Animal Feed Science and Technology 208 (2015) 23–32

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Intake, milk production and heat stress of dairy cows fed

a citrus extract during summer heat
J.M. Havlin, P.H. Robinson ∗
Department of Animal Science, University of California, Davis, CA, 95616, USA

a r t i c l e i n f o a b s t r a c t

Article history: This study determined effects of feeding a citrus extract (CE) to high producing dairy cows
Received 17 April 2015 during summer heat on measures of heat stress, as well as milk production and composition,
Received in revised form 23 June 2015 in a replicated 2 × 2 Latin square experiment with two 28 d periods on a dairy farm near
Accepted 24 June 2015
Hanford (CA, USA). Four ‘high group’ pens were used (i.e., cows which had cleared the fresh
pen but were not yet confirmed pregnant), each with ∼310 early lactation multiparity
Keywords: cows/pen. The two total mixed rations contained 171 g/kg dry matter (DM) crude protein
Limonene
(CP), 55 g/kg fat, 335 g/kg neutral detergent fiber (aNDF) and 135 g/kg starch, and were the
Vitamin C
same except for inclusion of the CE at 4 g/cow/d in the treatment diet (CED). Average daily
Citrus
Essential oils high temperatures during the study were 35 to 37 ◦ C with lows of 16–17 ◦ C. In general,
cows showed mild heat stress, but CE feeding had no effect on respiration rate, panting
score or rump temperature at any time of the day (i.e., 02:45, 09:15, 17:30 h). However at
02:45 h, a higher (P < 0.01) proportion of CED cows were lying (versus standing) compared
with Control cows (68.6 versus 53.7 cows/100 cows), which is an indicator of reduced heat
stress. Intake of DM (25.3 kg/d) and whole tract digestibility of CP (703 g/kg) and aNDF
(510 g/kg) did not differ between treatments. Milk production (47.3 kg/d) and its fat and true
protein levels (35.4, 28.6 g/kg) did not differ, and changes in body condition and locomotion
scores were also not impacted by treatment. However mammary health improved based
on lower SCC (somatic cell counts; P < 0.04) of CED versus Control cows (160,000 versus
196,000 cells/␮L), and lower linear SCC scores (P < 0.01; 2.12 versus 2.30). Feeding this CE
to lactating dairy cows during summer heat decreased SCC with no impact on other aspects
of performance.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction

A popular contemporary topic in ruminant nutrition is the study of naturally occurring dietary additives which modulate
rumen fermentation in order to improve nutrient utilization by rumen microorganisms, and/or reduce enteric production of
greenhouse gases such as methane. Essential oils (EO) encompass a wide range of naturally occurring secondary compounds

Abbreviations: AA, amino acids; ADF, acid detergent fiber expressed with residual ash; ADFom, ADF expressed without residual ash; aNDF, neutral
detergent fiber assayed with a heat stable amylase and expressed with residual ash; BCS, body condition score; CE, citrus extract; CED, CE diet; CP, crude
protein; DM, dry matter; EE, ether extract; EO, essential oils; LS, locomotion score; PS, panting score; RR, respiration rate; RT, rump temperature; SCC,
somatic cell count; THI, temperature/humidity index; TMR, total mixed ration.
∗ Corresponding author. Tel.: +1 530 754 7565; fax: +1 530 752 0175.
E-mail address: [email protected] (P.H. Robinson).

http://dx.doi.org/10.1016/j.anifeedsci.2015.06.022
0377-8401/© 2015 Elsevier B.V. All rights reserved.
24 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32

which occur in leaves, flowers, stems and seeds of many plants, and may have antimicrobial properties which modify rumen
microbial fermentation.
Reviews of various EO compounds, their possible modes of action and effects on rumen fermentation have been described
(e.g., Benchaar et al., 2008), and limonene, an EO found in citrus products, has been suggested to have beneficial effects on
rumen fermentation, and animal production, in ruminants (Calsamiglia et al., 2007). While few in vivo studies have examined
EO mixtures containing limonene, Tassoul and Shaver (2009) reported increased milk protein production, with an overall
trend to increased feed efficiency (i.e., kg of fat corrected milk/kg of DM intake), when an EO mixture including limonene was
fed to dairy cows. In contrast, Benchaar et al. (2006, 2007) had earlier reported that the same EO mixture used by Tassoul
and Shaver (2009) had no effect on milk production, dry matter (DM) intake or feed efficiency.
Vitamin C, found at high levels in citrus products, is not considered an essential nutrient for healthy cattle because their
liver can synthesize it from glucose at levels believed to be sufficient to meet their needs (Padh, 1990). However cattle under
stress, such as those in early lactation at high production levels and/or under summer heat, may not synthesize adequate
amounts of vitamin C to compensate for oxidative stress due to sub-clinical infection or illness (Weiss et al., 2004; Ranjan
et al., 2005), and feeding supplemental vitamin C can replace depleted reserves (Weiss, 2001). As high somatic cell counts
(SCC) in milk are the result of degraded mammary health, and have a negative impact on animal performance and milk
quality, the limonene and vitamin C in citrus products may help minimize SCC in dairy cows, thereby improving efficiency
of feed utilization as a result of improved immune function (Chaiyotwittayakun et al., 2002; Castillejos et al., 2006). Indeed
Jaramillo et al. (2009) and Giannenas et al. (2011) fed citrus pulp to ewes and reduced milk SCC.
In a review of citrus product feeding to ruminants, Bampidis and Robinson (2006) concluded that their feeding did not
affect DM digestibility, but decreased crude protein (CP) digestibility, while increasing neutral detergent fiber (NDF) and
acid detergent fiber (ADF) digestibility.
Our objective was to evaluate impacts of a CE on feed intake and productive performance of dairy cows during hot
weather.

2. Materials and methods

2.1. Animals, management, and experimental design

High group multiparity Holstein cows (i.e., those cows which had cleared the fresh pen but were not yet confirmed
pregnant) in 4 early lactation pens of ∼310 cows each, on a commercial dairy farm near Hanford (CA, USA), were used in
a study with two 28 d experimental periods in a replicated 2 × 2 crossover design during summer. Each pen had 300 head
gates and free stalls. The only source of cooling was bunk line misters which switched on automatically at 24 ◦ C, and were
on in each pen for 4 mins and then off for 12. Cows were assigned weekly to one of the 4 pens at random from a common
fresh cow pen, and moved to a mid pen by ∼200 days in milk once they were confirmed pregnant.
Cows were milked three times daily in a double 40 herringbone parlor. Pen 1 started milking at 04:00, 12:00 and 20:00 h.
Pens 2, 3, and 4 were milked in sequence after pen 1, at intervals of ∼45 min. The total mixed ration (TMR) was fed twice
daily, between 04:00 and 08:00 h, prior to cows returning to their pens from milking, and again between 11:30 and 12:30 h.
The amount of TMR delivered was determined from the previous days intake to create orts equal to ∼10 g/kg of total TMR
delivered (as fed). Orts were removed daily and weighed individually by pen while cows were in the parlor during the first
milking, just prior to the first TMR feeding.
Head-locks were set prior to cows returned from the morning milking, to facilitate artificial insemination, such that the
cows were head locked in the stanchions for 45–60 min daily. Cows were housed in covered barns with access to free stalls
bedded with dried manure, which was renewed weekly and groomed bi-weekly. Cows also had free access to an uncovered
manure pack drylot at all times. Inside alleyways were flushed with water three times daily while the cows were being
milked. Cows had ad libitum access to clean drinking water at all times.
At the start of the study, two pens were fed the CE containing diet (CED), while the two other pens were fed the Control
diet. After the first 28 d period, treatments were reversed. Each period contained a 21 d adjustment and 7 d collection period
during which all samples were collected.

2.2. Environmental conditions

Four portable weather data loggers (HOBO U23; Onset, Bourne, MA, USA) were recorded ambient temperatures and
relative humidity every 30 min throughout the study. One station was placed in each experimental pen on a pole at its
center ∼3 m above the floor and out of direct sunlight. The study took place in the 2 mo after the summer solstice to
minimize variation of day length and weather, while maximizing expected temperature/humidity indices (THI) which was
calculated as:
  .55 × RH 
THI = tdb − 0.55 − (tdb − 58.8)
100
where: tdb = dry bulb air temperature (◦ F) and RH = relative humidity (%). THI ≤ 74 is considered “normal”, 75–78 is “alert”,
79–83 is “danger”, and ≥84 is “emergency” according to the Livestock Weather Safety Index (LCI, 1970).
 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32 25

2.3. Diets

The TMR for the CED and Control groups was formulated identically except for inclusion of the CE at 4 g/cow/d in the
CED TMR. Rate of inclusion of the CE product (VéO Premium; Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac,
France) in TMR loads was calculated weekly based on the number of cows in the pen, and used to create bags of CE which
the feeding staff added to the CED TMR. The CE contained natural citrus extracts, natural and artificial flavoring as well as
corn flour, calcium carbonate and silicic acid as flow enhancers.

2.4. Sample collection

2.4.1. Feed and total mixed rations

At the start and end of the collection week of each period (i.e., days 21 and 27) all dietary ingredients were sampled. Hays
were sampled with a ‘golf club’ style hay probe (Sierra Testing Services, Acampo, CA, USA), with 12 core samples pooled
into a plastic bag. All other ingredients, with the exception of liquid whey, were sampled by hand (6 handfuls of each) and
composited to plastic bags. Silages were sampled from the loose pile which had been knocked off the silage face for use that
day and wet by-products (i.e., fresh chop alfalfa, tomato pomace) were sampled from the storage area. Samples of all other
feeds from the start and end of the collection weeks were combined to create one sample per ingredient prior for chemical
analysis.
Ten handfuls (∼200 g each) of each TMR by pen were collected according to Robinson and Meyer (2010) at pre-marked
posts with evenly spaced intervals along the bunk-line immediately after feeding and before the cows had access to it. These
TMR samples were pooled within period and pen to create 8 TMR samples for chemical analysis.

2.4.2. Milk
At the end of each collection week (i.e., day 28), the entire herd was tested for milk production and composition by Dairy
Herd Improvement Association (Kings County, CA, USA) personnel. Milk weights and representative milk samples collected
into tubes containing bronopol and natamycin as preservatives were collected from all cows using Tru-test milk meters
(Tru-Test Ltd., Auckland, New Zealand).

2.4.3. Body condition and locomotion scores

Body condition (BCS) was scored at the start of the study (i.e., day 0) and at the end (i.e., day 28) of each experimental
period while the cows were locked in stanchions immediately after morning milking. From each pen, 80 cows having the
lowest days in milk at the start of the study were selected. Cows were scored on the 1–5 scale of Edmonson et al. (1989),
with intermediate values between the 1/4 points if a 1/4 point score was not clear. One person scored all cows.
Locomotion was scored (LS) at the end (i.e., day 28) of each experimental period while the cows were returning from
the parlor from milking starting at 14:00 h. All cows in the pen were observed for lameness using the 1–5 scoring system of
Sprecher et al. (1997). One person scored all cows on both occasions.

2.4.4. Fecal
Fecal samples were collected from the same 18 cows during the morning lock-up on day 28 of each period. Feces were
manually collected (minimum 250 g) from the rectum of the cow into plastic containers, and immediately stored at −18 ◦ C
until processing for chemical analysis.

2.4.5. Heat stress behavior and skin temperature

Respiration rate (RR), panting score (PS), lying versus standing behavior, and rump temperature (RT) was measured three
times over a 24 h period on days 27 and 28 of each period. Skin temperature was recorded on days 26 and 28 during morning
lock-up during both periods, as they had not yet had access to the dry lot where they would be in direct sunlight at this time.
A subgroup of 50 cows which had been scored for BCS from each pen was observed for heat stress parameters. Identification
collars were placed on the cows during morning lock-up to facilitate easier identification of cows from a distance during
observations. Behavioral observations started ∼24 h after the collars were attached. The behavior observation periods for
pens 1 and 2 began at 08:00, 16:15 and 01:30 h, while observations for pens 3 and 4 began directly following observations
of pens 1 and 2. Observation periods lasted 75 mins/pen. The 08:00 h observation time was chosen to ensure that the cows
had been out of head-lock for at least 15 min prior to observations. The 16:15 h time was chosen to approximate the hottest
time of the day and, during the 01:30 h observation period, the sun had been down for ∼5.5 h and the temperature was
approaching the daily low. At this time the vast majority of the cows were in the outside dry lot.
The RR was determined by observing flank movements of the cows where the time it took a cow to take 20 breaths
was recorded and converted to breaths/min. A 0–5 PS was assigned to each cow (Gaughan et al., 2008) based on physical
signs including degree of chest movement and presence of drool. Cows in two pens were simultaneously observed for heat
stress behaviors by two individual scorers. Each scorer observed the same treatment and Control pen in each period. Scorers
trained together prior to the study, and between periods, to eliminate scorer bias.
26 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32

To obtain RT, two small spots of ∼40 × 60 mm each were shaved on each side of the rear flank, distal to where the udder
meets the leg. Temperatures from the right and left flank were measured using an infrared gun (Fluke-561, Everett, WA,
USA).

2.5. Analytical methods

2.5.1. Ingredient and total mixed ration chemical analysis

All wet ingredients and TMR samples were dried at 55 ◦ C for 48 h and air equilibrated for 24 h. Dried samples, with other
ingredients which did not require drying, were analyzed at the UC Davis Analytical Laboratory (Davis, CA, USA). All samples
were ground to pass a 0.4 mm screen on an Intermediate Wiley Mill or a 1 mm screen on a model 4 Wiley Mill (Thomas
Scientific, Swedesboro, NJ, USA). Moisture was determined by gravimetric loss of free water by heating to 105 ◦ C in a forced
air oven for 3 h, and ash was the gravimetric residue after heating to 550 ◦ C for at least 3 h. Total N and N in ADF were
determined with infrared detection and thermal conductivity (TruSpec CN Analyzers, St. Joseph, MI, USA) by AOAC (2006;
#990.03). Analysis of aNDF used a heat stable amylase (AOAC, 2006; #2002-04), and aNDF and ADF are expressed exclusive
of residual ash (i.e., aNDFom, ADFom) or with residual ash (i.e., aNDF). ADFom analysis was by boiling in acid detergent for
1 h (AOAC, 1997; #973.18).
Crude fat, as ether extract (EE), was determined by extraction in ethyl ether (AOAC, 2006; #2003.05). Free sugars are
the sum of glucose, fructose and sucrose as determined by HPLC (Johansen et al., 1996). Enzymatic hydrolysis was used to
determine the amount of total glucose, and free glucose was subtracted from total glucose, and the difference multiplied by
0.9 to calculate starch (Smith, 1969). Lignin(sa) was determined by the sulfuric acid procedure (AOAC, 1997; #973.18). The Ca,
Cu, Fe, Mg, Mn, P, K, Na, S and Zn contents were determined by microwave nitric acid/hydrogen peroxide digestion/dissolution
by inductively coupled plasma atomic emission spectrometry (Meyer and Keliher, 1992; Sah and Miller, 1992). The Cl content
was determined after water extraction and analysis by ion chromatography with conductivity detection (Jones, 2001). Total
Se was extracted by nitric/perchloric acid digestion/dissolution and determined by vapor generation using ICP-AES (Tracy
and Mueller, 1990).

2.5.2. Fecal
Fecal samples from the 18 cows/pen which had been sampled in each experimental period were divided into 3 subgroups
according to ear tag number order. Samples were individually dried at 55 ◦ C for 48 h and then 150 g of each sample within
a subgroup was pooled to create 3 fecal sample groups/pen/period. The pooled samples were analyzed by the UC Davis
Analytical Laboratory as described for the feed samples, as appropriate.

2.5.3. Milk
Fat, true protein and lactose, as well as SCC, were determined using infrared spectroscopy at the Dairy Herd Improvement
Association laboratory in Hanford (CA, USA).

2.6. Calculations

2.6.1. Dry matter intake

Intake of TMR for each pen was calculated during the collection week (i.e., day 22 through day 28, inclusive) of each
period based on the total weight of TMR delivered to each pen for that period corrected for the orts which were removed
prior to the first feeding of each day. The analyzed 105 ◦ C DM content of the TMR was used to determine DM intake. The
number of cows/pen during the collection period was the average of the number of cows in the pen on the electronic record
system (Dairy Comp 305, Valley Ag Systems, Tulare, CA, USA) on the first and last day of the final week of each period.

2.6.2. Energy calculations

Milk energy (MJ/kg) was calculated for individual cows according to Tyrrell and Reid (1965) using milk fat, crude protein
and lactose, and the energy (MJ/d) in BCS change was calculated by cow according to NRC (2001) as 1255.2 MJ/unit BCS.
Maintenance energy (MJ/d) was calculated as: (BW0.75 × 0.08 × 4.184) according to NRC (2001) for all groups assuming an
average body weight of 650 kg for all cows. Total energy output was calculated as the sum of milk energy (MJ/d), BCS change
energy (MJ/d) and maintenance energy (MJ/d). The NEL density of the TMR was calculated by pen and period as: Total Energy
Output (MJ/d)/DM intake (kg/d).

2.6.3. Somatic cell count and linear conversion

Because distribution of SCC in milk has a positive skewness with heterogeneous variance (Ali and Shook, 1980), log
transformation (Schukken et al., 2003) was used to normalize the data to create a linear SCC as: LnS = log2 (SCC/100) + 3,
where: SCC is expressed as cells/␮L.
 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32 27

Fig. 1. The 24 h pattern of ambient temperature and temperature humidity index (THI) during the 3 day period of behavioral observations in periods 1 and
2. Weather data was recorded every 30 min. Graphs show the averages of each weather data point.

2.6.4. Whole tract digestibility

Apparent digestibility of dietary components was calculated by the proportion of feed components remaining in the
feces from the diet, using lignin(sa) as the indigestible marker but assuming 0.95 fecal recovery of lignin(sa) (Stensig and
Robinson, 1997).

2.7. Statistical analysis

The chemical composition of the TMR fed was statistically analyzed using the GLM procedure of SAS (2012) with pen,
period and treatment as effects. The two TMR samples collected from each pen at the start and end of each collection week
(i.e., day 21 and 27 of each period) were combined prior to chemical analysis to be representative of the final week of the
collection period. The DM intake data was analyzed using the GLM option of SAS (2012) with pen, period and treatment as
fixed effects.
The eligibility criteria for cows to be included in the statistical analysis was that they had to have been in her ≤6 lactation,
and have remained in the same pen for the entire study (n = 670). However, cows with milk or milk component values
determined visually to be biological outliers were excluded from statistical analysis. Removal selection was completed blind
to treatment and pen. A total of 16 cows were so removed, leaving 654 cows which met the criteria for inclusion and were
included in the data set for statistical analysis. Milk yield and components, as well as LS, were analyzed using the MIXED
option of SAS with cow nested within pens as a random effect. The statistical model included fixed effects of pen, period and
treatment.
Data for cows included in the statistical analysis for BCS, which were a subset of the cows used for milk production data,
had to meet all criteria required for milk analysis, as well as having been scored for BCS at the start of the study and at the
end of both experimental periods. The BCS (n = 168) data was analyzed with the MIXED model of SAS with cows, pen, period
and treatment, as effects, as described above for analysis of milk parameters.
Statistical inclusion criteria of cows for digestibility of feed components were the same as the milk criteria for each cow,
and were a subset of these cows. Digestibility was analyzed with the MIXED model of SAS with fecal group, pen, period
and treatment as effects, as described above for milk parameter statistical analysis. Fecal group was a random effect in this
model.
Treatment differences were accepted if P ≤ 0.05, and tendencies to significance were accepted if 0.05 < P ≤ 0.10.

3. Results

3.1. Environmental conditions

Diurnal patterns of ambient temperature and THI were similar between experimental periods (Fig. 1). While the daily
temperature highs were similar to historical averages, the nighttime lows were slightly cooler than normal. During the study
period, highs were 36.5 ◦ C and 35.6 ◦ C with lows of 16.9 ◦ C and 15.8 ◦ C, for period 1 (July) and 2 (August), respectively.
28 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32

Table 1
Ingredient and chemical composition of Control and orange extract (CED) total mixed rations.

Control CED SEM P

Ingredient composition (g/kg DM)a

Alfalfa, hay 167 164
Cottonseed, whole uplandb 72 72
Cottonseed, cracked pimab 60 60
Almond, hullsb 159 160
Canola, meal-solventb 144 145
Wheat, strawb 33 34
Mineral, premixb 19 19
Limestone, crushedb 5 5
DDGSb 64 64
Corn gluten, pelletsb 71 71
Fat, rumen inertb , c 11 11
Molasses, liquidb 23 23
Alfalfa, fresh chop 36 36
Wheat, whole crop silage 22 21
Corn, flaked grain 33 33
Corn, whole crop silage 38 38
“Cristalfeed® Gold Rush”d – 0.16
Tomato, pomace-wet 14 14
Whey, liquid 29 29

Chemical composition (g/kg DM)

Dry matter (g/kg) 574 578 1.7 0.36
Crude protein 172 170 1.5 0.37
ADICP (g/kg CP)e 64 66 1.5 0.50
aNDF 335 334 2.3 0.88
aNDFom 316 317 2.0 0.69
ADFom 230 231 3.3 0.82
Lignin(sa) 5.0 5.2 0.4 0.09
Starch 125 144 7.3 0.25
Sugarsf 37 37 0.4 0.51
Crude Fat 54 55 0.4 0.31
Ash 94 93 0.2 0.07
Calcium 9.3 9.8 0.05 0.02
Phosphorus 5.4 5.5 0.06 0.63
Magnesium 3.0 3.1 0.02 0.27
Potassium 19.3 19.2 0.15 0.66
Sulfur 2.9 2.9 0.04 0.77
Sodium 2.8 2.8 0.04 0.92
Chloride 6.5 6.6 0.11 0.75
Zinc (mg/kg DM) 71.6 71.5 0.311 0.86
Manganese (mg/kg DM) 39.7 39.4 0.336 0.63
Iron (mg/kg DM) 245 212 17.1 0.35
Copper (mg/kg DM) 13.6 13.6 0.08 0.73
Cobalt (mg/kg DM) 0.43 0.41 0.018 0.70
Selenium (mg/kg DM) 0.43 0.42 0.005 0.31
a
TMR ingredients listed in the order in which they entered the mixer.
b
Ingredients combined to create a premix which was added to create the TMR.
c
EngerG II, Virtus Nutrition, Corcoran, CA, USA.
d
VéO Premium (Cristalfeed® Gold Rush; Reference ST 232 P2; Phodé, Terssac, France).
e
Acid detergent insoluble CP.
f
Free glucose, sucrose and fructose.

3.2. Chemical composition of feeds and total mixed ration

The chemical composition of feeds in the TMR were generally consistent with NRC (2001) values, and the chemical profile
of the TMR (Table 1) met or exceeded recommendations of the NRC (2001) for dairy cattle at similar production levels. There
were no differences between the chemical profile of the TMR fed to the Control and CED groups, except for a slightly higher
level of Ca (P = 0.02) of the CED diet.

3.3. Feed intake, milk production and body condition score

DM intake was not affected by feeding CE (Table 2), and milk and milk component yields also did not differ between
groups. Milk SCC (P = 0.04) and linear score of SCC (P < 0.01) were lower for cows fed CE. The BCS and LS, as well change of
BCS, did not differ between groups.
 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32 29

Table 2
Effects of feeding citrus extract (CE) on DM intake, milk yield, milk components and body condition score.

Control CED SEM P

DM intake (kg/d; n = 8) 25.39 25.18 0.390 0.62

Yield (kg/d; n = 654)

Milk 47.04 47.46 0.270 0.13
Fat 1.66 1.67 0.013 0.46
True Protein 1.34 1.35 0.007 0.08
Lactose 2.25 2.27 0.013 0.06
Energy (MJ/d) 135.3 136.6 0.826 0.19

Components (g/kg milk)

Fat 35.4 35.3 0.20 0.79
True protein 28.6 28.6 0.08 0.49
Lactose 47.8 47.9 0.06 0.14
SCC (cells/␮L) 196 160 16.5 0.04
SCC (linear score) 2.30 2.12 0.795 <0.01

Body Condition Score (n = 168)

Average (units) 2.59 2.59 0.016 0.53
Change (units/28 d) 0.028 0.027 0.0133 0.93

3.4. Heat stress parameters, body temperatures and locomotion score

The RR, PS and RT (Table 3) did not differ between treatment groups at any observation time. The proportion of cows
lying versus standing was higher (P < 0.01) for cows fed the CED (68.6 versus 53.7 cows/100 cows) at 02:45 h, but did not
differ at other times.

3.5. Whole tract apparent digestibility

Feeding the CE did not impact whole tract digestibility (Table 4), with values within normal ranges for cows with high
DM intake (Colucci et al., 1982; NRC, 2001).

4. Discussion

4.1. Effect of citrus extract on heat stress, body temperature and locomotion

Cows in the study were judged to have experienced mild heat stress because, even though daytime high THI levels were
often in the “alert” or “danger” levels, THI values of <74 (i.e., normal) occurred for ∼10 h/d. This judgement is consistent with
the measured RR of 58.7–64.0 breaths/min, which contrasts to RR of cattle of 100 breaths/min, or more, under extreme heat
stress (Turner et al., 1992). Likewise, the low PS of 0.89 to 1.27 of cows in both treatment groups suggests that they were not

Table 3
Effects of feeding citrus extract (CE) on respiration rates, panting scores, lying versus standing behavior at three times of the daya , as well as skin temperature
and locomotion scores in early lactation cows (n = 168).

Control CED SEM P

Respiration Rate (breaths/min)

2:45 h 63.2 61.6 1.18 0.36
9:15 h 62.9 63.9 1.34 0.59
17:30 h 59.8 58.7 1.14 0.50

Panting Score (unit)b

2:45 h 1.09 1.12 0.037 0.55
9:15 h 0.89 0.92 0.038 0.46
17:30 h 1.27 1.22 0.034 0.28

Lying versus standing (cow/100 cows)c

2:45 h 53.7 68.6 0.038 <0.01
9:15 h 41.1 39.7 0.038 0.79
17:30 h 33.7 27.3 0.034 0.18

Rump temperature (◦ C) 34.1 34.1 0.076 0.50

Locomotion score (unit; n = 654) 1.15 1.15 0.015 0.88

a
Midpoint of the period starting 75 min prior and ending 75 min after the listed time.
b
A measurement on a scale of 0–5 of the extent that a cow was is panting (Fig. 2).
c
The proportion of cows scored as lying versus standing based on the position in which they were first found.
30 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32

Table 4
Effects of feeding citrus extract (CE) on whole tract digestibility (g/kg) of some dietary nutrients (n = 24), and partial energy balance (MJ/d).

Control CED SEM P

Whole tract digestibility

Organic matter 724 715 4.7 0.18
aNeutral detergent fiberom a 513 508 6.3 0.62
aNeutral detergent fiberb 501 492 7.5 0.41
Crude Protein 711 695 7.3 0.14

Partial energy balance

Milk 135.3 136.6 0.83 0.19
Change in BCS 1.3 1.2 0.60 0.93
NEL c 179.7 180.9 –d –
NEL densitye (MJ/kg DM) 7.08 7.18 – –
a
aNDFom expressed exclusive of residual ash.
b
aNDF expressed inclusive of residual ash.
c
Net energy of lactation; calculated by summing maintenance, milk and change in BCS energy, where maintenance energy is 41.8 MJ/d assuming cow
average body weight was 650 kg.
d
Net energy of lactation divided by measured DM intake.
e
Reported as listed values with arithmetic means of treatment means.

substantively heat stressed, and rump temperatures were also within the range of mildly heat stressed cows (Di Costanzo
et al., 1997). This low level of observed heat stress is probably because there was consistent night-time cooling, and because
the cows were fully shaded from the sun with access to bunk line misters (West, 2003; Gaughan et al., 2008).
It is clear that feeding the CE had little or no effect on heat stress of our cows since RR, PS and RT were not influenced.
That the RR of both groups was lower at 17:30 than at 2:45 h differed from expectations due to temperatures at those times
of day (Di Costanzo et al., 1997). In contrast, the PS pattern for both groups was highest at 17:30 h, the hottest time of the
day, and the lowest at 09:15 h probably due to cows having recovered during the cooler night. At 2:45 h little TMR remained
in the feed bunks and very few cows were eating, with the majority in the open drylot where more CED cows were lying
versus standing. As Hillman et al. (2005) found that the body temperature of cows rises when they are lying, and that they
tend to stand and seek cooling when core body temperature rises above 38.9 ◦ C, the higher number of CED cows lying at
02:45 h suggests that CE may have reduced core body temperatures and heat stress, at least at this time.

4.2. Production responses to citrus extracts

Lack of a treatment difference on DM intake was not unexpected because studies with limonene fed in concert with
other EO have shown no DM intake effect (Benchaar et al., 2006, 2007), or a slight trend to a reduction (Santos et al., 2010).
Vitamin C supplementation also does not appear to effect DM intake (Weiss, 2001; Chaiyotwittayakun et al., 2002; Weiss
et al., 2004).
While there are no in vivo studies that have only fed limonene, there is little support from in vitro studies that limonene
affects microbial fermentation, feed efficiency or nutrient utilization to support milk production (Dorman and Deans, 2000).
This is consistent with our similar dietary NEL density (7.18 versus 7.08 MJ/kg) of the Control and CED TMR (Table 4). In
a review of citrus by-product feeding, Bampidis and Robinson (2006) reported that adding citrus by-products to diets of
lactating dairy cows did not change milk yield or composition if the diets had a similar nutrient profile, which is also
consistent with our finding that there were no production differences between the Control and CED groups.

4.3. Milk somatic cell count (SCC) response to citrus extracts

Based on our SCC differences between treatments, the CED diet appears to have reduced the incidence and/or extent of
mastitis. Indeed Lund et al. (1994) found a high genetic correlation between SCC and clinical mastitis (0.97), concluding that
SCC is an indicator of clinical mastitis. In addition, increased SCC has been shown to be positively correlated with increased
mastitis risk (Ward and Schultz, 1972; Kirk, 1984; Barkema et al., 1998), and Harmon (1994) found that, in cows with higher
SCC, lactose tends to leak out of the infected mammary gland into the blood, resulting in less milk lactose, which is consistent
with the slightly lower lactose output of Control cows, which also had higher SCC.

4.3.1. Effects of feeding citrus pulp or limonene in an EO blend on milk SCC

When lactating ewes were fed whole citrus pulp at 100, 200 and 300 g/kg DM of their ration, Jaramillo et al. (2009)
reported lower milk SCC. Giannenas et al. (2011) fed an EO blend, which included limonene, to dairy ewes at three levels.
At day 50, there was no change in SCC but, at day 100, ewes fed the highest EO treatment had lower SCC and, at day 150, all
EO treatments had a lower SCC, with ewes fed the highest EO level having a lower SCC than the other ewes. This suggests a
dose-dependent effect to the EO blend containing limonene which is magnified with longer feeding time.
 J.M. Havlin, P.H. Robinson / Animal Feed Science and Technology 208 (2015) 23–32 31

4.3.2. Effects of feeding vitamin C on milk SCC

In contrast to EO, there has been substantial amount of research on impacts of dietary Vitamin C supplementation on
udder health, especially mastitis. When pathogenic microorganisms enter the mammary gland through the streak canal an
inflammatory response is triggered in the infected gland, thereby increasing the number of reactive oxygen species (Ranjan
et al., 2005). Since two lines of defense are occurring at this time (i.e., sequestering of antioxidants and recruiting of leukocytes
(Ranjan et al., 2005; Harmon, 1994)), antioxidants such as vitamin C can be utilized in high quantities to inactivate reactive
oxygen species to decrease their concentrations in plasma and the infected mammary quarter (Harmon, 1994). Early studies
showed that vitamin C is quickly degraded in vitro and in vivo (Knight et al., 1941; Vavich et al., 1945), presumably by rumen
microbes. However Hidiroglou (1999) found that cows fed vitamin C or ethyl cellulose coated vitamin C (i.e., ruminally
protected), or had vitamin C infused abomasally, all had elevated plasma amino acid (AA) concentrations, but cows fed
uncoated vitamin C had the smallest increase. This supports the probability that not all dietary vitamin C is degraded in the
rumen. Weiss et al. (2004) and Ranjan et al. (2005) showed that infected mammary glands increase vitamin C utilization,
and Weiss et al. (2004) also showed that mammary quarters challenged with E. coli had decreased levels of vitamin C in
milk and plasma due to an inflammatory response which increased AA oxidation. This suggests that depletion of vitamin C
in plasma and milk occurs during infection, rather than a low vitamin C status per se, which allows a more severe infection
to occur.
Although cattle are thought to synthesize adequate vitamin C to maintain healthy mammary gland function, vitamin C
synthesis is not up-regulated when cows are under immunological stress (Weiss et al., 2004), but vitamin C uptake and
oxidation is increased in effected tissues. Thus it is possible that in a generally healthy mammary gland, such as was the case
of cows in our study based upon relatively low overall milk SCC counts, supplemental vitamin C may have helped maintain
a high antioxidant/pro-oxidant ratio, thereby aiding in prevention of oxidative damage.

5. Conclusions

Supplementing the diet of dairy cows with a commercially available citrus extract decreased SCC in generally healthy
dairy cows under mild heat stress conditions. However measures of heat stress and animal production were not substantively
treatment impacted.

Conflict of interest

The authors stipulate that they have no conflicts of interest in preparation or submission of this manuscript.

Acknowledgements

The authors thank all who volunteered to help: Grace Cun, Stacy Wrinkle, Nadia Swanepoel, Blanca Comacho and Emma
Robinson. We are grateful to Stacy Wrinkle and Pablo Chilibroste for sharing their knowledge on heat stress behaviors, and
to William Van Die for allowing us to conduct the study on his dairy.

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