
Glucose WB Ra, g/d 2,239  40.5 0.50
Lactose/glucose WB Ra   0.019 0.94
Glucoselactose IE, ape1 2.70 2.40 0.030 0.02
Galactoselactose IE, ape1 2.23 2.00 0.041 
Galactoselactose IE/Glucoselactose IE1 0.82 0.83  0.39
Glucoselactose IE/Glucoseplasma IE1 0.59 0.53 0.009 

12QVDPSOHFROOHFWHGKDIWHUWKHLQLWLDWLRQRIWKH'>2H ]glucose infusion.

2

of glucoselactose was still lower (P<0.01) than the IE of glucoseplasma, and this ratio was decreased
(3  E\($$inf.

Infusing EAA in cows fed to meet their protein requirement did not affect glucose availability and
GLGQRWDOWHUPLON\LHOG,QFRQWUDVWLQFRZVIHGDSURWHLQGH¿FLHQWGLHWDOWKRXJKLQIXVLRQRI($$
did not alter glucose Ra, but it did increase milk yield (Maxin et al., 2013). In the current study,
the IE of galactose averaged 82% of glucoselactose IE, suggesting that 18% of galactose did not
originate directly from intracellular glucose; this contribution was not affected by EAAinf. The ratio
of glucoselactose IE to glucoseplasma,(ZDVOHVVWKDQDIWHUKRIODEHOOHGJOXFRVHLQIXVLRQ
FRPSDUHGWRWKHREVHUYHGIRUWKHVSHFL¿FUDGLRDFWLYLW\RIODFWRVHRQSODVPDJOXFRVHDIWHUK
of infusion (Bickerstaffe et al.,ODFWRVHVSHFL¿FUDGLDRFWLYLW\ZDVQRWDQDO\]HGVHSDUDWHO\IRU
galactose and glucose) and this porportion was altered by EAAinf, suggesting either there are other
contributors to intracellular glucose than arterial glucose uptake or there is exchange of the deuterium
between intracellular metabolites or both. This in vivoVWXG\FRQ¿UPVWKDWJOXFRVHLVQRWWKHVROH
precursor of mammary lactose synthesis. The effects of AA supply on milk lactose synthesis needs
to be directly evaluated through infusion of labelled AA.

References
%HTXHWWH%-1(6XQQ\6:(O.DGLDQG6/2ZHQV$SSOLFDWLRQRIVWDEOHLVRWRSHVDQGPDVVLVRWRSRPHU
distribution analysis to the study of intermediary metabolism of nutrients. J. Anim. Sci. 84, Suppl. E, 50-59.
Bickerstaffe, R., E.F. Annison and J.L. Linzell, 1974. The metabolism of glucose, acetate, lipids and amino acids in
lactating dairy cows. J Agric Sci, Cambridge 82, 71-85.
Brockman, R.P., 1984. Validation of an equation for calculation of glucose appearance during nonsteady state in sheep.
&DQ-3K\VLRO3KDUPDFRO
Lapierre, H., C.E. Galindo, S. Lemosquet, I. Ortigues-Marty, L. Doepel and D.R. Ouellet, 2010. Protein supply,
glucose kinetics and milk yield in dairy cows. Pages 277-288. In EAAP publication No.127. ed. C.M. Crovetto.
Wageningen Academic Publishers. The Netherlands.
Maxin. G., D.R. Ouellet and H. Lapierre, 2013. Is there a relation between milk yield and glucose kinetics responses
to the supply of different amino acids mixtures? ISEP, Sacramento, CA.
NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC.

448 Energy and protein metabolism and nutrition in sustainable animal production
Mammary gland from lactating cows responded additively to individual
essential amino acids in casein synthesis rate
S.I. Arriola Apelo1, L.M. Singer1, X. Lin, W.K. Ray2, R.F. Helm2 and M.D. Hanigan1
1'HSDUWPHQWRI'DLU\6FLHQFH9LUJLQLD3RO\WHFKQLF,QVWLWXWHDQG6WDWH8QLYHUVLW\9$86$
[email protected]
2'HSDUWPHQWRI%LRFKHPLVWU\9LUJLQLD3RO\WHFKQLF,QVWLWXWHDQG6WDWH8QLYHUVLW\9$86$
Animal Science and Technology College, Shandong Agriculture University, Shandong Province
&KLQD35

Introduction
Dairy cattle only capture about 25% of dietary N in milk protein, with the remainder being excreted.
By coordinating AA supply with demand, mammary cells reduce recycling of AA to the splanchnic
WLVVXHVWKHUHE\LQFUHDVLQJHI¿FLHQF\6HYHUDOHVVHQWLDO$$UHJXODWHWKHUDWHRIV\QWKHVLVRIFDVHLQ
by altering the phosphorylation state of intracellular signaling proteins that affect the activity of
initiation (eIF) and elongation (eEF) factors (Appuhamy et al., 2012). Therefore, supplementing
WKHVSHFL¿F$$WKDWH[HUWWKHPRVWLQÀXHQFHRQSDWKZD\VFRQWUROOLQJH,)DQGH()VKRXOGDOORZD
UHGXFWLRQLQ&3VXSSO\WRWKHDQLPDOZKLFKZLOOLQFUHDVHSRVWDEVRUSWLYHHI¿FLHQF\DQGUHGXFH1
excretion. The objective of this study was to determine the quantitative responses in milk protein
synthesis to individual essential AA in mammary tissue.

Material and methods

Mammary tissue was obtained from 5 lactating dairy cows at slaughter. Each quarter was tested for
somatic cell count prior to slaughter to ensure the harvested tissue was not from an infected quarter.
$QLPDOVZHUHPLONHGDPD[LPXPRIKEHIRUHVODXJKWHU0DPPDU\WLVVXHVOLFHV “J 
were prepared from the excised tissue and incubated in 5 ml of treatment media at 37 °C while the
ÀDVNVZHUHRVFLOODWHGDWRVFLOODWLRQPLQIRUKXQGHUDQDWPRVSKHUHRI22:CO2. Preliminary
data showed that 2H5 Phe HQULFKPHQWRIWKHS+SURWHLQSUHFLSLWDWH increased linearly from 0.5
to 8 h (0.43±0.04% enrichment increase/h, adjusted R2=0.93). Following incubation, slices were
KRPRJHQL]HGLQO\VLVEXIIHUDQGFDVHLQVZHUHSUHFLSLWDWHGE\DFLGL¿FDWLRQWRS+$QDOLTXRWRI
the pellet was trypsinized and 2H5 Phe enrichment in the N[34]LLRFFVAPFPE peptide of alpha
S1 casein was measured by MALDI TOF-MS-MS. Fractional synthesis rate (FSR, %/h) of alpha
S1 casein was calculated as the ratio of area for labeled and unlabeled newly synthesized peptide
and the existent one:

where EtRNA represented the % enrichment in the Phe acylated-transfer RNA (Phe-tRNA) pool, m0
represented the area under the curve for peptides with no labeled Phe (m+0), and m15 represented
that for peptides with 3 labeled Phe (m+15). Phe-tRNA enrichment was estimated as the ratio of
the abundance of the m+15 peptides to m+15 plus a third of m+10 (2 labeled Phe) peptides. The
effect of Ile, Leu, Met and Thr on alpha S1-casein FSR was studied with a central composite design
consisting of four central runs, 2 axial runs per AA, and a complete 24 factorial. The central run
ZDVVHWDWRIWKHFRQFHQWUDWLRQRIHDFK$$LQ'XOEHFFR¶V0RGL¿HG(DJOH0HGLXP '0(0
PPROO,OHPPROO/HXPPROO0HWDQGPPROO7KU $[LDOUXQVZHUHVHW
at 0 and 100% (0.420 mmol/l Ile, 0.450 mmol/l Leu, 0.170 mmol/l Met and 0.220 mmol/l Thr) of
DMEM. Factorial runs were set at 20 (0.084 mmol/l Ile, 0.09 mmol/l Leu, 0.034 mmol/l Met and
0.044 mmol/l Thr) and 50% (0.210 mmol/l Ile, 0.225 mmol/l Leu, 0.085 mmol/l Met and 0.110
mmol/l Thr) of DMEM. The experiment was replicated in 5 cows. In each cow, treatments were

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 449
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_167, © Wageningen Academic Publishers 2013
replicated twice and the average of the two replicates was used for the analysis. Linear, quadratic and
one-way linear interaction parameters were estimated with the Glimmix procedure in SAS (Cary,
NC). Cow and residual were random effects.

Results and discussion

Phe-tRNA enrichment decreased by the addition of Ile and Met to the media (data not shown). Table
1 shows the linear, quadratic, and one-way interaction parameter values for individual AA effects on
casein FSR. The Leu, Met and Thr caused a quadratic response on FSR, and Ile tended to cause the
VDPHHIIHFW7KHTXDGUDWLFWHUPIRU0HWZDVJUHDWHUWKDQIRU,OHDQG/HX+RZHYHUWKH¿UVWGHULYDWLYH
of the term showed that all maximums were within the range of the data. Given this response, it is
possible that the tissue responded in a Michaelis-Menten fashion with a plateau starting before the
predicted maximum. Media Ile, Leu and Thr concentrations were lower than arterial concentrations
observed by Hanigan et al. (2004) at the highest level of casein infusion. In that study a quadratic
response in milk protein synthesis to increased AA supply was also observed. Therefore, studies at
the animal and the tissue level demonstrate that the mammary gland responds in a curvilinear fashion
WRVXSSO\RI$$ZKLFKLVLQFRQVLVWHQWZLWKWKH¿[HGSDUWLDOHI¿FLHQF\XVHGLQQXWULHQWUHTXLUHPHQW
PRGHOV2QHZD\OLQHDUDPLQRDFLGLQWHUDFWLRQVZHUHQRWVLJQL¿FDQW5HPRYLQJQRQVLJQL¿FDQW
LQWHUDFWLRQVIURPWKHPRGHOPDGHWKHIRXU$$WRKDYHDVLJQL¿FDQWHIIHFWRQ)65)XUWKHUPRUH
FRQ¿GHQWOLPLWVLQGLFDWHGWKDWWKHOLQHDUSDUDPHWHUVGLGQRWGLIIHUDPRQJ$$7KXVFXUYLOLQHDU
responses in casein FSR to each of the 4 AA studied were similar and additive rather than conditional.
7KHUHIRUHDFRZFOHDUO\GH¿FLHQWLQ0HWFRXOGEHVXSSOHPHQWHGZLWK/HXDVVXPLQJ/HXLVDOVR
PDUJLQDOO\GH¿FLHQWDQGDUHVSRQVHVKRXOGEHREVHUYHG0RUHRYHUWKHUHVSRQVHWR/HXZRXOGEH
independent of and additive to the response to Met. This observation is in agreement with Hanigan
et al. (2002)’s prediction using a modeling approach, and it contradicts the single limiting AA theory
assumed in nutrient requirement models.

7DEOH/LQHDUDQGTXDGUDWLFSDUDPHWHUHVWLPDWHVVWDQGDUGHUURUV 6( DQG3YDOXHVIRUWKHHIIHFW
RI,OH/HX0HWDQG7KURQIUDFWLRQDOV\QWKHVLVUDWHV )65 RIDOSKD6FDVHLQ

Int.1 I2 L M T I×I L×L M×M T×T I×L I×M I×T L×M L×T M×T

FSR, %/h
Param.3  -3.99   -0.31 -14.9 -23.0 -139 -74.4 7.50 94.2 79.4   92.8
SE4  7.52 7.05  14.0 7.79    25.4  51.3  48.3 125
P value 0.4  0.4 0.7 1.0  0.001 0.003 0.007 0.5 0.15 0.12 0.9  0.5

1 Intercept.
2 I = isoleucine; L = leucine; M = methionine; T = threonine.
3 Parameter.
4 Standard error.

References
Appuhamy, J.A.D.R.N., N.A. Knoebel, W.A.D. Nayananjalie, J. Escobar and M.D. Hanigan, 2012. Isoleucine and
leucine independently regulate mtor signaling and protein synthesis in mac-t cells and bovine mammary tissue
slices. J. Nutr. 142:484-491.
Hanigan, M.D., L.A. Crompton, B.J. Bequette, J.A. Mills and J. France, 2002. Modelling mammary metabolism in the
dairy cow to predict milk constituent yield, with emphasis on amino acid metabolism and milk protein production:
Model evaluation. J. Theor. Biol. 217:311-330.
Hanigan, M.D., C.K. Reynolds, D.J. Humphries, B. Lupoli and J.D. Sutton, 2004. A model of net amino acid absorption
DQGXWLOL]DWLRQE\WKHSRUWDOGUDLQHGYLVFHUDRIWKHODFWDWLQJGDLU\FRZ-'DLU\6FL

450 Energy and protein metabolism and nutrition in sustainable animal production
Effect of amino acid supply on whole body and tissue glucose kinetics in
postpartum dairy cows
C. Galindo1, M. Larsen2, D.R. Ouellet, G. Maxin, D. Pellerin1 and H. Lapierre
1'pSDUWHPHQW GH VFLHQFHV DQLPDOHV 8QLYHUVLWp /DYDO 4XpEHF 4& *9 $ &DQDGD
[email protected]
2'HSDUWPHQWRI$QLPDO6FLHQFH$DUKXV8QLYHUVLW\)RXOXP7MHOH'HQPDUN
$JULFXOWXUHDQG$JUL)RRG&DQDGD6KHUEURRNH4&-0&&DQDGD

Introduction
Previous results on dairy cows have shown that an increased amino acid (AA) supply imcreased net
hepatic glucose release, whole-body rate of appearance (WB Ra) of glucose, lactose secretion and
milk production. However, it is suggested that in the immediate postpartum period, the liver glucose
release is mainly supported by an important inter-organ transfer of glucogenic carbons such as lactate
from peripheral tissues to the liver instead of an increased utilization of AA for gluconeogenesis. The
objective of the study was to investigate the effects of supplementing AA on WB Ra of glucose, and
WLVVXHPHWDEROLVPRIJOXFRVHODFWDWHDQGȕ2+EXW\UDWH %+% LQpostpartum transition dairy cows.

Material and methods

1LQH+ROVWHLQFRZV¿WWHGZLWKUXPHQFDQQXODDQGLQGZHOOLQJFDWKHWHUVLQWKHSRUWDOKHSDWLFDQG
two mesenteric veins, and one artery, were used in a generalized randomized complete block design
with repeated measures. At calving, cows received the same lactation ration, were blocked according
to parity (2nd and 3rd+) and were divided in 2 treatments: abomasal infusion of water (Ctrl; n=4)
RUDERPDVDOLQIXVLRQRIIUHH$$ZLWKFDVHLQSUR¿OH $$&1Q  $$&1LQIXVLRQVWDUWHGZLWK
half the maximal dose on day in milk (DIM) 1 and then steadily decreased from 795±12 g/d to
“JGIURP',0WRWRFRYHUWKHHVWLPDWHG$$GH¿FLW2Q',0DQG'>2H2]
JOXFRVH “PPROK ZDVLQIXVHGLQWRDMXJXODUYHLQIRUKDQGEORRGVDPSOHVZHUHWDNHQ
from arterial, portal, hepatic, and mammary sources at 45 min-intervals, starting one hour after the
LQLWLDWLRQRIWKHJOXFRVHLQIXVLRQ7UDQVRUJDQÀX[ZHUHFDOFXODWHGDVYHQRDUWHULDOGLIIHUHQFHVWLPHV
SODVPDÀRZ VSODQFKQLFGRZQVWUHDPGLOXWLRQRIGHDFHW\ODWHGpara-aminohippurate; mammary: Fick
SULQFLSOHXVLQJ3KH7\U 'DWDZHUHDQDO\VHGZLWKDPL[HGPRGHOLQFOXGLQJWKH¿[HGHIIHFWVRI
block, treatment (Trt), DIM, and Trt × DIM and random effect of cow; DIM was tested as a repeated
measurement using the appropriate covariance structure.

Results and discussion

Milk production on the 3 days around sampling days increased numerically (PTrt 0.15) with
AA infusion (Table 1) and as DIM progressed, dry mater intake and milk production increased
(PDIM<0.01). Glucose and lactate arterial concentrations were not altered by AA infusion, but glucose
arterial concentration increased (PDIM<0.01) and lactate arterial concentration decreased numerically
(PDIM  DVODFWDWLRQSURJUHVVHG$UWHULDOFRQFHQWUDWLRQVIRUERWK%+%DQGQRQHVWHUL¿HGIDWW\DFLGV
ZHUHKLJKHULQ$$&1WKDQLQ&WUOFRZVDW',0 YVYVP0UHVSHFWLYHO\ EXW
QRWDW',0 YV6(0YV6(0P0UHVSHFWLYHO\PTrt×DIM<0.05).

Despite increased AA supply in AA-CN cows and increased demand for milk production, WB Ra
of glucose was not affected by AA-CN (PTrt  VLPLODUO\VSODQFKQLFWUXHÀX[ZDVXQDOWHUHG
by treatments and is numerically equivalent to WB Ra. Splanchnic utilization of glucose was
XQDIIHFWHGE\$$&1DQGUHSUHVHQWHGRQDYHUDJHRI:%5D0DPPDU\JOXFRVHXWLOL]DWLRQ
increased with AA-CN infusion (PTrt=0.03), but the proportion relative to WB Ra was unaltered
by treatment, averaging 78%, and increased gradually as lactation advanced (PDIM=0.01). Lactate

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 451
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_168, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIDPLQRDFLGLQIXVLRQ $$&1 RQPLON\LHOGJOXFRVHNLQHWLFVDQGJOXFRVHODFWDWH
DQGȕ2+EXW\UDWHWLVVXHPHWDEROLVPLQpostpartum dairy cows.1

Item Treatment SEM P-values

Ctrl AA-CN Trt DIM Trt × DIM

Dry matter intake, kg/d  19.4 1.0 0.42 <0.01 
Milk, kg/d 41.9 47.4 2.5 0.15 <0.01 
Body weight change, kg  -105 17 0.14 0.02 0.50
WB Ra2 of glucose, mmol/h  770 41 0.23 0.41 0.79
Arterial concentration, mM
Glucose 3.13 3.07 0.13 0.74 <0.01 0.52
Lactate 0.28 0.32 0.02 0.31 0.15 0.75
ȕ2+EXW\UDWH 0.83 1.14  <0.01 0.02 0.03
*OXFRVHÀX[3, mmol/h
3RUWDOQHWÀX[ -37  24 0.23 0.29 0.35
+HSDWLFQHWÀX[   81 0.77 0.21 0.39
6SODQKQLFQHWÀX[     0.07 0.47
0DPPDU\QHWÀX[ -549  28 0.09 0.01 0.28
Portal utilization4 -123 -117 23 0.84 0.73 0.85
Hepatic utilization 17  21 0.45 0.93 
Splanchnic utilization -105 -123 29  0.74 0.45
Mammary utilization -529   0.03 0.01 0.20
3RUWDOWUXHÀX[5  120 11 0.03 0.02 0.13
+HSDWLFWUXHÀX[   78 0.92 0.23 
6SODQFKQLFWUXHÀX[ 731 752 8  0.09 0.28
/DFWDWHÀX[PPROK
Portal QHWÀX[ 208 185 19 0.40 <0.01 
Hepatic QHWÀX[  -322  0.49 0.21 0.88
Splanchnic QHWÀX[ -78 -139 27 0.14 0.13 0.58
Mammary QHWÀX[ 9 -14 21 0.44 0.29 0.54
ȕ2+EXW\UDWHÀX[PPROK
Portal QHWÀX[ 193 222 37 0.58 0.03 0.85
Hepatic QHWÀX[ 330 408 45 0.24 0.12 0.23
Splanchnic QHWÀX[ 524  53  0.23 
Mammary QHWÀX[ -231 -271 22 0.22 0.02 0.79

1 Mean values of the 3 sampling days. 2 Whole body rate of appearance. 3$SRVLWLYHÀX[LQGLFDWHVUHOHDVHDQGDQHJDWLYH

ÀX[LQGLFDWHVXSWDNHE\WKHWLVVXH4 Utilization from plasma supply. 57UXHÀX[ QHWÀX[±XWLOL]DWLRQ

DQG%+%QHWÀX[HVZHUHQRWDIIHFWHGE\$$LQIXVLRQEXWVSODQFKQLFWLVVXHWHQGHGWRWDNHXSPRUH
lactate (PTrt=0.14) and to release more BHB (PTrt  ZKHUHDVWKHPDPPDU\XSWDNHRI%+%ZDV
numerically higher for AA-CN cows.

Conclusion
The increased supply of AA in postpartum dairy cows did not increase WB Ra of glucose but increased
glucose mammary glucose uptake in line with the increased milk yield. These results indicate that in
early lactation the AA have priority for metabolic pathways other than gluconeogenesis suggesting
that their contribution to hepatic gluconeogenesis may increase as lactation progresses. Consequently,
other glucogenic substrates such as lactate and the mobilization of body fat would play an important
role in this interval transition to support the energy demands.

452 Energy and protein metabolism and nutrition in sustainable animal production
(IIHFWVRIPHWDEROL]DEOHSURWHLQVXSSO\RQ1HI¿FLHQF\SODVPDDPLQR
acid concentrations in dairy cows
D.R. Ouellet, D. Valkeners and H. Lapierre
$JULFXOWXUH DQG $JUL)RRG &DQDGD  &ROOHJH 6KHUEURRNH 4& -0 & &DQDGD
[email protected]

Introduction
(I¿FLHQF\RI1XWLOL]DWLRQLPSURYHVZLWKORZ1LQWDNHEXWWKLVSUDFWLFHUHPDLQVDYLDEOHRSWLRQ
only if milk production is not compromised. With this strategy in mind, we studied in a broader
perspective the effect of reducing metabolizable protein (MP) supply on urea kinetics, endogenous
1VHFUHWLRQVPLON\LHOG1HI¿FLHQF\DQGSODVPDDPLQRDFLG $$ FRQFHQWUDWLRQV7KLVPDQXVFULSW
reports the three latter.

Material and methods

Four lactating cows were used in a replicated incomplete 3×3 Latin square design (28-day experimental
period). Three concentrates (50% of diet; corn and soybean meal-based) were formulated and fed in a
total mixed ration with 45% grass silage and 5% hay, to supply NELPHHWLQJUHTXLUHPHQW 0-G 
EXWLQFUHPHQWDODPRXQWVRI03 /RZ  0HG DQG +LJK J03GFRUUHVSRQGLQJ
to 72, 98 and 111% of estimated MP requirements (NRC, 2001). Cows were fed every other hour,
and blood samples were collected on day 27 at 8:00, 10:00, 12:00 and 14:00 h and on day 28 at
9:00, 11:00, 13:00 and 15:00 h. Concentrations of AA were determined on individual sample using
isotopic dilution technique (Calder et al., 1999). Dry matter intake (DMI), milk production, and
urinary purine derivatives were measured during the total collection of feces and urine (reported in
Valkeners et al., 2007) on days 13-20 of each period. Data were analyzed using the MIXED procedure
of SAS/STAT® using the mean of each cow × period. Linear and quadratic orthogonal polynomial
contrasts were used to delineate MP supply effect.

Results and discussion

The DMI did not differ between treatments (3 0.14) whereas, milk production decreased from
High to Low (Plinear 7DEOH 0LONSURWHLQFRQFHQWUDWLRQZDVQRWDIIHFWHG 3 0.49) by MP
supply, but milk protein yield decreased (Plinear=0.03) from High to Low. Proportion of non-protein
N in milk decreased (Plinear ZLWKGHFUHDVHG03VXSSO\(I¿FLHQF\RI1XWLOL]DWLRQLQFUHDVHG
(Plinear ZLWKGHFUHDVHG03VXSSO\7KHLQFUHDVHGHI¿FLHQF\RI1XWLOL]DWLRQDWORZ03ZDV
UHODWHGWRJUHDWHUXUHDUHF\FOLQJWRWKHJXWLQFUHDVLQJIURPWRRIXUHDHQWU\UDWH 9DONHQHUV
et al., 2007).

Decreasing MP supply decreased concentration of Lys (Plinear<0.08) and most branched-chain AA

(Val and Ile: Plinear<0.05; Leu: Pquadratic=0.11) whereas amongst Group 1 AA (His, Met, Phe+Tyr and
Trp), His (Plinear<0.01) and Trp (Plinear<0.07) decreased. This low His concentration at Low supply
PLJKWEHUHODWHGWRWKHKLJKSURSRUWLRQRIEDFWHULD1 6(0 RIWKHWRWDOGXRGHQDO1ÀRZ
HVWLPDWHGIURPXULQDU\SXULQHGHULYDWLYHVFRPSDUHGZLWKDQGIRU0HGDQG+LJKUHVSHFWLYHO\
Across the non-essential AA, only Ala (Plinear=0.08) and Glu (Plinear<0.001) concentrations were
altered by MP supply and they increased with decreased protein supply. These 2 non-essential AA are
known to be active contributors in inter-organ movement of N to maintain N homeostasis especially
during periods of low N supply.

In conclusion, peripheral concentrations of Group 1 AA, extracted in high proportion of hepatic

LQÀRZE\WKHOLYHUZHUHUHGXFHGWRVRPHGHJUHHIRU7USEXWODUJHO\IRU+LVE\ORZHUHG03VXSSO\

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 453
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_169, © Wageningen Academic Publishers 2013
This emphasizes the importance of monitoring His supply at low protein supply, as microbial protein
has a low proportion of His. On the other hand, the branched-chain AA which are poorly extracted
by the liver, responded to protein supply and could be related to a stimulation of protein synthesis
in peripheral tissues including the mammary gland (Lei et al., 2012).

7DEOH'U\PDWWHULQWDNHPLON\LHOGDQGFRPSRVLWLRQDQGSODVPDDPLQRDFLG $$ FRQFHQWUDWLRQ
RIFRZVUHFHLYLQJLQFUHPHQWDOPHWDEROL]DEOHSURWHLQ 03 VXSSO\

Item MP level SEM P value, contrast

Low Medium High Linear Quadratic

Dry matter intake, kg/d  17.2  0.4  0.11
Milk yield, kg/d  29.7 29.1 0.87  0.15
Milk N, % 3.13 3.15 3.22 0.08 0.34 0.52
Milk true protein yield, kg/d 0.83 0.89 0.89 0.02 0.03 0.27
Milk N: N intake 0.38 0.32 0.28 0.01 0.01 0.97
AA concentrations, μM
Histidine 18.2 42.2 42.2 4.2 0.009 0.17
Isoleucine 91.2 104.5  4.8 0.02 0.59
Leucine 132.1 127.0 151.9 7.1 0.20 0.11
Lysine 49.9 55.1  4.7 0.08 0.53
Methionine 15.8 17.7  0.7 0.24 0.23
Phenylalanine 37.8 39.4  1.8 0.39 0.34
Threonine  90.3 82.5 8.7 0.83 0.44
Tryptophan  49.4 48.1 1.5 0.07 0.19
Valine 150.1 188.3  13.4 0.02 0.70
Alanine 223.9 211.3 199.5 11.0 0.08 0.71
Aspartate 18.8 17.4 15.8 2.8 0.43 
Asparagine 38.2 42.7 37.3 1.9 0.89 0.08
Glutamate   58.5 2.4 0.001 0.15
Glutamine  277.9  15.4  0.53
Glycine  223.9 197.0   0.27
Proline 70.1 75.9 75.4 3.0 0.24 0.58
Serine 72.9 75.4 72.3 4.0 0.99 0.45
Tyrosine 48.0 43.7 44.4 2.2 0.50 

References
Calder, A.G., K.E. Garden, S.E. Anderson and G.E. Lobley, 1999. Quantitation of blood and plasma amino acids
using isotope dilution electron impact gas chromatography/mass spectrometry with U-13C amino acids as internal
standards. Rapid Commun. Mass Spectrom. 13, 2080-2083.
Lei, J., D. Feng, Y. Zhang, F.-Q. Zhao, Z. Wu, A. San Gabriel, Y. Fujishima, H. Uneyama and G. Wu, 2012. Nutritional
and regulatory role of branched-chain amino acids in lactation. Frontiers in Bioscience 17, 2725-2739.
National Research Council, 2001. Nutrient requirements of dairy cattle 7th rev. ed. Natl. Acad. Press, Washington, D.C.
Valkeners, D., H. Lapierre, J. Marini, and D.R. Ouellet, 2007. Effects of metabolizable protein supply on nitrogen
metabolism and recycling in lactating dairy cows. In Energy and protein metabolism and nutrition. EAAP
publication No. 124, Pages 417-418.

454 Energy and protein metabolism and nutrition in sustainable animal production
Small intestinal, stomach complex, and total gastrointestinal tract
PDVVHVDUHGHFUHDVHGUHODWLYHWRERG\ZHLJKWLQKLJKHI¿FLHQF\VWHHUV
A.M. Meyer, K.W. Christensen, W.J. Means, S.L. Lake and S.I. Paisley
'HSDUWPHQWRI$QLPDO6FLHQFH8QLYHUVLW\RI:\RPLQJ/DUDPLH:<86$DPH\HU#XZ\RHGX

Introduction
$OWKRXJKERWKVFLHQWL¿FDQGLQGXVWU\LQWHUHVWLQIHHGHI¿FLHQF\RIUXPLQDQWDQLPDOVKDVLQFUHDVHG
JUHDWO\LQUHFHQW\HDUVSK\VLRORJLFDOPHFKDQLVPVXQGHUO\LQJGLIIHUHQFHVLQLQGLYLGXDOIHHGHI¿FLHQF\
remain largely unknown. The gastrointestinal (GI) tract and liver are not only essential for nutrient
acquisition and utilization, but also use 40 to 50% of whole body energy expenditure (Ferrell, 1988).
Despite recent research in the area (Basarab et al., 2003; Mader et al., 2009; Meyer et al., 2012), the
UROHRIYLVFHUDORUJDQVHVSHFLDOO\WKDWRIWKH*,WUDFWLQIHHGHI¿FLHQF\LVXQFOHDU:HK\SRWKHVL]H
WKDWDSRUWLRQRIWKHLQGLYLGXDOGLIIHUHQFHVREVHUYHGIRUIHHGHI¿FLHQF\FDQEHDWWULEXWHGVSHFL¿FDOO\
to GI tract size and function through its energy use and ability to digest and absorb nutrients. The
objective of this study was to investigate GI tract and other visceral organ masses in market weight
VWHHUVFODVVL¿HGDVKLJKDQGORZHI¿FLHQF\EDVHGRQ¿QLVKLQJSHULRGUHVLGXDOIHHGLQWDNH 5), 

Material and methods

+HUHIRUG$QJXVFURVVEUHGVWHHUV Q IURPRQHFRQWHPSRUDU\JURXS ZHUHIHGDFRPPRQ¿QLVKLQJ
diet (11.4% CP, 2.0 Mcal NEm/kg, 1.35 Mcal NEg/kg; DM basis) for 57 d using the GrowSafe Feed
Intake System (model 4000E, GrowSafe Systems Ltd., Airdrie, AB, Canada). Twelfth rib fat thickness
was determined using ultrasound at the conclusion of the feed intake test. For all steers with at least
1.02 cm 12th rib fat thickness (n=40), RFI was calculated as the expected feed intake subtracted
from the actual feed intake. Expected feed intake was determined by regressing average daily gain
DQGPHWDEROLFPLGZHLJKWRQDFWXDOIHHGLQWDNH7KHPRVWHI¿FLHQW ORZ5),Q  DQG
OHDVWHI¿FLHQW KLJK5),Q  VWHHUVZHUHVODXJKWHUHGRUGDIWHUWKHHQGRIWKHIHHGLQWDNHWHVW
At slaughter, visceral organs were dissected, trimmed of fat, stripped of digesta, and weighed. Data
ZHUHDQDO\]HGZLWK352&0,;('RI6$6XVLQJ5),FODVV KLJKYVORZHI¿FLHQF\ DVD¿[HG
effect in the model.

Results and discussion

Omasal mass (kg) was less (3  IRUKLJKHI¿FLHQF\WKDQORZHI¿FLHQF\VWHHUVDOWKRXJKRWKHU
visceral organ masses (kg), body weight (BW), and hot carcass weight (HCW) were unaffected
(P>0.11) by RFI class. When expressed relative to BW, total GI tract mass (g/kg BW) was less
(3  IRUKLJKHI¿FLHQF\FRPSDUHGZLWKORZHI¿FLHQF\VWHHUV,QDGGLWLRQVPDOOLQWHVWLQDO
and omasal masses (g/kg BW) were less (P” DQGVWRPDFKFRPSOH[PDVV JNJ%: WHQGHG
(3  WREHOHVVLQKLJKHI¿FLHQF\VWHHUVWKDQORZHI¿FLHQF\VWHHUV&RQYHUVHO\KLJKHI¿FLHQF\
steers tended (3  WRKDYHJUHDWHUSDQFUHDWLFPDVV JNJ%: FRPSDUHGZLWKORZHI¿FLHQF\
steers. Similar results were observed when organ masses were expressed relative to HCW. Total GI
tract, small intestinal, and omasal masses (g/kg HCW) were less (P” DQGVWRPDFKFRPSOH[
mass (g/kg HCW) tended (3  WREHOHVVIRUKLJKHI¿FLHQF\VWHHUVWKDQORZHI¿FLHQF\VWHHUV
Despite this, other visceral organ (large intestine, liver, spleen, kidney, omental and mestenteric
fat, heart, and lung) relative masses (g/kg BW or HCW) were unaffected (P>0.10) by RFI class.

Results of this study suggest that GI tract masses relative to BW, especially those of the small intestine
DQGVWRPDFKFRPSOH[DUHGHFUHDVHGLQKLJKHI¿FLHQF\VWHHUV0RUHRYHUGLIIHUHQFHVLQVWRPDFK
complex mass appear to be driven by the omasum. Decreased tissue mass may result in reduced
HQHUJ\DQGQXWULHQWXVHH[SODLQLQJVRPHYDULDWLRQREVHUYHGLQHI¿FLHQF\RIQXWULHQWXWLOL]DWLRQLQ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 455
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_170, © Wageningen Academic Publishers 2013
IHHGORWVWHHUV$GGLWLRQDOGDWDIURPRXUODERUDWRU\VXJJHVWWKDWPRUHHI¿FLHQWFDWWOHKDYHOHVVVPDOO
intestinal mass, but more dense intestinal mucosa (Meyer et al., 2012), which may enable these
animals to maintain digestive and absorptive functions with less tissue mass. In conclusion, growth
and function of GI tract tissues are potential targets for development of strategies to improve feed
HI¿FLHQF\LQEHHIFDWWOH)XUWKHUUHVHDUFKLVSODQQHGWRLQYHVWLJDWHJURZWKYDVFXODULW\DQGJHQH
expression of these tissues to better understand differences in nutrient use and function that may
DIIHFWPHWDEROLFHI¿FLHQF\

7DEOH9LVFHUDORUJDQPDVVHVDQGUHODWLYHPDVVHVRIKLJKDQGORZHI¿FLHQF\VWHHUV

Item1 +LJKHI¿FLHQF\ /RZHI¿FLHQF\ SEM2 P-value

Total GI tract, kg 23.1 25.0 1.0 0.20

g/kg BW 42.7  0.9 0.02
g/kg HCW  73.9  0.01
Stomach complex, kg 17.2 18.4 0.8 0.31
g/kg BW 31.8 33.8 0.8 0.10
g/kg HCW 50.3 54.3 1.4 
Omasum, kg 4.38 5.49 0.31 0.02
g/kg BW 8.10 10.11 0.47 0.01
g/kg HCW 12.8  0.8 0.009
Small intestine, kg 4.13  0.20 0.12
g/kg BW  8.50 0.28 0.04
g/kg HCW 12.1 13.7 0.5 0.03
Large intestine, kg 1.80 2.01 0.12 0.24
g/kg BW 3.32 3.72 0.20 0.18
g/kg HCW 5.27 5.97 0.33 0.15
Liver, kg   0.31 0.53
g/kg BW 12.0 12.5 0.3 
g/kg HCW 19.0 20.1 0.4 0.11
Pancreas, kg  0.515 0.047 0.21
g/kg BW 1.10 0.94 0.07 0.10
g/kg HCW 1.75 1.50 0.10 0.13

1 Total GI tract = stomach complex + small intestine + large intestine; BW = body weight; HCW = hot carcass weight.
2 SEM for n=8 per RFI class.

References
Basarab, J.A., M.A. Price, J.L. Aalhus, E.K. Okine, W.M. Snelling and K.L. Lyle, 2003. Residual feed intake and body
composition in young growing cattle. Can. J. Anim. Sci. 83, 189-204.
)HUUHOO&/&RQWULEXWLRQRIYLVFHUDORUJDQVWRDQLPDOHQHUJ\H[SHQGLWXUHV-$QLP6FL
Mader, C.J., Y.R. Montanholi, Y.J. Wang, S.P. Miller, I.B. Mandell, B.W. McBride and K.C. Swanson, 2009. Relationships
DPRQJPHDVXUHVRIJURZWKSHUIRUPDQFHDQGHI¿FLHQF\ZLWKFDUFDVVWUDLWVYLVFHUDORUJDQPDVVDQGSDQFUHDWLF
digestive enzymes in feedlot cattle. J. Anim. Sci. 87, 1548-1557.
Meyer, A.M., K.M. Cammack, S.I. Paisley, P. Moriel, W.J. Means, M. Du, J.S. Caton and B.W. Hess, 2012. Correlation
RIIHHGHI¿FLHQF\DQGVPDOOLQWHVWLQDOJURZWKLQ¿QLVKLQJFDWWOHERUQWRGDPVIHGYDU\LQJOHYHOVRIQXWULHQWVGXULQJ
early to mid-gestation. J. Anim. Sci. 90, (Suppl. 2): 70.

456 Energy and protein metabolism and nutrition in sustainable animal production
Whole body oxidative metabolism in dairy cows with a different liver fat
content in early lactation
M. Derno, S. Börner, H.M. Hammon, M. Röntgen and B. Kuhla
,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 
'XPPHUVWRUI*HUPDQ\[email protected]

Introduction
During the transition from late pregnancy to early lactation, high-yielding dairy cows mobilize
ODUJHDPRXQWVRIDGLSRVHWLVVXHUHVXOWLQJLQLQFUHDVHGSODVPDFRQFHQWUDWLRQVRIQRQHVWHUL¿HGIDWW\
acids (NEFA). NEFA are mostly oxidized to CO2 whereas at excessive concentrations, NEFA are
UHHVWHUL¿HGWRIRUPWULDF\OJO\FHULGHVOHDGLQJWRWKHGHYHORSPHQWRIIDWW\OLYHU7KXVWKHFDSDFLW\
for fatty acid oxidation – among others – determines the fat load of the liver. We hypothesized that
whole-body fat oxidation adapts to the extent of fat mobilization during early lactation.

Material and methods

Fifteen German Holstein cows (2nd to 4th lactation, >10,000 kg/305 d in at least one previous
lactation) were fed ad libitum a total mixed ration (dry period: 5.8 MJ NEL/kg DM, 13% utilizable
protein, lactation period: 7.1 MJ NELNJ'0XWLOL]DEOHSURWHLQ IURPZHHNantepartum (ap)
until week 3 postpartum (pp). In week 3 ap and week 2 pp, body weight (BW) and ultrasonically
measured back fat thickness (BFT) were determined and animals were kept in respiratory chambers
(Derno et al., 2009) to which they were well adapted before. Therein, animals were fed ad libitum
for 24 h to determine dry matter intake (DMI) and milk yield. On the subsequent day, cows were
feed-deprived for 10 h and a blood sample was taken in an EDTA-containing tube via extension
tubing from the outside of the chamber to analyze plasma NEFA concentration. Concentrations of
O2, CO2 and CH4 PHDVXUHGFRQWLQXRXVO\LQPLQLQWHUYDOV ZHUHDQDO\]HGWRFDOFXODWHEDVDOKHDW
SURGXFWLRQ +3 DQGZKROHERG\IDWR[LGDWLRQ )2; DFFRUGLQJWR+3 N-  22 (l) + 5.02
CO2 (l) – 2.17 CH4 O ±1 J DQG)2; J  22 O ±&22 (metabolic) (l) using the last
measuring interval after 10 h feed deprivation as described previously (Derno et al., 2013). At the
end of the 3rd week pp, a liver biopsy was taken and based on liver fat content (LFC) as a measure of
body fat mobilization cows were allocated to high (HLFC, n=7) and low (LLFC, n=8) LFC groups
(G). Data were evaluated by the two-way repeated measure ANOVA procedure of SigmaPlot 11.0
including the effect of G, period (P), and the interaction of G × P followed by post-hoc comparison
using the Tukey-Kramer procedure. Linear correlations were calculated using SigmaPlot software
DQGWKH3HDUVRQFRUUHODWLRQFRHI¿FLHQWLVSURYLGHG

Results and discussion

Before and after parturition, BFT was greater (PG” LQ+/)&WKDQ//)&FRZVDQGGLIIHUHQFHV
were larger between G after than before parturition (PG×P<0.01), but DMI and energy corrected milk
yield were comparable between groups (Table 1). Furthermore, DMI, FOX, HP, and plasma NEFA
concentrations were lower (PP” GXULQJWKHGU\SHULRGWKDQLQHDUO\ODFWDWLRQLQDOODQLPDOV
(Table 1). Before parturition, NEFA concentrations were comparable between cow groups but pp,
HLFC cows developed higher (P<0.05) plasma NEFA concentrations as compared to LLFC cows
(PG×P=0.07), which parallels the fat content of the liver. Furthermore, ap HP was not different
between groups (PG=0.33) but in early lactation, HP was higher (P<0.05) in HLFC than in LLFC
cows (PG×P  &RQVLGHULQJWKHVDPH'0,EHIRUHWKHKIHHGGHSULYDWLRQSHULRGLQ+/)&DQG
LFFC cows, differences in pp HP are not attributable to dietary HP but should be of catabolic origin.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 457
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_171, © Wageningen Academic Publishers 2013
Table 1. Zootechnical parameters, heat production, whole-body fat oxidation and plasma NEFA
FRQFHQWUDWLRQVLQFRZVGHYHORSLQJDKLJK +/)& DQGORZ //)& OLYHUIDWFRQWHQWpostpartum.

Item antepartum postpartum Pgroup Pperiod Pgroup × period

LLFC HLFC LLFC HLFC

BFT1 12.1a 21.1b 13.0c 23.5b 0.01 <0.01 <0.01

Milk yield 42.8 40.4 0.4
DMI, kg/d 10.9a 10.2a b 15.9b  <0.001 
NEFA, μM2 459a 394a 1,432b 1,800c 0.123 <0.001 0.07
)2;JPLQ2 1.0a 5.8b 4.0c 12.0d 0.001 <0.001 0.1
HP, kJ/kg0.75d2 a a 748b c  <0.001 

1 Note: Back fat thickness still increased until parturition above pp values presented.
2 Determined after 10 hours feed deprivation.
a-d9DOXHVZLWKGLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\ P<0.05, Tukey-Kramer test).

FOX increased from the ap to pp period and strong correlations between ap FOX and pp FOX
(r=0.735; P<0.01) were found. These results indicate that increased FOX determined 10 h after feed
deprivation between ap and pp is primarily due to increased body fat mobilization and subsequent fatty
acid oxidation and less due to the higher pp DMI ingested before the 10 h-feed deprivation period.

Moreover, FOX was higher in HLFC than in LLFC cows not only pp but already ap despite there
were no indications on body fat mobilization in HLFC cows during week 3 ap (PGxP=0.1). Based
on the higher FOX in HLFC compared to LLFC cows already before parturition, we suggest that
a high ap BFT is accompanied with a high basal FOX, both subscribing the risk for overwhelming
fat mobilization and development of fatty liver pp.

Acknowledgements
Supported by DFG, Germany.

References
Derno, M., H.G. Elsner, E.A. Paetow, H. Scholze and M. Schweigel, 2009. Technical note: a new facility for continuous
respiration measurements in lactating cows. J. Dairy Sci. 92, 2804-2808.
Derno, M., G. Nürnberg, P. Schön, A. Schwarm, M. Röntgen, H.M. Hammon, C.C. Metges, R.M. Bruckmaier and B.
Kuhla, 2013. Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows. J. Dairy
6FL

458 Energy and protein metabolism and nutrition in sustainable animal production
Lipolytic capacity of visceral adipose tissue in the dairy cow
Á. Kenéz1, L. Locher1, A. Rizk2, S. Dänicke, J. Rehage and K. Huber1
1'HSDUWPHQWRI3K\VLRORJ\8QLYHUVLW\RI9HWHULQDU\0HGLFLQH+DQQRYHU%LVFKRIVKROHU'DPP
+DQQRYHU*HUPDQ\[email protected]
2Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Mansoura
8QLYHUVLW\(OJRPKHU\LD6W0DQVRXUD(J\SW
,QVWLWXWHRI$QLPDO1XWULWLRQ)HGHUDO5HVHDUFK,QVWLWXWHIRU$QLPDO+HDOWK%XQGHVDOOHH
Braunschweig, Germany
&OLQLFIRU&DWWOH8QLYHUVLW\RI9HWHULQDU\0HGLFLQH+DQQRYHU%LVFKRIVKROHU'DPP
Hannover, Germany

Introduction
Catecholamine-induced lipolysis in adipose tissue (AT) depots is considered to be the key metabolic
pathway for providing endogenous energy in times of high energy demand in the peripartal dairy cow
(McNamara, 1991; Koltes and Spurlock, 2011). It has been described in humans and rodents that
subcutaneous and visceral AT differ in their lipolytic activity (Tavernier et al., 1995; Reynisdottir et
al., 1997). However, in dairy cows, only the subcutaneous adipose tissue (SCAT) has been examined
UHJDUGLQJLWVOLSRO\WLFFDSDFLW\ 0F1DPDUDDQG+LOOHUV DQGOLWWOHLVNQRZQDERXWWKHERYLQH
visceral adipose tissues. Locher et al. (2011) have revealed that intrinsic expression of hormone-
sensitive lipase (HSL) in retroperitoneal adipose tissue (RPAT) is greater than in SCAT in dairy cows
antepartum; thus, it is hypothesized that the lipolytic response of RPAT is enhanced, too. Therefore,
this ex vivo study aimed to determine the rate of lipolysis in catecholamine-stimulated AT explants.

Material and methods

7KH6&$7DQG53$7ELRSV\VDPSOHVZHUHFROOHFWHGIURPGU\+ROVWHLQ)ULHVLDQFRZVZHHNV
before parturition. After 20 min of pre-incubation, triplicates of SCAT and RPAT slices were incubated
LQ'XOEHFFR¶V0RGL¿HG(DJOH0HGLXPFRQWDLQLQJIDWW\DFLGIUHH%6$IRUPLQDWƒ&LQWKH
absence (basal) or presence of 10 M isoproterenol (non-selective agonist of beta-adrenoceptors).
)UHHJO\FHURODQGIUHHQRQHVWHUL¿HGIDWW\DFLG 1()$ FRQWHQWRIWKHLQFXEDWLRQPHGLDZDVPHDVXUHG
and values were corrected for tissue wet weight. The NEFA:glycerol ratio was calculated. Data
were analyzed by two-way ANOVA for factor Tissue, factor Treatment and respective interactions.

Results and discussion

Release rates of glycerol and NEFA are shown in Figure 1 and Figure 2, respectively. Mean basal
release of glycerol and NEFA did not differ between RPAT and SCAT. In both AT, release of glycerol
and NEFA increased after isoproterenol-stimulation, but this increase was higher in RPAT than in SCAT.

2XUUHVXOWVVKRZHGIRUWKH¿UVWWLPHWKDW53$7DQG6&$7RIGDLU\FRZVGLGQRWUHVSRQGHTXDOO\DIWHU
challenging them with the same catecholamine stimulus. In particular, RPAT had a more pronounced
lipolytic responsiveness to catecholamine-stimulation than SCAT (Figure 1 and 2). This is most
OLNHO\GXHWRWKHGLIIHUHQFHLQWKHDPRXQWRI+6/SURWHLQLQ53$7DQG6&$7ZLWKVLJQL¿FDQWO\
higher HSL expression in RPAT in dairy cows antepartum (Locher et al., 2011). The higher lipolytic
response of RPAT indicated that RPAT was already able at day 42 antepartum to provide higher
lipolytic capacity and therefore was prepared to release more NEFA at times of peak energy demand
postpartum. Accordingly, being capable of releasing more NEFA, RPAT may contribute to a greater
extent to the plasma NEFA peak commonly seen in high yielding dairy cows postpartum.

Mean basal NEFA:glycerol release ratio did not differ between the two AT (Figure 3). After
isoproterenol-stimulation, the ratio increased in RPAT, but not in SCAT.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 459
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_172, © Wageningen Academic Publishers 2013
Figure 1. Glycerol release in adipose tissue explants.

Figure 2. NEFA release in adipose tissue explants.

)LJXUH1()$JO\FHUROUHOHDVHUDWLR

Our results showed that the ratio of NEFA release to glycerol release was constant under basal
FRQGLWLRQVLQERWKWLVVXHVDQGLQ6&$7DIWHULVRSURWHUHQROVWLPXODWLRQDVZHOOEXWZDVVLJQL¿FDQWO\
greater in isoproterenol-stimulated RPAT (Figure 3). Again, this suggests a high impact of RPAT
on the development of a plasma NEFA peak postpartum. The total hydrolysis and total release
of triglycerides would result in a NEFA:glycerol ratio of 3:1. The actual value of release ratio is
FRQVLGHUHGWREHDQLQGH[RI1()$UHHVWHUL¿FDWLRQZLWKLQWKHDGLSRF\WHVLQKXPDQVWXGLHV =LHUDWKet
al. +RZHYHUWKHUHLVQRHYLGHQFHFRQFHUQLQJWKHUROHDQGVLJQL¿FDQFHRIUHHVWHUL¿FDWLRQDQG
membrane transport mechanisms in altering NEFA and glycerol release in dairy cow AT. Therefore,
the exact causal factors which led to higher NEFA release rates in isoproterenol-stimulated RPAT
still have to be elucidated.

460 Energy and protein metabolism and nutrition in sustainable animal production
References
Koltes, D.A. and D.M. Spurlock, 2011. Coordination of lipid droplet-associated proteins during the transition period
of Holstein dairy cows. J. Dairy Sci. 94, 1839-1848.
Locher, L.F., N. Meyer, E.M. Weber, J. Rehage, U. Meyer, S. Dänicke and K. Huber, 2011. Hormone-sensitive lipase
protein expression and extent of phosphorylation in subcutaneous and retroperitoneal adipose tissues in the
periparturient dairy cow. J. Dairy Sci. 94, 4514-4523.
0F1DPDUD-35HJXODWLRQRIDGLSRVHWLVVXHPHWDEROLVPLQVXSSRUWRIODFWDWLRQ-'DLU\6FL
0F1DPDUD-3DQG-.+LOOHUV5HJXODWLRQRIERYLQHDGLSRVHWLVVXHPHWDEROLVPGXULQJODFWDWLRQ/LSRO\VLV
UHVSRQVHWRPLONSURGXFWLRQDQGHQHUJ\LQWDNH-'DLU\6FL
Reynisdottir S., M. Dauzats, A. Thörne and D. Langin, 1997. Comparison of hormone-sensitive lipase activity in
YLVFHUDODQGVXEFXWDQHRXVKXPDQDGLSRVHWLVVXH-&OLQ(QGRFULQRO0HWDE
Tavernier G., J. Galitzky, P. Valet, A. Remaury, A. Bouloumie, M. Lafontan and D. Langin, 1995. Molecular mechanisms
XQGHUO\LQJUHJLRQDOYDULDWLRQVRIFDWHFKRODPLQHLQGXFHGOLSRO\VLVLQUDWDGLSRF\WHV$P-3K\VLRO(
1142.
Zierath, J.R., J.N. Livingston, A. Thörne, J. Bolinder, S. Reynisdottir, F. Lönnqvist and P. Arner, 1998. Regional
GLIIHUHQFHLQLQVXOLQLQKLELWLRQRIQRQHVWHUL¿HGIDWW\DFLGUHOHDVHIURPKXPDQDGLSRF\WHVUHODWLRQWRLQVXOLQ
receptor phosphorylation and intracellular signalling through the insulin receptor substrate-1 pathway. Diabetologia
41, 1343-1354.

Energy and protein metabolism and nutrition in sustainable animal production 461
Effects of nutrient restriction on liver and small intestine energy use in
pregnant beef cows
L.D. Prezotto1, L.E. Camacho1, C.O. Lemley2, F.E. Doscher1, J.S. Caton1, K.A. Vonnahme1 and
K.C. Swanson1
1'HSDUWPHQW RI $QLPDO 6FLHQFH 1RUWK 'DNRWD 6WDWH 8QLYHUVLW\ )DUJR 1'  86$
[email protected]
2'HSDUWPHQWRI$QLPDO 'DLU\6FLHQFHV0LVVLVVLSSL6WDWH8QLYHUVLW\0LVVLVVLSSL6WDWH06
USA

Introduction
The extensive use of grazing systems for beef cattle and the high variation in forage quality throughout
the year has an important impact on production. Changes in forage quality and availability alter the
nutritional and physiological status of cows during gestation. Nutrient restriction during this crucial
time plays a large role on cow weight change and on fetal growth and development. The objectives
ZHUHWRGHWHUPLQHKRZQXWULHQWUHVWULFWLRQDQGUHDOLPHQWDWLRQGXULQJJHVWDWLRQLQÀXHQFHVHQHUJ\
utilization and oxygen consumption of intestinal and liver tissues of the cow and the developing fetus.

Material and methods

Forty six cross-bred multiparous gestating and non-lactating beef cows were individually-fed a
common hay diet using Calan gates to meet or exceed NRC recommendations until d 30 of gestation.
Pregnant cows were grouped by d of insemination and body weight, and randomly assigned to one
of three treatment groups: 1) 100% NRC recommendations throughout the experimental period
&&&Q   15&UHFRPPHQGDWLRQVIURPGXQWLORISUHJQDQF\WKHQUHDOLPHQWHGWR
RIUHFRPPHQGDWLRQV 5&&Q  RU RI15&UHFRPPHQGDWLRQVIURPGXQWLO
then realimented to 100% of recommendations (RRC; n=12). Cows were weighed and slaughtered
DWG Q DQGIRU5DQG&  Q DQGIRU555&DQG&& DQG Q DQGIRU
RRC, RCC and CCC) of gestation. Fetuses were immediately removed and weighed. Maternal and
fetal liver and small intestinal mass was recorded and tissues were sampled for immediate analysis.
For in vitro O2 consumption analysis, duplicate tissue subsamples (200±10 mg) were placed into
test chambers containing 3 ml of buffer and a Clarke polariographic electrode and O2 consumption
measured for 5 min. Maternal and fetal observations are expressed as absolute values and values
relative to maternal or fetal body weight (BW), respectively. Data were analyzed using a completely
randomized block (breeding group) design using PROC MIXED of SAS, with group used as a random
variable and treatment as the main effect. When greater than 2 means were tested and the overall
P<0.05, means were compared using the pdiff statement in SAS. Means followed by different lower
case letter (a, b) are different (P” 

Results and discussion

At d 85, maternal small intestinal mass increased (3  LQ5DVFRPSDUHGWR&FRZV 5 
C=4,127±148 g). Fetal small intestinal mass relative to BW decreased (3 0.02) in R as compared to
&FRZV 5 & “JNJ%: 0RUHRYHUIHWDOVPDOOLQWHVWLQDO22 consumption relative to BW
decreased (3 0.05) in R as compared to C cows (R=0.13; C=0.22±0.03 mol/min/kg BW). At d 140
of gestation, maternal liver weight decreased (3 0.01) in RR as compared to CC cows (RR=3,723a;
RC=4,143ab&& b±198 g). Total O2 consumption in the maternal liver decreased (3 0.01)
in RR as compared to CC cows (RR=28a; RC=43a&& b“PROPLQ 7RWDO22 consumption
in the maternal small intestine relative to BW also decreased (3”0.03) in RR cows as compared to
CC cows (RR=0.05a; RC=0.08ab; CC=0.09b±0.01 mol/min/kg BW). Fetal liver weight increased
(3  LQ5&DQG55FRZVZKHQFRPSDUHGWR&&FRZV 55 a5& a&& b±4.9 g).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 463
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_173, © Wageningen Academic Publishers 2013
Fetal small intestinal O2 consumption relative to BW increased (3 0.03) in the RC cows when
compared to RR and CC cows (RR=0.17b; RC=0.39a; CC=0.20b±0.05 mol/min/kg BW). At d 254
of pregnancy, maternal jejunal weight increased in RCC but decreased in RRC and CCC (3 0.04;
RRC=903b; RCC=1,343a; CCC=1,005b±130 g). All the other parameters measured did not differ
between the treatment groups (P>0.10) are not presented herein.

7LVVXHPDVVDQGPHWDEROLFDFWLYLW\LQÀXHQFHHQHUJ\XVHE\WLVVXHV,QUHVSRQVHWRPDWHUQDOQXWULHQW
restriction we observed an increase in small intestinal mass in cows and decrease in fetuses at d
85. This may indicate that cows are adapting to nutrient restriction by increasing intestinal mass in
an attempt to increase the digestion and utilization of feed. Moreover, realimentation appears to be
able to reverse such effects.

Oxygen consumption (and thus energy expenditure) in the gut accounts for a large proportion of
whole body oxygen consumption and it has been observed to vary depending on level of intake
(Webster, 1980), physiological status (Reynolds and Huntington, 1988), and animal age (Vatnick et
al., 1989). Our results show that intestinal and hepatic O2 consumption in maternal and fetal tissues
decreased in response to nutrient restriction. The decrease in O2 consumption with no change or an
LQFUHDVHLQPDVVPD\LQGLFDWHDQLPSURYHPHQWLQHQHUJHWLFHI¿FLHQF\RIWKHVHWLVVXHVGXULQJQXWULHQW
restriction. However, this change was reversed during the realimentation period.

In conclusion, nutrient restriction in pregnant cows during early and midgestation affects tissue mass
and energy utilization within the small intestine and liver. This may indicate that cows and fetuses
are adapting to restriction by increasing or decreasing mass in an attempt to increase the digestion
and utilization of feed to generate energy. However, maternal realimentation has the potential to
reverse these effects.

References
5H\QROGV&.DQG*%+XQWLQJWRQ3DUWLWLRQRISRUWDOGUDLQHGYLVFHUDOQHWÀX[LQEHHIVWHHUV%ORRGÀRZ
DQGQHWÀX[RIR[\JHQJOXFRVHDQGQLWURJHQRXVFRPSRXQGVDFURVVVWRPDFKDQGSRVWVWRPDFKWLVVXHV%U-1XWU

Vatnick, I., A.W. Bell, J.M. Kelly and B.W. McBride, 1989. Gestational changes in hepatic oxygen consumptioin of
the ovine fetus. In Energy metabolism of farm animals. Y. Van Der Honing and W.H. Close, comilers. Pudoc,
:DJHQLQJHQWKH1HWKHUODQGV
Webster, A.J.F., 1980. Energy cost of digestion and metabolism in the gut. In Digestive physiology and metabolism in
UXPLQDQWV<5XFNHEXVFKDQG37KLYHQGHGV0733UHVV/DQFDVWHU3$

464 Energy and protein metabolism and nutrition in sustainable animal production
The effect of pregnancy on weight change, visceral organ mass and
circulating serum metabolites in mature beef cows
K.M. Wood1, C.J. Fitzsimmons2, S.P. Miller1, B.W. McBride1 and K.C. Swanson
1Dept. of Animal and Poultry Science, University of Guelph. Guelph, ON, Canada; [email protected]
2Agriculture and Agri-Food Canada / University of Alberta, Edmonton, AB, Canada
Dept. of Animal Sciences, North Dakota State University, Fargo, ND, USA

Introduction
During the last trimester of gestation, fetal growth dramatically increases and results in increased
QXWULHQWGHPDQGLQRUGHUWRVXSSRUWJURZWK 15& )UHHWO\et al. (2008) suggested that cows
may be able to reduce maintenance energy costs in order to support the energetic demands of the
conceptus. Although visceral organs account for approximately 10% of bodyweight (BW) they
represent about 50% of total energy costs (Reynolds et al., 1991) and can be attributed to maintenance
energy costs and energy expenditures (McBride and Kelly, 1990). Little is known about the effect
of pregnancy in mature beef cows on visceral organ mass and metabolism. By better understanding
the metabolic role of pregnancy, there may be opportunities to better understand maintenance energy
FRVWVDQGLPSURYHRYHUDOOIHHGHI¿FLHQF\

Material and methods

Eighteen non-lactating, mature Simmental/Angus crossbred cows, pregnant (PREG; n=9) and non-
pregnant (OPEN; n=9) were used in a replicated randomize complete block design. Cows were
blocked (n=3) by date of parturition such that each block was slaughtered 4-5 wk prior to expected
parturition (approximately 250 d of gestation). Each block of the three blocks contained two cows
per treatment, with the exception of block three in the second replicate, which contained one cow
RQHDFKWUHDWPHQW7KHVHFRQGUHSOLFDWHFRQWDLQHGRQO\EORFNVDQGWKH¿UVWUHSOLFDWHFRQWDLQHG
FRZVDQGWKHVHFRQGUHSOLFDWHFRQWDLQHGFRZV7KH¿UVWEORFNZDVVODXJKWHUHGDWGRIWKH
trial and each block every 7 d thereafter.

Cows were individually fed a ration containing high grass haylage and 30% wheat straw for ad libitum
LQWDNH &3'0 1')'0 1(P 0FDONJ'015& ,QGLYLGXDOIHHG
intake was measured using Calan gates. Cows were weighed, ultrasounded for rib (over the 12th and
13thULE DQGUXPSIDWDQGDVHUXPVDPSOHREWDLQHGWKUHHWR¿YHGSULRUWRVODXJKWHU$WVODXJKWHU
organs were removed, trimmed of fat and weighed. Serum was analyzed for beta-hydroxybutyrate
%+%$ QRQHVWHUL¿HGIDWW\DFLGV 1()$ JOXFRVHXUHDWRWDOFKROHVWHURODQGWULLRGRWK\URQLQH 7 

Data were analyzed using Proc Mixed (SAS, 2008) as a replicated randomized complete block with
SUHJQDQF\FRZDJHDQGEORFNQHVWHGZLWKLQUHSOLFDWHDV¿[HGHIIHFWVDQGVLJQL¿FDQFHGHWHUPLQHG
at P”

Results and discussion

Liver (actual weights, relative to BW, and relative to HCW) were heavier (P”7DEOH LQ23(1
than in PREG cows. Rumen and kidney fat weight were also greater (P” LQ23(1WKDQLQ
PREG, but only when expressed relative to BW. As expected, uterus weight was greater (P” 
in PREG cows. Kidney, lung, heart, pancreas, spleen, lower gut, and total visceral fat weights
(actual, relative to BW or to HCW) were not different (P• EHWZHHQ35(*DQG23(1FRZV
Pre-slaughter PREG cows had greater BHBA, NEFA and urea concentrations (P” DQGORZHU
(3 0.04) total cholesterol concentration. These differences in liver mass and metabolites suggest that
pregnant cows alter their metabolism to meet the energetic demands of the growing fetus. However,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 465
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_174, © Wageningen Academic Publishers 2013
7DEOH9LVFHUDORUJDQDQGLQWHUQDOIDWPDVVLQSUHJQDQW 35(* DQGQRQSUHJQDQW 23(1 PDWXUH
beef cows.

Item OPEN PREG SEM P-value

Body weight pre-slaughter, kg 784 845  0.12

Liver, g 7,347  215.9 0.02
Liver/body weight, g/kg 9.42 7.82 0.201 <0.001
Liver/hot carcass weight, g/kg 18.53 15.97 0.457 0.002
Rumen, kg 15.5 13.9  0.08
Rumen/body weight, kg/kg 0.020 0.017 0.0009 0.01
Rumen/hot carcass weight, kg/kg 0.039 0.034 0.0019 
Kidney fat, g  10,812 1,077.4 0.13
Kidney fat/body weight, g/kg  12.73 1.231 0.04
Kidney fat/hot carcass weight, g/kg 32.47 25.99  

the following measurements did not differ between PREG and OPEN: pre-slaughter BW (3 0.12);
average daily gain (3 0.2); pre-slaughter ultrasound measures of rib or rump fat (P• DQGKRW
carcass weight or measured back fat (P• 'U\PDWWHULQWDNHGLGQRWGLIIHU 3 0.20) and averaged
NJ'0GIRU23(1DQGNJ'0GIRU35(* 6(0  

These data indicate that PREG cows may metabolize energy reserves and alter their metabolism in
order to meet the energetic demands of the growing fetus, without affecting their own growth. More
research is needed to determine the underlying metabolic processes involved in these differences
in circulating metabolites and liver, rumen and kidney fat mass and if these changes may lead to
XQGHUO\LQJGLIIHUHQFHVLQIHHGHI¿FLHQF\

References
Freetly, H.C., J.A. Nienaber and T. Brown-Brandl, 2008. Partitioning of energy in pregnant beef cows during nutritionally
LQGXFHGERG\ZHLJKWÀXFWXDWLRQ-$QLP6FL
McBride, B.W. and J.M. Kelly, 1990. Energy cost of absorption and metabolism in the ruminant gastrointestinal tract
DQGOLYHUDUHYLHZ-$QLP6FL
1DWLRQDO5HVHDUFK&RXQFLO1XWULHQW5HTXLUHPHQWVRIEHHIFDWWOH1DWLRQDO$FDGHP\
Press, Washington, DC.
Reynolds, C.K., H.F. Tyrrell and P.J. Reynolds, 1991. Effects of diet forage-to-concentrate ratio and intake on energy
metabolism in growing beef heifers: whole body energy and nitrogen balance and visceral heat production. J.
Nutr. 121, 944-1003.
SAS, 2008. Release 9.1.3. SAS Inst. Inc. Cary, NC, USA.

466 Energy and protein metabolism and nutrition in sustainable animal production
Skeletal muscle fatty acid oxidation in lactating dairy cows during early
lactation
C. Schäff, H.M. Hammon, M. Röntgen and B. Kuhla
,QVWLWXWHRI1XWULWLRQDO3K\VLRORJ\µ2VNDU.HOOQHU¶/HLEQL],QVWLWXWHIRU)DUP$QLPDO%LRORJ\ )%1 
'XPPHUVWRUI*HUPDQ\[email protected]

Introduction
7KHPRELOL]DWLRQRIDGLSRVHWLVVXHRIGDLU\FRZVGXULQJHDUO\ODFWDWLRQLVUHÀHFWHGE\LQFUHDVHGIDWW\
DFLG )$ SODVPDFRQFHQWUDWLRQV7KHFDSDFLW\RIWKHOLYHUIRUFRPSOHWHR[LGDWLRQRIQRQHVWHUL¿HG
)$ 1()$ LVOLPLWHGOHDGLQJWRDQLQFUHDVHGIRUPDWLRQRINHWRQHERGLHVUHHVWHUL¿FDWLRQDQG
accumulation of triacylglycerides in the liver. This is accompanied by a lower feed intake and a high
incidence of metabolic disorders (Drackley et al., 2005). The skeletal muscle may also oxidize FA
and thus relieve the liver from FA load. Here we hypothesized that skeletal muscle FA degradation
adapts to the extent of fat mobilization during early lactation.

Material and methods

German Holstein cows (2nd to 4th lactation, >10,000 kg/305 d in a previous lactation, n=19) were
fed twice daily a total mixed ration according to their physiological state. From 3 weeks before until
5 weeks after parturition weekly blood samples were taken for the photometric analysis of total
plasma NEFA and glucose concentrations (Abx Pentra 400; Horiba), and the determination of the
SODVPD)$SUR¿OHDQGPHWK\OKLVWLGLQH 0+ FRQFHQWUDWLRQE\+3/& *&DQG*&06
436KLPDG]X $VWKHH[WHQWRIIDWPRELOL]DWLRQLVUHÀHFWHGE\WKHOLYHUIDWFRQWHQW /)& 
liver biopsies were obtained on days 3, 18, 30 post-partum (PP). Based on the mean LFC, cows were
grouped into the ten highest (H) and nine lowest (L) mobilizing cows. Muscle biopsies were obtained
by shoot biopsy on days -17, 3, 30 relative to calving and used to quantify the phosphorylation of
AMP-activated protein kinase (pAMPK) by ELISA (ELISA DuoSet IC; R&D Systems) and the
mRNA expressions by quantitative real-time reverse transcriptase PCR (qRT-PCR). Total RNA was
extracted with Trizol Reagent. Integrity and quality were proofed by gel electrophoresis and on a
NanoPhotometer. RNA was reverse-transcribed and the obtained cDNA applied to qRT-PCR carried
out on a LightCycler 2.0 (Roche) for the following genes: peroxisome proliferator-activated receptor
IDPLO\ 33$5ĮȖįDQG33$5ȖFRDFWLYDWRUĮ 3*&Į FDUQLWLQHSDOPLWR\OWUDQVIHUDVHĮDQGȕ
&37Įȕ WKHȕR[LGDWLYHHQ]\PHVK\GUR[\DF\O&R$GHK\GURJHQDVH +$'+ YHU\ORQJFKDLQ
acyl-CoA dehydrogenase (ACADVL), 3-ketoacyl-CoA thiolase (ACAA2), and uncoupling protein
 8&3 P51$H[SUHVVLRQVZDVTXDQWL¿HGDFFRUGLQJWRWKHǻǻ&7 method using splicing factor
3 subunit 1 (SF3A1) as housekeeping gene. Back fat thickness (BFT) and m. longissimus dorsi
thickness (MLDT) were determined once a week. All data were analyzed by repeated measures
ANOVA using the mixed procedure of SAS (SAS/STAT software in the SAS System for Windows,
UHOHDVH ZLWKJURXSWLPHDQGJURXSîWLPHDV¿[HGHIIHFWV/HDVWVTXDUHPHDQVZHUHFDOFXODWHG
and presented with their standard error.

Results and discussion

BFT and MLDT were higher in H cows (Pgroup<0.05) and decreased PP as expected (Ptime<0.001).
Total NEFA concentration peaked in the third week PP (Ptime<0.001) and was higher in H than in
L cows (Pgroup<0.001). The ratio between unsaturated and saturated FAs was higher in H than in
L cows (Pgroup<0.01) and continuously increased over time (Ptime<0.001). Also 3-MH, considered
as a marker of muscle breakdown, peaked around calving (Ptime<0.001). Group × time interactions
were not observed for the variables analyzed. These results indicate that the mobilization of adipose
and muscle tissue PP is more pronounced in H as compared to L cows.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 467
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_175, © Wageningen Academic Publishers 2013
The activation (phosphorylation) of the energy sensor AMPK in muscle tissue was not different
EHWZHHQJURXSVDQGGLGDOVRQRWFKDQJHRYHUWLPH7KLV¿QGLQJLQGLFDWHVWKDWLQHDUO\ODFWDWLRQWKH
PXVFOHLVVXI¿FLHQWO\VXSSOLHGZLWK$73HQHUJ\GHVSLWHUHGXFHGSODVPDJOXFRVHFRQFHQWUDWLRQV
(Ptime<0.001) and muscle tissue degradation. To be able to maintain cellular energy homeostasis,
muscle tissue seems to switch from glucose to FA utilization in early lactation.

Long-chain FAs are transported into the mitochondria where they are oxidized. Several key regulators
DUHLQYROYHGLQWKHVHSURFHVVHVDQGZHUHDQDO\]HGIRUWKHLUP51$H[SUHVVLRQ&37ĮDQG&37ȕ
which facilitate the entrance of FAs from the cytosol into the mitochondria, were upregulated in early
ODFWDWLRQZLWK&37ȕEHLQJKLJKHVWRQG33DQG&37ĮRQG33 Ptime<0.001). The expression
of PPARs, which comprise important nuclear FA receptors and transcription factors regulating FA
oxidation, changed during the periparturient period (Ptime<0.05). In detail, we found increased
H[SUHVVLRQRI33$5ȖLQHDUO\ODFWDWLRQ Ptime<0.05) being higher in H than in L cows (Pgroup<0.05).
,QFRQWUDVWH[SUHVVLRQRI33$5ĮFRQWLQXRXVO\GHFUHDVHGZLWKWLPH Ptime 33$5į WKH
predominant form in muscle tissue) was highest expressed on day 3 PP and revealed an interaction
(Ptime×group<0.05). UCP3, which signals for the transport of FAs into and out of mitochondria and
so promotes fat oxidation in cattle (Brennan et al., 2009) was also increasingly expressed at day
3 PP and showed an interaction (Ptime×group ,QFRQWUDVW3*&ĮNQRZQWREHDFRDFWLYDWRU
RI33$5įDQGIRUWUDQVFULSWLRQRI8&3DQG&37ȕ 'H/DQJHet al., 2007), dropped markedly
after calving (Ptime 2QWKHRWKHUKDQGHQ]\PHVLQYROYHGLQȕR[LGDWLRQQDPHO\+$'+
(Ptime<0.07), ACAA2, and ACADVL (each Ptime<0.05) were highest expressed on day 3 PP.

Our results indicate that around calving transport and oxidation of FAs into muscle cells are highly
DFWLYDWHGZKLFKPD\RFFXUDVDGDSWDWLRQWRWKHRQVHWRIIDWPRELOL]DWLRQLQHDUO\ODFWDWLRQ33$5į¶V
SURPRWLQJHIIHFWRQ)$R[LGDWLRQLVOLNHO\QRWGHSHQGHQWRQWKHLQWHUDFWLRQZLWK3*&Į:LWKLQWKH
¿UVWZHHNVRIODFWDWLRQKRZHYHUPXVFXODU)$R[LGDWLRQGHFUHDVHVWRWKHDQWHSDUWXPOHYHODOWKRXJK
plasma NEFA concentrations are still elevated. Hence it seems likely that muscular FA oxidation is
generally more dependent on the stage of lactation than on the extent of fat mobilization. The latter
DVVXPSWLRQLVVXSSRUWHGE\WKH¿QGLQJWKDWRQO\PLQRUP51$H[SUHVVLRQGLIIHUHQFHVZHUHREVHUYHG
between groups. Consequently, FA oxidation in skeletal muscle may relieve the liver from too high
fat load already in the beginning of lactation but becomes less important in week 5 of lactation.

Acknowledgements
Supported by DFG, Germany

References
Brennan, K.M., J.J. Michal, J.J. Ramsey and K.A. Johnson, 2009. Body weight loss in beef cows: I. The effect of
increased beta-oxidation on messenger ribonucleic acid levels of uncoupling proteins two and three and peroxisome
SUROLIHUDWRUDFWLYDWHGUHFHSWRULQVNHOHWDOPXVFOH-$QLP6FL
Drackley, J.K., H.M. Dann, G.N. Douglas, N.A. Janovick-Guretzky, N.B. Litherland, J.P. Underwood and J.J. Loor,
2005. Physiological and pathological adaptations in dairy cows that may increase susceptibility to periparturient
diseases and disorders. Ital. J. Anim. Sci. 4, 323-344.
De Lange, P., M. Moreno, E. Silvestri, A. Lombardi, F. Goglia and A. Lanni, 2007. Fuel economy in food-deprived
skeletal muscle: signaling pathways and regulatory mechanisms. FASEB J. 21, 3431-3441.

468 Energy and protein metabolism and nutrition in sustainable animal production
7UDQVFULSWRPHSUR¿OHFRPSDULVRQEHWZHHQEHHIDQGGDLU\DGLSRVH
pooled mRNA reveals differences
J.M. Thomson1,2, P. Stothard2 and J.P. McNamara
10RQWDQD6WDWH8QLYHUVLW\$QLPDODQG5DQJH6FLHQFHV32%R[%R]HPDQ07
USA; [email protected]
28QLYHUVLW\RI$OEHUWD'HSDUWPHQWRI$JULFXOWXUH)RRGDG1XWULWLRQDO6FLHQFHV&ROOHJH
3OD]D(GPRQWRQ$%7*3&DQDGD
:DVKLQJWRQ6WDWH8QLYHUVLW\'HSDUWPHQWRI$QLPDO6FLHQFHV&ODUN+DOO3XOOPDQ:$86$

Introduction
Recent research has found evidence of selection in Bos taurus when compared to wild relatives
from the Bovina subtribe. The Bovina subtribe is thought to have diverged from B. taurus between
one and two million years ago (MacEachern et al., 2009). This research was also able to show large
deviations from neutrality in two distinct breeds of cattle: the Holstein which has been subjected to
DUWL¿FLDOVHOHFWLRQSULPDULO\RQWKHEDVLVRIPLONSURGXFWLRQDQG$QJXVDFRPPHUFLDOEHHIEUHHG
which has had broader selection on traits related to growth rate, meat quality, and suitability for a
more extensive production system (MacEachern et al., 2009; Hayes et al., 2008).

The uptake of next generation sequencing (NGS) technologies has expanded our ability to examine
the gene expression and transcriptome differences between these breeds. This technology allows a
EURDGHUORRNDWJHQHH[SUHVVLRQWKDQLWVSUHGHFHVVRUPLFURDUUD\VDVWKHQXPEHUDQGVSHFL¿FLW\RIWKH
probes on the array do not limit it. It provides a powerful tool to examine tissue characteristics and
improve our annotation and understanding of the relationship of gene expression and tissue function
(Harhay et al., 2010). Bioinformatic approaches have been developed that allow for comprehensive
functional analysis of the large gene lists generated by RNA-seq methods including gene set
enrichment analysis (GSEA) and modular enrichments analysis (MEA) (Huang et al., 2008). The
aim of the present experiment was to compare the functional transcriptome of beef and dairy cattle
adipose tissue in order to better understand the role of the tissue in the divergently selected breeds.

Material and methods

The RNA was isolated from adipose tissue samples from 8 North American Holstein-Friesian animals;
four mature lactating animals and 4 growing heifers and 10 crossbred beef animals. The crossbred
beef animals were primarily of Hereford and Angus genetics. The RNA was pooled within breed if
it had an RNA integrity number greater than 7.5. This was done to produce a single representative
sample and to reduce the background variation due to animal-to-animal genetic difference. The
pooled RNA was enriched for mRNA using poly-T oligo-attached magnetic beads and then cDNA
was synthesized. The cDNA was fragmented and sequencing adapters were added. The cDNA
libraries were sequenced according to manufacturer recommendations. The cDNA libraries from the
crossbred beef animals were sequenced on a Genome Anlyzer II (Illumina) and the adipose from the
Holstein animals was sequenced on a SOLiD 4 platform (Applied Biosystems). Even though two
1*6SODWIRUPVZHUHXVHGZHZHUHFRQ¿GHQWLQWKHFRPSDULVRQEHFDXVHRIWKHKLJKUHSHDWDELOLW\RI
RNA-seq experiments across platforms (Jiang et al., 2011; Nookaew et al., 2012) when a minimum
RIPLOOLRQPDSSHGUHDGVLVXVHG:HJHQHUDWHGPLOOLRQPDSSHGUHDGVIURPGDLU\DGLSRVHDQG
million mapped reads from beef adipose. The reads were assembled and normalized expression values
calculated using the Genesifter NGS pipeline (Geospiza). Both Genesifter (Geospiza) and DAVID
ELRLQIRUPDWLFVVRIWZDUHYHUVLRQZHUHXVHGWRSHUIRUPJHQHRQWRORJ\DQGSDWKZD\HQULFKPHQW
analyses. We used a Fisher’s Exact Test and a Bonferroni adjustment for multiple comparisons to
GHWHUPLQHGLIIHUHQWLDOH[SUHVVLRQ:HWKHQ¿OWHUHGRXUGDWDEDVHGRQDQDGMXVWHGP-value of <0.01
and at lease a 2 fold difference between breeds.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 469
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_176, © Wageningen Academic Publishers 2013
Results and discussion
:HIRXQGWUDQVFULSWVWKDWZHUHGLIIHUHQWLDOO\H[SUHVVHGLQWKHDGLSRVHWLVVXHRIWKHWZREUHHGV
Some of the most highly expressed genes in the Holstein adipose tissue included fatty acid binding
SURWHLQ )$%3 F\WRFKURPHFR[LGDVH &2; DQG$73V\QWKDVHVXEXQLW $73 )DWW\DFLG
binding protein 4 (FABP4) was also high in beef cattle but other highly expressed genes were not
shared including osteonectin (SPARC), and vimentin (VIM) and matrix Gla protein (MGP) which is
a growth inhibitor. In general beef cattle had enriched biosynthetic pathways when compared to dairy
and increased expression of genes related to chemokine signaling. Dairy cattle showed enrichment
of pathways related to oxidative phosphorylation and cellular remodeling when compared to beef
cattle adipose tissue.

This provides evidence that genetic selection can alter gene expression, tissue metabolism and
IXQFWLRQDQGPD\SURYLGHLQVLJKWLQWRKRZZHFDQLPSURYHJHQHWLFVHOHFWLRQHI¿FLHQF\

References
Harhay, G. P., T.P. Smith, L.J. Alexander, C.D. Haudeenschild, J.W. Keele, L.K. Matukumalli, S.G. Schroeder, C.P. Van
Tassell, C.R. Gresham, S.M. Bridges, S.C. Burgess and T.S. Sonstegard, 2010. An atlas of bovine gene expression
reveals novel distinctive tissue characteristics and evidence for improving genome annotation. Genome Biology
11:R102.
Hayes, B. J., A.J. Chamberlain, S. MacEachern, K. Savin, H. McPartlan, I. MacLeod, L. Sethuraman and M. Goddard,
$JHQRPHPDSRIGLYHUJHQWDUWL¿FLDOVHOHFWLRQEHWZHHQBos Taurus dairy cattle and Bos Taurus beef cattle.
$QLPDO*HQHWLFV
Huang, D.W., B.T. Sherman and R.A. Lempicki, 2008. Bioinformatics enrichment tools: paths toward the comprehensive
functional analysis of large gene lists. Nuc Acids Res 37:1-13.
Jiang, L., F. Schlesinger, C.A. Davis, Y. Zhang, R. Li, M. Dalit, T.R. Gingeras and B. Oliver. 2011. Synthetic spike-in
standards for RNA-seq experiments. Genome Res 21:1543-1551.
MacEachern, S., B. Hayes, J. McEwan and M. Goddard, 2009. An examination of positive selection and changing
effective population size in Angus and Holstein cattle populations using a high density SNP genotyping platform and
the contribution of ancient cattle polymorphism to genomic diversity in Domestic cattle. BMC Genomics10:181.
Nookaew, I., M. Papini, N. Pornputtapong, G. Scalcinati, L. Fagerberg, M. Uhlen and J. Neilsen, 2012. A comprehensive
comparison of RNA-seq based transcriptome analysis from reads to differential gene expression and cross-
comparison with microarrays. Nuc Acid Res 40:10084-10097.

470 Energy and protein metabolism and nutrition in sustainable animal production
Effects of omitting the dry period on plasma progesterone and prolactin
during lactogenesis and on colostrum IgG content in dairy cows during
the periparturient period
H.A. van Dorland1,2, R.S. Zbinden1, G. Remmelin, B. Kemp, A.T.M. van Knegsel and R.M. Bruckmaier1
1Veterinary Physiology, Vetsuisse Faculty University of Bern, Bern, Switzerland;
[email protected]
2Present address: School of Agricultural, Forest and Food Sciences, Bern University of Applied
Sciences, Zollikofen, Switzerland
Livestock Research, Wageningen University and Research Centre, Lelystad, the Netherlands
Adaptation Physiology Group, Wageningen University, Wageningen, the Netherlands

Introduction
Omitting the dry period represents a strategy that may reduce the metabolic stress in early lactating
cows (e.g. Andersen et al., 2005). A drawback of continuous milking with no dry period might
compromise colostrum quality in goats (Caja et al. KRZHYHULQVXI¿FLHQWVWXGLHVKDYHEHHQ
FDUULHGRXWWRFRQ¿UPWKLVLQGDLU\FRZV

The objective of this study was to evaluate the effects of omitting the dry period on key hormone
patterns during lactogenesis and the IgG content of colostrum in periparturient dairy cows.

Material and methods

Twenty Holstein-Friesian dairy cows were randomly assigned to two experimental groups of which
one group was managed without a dry period (0 d dry period; DRY0), and the other group was
PDQDJHGZLWKDGU\SHULRGRIGD\V '5< 3UHSDUWXPGU\FRZVUHFHLYHGDGU\FRZUDWLRQ
lactating cows received a lactation ration supporting 25 kg of milk. Postpartum, all cows received
a lactation ration. Dry cow ration consisted of grass silage, corn silage, wheat straw, rapeseed meal
DQGFRQFHQWUDWHLQDUDWLRRI '0EDVLV /DFWDWLQJUDWLRQFRQVLVWHGRIJUDVVVLODJH
FRUQVLODJHUDSHVHHGVWUDZVR\EHDQPHDODQGFRQFHQWUDWHLQDUDWLRRI'U\PDWWHU
intake was recorded daily between day -7 and day 3 relative to calving date. Milk yields were
recorded daily, and milk samples were collected daily for analysis of IgG concentration from day
7 pre- until day 3 postpartum for DRY0, and from day 0 until day 3 postpartumIRU'5<FRZV
Furthermore, from day 5 pre- until day 3 postpartum, blood was sampled daily from the coccygeal
vein and plasma concentrations of progesterone and prolactin were determined.

Data were analyzed with the MIXED procedure of SAS (SAS Institute, 2001), including treatment
GU\SHULRGOHQJWK VDPSOLQJWLPHSRLQWDQGWKHLULQWHUDFWLRQDV¿[HGHIIHFWV7KHVDPSOLQJWLPH
points were treated as repeated factor within subjects (animals).

Results and discussion

Dry matter intake did not differ (Ptreatment! EHWZHHQ'5<DQG'5<FRZV “YV
13.9±0.5 kg, resp.). Daily milk production between day 0 and day 3 post-calving averaged 22.3±1.9
NJLQ'5<FRZVDQG“NJLQ'5<FRZV Ptreatment<0.05).

3ODVPDSURODFWLQFRQFHQWUDWLRQGLGQRWGLIIHUEHWZHHQ'5<DQG'5<FRZVDQGIROORZHGD
similar pattern (Ptreatment×day! VWDUWLQJWRLQFUHDVHVLJQL¿FDQWO\IURPRQHGD\EHIRUHFDOYLQJ
in both groups (Pday<0.05; Figure 1A). Progesterone dropped prepartum (Pday<0.05) and followed
DVLPLODUSDWWHUQLQERWKJURXSVEXWZDVVLJQL¿FDQWO\ORZHURQRQHGD\EHIRUHFDOYLQJLQ'5<
compared to DRY0 (Figure 1B).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 471
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_177, © Wageningen Academic Publishers 2013
)LJXUH&RQFHQWUDWLRQVRISURODFWLQ $ DQGSURJHVWHURQH % LQFRZVZLWKRXWGU\SHULRG 
'5< DQGLQFRZVZLWKDGU\SHULRGRIGD\V Ɣ'5< $ UHSUHVHQWVDGLIIHUHQFHEHWZHHQ
'5<DQG'5< P< 

7KHVHREVHUYDWLRQVVKRZWKDWWKHHQGRFULQHSUR¿OHVRIKRUPRQHVVXSSRUWLQJODFWRJHQHVLVUHPDLQHG
ODUJHO\XQDIIHFWHGE\WKHRPLVVLRQRIWKHGU\SHULRG7KHRQO\VLJQL¿FDQWGLIIHUHQFHEHWZHHQ
'5<DQG'5<IRUSURJHVWHURQHRQHGD\EHIRUHFDOYLQJPD\SRLQWRXWWRDVWURQJHUGHFUHDVH
in progesterone concentration for DRY0 and could also imply a faster calving process after the
progesterone drop.

With regard to IgG concentration, IgG level decreased in both groups from calving up to day 3
postpartumDQGQRGLIIHUHQFHZDVREVHUYHGEHWZHHQWKH'5<DQG'5<FRZV,QFRQWUDVW
FDOFXODWHGWRWDO,J*PDVVLQ¿UVWFRORVWUXPZDVKLJKHU Ptreatment IRU'5<FRPSDUHGWR
'5<FRZV “JYV“J 

'5<FRZVFRPSDUHGWR'5<FRZVKDGDORZHUPLON\LHOG Ptreatment DWWKH¿UVWpostpartum

milking (13.5±2.0 kg vs. 21.8±1.7 kg). The continuous milking prevented accumulation of IgG mass
in the mammary gland in DRY0 cows, which was compensated by less dilution through less milk
synthesis. Therefore, the supply of colostrum to the newborn calf was not compromised in DRY0
FRPSDUHGWR'5<FRZVDVWKHQHZERUQFDOIZRXOGLQJHVWRQO\DVPDOODPRXQWRIWKHWRWDO¿UVW
FRORVWUXP JHQHUDOO\UHTXLUHPHQWLVNJRI¿UVWFRORVWUXP DQG,J*FRQFHQWUDWLRQLQPLONRQGD\
1 was similar in both groups.

References
Andersen, J.B., T.G. Madsen, T. Larsen, K.L. Ingvartsen and M.O. Nielsen, 2005. The effects of dry period versus
continuous lactation on metabolic status and performance in periparturient cows. J. Dairy Sci. 88, 3530-3541.
&DMD*$$.6DODPDDQG;6XFK2PLWWLQJWKHGU\RIISHULRGQHJDWLYHO\DIIHFWVFRORVWUXPDQGPLON\LHOG
in dairy goats. J. Dairy Sci. 89, 4220-4228.
SAS Institute. 2001. SAS User’s Guide: Statistics. Version 8 ed. SAS Inst. Inc., Cary, NC.

472 Energy and protein metabolism and nutrition in sustainable animal production
Part 8. Environmental sustainability
The contribution of animal production to agricultural sustainability
M. Doreau1, H.P.S. Makkar2 and P. Lecomte
1,15$805+HUELYRUHV6DLQW*HQqV&KDPSDQHOOH)UDQFH[email protected]
2Animal Production and Health Division, FAO, Rome, Italy
&,5$'8056(/0(70RQWSHOOLHU)UDQFH

Abstract
Due to the increasing demand for animal products for a growing world population, use of a large
amount of feed ingredients in competition with human food coupled with the urgent need for
mitigating environmental effects, livestock farming has to transform. New models of sustainability,
that are acceptable to all stakeholders, must be explored. Reducing negative environmental impacts
of various agricultural practices is a major global challenge. However, to avoid making inappropriate
decisions, sustainability should be understood and addressed by combining indicators that are
relevant at farm, country and world levels and not solely based on the emission of greenhouse
JDVHV6XVWDLQDELOLW\KDVWRJREH\RQGSURGXFWLYHHI¿FLHQF\HPEUDFLQJHFRHI¿FLHQF\FRQFHSWV
and including social equity, and ethical dimensions of development. There is a large diversity of
systems, from low-productive or extensive to high-productive or intensive, and countries are at
different stages of development, from developing, fast developing to developed. Hence solutions
are likely to be very different. Co-existence of different livestock production systems and addressing
their sustainability using multiple indicators is a key to success.

Introduction
Livestock farming enters a paradoxical situation. In a changing context, marked by escalating costs
of inputs, decreasing natural resources and ongoing climate change, livestock production systems
have to transform in the short- to medium-term. For the expected 9 billion people by 2050 the world
will need to produce much more food with the same agricultural area, or with a smaller area, if
the ongoing soil degradation is not abated and/or biofuel production keeps growing. Increases in
income and urbanization in emerging countries and resulting changes in food habits lead to inclusion
of a higher amount of animal-source foods (meat, milk, eggs) in the diet of humans (FAO, 2009).
7UDQVIRUPDWLRQRIELRPDVVLQWRHGLEOHDQLPDOIRRGVKDVDORZHUHI¿FLHQF\WKDQGLUHFWYHJHWDOIRRG
production. The former requires more natural resources per MJ of energy or kg of protein yield, and
thus leads to higher emissions. The desire to protect environment and biodiversity, and to improve
animal well-being are raising new questions regarding the manner by which animal foods are
being produced or will be produced. For example, how can the increase of the current production
be considered while reducing environmental effects and maintaining equity in the recognition and
sharing of economical and social impacts of the goods and services produced through livestock?
How can animal agriculture be made a driver for agricultural sustainability? In the future, countries
and societies will prioritize objectives of producing livestock differently, depending on factors such
as income levels, role of smallholders relative to large scale producers, importance of and prospects
for import or export, degree of pressure on, and degradation of, natural resources, and social and
ethical concerns. The objectives will be prioritized differently according to the country’s stage of
economic development. Addressing these divergent goals is a challenge for the next decades.

Livestock systems, which are very diverse throughout the world (Robinson et al., 2011), should
WDNHEHWWHULQWRDFFRXQWWKHHI¿FLHQF\ZLWKZKLFKQDWXUDOUHVRXUFHVFDUERQQLWURJHQDQGHQHUJ\
as well as human and animal resources are used to produce goods and provide multiple functions
in diverse landscapes. There is currently a major societal debate on both positive and negative
impacts of livestock production and their future capability to provide ecosystem services. The
differentiated role of intensive, mixed crop-livestock and extensive or low-input systems, their

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 475
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_178, © Wageningen Academic Publishers 2013
productive performances in context to their natural, renewable or non-renewable resource use, social
contributions and environmental impacts, are often controversial and challenging subjects. Across
the world, the diverse agro-ecological contexts present a wide spectrum of assets and constraints,
resulting in geographical diversity of livestock farming systems performing multiple functions in
the territories where they developed. Sustainability indicators for such diverse systems should be
broadened and balanced, to go beyond traditional assessments of productive performance.

6XVWDLQDELOLW\LQDJULFXOWXUH±DFRQFHSWGLI¿FXOWWRDUULYHDWDFRQVHQVXV
7KHFODVVLFDOGH¿QLWLRQRIVXVWDLQDELOLW\GHPDQGVEDODQFHDPRQJVWWKUHHSLOODUVFRUUHVSRQGLQJWR
environmental, economic and social issues. Criteria for their assessment are numerous, and there
is no consensus on their applicability. Environmental issues include the use of natural resources
and its impacts on the environment. An obvious natural resource capital is land, the use of which
for agriculture is increasingly becoming limiting, despite a recent and extensive deforestation of
primary forest and the presence of still large uncultivated areas, for example in Siberia. In addition,
on arable land there is an increasing competition between the production of human food, animal
feeds and biofuels. Other natural resources used by agriculture are either non-renewable (e.g. fossil
energy or phosphorus) or renewable but vulnerable (e.g. freshwater). In the latter case, a shortage
has consequences on global ecosystems and human activities. Environmental impacts may be global,
e.g. greenhouse gases (GHG) are emitted in the world’s atmosphere, wherever they are produced;
however, other impacts are restricted to a small territory or a watershed, e.g. soil and water pollutants.
Social issues do not have the same meaning in developed and developing countries, in rural and urban
societies. Lebacq et al. (2013) distinguished social issues based on farm-targeted criteria (working
conditions, education, way of life) and society-targeted criteria (multifunctionality, acceptability of
DJULFXOWXUDOSUDFWLFHVTXDOLW\RISURGXFWV $FRPPRQSRLQWWRDOOVRFLDOLVVXHVLVWKHGLI¿FXOW\LQ
¿QGLQJDQDJUHHPHQWDPRQJVWVWDNHKROGHUVIRUWKHFULWHULDDQGWKHQJLYLQJWKHPUHODWLYHZHLJKWV
(FRQRPLFHYDOXDWLRQLVJHQHUDOO\GRQHLQWHUPVRISUR¿WDELOLW\DVKRUWWHUPFULWHULRQZKHUHDVLW
should also consider macro-economic changes and future agricultural policies, as well as resilience to
climatic and market hazards. In addition, the functional expression of income is extremely variable,
and can be calculated per unit of product, or per hectare on-farm, or on-farm plus off-farm, etc. with
GLIIHUHQWFRQFOXVLRQVIRUSUR¿WDELOLW\DFFRUGLQJWRWKHXQLWFKRVHQ

The mandatory need to feed 9 billion people requires addressing trade-offs between sustainability at
IDUPOHYHODQGDWJOREDOOHYHO7KHHI¿FLHQF\RISURGXFWLRQLVQRWJLYHQGXHFRQVLGHUDWLRQLQVRPH
production systems. For example, it is unclear whether low-input diets with a low animal productivity,
which may provide a good income to the farmer (e.g. organic farming), are sustainable at a large
scale for providing food to fast developing countries. It is also necessary to integrate natural changes,
such as the effect of climate change on agriculture (change in crop yield, in crop species) and the
adaptive capacity of animals which may help to cope with these changes. Breeds/species which were
the most adapted may be less adapted to different natural conditions in future.

Addressing the environmental issues in many developing and transition countries is important but
it does not divert focus from the main objective which is to increase the national production for
meeting the increasing food needs. The social importance of family households (farmers) becomes
low at the expense of urban society in many developed countries, whereas in the least developed
countries smallholders who represent a major proportion of the population play a central role. Most
societal issues related to environment, security and welfare are of higher importance for countries
with consumers having a high standard of living. Attaining well-being of the agricultural community
is aimed throughout the world but with large differences among countries due to differences in their
developmental stage. There is a need to go beyond the sustainability assessment of existing systems,
and to redesign systems for the long term by anticipating economic, social and environmental trade-
offs and shocks that will come from future development of the global production. Table 1 proposes

476 Energy and protein metabolism and nutrition in sustainable animal production
Table 1. Criteria for agricultural sustainability: their nature and importance differ according to the
OHYHOZKHUHWKH\DUHFRQVLGHUHG RQO\DVHOHFWLRQRIFULWHULDKDVEHHQPHQWLRQHG 

Environment Social Economic

Farm level Use of organic manure Human well-being 3UR¿W

Increasing livelihood Resilience
Cultural role of animals
Country or territory National GHG inventory Employment Import-export balance
level Soil and water pollution Animal welfare Food sovereignty
Use of water Food security
World level Use of natural resources Reduction in poverty Achieving production
Air pollution target
Land use, deforestation

a set of sustainability criteria to address issues at different levels, from local level (the farmer) to
world level (planet and mankind).

Animal production and the environment

$VLJQL¿FDQWSDUWRIWKHHQYLURQPHQWDOLPSDFWDQGLQWHQVLYHXVHRIQRQUHQHZDEOHUHVRXUFHVDUH
related to food production. The role of livestock in greenhouse gases (GHG) emissions, water and
soil pollution, loss of natural resources and of biodiversity, among others, has been stressed by
FAO (Steinfeld et al. $VIDUPDQLPDOVFRQVXPHSODQWVDQLPDOVRXUFHIRRGVKDYHDKLJKHU
HQYLURQPHQWDOLPSDFWWKDQYHJHWDOIRRGSHUNJRIHGLEOHSURGXFW,QDGGLWLRQDVLJQL¿FDQWSDUWRI
GHG emissions comes from enteric methane produced by ruminants. Comparing impacts based on
GHG emission per kg of product might not be appropriate, because of differences in dry matter,
energy and protein contents of products. So making comparison based on energy or protein yield is
expected to give a better insight. Nevertheless, Vieux et al. (2012) surprisingly showed that replacing
meat with fruit and vegetables on an iso-caloric basis did not decrease total GHG emissions. A
FRPSDULVRQRQDQHQHUJ\EDVLVGRHVQRWDFFRXQWIRUDPDMRUVSHFL¿FLW\RIDQLPDOIRRGVZKLFKLV
protein supply. The expression per kg of protein is especially useful to compare milk, eggs and meat
of different origin. It has thus been shown that all impacts (GHG, eutrophication, energy and land
use) per kg protein are higher for beef than for other animal products (Gill et al., 2010, De Vries
and de Boer, 2010). What is often questioned about livestock production is that the products derived
from 35% of arable land are consumed by animals largely as feeds. This land could have been used
for producing food for direct consumption by humans. It is a simplistic view because a large part of
these products are co-products and by-products which are human non-edible. Moreover livestock
and their feeds also provide a buffer in national and international markets, which can be drawn
upon in case of food shortages. In the previous world food crises of the last 4 decades 1974/75 and
RYHUDOOJUDLQVXSSOLHVWRDQLPDOVHFWRUIHOOVLJQL¿FDQWO\DWZRUOGOHYHO )$2 ,Q
addition, many geographical areas, such as mountains and rangelands, are covered by grass because
they are not suitable for crop production.

High water demand of livestock is often cited as a major environmental issue. A frequently cited
¿JXUHLVOLWHUVRIZDWHUQHHGHGWRSURGXFHRQHNJRIEHHI7KLVFDOFXODWLRQLQFOXGHVµJUHHQ¶
water, i.e. water need for producing plants which are consumed by animals. Plant water need
corresponds to evapotranspiration, which is positively related with crop/forage yield and rainfall.
As a consequence, extensive systems require the largest amounts of water, because they need large
land areas. Such a calculation is made independent of water scarcity: when including green water, the
water requirement for world annual beef production is higher than the world’s freshwater reserves

Energy and protein metabolism and nutrition in sustainable animal production 477
(Doreau et al., 2012). In order to determine the effective impact of livestock on water resources,
recently improved methodologies have been developed. They use a life cycle assessment (LCA)
approach, and consider only blue water, i.e. water consumed as liquid by the animals and on-farm
water use for servicing and irrigation to produce feeds. Some of the authors propose a weighted
value accounting for the risk of water reserve depletion (Kounina et al., 2013).

Animal production has also positive effects on the environment. In developed countries, it often
adds to the quality of landscape for urban people. In developing countries, an adequate manure
management restores soil fertility in crop-livestock systems, and in pastoral areas management of
grasslands is a unique way to use soil and to protect it against erosion (Blanfort et al., 2011). These
HQYLURQPHQWDOVHUYLFHVDUHRIWHQGLI¿FXOWWRWDNHLQWRDFFRXQWEHFDXVHWKHYDOXHRIWKHTXDOLW\RI
ODQGVFDSHVZLWKQDWXUDOJUDVVODQGVFRPSDUHGWRFHUHDO¿HOGVIRUH[DPSOHLVGLI¿FXOWWRTXDQWLI\
e.g. in monetary terms. Recently, in a LCA approach, two groups of authors (Nguyen et al., 2012b,
for French beef; Ripoll-Bosch et al., 2013, for Spanish lamb) made an economic allocation between
income from selling meat and from subsidies targeted on the use of low-fertilized grasslands from this
production, and on the multi-functionality of livestock. These initial attempts, though commendable,
need to be improved.

As a partial solution to the livestock-related environmental issues, rationalisation of consumption

of animal products is an option. This would consist of a decrease in animal product consumption
by people in developed countries and by high- and middle-income classes in urban areas in the
fast developing economies and an increase in consumption by those living in developing countries
particularly in Africa where the animal product consumption is very low. However, this will not
solve the problem, as consumption is expected to increase in developing countries, resulting in a
substantial increase in GHG emissions from the livestock sector in these countries, although the
GHG emission will stabilize in developed countries (FAO, 2009). The need for improving production
HI¿FLHQF\IRUH[DPSOHE\LPSURYLQJDQLPDOEUHHGLQJQXWULWLRQDQGKHDOWKFRXOGKHOSWROLPLWWKH
environmental impact of the livestock sector (Thornton, 2010).

Challenges in reducing environmental impacts from animal production

'XULQJWKHODVWGHFDGHPDQ\VWXGLHVDLPHGDW¿QGLQJZD\VWRUHGXFHHQYLURQPHQWDOLPSDFWVXVLQJ
different approaches, among them LCA being the most used, have been conducted. These studies
were either on partial systems (fattening bulls or pigs, animals on pasture) or complete systems (beef
herd in a whole year, total pig production for a whole year). Results were very diverse, because of
differences in the system boundaries used in the LCA methodology, in the origin of the data, in the
emission allocation methods and in the choice of emission/pollution factors. Large difference among
systems or countries exist (FAO, 2010 for milk; Basset-Mens and van der Werf, 2005 for pig), but
within a system application of an array of options requiring important changes in the system are
necessary to considerably decrease impacts (Nguyen et al., 2013 for beef). However, between-farm
comparisons show large differences for the same system (Doreau and Dollé, 2011 for GHG in dairy
IDUPV DFTXLVLWLRQRIJRRGHQYLURQPHQWDOSUDFWLFHVE\IDUPHUVLVD¿UVWVWHS7KHUHDUHDORWRISXEOLF
recommendations for decreasing emissions especially GHGs (UNFCCC, 2008) but they are often
unstructured and do not account for interactions between options and trade-offs between impacts.
A valuable tool for policy makers is the establishment of marginal abatement cost curves which
FRPELQHWKHHI¿FLHQF\RIRSWLRQVDQGWKHLUFRVWRQDORQJWHUPSHUVSHFWLYH 0RUDQet al., 2008).
+RZHYHU¿QGLQJWKHEHVWDYDLODEOHRSWLRQVPD\EHKDPSHUHGE\WKHODFNRIDFFXUDWHNQRZOHGJH
RIWHFKQLFDOHI¿FLHQF\DQGWKHGLI¿FXOW\WRSUHGLFWHFRQRPLFWUHQGVDQGSULFHVLQWKHIXWXUH2QFH
RSWLRQVDUHLGHQWL¿HGSROLF\PDNHUVKDYHWZRPDMRULQVWUXPHQWVDWWKHLUGLVSRVDOFKDQJHVLQWD[HV
DQGVXEVLGLHVVWUXFWXUHGLIIHULQJLQQDWXUHDQGHI¿FDF\ *HUEHUet al., 2010).

478 Energy and protein metabolism and nutrition in sustainable animal production
For a few years, many countries focused their efforts on reducing GHG emissions, which emerged
as an enormous challenge due to the on-going global warming and need for an immediate answer.
The use of non-renewable energy and of the phosphorus, or localized degradation of water quality
also required urgent actions. An intervention that reduces one impact may increase another one in
production systems; for example cattle fattening diets are ranked differently for GHG emissions,
eutrophication and energy use (Nguyen et al., 2012a). Organic farming leads to lower changes than
conventional farming in GHG emissions per kg milk, because the higher methane emission due to
lower cow productivity is compensated for by lower nitrous oxide and carbon dioxide emissions
GXHWRORZHULQSXWV2WKHULPSDFWVVXFKDVHXWURSKLFDWLRQDQGDFLGL¿FDWLRQSRWHQWLDORUHQHUJ\XVH
are generally lower for organic farming in ruminants (De Boer, 2003); differences are less marked
for pig meat (Basset-Mens and van der Werf, 2005) because crop yields and pig growth rates are
lower in organic farming.

When impacts are expressed per ha instead of per kg of animal product, comparison between
farming systems give widely different results. Impacts per ha are lower for grass-based extensive
systems including organic farming. This unit is useful for a territorial approach, when the objective
is for example to reduce pollution at a watershed level, or to reduce GHG in a national inventory.
However, these systems result in an increase in land use. At a country level, increasing land use by
livestock may be positive for local management, for example in mountain areas; but at world level
it may result in deforestation of primary forest due to the pressure on land for agricultural activities
if the global increase in food production is aimed at.

A largely debated issue is the extent of carbon sequestration by soils, especially pastures, which
contributes to reducing net emissions of GHG. Permanent grasslands and no-tillage practices are
HI¿FLHQWWHFKQLTXHV$FFRUGLQJWR,3&&  WKHLQFUHDVHLQFDUERQVHTXHVWUDWLRQE\VRLOVPD\
contribute to 89% of GHG mitigation from agriculture by 2030. Although this estimate appears
excessive, integrating C sequestration in GHG balances should be more frequent; to our knowledge
it has been done only for 2 years (e.g. Doreau et al., 2011) but the amount of carbon storage per
ha of grasslands that has been measured in these studies is extremely variable (from 0.12 to 2.5 T
C/ha per year); moreover when crops or leys replace permanent grasslands there is a C release. In
order to account for both existence of C sequestration and criticisms relative to uncertainty, it may
EHUHFRPPHQGHGWRSUHVHQW¿UVW*+*UDZHPLVVLRQVWKHQ*+*EDODQFHGXHWRODQGXVHDQGODQG
use change, which comprises the effect of C storage or release, and the effect of deforestation of
primary forest when soybean is used.

Most agricultural LCA are established from the cradle to the farm gate; environmental cost of product
processing, distribution, domestic use and waste recycling are seldom taken into account. These
processes increase environmental costs, especially energy use and GHG emissions; post-farm gate
GHG are 17-22% and 1-10% of the total in industrialized and developing countries, respectively
(FAO, 2010). Besides the calculation of impact per kg of produced foods, it is necessary to assess
post-production losses. Food waste may go beyond 30% of production for dairy products and meat,
especially in developed countries. Post-harvest grain losses have long been mentioned in developing
countries; losses in animal-source foods have been less intensively studied but may be lower due
to the importance of family or local consumption. For example in India the losses in animal-source
foods are less than 5% of the production (Nanda et al., 2012). A recent paper stresses the risk of
LQFUHDVHLQZDVWHLQIDVWGHYHORSLQJFRXQWULHVGXHWRWKHUDSLGLQFUHDVHLQIRRGSURGXFWLRQ 3DU¿WW
et al., 2010). In developing countries, the main reason for waste is the lack of infrastructure and the
low organizational level for trade and transport; while in developed countries, the marketing push of
the supermarkets leading to excessive purchases by customers, the inability to sell foods before the
expiry date by supermarkets, the level of demand by the consumer and non-consumption within the
shelf life of the products are major sources of retail and consumer food losses (Hodges et al., 2011).

Energy and protein metabolism and nutrition in sustainable animal production 479
Sustainable animal production in the future
Many studies show there is still underutilized potential to reduce the burden of the sector on the
environment, and strengthen the positive role that the livestock sector may have in mitigating
climate change, nutrient recycling, protection of biodiversity and the provision of other environment
VHUYLFHV6LJQL¿FDQWSURJUHVVFDQEHPDGHWKURXJKWKHGHYHORSPHQWRIUHJXODWRU\IUDPHZRUNV
and incentives for environmental services. For extensive systems, the payment of environmental
services such as carbon sequestration and biodiversity protection (for example through silvopastoral
systems; see Murgueitio et al., 2011) are good examples. It has potential for application in many
UHJLRQVRIWKHZRUOGHJ/DWLQ$PHULFDDQG$IULFD,QLQWHQVLYHV\VWHPVPRUHHI¿FLHQWXVHRILQSXWV
(water, nutrients, energy) through innovations in technologies and adaptation and improvement in
the existing practices and/or restoration of strong linkages between livestock and crops can lead to
REYLRXVHQYLURQPHQWDOJDLQV,QHFRQRPLFWHUPVLQD¿QLWHVSDFHZKHUHWKHUHLVVWURQJFRPSHWLWLRQ
between agricultural and non-agricultural uses of land as well as among the crops themselves for use
as human food, animal feed or industrial transformation processes, improving animal performance
UHPDLQVFHQWUDOWRSURGXFWLYHHI¿FLHQF\7KHLPSURYHPHQWLQVNLOOVNQRZOHGJHWHFKQLTXHVDQG
tools in the areas of animal genetics, feed and feeding, animal health and management and their use
ZLOOKHOSDFKLHYHWKHSURGXFWLYHHI¿FLHQF\UHTXLUHGWRPDLQWDLQWKHFRPSHWLWLYHQHVVRISURGXFWVDQG
industries and to meet multiple expectations from livestock production.

In fast developing countries, aside from the traditional agricultural sector, the mobilization of public
and especially private funds for land acquisition and realization of investments in structures of very
large size, may help to develop new farming systems and contribute in these countries to increase
SURGXFWLRQFDSDFLW\DQGHI¿FLHQF\7KHVHV\VWHPVDUHGLIIHUHQWIURPWKHPRGHOWKDWSUHYDLOHG
during the twentieth century in developed countries (EU, US, Canada, Japan, etc.) wherein the
farm modernization and the growth in the quantity produced was made through specialization of
family size farms. These development models will certainly be capable of competing, and will
also be complementary to the dynamics that are created across the regions and the quality niches
WKDWWKH\HDFKDGGUHVV+RZHYHULWLVOLNHO\WKDWGXHWRWKHLUVL]HZKLFKFRQFHQWUDWHVHIÀXHQWVLQD
VDPHSODFHDQGVRPHWLPHVWRWKHLU¿QDQFLDOREMHFWLYHVWKHVHLQGXVWU\OLNHHQWHUSULVHVRIWHQUDLVH
environmental questioning.

Beyond the economic, environmental and societal assessments of the situations and of the stakes that
will dictate the future, transforming knowledge and skills into practice, and using them to improve
WKHHI¿FLHQF\RIUHVRXUFHDQGODQGXVHIRUWKHVHFWRUJURZWKLVDPDMRUFKDOOHQJH7RDGGUHVVWKH
complexity of issues raised for making changes in the operating practices in the livestock sector, it is
necessary that all actors (from the primary resource producer to the consumer) get better connected.
The different sources of knowledge to which they have access should lead to the adoption of new
technologies for production as well as of new modes of consumption of animal products. This will
UHTXLUHDQHYHQJUHDWHUÀRZRINQRZOHGJHDQGLQQRYDWLRQVDPRQJDOOVWDNHKROGHUVZKHWKHUWKHVH
are in developed or developing countries.

Sustainability of low-productive livestock systems

For low productive livestock systems, a major criticism is the environmental impact that they
produce, especially when expressed as GHG emission per kg of product, milk or meat. The FAO
(2010) compared GHG emissions for milk production in different countries. When annual milk yield
is lower than 2,000 kg, GHG emissions per kg increase rapidly. Since the full genetic potential of
DQLPDOVLQWKH¿HOGLVQRWUHDOL]HG &XQQLQJKDP LQFUHDVHLQSURGXFWLYLW\E\LPSURYLQJDQLPDO
nutrition, management and health practices will decrease GHG emission per unit of animal product.
Also rearing a high genetic potential cow with 8,000 kg milk production per year in the areas that
now produce 2,000 kg or less per year is also not a realistic option due to lack of feed resources and

480 Energy and protein metabolism and nutrition in sustainable animal production
XQVXLWDEOHFOLPDWLFIDFWRUV&UHDWLQJDUWL¿FLDOFRQGLWLRQVUHTXLUHGWRH[SUHVVIXOOJHQHWLFSRWHQWLDORI
such high producing animals is not expected to be sustainable and increase the GHG emission per
NJPLONZKHQ*+*HPLVVLRQLQWKHSURFHVVHVUHTXLUHGWRFUHDWHWKHDUWL¿FLDOFRQGLWLRQVDUHWDNHQ
into account. It may also be noted that low-productivity systems could also be economically viable
due to low input costs. For smallholder livestock farmers in developing countries, a major pillar
of sustainability is the social one. Indeed, livestock have a cultural role. Some societies as Peul or
0DVDLFDQQRWEHGLVVRFLDWHGIURPFDWWOHKHUGV%H\RQGWKHVHVSHFL¿FFDVHVOLYHVWRFNIDUPLQJ RU
crop-livestock farming) is essential for the livelihood of many rural communities in Africa, Asia or
/DWLQ$PHULFD+HUGVDUHDFDSLWDOZKLFKPD\EXIIHUFDVKÀRZSUREOHPV$QLPDOJLIWDQGOHQGLQJ
are a mechanism of solidarity for very poor people, and/or they contribute to a social network
favoring interdependency between communities (Alary et al., 2012). In addition, livestock farming
by smallholders also contributes to food security at country level, in addition to providing valuable
minerals and vitamins to pregnant women and growing children. Owing to the lack of infrastructures
for food transport, meeting locally the physiological needs and maintaining social equilibrium are of
utmost importance, which animal husbandry offers to farmers. There is a need to better value these
social dimensions the livestock play for such communities and bring them into the holistic equation
of sustainability rather than labeling such systems unsustainable based on only GHG emission per
kg of animal product.

Many farmers have very low income, either because of poor management of their farm (for example
excessive inputs with regard to output) or because of unfavorable natural conditions, for example
mountains or poor soils which lead to low crop and/or forage yields. The question is why to maintain
OLYHVWRFNLQVXFKSODFHVZKHUHWKHSURGXFWLRQLVOHVVHI¿FLHQWFRPSDUHGZLWKWKDWLQEHWWHUQDWXUDO
conditions. From a social point of view, livestock maintains human presence, which would not
occur in case of uncultivated land or forest. Moreover these areas do not compete with crops for
use as human food or for biofuel production due to low productivity. The challenge is to improve
HFRQRPLFSUR¿WZKHQIDUPVDUHYXOQHUDEOHWRERWKFOLPDWLFDQGHFRQRPLFVKRFNVVXFKDVGURXJKW
DQGZKHQWKHUHLVGURSLQSURGXFWSULFH)RUUXPLQDQWVWKH¿UVWVROXWLRQLVWRWDNHDGYDQWDJHRI
the high adaptive capacity of the animals, by alternating undernutrition and refeeding periods to
decrease global feed cost (Blanc et al. %LRHFRQRPLFDOPRGHOLQJDOORZV¿QGLQJWKHPRVW
appropriate strategies; for examples for beef farms increasing purchased feeds and grass surface for
haymaking are proposed for a better herd resilience (Mosnier et al., 2010). Darnhofer et al. (2011)
UHFRPPHQGGLYHUVL¿FDWLRQRIIDUPDFWLYLWLHVLQRUGHUWRLPSURYHUHVLOLHQFHRIWKHZKROHIDUP6SHFL¿F
systems related to a territory sometimes may give an opportunity for producing niche products
that consumers are ready to pay more, owing to a higher sensory quality or a unique geographical
origin. Appropriately managed grazing land and supportive institutional and policy frameworks can
HQKDQFHSURGXFWLYLW\DQGOLYHOLKRRGVLQDGGLWLRQWRSURYLGLQJODUJHEHQH¿WVLQWKHIRUPRIFDUERQ
sequestration, protection of biodiversity and water services.

Increasing sustainability in intensive systems

Faced with the need to increase meat, milk and egg production, the development of intensive
systems is often recommended (Steinfeld et al. 7KHLQWHQVLYHPRGHOXVHGIRUVHYHUDOGHFDGHV
aimed to increase production per animal or per unit of surface. However, it has become necessary
WRLPSURYHWKHHQYLURQPHQWDOSHUIRUPDQFH VRPHWLPHVFDOOHGHFRHI¿FLHQF\ E\GHFUHDVLQJLQSXWV
while maintaining or only slightly reducing productivity. For milk production, this is possible, for
example, by reducing grass N fertilization and stocking rate (Basset-Mens et al., 2009), by improving
HQHUJ\XVHHI¿FLHQF\WKURXJKPHDQVRIQXWULWLRQRUJHQHWLF 5H\QROGVet al., 2011) or by drastically
decreasing protein supply to animals (Fanchone et al. 7KLVLVPRUHGLI¿FXOWIRUSLJDQG
especially poultry production, but the use of synthetic amino acids decreases soybean use and thus
environmental costs and land use change (Mosnier et al., 2011). There are other examples showing
WKDWWHFKQRORJ\RUELRWHFKQRORJLHVPD\KHOS+RZHYHUVRPHWLPHVHI¿FLHQWELRWHFKQRORJLFDO

Energy and protein metabolism and nutrition in sustainable animal production 481
solutions are not acceptable from an ethical point of view or customer perception. This is the case
for instance for phytase-producing transgenic pigs which help to reduce phosphorus losses, whereas
phytase from fungal or bacterial origin is widely used. Another way to reduce inputs is to improve
animal fertility and health. Unfortunately, animals with higher productivity have a higher sensitivity
to various diseases; and in cows, there is a negative relation between animal productivity and fertility.
These undesirable effects are due to the past genetic selection which aimed at only increasing yield.
*HQHWLFVHOHFWLRQXVLQJRWKHUFULWHULDIRUH[DPSOHKLJKHUGLVHDVHUHVLVWDQFHUHSURGXFWLYHHI¿FLHQF\
DQGORQJHUSURGXFWLYHOLIHWKDWKHOSWRLQFUHDVHHI¿FLHQF\RISURGXFWLRQVKRXOGEHWDUJHWHG 6FROODQ
et al., 2010). Also precision agriculture i.e. on-farm development of monitoring techniques (e.g.
physical and chemical sensors, image and sound recordings, etc.) and provision of all inputs to the
places and at times (using computerized tools) required by the animal, thereby maximizing resource
XVHHI¿FLHQF\

7KHUHDOL]DWLRQRISUR¿WDELOLW\RIZHOOPDQDJHGLQWHQVLYHV\VWHPVOHDGVWRH[WUHPHVLWXDWLRQVIDUPV
having a huge number of animals, and exploitation of animal potential by maximizing productivity.
These farms are widespread in North America but are increasingly found in developing countries
as well. In developing countries they compete with smallholders who do not have the same access
to infrastructure and resources, and hence become extinct with time. Such farms limit imports of
DQLPDOSURGXFWVEXWSUR¿WJHQHUDOO\GRHVQRWEHQH¿WWKHORFDOSRSXODWLRQ,QDGGLWLRQWRUDLVLQJ
these major social issues in developing countries, these huge intensive farms concentrate manure
at one place with negative environmental consequences such as soil and water pollution if they are
not managed properly. According to Scollan et al. (2010) good management of manure and use of
biogas are attractive options to overcome these environmental problems.

It is worth noting that high productive farms may look sustainable based on GHG emission per kg
animal product but might not be sustainable in the future due to increasing cost of cereals and fossil
energy. Equally important is to consider animal welfare and ethical dimensions of feeding high grain
diets to animals. Also, such systems disrupt the nitrogen cycle through transport of high amounts of
soybean e.g. from Southern America to other parts of the world. In addition any disruption in trade
or volatility of the cost of these feed inputs can be catastrophic for such farms. In order to address
WKHVHLVVXHVDUHFHQWDSSURDFKSURPRWHVDJURHFRORJ\DZD\WRLPSURYHRUWRPDLQWDLQHI¿FLHQF\
by means of ecological solutions. Application of the principles of agro-ecology to livestock has been
extensively described by Dumont et al. (2013). In addition to a decrease in inputs, agro-ecology
advocates an increase in animal and vegetal biodiversity, the optimization of metabolic functioning
of farming systems and an improvement in management for maintaining animal health. These
techniques generally result in better sustainability and resilience to shocks (Thornton, 2010). A
FODVVLFDOLPSURYHPHQWRIHFRHI¿FLHQF\EDVHGRQWHFKQRORJ\DQGRSWLPL]DWLRQRIDQLPDOIXQFWLRQV
is not exclusive of an agro-ecological approach. It is applied in different systems. Some options are
presented in Table 2.

The dependence on cereals in many countries of the South will grow and their use for animals will
make the systems less resilient and more prone to food-feed-fuel competition. An improved use of
¿EURXVPDWHULDOVIRUDJHVRULQGXVWULDOE\SURGXFWV %RFTXLHUDQG*RQ]DOH]*DUFLD)$2
2012) could reduce the dependence on cereals. The dynamics of concentration of livestock farming
in peri-urban situations produces nutrient surplus and associated latent pollution in these areas. This
is in addition to the other challenge of nutrient depletion in the rural areas. In the cropping systems,
the loss of carbon as well as problems of fragility and fertility of tropical soils remains a major issue.
These issues together with the rising cost of fertilizers and energy, GHG emissions associated with
manufacturing and transportation of various inputs, and the scarcity and competition for various
resources (e.g. phosphorus, water) suggest re-designing the system to have closer integration between
livestock and crops, especially in developed countries.

482 Energy and protein metabolism and nutrition in sustainable animal production
7DEOH6RPHH[DPSOHVRIVROXWLRQVWRLPSURYHHFRHI¿FLHQF\E\GLIIHUHQWDSSURDFKHV

Technical and technological approach Agro-ecological approach

Genetics Increasing genetic merit (productivity, Choosing breeds adapted to the

SURGXFWLYHHI¿FLHQF\ORQJHYLW\IHUWLOLW\ecosystem and to the climate, resilient
multi-purpose (e.g. milk and meat) breeds
Nutrition Optimizing digestive functions, Using more feeds produced on farm,
LPSURYLQJPHWDEROLFHI¿FLHQF\IRUPLON diversifying feeds, use of co-products,
secretion and muscle accretion, feeding choosing reproduction periods according
balanced rations that meet nutrient to resources, providing adequate clean
requirements, using additives, adapting water
feeding to reproduction cycle
Animal health Conceiving healthy diets, monitoring Using natural products against parasites
animal activities, using quick and and diseases and for enhancing immune
HI¿FLHQWGLVHDVHGLDJQRVWLFPRQLWRULQJ response by animals, improving animal
and control methods and farm hygiene, taking care of animal
comfort
Use of resources Preferring feeds with low environmental Shifting from mineral to organic
impacts including low water and land fertilization, optimizing manure
use, managing herd to limit non- application, favoring plant biodiversity
productive animals
Manure management Improving slurry storage (type, duration) Better integrating crops and livestock
for lower losses, producing biogas

Conclusion
'H¿QLQJVXVWDLQDELOLW\IRUOLYHVWRFNIDUPLQJLVDFKDOOHQJHEHFDXVHLWUHTXLUHVEDODQFLQJPXOWLSOH
DQGFKDQJLQJREMHFWLYHVRIWKHWKUHHSLOODUVRIVXVWDLQDELOLW\SUR¿WSODQHWDQGSHRSOHLQFOXGLQJHWKLFV
in an array of dimensions from local to global, which is not easy to achieve. There is a need and
room for coexistence of very diverse systems, each of them being adapted to a set of environmental
and socio-economical conditions in different parts of the world. In this paper, the diversity of
these systems in terms of farm structure and natural resource use has been outlined, and the means
through which their sustainability could be enhanced have been discussed. In any system, key to
LPSURYLQJVXVWDLQDELOLW\OLHVLQLPSURYLQJWKHPXOWLSOHFULWHULDRIHI¿FLHQF\RIWKHDQLPDODQGWKH
herd. The challenge for policy makers is to elaborate with stakeholders the roadmaps for realization
RIOLYHVWRFNSURGXFWLRQV\VWHPVWKDWHI¿FLHQWO\XWLOL]HQDWXUDOUHVRXUFHVZKLOHUHVSHFWLQJHWKLFDODQG
socio-cultural dimensions of people, which may differ from region to region.

References
Alary, V., G. Duteurtre and B. Faye, 2011. Elevage et sociétés: les rôles multiples de l’élevage dans les pays tropicaux. In:
(OHYDJHHQUpJLRQVFKDXGHV &RXORQ-%/HFRPWH3%RYDO03HUH]-0HGV ,15$3URG$QLP
Basset-Mens, C. and H.M.G. van der Werf, 2005. Scenario-based environmental assessment of farming systems: the
case of pig production in France. Agric. Ecosyst. Environ. 105, 127-144
%DVVHW0HQV&6/HGJDUGDQG0%R\HV(FRHI¿FLHQF\RILQWHQVL¿FDWLRQVFHQDULRVIRUPLONSURGXFWLRQLQ
1HZ=HDODQG(FRO(FRQ
%ODQF))%RFTXLHU-$JDEULHO3'¶+RXUDQG7&KLOOLDUG$GDSWLYHDELOLWLHVRIWKHIHPDOHVDQGVXVWDLQDELOLW\
of ruminant livestock systems. A review. Anim. Res. 55, 489-510.

Energy and protein metabolism and nutrition in sustainable animal production 483
Blanfort, V., M. Doreau, J. Huguenin, J. Lazard, V. Porphyre, J.F. Soussana and B. Toutain, 2011. Impacts et services
environnementaux de l’élevage en régions chaudes. In : Elevage en régions chaudes (Coulon J.B., Lecomte P.,
Boval M., Perez J.M., eds). INRA Prod. Anim. 24, 89-112.
Bocquier, F. and E. González-Garcia, 2010. Sustainability of ruminant agriculture in the new context: feeding strategies
and features of animal adaptability into the necessary holistic approach. Animal 4, 1258-1273.
Cunningham, E. P., 2005. The genetic dimension. In: Knowledge agriculture: perspectives towards a new model of
milk production. R. Keenan & Co., Co. Carlow, Ireland, 9-11.
Darnhofer, I., S. Bellon, B. Dedieu and R. Milestad, 2010. Adaptiveness to enhance the sustainability of farming
systems. A review. Agron. Sustain. Dev. 30, 545-555.
De Boer, I.J.M., 2003. Review. Environmental impact assessment of conventional and organic milk production. Livest.
3URG6FL
De Vries, M. and I.J.M. de Boer, 2010. Comparing environmental impacts for livestock products: A review of life
cycle assessments. Livest. Sci. 128, 1-11.
Doreau, M. and J.B. Dollé, 2011. Strategies for reducing greenhouse gas emissions in dairy production. A European
perspective. Proc. 47th Eastern Nutrition Conference, Montréal, Canada, 55-77.
Doreau, M., H.M.G. van der Werf, D. Micol, H. Dubroeucq, J. Agabriel, Y. Rochette and C. Martin, 2011. Enteric
methane production and greenhouse gases balance of diets differing in concentrate in the fattening phase of a beef
production system. J. Anim. Sci. 89, 2518-2528.
Doreau, M., M.S. Corson and S.G. Wiedemann, 2012. Water use by livestock: a global perspective for a regional
LVVXH"$QLP)URQW  
Dumont, B., L. Fortun-Lamothe, M. Jouven, M. Thomas and M. Tichit, 2013. Prospects from agroecology and industrial
ecology for animal production in the 21st century. Animal, 7, 1028-1043.
Fanchone, A., P. Nozière, J. Portelli, B. Duriot, V. Largeau, M. Doreau, 2013. Effects of nitrogen underfeeding and
HQHUJ\VRXUFHRQGLJHVWLRQQLWURJHQSDUWLWLRQDQGPLONSURGXFWLRQLQGDLU\FRZ-$QLP6FL
)$27KHVWDWHRIIRRGDQGDJULFXOWXUH/LYHVWRFNLQWKHEDODQFH)$25RPH,WDO\S
FAO, 2010. Greenhouse gas emissions from the dairy sector: A Life Cycle assessment. FAO, Rome, Italy, 94 pp.
FAO, 2012. Biofuel co-products as livestock feed. Opportunities and challenges (H.P.S. Makkar, ed). FAO, Rome,
Italy, 534 p.
Gerber, P., N. Key, F. Portet and H. Steinfeld, 2010. Policy options in addressing livestock’s contribution to climate
FKDQJH$QLPDO
Gill, M., P. Smith and J.M. Wilkinson, 2010. Mitigating climate change: the role of domestic livestock. Animal 4,
323-333.
Hodges, R.J., J.C. Buzby and B. Bennett, 2011. Postharvest losses and waste in developed and less developed countries
: opportunities to improve resource use. J. Agric. Sci. 149 (S1), 37-45.
IPCC, 2007. Agriculture. In Climate Change 2007: Mitigation. Contribution of Working Group III to the Fourth
Assessment Report of the IPCC on Climate Change (B. Metz, O.R. Davidson, P.R. Bosch, R. Dave, L.A. Meyer,
eds), Cambridge University Press, UK.
Kounina, A., M. Margni, J.B. Bayart, A.M. Boulay, M. Berger, C. Bulle, R. Frischknecht, A. Koehler, L. Milà i Canals,
00RWRVKLWD01~xH]*3HWHUV63¿VWHU%5LGRXWW5YDQ=HOP)9HURQHVDQG6+XPEHUW5HYLHZ
of methods addressing freshwater use in life cycle inventory and impact assessment. Int. J. Life Cycle Assess.
18, 707-721.
Lebacq, T., P.V. Baret and D. Stilmant, 2013. Sustainability indicators for livestock farming. A review. Agron. Sustain.
Dev. 33, 311-327.
Moran, D., M. MacLeod, E. Wall, V. Eory, G. Pajot, R. Matthews, A. McVittie, A. Barnes, B. Rees, A. Moxey, A.
Williams, P. Smith, 2008. UK marginal abatement cost curves for the agriculture and land use, land-use change
and forestry sectors out to 2022, with qualitative analysis of options to 2050. Report to the Committee of Climate
Change, SAC, Edinburgh, UK.
Mosnier, C., J. Agabriel, M. Lherm and A. Reynaud, 2010. A dynamic bio-economic model to simulate optimal
adjustments of suckler cow farm management to production and market shocks in France. Agric. Syst. 102, 77-88.
Mosnier, E., H.M.G. van der Werf, J. Boissy and J.Y. Dourmad, 2011. Evaluation of the environmental implications
of the incorporation of feed-use amino acids in the manufacturing of pig and broiler feeds using Life Cycle
Assessment. Animal 5, 1972-1983.

484 Energy and protein metabolism and nutrition in sustainable animal production
Murgueitio, E., Z. Calle, F. Uribe, A. Calle and B. Solorio, 2011. Native trees and shrubs for the productive rehabilitation
RIWURSLFDOFDWWOHUDQFKLQJODQGV)RUHVW(FRO0DQDJ
Nanda, S.K., R. K. Vishwakarma, H.V.L. Bathla, A. Rai and P. Chandra, 2012. Harvest and post harvest losses of
major crops and livestock produce in India. All India Coordinated Research Project on Post Harvest Technology
(ICAR), Ludhiana. pp. 137, September 2012.
Nguyen, T.T.H., H.M.G. van der Werf and M. Doreau, 2012a. Life cycle assessment of three bull-fattening systems:
HIIHFWRILPSDFWFDWHJRU\RQUDQNLQJ-$JULF6FL
Nguyen, T.T.H., M. Doreau, M. Eugène, M.S. Corson, F. Garcia-Launay, G. Chesneau and H.M.G. van der Werf,
2012b. Effect of type of ration and allocation methods on the environmental impacts of beef-production systems.
Livest. Sci. 145, 239-251.
Nguyen, T.T.H., M. Doreau, M. Eugène, M.S. Corson, F. Garcia-Launay, G. Chesneau and H.M.G. van der Werf, 2013.
Effect of farming practices for greenhouse gas mitigation and subsequent alternative land-use on environmental
LPSDFWVRIEHHIFDWWOHSURGXFWLRQV\VWHPV$QLPDO
3DU¿WW-0%DUWKHODQG60DFQDXJKWRQ)RRGZDVWHZLWKLQIRRGVXSSO\FKDLQVTXDQWL¿FDWLRQDQGSRWHQWLDO
IRUFKDQJHWR3KLO7UDQV56RF%
5H\QROGV&./$&URPSWRQDQG-$10LOOV,PSURYLQJWKHHI¿FLHQF\RIHQHUJ\XWLOLVDWLRQLQFDWWOH$QLP
3URG6FL
Ripoll-Bosch, R., I.J.M. de Boer, A. Bernués and T. Vellinga, 2013. Accounting for multi-functionality of sheep farming
LQWKHFDUERQIRRWSULQWRIODPE$FRPSDULVRQRIWKUHHFRQWUDVWLQJ0HGLWHUUDQHDQV\VWHPV$JULF6\VW
Robinson, T.P., P.K. Thornton, G. Franceschini, R.L. Kruska, F. Chiozza, A. Notenbaert, G. Cecchi, M. Herrero, M.
Epprecht, S. Fritz, L. You, G. Conchedda and L. See, 2011. Global livestock production systems. FAO, Rome,
Italy, and ILRI, 152 pp.
Scollan, N.D., D. Moran, E.J. Kim, and C. Thomas, 2010. The environmental impact of meat production systems.
5HSRUWWRWKH,QWHUQDWLRQDO0HDW6HFUHWDULDWSKWWSZZZPHDWLPVRUJROGVLWH,065HYLHZ¿QDOSGI
6WHLQIHOG+3*HUEHU7:DVVHQDDU9&DVWHO05RVDOHVDQG&GH+DDQ/LYHVWRFN¶VORQJVKDGRZ
environmental issues and options. FAO, Rome, Italy, 390 p.
7KRUQWRQ3./LYHVWRFNSURGXFWLRQUHFHQWWUHQGVIXWXUHSURVSHFWV3KLO7UDQV56RF%
UNFCCC, 2008. Challenges and opportunities for mitigation in the agricultural sector. Technical paper FCCC/
TP/2008/8, 101 p.
Vieux, F., N. Darmon, D. Touazi and L.G. Soler, 2012. Greenhouse gas emissions of self-selected individual diets in
France: Changing the diet structure or consuming less? Ecol. Econ. 75, 91-101.

Energy and protein metabolism and nutrition in sustainable animal production 485
Environmental, social, and economic footprints of current and past beef
production systems
K.R. Stackhouse-Lawson1, J.O. Reagan1, B.J. Isenberg2, E.J. Pollak, T. Battagliese, B. Ulhman,
C. Barcan, I. Schulze, J. Silva and C.A. Rotz2
11DWLRQDO&DWWOHPHQ¶V%HHI$VVRFLDWLRQ&HQWHQQLDO&286$[email protected]
2USDA-ARS, Pasture Systems and Watershed Management Research Unit, University Park, PA
86$
86'$$5613$5RPDQ/+UXVND860HDW$QLPDO5HVHDUFK&HQWHU&OD\&HQWHU1(86$
%$6)&RUSRUDWLRQ1XWULWLRQDQG+HDOWK)ORUKDP3DUN1-86$
%$6)&RUSRUDWLRQ)XQGDomR(VSDoR(&26DR%HUQDUGRGR&DPSR%UD]LO

Introduction
7KHEHHILQGXVWU\KDVGH¿QHGVXVWDLQDELOLW\DVPHHWLQJWKHJURZLQJGHPDQGIRUEHHIE\EDODQFLQJ
environmental responsibility, economic opportunity, and social diligence. Measuring sustainability
is challenging, as the beef supply chain is one of the most complex food systems in the world. As
WKH¿UVWDQGODUJHVWUHVHDUFKSURMHFWRIWKLVNLQGWKLVVWXG\UHSUHVHQWVDQLQQRYDWLYHDSSURDFKWRZDUG
creating a more sustainable beef product. Our objective is to establish a sustainability baseline
(including environmental, economic, and social footprints) for the US beef industry by quantifying
life cycle inputs and outputs for beef production over time.

Material and methods

To determine the sustainability of beef production, a combination of models were used. The USDA-
ARS Integrated Farm System Model (IFSM) was used to simulate environmental and economic
IRRWSULQWVIURPFUDGOHWRIDUPJDWH7KHVRFLRHFRHI¿FLHQF\WRRO 6((%$/$1&(®) extends this
analysis by determining the environmental, economic, and social impacts of beef from cradle to
grave providing a comprehensive assessment of sustainability.

The IFSM is a process-level farm model that simulates crop growth, feed production and use,
animal growth, and returning manure nutrients to the land to predict the environmental impacts
and economics of agriculture production systems (Rotz et al., 2005). For the current study, relevant
information for the US Meat Animal Research Center (USMARC) beef operation was gathered
and used to establish model parameters. The USMARC farm, cow-calf and feedlot operations were
simulated to evaluate performance, environmental impact and economics.

The environmental impacts and economics of beef production at the USMARC were combined with
primary data from the packer, case ready, retail, and consumer segments of the beef value chain for
2005 and 2011 using SEEBALANCE®. The SEEBALANCE® analysis includes environmental,
social, and economic considerations as determined by method of life cycle analysis (Kölsh et al.,
 7KLVDSSURDFKTXDQWL¿HG86EHHIVXVWDLQDELOLW\FRQVLGHULQJHFRQRPLFVRFLDODQGHFRORJLFDO
impacts along all segments of the beef value chain.

Results and discussion

Integrated farm system model: USMARC

A 25-year simulation of the USMARC’s current production system gave a carbon footprint of 11
kg of CO2e per kg of live weight sold, which is consistent with other experiments (Johnson et al.,
2003; Capper, 2011; Stackhouse-Lawson et al., 2012). The energy required to produce that beef
(energy footprint) was 25.9 MJ/kg. The total water required (water footprint) was 21,300 l/kg of

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 487
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_179, © Wageningen Academic Publishers 2013
live weight sold, and the water footprint excluding that obtained through precipitation was 2,800 l/
kg. The simulated total cost of producing their beef was about $2.20/kg of live weight sold, which
agreed with USMARC production records.

SEEBALANCE®

7DEOHTXDQWL¿HVWKHHQYLURQPHQWDOVRFLDODQGHFRQRPLFFRQVLGHUDWLRQVRIWKHEHHIVXSSO\FKDLQ
expressed in 0.45 kg of minimally processed boneless edible consumed beef (UB). Overall, the
VXVWDLQDELOLW\RIWKH86EHHILQGXVWU\JLYHQWKHSUHVHQWDVVXPSWLRQVKDVLPSURYHGE\LQ\U

Table 1. Environmental, social and economic sustainability indicators for the beef supply chain.

Sustainability indicators 2005 2011 % change

Economic (expressed per UB1)

Consumer price ($) 5.24 5.55 
Environmental (expressed per UB)
Energy use (MJ) 521 511.0 -2
Resource consumption (mg silvere) 5.05  -2
Water consumption (L) 2,418  -3
Solid waste (kg municipal wastee) 0.19 0.18 -7
Greenhouse gases (kg CO2e) 23.7  -1
Photochemical ozone creation potential (g C2H4e)   0
$FLGL¿FDWLRQSRWHQWLDO J622e)  327 -3
Ozone depletion potential (g CFCe) 0.013 0.013 0
Water emissions (grey water (l diluted watere) 4,981 4,487 -10
Land use (m2a) 21.4 20.5 -4
Social (normalized and weighted2)
Occupation illnesses and accidents 0.90  -32
Toxicity potential 1.00 0.84 

18VHUEHQH¿W 8% NJRIPLQLPDOO\SURFHVVHGERQHOHVVHGLEOHFRQVXPHGEHHI
2 Social indicators are normalized and weighted based on severity of incident or chemical.

References
Capper, J.L, 2011. Replacing rose-tinted spectacles with a high-powered microscope: The historical vs. modern carbon
IRRWSULQWRIDQLPDODJULFXOWXUH$QLP)URQW
Johnson, D. E., H. W. Phetteplace, A. F. Seidl, U. A. Schneider, and B. A. McCarl, 2003. Management variations for
XVEHHISURGXFWLRQV\VWHPV(IIHFWVRIJUHHQKRXVHJDVHPLVVLRQVDQGSUR¿WDELOLW\3URFUG,QW0HWKDQHDQG
1LWURXV2[LGH0LWLJDWLRQ&RQI%HMLQJ&KLQD
Kölsh, D., P Saling, A. Kircherer, A. Grosse-Sommer, and I. Schmidt, 2008. How to measure social impacts? A socio-
HFRHI¿FLHQF\DQDO\VLVE\WKH6((%$/$1&(® method. Int. J. Sustainable Development. 11, 1-23.
Rotz, C.A., D.R, Buckmaster, and J.W. Comerford, 2005. A beef herd model for simulation of feed intake, animal
performance, and manure excretion in farm systems. J. Anim. Sci. 38, 231-242.
Stackhouse-Lawson, K.R., C.A. Rotz, J.W. Oltjen, and F.M. Mitloehner, 2012. Carbon footprint and ammonia emission
RI&DOLIRUQLDEHHISURGXFWLRQV\VWHPV-$QLP6FL

488 Energy and protein metabolism and nutrition in sustainable animal production
Effect of fat supplementation and stage of lactation on methane emission
in dairy cows
L. Alstrup, M.R. Weisbjerg and P. Lund
Department of Animal Science, AU Foulum, Aarhus University, Denmark; [email protected]

Introduction
Methane is produced in ruminants as a consequence of fermentation of organic matter in the rumen.
The methane produced represents an energy loss to the animal and will vary with feed composition
and intake; under extreme circumstances 2-12% of gross energy intake is converted into methane,
but in an intensive dairy production values between 3-7% are more realistic (Martin et al., 2008). It
has been shown that supplementation of fatty acids (FA) to the feed decreases methane emission on
a short term (Brask et al., 2012). Although supplementation with fat to the diet is the most promising
dietary strategy to reduce enteric methane emission (Grainger and Beauchemin, 2011), research in
long term effects of supplementing fat, and of days in milk (DIM) on methane production is lacking.
The aim of the present experiment was to study the effect of FA supplementation, effect of stage of
lactation, and their interaction on methane emission.

Material and methods

Six Holstein Friesian cows were blocked in three groups according to DIM and randomly assigned
to a ration with either no fat supplementation (NO FAT) or fat supplementation (FAT) in the form
of rolled rapeseed. Rations consisted of app. 55% forage (50% maize silage and 50% grass clover
silage) and 45% concentrate on dry matter (DM) basis. Content of fatty acids in rations were 18 and
47 g/kg DM for no fat and fat supplementation, respectively. Methane production was measured
for 2×48 h in 17 m3 open-circuit respiration chambers. Feed intake and milk yield was recorded in
the chamber period. The animals were housed individually and chambers were only opened twice
daily for milking and subsequent feeding. Methane was measured as the accumulated amount in l
over 24 hours. Corrections were made for the measurements during the openings of the chambers
for milking and feeding. Methane production was measured at approximately 50, 125, 170 and 220
DIM, and at 50 DIM all cows received the NO FAT ration. Experimental data were analysed with
the MIXED procedure of SAS, using recordings at 50 DIM as covariate and ration, cow and DIM
as class variables. Cow was set as random effect to account for repeated measurements.

Results and discussion

0HWKDQHHPLVVLRQLQOGD\LQFUHDVHGVLJQL¿FDQWO\ 3 0.03) from time point 125 DIM to 220 DIM;
IURPOGD\WROGD\IRUFRZVUHFHLYLQJ12)$7DQGIURPOGD\WROGD\IRUFRZV
UHFHLYLQJ)$7 )LJXUH 0HWKDQHDVONJ'0LQWDNH '0, DOVRLQFUHDVHGVLJQL¿FDQWO\ 3 0.002)
from time point 125 DIM to 220 DIM; from 29.0 to 31.8 and from 28.0 to 30.5 for NO FAT and
FAT respectively, and tended to be reduced for FAT (P=0.08).

In the same period DMI (kg/24 h) increased for cows receiving NO FAT from 21.9 kg/day to 23.1
NJGD\ZKHUHDVFRZVUHFHLYLQJ)$7KDGDGHFUHDVHLQIHHGLQWDNHIURPNJGD\WRNJ
GD\ )LJXUH 0LON\LHOGPHDVXUHGDVNJHQHUJ\FRUUHFWHGPLON (&0 GHFUHDVHGVLJQL¿FDQWO\
(3 0.009) throughout lactation. From time point 125 DIM to 220 DIM milk yield decreased from
32.9 kg/day to 25.9 kg/day and from 33.9 kg/day to 30.2 kg/day for NO FAT and FAT respectively.

The increase in daily methane emission throughout lactation was not only related to increased DMI,
as L methane/kg DMI also increased throughout lactation. According to Garnsworthy et al. (2012)
an increased methane emission despite an increase in DMI with stage of lactation may be due to

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 489
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_180, © Wageningen Academic Publishers 2013
)LJXUH0HWKDQHHPLVVLRQSHUGD\DQGSHUNJGU\PDWWHULQWDNH '0, WKURXJKRXWODFWDWLRQ

)LJXUH.JGU\PDWWHULQWDNH '0, DQGNJHQHUJ\FRUUHFWHGPLON (&0 SHUGD\WKURXJKRXW

lactation.

an increased proportion of forage in diet, leading to higher methane emission per kg DMI. In the
current study the proportion of forage was kept constant, so the increased methane emission cannot
be explained by changed forage/concentrate ratio.

In conclusion, this study indicates that methane production increases with days in milk, though
season could be confounded with DIM. Further it is suggested that supplementation with rapeseed
to the ration could reduce this increase in methane production, both as absolute methane production
in liters and as methane production related to DMI. Further, rapeseed supplementation could increase
milk yield.

References
Brask, M., P. Lund, M.R. Weisbjerg, A.L.F. Hellwing and T. Hvelplund. 2012. Methane Production and Digestion in
'DLU\&RZVIHG'LIIHUHQW3K\VLFDO)RUPVRI5DSHVHHGDV)DW6XSSOHPHQW-'DLU\6FLGRLMGV
Garnsworthy, P.C, J. Craigon, J.H. Hernandez-Medrano and N. Saunders. 2012. Variation among individual dairy cows
in methane measurements made on farm during milking. J. Dairy Sci. 95, 3181-3189
Grainger, C. and K.A. Beauchemin. 2011. Can enteric methane emissions from ruminants be lowered without lowering
WKHLUSURGXFWLRQ"$QLP)HHG6FL7HFKQRO
Martin, C., J. Rouel, J.P. Jouany, M. Doreau and Y. Chilliard. 2008. Methane output and diet digestibility in response
WRIHHGLQJGDLU\FRZVFUXGHOLQVHHGH[WUXGHGOLQVHHGRUOLQVHHGRLO-$QLP6FL

490 Energy and protein metabolism and nutrition in sustainable animal production
Methane emission and protein precipitating ability of condensed tannins
from warm-season perennial legumes
H.D. Naumann, L.O. Tedeschi1, A.E. Hagerman2, B.D. Lambert and J.P. Muir
1Texas A&M University, College Station, TX, USA; [email protected]
2Miami University, Oxford, OH, USA
Tarleton State University, Stephenville, TX, USA
Texas A&M AgriLife Research, Stephenville, TX, USA

Introduction
Enteric methane (CH4) emissions by ruminants represent a decrease in energy availability to the
animal. Forages containing condensed tannins (CT) may suppress enteric CH4 emissions and bind to
proteins, allowing protein to escape microbial degradation in the rumen resulting in ruminal bypass
protein. Objectives of this study were to evaluate the ability of CT from warm-season perennial
legumes commonly consumed by ruminants to suppress CH4 emissions and bind to protein, and to
assess the potential use of these forages in sustainable ruminant production systems.

Material and methods

Nine species of warm-season perennial legumes were evaluated (Table 1). Two separate extractions
were performed on oven-dried plant tissue from each species evaluated. Condensed tannins were
determined by a butanol-HCl method (Terrill et al., 1992). Protein-precipitating ability (PB) was
determined by reacting crude plant extracts with bovine serum albumin (Hagerman and Butler, 1978)
and analyzing protein-phenolic residues for N. Methane production was determined using an in vitro
gas production technique (Tedeschi et al., 2009). Replications consisted of two fermentation events,
12/7/2011 and 1/23/2012, where each plant species was fermented in each of two fermentation
FKDPEHUV)HUPHQWDWLRQFKDPEHUZDVFRQVLGHUHGDUDQGRPYDULDEOHZKHUHDVIHUPHQWDWLRQÀDVNV
within each fermentation chamber were considered random factors. Two ruminally-cannulated
VWHHUVXQDGDSWHGWRIRUDJHFRQWDLQLQJ&7ZHUHXVHGIRUUXPHQÀXLGFROOHFWLRQ)RUDJHVZHUH
LQGLYLGXDOO\IHUPHQWHGDQDHURELFDOO\LQUXPHQÀXLGIRUK0HWKDQHPHDVXUHPHQWVZHUHPDGHE\
gas chromatography following fermentation. Data were analyzed using the GLIMMIX procedure
of SAS (SAS Inst. Inc., Cary, NC, USA).

7DEOH7RWDOFRQGHQVHGWDQQLQ &7 FRQFHQWUDWLRQV 7&7'0 PHWKDQHSURGXFWLRQ &+; g/kg

'0 DQGDPRXQWRISURWHLQERXQG 3%JNJ'0 E\&7IURPZDUPVHDVRQSHUHQQLDOOHJXPHVin vitro.

Plant TCT CH4 PB

Acacia angustissima var hirta 8.9b e d

Arachis glabrata 0.0d 38.2a 0.0e
Desmanthus illinoensis 8.1b 24.9b 75.3bc
Desmodium paniculatum 12.5a 7.9de d
Lespedeza cuneata 8.3b 15.1cd 78.1ab
Lespedeza stuevei 11.7a 4.9e 72.3c
Leucaena retusa 3.2c 40.7a 4.4e
Mimosa strigillosa 11.7a de 70.9c
Neptunia lutea 8.3b 19.7bc 80.0a

a-e Within a column, LS means without a common superscript differ (P<0.05).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 491
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_181, © Wageningen Academic Publishers 2013
Results and discussion
Total CT (TCT), PB and CH4 are shown in Table 1. Total CT was greatest for D. paniculatum. L.
stuevei and M. strigillosa (P<0.05). Other than A. glabrata (CT negative control), L. retusa had the
least TCT (P<0.05). Condensed tannins from N. lutea and L. cuneata demonstrated the greatest PB
(P<0.05), whereas CT from L. retusa demonstrated almost no PB at all. Condensed tannins from L.
retusa did not suppress CH4 emissions in vitro. Fermentations containing A. angustissima suppressed
in vitro CH4 emissions to the greatest degree (P<0.05). Regressions of CH4 on TCT and PB on TCT
(Figure 1a-b) indicated that TCT could explain up to 44% of variation in CH4 production and up
WRRIYDULDWLRQDVVRFLDWHGZLWKSURWHLQSUHFLSLWDWLQJDELOLW\7KHQHJDWLYHFRUUHODWLRQEHWZHHQ
TCT and CH4 indicated that for every unit increase in TCT, CH4 decreased by 3.27 g/kg DM. Total
CT and PB were positively correlated such that for every unit increase in TCT, PB increased by
JNJ'03URWHLQSUHFLSLWDWLQJDELOLW\RI&7 )LJXUHF H[SODLQHGRQO\RIWKHYDULDWLRQ
associated with CH4 production.

The biological activity of CT relative to suppression of CH4 emissions and PB differed depending on
source of CT and were affected by TCT concentration. Results suggest that enteric CH4 suppression
is predominately affected by factors other than protein precipitation by CT. Of the species evaluated,
CT from L. cuneata demonstrated the greatest combined ability to bind to protein and suppress CH4
production in vitro.

)LJXUH7KHHIIHFWVRI D WRWDOFRQGHQVHGWDQQLQV 7&7 RQPHWKDQH &+JNJ'0 SURGXFWLRQ

E WRWDOFRQGHQVHGWDQQLQV 7&7 RQWKHDPRXQWRISURWHLQERXQG 3%JNJ'0 E\FRQGHQVHG
WDQQLQVDQG F WKHDPRXQWRISURWHLQERXQG 3%JNJ'0 E\&7RQPHWKDQH &+JNJ'0 
production in vitro.

References
Hagerman, A.E. and L.G. Butler, 1978. Protein precipitation method for the quantitative determination of tannins. J.
$JULF)RRG&KHP
Tedeschi, L. O., P. J. Kononoff, K. Karges, and M. L. Gibson, 2009. Effects of chemical composition variation on the
dynamics of ruminal fermentation and biological value of corn milling (co) products. J. Dairy Sci. 92, 401-413.
Terrill, T. H., A. M. Rowan, G. B. Douglas, and T. N. Barry, 1992. Determination of extractable and bound condensed
tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 58, 321-329.

492 Energy and protein metabolism and nutrition in sustainable animal production
Short-term dose effects of feeding monensin on methane emissions from
lactating Holstein dairy cattle
S.E. Place1, Y. Pan2, Y. Zhao2 and F.M. Mitloehner2
1'HSDUWPHQW RI $QLPDO 6FLHQFH 2NODKRPD 6WDWH 8QLYHUVLW\ 6WLOOZDWHU 2.  86$
VSODFH#JPDLOFRP
2'HSDUWPHQWRI$QLPDO6FLHQFH8QLYHUVLW\RI&DOLIRUQLD'DYLV&$86$

Introduction
0RQHQVLQLVDIHHGDGGLWLYHXVHGLQGDLU\FDWWOHGLHWVWRLPSURYHIHHGHI¿FLHQF\WKDWPD\UHGXFH
methane(CH4) emissions; however, past results have been variable, which could be due to the dose
of monensin included in the diet. The objective of this study was to test the short-term dose effects
of monensin on eructated CH4 emissions from lactating dairy cows.

Material and methods

7ZHQW\PXOWLSDURXVODFWDWLQJ+ROVWHLQFRZVZHUHVWUDWL¿HGE\GD\VLQPLONDQGUDQGRPO\DVVLJQHG
to one of four treatments provided in a pelleted supplemental top dress feed (CON, LOW, MED,
+,*+FRQWDLQLQJDQGPJFRZGD\RIPRQHQVLQUHVSHFWLYHO\ $OOFRZVZHUHIHG
the same basal total mixed ration (TMR) throughout the experiment and CON top dress for 19 d
(PRE period), then their respective treatment top dress for 21 d (MON period), and then returned
to the CON top dress for 21 d (POST period). Cows were milked and fed twice daily at 04:30 and
KZLWKIHHGRIIHUHGDGOLELWXP(UXFWDWHGDQGUHVSLUHGJDVHPLVVLRQVZHUHVDPSOHGIRUHDFK
cow individually on the last day of each period via a ventilated hood system described in detail by
Place et al. (2011).

All statistical analysis was conducted using Proc Mixed procedures in SAS version 9.3 (SAS Institute
Inc., Cary, NC, USA). Dry matter intake (DMI) was included as a covariate in the CH4 emissions models.

Results and discussion

Methane emissions, milk production, DMI, and milk composition were similar across treatments
in the MON period (Table 1).

The change in CH4HPLVVLRQVIURPWKH35(WR021SHULRGDFURVVWUHDWPHQWVYDULHG 

and 3.9 g/cow/h for the CON, LOW, MED, and HIGH treatments, respectively), with MED having
a lower change in CH4 emissions per cow and per kg of milk compared to CON (Figure 1). Changes
in CH4 emissions per cow and per kg of milk from the PRE to POST period were not different across
treatments, suggesting there were no carryover effects of monensin in the POST period.

Two of the previous studies feeding lactating dairy cows TMR have shown reductions in CH4
emissions with monensin fed at a rate of 24 mg/kg DM (Sauer et al., 1998 doses ranged from 348
to 427.2 mg/cow/d, Odongo et al., 2007 ranged from 307 to 708 mg/cow/d), while another found
no effect of monensin on CH4HPLVVLRQVZLWKPRQHQVLQIHGDWPJFRZG DSSUR[LPDWHO\
mg/kg DM) (Hamilton et al., 2010). The lower increase in CH4 emissions per cow and per unit of
PLONIRUWKHPJFRZG0('WUHDWPHQWLVZLWKLQWKHORZHUUDQJHVIHGE\6DXHUet al. (1998) and
Odongo et al. (2007). There may be an effective dose of saturation for monensin, over which all
rumen microorganisms are affected equally; however, further investigation is required. In conclusion,
during a short-term feeding period, monensin had over time dose effects on CH4 emissions, but the
effects do not seem to be linear in nature.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 493
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_182, © Wageningen Academic Publishers 2013
7DEOH7UHDWPHQWOHDVWVTXDUHVPHDQV Q  SHUFRZIRU&+ emissions and animal performance
by period.

Item Period Treatment SEM

CON LOW MED HIGH

CH4, g/h PRE  20.2 23.1 19.8 0.337

MON   24.7 23.7 0.417
POST 19.8a 23.0ab 28.7b 24.0ab 0.544
CH4: Milk yield, g/h: kg/d PRE 0.351  0.554 0.438 0.008
MON 0.512 0.599 0.581 0.534 0.011
POST 0.473a 0.542ab 0.701b 0.557ab 0.014
DMI, kg/d PRE 25.8a ab 29.0b ab 0.159
MON 27.4 29.2 29.2 27.5 0.135
POST 27.7 30.0 29.2 28.5 0.122
Milk yield, kg/d PRE 40.0 43.5 43.0 41.2 0.300
MON 43.4  44.3  0.343
POST 43.3 47.3 43.0 44.8 0.335
Milk fat % PRE 3.55 3.45 3.59  0.105
MON 3.39 3.85 3.55 3.50 0.118
POST 3.41 3.51  3.57 0.088
Milk protein % PRE  3.12  3.01 0.041
MON 2.94 3.08 3.12 2.98 
POST 3.14 3.32 3.33  0.048

a,b Within row least squares means without common subscript letters differ (P<0.05).

A 8.00 A A B 0.20 A
CH4 , g/cow per h

CH4:Milk, g/h: kg/d

A
6.00 AB 0.15 AB
4.00 B 0.10
B
2.00 0.05
0.00 0.00
CON LOW MED HIGH CON LOW MED HIGH
Figure 1. Changes in CHHPLVVLRQVRYHUWLPHIURP35(WR021SHULRGV 3DQHO$&+ g/cow/h,
Panel B: CHJKPLON\LHOGNJG $%LQGLFDWHVLJQL¿FDQWGLIIHUHQFHV P< 

References
Hamilton, S.W., E.J. DePeters, J.A. McGarvey, J. Lathrop, and F.M. Mitloehner, 2009. Gas emissions, animal
performance, and bacterial population structure responses to dietary monensin in lactating dairy cows. J. Environ.
4XDO
Odongo, N. E., R. Bagg, G. Vessie, P. Dick, M. M. Or-Rashid, S. E. Hook, J. T. Gray, E. Kebreab, J. France, and B.
W. McBride, 2007. Long-term effects of feeding monensin on methane production in lactating dairy cows. J.
Dairy Sci. 90, 1781-1788.
Place, S. E., Y. Pan, Y. Zhao, and F. M. Mitloehner, 2011. Construction and operation of a ventilated hood system for
PHDVXULQJJUHHQKRXVHJDVDQGYRODWLOHRUJDQLFFRPSRXQGHPLVVLRQVIURPFDWWOH$QLPDOV
Sauer, F. D., V. Fellner, R. Kinsman, J. K. Kramer, H. A. Jackson, A. J. Lee, and S. Chen, 1998. Methane output and
ODFWDWLRQUHVSRQVHLQ+ROVWHLQFDWWOHZLWKPRQHQVLQRUXQVDWXUDWHGIDWDGGHGWRWKHGLHW-$QLP6FL

494 Energy and protein metabolism and nutrition in sustainable animal production
Methane emission from sheep is related to concentrations of rumen
volatile fatty acids
C.S. Pinares-Patiño1, H. Kjestrup1, S. MacLean1, E. Sandoval1, G. Molano1, R. Harland1, S. Hickey2,
E. Young2, K. Dodds2, K. Knowler2, N. Pickering2 and J. McEwan2
1 $J5HVHDUFK *UDVVODQGV 3ULYDWH %DJ  3DOPHUVWRQ 1RUWK 1HZ =HDODQG
[email protected]
2$J5HVHDUFK,QYHUPD\3ULYDWH%DJ0RVJLHO1HZ=HDODQG

Introduction
Globally, ruminants are the most important source of emission of methane (CH4). Animal-to-animal
variation in CH4 emission has genetic basis (Pinares-Patiño et al., 2011), hence offering a potential
mitigation avenue through animal breeding. However, for this approach to progress to practical
application a rapid and reliable method of ranking animals for their CH4 emissions is required.
Microbial fermentation of feed in the rumen produces volatile fatty acids (VFA), hydrogen (H2),
carbon dioxide (CO2), ammonia and heat. A last step in the process is the reduction of CO2 to CH4 by
Archaea using H2 as a source of energy. Formation of both acetic and butyric acids is accompanied
by the production of H2 and CO2, whereas propionic production involves a net uptake of H2, hence
9)$SUR¿OHVPD\EHXVHGWRSUHGLFW&+4 emission rates (Benchaar et al., 2001). This controlled
study conducted with sheep explored the relationship between rumen VFA and CH4 emission.

Material and methods

2YHUWKUHH\HDUVODPEV LQFOXGLQJPDOHV RISURJHQ\WHVWSURJUDPVZHUHSKHQRW\SHGIRU
their CH4 emissions using respiration chambers (Pinares-Patiño et al., 2012) while fed on pelleted
OXFHUQH FUXGHSURWHLQQHXWUDOGHWHUJHQW¿EUHDQG0-0(NJ'0 )HHGLQJZDVDW
WLPHVPDLQWHQDQFHUHTXLUHPHQWVZLWKHTXDOVL]HPHDOVGHOLYHUHGDWDQGK7KHUHZHUH
UHVSLUDWLRQFKDPEHUVDYDLODEOH/DPEVZHUHPRQWKVROGDQGNJOLYHZHLJKW7KH\ZHUH
PDQDJHGLQORWVRIDQLPDOV$FFOLPDWLVDWLRQLQSHQV G ZDVIROORZHGE\WZRPHDVXUHPHQW
periods (P1 and P2, 10-15 d interval). In each period, individuals’ feed dry matter intake (DMI)
were measured in metabolic crates (4 d) and then in respiration chambers (2 d). Both in P1 and P2,
individuals were randomly allocated to groups (4, each of 24 animals) and respiration chambers. At
P1 and P2, soon after exit from the chambers (pre-feeding stage), rumen contents (20-50 ml) were
sampled by stomach-tubing for VFA analysis (Sun et al., 2012).

Methane yield (g/kg DMI) was calculated from daily CH4 emission and DMI. Data for VFAs were
analysed as their loge (x + 1), where x was concentration (mM) or molar %. Heritability (h2) and
repeatability of both CH4 yield and VFA were calculated. Fixed effects for CH4 yield were birth
ÀRFNDQGFRQWHPSRUDU\JURXS FJ UHFRUGLQJ\HDUORWJURXSSHULRGDQGIRU9)$VWKHFJELUWK
\HDUELUWKÀRFNVH[5DQGRPHIIHFWVZHUHDQLPDODQGWKHSHUPDQHQWHQYLURQPHQWDOHIIHFWVSHULRG
DQGUHFRUGLQJ\HDUZHUH¿WWHGWRHVWLPDWHUHSHDWDELOLW\ZLWKLQ\HDU 9)$DQG&+4 yield) and across
year (CH4 yield), respectively.

Results and discussion

The mean CH4 emission was 24.3 g/d, whereas the CH4 yield was 15.8 g/kg DMI. Heritability (±s.e.)
for CH4\LHOGZDV“ZLWKUHSHDWDELOLW\ZLWKLQDQGDFURVV\HDUVRI“DQG“
respectively. The mean of total concentration of VFA was 51.1 mM, whereas the concentrations
of acetic, propionic and butyric acids were 34.9, 8.8 and 4.0 mM. The mean molar % of acetic,
SURSLRQLFDQGEXW\ULFDFLGVZHUHDQGUHVSHFWLYHO\ZLWKDFHWDWHSURSLRQDWHUDWLRRI

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 495
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_183, © Wageningen Academic Publishers 2013
Concentrations (as loge) of total and major VFA had higher estimates of h2, repeatability and
genetic correlation (rg) with CH4 yield than when expressed on molar % (as loge) basis (Table 1).
7KLV¿QGLQJFRQWUDVWVZLWKWKRVHRI5RELQVRQet al. (2010), who from a study involving 12 rumen-
cannulated sheep concluded that VFA concentrations had poor relationships with both daily CH4
emission and CH4 yield. However, experimental conditions between the later study and this study
were largely different (e.g. feeding conditions, number animals, etc.). There is no agreement in the
literature on the validity and representativeness of sample of rumen contents collected via stomach
tube, but representativeness of stomach tube sample seems be related to feeding time and depth of
insertion (Shen et al., 2012). In the present study, sampling took place at fasting and the operation
was completed within one minute.

Results from this study in highly controlled conditions suggest that concentrations of VFA in rumen
samples obtained by stomach tubing in a fasting state are heritable and potentially useful to estimate
CH4\LHOG+RZHYHUWKLV¿QGLQJQHHGVWREHFRUURERUDWHG

7DEOH5XPHQ9)$ ORJe KHULWDELOLW\ K2 UHSHDWDELOLW\DQGFRUUHODWLRQ Ug ZLWK&+ yield.

mM molar %

h2 rep rg h2 rep rg

VFA 0.10±0.04 0.33±0.03 0.92±0.10

Ace 0.09±0.04 0.34±0.03 0.95±0.10 0.04±0.03 0.08±0.03 -0.01±0.28
Pro 0.10±0.04 0.31±0.03 0.78±0.15 0.09±0.04 0.15±0.03 -0.18±0.17
But 0.09±0.04 0.28±0.03 “ 0.04±0.04 0.18±0.03 “
Ace/Pro 0.10±0.04 0.11±0.03 0.08±0.18

Acknowledgements
This study was funded by the New Zealand Pastoral Greenhouse Gas Research Consortium

References
Benchaar, C., C. Pomar and J. Chiquette, 2001. Evaluation of dietary strategies to reduce methane production in
UXPLQDQWVDPRGHOOLQJDSSURDFK&DQ-$QLP6FL
Pinares-Patiño, C.S., J.C. McEwan, K.G. Dodds, E.A. Cárdenas, R.S. Hegarty, J.P. Koolaard and H. Clark, 2011.
5HSHDWDELOLW\RIPHWKDQHHPLVVLRQVIURPVKHHS$QLP)HHG6FLDQG7HFKQRO
Pinares-Patiño C.S. and G.C. Waghorn, 2012. Manual on respiration chambers designs. GRA, New Zealand (http://
www.livestockemissions.net/reports,listing,73,technical-manual-on-respiration-chamber-designs.html)
Robinson, D.L., J. Goopy and R.S. Hegarty, 2010. Can rumen methane production be predicted from volatile fatty
DFLGFRQFHQWUDWLRQV"$QLP3URG6FL
Shen, J.S., Z. Chai, L.J. Song, J.X. Liu and Y.M. Wu, 2012. Insertion depth of oral stomach tubes may affect the
IHUPHQWDWLRQSDUDPHWHUVRIUXPLQDOÀXLGFROOHFWHGLQGDLU\FRZV-'DLU\6FL
6XQ;='3DFKHFR6+RVNLQDQG':/XR5XPHQIHUPHQWDWLRQFKDUDFWHULVWLFVDUHLQÀXHQFHGE\IHHGLQJ
frequency in sheep fed forage chicory and perennial ryegrass at two feeding levels. Proc. N.Z. Soc. Anim. Prod.


496 Energy and protein metabolism and nutrition in sustainable animal production
(QHUJ\HI¿FLHQF\DQGPHWKDQHHPLVVLRQE\VKHHSIHGVRUJKXPVLODJHVDW
different maturation stage
F.S. Machado1, N.M. Rodríguez2, M.N. Ribas2, F.P. Pôssas2, L.C. Gonçalves2 and L.G.R. Pereira1
1 Brazilian Agricultural Research Corporation, Embrapa Dairy Cattle, Brazil;
[email protected]
2Department of Animal Science, Federal University of Minas Gerais, Brazil

Introduction
The importance of sorghum as a forage crop is growing in many regions of the world due to its high
SURGXFWLYLW\DQGDELOLW\WRXWLOL]HHI¿FLHQWO\ZDWHUHYHQXQGHUGURXJKWFRQGLWLRQV 6DQFKH]et al.,
2002). The introduction of calorimetric studies in tropical conditions is important for conceptual
advances in roughage evaluation, enabling the best way of utilization, optimizing livestock
performance (Rodriguez et al., 2007). The objectives of this study were to examine the effects
RIVRUJKXPJHQHWLFVDQGVWDJHVRIPDWXULW\DWKDUYHVWRQHI¿FLHQF\RIHQHUJ\XVHDQGPHWKDQH
production by sheep.

Material and methods

)RUW\¿YHPDWXUHZHWKHUVKHHSZHUHKRXVHGLQGLYLGXDOO\LQPHWDEROLFFDJHVDQGIHGDWJ'0
kg BW0.75SHUGD\7KHWUHDWPHQWVZHUHVLODJHVRIWKHVRUJKXPK\EULGV%56%5DQG%56
KDUYHVWHGDWWKUHHPDWXUDWLRQVWDJHV PLONVRIWGRXJKDQGÀRXU\ 7KHVRUJKXPK\EULGVZHUH
developed by Embrapa Maize and Sorghum (Sete Lagoas, Minas Gerais, Brazil). Diets were fed
for a 20 day adaptation followed by a 5 day collection period. Oxygen consumption, carbon dioxide
production, and methane emissions were monitored for a period of 24 h on individual sheep one after
the other, in an open-circuit respiration chamber for small ruminants, after adaptation period of two
days in another similar respiration chamber. The chambers were made of transparent acrylic resin
plates with external dimensions of 1.2 m (wide) × 2.0 m (height) × 2.1 m (length). The equipment
and procedure utilized for the respiration study were described by Rodriguez et al. (2007). Heat
SURGXFWLRQDQGIDVWLQJKHDWSURGXFWLRQZHUHFDOFXODWHGDVSHU%URXZHU¶VHTXDWLRQ %URXZHU 

The experimental design utilized was completely randomized in a 3×3 factorial arrangement. Analysis
of variance (ANOVA) was used to analyse data using the General Linear Model Procedure (SAS,
2001). Main effects and interactions of hybrid and maturation stage were evaluated. Treatments
means were differentiated using SNK test (SAS, 2001).

Results and discussion

There were no differences among the treatments for the apparent digestibility of gross energy
and metabolizability (P> $VLJQL¿FDQW P<0.005) interaction between sorghum hybrid and
PDWXUDWLRQVWDJHZDVREVHUYHGIRUWKHHI¿FLHQF\RI0(XWLOL]DWLRQIRUPDLQWHQDQFH .m). Silage
harvested at the soft dough stage had higher net energy to gross energy ratio (NE/GE) than that
KDUYHVWHGDWÀRXU\VWDJH P<0.05).

No differences were observed among the methane output, as liter per day, by sheep that received
VLODJHRIWKHK\EULG%56 ZLWKRXWWDQQLQ DQGWKRVHVKHHSIHGVLODJHVRIWKHK\EULGV%56DQG
%5 ZLWKWDQQLQ /LNHZLVH2OLYHLUDet al.  IRXQGQRHIIHFWRIGLHWVFRQWDLQLQJVRUJKXP
silage with low and high tannin content on methane emission by beef cattle. The methane emission
expressed as g per kg of digestible dry matter intake (g/kg DDM) and g per kg of digestible neutral
GHWHUJHQW¿EHULQWDNH JNJ'1') GLIIHUHGDPRQJPDWXUDWLRQVWDJHVZLWKVRIWGRXJKVWDJHKDYLQJ
ORZHUHPLVVLRQWKDQÀRXU\VWDJH P<0.05).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 497
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_184, © Wageningen Academic Publishers 2013
The lack of differences in total daily methane output indicates that sorghum genetics and maturity
at harvest should not be a strategy to reduce enteric methane emissions from ruminants.

7DEOH(QHUJ\HI¿FLHQF\DQGPHWKDQHHPLVVLRQE\VKHHSIHGVLODJHVRIWKHVRUJKXPK\EULGV%56
%5DQG%56LQWKUHHPDWXUDWLRQVWDJHV PLONVRIWGRXJKDQGÀRXU\ 

Hybrid MS ADGE qm Km NE CH4 CH4 CH4

(%GE) (l/d) (g/kg DDM) (g/kg NDFD)

%56 M 50.80 0.44 Aa 30.71 20.93 29.85 59.39

SD 57.97 0.52 Aa 40.12 19.15  
F 51.54  Aa  22.44  73.02
BR 700 M 55.77 0.50 Aa 34.51 18.01 23.88 49.42
SD 48.00 0.43 0.72Aa 31.38  24.23 51.57
F 52.07 0.45 0.71Aa 32.49   
%56 M 54.33 0.47 Ab 28.98 22.28 33.75 
SD   0.78Aa 35.99   
F  0.42 0.53Bc 22.58 20.32 32.10 
SEM  0.03 0.03 2.72  3.43 7.59
Main effects
Hybrids
%56 53.44 0.47 0.71 34.10 20.84  59.70
BR 700 51.95  0.70 32.79 19.73 27.49 
%56 51.72 0.45  29.18 18.75 30.04 
Maturation stage
M  0.47  31.40ab 20.41 ab ab
SD 52.55 0.47 0.75 35.83a  24.19b 48.13b
F 50.92 0.44  28.84b 22.41 31.15a a
3UREDELOLWLHV3”
H 0.7284 0.5182 0.0020 0.0871  0.5091 0.8482
MS 0.5122  0.0000  0.1049 0.0489 0.0088
H x MS  0.1113 0.0015 0.0583   0.7173

06 PDWXUDWLRQVWDJH0 PLON6' VRIWGRXJK) ÀRXU\$'*( DSSDUHQWGLJHVWLELOLW\RIJURVVHQHUJ\TP

PHWDEROL]DELOLW\.P HI¿FLHQF\RI0(XWLOL]DWLRQIRUPDLQWHQDQFH1( *(  QHWHQHUJ\WRJURVVHQHUJ\UDWLR
 ''0 GLJHVWLEOHGU\PDWHU1')' GLJHVWLEOHQHXWUDOGHWHUJHQW¿EHU6(0 VWDQGDUGHUURURIPHDQ+ 
VLJQL¿FDQFHIRUWKHK\EULGHIIHFW06 VLJQL¿FDQFHIRUPDWXUDWLRQVWDJHHIIHFW+î06 VLJQL¿FDQFHIRUK\EULGî
maturation stage interaction.
Means of the hybrids followed by the same capital letters in columns and means of the maturations stages followed by
WKHVDPHORZHUFDVHOHWWHUVLQFROXPQVGRQRWGLIIHUVLJQL¿FDQWO\E\61.WHVW P<0.05).

References
%URXZHU(5HSRUWRIVXEFRPPLWWHHRQFRQVWDQWVDQGIDFWRUV3URFUG6\PSRVLXPRQ(QHUJ\0HWDEROLVP
Eur. Assoc. Anim. Prod. Pub. No. 1, 441-443.
Oliveira, S. G., T. T. Berchielli, M. S. Pedreira, O. Primavesi, R. Frighetto, A. A. Lima. 2007. Effect of tannin levels
in sorghum silage and concentrate supplementation on apparent digestibility and methane emission in beef cattle.
$QLP)HHG6FL7HFKQRO
Rodríguez, N. M., W. E. Campos, M. L. Lachica, I. Borges and L. C. Gonçalves. 2007. A calorimetry system for
metabolism trials. Arq. Bras. Med. Vet. Zootec. 59, 495-500.
Sanchez, A. C.,P. K. Subudhi, D. T. Rosenow, H. T. Nguyen. 2002. Mapping QTLs associated with drought resistance
in sorghum (Sorghum bicolor /0RHQFK -3ODQW0RO%LRO
SAS, 2001. Release 8.01. SAS Inst. Inc., Cary, NC, USA.

498 Energy and protein metabolism and nutrition in sustainable animal production
Methane emission from lactating cows fed diets with different forage base
S. Colombini, L. Rapetti, G. Galassi, L. Malagutti, M. Pirondini and G.M. Crovetto
8QLYHUVLW\RI0LODQ'HSDUWPHQWRI$JULFXOWXUDODQG(QYLURQPHQWDO6FLHQFHVYLD&HORULD
Milan, Italy; [email protected]

Introduction
Enteric methane (CH4) emissions from ruminant livestock are major contributors to anthropogenic
emission of greenhouse gases (GHG) and diet manipulation is the most direct mean of lowering
CH4 emissions from ruminants (Beauchemin et al., 2008). Nutrition and feeding management is
a very broad area, with many opportunities for mitigating CH4 emissions (Knapp et al., 2011). A
typical diet fed to dairy cattle in Northern Italy is based on corn silage as main forage; however, corn
requires several agronomical inputs which also contribute to GHG emissions. Sorghum is growing in
popularity as a silage crop, primarily because it requires less water and agronomical inputs than corn.

Colombini et al. (2012) showed that whole plant grain sorghum silage can replace corn silage in
dairy cow TMR without negative effects on milk production. In the same study, the forage sorghum,
probably chopped too long, negatively affected dry matter intake (DMI) and milk production. The
aim of the present research was to study the effect of the TMR evaluated in the previous study
(Colombini et al., 2012), and with a different forage base: corn (CS), whole plant grain sorghum
(WPGS) or forage sorghum (FS) silages, on enteric CH4 emissions from dairy cows.

Material and methods

Six lactating primiparous Italian Friesian cows were used. Three diets with a different silage base
ZHUHWHVWHG&6:3*6RU)6'LHWVZHUHEDODQFHGWRKDYHDFRQWHQW '0 RIDQG
RIPHWDEROL]DEOHSURWHLQ1')DQGVWDUFKUHVSHFWLYHO\%HFDXVHRIWKHGLIIHUHQW¿EHUDQGVWDUFK
contents, the three forages were included in the experimental diets in different proportions, and corn
meal was needed at different proportions to compensate for the lower starch content of the WPGS
and FS silages. The cows were fed the experimental diets in a replicated 3×3 Latin square design
with 28-d period. In each period cows were placed in individual respiration chamber to register CH4
production for seven days continuously and to collect the daily amount of feces produced. Data were
statistically analyzed by the Mixed procedure of SAS (SAS Institute, 2001).

Results and discussion

Dry matter intake, milk yield and CH4 emission of cows fed the different experimental diets are given
in Table 1. A linear trend was observed for an effect of the kind of silage on DMI (3 0.07). Particularly,
the FS diet resulted in a lower DMI, probably due to the greater particle size of FS diet. Despite a
trend for a difference in DMI, total methane production (L/d) was not affected by diet. The passage
rates of the iNDF fraction (%/h), predicted according the equation of Krizsan et al. (2010), were:
2.19, 2.19 and 2.12 for diets CS, WPGS and FS, respectively. Consequently, a higher retention time
in the rumen for FS diet can be hypothesized which in turn increases the extent and rate of ruminal
GLHWDU\IHUPHQWDWLRQDVFRQ¿UPHGE\WRWDOWUDFW¿EHUGLJHVWLELOLW\ZKLFKZDVKLJKHU 3 0.01) for SF
GLHW  WKDQ&6  DQG:3*6  DVUHSRUWHGLQDSUHYLRXVSDSHU &RORPELQLet al.,
2012). As a consequence, there were differences among diets when CH4 production (l) was expressed
per kg of DMI (3 0.09) or per kg of NDF intake (3 0.02). Particularly, cows fed FS diet had the
KLJKHVWPHWKDQHSURGXFWLRQIRUNJRI1')LQWDNH YVDQGONJ1')LQWDNHIRU)6
CS and WPGS, respectively). Methane production per milk yield (l/kg) was: 18.7, 19.7 and 20.4 for
CS, WPGS and FS, respectively (3 0.12). Methane energy (% of the gross intake energy) was higher
for SF diet than CS and WPGS diets (3  6LJQL¿FDQWOLQHDUUHJUHVVLRQVZHUHIRXQGEHWZHHQ

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 499
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_185, © Wageningen Academic Publishers 2013
CH4 (l/d) and starch and NDF intakes (kg/d): CH4=388+17.3×starch intake (R2=0.18, 3 0.08) and
CH4 î1')LQWDNH 52=0.48, 3  6LJQL¿FDQWOLQHDUDQGTXDGUDWLFUHJUHVVLRQVZHUH
found between the percentage of CH4 gas energy loss on total gross energy (MGE) and the percentage
of the dietary particles retained on a 19 mm sieve (DP19): MGE=4.88+0.039×DP19 (3 0.002;
R2  0*( î'3±î'32 (3 0.002; R2=0.59).

Conclusions
Sorghum forage is an interesting crop for low agronomic inputs. However, in this study, despite a
ODFNRIVLJQL¿FDQWGLIIHUHQFHLQDEVROXWH&+4 emission, the SF diet increased the percentage of CH4
energy loss and CH4 emission per kg of NDF intake.

Table 1. Dry matter intake, milk yield and CH emission of the cows on experiment.

CS WPGS FS SE P-value

DMI kg/d 20.0 20.0 18.2 0.53 0.07

Milk yield kg/d 25.4a ab b 0.43 0.05
CH4 l/d  483 478  0.74
CH4/DMI l/kg DMI 23.8 24.3  0.94 0.09
CH4/NDF l/kg NDF intake b ab 71.3a 2.20 0.02
CH4/MILK l/kg milk 18.7 19.7 20.4 0.75 0.12
CH4 energy % gross energy 5.1b 5.2b 5.8a 0.21 0.04

a,b Means in the same rows with different superscript are different for P<0.05.

References
Beauchemin K.A, M. Kreuzer, F. O’Mara and T. A. McAllister, 2008. Nutritional management for enteric methane
abatement: a review. Aust. J. Exp. Agr. 48:21-27.
&RORPELQL6**DODVVL*0&URYHWWRDQG/5DSHWWL0LONSURGXFWLRQQLWURJHQEDODQFHDQG¿EHUGLJHVWLELOLW\
prediction of corn, whole plant grain sorghum, and forage sorghum silages in the dairy cow. J. Dairy Sci. 95:4457-

Knapp J.R., J.L. Firkins, J.M. Aldrich, R.A. Cady, A.N. Hristov, W.P. Weiss, A.D.G. Wright and M.D. Welch, 2011. Cow
of the future research priorities for mitigating enteric methane emissions from dairy. Working draft. Innovation center
for U.S. dairy. http://www.usdairy.com/Public%20Communication%20Tools/CowoftheFutureWhitePaper_7-25-11.
pdf
Krizsan S.J., S. Ahvenjärvi and P. Huhtanen, 2010. A meta-analysis of passage rate estimated by rumen evacuation
with cattle and evaluation of passage rate prediction models. J. Dairy Sci. 93:5890-5901.
SAS Institute. 2001. User’s Guide: Statistics. Release 8.01. SAS Inst. Inc., Cary, NC.

500 Energy and protein metabolism and nutrition in sustainable animal production
Effect of condensed tannins on methane emission and ruminal microbial
populations
M. Rira1,2, C. Marie-Magdeleine2, H. Archimède2, D.P. Morgavi1 and M. Doreau1
1,15$805+HUELYRUHV6DLQW*HQqV&KDPSDQHOOHDQG&OHUPRQW8QLYHUVLWp9HW$JUR
6XS805+HUELYRUHV%3&OHUPRQW)HUUDQG)UDQFH[email protected]
28QLWpGH5HFKHUFKHV=RRWHFKQLTXHV,15$3ULVHG¶(DX3HWLW%RXUJ*XDGHORXSH)UDQFH

Introduction
Enteric methane (CH4) produced by domestic ruminants represents approximately 15% of the global
emissions of this potent greenhouse gas. For reducing rumen CH4 emission various compounds
have been tested as feed additives. Among these compounds, tannins are considered a promising
group of natural additives. A meta-analysis by Jayanegara et al. (2012) showed that condensed and
hydrolysable tannins might reduce CH4 production. However, it is still unclear (1) whether tannin
supplementation reduces rumen CH4 in every situation and (2) to which extent this is associated with
adverse effects on digestibility and their potential toxicity to some rumen micro-organisms (Goel et
al., 2005). In this experiment we investigated the effect of tanniniferous tropical plants on enteric
CH4 production and on numbers of methanogens, protozoa, and total and main cellulolytic bacteria.

Material and methods

Two sheep breeds were used, Texel (T, n=4) of temperate origin and Blackbelly (B, n=4) of tropical
origin in two 4×4 Latin square designs. Diets, given ad libitum twice daily, consisted in tropical natural
grassland based on Dichanthium spp. fed alone (C) or in association with 3 different tanniniferous
forages given as pellets at 44% of the daily ration on average. The tanniniferous forages were leaves
of Leucaena leucocephala (L), Glyricidia sepium (G) or Manihot esculenta (M). Total contents in
condensed tannins measured by the vanillin-H2SO4 method were 75, 39 and 92 g/kg dry matter
(DM) for L, G and M, respectively. Intake, total tract digestibility and CH4 production using the
SF method were determined. For microbial parameters, rumen contents were sampled before the
morning feeding. Microbial groups (total bacteria, Fibrobacter succinogenes, Ruminococcus albus,
5XPLQRFRFFXVÀDYHIDFLHQVand total methanogens) were enumerated by quantitative PCR (qPCR)
XVLQJJURXSVSHFL¿FSULPHUVWDUJHWLQJWKHrrs gene for bacteria and the mcrA gene for methanogens.
Protozoa were counted by microscopy. Statistical analyses were performed using the mixed procedure
RI6$6ZLWKSHULRGGLHWEUHHGDQGWKHGLHW[EUHHGLQWHUDFWLRQDV¿[HGHIIHFWVDQGDQLPDODVUDQGRP
HIIHFW6WDWLVWLFDOGLIIHUHQFHVZHUHGHFODUHGVLJQL¿FDQWDW3”2UWKRJRQDOFRQWUDVWVEHWZHHQ
control diet and all tanniniferous forages were also determined.

Results and discussion

The addition of tannin-rich plants given as pellets increased DM intake probably due to the physical
presentation (Table 1). Within tannin-rich plants, it was higher for M than for G diet. Intake per kg
body weight was higher for Blackbelly than for Texel. Organic matter digestibility did not differ
among diets and breeds, although contrast analysis showed a higher digestibility for C than for
other diets. Daily CH4 production did not vary among diets and breeds, but CH4 production per kg
DM intake was higher with C diet compared with tannin-rich diets. Within these latter diets, CH4
production was higher for G than for M and L diets. Concentration of total bacteria and 5ÀDYHIDFLHQV
was higher for C and L diets than for G and M diets; concentration of R. albus was lowest for C diet.
The methanogens population was higher for Texel than for Blackbelly. In contrast the addition of
FRQGHQVHGWDQQLQVGLGQRWLQÀXHQFHWKHSRSXODWLRQRISURWR]RDDQGF. succinogenes.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 501
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_186, © Wageningen Academic Publishers 2013
Table 1. Intake, digestibility, methane emission and ruminal microbial populations in sheep fed
WURSLFDOJUDVVODQGKD\ & DORQHRUDVVRFLDWHGZLWKWDQQLQVFRQWDLQLQJSODQWVLeucaena leucocephala
/ Glyricidia sepium * DQGManihot esculenta 0 

Breed Texel Blackbelly SEM P value1

Diet C L G M C L G M

DM intake, g/kg body  24.45 20.72    24.49 31.54 1.877 B 0.04
weight/d D<0.01
Organic matter     70.12     NS
digestibility, %
CH4, g/kg DM intake  24.23 28.83 24.28 29.98 15.89 24.98  2.147 B 0.03
D<0.01
Protozoa, log10 cells/ml 4.97 5.01 5.04 5.03  5.01 4.97   NS
Total bacteria2 11.91 11.90 11.80 11.84 11.89  11.85 11.81 0.041 D 0.04
F. succinogenes2 9.71 9.79  9.72  9.85   0.079 NS
R. Albus2 8.00 8.79 8.33 8.49  8.42 8.34   D 0.04
5ÀDYHIDFLHQV2  8.71 8.53  8.94 9.03  8.85 0.154 D 0.05
Methanogens3 10.00 9.85 9.72 9.84  9.71 9.70 9.74 0.082 B 0.04

1% EUHHG' GLHW%UHHGîGLHWLQWHUDFWLRQZDVQHYHUVLJQL¿FDQW

2 rrs copy number /g DM (log10)
3 mcrA copy number /g DM (log )
10

2XUUHVXOWVFRQ¿UPWKDWWDQQLQULFKSODQWVFDQOLPLW&+4 production per kg DM intake. The low

effect of Glyricidia sepium on reduction of CH4 production could be explained by a low tannin
concentration or by the intrinsic characteristics of Glyricidia sepium tannins. The presentation of
tannin-rich plants as pellets probably decreased ruminal DM retention time which resulted in an
increase in DM intake and can partially explain the reduction in CH4 production. As methanogens
and protozoal numbers were not changed, further research is necessary to elucidate the relations
between methane production and microbial activity in tannin-rich diets.

Acknowledgements
This research is a part of the AnimalChange project funded by the European Community’s FP7
Programme. It was also funded by other EU funds (FEDER, FEADER) and by the Guadeloupe region.
7KH¿UVWDXWKRUUHFHLYHGDIHOORZVKLSIURPWKH$OJHULDQ0LQLVWU\RI+LJKHU(GXFDWLRQDQG5HVHDUFK

References
*RHO*$.3XQL\D&1$JXLODUDQG.6LQJK,QWHUDFWLRQRIJXWPLFURÀRUDZLWKWDQQLQVLQIHHGV
Naturwissenschaften 92, 497-503.
Jayanegara, A., F. Leiber, and M. Kreuzer, 2012. Meta-analysis of the relationship between dietary tannin level and
PHWKDQHIRUPDWLRQLQUXPLQDQWVIURPLQYLYRDQGLQYLWURH[SHULPHQWV-$QLP3K\VLRO$QLP1XWU

502 Energy and protein metabolism and nutrition in sustainable animal production
Methane emission by cattle supplemented with additives in Brazil
A.L.C.C. Borges1, M.P. Fonseca1, R.R. Silva1, H.F. Lage1, J.A.S. Rodrigues2, L.C. Gonçalves1, I.
Borges1 and E.O.S. Saliba1
1Department of Animal Science, Federal University of Minas Gerais, UFMG, Brazil;
[email protected]
2Brazilian Agricultural Research Corporation, Brazil

Introduction
Energy is a limiting factor to life and productive functions of animals. The knowledge of gas
SURGXFWLRQE\DQLPDOVDFFRUGLQJWRWKHGLHWGHPRQVWUDWHVWKHSDUWLDOHI¿FLHQF\RIHQHUJ\XWLOL]DWLRQDV
an important indicator of feed utilization. Virginiamycin is a non-ionophore antibiotic, with potential
effect on improvement of ruminal fermentation and features indicative of an increase in dry matter
intake. This antibibiotic acts by penetrating the bacterial cell wall, binding to the 50 S ribosomal
subunit and blocking protein synthesis through the inhibition of peptide bond formation (Cocito,
1979). Monensin, an ionophore antibiotic, has antimicrobial activity against Gram positive bacteria
and subsequent alterations in ruminal fermentation products, namely an increase in propionate at the
expense of acetate and methane (Nagaraja et al., 1997). This study aimed to evaluate the inclusion
of additives monensin, virginiamycin and its combination on methane emission determined by
respirometric chamber in F1 Crossbred Holstein x Gir bulls.

Material and methods

Twenty F1-crossbred (Bos taurus indicus) × Holstein (Bos taurus taurus) bulls (initial average body
ZHLJKWNJ¿QDODYHUDJHERG\ZHLJKWNJ ZHUHUDQGRPO\DVVLJQHGWRIRXUJURXSVRI¿YH
animals each and fed four diets: a control diet (C, with 50% sorghum silage with Tanzania grass
(Panicum maximum Jacq vr. Tanzânia l.), 9.89% soybean meal, corn meal 37.23%, 0.72% urea, in
dry matter basis), a diet with inclusion of monensin (M), a diet with inclusion of virginiamycin (VM)
and a diet with inclusion of monensin and virginiamycin (M+VM). Monensin and virginiamycin were
included in the formulation of concentrates at doses of 22 and 30 mg/kg dry matter, respectively.
Roughage and concentrate were supplied at a ratio of 50:50. The diets were fed twice daily as a total
mixture ration. Dry matter intake was monitored during all the experiment. The animals were fed ad
libitum. The production of methane was obtained individually by respirometric chamber through the
open circuit system available at the Animal Science Department of Veterinary School of UFMG and
described by Rodriguez et al. (2007). The experiment was conducted in a completely randomized
design. Data were analyzed by SAEG, described by Ribeiro (2001), and means were compared by
Tukey test at 5% probability (P<0.05).

Results and discussion

Table 1 reports the production of methane by the supplemented bulls. The methane emission was
lower (P<0.05) for animals supplemented with both additives, when expressed in liters by day (l/d),
liters by kilogram of dry matter (l/kg DM) and liters by kilogram of digestible dry matter (l/kg DIG
DM). The reduction in methane production with combined use of monensin and virginiamycin is
probably more related the reduction of the precursors of methane, H2 and formate. A direct effect
PLJKWKDYHUHVXOWHGIURPDFKDQJHLQUXPLQDOS+ %OD[WHU &ODSSHUWRQ EXWVLQFHQR
UHGXFWLRQZDVREVHUYHGLQIHHGLQWDNHDQGGLJHVWLELOLW\RIWKH¿EHUDQGGLHWVKDGQHXWUDO
GHWHUJHQW¿EHU 1') WKLVGRHVQRWVHHPOLNHO\5DWKHUFKDQJHVLQWKHJDVWURLQWHVWLQDOPLFURELRWD
PD\KDYHEHHQUHVSRQVLEOH)XUWKHUVWXGLHVKDYHWRVKRZZKHWKHUWKHNQRZQJUHDWHUHI¿FLHQF\LQ
converting to metabolizable energy and improving the utilization of metabolizable energy is also
realized under tropical conditions.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 503
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_187, © Wageningen Academic Publishers 2013
7DEOH0HDQYDOXHVSUREDELOLW\ PYDOXH DQGFRHI¿FLHQWRIYDULDWLRQ &9 RISURGXFWLRQRI
methane by supplemented bulls.

Variables1 Treatment P-value CV (%)

C M VM M+VM

CH4 (l/day) a 153.78ab 171.57ab 137.70b 0.0298 

l CH4/kg DM a 24.97ab 27.94ab 23.27b 0.0342 11.48
l CH4/kg DIG DM 48.21a ab 45.15ab 38.84b 0.0484 
l CH4/kg NDF a 58.01a a 57.27a  
l CH4/kg DIG NDF a a 135.10a 147.75a 0.2853 28.81
l CH4/kg OM a 25.74a 30.89a a 0.1520 15.40
LCH4/kg DIG OM 51.70a a 49.93a 45.07a 0.2909 
l/kg BW0,75 3.19a 2.53a 2.95a 2.42a 0.0548 
l CH4/kg gain a 107.25a 127.18a 108.24a  14.27

a,bP<0.05.
1Methane production expressed in liters per day (l/day), liters per kilogram of dry matter (l/g DM) liters per kilogram
RIGLJHVWLEOHGU\PDWWHU ONJ',*'0 OLWHUVSHUNLORJUDPRILQVROXEOH¿EHUQHXWUDOGHWHUJHQW ONJ1') OLWHUVSHU
NLORJUDPRIQHXWUDOGHWHUJHQW¿EHUGLJHVWLEOH ONJ',*1') OLWHUVSHUNLORJUDPRIRUJDQLFPDWWHU ONJ20 OLWHUVSHU
kilogram of digestible organic matter (l/kg DIG OM), liters per kilogram of metabolic weight (l/kg BW0.75) and liters
per kilogram of gain (l/kg gain).

Conclusion
The supplementation with 30 mg/kg DM of virginiamycin associated with 22 mg/kg DM of monensin
OLNHO\SURPRWHVHQHUJ\HI¿FLHQF\LPSURYHPHQWRIGLHWGXHWRWKHUHGXFWLRQLQPHWKDQHSURGXFWLRQ
without compromising nutrient intake.

Acknowledgment
The authors thank CNPq, CNPq-INCT, FAPEMIG, CAPES and EMBRAPA CORN AND SORGHUN,
for their cooperation in carrying out this work.

References
%OD[WHU./-/&ODSSHUWRQ3UHGLFWLRQRIWKHDPRXQWRIPHWKDQHSURGXFHGE\UXPLQDQWV%U-1XWU
511-522.
Cocito, C, 1979. Antibiotics of the virginiamycin family, inhibitors which contain synergistic components. Microb.
Rev. 43, 2, 145-198.
Hobson, P.N. and C.S. Stewart, 1997. Rumen Microbial Ecosystem. In: Nagaraja, T.G., C.J. Newbold, C.J. Van Nevel
DQG','HPH\HU0DQLSXODWLRQRIUXPHQDOIHUPHQWDWLRQ%ODFNLH$FDGHPLFDQG3URIHVVLRQDO/RQGRQ
Ribeiro Jr. J.I. SAEG (A system for statistical analysis), 2001. Federal University of Viçosa, Viçosa, MG, Brazil.
Rodríguez, N.M., Campos, W.E., Lachica, M.L., Borges, I., Gonçalves, L.C., Borges, A.L.C. C. Saliba, E.O.S, 2007.
A calorimetry system for metabolism trials. Arq. Bras. Med. Vet. Zoot. 59, 2, 495-500.

504 Energy and protein metabolism and nutrition in sustainable animal production
Effect of paddy rice diets on performance in chickens under
thermoneutral and heat stress conditions
F. Nanto, C. Ito, M. Kikusato, S. Ohwada and M. Toyomizu
Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, Japan;
[email protected]

Introduction
The global demand for corn to be used in the production of feed and fuel is increasing at a rapid
rate. Many types of grain have been proposed as substitutes for corn in broiler chicken diets, thereby
serving as alternative sources of dietary carbohydrates. We have already demonstrated that paddy rice
show some potential for use as a substitute for corn in poultry feed under thermoneutral conditions
(Nanto et al 7RWKHDXWKRUV¶NQRZOHGJHYLUWXDOO\QRLQIRUPDWLRQLVDYDLODEOHWRFRQ¿UPD
direct effect of the rice feeding on performance on chickens exposed to heat stress. Heat stress is a
major issue for the poultry industry because of the growth retardation and high mortality. We have
previously found that heat stress induces oxidative damage, resulting in growth performance in birds
(Azad et al., 2010). Furthermore, it has been reported that intestinal morphology was altered by
heat stress (Quinteiro-Filho et al )URPWKHVH¿QGLQJVZHFRXOGK\SRWKHVL]HWKDWR[LGDWLYH
stress and intestinal morphology might be factor responsible for growth retardation under heat
stress conditions. Therefore, the present study was conducted to not only determine the effects of
whole-grain paddy rice-based diets on growth performance in birds under heat stress conditions,
but also to clarify the involvement of intestinal morphological alterations and oxidative stress in the
performance under chronic heat stress conditions.

Material and methods

Forty-eight chicks (ROSS) at 0 day old were divided into 3 groups that were fed one of the following
3 experimental diets ad libitum for 28 day: one of corn-based diets (CP: 20%, ME:3.1 kcal/g, fat
content:5.5%), another two ME levels of whole-grain paddy rice-based diets: standard ME rice diet
(CP: 20%, ME: 3.1 kcal/g, fat content: 11.5%) and low ME rice diet (CP: 20%, ME: 2.8 kcal/g, fat
content: 5.5%). At 21 d of age, birds in the each groups were randomly divided into two sub-groups
(n=8), and then one of the two groups were exposed to heat stress (33 °C for 7 days) while the
UHPDLQLQJJURXSZDVPDLQWDLQHGDWƒ&$WWKHHQGRIWKHH[SHULPHQWDOOFKLFNHQVZHUHVDFUL¿FHG
by decapitation. Tissues were immediately frozen, powdered in liquid nitrogen and stored at -80 °C
until analyzed.

A one cm length of intestinal tissue from each chicken was collected from the midpoint of the
duodenum and stored in 10% formalin neutral buffer solution prior to morphological analysis,
ZKLFKZDVSHUIRUPHGRQȝPWKLFNVHFWLRQVFXWRQDPLFURWRPH6HFWLRQVZHUHVWDLQHGXVLQJWKH
hematoxylin-eosin method. Villus height and crypt depth were measured with the aid of a microscope
from 10 randomly selected villi and associated crypts on two sections per chicken. Plasma endotoxin
concentrations were determined by a chromogenic Limulus amoebocyte lysate (LAL) end-point
assay (QCL-1000, Lonza Group Ltd., Basel, Switzerland). Lipid peroxidation levels in tissues were
determined colorimetrically in terms of the production of TBARS. Chemiluminescence (CL) intensity
of the pectoralis major muscle was measured as free radical reaction and lipid peroxidation after
incubation for 10 minutes at 100 °C with N2ÀRZ3ODVPDFHUXORSODVPLQ &HU FRQFHQWUDWLRQWKDWLVDQ
LQGLFDWRURILQÀDPPDWRU\UHVSRQVHZDVPHDVXUHGZLWKWKHp-phenylenediamine colorimetric method.

'DWDZHUHDQDO\]HGXVLQJWKHVWDWLVWLFDODQDO\VLVV\VWHP'DWDZHUH¿UVWDQDO\]HGE\DJHQHUDOOLQHDU
PRGHODQDO\VLVRIYDULDQFHSURFHGXUHDQGWKHPHDQVZHUHFRPSDUHGXVLQJ'XQFDQ¶VOHDVWVLJQL¿FDQFH
PXOWLSOHUDQJHWHVW&RUUHODWLRQDQDO\VLVZDVDVVHVVHGXVLQJWKH3HDUVRQFRUUHODWLRQFRHI¿FLHQW

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 505
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_188, © Wageningen Academic Publishers 2013
Results and discussion
Under thermoneutral condition, body weight gain (BWG) of birds fed both rice-based diets was
VLJQL¿FDQWO\GHFUHDVHG P<0.05) compared with corn-based diet, with the standard-ME rice diet-
fed chickens showing a reduced tendency to lose weight compared with low-ME diet-fed animals
)LJXUH$ 2QH[SRVXUHWRFKURQLFKHDWVWUHVV%:*RIELUGVIHGFRUQEDVHGGLHWZHUHVLJQL¿FDQWO\
decreased. Heat-stressed birds fed both standard- and low-ME rice diets showed a decrease in the
BWG compared with control diet-fed heat-stressed birds, but birds fed low-ME rice diet showing
a tendency of improved growth compared with standard-ME diet-fed birds. On exposure to heat
VWUHVVWKHYLOOXVKHLJKWFU\SWGHSWKUDWLRZDVVLJQL¿FDQWO\GHFUHDVHGLQELUGVIHGDFRQWUROGLHW
(Figure 1B). Heat-stressed birds fed both standard- and low-ME rice diets showed slightly decrease
in the villus height: crypt depth ratio compared with control diet-fed heat-stressed birds. Meanwhile,
plasma endotoxin concentration (which is one of indexes of intestinal barrier dysfunction) was
VLJQL¿FDQWO\LQFUHDVHGE\KHDWVWUHVVLQELUGVIHGHLWKHUFRUQRUULFHGLHWDQGWKHGHJUHHVRIWKH
increase due to heat stress was enhanced in both standard- and low-ME rice diet groups compared
to control diet-fed heat-stressed ones (Figure 1C). We further investigated the relationship between
the intestinal morphology and plasma endotoxin concentration of birds under thermoneutral and
heat-stressed conditions. As a result, a negative correlation between plasma endotoxin concentration
and the villus height: crypt depth ratio was observed (r=-0.534, P<0.0001; Figure 2), suggesting that
LQWHVWLQDOPRUSKRORJ\GDPDJHPLJKWWULJJHULQÀX[RIHQGRWR[LQIURPLQWHVWLQHLQWREORRGXQGHUKHDW
stress conditions. Moreover, negative correlation between the plasma endotoxin concentration and
CL intensity of muscle (r 3 0.0029) or TBARS content of the liver (r 3 0.020)
LPSOLHGWKDWWKHSODVPDHQGRWR[LQPLJKWLQGXFHR[LGDWLYHGDPDJH7KHUHLVQRVLJQL¿FDQWFRUUHODWLRQ
between the plasma endotoxin concentration and plasma Cer concentration among all the groups.

A. Body weight gain

800 a
(g/7 d)

b b
400 c
d cd

0
B. Villus height: Crypt depth
15 a a
a b
10 b b

5
0
C. Plasma endotoxin concentration
50 a
a
ab
bc
(EU/ml)

25 c c

0
Control Standard- Low- Standard- Low-
ME rice ME rice Control ME rice ME rice
Thermo neutral Heat stress
Figure 1. Effects of corn-based diet and two ME levels of whole-grain paddy rice-based diets
VWDQGDUGDQGORZ RQERG\ZHLJKWJDLQ $ YLOOXVKHLJKWFU\SWGHSWK % DQGSODVPDHQGRWR[LQ
FRQFHQWUDWLRQ & RIEURLOHUFKLFNHQVH[SRVHGWRWKHUPRQHXWUDODQGFKURQLFKHDWVWUHVV GHJUHHV
G 9DOXHVDUHPHDQV“6(Q SHUJURXS
a,b,c P<IRUHDFKWUHDWPHQWYDOXHVZLWKGLIIHUHQWOHWWHUVDUHVWDWLVWLFDOO\GLIIHUHQW

506 Energy and protein metabolism and nutrition in sustainable animal production
 16

Villus hight: crypt depth ratio

12

4
r =-0.534 , P< 0. 0001
0
0 20 40 60 80
Plasma endotoxine concentration (IU/ml)

Figure 2. The relationship between the villus height and plasma endotoxin concentration.

Nevertheless, heat-exposed birds fed standard ME diet showed remarkable increases of plasma
endotoxin and Cer compared with control diet-fed heat-stressed birds.

Taken together, the present study demonstrated that the growth performance due to chronic heat
stress in the standard-ME rice diet-fed chickens was further decreased compared to that of the corn-
fed chickens, but this decrease was ameliorated in the low ME rice diet-fed chickens. Furthermore,
we suggest that heat stress-induced intestinal morphology damage might be partly responsible for
WKHLQFUHDVHVLQR[LGDWLYHGDPDJHDQGLQÀDPPDWRU\UHVSRQVHSUREDEO\YLDDQHQGRWR[LQLQÀX[
into blood.

References
Azad, M.A.K., Kikusato, M., Maekawa, T., Shirakawa, H. and Toyomizu M, 2010. Metabolic characteristics and
oxidative damage to skeletal muscle in broiler chickens exposed to chronic heat stress. Comp. Biochem. Physiol.
$
Nanto, F., Kikusato, M., Ito, C., Sudo, S. and Toyomizu M, 2012. Effects of Dehulled, Crushed and Untreated Whole-
Grain Paddy Rice on Growth Performance in Broiler Chickens. The Journal of Poultry Science, 49: 291-299.
Quinteiro-Filho, W.M., Ribeiro, A., Ferraz-de-Paula, V., Pinheiro, M.L., Sakai, M., Sá. L.R., Ferreira, A.J. and Palermo-
Neto J, 2010. Heat stress impairs performance parameters, induces intestinal injury, and decreases macrophage
activity in broiler chickens. Poultry Science, 89: 1905-14.

Energy and protein metabolism and nutrition in sustainable animal production 507
Effect of invertebrates on growth performance and feeding behavior of
red-legged partridge (Alectoris rufa) chicks
M. Lachica and I. Fernández-Fígares
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
Red-legged partridge (Alectoris rufa) has a big ecological importance in Spain and it is under huge
hunting pressure. So it is bred intensively in game farms for restocking areas where the number of
birds is low or even it has been exterminated. About 4 mill birds/year are released. Their natural
GLHWLQFOXGHVSODQWVPDLQO\*UDPLQHDHDQGDOVRDKLJKSURSRUWLRQRILQYHUWHEUDWHVLQWKH¿UVWZN
of life, which is linked with the bird survival index (Green, 1984). The sudden diet change from the
IDUPWRWKH¿HOGVHHPVWREHUHVSRQVLEOHRIDORZVXUYLYDOLQGH[ LQWKH¿UVW\HDU ,QWKLV
phase, studies about nutrition and appetency for animal vs. vegetal feed are scarce or non-existent.
The aim of this work was to determine the impact of the inclusion of invertebrates in a commercial
diet for partridge chicks over its metabolic utilization by obtaining the feed conversion ratio (FCR),
the total protein and gross energy (GE) intake, and the voluntary intake of animal vs. vegetal food
GXULQJWKH¿UVWZNRIOLIH

Material and methods

Twenty-eight 1-2 d old (11.8±0.2 g body weight (BW)) chicks hatched in our lab were allocated
at random by pairs in metabolic cages and fed ad libitum. A control (C; n=14) group was fed with
a commercial starter diet (908 g/kg dry matter (DM), 279 g/kg crude protein (CP) and 18.0 MJ/kg
GE), and a larvae (L; n=14) group could choose between the starter diet and alive Calliphora sp.
ODUYDH JNJ'0JNJ&3DQG0-NJ*( ,QWDNHDQG%:ZHUHUHFRUGHGHYHU\G
for 45 d for determining FCR (g DM feed/g BW gain). Analysis of variance was used to determine
the diet and age effect on FCR, the diet effect on total protein and GE intake, and the proportion
of larvae intake in the group L. Bonferroni multiple range test was used to ascertain the statistical
VLJQL¿FDQFHRIGLIIHUHQFHV

Results and discussion

Table 1 shows the results obtained. BW, similarly as observed by Liukkonen-Anttila et al. (2002)
with Perdix perdix, and FCR were improved in the L (2.05 vs. 2.31). FCR increased from day 30 until
WKHHQGZKHQODUYDHLQWDNHGHFUHDVHGLQGLFDWLQJDQLQDGHTXDWHSURWHLQHQHUJ\UDWLR$VLJQL¿FDQW
GLHWîDJHLQWHUDFWLRQZDVIRXQG)RUWKH&JURXSWKH)&5UDQJHGIURP2]HN  
reported FCR ranging 2.2-3.1 in Alectoris chukar fed diets of similar CP content and growth period.
)RUWKH/JURXS)&5UDQJHGIURP1RVLJQL¿FDQWGLIIHUHQFHVZHUHREVHUYHGEHWZHHQ
&DQG/JURXSVLQSURWHLQ YVJG DQG*(LQWDNHV YVN-G LQGLFDWLQJDEHWWHU
protein quality for the L diet since Met and Cys level is higher in larvae than in plants (Anonymous,
 7KHODUYDHFRPPHUFLDOIHHGLQWDNHUDWLRZDVDQGIRUWKHst, 2nd and 3rd wk,
respectively. Rueda et al.  REWDLQHGUDWLRVRIDQGIRUWKHVDPHSHULRGVLQ
Alectoris rufa in the wilderness. The ratio decreased and reached a steady ratio (0.35) from the 4th
wk until the end of the experiment indicating that birds would always eat invertebrates if they are
available in the habitat.

7KHLQFOXVLRQRILQYHUWHEUDWHVLQWKHGLHWRIKDQGUHDUHGSDUWULGJHFKLFNVGXULQJWKHLU¿UVWZNRIOLIH
improved their growth may be attributable to the intake of protein of better quality. This could be
of importance for the survival of the red-legged partridge in restocking areas.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 509
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_189, © Wageningen Academic Publishers 2013
7DEOH(IIHFWRIGLHW JURXS VDPSOLQJSHULRG 631 DJH DQGLWVLQWHUDFWLRQRQWKHIHHG
FRQYHUVLRQUDWLR )&5J'0IHHGJ%:JDLQ RIAlectoris rufa chicks fed ad libitum with a
FRPPHUFLDOVWDUWHUGLHW &RQWURO&Q  RUEDVHGRQWKHVWDUWHUDQGDOLYHCalliphora sp. larvae
/DUYDH/Q  %RG\ZHLJKW %:J LQWDNH J DQGSHUFHQWDJHRIODUYDHLQWDNH  LQHYHU\63

FCR BW2 Intake2 FCR Larvae

intake
Diet C L

C 2.31a
L 2.05b
SP1
1 1.37ab 14.5  1.88ab 0.85a def
2 1.54ab 18.4 10.8 1.74a 1.33ab 72.0f
3 1.37a 27.5 18.1 1.48a ab 70.1ef
4 abc 37.7 27.7 1.93ab 1.40abc 71.1f
5 1.48ab 51.7 29.9 a 1.31ab def
 abc   a 1.57abc 51.2bcdef
7 abc  40.9 1.85a 1.50abc 47.8abcde
8 1.98abc 105.2 50.5 ab 1.90bcd 53.8cdef
9 2.18cd  51.5 2.22abc 2.14cd ab
10 3.11ef 141.0 71.0 3.20de 3.01ef 30.9abc
11 2.04bc  47.9 2.04ab 2.04bcd 40.5abcd
12 2.71de 179.0 52.1 2.93cde 2.49de 27.8a
13 3.03ef 195.3  2.70bcd f 38.9abc
14 3.41f 208.5  e ef 38.4abc
15 3.51f 227.4  e 3.37f 38.0abc
Diet×SP 0.0073
SEM3 0.029 0.043 0.040 

1 Sampling period every 3 d.

2 Measured at the end of every 3 d period.
3 Standard error of mean; a,b,c,d,e,fZLWKLQDURZYDOXHVZLWKGLIIHUHQWVXSHUVFULSWVGLIIHUVLJQL¿FDQWO\ P<0.05).

Acknowledgements
7KLVUHVHDUFKZDVVXSSRUWHGE\JUDQWQR$*5IURP-XQWDGH$QGDOXFtD 6SDLQ 

References
Anonymous, 1970. Amino-acid content of foods and biological data on proteins. In: F.A.O. Nutritional Studies No.
24. Rome, 285 pp.
Green, R.E., 1984: The feeding ecology and survival of partridge chicks (Alectoris rufa and Perdix perdix) on arable
farmland in East Anglia. J. Appl. Ecol. 21, 817-830.
Liukkonen-Anttila, T., A. Putaala and R. Hissa, 2002. Feeding of hand-reared grey partridge Perdix perdix chicks-
importance of invertebrates. Wildl. Biol. 8, 11-19.
2]HN.7KHRSWLPXPSURWHLQFRQWHQWLQKLJKHQHUJ\VWDUWHUGLHWIRUFKXNDUSDUWULGJH Alectoris chukar chukar).
Int. J. Poult. Sci. 5, 522-525.
Rueda, M.J, J.R. Baragaño and A. Notario, 1992. Alimentación natural de la perdiz roja (Alectoris rufa L.). In: La
perdiz roja, gestión del hábitat, 1ª ed. Fundación ‘La Caixa’, Barcelona, 27-39.

510 Energy and protein metabolism and nutrition in sustainable animal production
Approach to determine the amino acid composition of the natural diet of
red-legged partridge (Alectoris rufa)
I. Fernández-Fígares and M. Lachica
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
$UPLOOD*UDQDGD6SDLQ[email protected]

Introduction
Red-legged partridge (Alectoris rufa) plays a very important role in Spanish ecosystems being part
of the diet of 32 predators (3 reptiles, 9 birds and 20 mammals; Yanes et al., 1998). Also, it has a
big economic importance and, as the favorite game species, it is subjected to an enormous hunting
pressure. As a result, it is bred intensively in game farms to satisfy the hunters’ demand and restock
areas where its number is too low. About 4 millions of birds/yr are released. Hunting season is
performed during the reproductive rest when partridges are in maintenance conditions. NRC (1994)
UHSRUWHGWKHQXWULHQWVUHTXLUHPHQWVIRUJDPHELUGVZLWKRXWVSHFL¿FGDWDIRUSDUWULGJHVDQGLQPDQ\
cases the reader is addressed to requirements of turkeys. Nevertheless, the requirements of free-
OLYLQJELUGVSRWHQWLDOO\GLIIHUVLJQL¿FDQWO\IURPWKRVHRIGRPHVWLFVSHFLHVWKDWKDYHEHHQLQEUHG
for improvement of meat and egg production (Murphy and King, 1982). Therefore, Alectoris rufa
requirements still remain essentially unknown with no information about the amino acids (AA)
composition of the natural diet or differences related to gender. The objective of this work was to
HVWDEOLVKD¿UVWDSSURDFKWRGHWHUPLQHWKH$$FRPSRVLWLRQRIWKHQDWXUDOGLHWRIDGXOWUHGOHJJHG
partridge.

Material and methods

The content of crops and gizzards of 17 birds (7 females and 10 males) hunted during the same
hunting season (October-February; nonbreeding season and postbreeding moult passed) in ‘Sierra
de Baza’ Natural Park (Granada, Spain) was analyzed for dry matter, organic matter (OM) and AA.
After protein hydrolysis, AA were determined by HPLC according to the Waters Pico Tag method.
AA concentration was expressed as mg/g OM to avoid interference by inorganic compounds. To
GHWHUPLQHWKHJHQGHUHIIHFWRQ$$SUR¿OHLQFOXGLQJHVVHQWLDO ($$ QRQHVVHQWLDO$$ 1($$ 
and total AA (TAA), ANOVA-I was used. Bonferroni multiple range test was used to ascertain the
VWDWLVWLFDOVLJQL¿FDQFHRIGLIIHUHQFHV

Results and discussion

Table 1 shows the results obtained. Diet content of females had a higher (P<0.05) concentration of
individual EAA than males (Arg, Ile, Leu, Lys and Phe) and a tendency for higher (P< 0HW
His, Thr and Val; the same occurred with individual NEAA (P<0.09). Likewise females presented
greater values than males for EAA (P<0.05), NEAA (P<0.07) and TAA (P<0.05). Except for Arg,
dietary AA concentration of females was more than twice the one of males.

5HDGLQHVVRIIHDWKHUORVVLVDQHVFDSHEHKDYLRUIRUFDSWXUHGELUGVZKLFKLVVLJQL¿FDQWO\UHODWHGWR
susceptibility to predation (Møller et al. ,WGLIIHUVVLJQL¿FDQWO\EHWZHHQPDOHVDQGIHPDOHV
The role of females in parental care is more important than males in dichromatic species, which may
promote their escape behavior (Moller et al., 2011). On the other hand, the metabolizable energy
HI¿FLHQF\IRUIHDWKHUV\QWKHVLV DURXQG LVORZHUWKDQWKHRQHHVWLPDWHGIRUERG\SURWHLQ 
 LQJURZLQJKRPHRWKHUPV0RUHRYHUGLHWDU\$$SOD\DQLPSRUWDQWUROHLQWKHGHYHORSPHQW
of feathers (Murphy and King, 1984a,b). Females depend entirely on exogenous sources of protein
to satisfy their AA requirements for molting (Hipes and Hepp, 1995). There is a reduction of S
retention during molt by a loss of bone chondroitin sulfate producing a cyclic osteoporosis (Meister,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 511
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_190, © Wageningen Academic Publishers 2013
7DEOH&RQFHQWUDWLRQ PJJ20 RILQGLYLGXDOHVVHQWLDO ($$ QRQHVVHQWLDO 1($$ DQGWRWDO
DPLQRDFLGV 7$$ LQIHPDOHV Q  DQGPDOHV Q  RIAlectoris rufa.

EAA His Arg Thr Val Met Ile Leu Phe Lys

)HPDOHÛ 25.8ª  5.08  2.11  1.57ª 3.24ª 1.98a 
Male b 0.57   0.94 0.30 b 1.30b 0.78b 1.09b
SEM   0.532 0.239    0.438 0.270 

NEAA Ala Asp Cys Glu Gly Pro Ser Tyr TAA

Female 14.1 2.45  0.39 5.22 1.85 2.24 1.94  39.9a
Male 4.9 1.17 1.23 0.18 1.83 0.90 0.80 0.70 0.73 b
SEM 2.34 0.330 0.517 0.052 0.802 0.237 0.387 0.300 0.255 5.41
ab9DOXHVZLWKLQDFROXPQZLWKGLIIHUHQWVXSHUVFULSWOHWWHUVZHUHVLJQL¿FDQWO\GLIIHUHQW P<0.05).

1951). Thus, the protein for feather replacement relies on food protein sparing body proteins from
depletion (Murphy and King, 1984b).

There were differences in the concentration of AA in the natural diet of females and males. It seems
important for the females to have a greater intake of AA than males to maintain a pool of available
$$WRUHSODFHIHDWKHUSURWHLQORVVHYHQRXWRIWKHVSHFL¿FPROWVHDVRQ,WPD\EHDGYLVDEOHWRXVH
diets differing in the proportion of individual AA in the game farms during the maintenance phase
for breeding birds.

Acknowledgements
7KLVUHVHDUFKZDVVXSSRUWHGE\JUDQWQR$*5IURP-XQWDGH$QGDOXFtD 6SDLQ 

References
+LSHV'/DQG*5+HSS1XWULHQWUHVHUYHG\QDPLFVRIEUHHGLQJPDOH:RRG'XFNV&RQGRU
Hohman W.L. and R.D. Crawford, 1995. Molt in the annual cycle of Ring-necked Ducks. Condor 97, 473-483.
Meister W., 1951. Changes in histological structure of the long bones of birds during molt. Anat. Rec. 111, 1-21.
0¡OOHU$3-71LHOVHQDQG-(UULW]¡H/RVLQJWKHODVWIHDWKHUIHDWKHUORVVDVDQDQWLSUHGDWRUDGDSWDWLRQLQ
ELUGV%HKDY(FRO
Møller A.P., S.S. Christiansen and T.A. Mousseau, 2011. Sexual signals, risk of predation and escape behavior. Behav.
Ecol. 22, 800-807.
0XUSK\0(DQG-5.LQJ6HPLV\QWKHWLFGLHWVDVDWRROIRUQXWULWLRQDOHFRORJ\$XN
Murphy M.E. and J.R. King, 1984a. Sulfur amino acid nutrition during molt in the white-crowned sparrow. 1. Does
dietary sulfur amino acid concentration affect the energetics of molt as assayed by metabolized energy? Condor

Murphy M.E. and J.R. King, 1984b. Sulfur amino acid nutrition during molt in the white-crowned sparrow. 2. Nitrogen
DQGVXOIXUEDODQFHLQELUGVIHGJUDGHGOHYHOVRIWKHVXOIXUFRQWDLQLQJDPLQRDFLGV&RQGRU
NRC, 1994. Nutrient requirement of poultry. National Academy Press, Washington, DC.
Yanes, M., J. Herranz, J. de la Puente and F. Suárez, 1998. La perdiz roja: Identidad de los depredadores e intensidad
de la depredación. In: I Curso sobre la perdiz roja. FEDENCA-GRUPO Editorial V, 135-147.

512 Energy and protein metabolism and nutrition in sustainable animal production
Part 9. Baldwin symposium
The life and legacy of Dr. Ransom Leland (‘Lee’) Baldwin V
September 21, 1935 – November 30, 2007

R.L. Baldwin, VI1 and C.C. Calvert2

1Bovine Functional Genomics Laboratory, USDA ARS, Beltsville, MD, USA;
[email protected]
2Department of Animal Sciences, University of California, Davis, CA, USA

Professor Ransom Leland (‘Lee’) Baldwin was born on September 21, 1935, in Meriden, Connecticut.
Lee was the eldest of three children raised on the family dairy farm. He grew up milking dairy cows
and this background led to his lifelong commitment to the dairy industry. Lee attended the University
of Connecticut and earned a B.S. in animal industries.

Subsequently, he attended Michigan State University and earned a M.S. in dairy nutrition before
KLV3K'LQELRFKHPLVWU\DQGQXWULWLRQLQ+HZDVD1DWLRQDO6FLHQFH)RXQGDWLRQIHOORZIURP
WR/HHMRLQHGWKHIDFXOW\DWWKH8QLYHUVLW\RI&DOLIRUQLDDW'DYLVLQDWWDLQLQJWKH
rank of professor in 1970. From 1992 to 2000 he served as Sesnon Professor of Animal Science.
/HH¿QDOO\UHWLUHGLQ$SULOIROORZLQJD\HDUFDUHHU+HPHWKLVZLIH0DU\(OOHQXSRQ
graduation from High School and they were married on June 1, 1957, following completion of his
bachelor’s degree from the University of Connecticut. Together they raised their three children Cheryl
Choate, Randy Baldwin, and Robert Baldwin and a foster child, Angel Starr, in Davis, California
and had six grandchildren.

Lee was truly a Professor and intertwined his research and teaching endeavors during his long career
at UC Davis. He was a passionate educator with an exceptional gift for challenging his students to
integrate knowledge from different disciplines. His primary undergraduate course was lactational
biology where he enthusiastically challenged his students and taught critical-thinking skills. His
ODFWDWLRQFRXUVHZDVVSHFL¿FDOO\GHVLJQHGWRLPSDUWDQLQWHJUDWHGXQGHUVWDQGLQJRIELRFKHPLFDO
genetic, nutritional, physiological, and structural factors relating to mammary gland development,
the initiation and maintenance of lactation, the composition of milk and limits to productivity. A
strong emphasis was placed on using knowledge from basic mathematics, chemistry, and biology
to solve problems in animal production. Lee also taught the graduate level nutritional energetics
course, which began during a time of historic change. The nutritional energetics course evolved from
the classical approach of ‘The Fire of Life’ .OHLEHU WRIRFXVLQJRQQXWULWLRQDOHQHUJHWLFV
IURPDQLPDOOHYHOLQSXWRXWSXWUHODWLRQVKLSVWRRQHWKDWHPSKDVL]HGWKHVSHFL¿FELRFKHPLFDODQG
physiological bases for energy expenditures. While the graduate students were learning metabolism
and energetics, Lee was teaching them how to think. His teaching developed his research, just as
his research added to his teaching. These teaching and research efforts became the foundation for
the development of integrated systems approach to research and application in animal nutrition, an
approach that has been adopted in most courses in nutritional energetics across the world.

An example of Lee’s early commitment to the science of integrative biology and to training future
generations of students was his paper published rather early in his career entitled: ‘Estimation of
7KHRUHWLFDO&DORUL¿F5HODWLRQVKLSVDVD7HDFKLQJ7HFKQLTXHD5HYLHZ¶  7KLVSDSHULVRQHRI
WKH¿UVWWRIRFXVRQWKHDFWXDOELRFKHPLFDOSURFHVVHVDQGFRQWUROHOHPHQWVWKDWPDNHXSWKHSUDFWLFDO
animal-level outcomes in growth and lactation.

/HHPDLQWDLQHGDQH[WUHPHO\SURGXFWLYHUHVHDUFKDQGJUDGXDWHWUDLQLQJSURJUDPVSDQQLQJ¿YH
decades. From his earliest efforts as an assistant professor of animal sciences at UC Davis to his
retirement as Sesnon professor (above rank) and member of the National Academy of Sciences (in
1993), Lee never lost sight of the importance of a rationally thought-out and followed research plan,

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 515
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_191, © Wageningen Academic Publishers 2013
integrated seamlessly with a similar graduate training program. Today dozens of graduate students
PHQWRUHGE\/HHPDNHXSDVLJQL¿FDQWFRUHRISURGXFWLYHDQLPDOVFLHQWLVWVLQHGXFDWLRQDQGLQGXVWU\
around the world. He wrote in the introduction to ‘Modeling Ruminant Digestion and Metabolism’
(1995) that his research program is best characterized as one which couples experimental reduction
and analysis with the use of mathematical modeling to achieve synthesis, integration and effective
utilization of knowledge of underlying function in the solution of problems in ruminant animal
production.

He started his career as a basic ruminant microbiologist, subsequently moving his focus to whole
DQLPDODQGWLVVXHOHYHOSDWKZD\HQHUJHWLFVWKHQRQWRWLVVXHJURZWKDQGGHYHORSPHQWDQG¿QDOO\
to practical animal feeding strategies using integrative approaches. The results of his studies of
ruminal microbiology and tissue metabolism are the material upon which microbial and digestive
and metabolic elements of all lactating cow-feeding systems worldwide are based, including the
National Research Council, the Cornell Net Carbohydrate and Protein System, CPM Dairy, and the
various systems in use in Europe and Australasia. An innovative feature of his research on tissue
metabolic functions has been the use of experimental protocols that reveal kinetic properties of
tissues and enable quantitative and time-dynamic evaluations of factors that determine patterns of
QXWULHQWXWLOL]DWLRQ/HHZDVRQHRIWKH¿UVWWRFKDPSLRQWKHFRPELQDWLRQRIin vivo and in vitro
experiments to extract the greatest possible knowledge and quantitative descriptions from every
experiment. Clearly he is generally considered among the founding fathers of modeling in animal
science, for introducing process-based simulation modeling using differential equations. Starting
LQWKHVWKURXJKWKHVWFHQWXU\KLVIRFXVUHPDLQHGRQWKHGHYHORSPHQWRIELRFKHPLFDO
mechanistic, dynamic, computer-assisted models of ruminal function and tissue metabolism, primarily
in lactating cattle. Lee’s summation of this work led to the development of ‘Molly’, a computer
model of rumen and tissue metabolism in the lactating cow, which he named after a particularly
patient cow of his childhood. The uniqueness of Lee’s approach is that it accommodates effects of
FXUUHQWIHHGLQJSUDFWLFHVLQSUDFWLFDOXVHXSRQVXEVHTXHQWSHUIRUPDQFHWKDWLQÀXHQFHPLON\LHOGV
LQIXOOODFWDWLRQVLQFOXGLQJUHVXOWDQWHFRQRPLFEHQH¿WVDQGWKDWLWWUDFHVPHWDEROLVPRILQGLYLGXDO
nutrients rather than aggregate entities such as metabolizable energy. Because different nutrients are
XVHGIRUGLIIHUHQWSXUSRVHVDWGLIIHULQJHI¿FLHQFLHVWKLVPRGHOSUHGLFWVHIIHFWVRIGLHWVRIJUHDWO\
differing composition upon lactational performance in a superior fashion. Today Lee’s model and,
PRUHLPSRUWDQWO\KLVLQWHJUDWHGDSSURDFKFRQWLQXHVWRGH¿QHUXPLQDQWIRRGSURGXFWLRQLQ(XURSH
the United States, Australia, Canada, and New Zealand.

Through his pioneering animal research and teaching activities Baldwin created an entire generation
of nutritional scientists, and altered the basic philosophy and application of agricultural research and
HGXFDWLRQIXQGLQJDQGFRQGXFWZRUOGZLGH,QDGGLWLRQWRWKHVFLHQWL¿FDQGHGXFDWLRQDOLPSDFWLWLVD
PDWWHURIKLVWRULFDOUHFRUGWKDWDSSOLFDWLRQRIKLVUHVHDUFKDQG¿QGLQJVKDVOHGWRDPDMRULQFUHDVHLQ
HI¿FLHQF\RIDQLPDOSURGXFWLRQDQGDQLPSURYHPHQWLQWKHQXWULWLRQDOKHDOWKRIPLOOLRQVRISHRSOH

References
Baldwin R. L., 1995. Modeling Ruminant Digestion and Metabolism. London: Chapman & Hall.
.OLHEHU07KH¿UHRIOLIHDQLQWURGXFWLRQWRDQLPDOHQHUJHWLFV-RKQ:LOH\DQG6RQ1HZ<RUN86$

516 Energy and protein metabolism and nutrition in sustainable animal production
Application of mathematical modelling in animal nutrition, physiology
and energy balance
J. France
Centre for Nutrition Modelling, Department of Animal and Poultry Science, University of Guelph,
Guelph N1G 2W1, ON, Canada; [email protected]

Lee Baldwin was a pioneer of mathematical modelling in animal science, and is widely regarded
within the subject as the founding father of process-based simulation modelling using differential
HTXDWLRQV$QGULJKWO\VR+HZDVDQLQWHJUDWLRQLVWDQGDYLVLRQDU\/HHZDVWKH¿UVWWRFKDPSLRQ
the integration of in vivo and in vitro experimentation and mathematical modelling to extract as
much knowledge as possible from an experimental programme. Indeed his mantra was in vivo,
in vitro, in silico. He saw beyond statistics and biometrics into the wider realms of mathematics.
He believed that mathematics as employed in the physical and engineering sciences had much to
offer the natural sciences, and that the methods of operations research and applied mathematics
(mathematical physics) in particular should be actively embraced in applied biology. He was, for
H[DPSOHDPRQJWKH¿UVWWRDSSO\OLQHDUSURJUDPPLQJWREDVLFUXPLQDQWGLJHVWLRQVWXGLHV 5HLFKODQG
Baldwin, 1975), and a great admirer of Sir Kenneth Blaxter’s work, not only on energy metabolism,
EXWRQKLVXVHRIGLIIHUHQWLDOHTXDWLRQVWRLQWHUSUHWGLJHVWDÀRZPHDVXUHPHQWV HJ%OD[WHUet al.,
 ,QGHHG/HHWHQGHGWRVXEVFULEHWRWKDWZHOOZRUQFOLFKpRIGLIIHUHQWLDOHTXDWLRQVEHLQJ6LU
Isaac Newton’s key to the universe.

This summary gives a synoptic account of Lee’s evolution as a mathematical modeller, much of it
LQKLVRZQZRUGV/HHXQGHUWRRNJUDGXDWHVWXGLHVDWWKH8QLYHUVLW\RI0LFKLJDQLQWKHHDUO\V
DQGZDVSURIRXQGO\LQÀXHQFHGE\KLVWZRSULQFLSDODGYLVRUV

Professor Roy S. Emery, major professor for my M.S. degree and co-mentor during my Ph.D:
his training encouraged me to develop as a quantitative scientist – ‘Do your arithmetic and
take a course in differential equations.’ Professor W. A. Wood wondered in my interview with
him as a prospective student why I used statistical techniques in my M.S. thesis – ‘What was
wrong with your data?’ – and taught me biochemistry, biochemical methods and theology,
and most importantly perhaps, to take students for what they are and help them develop. His
advice in this regard is the reason that I have helped train many successful Ph.D. students,
who, in turn, have contributed so much to our research program over the years.

7KXV/HH¶VFUHGR±TXDQWL¿FDWLRQDQGLQWHJUDWLRQ±ZHUH¿UPO\HVWDEOLVKHGE\WKHYHU\RXWVHWRI
his career.

His development as a mathematical modeller was marked by his belief in taking sabbatical
VWXG\SHULRGV+LV¿UVWVDEEDWLFDOZDVLQWKHODWHVZLWK'DYLG*DU¿QNHODWWKH8QLYHUVLW\RI
Pennsylvania in Philadelphia to learn-in-depth mathematical descriptions of biochemical equations,
SDUWLFXODUO\WKRVHRIFDUERK\GUDWHPHWDEROLVP+HZDVKLJKO\LPSUHVVHGZLWKDQGLQÀXHQFHGE\
*DU¿QNHO¶VZRUNHVSHFLDOO\KLVZRUNRQPRGHOOLQJEUDLQPHWDEROLVP *DU¿QNHO ZKLFKWUXO\
was the cornerstone of his devotion to mathematical aspects of biology and the genesis of Lee’s
efforts to model the rumen.

In the 1970s he took sabbatical periods ‘down under’ with Marc Ulyatt at DSIR in Palmerston North,
New Zealand, and with John Black at CSIRO in Blacktown near Sydney, Australia. In Palmerston
North he worked on modelling rumen function (Baldwin et al., 1977) and in Sydney focused on
modelling the nutritional and physiological status of different organs and tissues (Baldwin and Black,
1979). His work in Australia laid the foundations for the growth modelling of Oltjen et al.  
and the current California beef model (Garcia et al., 2008). By the end of the 1970s, Lee (along with

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 517
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_192, © Wageningen Academic Publishers 2013
John Black) was generally recognised as the world’s leading animal modeller, and his precepts for
PRGHOOLQJUXPHQIXQFWLRQDQGSRVWDEVRUSWLYHPHWDEROLVPZHUH¿UPO\HVWDEOLVKHG

,Q6HSWHPEHU/HHWRRNKLV¿QDOVDEEDWLFDODWWKH*UDVVODQG5HVHDUFK,QVWLWXWHLQ+XUOH\QHDU
Henley-on-Thames, England. The Hurley group (John Thornley, David Beever, Maggie Gill and
myself), in collaboration with John Black, were actively involved in modelling ruminant digestion
and metabolism at the time. Lee’s primary goal was to fuse the Davis (Baldwin et al., 1977) and
Hurley (France et al., 1982) rumen models, and the Davis (Baldwin et al., 1980) and Hurley (Gill
et al., 1984) metabolism models, to produce new rumen and metabolism models for subsequent
integration into a whole cow model. The purpose of the rumen model was to transform feed inputs
into nutrient supply, and was parameterised for North American diets; it was not originally intended
for grass-based diets or to account for microbial interactions. The objective of the metabolism model
was to provide a detailed description of partition of nutrients and energy balance within the cow. His
other goal was to learn the computer simulation language ASCL, recently developed at Cambridge
University by George Mitchell with the capacity to handle non-linear kinetics easily, which we
ZHUHXVLQJ%\$SULOWKH¿UVWLQGHSWKPDWKHPDWLFDOPRGHORIWKHODFWDWLQJGDLU\FRZZDV
completed, and a series of papers written – one on the metabolism model, one on the rumen model,
one on the whole cow model, and one on solving stiff equations (Baldwin et al., 1987a,b,c; France
et al., 1992). The last paper, which illustrates Lee’s life-long interest in mathematics, arose because
the cow model originally exhibited stiffness (due to the rate parameters having different orders of
magnitude), a problem not easily handled back then, and this issue had to be resolved in order to run
it speedily. It is interesting to note the gap between the papers being written and their publication.
Mainstream nutrition journals were highly reluctant to publish non-experimental papers at that time.

7KH¿UVWZRUNLQJYHUVLRQRIWKHZKROHFRZPRGHOZDVXQLPDJLQDWLYHO\FDOOHGFRZFVOEHIRUH
HYHQWXDOO\EHFRPLQJ0ROO\'LVFXVVLQJWKH¿UVWRXWSXWVIURPFRZDQGSRVVLEOHFKDQJHVWRWKH
coding, I remember Lee becoming a little agitated:

‘We can’t call it cow1, she must have a name’. OK Lee, what shall we call it? ‘We’ll name
her after one of the cows on my father’s farm; let’s call her Molly.’ No Lee, that would be
rather imprudent around here at the moment. ‘I guess so; what about Daisy then?’ No, that’s
the acronym Reading University use for their computerised dairy management information
system and it will cause confusion. ‘Myrtle then?’ OK Lee, Myrtle it is!

So Molly started life as Myrtle. Two years later, back in California, Lee made several alterations to
the computer code and Myrtle became Daisy. Subsequently, and in Lee’s words:

2YHUWKH\HDUSHULRG  GXULQJZKLFK'$,6<UHLJQHGPDQ\SLHFHPHDOFKDQJHV

ZHUHLQWURGXFHGDQGWKHÀRZRIELRORJLFDOORJLFEHFDPHGLVMRLQWHGHJ'$,6<JRWROGDIWHU
all, most cows are culled before they complete six lactation cycles. Therefore the program
was re-organized, corrected, and formatted to form MOLLY, named after the very docile,
patient cow to which my father assigned the task of teaching me to milk by hand when
I was 8 or 9 years old. Perhaps Molly will provide me and associates with a continuing
opportunity to learn.

Lee’s capstone achievement in modelling came with the publication of his seminal book Modelling
Ruminant Digestion and Metabolism, in which a detailed account of Molly was presented (Baldwin,
1995). Since his retirement, improvements to the cow model have continued and the current version
LVPRUHXVHUIULHQGO\0ROO\¶VDGRSWLRQLVÀRXULVKLQJDQGVKHLVFXUUHQWO\SRSXODUQRWRQO\LQ1RUWK
America but also in Australasia and Europe. But much more importantly, the integrated approach
and vision that Lee championed have become accepted practice throughout animal science globally.
His legacy is therefore an outstanding one.

518 Energy and protein metabolism and nutrition in sustainable animal production
A fuller appreciation of Lee’s life and achievements can be found in the memoir by Baldwin VI et
al. (2010).

References
Baldwin R.L., 1995. Modeling Ruminant Digestion and Metabolism. London: Chapman & Hall.
Baldwin R.L., J. France and M. Gill, 1987a, Metabolism of the lactating cow: 1. Animal elements of a mechanistic
model. J. Dairy Res. 54:77-105.
Baldwin R.L., J.H.M. Thornley and D.E. Beever, 1987b. Metabolism of the lactating cow: 2. Digestive elements of a
mechanistic model. J. Dairy Res. 54:107-131.
Baldwin R.L., J. France, D.E. Beever, M. Gill and J.H.M. Thornley, 1987c. Metabolism of the lactating cow: 3. Properties
of mechanistic models suitable for evaluation of energetic relationships and factors involved in the partition of
nutrients. Journal of Dairy Research 54:133-145.
Baldwin R.L. and J.L. Black, 1979. Simulation of the effects of nutritional and physiological status on the growth
of mammalian tissues: description and evaluation of a computer program. CSIRO Aust. Anim. Res. Lab. Tech.
3DS1R
Baldwin R.L., L.J. Koong and M.J. Ulyatt, 1977. A dynamic model of ruminant digestion for evaluation of factors
affecting nutritive value. Agric. Systems 2:255-288.
Baldwin, R.L., N.E. Smith, J. Taylor and M. Sharp, 1980. Manipulating metabolic parameters to improve growth rate
DQGPLONVHFUHWLRQ-$QLP6FL
Baldwin R.L. VI, C.C. Calvert, J.G. Fadel, J. France and J.P. McNamara. 2010. Ransom Leland (‘Lee’) Baldwin V
1935-2007: A biographical memoir. National Academy of Sciences, Washington, D C.
%OD[WHU./10F&*UDKDPDQG)::DLQPDQ6RPHREVHUYDWLRQVRQWKHGLJHVWLELOLW\RIIRRGE\VKHHSDQG
RQUHODWHGSUREOHPV%U-1XWU
France J., J.H.M. Thornley and D.E. Beever, 1982. A mathematical model of the rumen. J. agric. Sci., Camb: 99:343-353.
France J., J.H.M. Thornley and K.A. Crist, 1992. On solving stiff equations with reference to simulating ruminant
PHWDEROLVP-7KHRUHW%LRO
Garcia, F., R.D. Sainz, J. Agabriel, L.G. Barioni and J.W. Oltjen, 2008. Comparative analysis of two dynamic mechanistic
models of beef cattle growth. Anim. Feed Sci. Tech. 143:220-241.
*DU¿QNHO'$VLPXODWLRQVWXG\RIWKHPHWDEROLVPDQGFRPSDUWPHQWDWLRQLQEUDLQRIJOXWDPDWHDVSDUWDWHWKH
Krebs cycle, and related metabolites. J. Biol. Chem. 241:3918-3929.
Gill M., J.H.M. Thornley, J.L. Black, J.D. Oldham and D.E. Beever, 1984. Simulation of the metabolism of absorbed
HQHUJ\\LHOGLQJQXWULHQWVLQ\RXQJVKHHS%U-1XWU
2OWMHQ-:$&%\ZDWHU5/%DOGZLQDQG:1*DUUHWW'HYHORSPHQWRIDG\QDPLFPRGHORIEHHIFDWWOH
JURZWKDQGFRPSRVLWLRQ-$QLP6FL
Reichl J.R. and R.L. Baldwin, 1975. Rumen modelling: rumen input-output balance models. J. Dairy Sci. 58:879-890.

Energy and protein metabolism and nutrition in sustainable animal production 519
Contributions of Lee Baldwin to lactation biology
J.P. Cant
Centre for Nutrition Modelling, Department of Animal and Poultry Science, University of Guelph,
Canada; [email protected]

/HH%DOGZLQEHJDQKLVVWXGLHVRIODFWDWLRQLQWKHHDUO\¶VE\FKDUDFWHUL]LQJPHWDEROLFFKDQJHV
that occurred in the mammary glands before and after parturition. He considered the mammary
glands a rare and fascinating experimental model of organ development and differentiation that
could be induced to occur repeatedly in the adult animal. His ambitious undertaking was to catalog
the expression levels of literally dozens of mammary enzymes and their associated intermediary
metabolites at various stages of pregnancy and lactation in the rat, cow, and guinea pig (Baldwin,
0LOOLJDQDQG%DOGZLQ +HZDVFRQGXFWLQJSURWHRPLFVDQGPHWDERORPLFVGHFDGHV
before the terms were invented.

In the quest to understand regulation of milk synthesis, Baldwin developed an experimental approach
that was to remain a cornerstone of his style. The approach was to apply perturbations to the animal,
measure lactational performance, collect tissues at slaughter and measure an array of metabolite and
enzyme levels, and incubate tissues in vitro with a range of radiolabelled substrates to assess treatment
HIIHFWVRQÀX[HVWKURXJKPHWDEROLFSDWKZD\V%HFDXVHRIWKHODUJHQXPEHURIPHDVXUHPHQWVDQG
the complexity of the system, contradictions and anomalies would often arise in the interpretation
of data. Baldwin saw these discrepancies as opportunities to advance knowledge because obviously
something was missing in the explanation. He sought to improve the explanations with the help of
mathematical modelling.

Arguably, Lee Baldwin’s greatest contribution to the discipline of lactation biology was the school
of thought he espoused. His approach was one of perturbation, analysis, and synthesis, where
analysis and synthesis refer, respectively, to the breaking of a whole into parts, and the reassembly
of a whole from its parts. Baldwin was an admirer and a scholar of Antoine Lavoisier, who wrote in
1790, ‘Chemistry affords two general methods of determining the constituent principles of bodies,
the method of analysis, and that of synthesis… In general it ought to be considered as a principle
LQFKHPLFDOVFLHQFHQHYHUWRUHVWVDWLV¿HGZLWKRXWERWKWKHVHVSHFLHVRISURRIV¶:KHQ,UHDGWKHVH
words, I cannot help but think of Baldwin’s constant reminders of the need to practice synthesis
after analysis.

At the time Baldwin started his career, it was just becoming known which hormones were responsible
for the onset and maintenance of lactation, while the intracellular second messengers of these
lactogenic hormones and their mechanisms of action were deep in the fog. The accepted metabolic
pathways by which lactose, short- and long-chain fatty acids, and triacylglycerol were synthesized
in mammary epithelial cells contained large gaps. By now, most of the metabolic pathways by
which milk components are synthesized have been worked out and our horizons have expanded with
the use of the modern tools of molecular biology that capture a wider panorama of the mammary
ODQGVFDSHWKDQLQWKHSDVW6RPHRIWKHNH\¿QGLQJVIURPDQDO\VLVRIUHFHQWO\FROOHFWHGGDWDDUH
that: nutritive effects on milk synthesis are due in large part to changes in secretory cell number
through proliferative or apoptotic mechanisms (Capuco et al., 2001); prolactin affects mammary
gene expression through a JAK-STAT signalling pathway (Rui et al., 1994); activation of the ser/
thr kinase Akt by lifting of the repressive effects of progesterone at the end of gestation is central to
the initiation of copious milk secretion (Rudolph et al. JURZWKKRUPRQHLQÀXHQFHVLQVXOLQ
sensitivity and fat mobilization from adipose tissues via synthesis of an inhibitory subunit of the
PI3-kinase (Del Rincon et al., 2007); the decline in milk production post-peak is due to a limited
capacity of the secretory cell to perform any function other than milk synthesis (Lemay et al., 2007).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 521
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_193, © Wageningen Academic Publishers 2013
Many of these hypotheses were developed from an analysis of large volumes of data generated
from modern transcriptomics or proteomics studies. There is widespread optimism that the ability
to measure expression levels of thousands of genes at once is going to transform our understanding
of biology, and yet just under the surface lies a vein of skepticism that anything meaningful can
be extracted from the reams of data that are so easily obtained. Baldwin, as an early practitioner in
WKH¿HOGVHWRXWVRPHLPSRUWDQWWHQHWVDVWRKRZWRSURFHHGWKURXJKWKHWDQJOH+HWDXJKWWKDW  
QXWULHQWÀX[HVRURWKHUVXFKFKDQJHVRYHUWLPHPXVWEHUHFRUGHGLQDVVRFLDWLRQZLWKWKHSDQRUDPLF
snapshot of the gene expression levels, and (2) hypotheses developed analytically from the expression
data should be written in mathematical form so that they can generate predictions of the observed
ÀX[HVDQGFRPSOHWHWKHDQDO\VLVV\QWKHVLVSURRI,WLVLQWHUHVWLQJWRVHHWKDWV\VWHPVELRORJ\LQ
which biological hypotheses are expressed mathematically for synthetic purposes, is experiencing
a rebirth under the umbrella of bioinformatics in an attempt to extract understanding from high-
density biological data.

It is exactly as Baldwin taught.

References
%DOGZLQ5/(Q]\PDWLFDFWLYLWLHVLQPDPPDU\JODQGVRIVHYHUDOVSHFLHV-'DLU\6FL
%DOGZLQ5/DQG/30LOOLJDQ(Q]\PDWLFFKDQJHVDVVRFLDWHGZLWKWKHLQLWLDWLRQDQGPDLQWHQDQFHRIODFWDWLRQ
LQWKHUDW-%LRO&KHP
Capuco, A.V., D.L. Wood, R. Baldwin, K. McLeod and M.J. Paape, 2001. Mammary cell number, proliferation and
apoptosis during a bovine lactation: relation to milk production and effect of bST. J. Dairy Sci. 84, 2177-2187.
Del Rincon, J.-P. K. Iida, B.D. Gaylinn, C.E. McCurdy, J.W. Leitner, L.A. Barbour, J.J. Kopchick, J.E. Friedman, B.
Draznin and M.O. Thorner, 2007. Growth hormone regulation of p85 expression and phosphoinositide 3-kinase
DFWLYLW\LQDGLSRVHWLVVXHPHFKDQLVPIRUJURZWKKRUPRQHPHGLDWHGLQVXOLQUHVLVWDQFH'LDEHWHV
Lavoisier, A.L. 1790. Elements of chemistry, in a new systematic order, containing all the modern discoveries. transl.
Kerr, William Creech, Edinburgh.
Lemay, D.G., M.C. Neville, M.C. Rudolph, K.S. Pollard and J.B. German, 2007. Gene regulatory networks in lactation:
LGHQWL¿FDWLRQRIJOREDOSULQFLSOHVXVLQJELRLQIRUPDWLFV%0&6\VW%LRO
Rudolph, M.C., J.L. McManaman, L. Hunter, T. Phang and M.C. Neville, 2003. Functional development of the mammary
JODQGXVHRIH[SUHVVLRQSUR¿OLQJDQGWUDMHFWRU\FOXVWHULQJWRUHYHDOFKDQJHVLQJHQHH[SUHVVLRQGXULQJSUHJQDQF\
lactation, and involution. J. Mam Gl. Biol. Neoplas. 8, 287-307.
Rui, H., R.A. Kirken and W.L. Farrar, 1994. Activation of receptor-associated tyrosine kinase JAK2 by prolactin. J.
%LRO&KHP

522 Energy and protein metabolism and nutrition in sustainable animal production
Modeling animal growth with Lee Baldwin
Roberto D. Sainz
'HSDUWPHQWRI$QLPDO6FLHQFH8QLYHUVLW\RI&DOLIRUQLD'DYLV&$86$[email protected]

Lee Baldwin’s research career focused on elucidating the physiological functions underlying animal
performance. Early on, he realized that the number of metabolic variables and the interactions among
them were too great to be fully understood and manipulated without the use of quantitative modeling
WHFKQLTXHV$W¿UVWWKLVHQWDLOHGWKHFRQVWUXFWLRQDQGXVHRIVWDWLFDQDO\WLFDOPRGHOVWKDWHYROYHG
into more complex dynamic forms. Always, the emphasis was on improving our understanding, or
DVKHSXWLWµDGYDQFLQJWKHVFLHQFH¶UDWKHUWKDQVLPSO\DFKLHYLQJWKHEHVW¿WWRGDWD%DOGZLQQHYHU
considered himself a modeler, rather a scientist who used modeling as part of the overall research
program. For Baldwin, models were most useful for:
1. integration of existing concepts and data regarding animal function into a consistent mathematical
framework;
2. evaluation of those same concepts and data for adequacy in describing growth and body
composition under different feeding regimes, growth trajectories, etc.;
 LGHQWL¿FDWLRQRIFULWLFDOH[SHULPHQWVDQGPHDVXUHPHQWV
4. evaluation of alternative hypotheses for probable adequacy in explaining the effects of genotype,
nutrition, and other growth manipulations.

Nutritional models for growing animals should enable accurate predictions of nutrient requirements,
FDOFXODWLRQRIUHVSRQVHVRIGH¿QHGDQLPDOVWRGH¿QHGIHHGVDQGFDOFXODWLRQRIRSWLPDOQXWULWLRQDO
strategies. Baldwin recognized that the traditional approach applied in development of feeding
V\VWHPVEDVHGXSRQHPSLULFDO¿WVWRH[WHQVLYHGDWDVHWVZRXOGQRWEHDEOHWRH[WHQGRXUNQRZOHGJH
of nutritional biology. He quoted Sir Kenneth Blaxter’s view that ‘we must advance through
utilization of ‘First Principles’’. This led him to adopt the approach espoused by Robinson (1971)
that ‘quantitative representation of the growth process and effects of endogenous and exogenous
agents thereupon must accommodate the contributions of hyperplasia, hypertrophy and accretion
to the growth process’ (Baldwin, 1995). Underlying that notion is the principle that understanding
of governing biological mechanisms coupled with appropriate mathematical modeling tools should
result in greater accuracy and wider application than can be achieved with the traditional, empirical
approach.

%DOGZLQ¶V¿UVWHIIRUWDWPRGHOLQJDQLPDOJURZWKRFFXUUHGGXULQJDVDEEDWLFDOOHDYHLQ$XVWUDOLD
when he was able to collaborate with John Black at CSIRO. The resulting model (Baldwin and
Black, 1979) of growth of tissues and organ systems was based on the realization that relative
organ weights contribute to differences in fasting heat production and that nutritional, physiological
and environmental interactions determine patterns of nutrient utilization, growth rates and body
composition. Initially, allometric equations were used to estimate tissue and organ weights in
growing animals, based on published data. No consistent relationship was found in the values of a
and bDPRQJDQGZLWKLQVSHFLHV7KLVODFNRILQWHUVSHFL¿FDSSOLFDELOLW\RIQXPHULFDOLQSXWVWRWKH
allometric equation was considered unacceptable.

5RELQVRQ  GHYHORSHGWKHFRQFHSW¿UVWSURSRVHGE\(QHVFRDQG/HEORQG  WKDWJURZWK

of tissues, organ systems and whole animals occurs in three phases: hyperplasia, hypertrophy and
DFFUHWLRQ+\SHUWURSKLFJURZWKZDVGH¿QHGE\5RELQVRQ  DVHVVHQWLDOO\LQFUHDVHVLQWKH
DPRXQWRISURWHLQSHUXQLWRI'1$WRVRPHJHQHWLFDOO\GH¿QHGPD[LPXP7KHVHFRQFHSWVOHGWR
three premises that apparently determine growth in mammals:
 7KHSULPDU\JHQHWLFGHWHUPLQDQWRIRUJDQVL]HLVWKH¿QDOQXPEHURIQXFOHLUHSUHVHQWHGE\WKH
amount of deoxyribonucleic acid (DNA).

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 523
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_194, © Wageningen Academic Publishers 2013
 (DFKXQLWRI'1$VSHFL¿HVRQDJHQHWLFDOO\GH¿QHGEDVLVIRUHDFKWLVVXHDQGHDFKVSHFLHV
LQIRUPDWLRQUHTXLUHGIRUWKHXOWLPDWHIRUPDWLRQRIDVSHFL¿FDPRXQWRIFHOOPDWHULDO:KHWKHU
LQIRUPDWLRQVSHFL¿HGE\DXQLWRI'1$OHDGVWRWKHIRUPDWLRQRIFHOOPDWHULDOLVGHSHQGHQWXSRQ
the nutritional and physiological status of the animal.
 7KHVSHFL¿FDFWLYLWLHVRIHQ]\PHVUHVSRQVLEOHIRUWLVVXHPHWDEROLVPDQGJURZWKYDU\DVDQ
exponential function of organ size. Also, the kinetic properties of enzymes are reasonably constant
across species.

Using a model based upon these three premises, Baldwin and Black (1979) simulated the effects
of nutrition on various organs in different species and concluded that they were adequate bases for
mechanistic and dynamic representations of the growth process. The concepts and equation forms
used by Baldwin and Black (1979) served as the starting point for the beef cattle growth model
of Oltjen et al.  QRZFDOOHGWKH'*0 'DYLV*URZWK0RGHO 7KH'*0LVG\QDPLFWKXV
differential equations were integrated to estimate gain (or loss) of DNA and body protein. Body fat
gain is estimated from the difference between energy intake and energy used for maintenance and
protein gain. Since the model requires initial estimates of whole-body DNA, protein and fat, empirical
relationships between these and animal weight, mature size and condition score were developed to
VHWEHJLQQLQJYDOXHVIRUPRGHOLPSOHPHQWDWLRQ/DWHUDGGLWLRQDOUHVHDUFKZDVFRQGXFWHGWR¿OOLQ
gaps of knowledge regarding cell number and sizes in various tissues of the bovine (DiMarco et al.,
1987; Sainz and Bentley, 1997), an example of the interplay between modeling and experimentation
that was the hallmark of Baldwin’s research.

The DGM has been used widely in beef cattle management. For example, this model, coupled with
frame, weight and ultrasound measurements of cattle entering the feedlot, is being used to sort cattle
into more uniform outcome groups (Sainz and Oltjen, 1994; Sainz et al., 1995, 2009). The DGM has
also been reparameterized for Bos indicus cattle (Sainz et al. DQGVHUYHGDVWKHEDVLVIRUD
practical management tool for the Australian beef industry (Oddy et al., 2008). Despite its simplicity
and practical applicability, the DGM’s representation of fundamental mechanisms has also enabled
LWVXVHLQUHVHDUFKIRUH[DPSOHWRHYDOXDWHK\SRWKHVHVUHJDUGLQJYDULDWLRQVLQIHHGHI¿FLHQF\ &UX]
et al., 2007), and as the basis for a sheep growth model (Oltjen et al., 2010). More recent versions of
the DGM include the introduction of variable maintenance functions (Oltjen and Sainz 2001; Oltjen
et al. DQGGLVWULEXWLRQRIERG\IDWDPRQJGLIIHUHQWGHSRWV 6DLQ]DQG+DVWLQJ0F3KHHet
al., 2008). A study (Garcia et al., 2008) comparing the DGM with a model of cattle growth and body
composition based upon explicit representation of protein and fat metabolism (Hoch and Agabriel,
2004) demonstrated that each model adequately described accretion of protein and fat, but the DGM
was superior under discontinuous feeding and with different diet qualities.

Many other models of animal growth have been developed and are in use today for practically every
livestock species. These models were developed for different purposes and using different approaches,
and so may or may not derive directly from the growth modeling work of Baldwin and coworkers.
Indirectly, of course, the notion that mathematical models can help us solve research and practical
problems had no greater champion. In his words (Baldwin, 1995):

In our view, dynamic models are absolutely essential to economic evaluations of risks
DVVRFLDWHGZLWKFXUUHQWGHFLVLRQVEHFDXVHWKHVHFOHDUO\LQÀXHQFHVXEVHTXHQWSHUIRUPDQFH
When a proven, dynamic, mechanistic model becomes available to students and practitioners
... to enable cause-and-effect analyses of underlying reasons for the observed responses, we
will have created a superior instrument for both the teaching and application of our science.
This is the challenge we pose.

524 Energy and protein metabolism and nutrition in sustainable animal production
References
Baldwin, R. L. and J. L. Black, 1979. Simulation of the effects of nutritional and physiological status on the growth of
mammalian tissues: description and evaluation of a computer program. Animal Research Laboratories Technical
3DSHU&RPPRQZHDOWK6FLHQFHDQG,QGXVWULDO5HVHDUFK2UJDQL]DWLRQ0HOERXUQH$XVWUDOLD
%DOGZLQ5/0RGHOOLQJ5XPLQDQW'LJHVWLRQDQG0HWDEROLVP&KDSPDQ +DOO/WG6FLHQWL¿F/RQGRQ
Cruz, G. D., J. W. Oltjen and R. D. Sainz, 2007. Evaluation of hypotheses about residual feed intake in beef steers
XVLQJWKH'DYLV*URZWK0RGHO&DQ-$QLP6FL
DiMarco, O. N., R. L. Baldwin and C.C. Calvert, 1987. Relative contributions of hyperplasia and hypertrophy to
JURZWKLQFDWWOH-$QLP6FL
(QHVFR0DQG&3/HEORQG,QFUHDVHLQFHOOQXPEHUDVDIDFWRULQWKHJURZWKRIWKHRUJDQVDQGWLVVXHVRIWKH
\RXQJPDOHUDW-(PEU\RO
Garcia, F., R. D. Sainz, J. Agabriel, L. G. Barioni, and J. W. Oltjen, 2008. Comparative analysis of two dynamic
mechanistic models of beef cattle growth. Anim. Feed Sci. Technol. 143, 220-241.
Hoch, T. and J. Agabriel, 2004. A mechanistic dynamic model to estimate beef cattle growth and body composition:
1. Model description. Agric. Syst. 81, 1-15.
McPhee, M. J., J. W. Oltjen, J. G. Fadel, D. Perry, and R. D. Sainz, 2008. Development and evaluation of empirical
equations to interconvert between twelfth-rib fat and kidney, pelvic, and heart fat respective fat weights and to
SUHGLFWLQLWLDOFRQGLWLRQVRIIDWGHSRVLWLRQPRGHOVIRUEHHIFDWWOH-$QLP6FL
Oddy, V. H., R.C. Dobos, M. J. McPhee, W. McKiernan, J. W. Oltjen and R. D. Sainz, 2008. A new tool to predict beef
FDWWOHIDWQHVVLQWKH¿HOG&DQ-$QLP6FL
Oltjen, J. W. and R. D. Sainz, 2001. Alternate forms for heat production in ruminant growth and composition models.
In: Chwalibog, A. and K. Jakobsen (editors), Energy Metabolism in Animals. Wageningen Pers, Wageningen, The
Netherlands, EAAP Pub. 103, 39-42.
2OWMHQ-:$&%\ZDWHU5/%DOGZLQDQG:1*DUUHWW'HYHORSPHQWRIDG\QDPLFPRGHORIEHHIFDWWOH
JURZWKDQGFRPSRVLWLRQ-$QLP6FL
Oltjen, J. W., A. Cannas, A., A. S. Atzori, L. O. Tedeschi, R. D. Sainz and D. G. Fox, 2010. Integration of the Small
Ruminant Nutrition System and of the UC Davis sheep growth model for improved predictions. In: Crovetto, G.
M. (editor), Energy and Protein Metabolism and Nutrition. EAAP Pub. 127, 553-554.
2OWMHQ-:5'6DLQ]$%3OHDVDQWV7.6REROHYDDQG9+2GG\5HSUHVHQWDWLRQRIIDWDQGSURWHLQ
gain at low levels of growth and improved prediction of variable maintenance requirement in a ruminant growth
and composition model. In: Kebreab, E., J. Dijkstra, A. Bannink, W. J. J. Gerrits, and J. France (editors), Nutrient
Digestion and Utilization in Farm Animals: Modelling Approaches. CAB International, Wallingford, U.K., pp.

5RELQVRQ':&HOOXODUEDVLVIRUFKDQJHVLQERG\FRPSRVLWLRQ-$QLP6FL
Sainz, R. D. and B. E. Bentley, 1997. Visceral organ mass and cellularity in growth-restricted and refed beef steers.
-$QLP6FL
Sainz, R. D. and J. W. Oltjen, 1994. Improving uniformity of feeder steers using ultrasound and computer modelling.
Proc. Am. Soc. Anim. Sci. Western Section 45, 179-181.
Sainz, R. D., and E. Hasting. 2000. Simulation of the development of adipose tissue in beef cattle. In: McNamara,
J. P., J. France, and D. E. Beever (editors), Modelling Nutrient Utilization in Farm Animals. CAB International,
Wallingford, U.K., pp. 175-182.
6DLQ]5'%DULRQL/*3DXOLQR399DODGDUHV)LOKR6&DQG2OWMHQ-:*URZWKSDWWHUQVRI1HOORUH
vs. British beef cattle breeds assessed using a dynamic, mechanistic model of cattle growth and composition. In:
Kebreab, E., J. Dijkstra, A. Bannink, W. J. J. Gerrits, and J. France (editors), Nutrient Digestion and Utilization in
)DUP$QLPDOV0RGHOOLQJ$SSURDFKHV&$%,QWHUQDWLRQDO:DOOLQJIRUG8.SS
Sainz, R. D., J. G. Smith, I. Garnett and Y. B. Lee, 1995. Use of ultrasound and computer modeling to predict days on
IHHGDQGLPSURYHEHHIFDUFDVVXQLIRUPLW\3URF$P6RF$QLP6FL:HVWHUQ6HFWLRQ
Sainz, R. D., L. G. Barioni, F. R. C. Araujo, and J.W. Oltjen. 2009. Development and evaluation of a tool to sort feedlot
beef cattle into uniform outcome groups. Can. J. Anim. Sci. 89, 533.

Energy and protein metabolism and nutrition in sustainable animal production 525
Advances in rumen microbiology
M.R. Murphy
Department of Animal Science, University of Illinois, Champaign, IL, USA; [email protected]

With the publication of his MSc thesis at Michigan State University in 1958, R. Lee Baldwin began
contributing to advances in rumen microbiology by reporting on an ‘Investigation of the oxidation-
UHGXFWLRQSRWHQWLDORIUXPHQFRQWHQWV¶+LV3K'GLVVHUWDWLRQWKHUHLQH[DPLQHGWKHFRQYHUVLRQ
RIODFWDWHWRSURSLRQDWHµLQDQDWXUDOELRORJLFDOV\VWHP¶ERYLQHUXPHQÀXLG7KRVHUHVXOWVZHUH
GHWDLOHGLQ-RXUQDORI%DFWHULRORJ\DUWLFOHVSXEOLVKHGLQDQG7KHQH[W\HDU%DOGZLQ
presented a paper at the Second International Symposium on the Physiology of Digestion in the
Ruminant. His topic was pathways of carbohydrate metabolism in the rumen; however, he also called
attention to the fact that very few data were available ‘regarding the quantitative contributions of
GLIIHUHQWPHWDEROLFSDWKZD\VRUPLFURRUJDQLVPVDQGWKHVSHFL¿FIDFWRUVZKLFKFRQWUROWKHJURZWK
and metabolic activity of the microorganisms in the rumen.’

By the time that the Third International Symposium on the Physiology of Digestion and Metabolism
LQWKH5XPLQDQWRFFXUUHG¿YH\HDUVODWHU  %DOGZLQZDVDEOHWRGHPRQVWUDWHKRZDUHODWLYHO\
QHZWHFKQLTXHFRPSXWHUVLPXODWLRQFRXOGEHXVHGWRPRGHOUXPLQDQWV\VWHPV,GHQWL¿HGDGYDQWDJHV
RIWKLVDSSURDFKLQFOXGHGWKHXVHRIIDPLOLDUPDWKHPDWLFDOIRUPXODWLRQVDSSOLFDELOLW\WRVSHFL¿F
H[SHULPHQWDOGDWDDQGÀH[LELOLW\DQGFDSDFLW\WRUHSUHVHQWPHWDEROLFDQGUHJXODWRU\PHFKDQLVPVDW
various levels. This modeling effort focused on energetic relationships in the formation and utilization
of fermentation end-products in the rumen. Notably, it was both quantitative and dynamic; i.e. based
RQGLIIHUHQWLDOHTXDWLRQVUHSUHVHQWLQJVSHFL¿FPHWDEROLFUHDFWLRQV

Rumen fermentation characteristics were considered to be largely determined by the microbial

species present but representing each species explicitly in a model of rumen function was likely
neither feasible nor necessary. What was essential was that species characteristics and functions be
considered, and represented implicitly in the model. To that end, three composite microbial groups
ZHUHGH¿QHGWKHFHOOXORO\WLFFRPSOH[DQGPHWKDQRJHQLFVSHFLHVDP\ORO\WLFDQGDVVRFLDWHGVSHFLHV
and species utilizing a broad range of substrates. Another important element of the approach was
that the validity of the model was tested by simulating data collected in experiments that were not
considered in its construction. The conclusion of this article also extended the exercise by addressing
philosophical concerns related to mathematical modeling and computer simulation. The point was
that, properly applied, these approaches could contribute to our understanding of biological and
physiological systems which are both complex and dynamic.

These results were then utilized, along with a separate analysis of energy metabolism in anaerobes,
to develop two input-output balance models based on systems of simultaneous linear equations.
7KH¿UVWH[DPLQHGUHODWLRQVKLSVDPRQJHOHPHQWDU\FRPSRVLWLRQVRIPLFURELDOFHOOV EDFWHULDDQG
protozoa), digestible feed nutrients, and fermentation products. Another calculated balances for
biosynthetic and fermentation equations for rumen microbiota.

Because relative proportions of fermentation products differed depending on the type of substrate,
stoichiometric and precursor-product relationships used in previous studies of rumen fermentation
balance then had to be extended to provide more detailed evaluations of nutrient digestive patterns.
7KLVLQYROYHGWKHFRPSXWDWLRQRIPHWDEROLFÀX[DQGVWRLFKLRPHWULFSDUDPHWHUVIRUVROXEOHVXJDUV
starch, cellulose, hemicellulose, and protein using statistical methods. Expected products of rumen
digestion were then predicted quantitatively and compared to results observed in a sample problem.
This framework was adapted to estimate these relationships for a wider range of diets and input-
output data, results of which were then utilized in later models of rumen function.

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 527
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3_195, © Wageningen Academic Publishers 2013
The next major development was a dynamic model of ruminant digestion for evaluation of factors
affecting nutritive value. This effort integrated aspects of earlier models with recent experiments
in his laboratory on the dynamics of fermentation and microbial growth, and results of animal
studies from the literature and by a coauthor. Representations of rumen function remained similar
in subsequent models; however, the models incorporated many other elements.

Recent advances and future directions discussed in, and inspired by, Baldwin’s work on rumen
microbiology are many but only a few are mentioned here. One is that data for estimating
VWRLFKLRPHWULFFRHI¿FLHQWVRIVXEVWUDWHIHUPHQWDWLRQLQWKHUXPHQIRUDQLPDOVIHGDUDQJHRIGLHWVDW
YDULRXVLQWDNHVUHPDLQOLPLWLQJ6SHFL¿FDOO\PDQ\PRUHPHDVXUHPHQWVRIYRODWLOHIDWW\DFLGUDWHVDUH
needed; perhaps a way forward is determination using stable isotope techniques (e.g. 13C-propionate)
or estimation via in vitro fermentation methods.

$QRWKHUSRWHQWLDOO\IUXLWIXOWRSLFZDVLGHQWL¿HGHDUO\EXWKDVUHFHLYHGOLWWOHDWWHQWLRQLHWKH
interaction between comminution of feed and digesta particles by chewing during eating and
rumination, and the kinetics of microbial digestion and passage from the reticulorumen. It has been
demonstrated, at least for two forages, that synergism exists between animal and microbial effects:
mastication during eating enhances microbial fermentation, which increases the effectiveness of
FRPPLQXWLRQGXULQJUXPLQDWLRQ2EYLRXVO\VXFKHIIHFWVQHHGWREHTXDQWL¿HGIRUPRUHIRUDJHVDQG
mixed diets to better understand the dynamics of this interaction.

The structure of microbial populations in the rumen and interactions among species has received
increased attention. Of particular interest is how bacterial communities in the rumen are affected by
FKDQJHVLQGLHWDQGSK\VLRORJLFDOVWDWH&KDOOHQJHVLQFOXGHTXDQWL¿FDWLRQRIWKHLUPHWDEROLFLPSDFW
and assessment of the level of detail needed to adequately describe rumen function. Basically, how
PDQ\µFRPSRVLWHPLFURELDOJURXSV¶DUHMXVWL¿HG"6RPHZRUNVXJJHVWVWKDWRQO\DIHZFODVVHVRI
¿EURO\WLFEDFWHULDDQGVSHFLHVDUHQHHGHGWRH[SODLQPXFKRIWKHYDULDWLRQDVVRFLDWHGZLWKVXEVWUDWH
solubilization and utilization.

528 Energy and protein metabolism and nutrition in sustainable animal production
Author index
A Bee, G. 
Abdelqader, M.M.  Bendezu, H.C.P. 333
Abreu, M.L.T. 245 Bequette, B.J. 421
Acetoze, G. 393 Berchielli, T.T. 139
Acharya, S. 385 Berends, H. 79, 223
Agabriel, J. 71, 319 Berri, C. 209, 
Agarwal, U. 421 Bertolini, F. 297
Aguilar, M. 437 Biagioli, B. 135
Aguilar-Pérez, C.F. 121, 123 Bielecki, W. 423
Aguilera, J.F. , 177, 403, 431 Bikker, P. 
Ahnert, S.  Blaabjerg, K. 191
Ahola, J.K. 385 Blache, D. 201
Albino, R.L. 107 Blanc, F. 71
Albrecht, E.  Boaventura Neto, O. 135
Aldrich, J.M. 83 Boer, M. 321
Al-Jammas, M. 319 Bompadre, T.F.V. 119, 135
Almeida, P.J.P. 93, 95 Bonneau, M. 185
Alstrup, L. 489 Borges, A.L.C.C. 91, 117, 247, 503
Amaral, H.F. 325 Borges, I. 113, 503
Ampe, B.  Börner, S. 283, 457
Andrade, F.T. 245 Bosi, P. 295, 297
Andretta, I. 205 Boutry, C. 415, 427
Anjema, D. 279 Brøkner, C. 201
Araujo, F.R.C.  Brossard, L. 339
Archimède, H. 75, 501 Bruckmaier, R.M. , 471
Arriola Apelo, S.I. 449 Bruininx, E.M.A.M. 159
Ashihara, A. 253, 395 Bush, L.P. 
Assadi, E. 191 Buyse, J. 301
Austbø, D. 201
Ayala-Burgos, A.J. 105, 121, 123 C
Aziani, W.L.B. 125 Cabon, G. 231
Cailleau-Audouin, E. 391
B Caldas, J. 199
Bach Knudsen, K.E. 201 Calvert, C.C. 515
Baéza, E. 271 Camacho, L.E. 285, 
Bahloul, L. 347 Campos, D.M.B. 197
Balcells, J. 85 Campos, M.M. 341
Baldwin, VI, R.L. 515 ýDQGHN3RWRNDU0 185
Ball, R.O. 173, 203 Canibe, N. 191
Bannink, A. 47, 227 Cannas, A. 77
Barbosa, G.S.S.C. 247 Cantalapiedra-Hijar, G. 435
Barcan, C. 487 Cant, J.P. 521
Barea, R. , 313, 403 Carneiro, P.R.O. 197
Barratt, C.E.S. 407 Carstens, G.E. 371
Barsila, S. 73 Carvalho, A.U. 91
Barszcz, M. 375, 377, 379 Carvalho, P.H.A. 117
Bateman, II, H.G. 83 Casadio, R. 297
Batorek, N. 185 Casper, D.P. 327
Battagliese, T. 487 Castagnino, D.S. 129
Baumont, R. 231 Castro, M.M.D. 341

J.W. Oltjen et al. (eds.), Energy and Protein metabolism and nutrition is sustainable animal protection, 529
EAAP publication No. 134, DOI 10.3920/978-90-8686-781-3, © Wageningen Academic Publishers 2013
Caton, J.S. 285,  Doreau, M. 475, 501
Cavalcanti, L.F.L. 113, 249 Dorigam, J.C.P. 333, 343
Cerrate, S. 199, 239, 305, 307 Doscher, F.E. 285, 
Chartrin, P. , 271 Dubois, S. 185, 313
Chaves, A.S. 127 Duclos, M.J. , 391
Chay-Canul, A.J. 105 Dupont, J. 391
Chizzotti, F.H.M. 125, 291 Dusel, G. 
Chizzotti, M.L. 89, 105, 125, 291, 325
Christensen, K.W. 455 E
Chwalibog, A. , 417 Egal, D. 71
Cnossen, A.J. 279 Eising, I. 159
Cobo-Ortega, C. 179, 429 Ekmay, R.D. 199, 239, 305, 307
Coertze, R. 99 El-Kadi, S.W. 415, 427
Collin, A.  Ellis, J.L. 47, 227
Colombini, S. 499 England, J. 199, 239, 305, 307
Colombo, M. 295, 297 Enishi, O. 81
Columbus, D. 413 Espinoza-Hernández, J.C. 123
Conde-Aguilera, J.A. 179, 429 Everaert, N. , 301
Coon, C.N. 199, 239, 305, 307
Corrent, E. , , 195, 209, 339 F
Costa, D.S. 131 Facury Filho, E.J. 117
Costa, F.G.P. 345 Fadel, J.G. 327
Coudert, E. 391 Farjalla, Y.B. 
Cox, C. 399 Férard, A. 231
Crochet, S. 391 Fernandes, M.H.M.R. 77, 119, 131, 135, 137
Crompton, L.A. 407 Fernandez-Alarcon, M.F. 197
Crovetto, G.M. 499 Fernández, C. 109, 111
Cruz, G.D.  Fernández-Fígares, I. 177, 183, 293, 411,
Cruz, J.F. 93, 95 433, 509, 511
Ferreira, J.P. 97
D Ferreira, R.A. 291
Dänicke, S. 459 Fiedorowicz, S. 423
Da Silva, T.E. 107 Figueiredo, F.O.M. 137
Davis, T.A. 415, 427 Fiorentini, G. 139
De Alencar, M.M. 127 Fiorotto, M.L. 427
De Almeida, A.K. 131 Fiszdon, K. 423
De Campeneere, S.  Fitzsimmons, C.J. 
Decuypere, E. 301 Fonseca, M.A. 249
De Lange, C.F.M. 143, 313, 413 Fonseca, M.P. 503
De La Torre, A. 71 Fontanesi, L. 297
De Medeiros, S.R. 127 Foote, A.P. , 235
Derno, M. 157, 275, 283, 457 Forbes, T.D.A. 371
Detmann, E. 89 France, J. 47, 327, 517
Devkota, N.R. 73 Franssens, L. 301
D’Heer, B.  Fraysse, P. 209
Dickhoefer, U.  Freire, M.M. 137
Dijkstra, J. 47, 227, 327 Frighetto, R.T.S. 119
Dodds, K. 495 Furbeyre, H. 189
Doepel, L. 115 Furtado, T. 325
Dohme-Meier, F. 225
Domingues, S.S. 291 G
Donato, D.C.Z. 345 Galassi, G. 499

530 Energy and protein metabolism and nutrition in sustainable animal production
Galindo, C. 409, 451 Htoo, J.K. , 413
Galvão, M.C. 291 Huber, K. 399, 459
Gangnat, I.D.M. 87 Hulshof, T.G. 
Gao, J.  Humphries, D.J. 407
Garcia-Launay, F. 319 Hu, Q. 421
Gazzaneo, M.C. 427
Gerrard, D.E. 387 I
Gerrits, W.J.J. 59, 79, 159, 223, 233, 237, Illovies, A. 435
289,  Iroshan, I.H. 115
Ghimire, S. 323 Isenberg, B.J. 487
Giggel, K.  Iseri, V.J. 353
Gilbert, M.S. 59, 233, 289 Ishida, A. 253, 395, 419
Girish, C.K. 175 Isola, R. 205, 207
Gloaguen, M. , 339 Ito, C. 505
Gomes, B.C. 107
Gomes, R.C.  J
Gonçalves, L.C. 497, 503 Jahromi, M.F. 85
Gonçalves, N.C. 247 Jansman, A.J.M. , 195, 237, 295, 
González-Valero, L. 183, 293, 411 Jaworski, S. 
Górka, P. , 373, 397 Jensen, B.B. 191
Görs, S. 157, 225,  Jensen, R.B. 241, 243
Green, C. 407 Ji, S. 385
Gregorini, P. 323 Johnson, K.A. 371
Grodzik, M.  Junghans, P. 241, 243
Gross, J.J. 
K
H Kalscheur, K.F. 
Hada, F.H. 197 Kampman-Van de Hoek, E. 237, 
+DÀD$1 371 Katsumata, M. 253, 395, 419
Hagerman, A.E. 491 Kaufmann, L.D. 225
Hammon, H.M. 157, , 275, 277, 283, Kautzsch, U. 275
, 457,  Kebreab, E. 47, 327
Hanigan, M.D. 323, 387, 437, 449 Kemp, B. 471
Harland, R. 495 Kenéz, Á. 459
Harmon, D.L. , 235 Khan, D.R. 329
Haro, A. 431, 433 Kikusato, M. , 309, 505
Härter, C.J. 129 Kim, D.H. , 235
Hassen, A. 99, 101 Kipper, M. 205
Hauschild, L. 205, 207, 333, 343 Kjestrup, H. 495
Heiderich, D. 125 Klasing, K.C. 353, 393
Helm, R.F. 449 Klop, A. 279
Hennequet-Antier, C.  Klotz, J.L. , 235
Heo, J.M. 187 Knowler, K. 495
Hermansen, J.E. 35 Knudsen, M.T. 35
Hermier, D. 271 Kobayashi, H. 253
Herrero, M. 27 Kobayashi, Y. 81
Hickey, S. 495 Kohn, R.A. 323
Higuchi, K. 81 Koolaard, J. 405
Hill, R.A. 385 Koontz, A.F. , 235
Hill, T.M. 83 Koopmans, S.J. 295
Hinders, R. 229 Koppenol, A. 301
Horn, G.W. 389 Kosieradzka, I. 381, 423

Energy and protein metabolism and nutrition in sustainable animal production 531
Krehbiel, C.R. 389 Lourençoni, D. 125
Kreuzer, M. 73, 87,  Louveau, I. 189
Kristensen, N.B. 409, 437, 439 Loyau, T. 
Kristensen, T. 35 Lund, P. 489
Krüger, R. 157, 
Kruijt, L. 279 M
Kuhla, B. 213, 273, 275, 277, 283, , 457, Macari, M. 197
 Macedo Júnior, G.L. 113
Kurzbard, R. 393 Machado, F.S. 341, 497
Ku-Vera, J.C. 105, 121, 123 Machado, P.A.S. 325
Kuwayama, H. 283 MacLean, S. 495
Kyoya, T. 253 Magaña-Monforte, J.G. 105, 123
Magnabosco, C.U. 
L Makkar, H.P.S. 475
Labussière, E. 185, 313, 401 Malagutti, L. 499
Lachica, M. 109, 111, 177, 183, 293, 411, Mansilla, W. 413
433, 509, 511 Marcondes, M.I. 107, 341
Ladeira, M.M. 125, 291 Marie-Magdeleine, C. 501
Laeger, T. 273 Marquardt, S. 73
Lage, H.F. 91, 117, 503 Márquez, R.A. 403
Lake, S.L. 455 Martelli, P.L. 297
Lambert, B.D. 491 Martens, K. 157
Lancaster, P.A. 389 Martínez-Ramírez, H.R. 143
Lanferdini, E. 245 Matthiesen, C.F. 299
Lanna, D.P.D. 127 Matusiewicz, M. 381, 423
Lapierre, H. 115, 347, 409, 443, 447, 451, Maxin, G. 409, 443, 451
453 McBride, B.W. 
Lara, L. , 403, 433 McCann, M.A. 387
Larsen, M. 409, 437, 439, 451 McCoard, S. 405
Larsson, C. 241, 243 McEwan, J. 495
Lasnier, J. 313 McGee, M. 385
Lecomte, P. 475 McKinnon, J.J. 373
Le Floc’h, N. , 189, 339, 401, 429 McLeod, K.R. , 235
Lehnen, C.R. 205 McNamara, J.P. 321, 
Leiber, F. 87 Means, W.J. 455
Leite, R.F. 137 Mendes, E. 
Leme, P.R.  Mendonca, A.N. 135
Lemley, C.O. 285,  Mentz, A.M. 99
Le Morvan, A. 231 Mercier, Y. 179, 429
Lemosquet, S. 447 Merlot, E. 401
Lessire, M. 179, 209, 271 Messad, F. 435
Lesuisse, J. 301 Métayer-Coustard, S. , 271, 391
Levesque, C.L. 143 Metges, C.C. 157, 213, 225, , 273, 275
Liang, J.B. 85 Metzler-Zebeli, B.U. 157, 
Liebert, F. 193, 329, 335, 425 Meyer, A.M. 455
Lima, A.R.C. 119 Mezzomo, R. 97
Lima, L.D. 129 Michal, J.J. 371
Lin, X. 449 Mignon-Grasteau, S. 
Locher, L. 459 Miller, J. 229
Loewen, M.E.  Miller, S.P. 
Lopes, J.B. 245 Mitloehner, F.M. 493
López, M.C. 109, 111 Modesto, V.C. 139

532 Energy and protein metabolism and nutrition in sustainable animal production
Moehn, S. 173, 203 Pantophlet, A.J. 59, 233, 289
Molano, G. 495 Pan, Y. 493
Monnerat, J.P.I.S. 97 Pardo, C.E. 
Moraes, L.E. 327 Pastor, A. 193, 335, 425
Moreira, G.R. 247 Paulino, M.F. 89
Morgavi, D.P. 501 Paulino, P.V.R. 97
Muir, J.P. 491 Payne, R.L. 175
Münger, A. 225 Peixoto, C.A.M. 93, 95
Murphy, M.R. 527 Pellerin, D. 451
Mutsvangwa, T. , 287 Pencharz, P.B. 173, 203
Mynhardt, H. 99 Penner, G.B. , 287, 373, 397
Pereira, G.T. 139
N Pereira, L.G.R. 107, 497
Næsset, J.A. 201 Pereira, M.L.A. 93, 95
Nakashima, K. 253, 395, 419 Pereira, T.C.J. 93, 95
Nanto, F. 505 Peyrat, J. 231
Nascimento, M.L. 127 Peyraud, J.L. 315
Naumann, H.D. 491 Peyronnet, C. 271
Nayananjalie, W.A.D. 387 Pfuhl, R. 
Nebendahl, C. 157,  Pickering, N. 495
Nere, E.A. 285 Pilgrim, R.D. 407
Nguyen, H.V. 415, 427 Pinares-Patiño, C.S. 495
Nielsen, B. 191 Pineda, L. 417
Nieto, R. , 177, 403, 431 Pirondini, M. 499
Nishimura, K. 441 Place, S.E. 493
Noblet, J. 185, 313, 401 Plöntzke, J. 321
Nogueira, W.C.L. 197 Pluschke, A. 289
Nonaka, I. 81 Pollak, E.J. 487
Nørgaard, J.V. 191 Pôssas, F.P. 497
Nozière, P. 231, 315, 317, 319, 347 Poulsen, H.D. 191
Nyachoti, C.M. 187 Prasek, M. 
Praud, C. 
O Prezotto, L.D. 285, 
Obitsu, T. 441 Priebe, M.G. 289
Odongo, N.E. 101 Primot, Y. , 209, 339
Ohanesian, N. 229 Priori, D. 295
Ohtani, F. 81 Protin, P.V. 231
Ohwada, S. 505 Prunier, A. 189
Old, C. 229
Old, C.A. 331 Q
Oliveira, C.F.S. 345 Quigley, J.D. 83
Oliveira, C.V.R. 291
Oliveira, I.M. 97 R
Oliver, M. 405 5D¿L0 173, 203
Oltjen, J.W. 23, 229 Rama Rao, S.V. 175
Orellana, R.A. 415, 427 Ramsey, J.J. 393
Ortigues-Marty, I. 319, 347, 435 Ranathunga, S.D. 
Ouellet, D.R. 409, 443, 447, 451, 453 Rapetti, L. 499
Ray, W.K. 449
P Reagan, J.O. 487
Paes, P.R.O. 117 Recoules, E. 71
Paisley, S.I. 455 Regadas Filho, J.G.L. 249

Energy and protein metabolism and nutrition in sustainable animal production 533
Rehage, J. 459 Schlotterbeck, R.L. 83
Rei, A.J. Del 95 Schneider, C.S. 385
Reiko, C. 275 Schols, H.A. 59, 289
Reis, R.A. 139 Schönhusen, U. 277
Remmelin, G. 471 Schulze, I. 487
Remus, A. 205, 207 Schurmann, B.L. , 397
Resende, K.T. 77, 119, 129, 131, 135, 137 Schwarm, A. 
Reynolds, C.K. 47, 407 Schwarz, F.J. 
Ribas, M.N. 497 Schweigel-Röntgen, M. 
Rideau, N.  Sciascia, Q. 405
Rira, M. 501 Scotti, E. 297
Rivera, A.R. 129 Seiquer, I. 431
Rivera, V. 313 Sharman, E.D. 389
Rizk, A. 459 Shenkute, B. 101
Rodrigues, A.C. 291 Shields, S.L. 321
Rodrigues, J.A.S. 503 Silva, A.L. 341
Rodriguez-Lopez, J.M. 435 Silva, E.P. 333, 343, 345
Rodríguez-López, J.M. 183, 293, 411 Silva, F.A. 247
Rodriguez, N.M. 91, 117, 247, 497 Silva, H.G.O. 93, 129
Röeblitz, S. 321 Silva, J. 487
Roelofsen, J. 289 Silva, L.H.P. 97
Rojas-Cano, M.L. 433 Silva, P.P. 97
Røjen, B.A. 79 Silva, R.R. 91, 117, 503
Röntgen, M. 275, 277, 283, 457,  Silva, S.P. 131
Rosa, A.N. 127 Simon, J. 391
Rossow, H.A. 331, 393 Simon, N. 271
Rotz, C.A. 487 Singer, L.M. 449
Rovers, M. , 195 6NRPLDá- 375, 377, 379
Ruas, J.R.M. 91 Souma, K. 309
Ruiz-González, A. 121 Souza, A.P. 77
Souza, A.R.D.L. 127
S Souza, F.A. 113, 197
Sainz, R.D. , 523 Spachmann, S.K. 277
Sakkas, P. 159,  Spek, J.W. 227
Sakomura, N.K. 207, 333, 343, 345 Srivastava, N. 427
Salah, N. 75 Stackhouse-Lawson, K.R. 487
Salas, C. 305, 307 Starke, S. 399
Sales, F. 405 Starkey, J.D. 389
Saliba, E.O.S. 91, 247, 503 6WDĞNLHZLF]à 375, 377, 379
Sandoval, E. 495 Storm, A.C. 437, 439
Santana, M.C.A. 139 Stothard, P. 
Santos, A.B. 93, 95 Stötzel, C. 321
Santos, E.J. 93, 95 St-Pierre, N. 77
Sapienza, D. 229 Strathe, A.B. 327
Saraiva, A. 245 Südekum, K.-H. 399
Sato, J. 333 Sugino, T. 441
Sauerwein, H. 273 Sünder, A. 425
Sauvant, D. 75, 315, 317, 347 Suryawan, A. 415, 427
Sawosz, E. , 417 Susenbeth, A. 
Schäff, C.  Sutoh, M. 81
Schiassi, L. 125 Swanson, K.C. 285, , 
Schiavo, G. 295, 297 ĝZLĊFK( 375, 377, 379

534 Energy and protein metabolism and nutrition in sustainable animal production
Szopa, J. 381 Viergutz, T. 
Vignale, K. 239, 305
T Villetelle, C. 319
Taciak, M. 375, 377, 379 Vivenza, P.A.D. 117
Taniguchi, K. 441 Vogt, W. 229
Tauson, A.-H. 201, 241, 243, 299 Vonk, R.J. 289
Tedeschi, L.O. 105, 113, 249, 491 Vonnahme, K.A. 285, 
Teixeira, I.A.M.A. 77, 119, 129, 131, 135,
137 W
Tesseraud, S. 179, 209, , 271, 391 Walpole, M.E. , 287, 397
Thanthan, S. 283 Wang, Y. 301
Thibault, J.-N. 401 Wards, N. 405
Thomaz, M.C. 207 Warner, D. 87
Thomson, J.M.  Warpechowski, M. 313
Toyomizu, M. , 309, 505 Watremez, E. 231
Trautwein, J.  Wecke, C. 193, 329, 335
Trece, A.S. 107, 341 Weisbjerg, M.R. 489
Trevisi, P. 295, 297 Welch, C.M. 385
Troni, A.R. 345 Wheatley, S.M. 415
Tullio, R.R. 127 Widjaja, H.C.A. 279
7XĞQLR$ 375, 377, 379 Wierzbicki, M. 
Wiles, T.R. 387
U Willems, E. 301
Udaka, Y. 441 Woelders, H. 321
Ulhman, B. 487 Wood, K.M. 

V Y
Vadalasetty, K.P. 417 Yahaghi, M. 85
Valadares Filho, S.C. 89, 325 Yahav, S. 
Valente, E.E.L. 89, 125, 291 Yanagi Junior, T. 125
Valizadeh, R. 85 Yayou, K. 81
Valkeners, D. 453 Yoshida, H. 309
Van Baal, J. 79, 279 Young, E. 495
Van Beers, H. 237,  Yunusova, R.D. 285
Vandaele, L. 
Van den Borne, J.J.G.C. 59, 79, 159, 223, Z
233, 237, 289,  Zbinden, R.S. 471
Van der Linden, D. 405 Zeitz, J.O. 87
Van der Peet-Schwering, C.M.C. 237,  Zhao, Y. 493
Van der Poel, A.F.B.  ĩXN0 381
Van Diepen, H. , 195
Van Dorland, H.A. , 471
Van Eys, J. 
Van Knegsel, A.T.M. 471
Van Milgen, J. , 179, 189, 313, 339, 401,
429
Van Niekerk, W.A. 99
Van Vuuren, A.M. 47, 279
Vaughn, M.A. 389
Velayudhan, D.E. 187
Vernet, J. 347
Vervenne, P. 159

Energy and protein metabolism and nutrition in sustainable animal production 535