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Sturkie’s Avian Physiology
Seventh Edition

Edited by
Colin G. Scanes
Department of Biological Science, University of Wisconsin, Milwaukee, WI, United States;
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR, United States
Sami Dridi
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR, United States
Academic Press is an imprint of Elsevier
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To our mentors who have guided and inspired us and our
families who have given us so much support.
Contributors

N.J. Beausoleil, Animal Welfare Science and Bioethics Environment, The Hebrew University, Rehovot, Israel;
Centre, School of Veterinary Science, Massey Univer- Miloubar Feedmill, Ashrat Industrial Area, Israel
sity, Palmerston North, Manawatu, New Zealand Hans H. Cheng, USDA, ARS, Avian Disease and
Charles M. Bishop, School of Natural Sciences, Bangor Oncology Laboratory, East Lansing, MI, United States
University, Bangor, Gwynedd, United Kingdom Helen E. Chmura, Institute of Arctic Biology, University
Julio Blas, Department of Conservation Biology, Estación of Fairbanks, Fairbanks, AK, United States
Biológica de Doñana, Consejo Superior de Inves- Larry Clark, METIS, Ltd., Fort Collins, CO, United States
tigaciones Científicas (CSIC), Seville, Spain
Mark A. Cline, Department of Animal and Poultry Sci-
Walter Gay Bottje, Department of Poultry Science, Center ences, Virginia Tech, Blacksburg, VA, United States
of Excellence for Poultry Science, Division of Agri-
culture, University of Arkansas, Fayetteville, AR, Jamie M. Cornelius, Department of Integrative Biology,
United States Oregon State University, Corvallis, OR, United States

Kathleen R. Brazeal, School of Biological Sciences, Dane A. Crossley, Department of Biological Sciences,
University of Nebraska-Lincoln, Lincoln, NE, United University of North Texas, Denton, TX, United States
States Veerle M. Darras, Department of Biology, KU Leuven,
Lindsay P. Brown, Department of Chemistry, University Leuven, Belgium
of Tennessee, Knoxville, TN, United States Karen D.M. Dean, University of Lethbridge, Lethbridge,
Shane C. Burgess, College of Agriculture & Life Sciences, AB, Canada
The University of Arizona, Tucson, AZ, United States Eddy Decuypere, Laboratory of Livestock Physiology,
Warren W. Burggren, Developmental and Integrative Department of Biosystems, Faculty of Bioscience
Biology, Department of Biological Science, University Engineering, KU Leuven, Leuven, Belgium
of North Texas, Denton, TX, United States Mike Denbow, Department of Animal and Poultry Sci-
Johan Buyse, Laboratory of Livestock Physiology, ences, Virginia Tech, Blacksburg, VA, United States
Department of Biosystems, Faculty of Bioscience En- Pierre Deviche, School of Life Sciences, Arizona State
gineering, KU Leuven, Leuven, Belgium University, Tempe, AZ, United States
Shawn R. Campagna, Department of Chemistry, Univer- Sami Dridi, Center of Excellence for Poultry Science,
sity of Tennessee, Knoxville, TN, United States; University of Arkansas, Fayetteville, AR, United States
Biological and Small Molecule Mass Spectrometry Joëlle Dupont, PRC (UMR 6175), INRA, Nouzilly,
Core, University of Tennessee, Knoxville, TN, United France; Unité de Physiologie de la Reproduction et des
States Comportements, Institut National de la Recherche
Rocco V. Carsia, Department of Cell Biology and Neu- Agronomique, Nouzilly, France
roscience, Rowan University School of Osteopathic Vijay Durairaj, Huvepharma Inc., Lincoln, NE, United
Medicine, Stratford, NJ, United States States
Vincent M. Cassone, Department of Biology, University Edward M. Dzialowski, Department of Biological
of Kentucky, Lexington, KY, United States Sciences, University of North Texas, Denton, TX,
Natalia Cerón-Romero, Food and Animal Sciences, Ala- United States
bama A&M University, Huntsville, AL, United States Nima K. Emami, Center of Excellence for Poultry
Shira L. Cheled Shoval, Department of Animal Science, Science, University of Arkansas, Fayetteville, AR,
The Robert H. Smith Faculty of Agriculture, Food and United States

xxvii
xxviii Contributors

Nadia Everaert, Precision Livestock and Nutrition Unit, Michael H. Kogut, Southern Plains Agricultural Research
TERRA Teaching and Research Centre, Gembloux Center, USDA-ARS, College Station, TX, United States
Agro-Bio Tech, University of Liège, Gembloux, Daniel T. Ksepka, Bruce Museum, Greenwich, CT, United
Belgium States
Graham D. Fairhurst, School of Environment and Sus- Christine Köppl, Cluster of Excellence “Hearing4all”,
tainability, University of Saskatchewan, Saskatoon, SK, Carl von Ossietzky University, Oldenburg, Germany;
Canada Research Center Neurosensory Science, Carl von
Alison Ferver, Center of Excellence for Poultry Science, Ossietzky University, Oldenburg, Germany; Department
University of Arkansas, Fayetteville, AR, United States of Neuroscience, School of Medicine and Health Science,
Alexander R. Fisch, Department of Chemistry, University Carl von Ossietzky University, Oldenburg, Germany
of Tennessee, Knoxville, TN, United States Wayne J. Kuenzel, Poultry Science Center, University of
Joel Gautron, BOA, INRAE, Université de Tours, Arkansas, Fayetteville, AR, United States
Fonction et Régulation des protéines de l’œuf, Vinod Kumar, Department of Zoology, University of
Développement de l’œuf, Valorisation, Évolution, Delhi, Delhi, India
France H. Lehmann, Animal Welfare Science and Bioethics
Elizabeth Gilbert, Department of Animal and Poultry Centre, School of Veterinary Science, Massey Univer-
Sciences, Virginia Tech, Blacksburg, VA, United States sity, Palmerston North, Manawatu, New Zealand
David L. Goldstein, Department of Biological Sciences, Scott A. MacDougall-Shackleton, Departments of Psy-
Wright State University, Dayton, OH, United States chology and Biology, University of Western, London,
Elizabeth S. Greene, Center of Excellence for Poultry ON, Canada
Science, University of Arkansas, Fayetteville, AR, Graham R. Martin, School of Biosciences, University of
United States Birmingham, Birmingham, United Kingdom
Christopher G. Guglielmo, Department of Biology, J.E. Martin, Royal (Dick) School of Veterinary Studies
Advanced Facility for Avian Research, Western Uni- and the Roslin Institute, University of Edinburgh,
versity, London, ON, Canada Edinburgh, Scotland
Thomas P. Hahn, Department of Neurobiology, Physiol- Amanda L. May, Center for Environmental Bio-
ogy and Behavior, University of California, Davis, CA, technology, University of Tennessee, Knoxville, TN,
United States United States
Orna Halevy, The Hebrew University of Jerusalem, Andrew E. McKechnie, South African Research Chair in
Rehovot, Israel; The Ohio State University, Wooster, Conservation Physiology, South African National Bio-
OH, United States diversity Institute, Pretoria, Gauteng, South Africa;
Maxwell Hincke, Department of Innovation in Medical DSI-NRF Centre of Excellence at the FitzPatrick
Education; Department of Cellular and Molecular Institute, Department of Zoology and Entomology,
Medicine, Faculty of Medicine, University of Ottawa, University of Pretoria, Hatfield, Pretoria, Gauteng,
Ottawa, ON, Canada South Africa

S.E. Holdsworth, Animal Welfare Science and Bioethics D.E.F. McKeegan, Institute of Biodiversity, Animal
Centre, School of Veterinary Science, Massey Univer- Health and Comparative Medicine, University of
sity, Palmerston North, Manawatu, New Zealand Glasgow, Glasgow, Scotland

Christa F. Honaker, Department of Animal and Poultry Scott R. McWilliams, Department of Natural Resources
Sciences, Virginia Tech, Blacksburg, VA, United States Science, University of Rhode Island, Kingston, RI,
United States
Anna Hrabia, Department of Animal Physiology and
Endocrinology, University of Agriculture in Krakow, Henrik Mouritsen, Institut für Biologie und Umweltwis-
Krakow, Poland senschaften, Universität Oldenburg, Oldenburg, Ger-
many; Research Centre for Neurosensory Sciences,
Alexander Jurkevich, Advanced Light Microscopy Core, University of Oldenburg, Oldenburg, Germany
University of Missouri, Columbia, MO, United States
Casey A. Mueller, Department of Biological Sciences,
John Kirby, College of the Environment and Life Sci- California State University San Marcos, San Marcos,
ences, The University of Rhode Island, Kingston, RI, CA, United States
United States
 Contributors xxix

Yves Nys, BOA, INRAE, Université de Tours, Fonction et Cynthia A. Smeraski, Science Education and Literacy
Régulation des protéines de l’œuf, Développement de Foundational Program, Fort Collins, CO, United States
l’œuf, Valorisation, Évolution, France Nurudeen Taofeek, Food and Animal Sciences, Alabama
Mary Ann Ottinger, Department of Biology and Bio- A&M University, Huntsville, AL, United States
chemistry, University of Houston, Houston, TX, United Hiroshi Tazawa, Developmental and Integrative Biology,
States Department of Biological Science, University of North
Barbara J. Pierce, Department of Biology, Sacred Heart Texas, Denton, TX, United States
University, Fairfield, CT, United States Zehava Uni, Department of Animal Science, The Robert
Tom E. Porter, Department of Animal and Avian Sci- H. Smith Faculty of Agriculture, Food and Environ-
ences, University of Maryland, College Park, MD, ment, The Hebrew University, Rehovot, Israel
United States Sandra G. Velleman, The Hebrew University of Jerusa-
Frank L. Powell, Department of Medicine Division of lem, Rehovot, Israel; The Ohio State University,
Pulmonary, Critical Care, Sleep Medicine, University of Wooster, OH, United States
California, San Diego La Jolla, CA, United States Jorge A. Vizcarra, Food and Animal Sciences, Alabama
Monika Proszkowiec-Weglarz, United States Department A&M University, Huntsville, AL, United States
of Agriculture, Agricultural Research Service, Animal Brynn H. Voy, Department of Animal Science, University
Biosciences and Biotechnology Laboratory, Beltsville, of Tennessee, Knoxville, TN, United States
MD, United States
Yajun Wang, Key Laboratory of Bio-resources and Eco-
Marilyn Ramenofsky, Department of Neurobiology, environment of Ministry of Education, College of Life
Physiology and Behavior, University of California, Sciences, Sichuan University, Chengdu, Sichuan, PR
Davis, CA, United States China
Narayan C. Rath, USDA/Agricultural Research Service Wesley C. Warren, Department of Animal Sciences, Bond
and Department of Poultry Science, University of Life Sciences Center, University of Missouri, Colum-
Arkansas, Fayetteville, AR, United States bia, MO, United States
Nicole Rideau, Recherches Avicoles, (UR 83), INRA, Heather E. Watts, School of Biological Sciences,
Nouzilly, France; Unité de Recherches Avicoles, Washington State University, Pullman, WA, United
Institut National de la Recherche Agronomique, Nouzilly, States
France
J. Martin Wild, Department of Anatomy and Medical
Alejandro B. Rodriguez-Navarro, Departmento de Min- Imaging, Faculty of Medical and Health Sciences,
eralogia y Petrologia, Universidad de Granada, Spain University of Auckland, Auckland, New Zealand
Colin G. Scanes, Center of Excellence for Poultry Science, John C. Wingfield, Department of Neurobiology, Physi-
University of Arkansas, Fayetteville, AR, United States; ology and Behavior, University of California, Davis,
Department of Biological Sciences, University of CA, United States
Wisconsin, Milwaukee, WI, United States
Takashi Yoshimura, Laboratory of Animal Integrative
Elizabeth M. Schultz, Department of Biology, Wittenberg Physiology, Graduate School of Bioagricultural Sci-
University, Springfield, OH, United States ences, Institute of Transformative Bio-Molecules,
Paul B. Siegel, Department of Animal and Poultry Nagoya University, Nagoya, Aichi Prefecture, Japan
Sciences, Virginia Tech, Blacksburg, VA, United States Huaijun Zhou, Department of Animal Science, University
Jean Simon, Recherches Avicoles, (UR 83), INRA, of California, Davis, CA, United States
Nouzilly, France
Chapter 1

The importance of avian physiology

John C. Wingfield
Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA, United States

The 20th century saw many revolutionary advances in the well as the complex interplay of physiology, behavior,
biological sciences. In 100 years, biology progressed from cells, and molecules by which individuals function and
cataloging and describing species to sequencing the human interact. Management and control of these issues in relation
genome. Along the way, astounding advances were made to natural resources will also be difficult unless there are
in cell and molecular biology, biomechanics, physiology, incentives to foster interdisciplinary research, integrate
theoretical ecology, genetics, behavioral neurobiology, etc. education, and maximize our potential to address problems
These and other areas of the biological sciences continue to by bringing to bear all of our knowledge in an effective
develop with Aves an important model group for links way. Citing a report from the National Academy of Sci-
between environment and gene expression (e.g., Konishi ences, Jasanoff et al. (1997) assert that in the next decades,
et al., 1989). Environmental change including degradation, young scientists will be impeded in their advancement
population, public health, food and energy production, unless they are trained from an early age to diversify their
education, and especially the public’s understanding of expertise and career objectives.
science and technology are some of the most critical issues As an example of the need to integrate ecology,
facing science and society. All of these pertain to biology. behavior, and evolutionary biology with mechanisms at
Technology and basic research in biological sciences have physiological, genetic, cell, and molecular levels, we can
the potential to address the above issues, but they cannot be consider the functions of a differentiated cell in which
understood along traditional disciplinary lines. One enor- complex interactions of many proteins occur. These func-
mous hurdle awaits 21st century biological research: how tions can be modified by hormones that coordinate gene
to integrate our knowledge of biology at all levels so we activity among various cells and tissues of the organism
can pave the way for a deeper and broader understanding of leading to the ultimate responses to internal and external
how life on this planet works? This will also entail how to environmental signals. A critical question is then how do
feed a still burgeoning population and educate future gen- hormones orchestrate transitions in morphology, physi-
erations of undergraduates while conserving as much of the ology, and behavior of individual organisms in relation to a
natural world as possible? If this were not enough, we must changing, and sometimes capricious environment? We can
also deal with potentially catastrophic environmental imagine observing an ecosystem and focusing on certain
problems resulting from climate change. populations of individuals within it that have problems
A major recurring problem with the spectacular success dealing with change in the environment triggered by, for
biology has experienced is that individual investigators example, human disturbance. It quickly becomes apparent
have become so specialized and focused that in many cases that some populations, and individuals within them, deal
they have, understandably, lost track of other disciplines. with the environmental challenges better than others. It is
There is a growing consensus that biologists within disci- reasonable to then ask what aspects of their physiology,
plines have difficulty communicating with colleagues in behavior, and morphology are the causes of this failure, or
other branches of biology. Clearly, the problems we face in success, and what the cell, molecular, and genetic bases of
the next decades will be solved only by taking broad these differences might be. The investigation of interindi-
integrative approaches involving expansive, multidisci- vidual variation will be another foundation for under-
plinary collaborations. In other words, molecular biology or standing how populations will evolve in response to
theoretical ecology in isolation will not resolve problems environmental change. It is relatively simple to construct
that involve populations, their component individuals, as hypothetical scenarios whereby we traverse the spectrum of

Sturkie’s Avian Physiology. https://doi.org/10.1016/B978-0-12-819770-7.00004-9

Copyright © 2022 Elsevier Inc. All rights reserved. 3
4 PART | I Undergirding themes

biological science from ecosystem to molecule in either physiology will be a central focus for such integration. The
direction, but it is not so intuitively obvious how we do this examples given below are by no means all inclusive and
in practical terms. If such an interdisciplinary and integra- new concepts and research directions are inevitable.
tive approach can be achieved, it will be possible to
determine how we will deal with global changes already
underway and ultimately prepare future generations to cope 1.1.1 Physiology and poultry production
with ongoing changes. Physiological functions of domestic fowl from reproduction
Avian physiology has a long history of providing and growth to responses to diseases and stress such as
models for integration of disciplines, and it will continue in weather factors (especially heat) also triggered thorough
this role in the future (Konishi et al., 1989). In general, investigations of environmental biology and hormonal
there are exceptionally broad data bases for birds on ecol- control of these processes (see Sturkie’s Avian Physiology
ogy and evolution across the globe. Match this with rapidly revised editions from 1954 to 2020). The domestic fowl
developing tools at genomic, transcriptomic, and epigenetic and some other domesticated species (e.g., quail. duck,
levels, then avian physiology is uniquely poised midway turkey) provide phenomenally broad data bases of physi-
along the spectrum from genes to environmental to explore ology that rival those of mammals and fish. These in turn
the molecular bases of adaptation and the integration of provide endless opportunities for other studies on wild
ecological and evolutionary aspects at the interfaces of species and gene-environment interactionsdthe founda-
morphology, physiology, and behavior. In addition, many tions of understanding adaptation to a changing world.
wild avian species are abundant and easy to study making
them ideal subjects for field observations and experiments
in their natural environment, as well as in laboratory 1.1.2 Physiological ecology and birds, marine,
experimentation. Specific examples how avian physiology freshwater, and terrestrial
has played a major role in the development and integration Birds have played a central role in the development of
of biological processes at many functional levels are dis- physiological ecology, especially building on the vast array
cussed next. of data, techniques and tools made available from agricul-
tural research (Phillips et al., 1985; Konishi et al., 1989).
1.1 Specific examples of the Avian physiological ecology is a thriving branch of biology
that has provided a framework for other emerging topics
importance of avian physiology such as environmental endocrinology, the regulation of
Avian physiology has its roots in over 100 years of research seasonal changes in physiology, and individual differences
on domesticated species, particularly the domestic fowl and fitness. (e.g., Hau et al., 2010; Taff and Vitousek,
(Gallus gallus) as presented in the original version of 2016). Avian migration is a prime example of the integra-
Sturkie’s Avian Physiology (Sturkie, 1954). Over the de- tion of physiological systems that has benefitted enor-
cades since there have been huge advances in avian phys- mously from avian physiology in general (Newton, 2010;
iology focused primarily on the poultry industry and food Dingle, 2014; Ramenofsky and Wingfield, 2007). This has
production. Then in the early 21st century, the chicken fueled investigations of one of the most integrative bio-
genome was sequenced and annotated (e.g., Burt, 2005; logical processesdthe evolution, ecology, morphology,
Burt and Pourquie, 2003). This opened up a new generation physiology, and behavior of movements across the envi-
of biological studies at many of the interfaces outlined ronment in general.
above, and was followed by sequencing the genome of a Fundamental and applied research on domestic fowl has
songbird, the zebra finch, Taeniopygia guttata (Warren enriched development of avian physiology in relation to the
et al., 2010). As technologies developed to sequence comparative biology of environmental stress (Romero and
genomes more quickly and cheaply, the genomes of many Wingfield, 2016). All organisms must be able to cope with
other species have been sequenced as part of ambitious and perturbations of the environment that are largely unpre-
exciting projects to eventually sequence the genomes of all dictable, but require individuals to express facultative
the extant species of birds and some extinct ones as well physiological and behavioral responses to cope. The “stress
(e.g., Zhang et al., 2014). If successful, such an unprece- response” is common to most, if not all, organisms from
dented data base will allow analyses at so many levels of bacteria and plants to invertebrate as well as vertebrate
biological function. However, it should be borne in mind animals. Although the mechanisms involved vary funda-
that the computational technologies to analyze such enor- mentally from plants to animals, they are well conserved
mous amounts of data and integrate them with other diverse within the vertebrates making avian systems excellent
and unique data bases on morphology, physiology, models. One aspect of climate change is the increase in
behavior, and environment are huge challenges. Avian frequency, duration, and intensity of extreme weather
 Importance of avian physiology Chapter | 1 5

events such as unseasonal storms, hurricanes, and ty- fundamental biology are vast. In the 21st century, with
phoons. Understanding how some populations cope with technologies until recently unimagined, these contributions
these unpredictable events and others do not will have will continue. What could not have been envisaged all
profound implications for understanding adaptation and those decades ago is how avian physiology has helped
consequences for conservation and agriculture. pioneer, initiate and develop new environmental aspects of
New emerging avian models such as the zebra finch, biological sciences such as morphology and behavior at the
great tit (Parus major, Santure et al., 2011), white-throated interfaces with gene-environment interactions and coping
sparrow (Zonotrichia albicollis, Tuttle, 2003), and dark- with global change from urbanization, endocrine disrupting
eyed junco (Junco hyemalis, Ketterson and Atwell, 2016) pollutants, and invasive species to the burgeoning effects of
and others, provide field and laboratory contexts for climate change. There is no question that avian physiology
understanding physiological ecology as well as advancing is very important and will continue to be at the cutting edge
basic research in neurobiology such as the physiology of so many branches of biological sciences. It will continue
underlying behavioral patterns and avian song control to influence not only basic research but also policy in
systems (see Konishi et al., 1989; Pfaff and Joëls, 2017). managing planet Earth for future generations.
These approaches using experiments both in the laboratory
and the field have allowed the beginning of investigations
of coping with climate change, shifts in habitat range, References
urbanization etc. (e.g., Partecke et al., 2006; Fokidis et al., Bonier, F., 2012. Hormones in the city: endocrine ecology of urban birds.
2009; Martin et al., 2010; Bonier, 2012; Caro et al., 2013; Horm. Behav. 61, 763e772.
Visser and Gienapp, 2019). Birds have become important Burt, D., 2005. Chicken genome: current status and future opportunities.
examples of models for conservation physiology (Wikelski Genome Res. 15, 1692e1698.
and Cooke, 2006; Madlinger et al., 2016). For example, Burt, D., Pourquie, O., 2003. Chicken genome- science nuggets to come
avian physiology is providing critical insights into one of soon. Science 300, 1669.
Carere, C., Costantini, D., Sorace, A., Santucci, D., Alleva, E., 2010. Bird
the most important aspects of conservationdthe impact of
populations as sentinels of endocrine disrupting chemicals. Ann. 1st.
invasive species resulting from human activity as well as Super Sanita 46, 81e88. https://doi.org/10.4415/ANN_10_01_10.
range expansions and contractions that have important and Caro, S.P., Schaper, S.V., Hut, R.A., Ball, G.F., Visser, M.E., 2013. The
often devastating impacts on the survival of indigenous case of the missing mechanisms: how does temperature influence
species from plants to mammals. seasonal timing in Endotherms? PLoS Biol. 11, e1001517.
Another spin-off from avian physiology is the use of Dawson, A., 2000. Mechanisms of endocrine disruption with particular
birds as sentinels of endocrine disruptiondphysiology, reference to occurrence in avian wildlife: a review. Ecotoxicology 9,
morphology, and behavior (National Research Council, 9e69.
1999; Dawson, 2000; Norris and Carr, 2006; Carere et al., Dingle, H., 2014. Migration: the Biology of Life on the Move, second ed.
2010). Although the responses of plants, invertebrates, as Oxford Univ Oxford.
well as vertebrates, in general, are all important foci to Fokidis, H.B., Orchinik, M., Deviche, P., 2009. Corticosterone and
corticosterone binding globulin in birds: relation to urbanization in a
document toxicology and endocrine disruption, the use of
desert city. Gen. Comp. Endocrinol. 160, 259e270.
birds as models, and their well-known physiology both in
Hau, M., Ricklefs, R.E., Wikelski, M., 2010. Corticosterone, testosterone
the laboratory and field once again provide a useful model and life history strategies of birds. Proc. R. Soc. B 277, 3203e3212.
for understanding how organisms respond to disasters such Jasanoff, S., Colwell, R., Dresselhaus, M.S., Goldman, R.D.,
as leakage of toxic chemicals, oil spills, etc. An example of Greenwood, M.R.C., Huang, A.S., Lester, W., Levin, S.I., Linn, M.C.,
a remaining critical issue of endocrine disruption is how Lubchenco, J., Novacek, M.J., Roosevelt, A.C., Taylor, J.E.,
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distribution of chemical mixtures in varying concentrations millennium. Science 278, 2066e2067.
and how we may be able to ameliorate some of the effects Ketterson, E.D., Atwell, J.W., 2016. Snowbird: Integrative Biology and
of endocrine disruption by broad ranging “clean up” pro- Evolutionary Diversity in the Junco. Univer Chicago Press, Chicago.
grams to levels of chemicals that organisms can at least Konishi, M., Emlen, S., Ricklefs, R., Wingfield, J.C., 1989. Contributions
of bird studies to biology. Science 246, 465e472.
tolerate?
Madlinger, C.L., Cooke, S.J., Crespi, E.J., Funk, J.L., Hultine, K.R.,
Hunt, K.E., Rohr, J.R., Sinclair, B.J., Suski, C.D., Willis, C.K.R.,
1.2 Conclusions Love, O.P., 2016. Success stories and emerging themes in conserva-
tion physiology. Cons. Physiol. 4, 206. https://doi.org/10.1093/con-
Reading Sturkie’s original avian physiology text (Sturkie, phys/cov/057.
1954), it is astonishing to learn how avian physiology has Martin, L.B., Hopkins, W.A., Mydlarz, L.D., Rohr, J.R., 2010. The effects
developed, expanded, and progressed in almost 70 years. of anthropogenic global changes on immune functions and disease
The contributions of avian physiology to agriculture, resistance. Annals NY Acad. Sci. https://doi.org/10.1111/j.1749-
biomedicine, veterinary science, and practice, as well as 6632.2010.05454.x.
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National Research Council, 1999. Hormonally Active Agents in the Sturkie, P.D., 1954. Avian Physiology. Cornell University Press, Ithaca.
Environment. National Academy Press, Washington DC. Taff, C.C., Vitousek, M.N., 2016. Optimizing phenotypes in a dynamic
Newton, I., 2010. The Migration Ecology of Birds. Academic Press world? Trends Ecol. Evol. 31, 476e488. https://doi.org/10.1016/
(Elsevier), New York. j.tree.2016.03.005.
Norris, D.O., Carr, J.A. (Eds.), 2006. Endocrine Disruption. Oxford Univ Tuttle, E.M., 2003. Alternative reproductive strategies in the white-
Press, New York. throated sparrow: behavioral and genetic evidence. Behav. Ecol. 14,
Partecke, J., Schwabl, I., Gwinner, E., 2006. Stress and the city: urbani- 425e432.
zation and its effects on the stress physiology in European blackbirds. Visser, M., Gienapp, P., 2019. Evolutionary and demographic conse-
Ecology 87, 1945e1952. quences of phonological mismatches. Nat. Ecol. Evol. 3, 879e885.
Pfaff, D.W., Joëls, M. (Eds.), 2017. Hormones, Brain and Behavior, third https://doi.org/10.1038/s41559-019-0880-8.
ed. Academic Press, New York. Warren, W.C., Clayton, D.F., Ellegren, H., Arnold, A.P., et al., 2010. The
Phillips, J.G., Butler, P.J., Sharp, P.J., 1985. Physiological Strategies in genome of a songbird. Nature 464. https://doi.org/10.1038/
Avian Biology. Blackie and Son, Glasgow and London. nature08819.
Ramenofsky, M., Wingfield, J.C., 2007. Regulation of migration. BioSci Wikelski, M., Cooke, S.J., 2006. Conservation physiology. Trends Ecol.
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by next generation sequencing. BMC Genom. 12, 283. Sci. 3, 26. http://www.gigasciencejournal.com/content/3/1/26.
Chapter 2

Avian genomics
Hans H. Cheng1, Wesley C. Warren2 and Huaijun Zhou3
1
USDA, ARS, Avian Disease and Oncology Laboratory, East Lansing, MI, United States; 2Department of Animal Sciences, Bond Life Sciences
Center, University of Missouri, Columbia, MO, United States; 3Department of Animal Science, University of California, Davis, CA, United States

2.1 Introduction For example, many studies now start with transcriptome
profiling (RNA sequencing) with the goal of generating
All fields of biology have been greatly influenced by the hypotheses on what relevant genes and pathways to
generation of complete genome assemblies. This impact is investigate further. Without genomics and its accompa-
most apparent with the findings and resulting applications nying tools and resources, this approach would not have
from the Human Genome Project (HGP), which has been possible. It is likely that this trend will only increase
transformed biomedical science (Green et al., 2015). Hop- as more genomes are sequenced and functionally under-
ing for a similar transformation in agriculture, a genome stood, along with the continuing development of more
assembly was first made for chicken in 2004 (International empowering and economical genomic-based technologies,
Chicken Genome Sequencing Consortium, 2004). More and the desire to have and integrate multidimensional types
recently, as the feasibility and economics of generating a of data to help understand complex biological problems.
genome assembly increased, the number of species across Having stated this, traditional experiments and the detailed
all avian orders has increased dramatically (e.g., 48 were information that they provide at many levels are still critical
produced and analyzed in the landmark Zhang et al. (2014) needs and inputs to truly empower avian genomes. In short,
paper), which strongly suggests that the field of genomics genomics adds another powerful approach to understanding
will only grow in its ability to shape the future direction for biology.
all fields of avian biology. Our target audience of this chapter is avian physiologists
The original justification for having a genome assem- who might not be aware of the field of genomics. Specif-
bly was to get a complete “parts list” of elements that ically, we briefly summarize the current status of avian
contribute to an organism’s phenotype with the primary genome assemblies with the focus on chicken, as this is the
goal being the identification and location of all genes. most developed avian species to date, and common
However, it soon become readily apparent that genomes approaches that focus on DNA-based genomics. Key refer-
were much more than just sequences that code for pro- ences are provided in each subsection to aid further reading.
teins; protein-coding regions account for w1% of the
human genome. Thus, current efforts have been focused
on finding other relevant functional elements, such as 2.2 Genome
noncoding elements that regulate when, where, and how
2.2.1 Size
much specific genes and/or particular isoforms are
expressed. As these annotation efforts become more Eukaryotic genomes vary immensely, over 60,000-fold,
complete, the power and utility of genomes to inform from as small as 2.3 Mb for Encephalitozoon intestinalis,
other biological fields, like physiology, will become more an intestinal parasite of humans and animals, to as large as
apparent and enhanced. 150 Gb found in the plant Paris japonica (Elliott and
Besides providing an incredible resource, genome Gregory, 2015). This range in genome size, which does not
assemblies and the field of genomics have fundamentally correlate with organismal complexity (commonly referred
changed how experimental biology is conducted. It is now to as the C-value paradox), has been recognized for a long
common to begin with discovery-based genomics science time (Swift, 1950). However, genome size does seem to
to build agnostic large datasets to analyze, which contrasts correlate positively with cell size and negatively with cell
with traditional, reductionist hypothesis-based experiments. division rate (Elliott and Gregory, 2015). When considering

Sturkie’s Avian Physiology. https://doi.org/10.1016/B978-0-12-819770-7.00047-5

Copyright © 2022 Elsevier Inc. All rights reserved. 7
8 PART | I Undergirding themes

only vertebrates, the size range (0.35e133 Gb) is less but remaining autosomes (11 to 38). The microchromosomes
still large (333-fold) (www.genomesize.com). In contrast to can be identified only through fluorescent in situ hybridi-
most other taxa, the interspecific variation for bird genome zation using specific probes (e.g., Romanov et al., 2005;
size is quite narrow e 0.9 Gb for the black-chinned hum- Delany et al., 2009; Zhang et al., 2011). Due to their small
mingbird to 2.1 Gb for the ostrich; see Damas et al. (2019) size and the need for at least one recombination event during
for a more comprehensive review. The likely explanation meiosis, microchromosomes are more gene dense, GC-rich
for the relatively small genome size and stability of avian in sequence content, and have higher recombination rates
genomes is the low occurrence of transposable elements compared to macrochromosomes (International Chicken
(TEs) and the loss of large segments that could counteract Genome Sequencing Consortium, 2004).
any TE expansion, which is the main driving force for the
increase (Kapusta et al., 2017). Of interest to physiologists
is the observation that genome size variation within bird 2.3 Genome assemblies
species is associated with metabolism with smaller
2.3.1 Chicken legacy genomes
genomes having higher metabolic rates (Gregory, 2002).
Since the first assembled chicken genome in 2004 (Inter-
national Chicken Genome Sequencing Consortium, 2004)
2.2.2 Karyotype
that used Sanger sequencing efforts, improvements in qual-
Avian species are unique in having fairly high number of ity have closely followed the technological advancements in
chromosomes. The majority of the species have 74 to 86 sequencing and mapping (Schmid et al., 2015). Furthermore,
total chromosomes, which is equivalent to 37 to 43 pairs, multiple upgrades (Gallus_gallus-2.1, 4.0, 5.0, GRCg6a)
though the reported range is 40 to 126 chromosomes have greatly benefited from the better assembly of segmental
(Griffin et al., 2007). The chicken karyotype is fairly duplications (SDs) (Eichler et al., 2004), closure of scaffold
typical (Fig. 2.1). It possesses 39 pairs of chromosomes, 38 gaps, specialized approaches to resolve the complex
pairs of autosomes and the Z and W sex chromosomes; sequence structure of the avian sex chromosomes (Bellott
unlike mammals, males are the homogametic sex (ZZ). An et al., 2018), and traditional bacterial artificial chromosome
unusual feature of avian chromosomes is the variation (BAC) clonal sequencing efforts that have improved
in size distribution. Although arbitrarily defined as sequence representation of chicken chromosome 16
(1) macrochromosomesdautosomes one to five plus the Z (GGA16), which contains the major histocompatibility
chromosome are comparable in size to mammalian chro- complex (MHC) (Miller and Taylor, 2016). Many of the
mosomes, (2) intermediate microchromosomesdautosomes genes, critical in governing immune responses, across spe-
6 to 10 and the W chromosome are similar in size to smaller cies are clustered, multigenic families containing individual
human chromosomes, and (3) microchromosomesdall the members that vary across haplotypes with intricate differ-
ences in sequence.
As with other vertebrate genomes, in chicken, a recur-
ring hurdle to accurate computational experimentation is
falsely collapsed or redundant SDs, repetitive elements and
fragmented sex chromosome assemblies, mostly due to the
organization of highly identical sequences that confuse de
novo assembly attempts at constructing simple bifurcating
graphs of each haplotype. Bellott et al. (2010) provide an
exemplary case of the difficulties encountered where
identification of a tandem array of four testis-expressed
genes that constitutes w15% of the Z chromosome, one-
fifth of all chicken SDs and in total about a third of the
protein-coding genes on Z. Study of the W sex chromo-
some illustrates even more extreme heterochromatin
stretches and a tandemly arranged gene with 40 copies
(Bellott et al., 2017). It is well known that such duplications
are frequently missed (“over-collapsed”) in draft quality
FIGURE 2.1 Mitotic metaphase chromosomes of a male (ZZ) chicken assemblies that they are common sites of copy number
illustrating macro- and microchromosomes and the sex chromosomes. The variation (CNV), and that such variation frequently have
cell shown was stained with the fluorochrome DAPI, which stains DNA
and the image then inverted to black/white. Chromosome pairs one to nine
major phenotypic consequences (Conrad et al., 2010).
and the Z are indicated by numbers. Figure contributed by M.E. Delany, Another example is that of the MHC-B complex in chicken
University of California, Davis, CA. on GGA16 (one of the smallest microchromosomes), a
 Avian genomics Chapter | 2 9

region of great immunological importance with many gene 2.3.2 Future chicken genome assembly
duplications and CNVs. Directed sequencing of selected
A need to represent the genome variation that exists within
chicken BACs was critical to elucidate the partial organi-
species has recently led to pangenome reference efforts
zation of this region (Shiina et al., 2007), but in the future
(Computational Pan-Genomics Consortium, 2018). In
extreme sequence lengths (>500 Kb), such as the outcome
chicken, a pangenome reference build must include main-
of Oxford Nanopore sequencing technology, will be needed
stream and rare breeds, as well as commercially selected
for gap-free genome sequence characterization.
lines, both layers and broilers. De novo assembly methods,
Over the years, genetic linkage maps comprised of
continue to evolve rapidly, where best practice is now,
ordered and oriented molecular markers have helped to
when possible, long-read sequencing of an F1 offspring and
guide the genome assembly chromosome build. However,
use of short reads from the parents to near fully phase
even though the consensus chicken genetic linkage map
haploid genomes of their offspring (Koren et al., 2018). In
contains 50 linkage groups (Groenen et al., 2000), several
fact, the success of this approach has been recently docu-
microchromosomes were missing sequence marker align-
mented for 16 vertebrate genomes, including bird species
ments to a linkage group and still today chicken genome
(Rhie et al., 2021). Assemblies created with this method
references have not captured the full autosome composi-
offer more accurate assessments of complex structural
tion. This is of primary importance to the avian genome
variation (Kronenberg et al., 2018) and for some chromo-
analysis process, especially given the high gene density per
somes gap-free starting points, such as human X that was
microchromosome. Recently improved sequencing and de
spanned telomere-to-telomere (Miga et al., 2020).
novo assembly approaches or alternatively the use of
In chicken, the primary objectives are to reduce sequence
advancing cytogenetic techniques will be crucial in efforts
gaps and correct misassemblies that exist in the GRCg6a
to assemble and assign these missing microchromosomes.
reference. Currently single haplotype genome references for
In the current reference, GRCg6a, many of these unas-
an F1 cross of a commercial layer (paternal) and broiler
signed sequences are likely found within the unplaced bin
(maternal) line are under construction using this technique.
of chicken assembly reference files. However, in GRCg6a,
Preliminary measures of sequence contiguity completeness
there are only 14 Mb of unplaced contigs and scaffolds
show N50 contig “ungapped” lengths of 16 and 14 Mb in
indicating there is also an inability to sequence or assemble
paternal and maternal genomes, respectively. After iterative
some of these microchromosomes.
scaffold builds with a long fragment physical map (Bio-
Ideally, the goal is to have complete sequence repre-
Nano) and chromosomal proximity ligation data, the near
sentation of all 40 chicken chromosomes, this includes Z
theoretical chromosome N50 scaffolds lengths of 60 and
and W sex chromosomes, but, of course, today this is not
89 Mb for paternal and maternal genomes is achieved,
technically feasible for any vertebrate genomes, even
respectively. Further curation of these single haplotype as-
human, although a recent telomere to telomere assembly of
semblies promises to advance chicken genetic research by
human X is encouraging (Miga et al., 2020). This study
resolving much of the sequence structure presently frag-
also highlights the incredible amount of manual curation
mented and misappropriated in the GRCg6a reference.
that is needed to achieve this feat. Basically, automated
methods to stitch together complex sequence structures still
requires significant human intervention. Although the 2.3.3 Genes
reasons for the breaks in vertebrate assembly contiguity
“gaps” aren’t always apparent, the usual culprits are repeats The original chicken genome assembly estimated there
and base composition distortions, such as high GC homo- were 20,000e23,000 protein-coding genes (International
polymers. In the chicken, we have observed assembly gap Chicken Genome Sequencing Consortium, 2004). How-
edges including elevated GC content approaching 75% in ever, more recent estimates from the comparison of 48
some regions and low-complexity sequences (International avian genomes indicates the number for bird genomes is
Chicken Genome Sequencing Consortium, 2004). Collec- lower at 15,000e16,000 (Zhang et al., 2014). Based on this
tively this loss of sequence knowledge demonstrates more number, this would reflect a w30% reduction compared to
work is essential to not limit impending experiments. Of the number found in mammals. At least 1241 of the loss in
late, through longer read lengths, improved de novo as- genes can be explained by large segmental deletions during
sembly algorithmic methods and evolving mapping tech- the evolution of birds. However, w70% of the lost genes
nology the ability to close w80% of existing spanned gaps show paralogs suggesting functional compensation. In
within assembled scaffolds, scaffolds defined as an ordered addition, some of the gene loss may not be true but rather
collection of contigs, are feasible (Bickhart et al., 2017; the inability to detect genes in high GC-rich regions
Koren et al., 2018). (Bornelov et al., 2017).
10 PART | I Undergirding themes

Avian genes are w50% smaller than their mammalian and their associated functional features. Future iterations of
counterparts, mainly due to the shortening of introns and these browsers and new ones, such as FAANGMine.org for
reduced distances between genes, which helps to account exploring sequence structures associated with gene regu-
for the reduced size of avian genomes. Interestingly, with lation, will be of great value for researchers seeking
respect to avian-specific highly conserved elements, most information to design functional experiments in poultry.
are significantly associated with transcription factors asso- Another portal created by the Genome Reference Con-
ciated with metabolism. Having a complete list of genes in sortium (GRC; https://www.ncbi.nlm.nih.gov/grc) exists to
many avian species has also enabled hypotheses on the report errors in the underlying chicken genome reference by
evolution of flight, diets, vision, and reproductive traits the community. The GRC will ensure this resource con-
(Zhang et al., 2014). tinues to attain higher levels of reference quality and
reporting accessibility with its readily useable web inter-
2.3.4 Transposons and endogenous viral face. All these public access points bode well for the
continued use of this resource and advancing the knowl-
elements
edge of avian genome biology.
Avian genomes have a relatively low amount of TEs. In
chicken, the most abundant TE is chicken repeat 1 (CR1), a
long interspersed nucleotide element that with 200,000þ
2.4 Connecting genome sequence to
copies, comprises over 80% of all interspersed repeats in phenotype
the chicken genome (International Chicken Genome
2.4.1 Connecting genotype to phenotype
Sequencing Consortium, 2004). A complete CR1 element
is 4.5 kb in length, however, more than 99% of CR1 ele- As discussed previously, a major driving force for gener-
ments are truncated from their 50 end, which is necessary ating genome assemblies was to accelerate the power of
for retrotransposition. Short interspersed nuclear element biology, and especially in the ability to identify the ele-
transposons are extremely rare, which contrasts to all other ments that define the makeup of an organism. Put another
vertebrate genomes. way, for few exceptions, while every cell in a chicken has
With respect to endogenous viral elements, analysis of the same DNA sequence, these cells vary greatly in many
48 avian genomes has shown that there are five families of biological attributes primarily due to differences in gene
endogenous virusesdRetroviridae, Hepadnaviridae, Circo- expression. Couple this existing variation with genetic
viridae, Parvoviridae, and Bornaviridae (Cui et al., 2014). variation, then one begins to understand how the genome
However, over 99þ% of these are endogenous retroviruses contributes to the wide diversity of traits observed within
(ERVs), and the copy number of these elements range from and between bird species. A mechanistic understanding of
132 to 1032. There is great interest in ERVs as they are these processes can be harnessed to solve many important
likely to contribute to evolution of gene expression and, questions such as improvement of health, growth, repro-
thus, contributing to complex traits including ones for duction, etc.
physiology. In humans, there are w110,000 ERVs that In the following sections, we briefly discuss the major
contain over 300,000þ transcription factor binding sites approaches used to identify features in avian genomes that
(Buzdin et al., 2017). As many chicken ERVs are found in define trait variation observed in organisms.
promoters and introns, they are likely to play a similar role.
2.4.2 Genome wide association study
2.3.5 Genome browsers It’s been known for a long time that many traits have a
The chicken genome can be queried multiple ways using genetic basis. A prime example is the tremendous progress
the major established genome browsers: Ensembl (https:// made by poultry breeders to improve agronomic traits.
useast.ensembl.org/index.html), NCBI (https://www.ncbi. However, prior to the genome sequence, identifying the
nlm.nih.gov), and UCSC (https://genome.ucsc.edu/). Each underlying causative gene or genes was a very difficult
genome browser offers a gateway to varied data types and task. Two contributing reasons for the lack of precision
search abilities for your sequence of interest. For example, were (1) most traits are complex and controlled by many
using the UCSC genome browser any chicken chromosome genes, each of which have can only have a small effect, and
can feature Ensembl or NCBI gene annotation, aligned (2) there was a lack of known genetic markers that could
mRNAs, conserved sequence chains against other verte- survey the majority of the chicken genome.
brates, simple repeats, CRISPR targets, and much more The second deficiency was largely removed with the
(Fig. 2.2). One can display a gene of interest, for example, advent of the chicken genome assembly. More specifically,
the T cell receptor CD3E, and evaluate all putative targets by sequencing additional chickens, millions of single
for editing its gene structure as well as surrounding genes nucleotide polymorphisms (SNPs) were identified that
 Avian genomics Chapter | 2 11

FIGURE 2.2 A UCSC Genome browser representation of GRCg6a chromosome 24. (A) regional overview at 44 kb window size that highlights the
T cell receptor gene CD3E with other annotation tracks, (B) a higher resolution overview (4.9 kb) of the CD3E gene where the track is shown for CRISPR
gene editing targets in this gene are represented by multicolor boxes.

could serve as genetic markers (International Chicken causative genetic variant. Thus, further experimentation is
Polymorphism Map Consortium, 2004). Combined with needed to validate and better refine the association in order
affordable arrays that could determine the genotype tens to to identify the relevant gene or regulatory element.
hundreds of thousands genetic variants, it was now feasible Besides identifying trait associations, large-scale geno-
to survey the entire chicken genome. Studies that survey typing efforts can have other applications. One that might
large numbers of birds with SNP chips are known as be more relevant to surveying avian diversity is the ability
genome wide association studies or GWAS, for short; for to define population structure. One of the first examples in
more comprehensive reviews, see Uitterlinden (2016), birds was the study to examine whether heavily selected
Dehghan (2018), Tam et al. (2019). As a result, currently chicken populations were losing alleles (Muir et al., 2008).
Animal QTLdb (animalgenome.org) lists 11,818 trait as-
sociations from 318 publications for chicken alone. 2.4.3 Resequencing
A typical GWAS experiment to dissect a complex trait
requires thousands, if not tens of thousands of individuals, Another major technological advancement that greatly
to have both genotype data from SNP chips and phenotypic impacted genomics was the development of next generation
data. Then statistical analysis is used to determine whether sequencing (NGS); for reviews on the NGS technologies,
each SNP contributes to the trait of being studied. GWAS impact, and history see Koboldt et al. (2013), Goodwin
power is greatly enhanced by having more individuals et al. (2016), Heather and Chain (2016), Levy and Myers
surveyed and accurately measured phenotypes. (2016), Slatko et al. (2018). Largely driven by the needs of
Like any genomic screen, the primary objective of the HGP, NGS is defined by highly parallel methods that
GWAS is to identify candidate genes to interrogate further. produce extremely large amount of data. Existing platforms
Stated differently, while GWAS has revolutionized the can easily provide reads that, when combined, cover the
genetic analysis of complex traits, any associated SNP is entire chicken genome many fold times at economical
unlikely to be causative and only linked to the underlying costs. With the widespread distribution of sequencing
12 PART | I Undergirding themes

machines, like SNP chips, NGS has enabled almost all (H3K27me3), H3 lysine 27 acetylation (H3K27ac), and H3
laboratories to conduct large-scale sequencing efforts. This lysine 4 monomethylation (H3K4Me1) have been utilized
power continues to increase with the continuing declines in to characterize chromatin state (ENCODE Project
costs and, in particular, the increasing capability of “long Consortium, 2012). An abundance of H3K4me3 correlates
read” platforms. And compared to SNP arrays, NGS allows with promoters of active genes (Bernstein et al., 2002;
for the interrogation of the entire genome and provides Santos-Rosa et al., 2002; Xiao et al., 2012), while increased
single base resolution. On the other hand, this increased levels of H3K27me3 are associated with promoters of
power and sequence information comes at a cost mainly inactive genes (Heintzman et al., 2007). H3K27ac is a mark
with the increased need for computational biology skills. of active regulatory elements, and may distinguish active
The great challenge in resequencing is in interpreting enhancers and promoters from their inactive counterparts
the results. This is because there are a large number of (ENCODE Project Consortium, 2012). H3K4me1 is a mark
polymorphisms between any two individuals, of which of regulatory elements associated with enhancers and other
only a small fraction will be associated with the trait of distal elements, but also enriched downstream of tran-
interest. For this reason, most resequencing efforts have scription starts (ENCODE Project Consortium, 2012). High
been most successful in identifying genes of large effect, levels of H3K27ac and H3K4me1 are associated with
e.g., dermal hyperpigmentation (Dorshorst et al., 2011), enhancer regions and also correlate with areas of chromatin
polydactyly (Dunn et al., 2011), comb phenotypes (Wright accessible sites (Greer and Shi, 2012). Insulators also play
et al., 2009; Dorshorst et al., 2015), sex-linked barring an essential role in regulating gene expression. The
(Schwochow Thalmann et al., 2017), and talpid 2 (Chang insulator-binding protein CCCTC-binding factor (CTCF) is
et al., 2014). Typically, these efforts relied on mapping the the main insulator protein (Ong and Corces, 2014) for an-
mutations to a locus or having comparative knowledge imal species. Chromatin accessibility provides a direct
from similar mutations in other organisms, which was then interpretation of epigenetic state, since chromatin organi-
further characterized by whole genome resequencing. zation and transcription factor binding together dictate the
Analysis of the data revealed likely causative alleles. impact of regulatory elements on gene expression, deter-
mining cell-specific expression patterns and cell fate (Riv-
2.4.4 Annotation era and Ren, 2013). Assay for transposase-accessible
chromatin (ATAC-seq) can be used to profile chromatin
Functional characterization of genetic variants is often accessibility. Using the histone marks and CTCF binding
required to move from statistical association to causal sites in concert with ATAC-seq and RNA-seq can provide
variants and genes, especially in the noncoding genome. an unprecedented view of regulatory elements in the
Recent studies have revealed that regulatory mutations in chicken genome. Active and inactive regulatory elements
the noncoding regions are one of major drivers of pheno- including promoters, enhancers, and insulators can be
typic variations in complex traits (e.g., She and Jarosz, identified and annotated based on combinations of the as-
2018). Therefore, functional annotation of regulatory ele- says above (Table 2.1).
ments in the chicken genome is essential in understanding Based on these assays of eight tissues in chickens, our
biological mechanisms of physiological traits. In order to results suggest that chicken genome has about half of the
functionally annotate regulatory elements of genes, one number of active regulatory elements including enhancers
must know the locations and functions of various sequence and promoters compared to mammals such as cattle and
features such as promoters, enhancers, insulators, and pig, especially on the number of enhancers, which may be
silencers. In the past few years, the International Functional due to same size of the chicken genome, one third that of a
Annotation of Animal Genomes Consortium (FAANG.org) typical mammal (Kern et al., 2021).
has developed core assays and bioinformatic pipelines that
can be used to annotate these regulatory elements in the
2.4.5 CRISPR
genomes of farm animals including chicken (Andersson
et al., 2015; Giuffra et al., 2019). Clustered, regularly interspaced, short palindromic repeats
Assays for characterizing chromatin accessibility and (CRISPR) is another revolutionary tool for genomic
posttranslational histone modification can be used to research that has very quickly become an indispensable tool
identify and annotate regulatory elements. These post- for use in many organisms. Originally discovered in bac-
translational histone modifications can affect chromatin teria as a defense mechanism against invading viruses,
accessibility and gene expression. Chromatin immunopre- CRISPR loci express small RNAs that complement viral
cipitation sequencing (ChIP-seq) has been widely used to genome sequences and when combined with associated
characterize histone modifications such as methylation and CRISPR-associated nucleases (Cas) result in the enzymatic
acetylation. For example, assays for histone H3 lysine 4 cleavage of both strands of the targeted viral DNA. Thus,
trimethylation (H3K4me3), H3 lysine 27 trimethylation only two components are required for DNA cleavage:
 Avian genomics Chapter | 2 13

TABLE 2.1 Identification of promoter, enhancer, and insulator elements based on activation marks on ATAC-seq
and ChIP-seq.

Activation marks

Regulatory element ATAC-seq H3K4me3 H3K27me3 H3K27ac H3K4me1 CTCF

Active promoter þ þ  þ  þ/
Inactive promoter þ  þ  þ þ/
Active enhancer þ þ/ þ/ þ þ þ/
Inactive enhancer þ þ/ þ/  þ þ/

Insulator þ     þ

þ, indicates peaks on the marks; , indicates no peaks on the marks; þ/, indicated poised peaks on marks depends on tissues, developmental or physio-
logical status.

(1) an RNA molecule, called guide RNA (gRNA), which three components: (1) the perturbation, (2) the model, and
includes 17e20 bases that are complementary to the target (3) the assay. With barcoded gRNAs and pooled cells,
DNA and (2) a Cas nuclease. perturbations are clean and thorough as one can knockout
Recognizing the simplicity of the system, innovative the gene in every cell. The key is to have relevant cell types
scientists (Jinek et al., 2012; Cong et al., 2013) harnessed and ability to measure relevant phenotypic changes asso-
this system for widespread use in many other organisms ciated with loss of function.
including avians that allows for a programmable endonu-
clease, a feat that was previously difficult, if not impossible
to achieve (Adli, 2018). After inducing double-stranded
2.5 Conclusions
breaks in the genome, the resulting DNA damage must Since the generation of the chicken genome assembly in
be repaired either by nonhomologous end-joining (NHEJ) 2004, the pace of genomics continues unabated. Due to the
or homology-directed recombination (HDR), resulting in a continued decline in sequencing costs and technologies, the
modified sequence. NHEJ can be used to cause small, ability to produce genome assemblies in other avian species
random indel mutations, while HDR can generate precise or members within a species has grown immensely (Jarvis
replacement of targeted DNA or insertion of novel trans- et al., 2014). These improvements have and will continue to
gene cassettes up to many Kb in length. Given the power greatly enhance of the power of genomics. Having stated
and simplicity of the system and different Cas proteins, this, the challenge is how to get parallel increases in other
there are many other new uses that are being employed fields such as physiology. Although obtaining and
including the ability to regulate gene expression, modify accurately measuring many phenotypes is a difficult and
epigenetics, etc (Pickar-Oliver and Gersbach, 2019). In time-consuming process, this new information will surely
short, CRISPR allows for all groups to conduct gene edit- spur massive gains in our fundamental knowledge of
ing and more in their cells or organism of interest. genome structure, genetic variation, gene regulation,
With respect to avian systems, there are likely to be at genome evolution, and many other areas of study.
least two key uses in the immediate future. The first is
validation of the function of predicted genomic sites by
knocking out the hypothesized DNA sequence and
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Chapter 3

Transcriptomic analysis of physiological

systems
Tom E. Porter
Department of Animal and Avian Sciences, University of Maryland, College Park, MD, United States

Abbreviations cell responses to pathogens? What are the mechanisms

underlying muscle differentiation and development? How
ACTH Adrenocorticotropic hormone gene
do hepatic, gastrointestinal, and adipose tissues respond to
ADRB2 Beta 2 adrenergic receptor gene
ALD Anterior latissimus dorsi
mediate nutrient uptake and metabolism? How does the
BDNF brain-derived neurotrophic factor gene cardiovascular system adjust to systemic and environmental
cDNA Complementary DNA needs? These questions have been asked by physiologists
CGA Glycoprotein hormones, alpha subunit gene for decades. Multiple approaches have been taken to
DIO2 Thyroid hormone deiodinase 2 gene advance our knowledge in these areas. With the sequencing
DNA Deoxyribonucleic acid of the genomes for multiple avian species and the devel-
ESTs Expressed sequence tags opment of genome-wide tools for analysis of mRNA levels,
FSH Follicle-stimulating hormone gene physiologists have begun to address these still open ques-
FSHB FSH beta subunit gene tions on a genomic scale. Transcriptomics, also known as
FSHR FSH receptor gene
transcriptional profiling and functional genomics, involves
GLUT1 Glucose transporter 1 gene
large-scale and in many cases genome-wide analysis of
GRM8 Type 8 glutamate receptor gene
LH Luteinizing hormone
mRNA levels in samples using DNA microarrays and
LHB LH beta subunit gne massively parallel sequencing of RNA (RNAseq). The
LPS Lipopolysaccharide discussion below will highlight some of the efforts by avian
MAPK Mitogen-activated protein kinase physiologists to apply transcriptomics to the open questions
miRNA Micro-RNA noted above.
MR mineralocorticoid receptor gene
mRNA Messenger RNA
NFkB Nuclear factor kappa B gene 3.2 Early efforts
NPYR5 Neuropeptide Y receptor type 5 gene The first major effort in transcriptional profiling in avian
PLD Posterior latissimus dorsi
species occurred with the chicken. A dramatic increase in
POMC Proopiomelanocortin gene
the number of expressed sequence tags (ESTs) for chicken
PPARg Peroxisome proliferator-activated receptor gamma gene
RNA Ribonucleic acid
submitted to GenBank occurred between 1999 and 2002,
RNAseq Massively parallel sequencing of RNA prior to the release of the chicken genome sequence in
TTR Transthyretin gene 2004. During that four-year period, the number of ESTs for
VIP vasoactive intestinal peptide gene the chicken increased from a few hundred to more than
400,000. Assembly of these ESTs allowed for the pro-
duction of more than a dozen DNA microarray platforms.
3.1 Introduction Details on the production of these microarrays have been
How does the brain integrate environmental cues to control reviewed in detail (Cogburn et al., 2007; Gheyas and Burt,
behavior, vocalization, and reproduction? How do the 2013). In 2001, three papers were published that marked
endocrine tissues respond to internal and external signals to the beginning of transcriptomics in birds (Liu et al., 2001;
coordinate physiological responses? What governs immune Morgan et al., 2001; Neiman et al., 2001). These first avian

Sturkie’s Avian Physiology. https://doi.org/10.1016/B978-0-12-819770-7.00006-2

Copyright © 2022 Elsevier Inc. All rights reserved. 17
18 PART | I Undergirding themes

cDNA microarrays contained cDNAs representing

approximately 2000 genes expressed in lymphoid tissues
that were printed on nylon membranes. Several other
tissue-specific cDNA microarrays were developed in the
following few years (Bailey et al., 2003; Carre et al., 2006;
Cogburn et al., 2003, 2004; Ellestad et al., 2006), and two
groups produced cDNA microarrays representing more
than half the expressed genes in the chicken (Burnside
et al., 2005; Cogburn et al., 2004). These systems-wide
cDNA microarrays were followed by oligonucleotide ar-
rays representing the majority of expressed genes in the
chicken (Affymetrix, ARK-Genomics/Operon, Agilent).
Within five years of the foundation of functional genomics
in the chicken and shortly after the release of the chicken
genome sequence, tools were established for near genome-
wide analysis of mRNA levels in chicken samples. Sub-
sequent to these early efforts in the chicken, similar projects
established resources for genomics studies in the zebra
finch (Li et al., 2007) and Northern bobwhite quail (Rawat
et al., 2010). These genomics tools were rapidly put to use
in studies of physiological systems, which will be discussed
below.
Early studies on transcriptomics in birds frequently FIGURE 3.2 Self-organizing maps (SOMS) clustering of mRNA levels
resulted in long lists of genes that were either up-regulated in the anterior pituitary gland during embryonic development. Pituitary
or down-regulated in one set of samples relative to another. gland mRNA samples from embryonic days 12, 14, 16, and 18 were
While these lists represented candidate genes for potential analyzed using cDNA microarrays, and the results were clustered into
SOMS based on mRNA level profiles during development. Cluster 2 (C2)
involvement in physiological responses, they did not add outlined in yellow contains 15 genes whose expression increased on
substantially to our understanding of the gene interactions embryonic day 16. Growth hormone (GH) was among the genes in this
mediating those responses. Many studies used hierarchical cluster. These results have been published previously in a different format
clustering, heat maps, or self-organizing maps to group (Ellestad et al., 2006).
genes based on their expression patterns in responses to
treatments or during time-course studies. Examples of approaches to analyze transcriptomic results can lead to a
heat map and SOMS clustering are presented in Figs. 3.1 broader understanding of the cellular pathways, gene net-
and 3.2, respectively. The rationale used was that genes that works, and transcriptional regulation of gene expression
respond with similar patterns are likely regulated by shared involved in physiological processes.
mechanisms or even common transcription factors. More
recent transcriptomic studies have placed differentially
expressed genes into known or predicted gene networks
3.3 Nervous system
and pathways based on reports in the literature (see Vocalization in songbirds is part of the courtship ritual and
example in Fig. 3.3). One commonly used pathway analysis an important neuroscience model for speech in humans and
software is Ingenuity Pathway Analysis. Taking multiple for sexual dimorphism in the central nervous system.
However, the genes involved in establishing differences in
vocalization between male and female songbirds were not
D ay 2 Fed known. Using microarray technology, differences in hy-
D ay 2 Fasted pothalamic gene expression in response to territorial
D ay 1 Fasted intrusion were found between the spring and autumn in
D ay 1 Fed free-living song sparrows (Mukai et al., 2009). Among the
D ay 0 genes that were differentially expressed between the sea-
FIGURE 3.1 Heat map illustrating effects of fasting or feeding of newly sons were genes involved in thyroid hormone regulation
hatched chickens on mRNA levels in the hypothalamus. Hypothalamic and action, including genes encoding for the alpha subunit
mRNA samples were analyzed using cDNA microarrays, and the results
of thyroid-stimulating hormone (CGA) and transthyretin
were clustered. Shown is a cluster of genes for which mRNA levels were
increased (red) in response to fasting. Among these were DIO2 and neu- (TTR), supporting a role for thyroid hormones in modu-
ropeptide Y receptor type 5 (NPYR5). The data presented have been lating hypothalamic control of territorial aggression during
published previously (Higgins et al., 2010), but not in this format. seasonal reproduction. Gene express ion in the high-vocal
 Transcriptomic analysis of physiological systems Chapter | 3 19

K E G G M etabolism
N etw orks

G lyc o lys is

O x id a tiv e
p h o s p h o ryla tio n

FIGURE 3.3 Network of genes that were differentially expressed in the hypothalamus of chickens genetically selected for high or low-body fat.
Hypothalamic RNA samples from the two genetic lines were analyzed using cDNA microarrays, and the results were further analyzed using Ingenuity
Pathway Analysis software. Genes involved in glycolysis and oxidative phosphorylation were among those that were differentially expressed in the
hypothalamus of the fat and lean chickens. Additional details on these finding can be found in a previous publication (Byerly et al., 2010).

center (HVC) of zebra finches and canaries was similarly males of both passerine species. Transcriptional profiling of
characterized (Li et al., 2007). Relative to a whole-brain the auditory lobe of zebra finches identified genes whose
reference RNA sample, expression of 190 genes was expression changed with the introduction of a novel song
greater in the high vocal center of both zebra finch and and reverted upon habituation of the birds to the introduced
canaries, suggesting that these genes might function in song (Dong et al., 2009; London et al., 2009). Interestingly,
controlling vocalization. Genes expressed specifically RNAseq revealed micro-RNAs (miRNA) in the auditory
within the song control nucleus (HVC) were also identified forebrain responsive to song that target genes whose
in the Bengalese finch using microarrays (Kato and expression changes with song (Gunaratne et al., 2011),
Okanoya, 2010). In a more comprehensive study, micro- indicating that expression of miRNAs likely contributes to
array analysis of the basal ganglia identified thousands of song in birds.
genes differentially expressed in area X of singing zebra The central nervous system plays an essential role in
finches (Hilliard et al., 2012). Microarray analysis was also regulating metabolism, growth, and body composition.
used to identify genes located on the Z chromosome that However, the full complement of genes expressed within
are expressed within the song control nucleus of the male the central nervous system that function in regulating these
zebra finch that are involved in cell survival (Tomaszycki processes is not known. Transcriptomics has been used to
et al., 2009), supporting their role in formation of the identify genes and gene networks involved in these pro-
sexually dimorphic nucleus involved in masculinization of cesses. Transcriptional profiling of the telencephalon of the
song. A similar microarray analysis of gene expression in white-crowned sparrow during the migratory and nonmi-
the telencephalon of zebra finch and whitethroat indicated gratory seasons revealed differences in expression of genes
that most of the genes differentially expressed in males involved in glucose transport, including glucose transporter
were linked to the Z chromosome (Naurin et al., 2011). 1 (GLUT1) (Jones et al., 2008). These findings support an
However, only half of these were differentially expressed in increased need by the nervous system for glucose during
20 PART | I Undergirding themes

the migratory season. In a study in which metabolism of system and that the effects are brain region and develop-
newly hatched chickens was perturbed by fasting, micro- mental stage dependent.
array analysis revealed that fasting altered expression in the Maternal care of offspring is coordinated by the central
hypothalamus of genes involved in the regulation of nervous system, with particular involvement of the hypo-
metabolic rate, including thyroid hormone deiodinase 2 thalamus. Neuroendocrine regulation of brooding behavior
(DIO2) and proopiomelanocortin (POMC; Fig. 3.1), sug- in birds has been studied extensively. However, details on
gesting hypothalamic modulation of metabolic rate in order the molecular regulation of brooding behavior remain
to compensate for decreased feed intake (Higgins et al., elusive. Transcriptional profiling of the hypothalamus of
2010). Genes not previously associated with hypothalamic laying and brooding Muskovy ducks was performed using
regulation of feed intake and metabolism were also iden- RNAseq analysis (Ye et al., 2019). Genes in the dopami-
tified, including the beta 2 adrenergic receptor (ADRB2) nergic and serotonergic pathways, along with the principle
and the type 8 glutamate receptor (GRM8). Functional re- regulator of prolactin secretion vasoactive intestinal peptide
lationships between ADRB2, GRM8, and POMC were (VIP), were up-regulated in brooding birds. In contrast,
confirmed in cultures of hypothalamic neurons, and effects genes in the glutamatergic pathway were down-regulated in
were dependent on whether the neurons were derived from brooding birds. Interestingly, DIO2 and TTR mRNA levels
chicks that were previously fed or fasted. In two other re- were decreased in the hypothalamus of brooding ducks,
ports, hypothalamic gene expression was profiled in genetic implicating a potential role for thyroid hormones in the
lines of chickens divergently selected for high or low-body transition from laying to brooding states. Not all avian
fat (Byerly et al., 2010) or high or low-body weight (Ka species brood their young. Some species are brood para-
et al., 2011). Differences in expression of genes associated sites, laying their eggs in the nests of another species to be
with glucose sensing, transport, and metabolism were brooded by the host. In a comparison of brood parasitic
detected in the hypothalamus of birds selected for low or and nonparasitic blackbirds, transcriptional profiling of
high-body fat, suggesting that differences in hypothalamic the preoptic area of the brain was conducted using RNAseq
regulation of body fat in birds might involve the capacity of (Lynch et al., 2019). Two brood parasitic Icterids, brown-
the hypothalamus to sense and metabolize glucose headed (Molothrus ater) and bronzed cowbirds (Moloth-
(Fig. 3.3). In contrast, selection for body weight did not rus aeneus), were compared with juvenile and adult
affect hypothalamic expression of genes known to regulate red-winged blackbirds (Agelaius phoeniceus), a nonpara-
feed intake and metabolism, even though the high body sitic Icterid. Gene expression profiles for the parasitic birds
weight birds are hyperphagic. These studies demonstrated more closely resembled those of the juvenile nonparasitic
how involvement of novel gene pathways within the central birds than the adult nonparasitic birds. These findings raise
nervous system in physiological processes can be identified the intriguing possibility that the evolution of brood para-
using transcriptomics. sitism in birds might have involved a failure of the preoptic
The central nervous system plays an integral role in area to transition from the juvenile state to the reproduc-
stress responses. In an effort to define divergent mecha- tively mature state, a neotenic phenomenon.
nisms regulating long-term responses to stress occurring
during embryonic and early posthatch development, corti-
costerone was administered to Japanese quail during pre- or
3.4 Endocrine system
postnatal development, and the effects on gene expression Transcriptomics has been used to characterize gene
within the hippocampus and hypothalamus was assessed at expression within endocrine tissues and the responses to
adulthood using RNAseq (Marasco et al., 2016). Results hormonal treatments in birds. In one of the first microarray
indicated that effects depended on the developmental age of analyses in birds reported, transcriptional profiles were
corticosterone treatment and on the region of the brain. defined within the pineal gland of chicks during a light-dark
Corticosterone-treated birds exhibited increased levels of circadian cycle (Bailey et al., 2003). Expression of hun-
mRNA for brain-derived neurotrophic factor and mineral- dreds of genes in the pineal gland oscillated during the
ocorticoid receptor (MR) in the hippocampus and light-dark cycle, including genes involved in melatonin
corticotrophin-releasing hormone and serotonin receptors synthesis. Importantly, this transcriptomics analysis
in the hypothalamus. Interestingly, mRNA levels in the revealed many genes associated with immune responses,
hippocampus for the thyroid hormone transporter TTR were stress responses, and hormone binding, suggesting other
increased in response to prenatal but not postnatal roles for these genes within the pineal gland or for the pi-
corticosterone treatment. Conversely, levels of mRNA for neal gland within these other physiological systems. A
the serotonergic system were up-regulated in the hypo- similar analysis was subsequently performed in the chick
thalamus following postnatal but not prenatal corticoste- retina (Bailey et al., 2004). Although some overlap in
rone treatment. These findings indicate that early life stress oscillating gene expression was found between the retina
can have prolonged effects within the central nervous and the pineal gland, distinct differences were also noted,
 Transcriptomic analysis of physiological systems Chapter | 3 21

suggesting that differences might exist in circadian regu- expressed specifically in the magnum or the isthmus (Jon-
lation at the molecular level within the two tissues. Effects chère et al., 2010). Among these were genes encoding for
of thyroid hormone and growth hormone on hepatic gene antimicrobial proteins and ion transporters, respectively,
expression have been characterized in the chicken using supporting their role in antibacterial properties of the egg
microarrays (Wang et al., 2007b). Dozens of thyroid hor- albumen and for eggshell formation. An analysis of the
mone- and growth hormone-regulated genes were identi- effects of a synthetic estrogen on gene expression in the
fied. Interestingly, cross talk between the two systems was oviduct demonstrated that estrogen affects expression of
noted, as thyroid hormone status affected mRNA levels for genes associated with epithelial differentiation and tissue
growth hormone receptor and insulin-like growth factor remodeling (Song et al., 2011), consistent with the dramatic
binding protein 1. Microarrays were used to characterize effects of estrogens on oviduct size and glandular devel-
the response of the avian adrenal gland to adrenocortico- opment. Microarrays were used to identify genes expressed
tropic hormone (ACTH). Injection of ACTH increased specifically within the germinal disk of the developing
mRNA levels for several steroidogenic genes but also oocyte (Elis et al., 2008). These genes are likely to play a
genes with other functions, such as transcription, cell di- role in oocyte maturation or early embryonic development.
vision, and electron transfer (Bureau et al., 2009). The ef- Other genes found to be expressed in the granulosa cells are
fects of insulin immunoneutralization through more likely involved in follicular maturation. Within the
administration of antiserum to insulin were evaluated in the developing gonad of the chicken, miRNAs that were spe-
chicken (Simon et al., 2012). Microarray analysis of mRNA cific to the testes or ovary were identified using microarrays
samples from liver and muscle revealed that expression (Bannister et al., 2009), suggesting a role for miRNA
levels for more than 1000 genes were affected by decreased expression in gonadal differentiation. One reproductive
insulin levels or the elevated glucose levels associated with organ that is often overlooked is the pigeon crop that pro-
insulin immunoneutralization. The results demonstrated the duces “crop milk” for the nutritional supply of offspring. A
wide range of effects of insulin on two of its target tissues. comparison of gene expression in nonlactating and
Microarrays were used to characterize changes in gene lactating pigeon crop using oligonucleotide arrays identi-
expression in the pituitary gland during chicken embryonic fied genes that are differentially expressed in the lactating
development (Ellestad et al., 2006). Numerous genes were crop (Gillespie et al., 2011). Genes associated with extra-
identified with expression profiles that suggested involve- cellular matrix receptors, adherens tight junctions, and Wnt
ment in differentiation of pituitary thyrotrophs, somato- signaling were found. This finding supported hyperplasia
trophs, and lactotrophs (Fig. 3.2). Effects of glucocorticoids and cellular release into the crop lumen in the formation of
on pituitary gene expression were also identified using pigeon crop milk.
cDNA microarrays. Treatment with corticosterone of Several studies using transcriptional profiling of the
chicken embryonic pituitary cells affected mRNA levels for hypothalamo-pituitary-ovarian axis have been reported.
hundreds of genes, and these were placed into networks of Transcriptional profiling of prehierarchical, preovulatory,
affected genes (Jenkins et al., 2013). The results were also and postovulatory ovarian follicles in the chicken using
used to identify putative glucocorticoid receptor targets in RNAseq identified genes involved in adherens junctions,
the chicken, demonstrating the power of pathway and apoptosis, and steroid biosynthesis (Zhu et al., 2015).
network analysis of transcriptional profiles. RNAseq analysis was conducted on small white, large
white, and small yellow ovarian follicles of laying and
broody Zhedong white geese (Yu et al., 2016). Differen-
3.5 Reproductive system tially expressed genes identified included many involved in
Development and function of the reproductive system in- hormone responses, follicular development, autophagy, and
volves gonadal differentiation and hormonal effects on oxidation. Interestingly, those related to autophagy were
reproductive tissues, including testes, ovary, and oviduct. up-regulated in ovarian follicles from broody hens, indi-
However, the full extent of the genetic mechanisms un- cating that autophagy might play a significant role in
derlying these processes is not known. A number of studies follicular atresia associated with broodiness. RNAseq
have been reported in which investigators used tran- analysis of individual small yellow follicles with different
scriptomics to shed light on the underlying mechanisms levels of mRNA for the follicle-stimulating hormone (FSH)
controlling development and function of the reproductive receptor (FSHR) was used to identify a novel mechanism
system in birds. A comparison of gene expression in the for follicular recruitment (Wang et al., 2017). The Wnt
shell glands of juvenile and laying chicken hens using signaling pathway was significantly up-regulated in the
cDNA microarrays identified hundreds of genes that are follicles with the highest levels of FSHR expression. Wnt4
differentially expressed in the mature shell gland (Dunn was shown to stimulate proliferation of follicular granulosa
et al., 2009). Similar transcriptional profiling of the uterus cells and increase granulosa cell FSHR mRNA and
of the chicken using cDNA microarrays revealed genes decrease anti-Müllerian hormone mRNA levels, while FSH
22 PART | I Undergirding themes

was found to increase Wnt4 mRNA. These findings, based macrophage responses to lipopolysaccharide (LPS) or
initially on transcriptional profiling, suggest that Wnt4 E. coli, downstream targets of the Toll-like receptor
plays an important role in follicle selection in the chicken pathway were affected (Bliss et al., 2005). Transcriptional
ovary. Transcriptomic analysis using RNAseq was per- profiling of cecal gene expression in Salmonella-challenged
formed on the hypothalamus and anterior pituitary gland of neonatal chicks followed by pathway analysis revealed that
domestic turkey hens producing high or low numbers of expression of genes associated with the nuclear factor
eggs in a reproductive season. Pathway analysis of the kappa B (NFkB) complex and apoptosis were affected by
differentially expressed genes revealed differences in thy- Salmonella administration (Higgins et al., 2011). In each of
roid hormone and estradiol signaling between the two these studies, other genes not previously associated with
groups of hens (Brady et al., 2020). Treatment of pituitary immune responses were identified that might play a role
cells in culture confirmed that T3 suppressed mRNA levels in immunological responses to pathogens. In an analysis
for the beta subunits of luteinizing hormone (LHB) and of miRNA expression within the spleen and the bursa of
FSH (FSHB), with the effect being greater for cells from the Fabricius of embryonic chicks, divergent expression of
high egg-producing hens. In contrast, treatment with E2 numerous miRNAs was noted, suggesting that these miR-
increased mRNA levels for LHB and FSHB, with the effect NAs might play diverse roles in the functions of the various
being more pronounced for cells from the low egg- tissues of the immune system (Hicks et al., 2009).
producing hens than the high egg-producing hens. These
findings indicate that differences in thyroid hormone and 3.7 Muscle, liver, adipose, and
estradiol signaling might account, in part, for differences in
egg production rates. Moreover, they highlight how tran-
gastrointestinal tissues
scriptional profiling can uncover novel mechanisms gov- Multiple tissues are involved in nutrient absorption, meta-
erning physiological processes. bolism, and partitioning into tissues for animal growth or
energy storage. Among these are the intestine, liver, skel-
etal muscle, and adipose. However, all of the genes
3.6 Immune system expressed in these tissues to regulate growth and nutrient
Differences exist in immunological responses to pathogens partitioning are not known. In a comparison of skeletal
among individuals in a species. However, the genes muscle from slow-growing layer and fast-growing broiler
expressed within cells of the immune system that account chickens, transcriptional profiling using microarrays
for responses to pathogens and differences in responses revealed differences in expression of genes encoding for
among individuals are not entirely known. Microarray muscle fiber proteins and regulators of satellite cell prolif-
analysis was used to study gene expression in one eration and differentiation (Zheng et al., 2009). Genes
lymphoid organ, the spleen, of susceptible and resistant associated with slow muscle fibers were expressed at a
lines of chickens in response to Campylobacter jejuni greater level in breast muscle of layer than broiler chickens,
infection (Li et al., 2012b). Not surprisingly, expression of while mRNA levels for genes associated with satellite cell
genes for lymphocyte activation and humoral responses, growth were greater in muscle of broiler than layer
including immunoglobulin heavy and light chains, was chickens. In a similar analysis of gene expression in muscle
increased following infection in the resistant line. Surpris- types, microarray analysis was used to identify differen-
ingly, expression of genes related to erythropoiesis and tially expressed genes between the anterior latissimus dorsi
apoptosis was affected in the susceptible line. These dif- (ALD) and posterior latissimus dorsi (PLD) muscles of
ferences in genetic responses within the spleen to turkeys (Nierobisz et al., 2011). Expression of genes
Campylobacter jejuni infection could contribute to sus- encoding for extracellular matrix proteins was greater in the
ceptibility or resistance of individual birds to infection. In a slow twitch, red ALD muscle than in the fast twitch, white
similar study, transcriptional responses of the spleen to PLD. In contrast, expression of genes involved in glycol-
Escherichia coli infection were profiled (Sandford et al., ysis was greater in the PLD than ALD. In a comparison of a
2011). Immunological pathways including cytokine random-bred turkey line with a line selected for increased
signaling and Toll-like receptors were affected by E. coli body weight, microarray analysis of mRNA levels in breast
challenge, and the magnitude of transcriptional changes muscle revealed alterations in expression of genes associ-
was correlated with the severity of infection. Surprisingly, ated with extracellular matrix, apoptosis, Ca2þ signaling,
immunization prior to E. coli challenge had no significant and muscle function. Transcriptomics has been used to
effects on transcriptional profiles in response to E. coli. identify genes associated with meat quality of chicken
Similarly, transcriptional profiling of macrophage re- breast muscle. Genes associated with lipid and carbohy-
sponses to Salmonella-derived endotoxins revealed effects drate metabolism were associated with meat quality (Sibut
on expression of genes for multiple cytokines and Toll-like et al., 2011). In a similar study aimed at identifying genes
receptors (Ciraci et al., 2010). In an earlier study of involved in deposition of intramuscular fat in chickens,
 Transcriptomic analysis of physiological systems Chapter | 3 23

transcriptional profiling of breast muscle from broiler in poultry are not known. Transcriptional profiling of adi-
chickens and a slow-growing Chinese breed using DNA pose tissue from chicken lines divergently selected for low
microarrays revealed differential expression of genes or high-body fat revealed that genes involved in lipid
involved in lipid metabolism and muscle development (Cui metabolism and endocrine function were differentially
et al., 2012). DNA microarrays for chicken were also used expressed between the genetic lines (Wang et al., 2007a).
to study pectoralis gene expression in juvenile and sea Genes identified included lipoprotein lipase (LPL), fatty
acclimated king penguins (Teulier et al., 2012). Genes acid binding protein, thyroid hormone-responsive protein
associated with lipid metabolism were up-regulated, while (Spot14), and leptin receptor. In a more comprehensive
genes associated with carbohydrate metabolism were study of a different pair of chicken lines genetically
down-regulated in older, sea acclimated penguins. selected for low or high-abdominal fat, microarray analysis
High environmental temperatures impair growth per- of adipose tissue was used to identify genes and gene
formance in chickens, but the molecular mechanisms networks involved in the observed differences in adiposity
involved have not been elucidated. Microarray analysis of (Resnyk et al., 2013). Many genes involved in adipogenesis
gene expression in breast muscle of chickens exposed to and lipogenesis were up-regulated in the fat line. Again,
chronic heat stress revealed changes in expression of genes many genes involved in endocrine signaling were also
involved in protein turnover, tumor necrosis factor differentially expressed between the two genetic lines,
signaling, and mitogen-activated protein kinase (MAPK) including TTR, DIO1, DIO3, Spot14, and chemerin. These
signaling (Li et al., 2011). Effects of acute and chronic heat findings suggest that differences in adiposity among indi-
stress on gene expression in the liver of broiler chickens vidual birds might be related to differences in endocrine
were compared using RNAseq (Lan et al., 2016). The regulation of adipocyte differentiation and growth and lipid
transcriptomic results indicated that acute heat stress had a metabolism.
greater impact on the broiler liver than chronic heat stress. Lipogenesis in birds occurs primarily in the liver, and
Moreover, pathway analysis resulted in a novel network this process is regulated by the energy needs of the animal.
that combined the heat shock protein genes with immune Transcriptional profiling of newly hatched chicks that were
response genes. In another study examining effects of heat fed or fasted revealed that the metabolic perturbation of
stress on liver function in broiler chickens, transcriptomic fasting delayed the up-regulation of lipogenic genes in the
and metabolomic analyses were combined (Jastrebski et al., liver (Richards et al., 2010). Expression of one gene that
2017). Both analyses indicated that glycogenolysis and encodes for a transcription factor that regulates expression
gluconeogenesis were elevated in the liver in response to of the lipogenic genes, peroxisome proliferator-activated
heat stress. RNAseq was used to compare effects of acute receptor gamma was also delayed, supporting coordinated
and chronic heat stress on cardiac and skeletal muscle in regulation of lipogenesis. A similar analysis of transcrip-
two indigenous chicken ecotypes, from the lowland and tional responses of the liver to fasting of older chickens has
highland regions of Kenya (Srikanth et al., 2019). The p53 also been reported (Désert et al., 2008). In this study,
and PPAR signaling pathways were enriched in both low- fasting resulted in up-regulation of genes involved in
land and highland chickens, while MAPK signaling and ketogenesis, gluconeogenesis, and fatty acid beta-
protein processing in the endoplasmic reticulum were oxidation, while genes involved in fatty acid synthesis
enriched only in the more heat-sensitive highland chickens. were down-regulated. These findings demonstrated the
These results indicate that the ecotypes activated or sup- coordinated regulation of genes involved in nutrient parti-
pressed different genes, demonstrating different underlying tioning by the liver in response to the metabolic perturb-
mechanisms in heat stress responses between the two ance of fasting.
ecotypes. Development of the intestine has also been studied us-
Domestic turkeys and broiler chickens have been bred ing transcriptomics. Transcriptional profiles of the duo-
for increased feed conversion and muscle growth. In studies denum during embryonic development of the turkey were
aimed at identifying mechanisms responsible for differ- characterized using microarrays (de Oliveira et al., 2009).
ences in feed efficiency in broiler chickens, transcriptional Results indicated that expression of peptidase and lipase
profiling of breast muscle mRNA levels using microarrays genes (LPL) decreased toward hatch, while expression of
indicated that an up-regulation of genes involved in genes encoding for peptide and glucose transporters
anabolic processes and energy sensing and a down- (e.g., PEPT1 and SLC5AP) increased toward hatch. Tran-
regulation of genes involved in muscle fiber development scriptional profiles of the chicken jejunum were character-
and function are associated with high-feed efficiency ized during the first three weeks after hatch (Schokker et al.,
(Bottje et al., 2012; Kong et al., 2011). Excessive deposi- 2009). Microarray analysis indicated that genes involved in
tion of body fat in commercial poultry leads to decreased morphological and functional development were highly
conversion of feed into muscle for meat. However, the expressed immediately after hatch, while expression
genetic mechanisms involved in accumulation of body fat declined during later juvenile development.
24 PART | I Undergirding themes

3.8 Cardiovascular system mRNA and miRNA levels in the chicken (Goher et al.,
2013; Hicks et al., 2010; Kang et al., 2013; Nie et al., 2012;
Chicken embryos are a widely used model for studies of Wang et al., 2011). Importantly, RNAseq has been used in
cardiac development. As such, much is known about nonmodel species to sequence and annotate the tran-
development of the heart in chickens. However, the com- scriptome in the dark-eyed junco (Peterson et al., 2012) and
plex relationships among genes necessary for cardiac song sparrow (Srivastava et al., 2012), identify genes
development are not known. Gene expression profiling of involved plumage coloring in ducks (Li et al., 2012a), study
early embryonic heart tissues was used to identify genes gene dosage compensation in the European crow (Wolf and
associated with cardiac development (Buermans et al., Bryk, 2011), compare gene expression between the black
2010). Results from this analysis included components of carrion crow and the gray coated crow (Wolf et al., 2010),
Wnt signaling. In another microarray analysis of heart and identify genes differentially expressed between the
development, differences in gene expression between the ovaries of laying and broody geese (Xu et al., 2013). These
left and right ventricle were defined (Krejcí et al., 2012). studies using RNAseq were among the first transcriptomics
Not surprisingly, the set of differentially expressed genes studies in nonmodel avian species, and they demonstrate
included genes associated with cardiac cell differentiation, the utility of RNAseq for transcriptional profiling in
heart development, and morphogenesis. However, many physiological systems of any avian species. However, one
other genes not associated with these processes were also major hurdle remains for comparative physiologists. Rela-
identified, providing a novel list of candidate genes for tively few of the thousands of animal genomes now
future research on the mechanisms underlying cardiac sequenced are well annotated. Prediction and annotation of
development. The cardiovascular system acclimates to life genes in most genomes is based on sequence homology.
at higher altitudes and the associated hypoxia. However, With more than 10,000 avian species characterized, the
the genes involved in cardiac acclimation in birds are not possibilities for incorrect annotation of differentially
known. Microarray analysis of gene expression in the heart expressed genes and, therefore, interpretation of transcrip-
of the Tibetan chicken and chickens not adapted to life at tional profiling results, are potentially equally as large as
high altitudes has been performed (Li and Zhao, 2009). the number of species. Similarly, most commonly used
Results provided a list of candidate genes that might pathway tools are based on the functions of genes deter-
function in chronic acclimation to high altitudes. Although mined from studies in mice and humans. Investigators and
not part of the cardiovascular system, microarray analysis readers should use caution when interpreting transcriptomic
of pectoral muscle was performed on rufous-collared results in avian species that have not been confirmed
sparrows sampled at 2000 and 4000 m above sea level in experimentally.
the Andes Mountains (Cheviron et al., 2008). Differentially
expressed genes included those involved in oxidative
phosphorylation and oxidative stress. Interestingly, none of References
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Wang, X., Carre, W., Saxton, A.M., Cogburn, L.A., 2007b. Manipulation of Zhedong white goose. Sci. Rep. 6, 36877.
of thyroid status and/or GH injection alters hepatic gene expression in Zheng, Q., Zhang, Y., Chen, Y., Yang, N., Wang, X.-J., Zhu, D., 2009.
the juvenile chicken. Cytogenet. Genome Res. 117, 174e188. Systematic identification of genes involved in divergent
Wang, Y., Ghaffari, N., Johnson, C.D., Braga-Neto, U.M., Wang, H., skeletal muscle growth rates of broiler and layer chickens. BMC
Chen, R., Zhou, H., 2011. Evaluation of the coverage and Genom. 10, 87.
depth of transcriptome by RNA-Seq in chickens. BMC Bioinf. 12 Zhu, G., Mao, Y., Zhou, W., Jiang, Y., 2015. Dynamic changes in the
(Suppl. 10), S5. follicular transcriptome and promoter DNA methylation pattern of
Wang, Y.Y., Chen, Q.Y., Liu, Z.M., Guo, X.L., Du, Y.Z., Yuan, Z.J., steroidogenic genes in chicken follicles throughout the ovulation cy-
Guo, M., Kang, L., Sun, Y., Jiang, Y.L., 2017. Transcriptome analysis cle. PLoS One 10, e0146028.
Chapter 4

Avian proteomics
Alison Ferver1, Shane C. Burgess2, Colin G. Scanes1 and Sami Dridi1
1
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR, United States; 2College of Agriculture & Life Sciences, The
University of Arizona, Tucson, AZ, United States

Abbreviations Meq MDV EcoQ protein

mRNA messenger ribonucleic acid
ACAA2 acetyl-CoA acyltransferase 2 MS mass spectrometry
ACE angiotensin I converting enzyme mTOR mechanistic target of rapamycin
ACOX1 acyl-CoA oxidase 1 MudPIT multidimensional protein identification technology
AGR2 anterior gradient homolog 2 NMR nuclear magnetic resonance
APEX ascorbic acid peroxidase PAGE polyacrylamide gel electrophoresis
ATP adenosine triphosphate PCR polymerase chain reaction
AvBD10, AvBD9 avian b-defensin 10, avian b-defensin 9 PGAM1 phosphoglycerate mutase 1
BCO bacterial chondronecrosis with osteomyelitis pI isoelectric point
BirA bifunctional ligase/repressor A qPCR quantitative polymerase chain reaction
Cas9 CRISPR associated protein 9 QTL quantitative trait locus
CD cluster differentiation RNA ribonucleic acid
CECs chicken embryo cells RP reversed-phase
cGMP-PKG cyclic guanosine 30 , 50 -monophosphate-protein SCX strong cation exchange
kinase G SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
CORT corticosterone electrophoresis
CPT1A carnitine palmitoyltransferase 1A SILAC stable isotope labeling with amino acids in cell culture
CRMP2 collapsin response mediator protein 2 SLC25A5 Solute carrier family 25 member 5
CST3 Cystatin C SM spaghetti meat
DNA deoxyribonucleic acid SPINK7 serine peptidase inhibitor kazal type 7
eIF-2/eIF4 eukaryotic initiation factor 2 T-cells thymus cells
EOC epithelial ovarian cancer T-reg thymus cell regulatory
FHN femur head necrosis TD tibial dyschondroplasia
FSH follicle stimulating hormone Th thymus cell helper
GaHV-2 Gallid herpesvirus 2 TSPO translocator protein
H5N1 hemagglutinin type 5 and neuraminidase type 1 VEGFA vascular endothelial growth factor A
HIF-1 alpha hypoxia-inducible-factor-1 alpha VLDLR very low density lipoprotein receptor
HPLC high-performance liquid chromatography WB wooden breast
Iba57 Iron-sulfur cluster assembly factor WS white striping
ICATs isotope coded affinity tags XIC extracted-ion chromatogram
IFN interferon
IPA ingenuity pathway analysis
iTRAQ isobaric tags for relative and absolute quantification 4.1 Introduction
LC liquid chromatography
LC-MS liquid chromatography tandem mass spectrometry
Whereas genomics provides and analyzes the entire set of
MALDI matrix assisted laser desorption/ionization functional elements encoded in a genome, and tran-
MALDI-TOF-MS matrix-assisted laser desorption ionization time of scriptomics studies gene expression by measuring RNA
flight mass spectrometry levels, proteomics analyzes protein expression, modifica-
MD Marek’s disease tion, structure, localization, interaction, and function.
MDV Marek’s disease virus Having a completed genome sequence of an organism is a

Sturkie’s Avian Physiology. https://doi.org/10.1016/B978-0-12-819770-7.00001-3

Copyright © 2022 Elsevier Inc. All rights reserved. 29
30 PART | I Undergirding themes

key step toward understanding how that organism is built behavior. Although not absolutely essential, a sequenced
and maintained, and thus its complex biology. This in- and structurally annotated genome greatly facilitates pro-
formation is stored in the genome in the form of genes, teomics. The more accurate the genome assembly and
which are transcribed into RNA, and RNA is translated annotation, the more accurate the proteomics methodolo-
into proteins. The entire set of RNA transcripts and pro- gies can be; this extends to the individualdthe most ac-
teins encoded by the genome is called transcriptome and curate proteomics experiments will be done using the
proteome, respectively (Velculescu et al., 1997; Wilkins individual’s own genome sequence and, to be more ac-
et al., 1996). Although genes provide instructions, pro- curate still, the transcriptome that corresponds to the
teins are the functional units of almost all biological proteome. Conversely, proteomics can be used to improve
processes and the principal structural building blocks of the structural annotation of genomes (Nanduri et al., 2010;
all living organisms. Systems-level understanding of cell Jaffe et al., 2004). Red jungle fowl (Gallus gallus), the
physiology is thus inevitably based on understanding the major wild ancestor of the domestic chicken, was the first
multifaceted interplay of gene expression and protein avian and nonmammalian amniote to have its genome
functional networks. An individual’s genome sequence sequenced (International Chicken Genome Sequencing
(with the exception of some regions dedicated to the Consortium, 2004). The chicken is the principal non-
adaptive immune system) is static, its epigenome (the mammalian vertebrate animal model for studying devel-
methylation patterns on DNA) less so, and the tran- opment, infectious disease, immunology, oncogenesis,
scriptome and proteome are extremely dynamic. These and behavior. It is also one of the most important agri-
latter two differ from cell to cell, and change dramatically cultural species for production of meat and eggs. Until
according to conditions that cells are exposed to. The additional avian complete genomes became available, the
transcriptome is more complicated than the genome chicken genome served as de facto model bird genome,
because of both frame-shifting and alternative splicing. and most of the proteomics studies have utilized this
The proteome is even more complex because most pro- model to study various aspects of bird biology. Complete
teins are co- and posttranslationally modified (Walsh, or draft genomes of several other avian species have
2006). More than 200 different types of protein modifi- become available, including several lines of domestic
cations are documented in vertebrates, and more than one chicken (Gallus gallus domesticus), zebra finch (Taenio-
of these modifications routinely occurs on most proteins pygia guttata), domestic turkey (Meleagris gallopavo),
(Walsh, 2006). Measurement of the proteome is also more collared flycatcher (Ficedula albicollis), pied flycatcher
challenging than that of the transcriptome because the (Ficedula hypoleuca), large ground finch (Geospiza
dynamic range of proteins in tissues is higher than of magnirostris), scarlet macaw (Ara macao), mallard duck
transcripts, and can span over 11 orders of magnitude in (Anas platyrhynchos), ground tit (Pseudopodoces
body fluids (Anderson and Anderson, 2002), and most humilis), Puerto Rican parrot (Amazona vittata), and
importantly, from a technical perspective, there is no budgerigar (Melopsittacus undulatus) (Ellegren et al.,
equivalent of the polymerase chain reaction (PCR) for 2012; Warren et al., 2010; Oleksyk et al., 2012; Dalloul
proteinsdwe must use very expensive machinery to et al., 2010; Rands et al., 2013; Rubin et al., 2010; Huang
directly identify proteins. Although mRNA quantities et al., 2013; Cai et al., 2013; http://aviangenomes.org/,
measured by quantitative (q)PCR, microarrays, or 2013). The turkey, duck, and domestic chicken were
sequencing are often used as surrogates for protein sequenced because they are economically important
quantities, and indirectly, protein activity, there is no or (Dalloul et al., 2010; Rubin et al., 2010; Rao et al., 2012).
little correlation between mRNA and levels of its corre- These species are also used as biomedical models, which
sponding protein (Gygi et al., 1999a; Cullen et al., 2004; is the reason that the zebra finch, scarlet macaw, and
Nagaraj et al., 2011; Marguerat et al., 2012). This means Puerto Rican amazon were sequenceddthey are important
that the presence or quantities of proteins in biological in neuroscience for studying their behavioral, cognitive,
samples cannot be satisfactorily estimated solely through and speech abilities (Warren et al., 2010; Oleksyk et al.,
their mRNA levels. In addition, posttranslational modifi- 2012; Seabury et al., 2013). Darwin’s finches are model
cations often profoundly affect protein activities. Though organisms to study of various aspects of evolution and
arguably less sensitive, proteomics methods are more development (Rands et al., 2013). Flycatchers are impor-
specific for determining what is happening at the protein tant models for speciation (Ellegren et al., 2012), and the
leveldthey can identify and quantify protein amounts and genome of the ground tit provides new opportunities to
posttranslational modifications as well as be combined study adaptation mechanisms to extreme conditions (Cai
with other omics data to produce a complete biological et al., 2013). Collectively, the availability of these addi-
picture of the organism or physiological state in question. tional genomes is opening up new vistas for genome-wide
Proteomics thus provides a direct measure of the pre- research and studies of various aspects of bird biology
dominant functional units responsible for cellular both on the RNA and protein levels. It is expected that
 Avian proteomics Chapter | 4 31

large-scale analysis of the avian genome, transcriptome, occasionally NMR, are now being utilized in conjunction
and proteome will increase our understanding of complex with bioinformatics for larger pathway annotations in high-
molecular processes that determine phenotype. throughput protein profiling and analysis (Aslam et al.,
2017). NMR is less sensitive than MS; however, it benefits
from more stable spectrometer, an absence of spectrum
4.2 Protein identification and analysis quenching, and its ability to use in a large range of ex-
Although traditional protein biochemistry focuses on periments (Dona et al., 2016). The versatility of MS and
studying properties of individual proteins, proteomics en- modifications to its ionization method has made MS the
compasses nearly any type of technology that enables most commonly used technique in large-scale proteomic
studying proteins on a large scale. There have been and high-throughput analysis. In addition to its versatility,
numerous tools developed for identification of proteins, MS has also the ability to handle the difficulties associated
including two-hybrid systems (Fields and Song, 1989), with the complex and dynamic nature of proteomes (Han
protein/peptide microarrays (Haab, 2003; Panse et al., et al., 2008). MS simplifies and accelerates the analysis and
2004), nuclear magnetic resonance (NMR) spectrometry, characterization of proteins. MS-based proteomics refers to
and mass spectrometry (MS)-based approaches (Fenn et al., approaches that use MS for identifying, characterizing, and/
1989). The two-hybrid systems and peptide/peptide or quantifying proteins in biological samples (Fig. 4.1).
microarrays had limited applicability which although was Mass spectrometers are used to detect, identify, and quan-
alleviated through analytical capabilities of MS, resulted in tify small molecules based on their mass and charge (m/z)
more MS-based approaches taking over. MS, and ratios with high precision, sensitivity, and speed. Many

FIGURE 4.1 The workflow of

mass spectrometry-based prote-
omics. The goal of proteomics is
physical (protein identity, modifi-
cations, structure, localization) and
functional (protein interactions,
composition of protein complexes)
characterization of the proteome.
Proteomics denotes the collection of
diverse technologies that enable the
study of proteins on a large scale. In
current proteomic research, mass
spectrometry (MS) plays prime and
indispensable role because several
thousand proteins can be rapidly
analyzed, correctly identified, and
accurately quantified in a single
MS-based experiment. Proteins
(A) are typically too large for ac-
curate mass determination by mass
spectrometry, therefore, they are
first digested into peptides (B),
which are then analyzed by a mass
spectrometer. The obtained peptide
masses, peptide (tandem) mass
spectra (C), are then searched
against predicted peptide masses
derived from DNA or protein
sequence databases by using one or
several different mass spectral
search algorithms and their amino
acid sequence is determined. The
amino acid sequence of an identified
peptide is often unique to its parental protein, and such peptides are used for unambiguous identification of proteins that were present in the analyzed
sample. In addition to protein identification, MS enables accurate relative or absolute quantification of proteins. As a result, MS is commonly used to
identify proteins that are qualitatively and/or quantitatively differentially expressed between studied conditions. The obtained proteomic information is
integrated with existing knowledge and/or data from other large-scale studies (mRNA profiling, siRNA screens) to better understand cell or tissue biology
at the system level. For example, protein expression levels are compared with mRNA levels (D), and differences in protein expression are analyzed in the
context of biological pathways or interaction networks (E) in effort to identify the underlying causalities and mechanistic principles that give rise to a
studied phenomenon or phenotype.
32 PART | I Undergirding themes

excellent reviews have been written that cover instrumen- peptides, in which the connection between the peptide and
tation and principles of protein identification by MS (Yates the originating protein is lost. Peptides detected and iden-
et al., 2009; Yates, 1998; Steen and Mann, 2004; Cottrell, tified by MS are then used to infer the presence of all
2011), and we will not replicate this information. Rather, original proteins in the sample, which is the principle of the
we will introduce the key methods. A typical biological “shotgun” proteomics named by an analogy to shotgun
sample contains extremely complex proteomes. Because DNA sequencing. Multidimensional HPLC combines
mass spectrometers can analyze only a limited number of several separation steps to improve resolution of complex
different peptides at a time, the sample complexity must be mixtures of peptides. One of the most popular multidi-
reduced before MS. This has been historically done on the mensional separation methods utilizes strong cation ex-
protein level by gel electrophoresis and more commonly change and reversed-phase (RP) chromatography to
done on the peptide level by various chromatography separate peptides in two dimensions: first peptides are
techniques. separated based on charge, and then on hydrophobicity
(Washburn et al., 2001). This separation method is the basis
of the shotgun proteomic strategy known as multidimen-
4.2.1 Historical to current techniques
sional protein identification technology (MudPIT)
Two-dimensional (2D) polyacrylamide gel electrophoresis (Washburn et al., 2001). Another important chromato-
(PAGE) was the first technique that allowed truly complex graphic technique that is often used in gel-free approaches
proteomic analysis and was instrumental for the develop- is affinity purification. Selective enrichment affinity mate-
ment of proteomics (O’Farrell, 1975; Rabilloud et al., rials are either used to enrich for peptides that contain
2010). 2D PAGE is the most widely used technique in gel- certain posttranslational modifications, such as phosphor-
based proteomics; it simply deconvolutes a protein mixture ylation (Ficarro et al., 2002; Cao and Stults, 1999) or
in two dimensions. Proteins are first separated in the first glycosylation (Geng et al., 2000; Durham and Regnier,
dimension by their isoelectric point (pI), and then in the 2006), or peptides that contain a specific selectable amino
second dimension according to their electrophoretic acid residue, such as cysteine (Wang and Regnier, 2001) or
mobility (which is a function of molecular weight and histidine (Wang et al., 2002). Affinity-enriched peptide
charge of a protein) in a polyacrylamide gel. Separated mixtures are usually directly (online) or offline transferred
proteins are then stained and appear as spots in the gel. The to RP HPLC columns and further analyzed by MS. Gel-free
amount of protein in a spot is determined by measuring the approaches overcome many of the drawbacks that are
spot volume. This quantification is used as a screening inherent to gel-based methods (for example, proteins with
process to select a limited number of corresponding spots extreme size, pI, or hydrophobicity are amenable for
that contain different amounts of protein on related gels. analysis); but more importantly, they allow a large number
This technique is very useful for comparing two samples of proteins to be identified and quantified in a high-
that have similar protein expression profiles in order to find throughput manner and short time. The ability for high-
proteins that differ between the samples in their expression throughput proteomics to detect low-abundance proteins
levels or posttranslational modifications (Rabilloud et al., was heavily investigated as it was a major drawback
2010). The major advantage of this method is that it is compared to gel-based approaches. Techniques on the front
intrinsically quantitative. The major drawbacks are the end of MS analysis that improve sensitivity include frac-
limited capacity of protein separation; poor reproducibility tionation, affinity enrichment, and immune-depletion,
of 2D gels; and low sensitivity, dynamic range, and depending on the sample (Shi et al., 2012). Improvements
throughput. Although efforts to overcome some of the in statistical modeling of liquid chromatography tandem
shortcomings inherent to gel-based approaches resulted in mass spectrometry (LC-MS/MS) in the MS-spectrum count
the development of improved 2D gel methods, such as 2D label-free quantitative proteomic approach have also
fluorescence gel electrophoresis (Unlu et al., 1997), gel-free resulted in improved detection and attenuated bias toward
chromatographic approaches, which offer a number of low-abundance proteins (Lee et al., 2019). Recently, a
advantages over gel-based methods, now completely combined approach of nanoparticles and MS, called
dominate the field of proteomics. One of the most common Nanoproteomics, has shown to be an antibody-free, scal-
approaches being multidimensional high-performance able and reproducible strategy to enable analysis of low-
liquid chromatography (HPLC). Multidimensional HPLC abundance proteins directly form samples such as serum
quickly became the technique of choice for large-scale (Tiambeng et al., 2020).
proteomic studies. Here, in contrast to gel-based ap-
proaches, the entire analyzed proteome is first digested, and
the resulting peptides are then separated by multidimen-
4.3 Quantitative proteomics
sional HPLC and further analyzed by MS. Digestion of a In addition to protein identification, mass spectrometry can
protein mixture generates a highly complex mixture of quantify proteins in complex biological samples. The
 Avian proteomics Chapter | 4 33

proteome of a cell is highly dynamic and expressed proteins of the protein that is exposed to the solvent by exchanging
often change their locations, interactions, and modifications amide protons with heavier deuterium atoms (Wales and
in response to different stimuli. The goal of quantitative Engen, 2006) or by different covalent modifications (Stocks
proteomics is to obtain a snapshot of a proteome at a and Konermann, 2009). The labeling changes the molecular
particular time. Accurate quantification of proteins is weight of a protein, and this enables MS to identify the
important for understanding of physiological or patholog- modified sites. Protein foot-printing methods are used to
ical phenomena, and for identification and modeling of investigate protein conformation in solution. Chemical
functional networks. Traditionally, protein quantification cross-linking covalently couples parts of a protein(s) that
has been based on the 2D PAGE approaches, but several are close in space under native conditions. Subsequent MS
gel-free methods allow accurate protein quantification analysis identifies the location and identity of the cross-
solely by MS. Methods of quantitative proteomics are linked sites, which provides important clues about the
classified into two major categories: those that use stable structural topology of the protein or protein complexes
isotopes and those that do not. The most popular stable (Young et al., 2000; Petrotchenko and Borchers, 2010).
isotope-labeling techniques either label peptides with Determining interacting partner proteins has become of
isobaric tags for relative and absolute quantitation (iTRAQ) increasingly high interest and poses additional challenges
(Ross et al., 2004), or label proteins metabolically by and is especially time consuming in in-vitro models. Pre-
incorporation of stable isotope labels with amino acids in dictive protein interaction software enables predictive
cell culture (Ong et al., 2002) or enzymatically with models that alleviate the difficulty of protein interaction
isotope-coded affinity tags (Gygi et al., 1999b). Protein prediction in vitro. A fusion of experimental and predictive
quantification by label-free approaches is based on the models allows the use of sparse experimental data to pro-
observation that the chromatographic peak area for any duce more accurate protein interactions. Numerous bio-
given peptide in an LC run (Bondarenko et al., 2002; informatic software have been developed to aid in
Chelius and Bondarenko, 2002) and the number of tandem predictive modeling approaches to protein structure and
MS spectra of a given peptide (Liu et al., 2004) are pro- protein interactions, and there are extensive reviews
portional to peptide concentration in the analyzed sample. covering the differences and uses of the software (Pagadala
Thus, relative quantification by label-free approaches can et al., 2017; Nealon et al., 2017; Aslam et al., 2017). Spatial
be done by measuring and comparing the intensities of proteomics is a relatively new method of determining
precursor ions, or by counting and comparing the number proteins in the vicinity of a bait without a high affinity
of tandem MS spectra derived from a particular protein in between interactors as long as they are in a defined region.
different experiments. Current techniques include an engineered version of
ascorbic acid peroxidase (APEX) fused to proteins or
involved in epitope tagging of Cas9. APEX fusion results
4.4 Structural proteomics in a hydroxyl radical when hydrogen peroxide and phenoxy
A protein’s function is determined by its structure. The biotin are added, labeling protein. In conjunction with
major goals of structural proteomics are to elucidate the Cas9, proteins surrounding a gene found using the Cas9
three-dimensional (3D) protein structures and to determine guide RNA can be labeled. Another strategy is BioID
the relationship between protein structure and function. which uses biotin ligase enzyme BirA for similar data
Traditionally, static 3D protein structures are determined by (Yates, 2019; Kim and Roux, 2016; Myers et al., 2018).
X-ray crystallography (Sherwood et al., 2011) or NMR
spectroscopy (Wuthrich, 1990). However, experimental
determination of protein structure by these methods re- 4.5 Application of proteomics in avian
mains a difficult and laborious task in vitro but can be
improved upon by utilizing current software for predictive
research
models (Sherwood et al., 2011; Hyung and Ruotolo, 2012; Until recently, red junglefowl, the ancestor of domestic
Nealon et al., 2017). Alternatively, protein structures can be chickens, was the only avian species with a sequenced
predicted computationally by homology modeling or ab genome (2004) (International Chicken Genome Sequencing
initio (Flock et al., 2012). However, and despite decades of Consortium, 2004). Because proteomics greatly depends on
intensive research, these approaches do not always produce a complete and well-annotated genome sequence, most of
reliable models (Flock et al., 2012). Hydrogen-deuterium the proteomics studies in birds have been done on this
exchange (Wales and Engen, 2006), covalent labeling animal model. Since then, the complete genomes of zebra
(Chance, 2001), or chemical cross-linking (Young et al., finch (2010) (Warren et al., 2010), turkey (2011) (Dalloul
2000; Petrotchenko and Borchers, 2010) have been coupled et al., 2010), and two flycatchers (2012) (Ellegren et al.,
with MS to emerge as viable methods to probe 3D protein 2012) have been sequenced and assembled and many more
structure. Protein foot-printing methods modify the surface are underway (Oleksyk et al., 2012; Rands et al., 2013;
34 PART | I Undergirding themes

Rubin et al., 2010; Huang et al., 2013; Cai et al., 2013; light and 82 upregulated in dark egg layers. Functional
Seabury et al., 2013). The increasing number of sequenced analysis indicated that light brown egg layers produced less
avian genomes expands the range of unique bird phenom- protoporphyrin IX for brownness, potentially via down-
ena that can be studied on a global protein level, but more regulated Iba57 and up-regulated SLC25A5 with down-
importantly, makes it possible to study avian proteomes regulated TSPO (Li et al., 2016).
directly through their genomes, obviating the need for The chicken embryo is one of the most useful and
cross-species peptide matching. A number of proteomic investigated comparative and biomedical models for
studies have been done to study various aspects of bird studying development, physiology, and pathogenesis. Pro-
biology including egg production, embryogenesis, devel- teomics has been used to characterize the proteome of a
opment, metabolism, behavior, cognition, immunity, can- chicken embryonic cerebrospinal fluid (CSF) (Parada et al.,
cer, disease, and infection. 2006). This study identified, among others, 14 proteins that
are also present in human CSF, and 12 of them are altered
in neurodegenerative diseases and/or neurological disor-
4.5.1 Proteomics of egg physiology, embryonic
ders. Bon and co-workers identified and quantified selected
development, and reproduction
proteomes of three different heart tissues and studied them
A number of studies have focused on initial description and at three different developmental stages (Bon et al., 2010).
functional characterization of proteomes of different avian By comparing the identified proteomes, it was possible to
tissues, anatomical structures, or entire organs. The avian study the changes in proteome expression and to identify
egg is a reproductive cell and a highly elaborate biological proteins that were specific for particular heart structures or
structure that protects and nourishes the developing em- developmental stages. Grey et al. (2010) used an alternative
bryo. The major components of the eggdthe crystalline approach, based on MALDI tissue imaging MS, to study
shell (Mann et al., 2006, 2007; Ahmed et al., 2017), spatial distribution of proteins in chicken heart structures
albumen (egg white) (Mann and Mann, 2011; D’Ambrosio such as vessels, valves, endocardium, myocardium, and
et al., 2008; Mann, 2007; Sun et al., 2017), yolk (Farinazzo septa. To better understand embryonic chicken bone for-
et al., 2009; Mann and Mann, 2008; Liu et al., 2018), and mation, Balcerzak et al. applied proteomics to identify
the vitelline membrane (Mann, 2008)dhave been exten- protein machinery of matrix vesicles, which is essential for
sively characterized by various proteomic approaches. the formation of hydroxyapatite (Balcerzak et al., 2008).
Recent work combining transcriptome, QTL, and proteome Functional analysis of the matrix vesicle constituents sug-
data determined differentially expressed and significant gested what roles these proteins might have in the miner-
genes and proteins from six different bird species (chicken, alization process. The embryonic development of skeletal
duck, goose, turkey, pigeon, and quail) in egg formation. muscle has also been investigated in chicken using iTRAQ
Eleven common differentially expressed proteins were analysis and protein interaction network analyses (Ouyang
found in the egg white proteomes of all six species, but et al., 2017). There were 19 up-regulated and 32 down-
numerous genes and proteins were found to be uncommon regulated proteins in embryonic age 11 versus 16, 238
in either the egg proteome of oviducts of the species (Zhang up-regulated and 227 down-regulated proteins in embry-
et al., 2020a,b,c). This type of large-scale, multifaceted onic age 11 and day 1 of age. The differentially expressed
research reveals key differences in proteomes across spe- proteins were involved in pathways of protein synthesis,
cies while also highlighting the conservation of certain muscle contraction, and oxidative phosphorylation. By
proteomes in egg formation. A key difference in Guinea combining transcriptome data, results found 189 differen-
Fowl compared to chicken eggs is the hardness of shell. A tially expressed proteins correlating to mRNA levels and
quantitative proteomic study using nano-LC-MS/MS these proteins also were involved in muscle contraction and
identified 149 proteins, nine unique to Guinea Fowl, in the oxidative phosphorylation. A recent study on haptic pro-
eggshell organic matrix at key stages of bio-mineralization. teins in embryonic and newly hatched chicks revealed 41%
61 of the proteins were found in the zone of microstructure of the 3409 differentially expressed proteins were involved
shift, 17 were abundant in all zones, and some were in metabolic processes and significant differences in haptic
involved in the control of calcite precipitation (Le Roy proteins from embryonic development to hatching (Peng
et al., 2019). This study provides potential biomarkers for et al., 2018). Most importantly, key factors controlling fat
selection of layers producing eggs with improved shell deposition during chicken embryonic development were
mechanical properties and therefore enhance food safety elucidated to be ACAA2, CPT1A, and ACOX1. This study
and potential decrease in vertical transmission of bacteria. also revealed an alternative model using chicken for human
Proteins involved in eggshell brownness were investigated obesity and insulin resistance. To better understand how
via iTRAQ analysis with proteomics of the shell gland birds are prepared for transition from a fat-rich diet in ovo
epithelium of hens laying dark and light brown eggs. About to saccharide- and protein-based diet after hatch, Gilbert
147 differentially expressed proteins with 65 upregulated in et al. (2010) analyzed the proteome of chicken small
 Avian proteomics Chapter | 4 35

intestine at hatch and during the early posthatch period in AvBD10, and AvBD9. Selection for higher fertility was
two different broiler lines (Gilbert et al., 2010). This study shown to impact proteomic profile of seminal fluid in white
identified differences in expression of digestion and leghorn males compared to the red junglefowl ancestor
absorption-related proteins between different genetic lines. (Atikuzzaman et al., 2017). 2DE and immunoassays were
A proteomic approach was also used to identify hypo- conducted on the seminal fluid of pooled males from each
thalamic biomarkers associated with high-egg production in breed with statistics to cover intra- and intergroup vari-
chickens (Kuo et al., 2005). Comparison of the hypotha- ability. The results showed conserved proteins being serum
lamic proteomes from two related chicken lines selected for albumin and ovotransferrin, but clear enrichment of pro-
meat and high-egg production resulted in identification of teins related to immune response regulation for sperm
six proteins that differed in their expression between the survival in female reproductive tract including less proin-
lines, and some of these proteins are involved in regulation flammatory cytokines and an abundance of immunomod-
of gene expression, signal transduction, and lipid meta- ulatory/anti-inflammatory peptides. Taken together, these
bolism. The heterogeneous nuclear ribonucleoprotein H3 studies demonstrate the diverse application of proteomics in
was suggested as novel biomarker for high-egg production. elucidating key pathways and proteins involved in avian
A contributing factor in egg production is follicle selection. reproduction and development.
A study comparing transcriptome and proteome of small
yellow follicles and hierarchical follicles in laying hens
4.5.2 Proteomics of behavior and plumage
identified nine proteins and seven genes with VLDLR gene
and protein being higher in hierarchical follicles as well as The zebra finch is the dominant animal model for studying
being stimulated by FSH in granulosa cells (Chen et al., molecular mechanisms underlying learning, memory,
2020b). This study has implications for both laying hens vocalization, and social behavior. A natural perceptual
and broilers as broiler breeders often undergo multiple experience, such as a sound of another bird singing, triggers
follicle selection (Hocking and Robertson, 2000). 2D rapid changes in expression of specific genes in the audi-
PAGE MS was used to compare expression profiles of tory region of the zebra finch brain (Mello et al., 1992).
proteins in the oviduct in chicken hens of different ages Repeated exposure to the same song leads to stimulus-
during the egg-laying period (Kim et al., 2007). The anal- specific habituation of the original response (Petrinovich
ysis revealed that anterior gradient homolog 2 (AGR2) and Patterson, 1979). To understand the process of habit-
protein was among the most differentially expressed pro- uation better, Dong et al. (2009) used DNA microarrays
teins. Analysis of the mRNA showed that expression of and 2D PAGE MS approaches to analyze global changes of
AGR-2 was limited to the magnum and isthmus of the gene expression at different stages in the development of
oviduct, and that this expression was approximately 900- habituation. This study showed that exposure to a song
fold higher in the mature oviduct in comparison to the induces massive changes in gene expression, and that song
premature one. Because AGR-2 is a secreted protein that response habituation is not a simple loss of the original
shows estrogen dependent expression, and egg laying is responses but rather a change of neuronal responses
strongly affected by estrogen, it was suggested that AGR-2 underpinned by a novel and different gene expression
might be important for the development of the epithelial profile. Analysis of protein expression showed that habit-
cells in the oviduct during the egg-laying period in uation is accompanied by a decrease in expression of
chickens. 2D PAGE and MudPIT have been used to cellular and mitochondrial proteins that are involved in
discover genetic and molecular mechanisms that compro- biosynthesis and energy metabolism. Neuropeptides are
mise sperm mobility in chickens (Froman et al., 2011). signaling peptides found in neural tissue that modulate a
Analysis of the sperm proteome from chicken lines of low wide range of physiological and behavioral processes
or high-sperm mobility allowed deduction of a proteome- including metabolism, reproduction, learning, and memory.
based model that explained well the differences in sperm Xie et al. (2010) used a combination of bioinformatics, MS,
mobility between lines, and confirmed the initial hypothesis and biochemistry for prediction, identification, and locali-
that defects in ATP metabolism and glycolysis are zation of neuropeptidome of the zebra finch. Computational
responsible for premature mitochondrial failure, which re- analysis of the zebra finch genome predicted 70 putative
sults in sperm immobility. MALDI and top-down MS of prohormones and 90 peptides derived from 24 putative
chicken spermatozoa and seminal plasma along with pheromones identified in the zebra finch brain by two
comparative analysis using spectral count and XIC revealed different MS approaches. The power of MS was further
unique profiles of most fertile males compared to least used for localization of a subset of peptides in the major
fertile male chickens (Labas et al., 2015). Over-represented song control nuclei of the zebra finch brain. Furthermore,
enzymes in the most fertile males were involved in energy gene expression of a subset of pheromone genes was
metabolism and respiratory chain activity and least fertile anatomically mapped in selected zebra finch brain sections
males differed in proteins including acrosin, ACE, by in situ hybridization.
36 PART | I Undergirding themes

Birds display an enormous range of plumage colors, and deficiency. The analysis revealed that lysine deficiency
this diversity rivals or exceeds that of plants (Stoddard and might not result in a simple overall reduction of protein
Prum, 2011). Still, bird plumage occupies only about 30% synthesis in chickens fed with a lysine-deficient diet, but
of the possible colors that birds can see (Stoddard and rather in reduced anabolism of specific proteins. Corzo
Prum, 2011). The molecular mechanisms that determine et al. (2006) and Zhai et al. (2012) also evaluated the effect
and drive the development of this diversity are largely of dietary methionine on breast muscle accretion in broiler
unknown. The breeding plumage of male pied flycatchers chickens (Corzo et al., 2006; Zhai et al., 2012). This study
varies from a brown to dark black. Leskinen et al. (2012) showed that four canonical pathways related to muscle
characterized and quantified the proteome of developing development (citrate cycle, calcium signaling, actin cyto-
pied flycatcher feathers to advance understanding of skeleton signaling, and clathrin-mediated endocytosis
physiological processes that underlay the variation in signaling) were differentially regulated between chickens
pigmentation. In total, 294 proteins were identified in the that were fed with low- and high-methionine diets (Zhai
developing feathers. About 65 proteins were linked with et al., 2012). In addition, this study suggested that a
epidermal development and/or pigmentation in the devel- methionine-rich diet preferably induced muscle accretion
oping feathers, and 23 proteins were associated with by sarcoplasmic over myofibrillar hypertrophy. A recent
pigment-containing organellesdmelanosomes. The com- study concluded that changes in the diet composition can
parison of the brown- and black-specific proteomes have large impacts on protein origins leaving the ileum by
revealed several proteins and functional networks that analyzing 160 Ross PM3 broilers fed either maize/soy-
differed in expression between the two phenotypes and that based diet as reference or the addition of 20% raw soy-
are candidates for further studies. The most pronounced meal to putatively cause changes in protein flow from the
differences were detected in immunological signaling, ileum (Cowieson et al., 2017). The raw soy-meal addition
oxidative stress, energy balance, and protein synthesis caused an increase in endogenous protein relative abun-
networks, and these differences might be responsible for dance and decrease in protein from soy and no effect on
differential feather growth and color pigmentation. A recent protein from maize. This study describes the ability of
study tried to determine the molecular mechanism behind proteomics to be used to determine changes in digestion
Columbian plumage feathers in the H line of the “Yufen I” and secretion in chickens for a more in depth understanding
commercial egg-layer (Wang et al., 2019). Both tran- of diet effects on the digestive tract. The blood plasma is an
scriptome and proteome were utilized in the analysis with extremely complex tissue that contains thousands of
209 genes and 382 proteins differentially expressed in two distinct proteins. It is also the most common tissue used in
locations. The melanogenesis pathway and melanin syn- diagnosis of disease and nutritional status. Several authors
thesis were involved via nine proteins that were differen- analyzed blood plasma protein composition to gain better
tially expressed. Melanogenesis, cardiomyocyte adrenergic, understanding of protein dynamics during chicken devel-
calcium, and cGMP-PKG were all activated pathways in opment (Huang et al., 2006) or for discovery of plasma
Columbian plumage. biomarkers that reflect nutritional conditions (Corzo et al.,
2004, 2006). Muscle meat food products derived from
4.5.3 Proteomics of performance and birds, and in particular chickens, are important sources of
essential nutrients and energy intake in the human diet.
physiology
Proteomics has an obvious potential to study a broad range
Food products derived from farm animals, birds, and fish of aspects related to the meat production including nutri-
represent a significant part of the human diet. Under- tion, muscle formation, breed differentiation, meat quality,
standing the nutrient metabolism, muscle accretion, and fat and meat contamination (Paredi et al., 2013; Schilling et al.,
deposition in food birds provides practical knowledge that 2017). Chicken strains selected for meat production show
can be used to improve feed conversion efficiency, food dramatic growth rates and accelerated accretion of the
quality, and the health and welfare of animals. Animal feed pectoralis (breast) major and minor muscles. The proteome
represents the major cost of poultry production. A balanced of the chicken pectoralis muscle has been extensively
poultry diet is reflected in optimal growth and production at profiled (Corzo et al., 2006; Zhai et al., 2012; Doherty
minimal nutrient expense. Because of the composition of et al., 2004; Teltathum and Mekchay, 2009), and another
poultry diet, where corn and soybeans are used as major study identified over 5000 unique proteins in the pectoralis
sources of energy and proteins, respectively, some amino muscle of studied birds (Zhai et al., 2012). A comple-
acids become more limited than others. Corzo et al. (2005) mentary study identified the proteome of the pipping
utilized the power of MS to understand amino acid re- muscle, which is primarily used for breaking the egg’s
quirements in chickens. Blood plasma proteome from surface during hatching (Sokale et al., 2011). A recent
chickens fed an adequate or lysine-deficient diet was study identified the potential role of mitochondria in the
analyzed to identify potential biomarkers of dietary lysine breast meat of feed efficient broilers through analysis of
 Avian proteomics Chapter | 4 37

152 differentially expressed proteins using IPA (Kong stages of retina development (Lam et al., 2006; Mizukami
et al., 2016). A complementary study involving the pro- et al., 2008; Finnegan et al., 2008). These studies identified
teogenomic analysis of breast meat in high-feed efficiency known and novel proteins that play roles in early ocular
broiler males found those birds showed enrichment of growth and neural development. The retinal dysplasia and
ribosome assembly as well as proteasomes and autophagy degeneration (rdd) chick was used as a model to identify
processes for quality control and nuclear transport and proteins that are differentially expressed during the onset of
protein translation compared to low-feed efficiency males degeneration of retina (Finnegan et al., 2010). Mangum
(Bottje et al., 2017). This study is also a basis for pheno- et al. (2005) studied the development of the first pharyngeal
typing feed efficiency from a proteogenomic perspective arch, an embryonic structure that is crucial for the forma-
and improving selection through a more well-defined tion of the face, as a model for the craniofacial defects in
phenotype. The identified proteins, 676 in all, were humans (Mangum et al., 2005). This study showed that
analyzed using the assigned Gene Ontology categories for expression of molecular chaperones, cytoskeletal proteins,
molecular function, biological process, or cellular compo- and plasma proteins associated with vascularization was
nent. This analysis revealed which protein functions and altered the most between the different stages of craniofacial
cellular activities are important for rapid development of development. Initial characterization of the zebra finch
pipping muscle during embryogenesis. retina and optic tectum, a major structure of the midbrain,
McCarthy et al. (2006) analyzed the proteomes of the proteomes have been done using the one-dimensionl PAGE
supporting stromal and B cells isolated from the chicken approach coupled with MS (Sloley et al., 2007a,b). Because
bursa of Fabricius, a unique bird organ and a common these studies were done before the complete zebra finch
experimental system for B-cell development (McCarthy genome was available, potential zebra finch proteins had to
et al., 2006). Proteins were isolated from the two major be identified by cross-species matching using the nonre-
functional cell types of bursa by a sequential detergent dundant NCBI, Ensemble, and Swissprot protein databases.
extraction procedure that increased proteome coverage and
helped to localize known and previously unknown proteins
to different cellular compartments. Functional modeling of 4.5.4 Proteomics of disease, myopathy, and
the identified proteins provided insights about signaling infection
pathways involved in programmed cell death, proliferation,
The recent explosion of animal and pathogen genomes has
and differentiation. In a similar study, van den Berg et al.
not only enabled identification of genes involved in the
(2007) applied whole organ proteomics to study frozen
etiology and pathology of diseases (such as mutant gene
spleen. To gain a better understanding of B-cell develop-
variants or virulence factors) but also has opened up the
ment in the bursa of Fabricius, Korte et al. used a quanti-
door for proteomics to probe the pathogenesis and
tative 2D PAGE approach to study bursal proteomes from
pathogenehost interactions on a global protein level. Pro-
the embryonic and posthatch developmental stages. They teomics has greatly improved understanding of diseases; it
showed that enzymes of the retinoic acid metabolism play a
has been very valuable in diagnostic marker discovery, and
crucial role in the early development of the primary avian
it has a great potential in drug discovery. Despite its great
B-cell organ (Korte et al., 2013). Similar observations were
value, disease proteomics remains to be one of the least-
done in mammals, where vitamin A plays a similarly
developed areas in avian research. Nevertheless, prote-
important role in the development of secondary lymphoid
omics has been used to study the pathogenesis, etiology,
organs (van de Pavert et al., 2009). Proteomic analysis of
and pathology of several avian (infectious) diseases. In
the Harderian gland showed that Harderian gland is a site of
addition, proteomics has been used to study various human
active mucosal immunity also due to expression of he- diseases on chicken experimental models (Andrews Kingon
matopoietic prostaglandin D synthase (Scott et al., 2005),
et al., 2013).
which is necessary for production of prostaglandin D2, the
potent activator of inflammatory responses (Serhan et al.,
4.5.4.1 Disease proteomics
2008). Several proteomic studies have used chicken em-
bryos to study embryonic development of retina (Lam The chicken is an ideal and unique animal model to probe
et al., 2006; Mizukami et al., 2008; Finnegan et al., 2008, the etiology and progression of spontaneous human
2010), face (Mangum et al., 2005), CSF (Parada et al., epithelial ovarian cancer, largely because the domestic
2005, 2006), liver (Jianzhen et al., 2007), cardiovascular chicken has a high prevalence of spontaneous ovarian
system (Bon et al., 2010), and vasculature (Soulet et al., carcinomas. EOC remains the most lethal gynecologic
2013). Lam et al. (2006), Mizukami et al. (2008), and malignancy in part because early detection and therapeutic
Finnegan et al. (2008) used 2D PAGE to catalog the most strategies have been largely unsuccessful (Kurman and
abundant proteins in young chicken retina and to identify Shih, 2010). Hawkridge et al. (2010) used this model to
those that were differentially expressed between different study the onset and progression of EOC by a large-scale
Other documents randomly have
different content
 Pero ¿no podia ser que sólo hubiese muerto su
enfermedad? ¿No podia ser que Manuel Venegas acabase
de revivir á la razon, á la justicia, á la dignidad humana, á la
vida de la conciencia?
En esta duda, el Sacerdote desistió de la idea (que tuvo
un momento) de coger una luz y entrar en la sala.
Pronto se alegró de haber sabido esperar; pues no tardó
en advertir una cosa que le pareció fausta, simbólica y de
mucho alcance, en medio de su vulgarísima sencillez, por
cuanto le trajo á la imaginacion la humilde ceremonia con
que se enciende fuego nuevo en la Iglesia la mañana del
Sábado de Gloria...
Fué el caso que Manuel dió repentinamente señales de
estar vivo y despierto, poniéndose á encender luz por medio
de eslabon, pedernal, yesca y alcrebite, al uso de aquella
época.
—Lumen Christi...—murmuró D. Trinidad, santiguándose.
Obtenido que hubo nueva luz, el jóven la aplicó á las
velas que apagara ántes, con lo que el Niño de la Bola tornó
á verse profusamente alumbrado y tan clara como de dia
toda la espaciosa habitacion.
Sentóse entónces nuestro héroe enfrente de la Imágen,
y púsose á contemplarla con honda y pacífica tristeza.—La
tempestad habia pasado, dejando en la ya sosegada
fisonomía de aquel hombre de hierro profundas é indelebles
señales.—Dijérase que habia vivido diez años en dos horas.
Sin ser viejo, ya no era jóven. Sus facciones habian tomado
aquella expresion permanente de ascética melancolía que
marca la faz de los desengañados.
 En cuanto á la triste mirada con que parecia acariciar la
Efigie del Niño Jesus, no tenía tampoco la dulzura del
consuelo. Era una mirada de tranquilo, incurable dolor,
como la que, pasados muchos años de la cruel pérdida y del
agudo padecer, posamos en el retrato de un hijo muerto, de
los padres que nos dejaron en la orfandad ó de un antiguo
amor que se llevó consigo las más bellas flores de nuestra
alma...
—¡No reza! ¡no llora!—pensó amargamente don
Trinidad, formulando á su modo las mismas ideas que
acabamos de emitir.
Y se alejó de su acechadero con mucha más inquietud
que alegría le causara al principio el ver que el jóven
contemplaba á su antiguo Patrono.
—¡No hacen las paces! (añadió luégo, expresando en
otra forma su disgusto.)—¡Y la verdad es que el pobre
Manuel está dando muestras clarísimas de querer hacerlas!
—¡Misterios de Dios! ¿Qué trabajo le costaba ahora á ese
Chiquito tender los brazos á mi ahijado, como se los tendió
antiguamente á San Antonio de Padua?—¡Nada más que
con esto saldríamos todos de apuros!
Y tornó á acercarse á la rendija de la puerta, y comenzó
á rezar fervorosamente á la primorosa Efigie, como
arengándola á realizar un milagro indudable.
—¡Nada! ¡No me hace caso! (se dijo, por último, viendo
que el Niño Jesus no pestañeaba.)—¡Sin duda no conviene!
¡Respetemos la voluntad de Dios!—Ni ¿quién soy yo,
pecador miserable, para meterme á dar consejos á las
Imágenes de mi Parroquia? ¡Si los siguiesen, yo sería el
Santo, que no ellas!—¡Haces bien, Niño mio! ¡Haces muy
bien en desobedecerme!
Manuel se habia puesto de pié entretanto.
La tristeza de su semblante era mayor que nunca. Un
profundo suspiro salió de su pecho, y pasóse ambas manos
por la frente, como para echar de su imaginacion renovadas
angustias...
Parecia un reo en capilla, la noche que precede al
suplicio.—La conformidad de la desesperacion iba
envolviéndole en su fúnebre velo...
En el fondo de la sala veíanse algunos de los grandes
cofres que habia traido de América... Manuel abrió el mayor
de ellos, y sacó una preciosa caja de madera, que puso
sobre el velador...
D. Trinidad temió que el jóven fuese á suicidarse, y se
apercibió á entrar en el aposento...
Pero tranquilizóse en seguida, al observar que lo que de
allí sacaba Manuel no eran pistolas, sino vistosísimas
alhajas, collares, pendientes, brazaletes, sortijas, alfileres...;
—un tesoro, en fin, de perlas, brillantes, esmeraldas y otras
piedras preciosas...
—¡Son las donas que pensaba ofrecer á Soledad el dia
que se casase con ella! ¡Son los regalos de boda que le
traia el desgraciado!...—pensó el Sacerdote, lleno de
conmiseracion.
Manuel fué contemplando una por una aquellas galas
póstumas, aquellas joyas sin destino, aquellos emblemas de
su infortunio...; y, ejecutando luégo la idea que sin duda le
habia movido á tan penosa operacion, comenzó á ponerle
las alhajas á la Sagrada Efigie de que era Mayordomo y á
quien estaba obligado á agasajar...
D. Trinidad Muley no pudo contener su entusiasmo y su
regocijo, y corrió de puntillas á llamar á las ancianas, para
que contemplasen aquella piadosísima escena.
Imagínese, pues, el que leyere la emocion, los
comentarios en voz baja y los dulces lloros que habria al
otro lado de la puerta, en tanto que Manuel prendia á las
ropas del Niño Jesus, ó colgaba de su cuello y de sus
brazos, los restos del naufragio de sus esperanzas...—Estas
cosas se sienten ó no se sienten; pero no se explican.
Baste decir (como resúmen de sus impresiones, palabras
y pensamientos) que todos decian en voz baja, con religioso
júbilo, y abrazándose cariñosamente:
—¡Se ha salvado! ¡Ha resuelto perdonar!—¡Dentro de
pocas horas se habrá marchado para siempre!—¡Dios lo
haga más venturoso que hasta ahora!
Miéntras D. Trinidad y las tres virtuosas ancianas
hablaban así, la pérfida Volanta (que todo lo habia visto y
oido) deslizóse por la escalera abajo como una sabandija,
sin que nadie reparara en ello, y marchóse á la calle,
cuidando de no despertar al improvisado conserje...
Ni ¿cómo habian de advertir aquel suceso los que arriba
seguian con el alma las operaciones de Manuel, cuando éste
acababa de ejecutar otro acto que ya no dejaba ni asomos
de duda acerca de sus nobles y pacíficas intenciones?
Tal fué el sublime arranque de humildad con que,
sacando del bolsillo el primoroso puñal indio que aquella
tarde habia llevado á la Procesion, lo desnudó, alzólo á la
altura de su cara, contempló su luciente hoja y rica
empuñadura, lo besó luégo, y lo colocó á los piés del Niño
Jesus...
Sin la fe ciega que D. Trinidad Muley tenía ya en la
redencion del jóven, hubiera temblado por su vida, como
temblaron las mujeres, al verlo levantar el puñal, y no
habria estorbado, como estorbó, que se precipitasen en la
sala... Y tambien fué necesaria en seguida toda la autoridad
del Sacerdote para impedir que estallasen en gritos de
santo alborozo al contemplar aquella solemne abdicacion de
la mayor soberbia que jamás cupo en corazon humano.
—¡Callad! ¡callad!... (les decia al oido el autor de tan
prodigiosa obra.) ¡Callad!... ¡Dejadlo!...—¡Dios está con él!—
¡No despertemos al demonio del orgullo, que ya duerme, y
pronto habrá muerto, en el corazon de mi buen hijo!
Manuel consideró lo que habia hecho, y su grave rostro
expresó una reflexiva y triste complacencia; pero no en
modo alguno aquella devocion activa, directa, personal, que
suponian las buenas mujeres y cuyos resplandores de
triunfo y de esperanza hubiera querido hallar D. Trinidad
Muley en los ojos del leon vencido...
—¡Eso no es fe! ¡Eso no es más que caridad! (dijo el
indocto Padre de almas, dando crédito, como siempre, á su
leal corazon.)—¡Mi obra puede quedar incompleta!—
¡Malhaya los hombres que han secado las fuentes de la
alegría en un espíritu tan bueno! ¡Miéntras Manuel no crea,
no tendrá dicha propia, y sólo gozará en ver que los demas
son venturosos!
El hijo de D. Rodrigo sacó en esto el reloj y miró la hora.
—Pero debió de hallarlo parado; pues en seguida abrió un
balcon que daba á Oriente y dominaba toda la vega; y
consultó la posicion de los astros...
Corrió entónces á la puerta del salon, y, sin abrirla, dió
dos palmadas, como llamando...
—Dejadme á mí...—murmuró D. Trinidad, haciendo
señas á las mujeres para que se alejasen.
Y penetró en el vasto aposento.
—¿Quieres algo?—preguntó dulcemente á Manuel.
Fuese modestia, fuese cansancio, fuese aquel pueril
resentimiento que el amputado guarda algunas horas al
operador que en realidad le ha salvado la vida, nuestro
jóven bajó los ojos, esquivando la mirada del Sacerdote, y
dijo rápidamente:
—Que venga Basilia.
Don Trinidad se retiró sin enojo alguno.
Basilia entró á los pocos momentos.
—¿Está ahí el arriero de Málaga?—le preguntó Manuel
con la sequedad de quien desea pronta y breve
contestacion.
—Abajo está...—respondió temblando el ama de
gobierno.
—Pues dígale que cargue todo mi equipaje y ensille mi
caballo.—Son las tres y media... Partiré á las cinco.—Que
entren por estos cofres... Pero que no me hable nadie.—
Ruegue usted á D. Trinidad, de parte mia, que tome algo y
se acueste.—Necesito estar solo.
Y, dicho esto, se salió al balcon que acababa de abrir,
donde permaneció, vuelto de espaldas al aposento,
miéntras que Basilia y Polonia, llorando silenciosamente,
sacaban los baules, y miéntras que D. Trinidad y la señá
María Josefa lloraban tambien en el próximo corredor y
dirigian desde allí fervientes acciones de gracias y tiraban
cariñosos besos á la Imágen del Niño Jesus.
Al cabo de una hora comenzó á clarear el dia...
Manuel se quitó entónces del balcon, y, cogiendo una
silla, sentóse en medio de la ya solitaria estancia, y siguió
mirando al cielo, con la resignada expectativa del héroe
condenado á muerte que ve nacer la última luz de su
existencia.
Así estuvo mucho tiempo, sumido en un éxtasis de dulce
dolor que iba hermoseando cada vez más su noble rostro...
—La fiera habia llegado á tener cara de hombre... El
hombre no tardó en tener cara de ángel.—Dijérase que su
alma habia entablado un largo coloquio con lo infinito...
Ya era enteramente de dia... Ya habian dado las cinco, y
las cinco y media...—Ya estaban listas las cargas y ensillado
el caballo...—¡Y nadie se atrevia á decírselo: nadie se
atrevia á interrumpir aquel inefable arrobamiento en que el
jóven parecia gozar anticipadamente la recompensa de su
abnegacion, el premio de su sacrificio!
Salió, al fin, el sol, y su primer rayo penetró en la sala,
bañando de fúlgida luz la plácida figura de Manuel
Venegas...
—«Soledad»...—gritó entónces el loro en el balcon,
donde lo habian dejado olvidado...
Manuel se estremeció convulsivamente al oir aquel
nombre con que el pájaro americano saludaba todos los
dias, hacía muchos años, la salida del sol, y un mundo de
recuerdos y de fallidas esperanzas reapareció ante sus ojos,
haciéndole volver del cielo á la tierra, de la eternidad al
tiempo, del olvido á la realidad...—Pero, falto ya de soberbia
para luchar con su enemiga suerte, una mortal congoja
oprimió su corazon; un desfallecimiento nunca sentido
aniquiló todo su sér; extendió los brazos como quien se
ahoga (y áun pareció que efectivamente pedia auxilio),
hasta que, por último, estalló en amargos sollozos, seguidos
de copiosísimo llanto...
Y, roto por primera vez en toda su vida el dique de las
lágrimas, desbordáronse éstas con tal ímpetu que pronto
bañaban su faz, sus manos y su agitado pecho...—Al
principio, fueron ardiente lava...; luégo, benéfica sangría y
salvador desahogo de su corazon..., y, al fin, blando rocío
que bajaba del cielo á templar la sed de su alma sin
ventura...
D. Trinidad corrió á él y lo envolvió piadosamente en su
manteo, diciéndole:
—¡Llora, llora, hijo mio! ¡llora cuanto quieras! ¡Llora en
los brazos de tu padre!
Manuel se colgó del cuello del Sacerdote y le llenó la
cara de besos, diciéndole entre dulces gemidos:
—¡Perdon! ¡Perdon!...
—¡Perdóname tú á mí!—sollozaba D. Trinidad.
Y las mujeres lloraban tambien desatadamente,
comenzando á invadir la sala, y el mismo arriero (que habia
entrado por el loro) se daba puñetazos en la cabeza,
diciendo con profunda emocion:
—¡Qué lástima de hombre! ¡Maldita sea la primera
mujer!
—¡Padre mio! ¡la adoro!—exclamaba entretanto Manuel,
incomunicado con los espectadores por el manteo de D.
Trinidad.
—¡Y yo á tí!—le respondió el Párroco, besándolo
reiteradas veces.—¿Quieres que me vaya contigo?
—No... no...—Me iré yo solo...
—Pues bien: sé muy bueno: haz muchas limosnas, y
verás qué feliz eres...—Toma... (añadió luégo en voz más
baja.) Aquí tienes esto... Llévate tu caudal... En todas partes
hay pobres...
—No... no... (le respondió Manuel al oido.) Guarde usted
eso... Y haga lo que ya tenemos hablado... En esos papeles
lo encontrará explicado todo...
—Está confesando...—dijeron las mujeres, retirándose al
corredor.
—Pero tú vivirás... Tú me escribirás esta vez...
(murmuró D. Trinidad.) ¿No es cierto?
—Sí, señor... ¡Yo viviré cuanto me sea posible!—contestó
el jóven, enjugándose las lágrimas.
Y, abrazando por última vez al Cura, se levantó y dijo:
—¡Vamos!
Entónces se le acercó Polonia, con las puntas del
delantal sobre los ojos.
—¡Perdon, Polonia!—exclamó el jóven, abrazándola.
—Anda con Dios, hijo mio... (respondió la anciana:) ¡Ya
estás curado, y puedes ser dichoso!—¡Tu enfermedad
consistia en no haber llorado nunca!
—Señor... ¡Buen viaje!—le dijo Basilia, besándole la
mano...
—¡Venga usted tambien, señá María Josefa! (gritó al
mismo tiempo D. Trinidad.)—Pero no suelte usted el niño...
—¡Hoy hay perdon para todos!
 —¡Oh!... ¡no!—pronunció Manuel, retrocediendo.
—¡Manuel, castígate! (exclamó el Sacerdote.) ¡Cuanto
más te humilles hoy, más dichoso serás mañana con el
recuerdo de este dia!—¡Arranca de tu corazon, ahora que
están blandas, las raíces de tu soberbia, á fin de que nunca
retoñen!—¡No te lleves en la conciencia ningun veneno, hoy
que la has lavado con tus lágrimas!
—¡Manuel! (dijo la señá María:) ¡Yo hubiera sido muy
dichosa en llamarme tu madre!—¡Harto lo sabe el señor
Cura!
Manuel se quitó el reloj, y se lo entregó al niño,
colgando de su cuello la larga cadena de oro de que pendia,
y pronunció estas palabras:
—¡Perdono á tu madre!...—¡Dios te haga más feliz que á
Manuel Venegas!
Y volvió la espalda, y se apartó algunos pasos, como
despidiendo á la madre y al hijo de Soledad.
La pobre abuela se alejó hecha un mar de lágrimas,
miéntras que el niño iba dando besos al reloj y sonriendo
como un ángel.
D. Trinidad siguió á Manuel al promedio de la sala, y,
señalándole al Niño Jesus, que refulgia á la luz del sol como
un ascua de oro, con tanta rica presea como adornaba su
graciosa figura, preguntóle en són de dulce ruego:
—¿Y á Éste? ¿qué le dices por despedida?
—¡Á Éste le pediria que resucitase dentro de mi corazon,
si tal milagro fuese posible!—contestó Manuel
melancólicamente.
—¡Dios querrá! (dijo el Sacerdote, levantando los ojos al
cielo.)—Las raíces de tu antigua Fe están vivas, y ya ha
comenzado á correr por ellas la savia de la regeneracion.—
Las máximas que tu padre y yo sembramos en tu corazon
de niño han vuelto á germinar esta noche bajo los auspicios
de esta Efigie del Redentor del mundo...—Debes, pues,
agradecimiento al Amigo de tu niñez, y, aunque hoy no veas
en su dulce Imágen más que una sombra, un retrato, un
recuerdo del cariño que le tuviste (y que Él no ha dejado de
tenerte); aunque todavía no haya penetrado en tu nublada
razon la nueva luz que ya ilumina las más altas cumbres de
tu espíritu..., ¡bésalo, Manuel!... (¡Nada pierdes con
besarlo!) ¡Bésalo, y verás cómo toda la soberbia que te
queda en el cerebro se desbarata en lágrimas, del propio
modo que se ha desbaratado la que tenías en el corazon!
¡Verás cómo, al poner tus labios en los descalzos piés del
Niño en cuya divinidad creian tu padre y tu madre, conoces
que estás haciendo una cosa muy santa, y vuelves á llorar
de dicha!—¿Qué te cuesta probar? ¿Por qué no te atreves á
ello?—¿No te dicen ese miedo y ese respeto, que el acto de
sumision que te propongo es de maravillosas
consecuencias?—Ven... mira... ¡Yo te daré el ejemplo, como
cuando eras chico!...—Yo lo besaré ántes que tú...—¡Así se
hace!... ¡así!—Y luégo se dice (llorando, como lloro yo):
«¡Bendito seas, Jesus crucificado! ¡Bendita sea tu Santísima
Madre! ¡Bendito sea tu Padre Celestial, que te envió á la
tierra á redimirnos!»
Manuel cerró los ojos y cayó de rodillas, como una torre
que se desploma...
De rodillas estaban tambien las dos ancianas y el
malagueño, y con fervientes oraciones daban gracias á
Dios, al ver que el jóven se abrazaba á los piés del Niño de
la Bola y los cubria de besos y de lágrimas...
De rodillas, en fin, estaba D. Trinidad Muley, á quien de
seguro hubieran abrazado gustosos en aquel momento
hasta los incrédulos más empedernidos...; ¡porque la
verdad es que en todo aquello no habia nada malo para
nadie ni para nada, y sí mucho bueno para todos y para
todo, ó nosotros no sabemos lo que es bueno ni lo que es
malo en esta miserable vida!

* * * * *

No intentaremos describir los últimos minutos que

Manuel Venegas permaneció todavía en su casa, ni los
renovados, tristísimos adioses que allí se dieron aquellos
séres de tan sencillo y tierno corazon...—Temeríamos afligir
demasiado á nuestros lectores, que, pues todavía no han
soltado esta obra en que se rinde culto á la pobreza de
espíritu, seguramente tienen la dicha de pensar y sentir
como ellos.—Preferimos, pues, salir á la Plaza, y
confundirnos con la generalidad del público, en cuya
compañía podremos ver con más frescura la solemne
marcha de Manuel Venegas y los dramáticos lances que
acontecieron con este motivo.
 VI.
MARCHA TRIUNFAL.

Hacía una mañana hermosísima, sobre todo para

aquellos felices mortales que no tuvieran fijos sus ojos en la
negrura del revuelto mar de las pasiones, sino que hubiesen
preferido salir al campo á espaciar su vista y su alma por el
sublime templo de la Naturaleza, por la pintada Tierra, llena
de prodigios, por la rutilante bóveda del Cielo, y por el
propio cielo de una conciencia suficientemente limpia para
poder reflejar las misteriosas visiones de lo Infinito...
No estaban de este humor aquel funesto lúnes, 6 de
Abril de 1840, las muchas personas que acudian á la Plaza
Mayor de la Ciudad á enterarse de los adelantos que el
dolor y la ira habian hecho durante la noche en el corazon
de Manuel Venegas y Antonio Arregui. Ni hay que decir que
el grupo en que más excitados estaban los ánimos, por
cuenta ajena, era el formado, como de costumbre, á la
puerta de la Botica, ¡terrible aduana, por donde tenía que
pasar el infortunado Niño de la Bola al marcharse del
pueblo!
Vitriolo estaba más acerbo y feroz que nunca, sin poder
callarse (aunque no dejaban de aconsejárselo sus
discípulos), y, si por acaso interrumpia sus discursos, era
para decir á los que iban á comprar medicinas:
—«¡No hay de eso!»...—ó—«¡Vuelva usted más
tarde!»—ó—«¡Dígale al enfermo que se muera; que esto
que le han mandado no sirve para nada!»
 Ello es que no se apartaba del mencionado grupo,
donde ya habia tronado largamente contra la imbecilidad de
Manuel,—«cuya casa, dijo, habia llenado de Santos y de
viejas el Cura de Santa María, á fin de separarlo del camino
de la decencia y del honor y hacerle faltar á sus famosos
juramentos.»
Luégo añadió:
—Segun mis informes, á las tres de la madrugada lo
llevaban ya de vencida, y el cuitado estaba rezando el
confiteor á los piés del Niño Jesus, despues de haberle
regalado una porcion de joyas, á ruegos de D. Trinidad, que
es una hormiguita para su Iglesia...—¡Pobre Manuel! ¡Si su
animoso padre levantase la cabeza!
El auditorio se miró, como dudando de la congruencia
de aquella invocacion, y Vitriolo, que lo advirtiese, dobló la
hoja y pasó á otro asunto.
—En cuanto al marido de Soledad (exclamó con enfático
tono), ¡hay que reconocer que es un valiente! ¡Ya vieron
ustedes lo que hizo ayer! ¡Ir, sin quitarse las espuelas, á la
Ermita de Santa Luparia, en busca del célebre maton, á
quien D. Trinidad Muley habia escondido en una especie de
escaparate!—¡Yo no dudo de que cuando sepa (como ya lo
sabrá á estas horas) que su madre política y su hijo han
pasado la noche en casa del amante de su mujer, vendrá á
pedir satisfaccion á éste y echará por tierra todas las
artimañas del fanatismo y la cobardía!
Muchas personas se apartaron muy disgustadas de
aquel energúmeno, y fuéronse en busca de otros corrillos
donde se comentasen más piadosamente las maravillosas y
ya públicas escenas ocurridas aquella noche en la antigua
Casa del Chantre... Pero Vitriolo no se desconcertó por ello,
sino que se rió de los que le dejaban, y continuó hablando
de esta manera:
—¡Por supuesto, que Antonio Arregui irá de todos modos
esta tarde á la Rifa, á recoger el guante de su rival!—Así lo
juró ayer, cuando se enteró de que el hijo de D. Rodrigo
tuvo anteanoche el atrevimiento de ir á llamar á la puerta
de su casa, estando él en la Sierra...—¡Lo sé de muy buena
tinta!—¡Por consiguiente, si el Niño de la Bola, el de las
amenazas de hace ocho años, se marcha del pueblo, sin
acudir á la palestra, tanto peor para su honra y fama!—
¡Verdad es que puede que todavía ignore nuestro pobre
paisano (y se le haria un gran favor en contárselo) que
Antonio Arregui fué ayer tarde á buscarle en són de desafío
á la Capilla de Santa Luparia!...—¡Honor es de este pueblo
que el asunto no se haga tablas de la manera indecorosa
que se propone D. Trinidad Muley! ¿Qué dirian los riojanos,
si el héroe de la Ciudad huyese de uno de ellos? ¡Dirian que
los andaluces no tenemos sangre en las venas!—Y todo
¿por qué? ¡Porque los curas han sorbido los sesos á una
especie de salvaje cargado de millones, á fin de sacarle el
dinero!—¡Digo á ustedes que me abochorno de tan groseras
supercherías!
—¡Y yo me abochorno de que usted vista el uniforme de
persona humana! (exclamó el Capitan, que habia llegado
momentos ántes.) ¡Usted es un bicho!
Vitriolo se echó á reir.
—¡No se ria usted! (añadió el veterano, temblando de
cólera) ¡Mire que hoy vengo resuelto á aplastarlo, si no deja
de corromper el aire con sus viles calumnias!
 —¡Amenazas y todo! (replicó el boticario
despreciativamente.)—¿Lo han comprado tambien á usted?
¿Le ha tocado alguna joya de las regaladas al Niño de
madera?—¡Pues me alegraré de que la disfrute!
Y le volvió la espalda, asustado de lo que acababa de
decir.
—¡Lo que me ha tocado va usted á verlo ahora mismo!
(rugió el Capitan.) ¡Tome usted! ¡en nombre del Ejército!
Y arrimó al insolente materialista un soberano puntapié
en la parte más vil de su materia propia.
El pobre ateo se llevó las manos á la parte contusa, y
huyó diciendo:
—¡Ah! ¡lo de siempre! ¡el militarismo! ¡el cesarismo! ¡la
fuerza bruta! ¡el brazo secular de la tiranía!
—No ha habido tal brazo, mi buen Papaveris... (díjole
Paco Antúnez, negándole el auxilio que fué á pedirle.) ¡La
caricia ha sido con el pié, y de las buenas!
Y se alejó de él desdeñosamente.
Este lance, que hizo reir mucho á cuantos lo
presenciaron, fué como la señal y comienzo de la gran
derrota que habia de sufrir Vitriolo aquella mañana á la
vista de todos sus discípulos.
Decímoslo, porque en tal momento comenzaron á salir
de casa de Manuel las famosas cargas de equipaje,
precedidas del arriero de Málaga,—que estaba contentísimo,
creyéndose ya, sin duda, camino de las Indias.
La emocion del público, al ver aquella prueba material
de que Manuel se iba, de que D. Trinidad habia triunfado,
de que la fiera perdonaba..., fué grandísima, al par que
noble y jubilosa, con muy escasas excepciones.
 —¡Manuel se va! (decian unos.) ¡D. Trinidad no tiene
precio! ¡Eso es lo que se llama un buen cristiano!
—¡Manuel se va! (exclamaban otros.) ¡La verdad es que
este desenlace tiene algo de prodigio!
—¡Los Venegas fueron siempre así! (expuso el viejo
buñolero de la Plaza.) ¡Parece que poseen el don particular
de entusiasmar al pueblo!—La mañana de hoy me recuerda
aquella otra en que don Rodrigo salvó los papeles de D.
Elías del incendio que nadie queria apagar...—¡Todos
aplaudimos entónces sin saber por qué..., y ya está
pasando ahora lo mismo!...—¡Miren ustedes!—La gente
llora...; los chicos bailan de contento...; las mujeres se
asoman á los balcones...—Voy á avisar á la mia...
—¡Lástima de dinero, que sale de la Ciudad! (decian al
mismo tiempo los de otro corrillo, aludiendo á las tres
voluminosas cargas.) ¡Cuidado que ahí caben onzas!
En el ínterin, Vitriolo, olvidado de su percance, como se
olvida el General de sus heridas hasta que concluye la
batalla, acercábase desesperado y medio convulso al
triunfante arriero, y le preguntaba con indecible angustia:
—¿Á qué hora se marcha su amo de usted? ¿Tardará
todavía algo? ¿Habrá tiempo de hablarle?
—¡Qué ha de haber, hombre! (respondió el malagueño,
con voz descompasada.) ¡Lo que hay en este pueblo es un
Cura que vale más que Dios!
Y, quitándose el calañés, y tremolándolo por alto,
exclamó en medio de la Plaza, con un fervor y un gracejo
indescriptibles:
—¡Caballeros! ¡Viva D. Trinidad Muley!
—¡Viva!—respondieron más de mil voces.
 Y tampoco faltó quien convidara, en el acto, á
aguardiente y buñuelos al señor Frasquito Cataduras, en
pago de «la justicia que acababa de hacer á un hijo de tan
calumniada ciudad.»
Desde aquel instante, la batalla estaba completamente
perdida para Vitriolo.—Todo el público era del Cura, aplaudia
su obra, respiraba la grata atmósfera del bien, daba su
sancion á la pacífica retirada de Manuel Venegas.
Y tal fué el momento en que nuestro héroe apareció á
caballo en la puerta de la que tan pocas horas habia sido su
casa.
Un murmullo de honda conmiseracion lanzó la apiñada
muchedumbre.
Manuel avanzaba rígido, cárdeno, silencioso, mirando al
cielo, por no mirar al mundo, y acompañado de D. Trinidad
Muley, que marchaba á pié, á su derecha, y le dirigia de vez
en cuando alguna palabra consoladora.
Era, exactísimamente, el luctuoso cuadro de un reo
marchando al patíbulo.
El gentío empezó por saludarlo grupo á grupo, segun
que iba pasando por delante de cada uno de ellos; pero al
fin acabaron descubriéndose todos de golpe, como cuando
se está en presencia de un rey.
Ocurrió entónces un incidente en que repararon muy
pocos.—La célebre Volanta trató de acercarse á Manuel
Venegas, por el lado opuesto al en que iba D. Trinidad, y
áun se vió en sus manos un papel, que pudo suponerse una
peticion de limosna.—Pero el Sacerdote, que lo observara,
pasóse con rapidez á aquel lado; y miró y habló á la indigna
vieja con tal furia, que la hizo huir y esconderse entre la
muchedumbre.
Manuel no advirtió nada, sino que prosiguió su marcha
triunfal, mudo, inmóvil, indiferente, clavado en el caballo,
como el cadáver del Cid, y ganando, como él, aquella
batalla póstuma á que no asistia su espíritu.
De este modo pasaba ya por delante de la puerta de la
botica, no sin profundo dolor de Vitriolo, que iba á
encerrarse en ella con su derrota, cuando notóse gran
agitacion al otro lado de la Plaza, y vióse que Antonio
Arregui, lívido de furor, corria primero hácia la casa en que
Venegas habia vivido, y luégo en seguimiento de él,—
indicado que le hubo álguien que aquel jinete era la
persona á quien buscaba.
Pero D. Trinidad estaba en todo; y, abandonando á
Manuel, voló al encuentro del indignado Arregui, al cual
(justo es decirlo) detenian aquella vez muchas personas
bien intencionadas, de cuyas manos iba desasiéndose á
duras penas.
Pocas palabras le habló D. Trinidad para explicarle
satisfactoriamente cómo y por qué su suegra y su hijo
habian pasado la noche en casa del indiano, y pocas
tambien para convencerle de lo extemporáneo y hasta
sacrílego del paso que queria dar, provocando á un hombre
arrepentido y valeroso, que huia del combate por creerlo
injusto, y se marchaba para siempre de su patria.
Arregui quedó absorto, al hacerse cargo de aquellas
inopinadas novedades; y, como tenía mucho y excelente
corazon, y D. Trinidad era el grande hombre que ya
conocemos, y el mudable público echaba aquel dia todo su
peso en el platillo del bien, ocurrió una cosa que de otro
modo hubiera sido incomprensible...
Pero digamos qué le habia pasado entre tanto á Manuel
Venegas.
Tan luégo como D. Trinidad se apartó de él, corrió á
reemplazarle Vitriolo, el cual tuvo la audacia de coger la
brida y parar el caballo, miéntras que alargaba la otra mano
al Niño de la Bola y le decia á media voz:
—¡Buen viaje, vecino!—¿No queria usted conocer á D.
Antonio Arregui?—¡Pues ahí detras lo tiene, luchando con el
señor Cura, que no puede ya sujetarlo!
El aborrecido nombre del marido de Soledad despertó á
Manuel de su estupor y le hizo oir las demas palabras de
Vitriolo.—Volvió, pues, rápidamente el caballo, y preguntó,
echando fuego por los ojos:
—¿Cuál? ¿Cuál es?
Y se encontró con D. Trinidad Muley, que tornaba ya en
su busca, diciéndole:
—Hijo mio: completa tu obra...—Acuérdate de lo que
hemos hablado...—Aquí tienes á D. Antonio Arregui...—Te
suplico que le pidas perdon...
Arregui estaba dos ó tres pasos más atras, altivo, digno,
dispuesto á todo, bien que no pudiendo ménos de admirar
aquella noble, hermosa y dolorida figura, que veia por
primera vez, y acaso, acaso compadeciendo tan inmerecido
infortunio.
Manuel contempló amargamente al esposo de Soledad,
y vaciló algunos instantes entre los dos abismos que volvia
á presentarle la desventura.
 Reinó, pues, en toda la Plaza un hondo silencio, preñado
de horrores.—Los segundos parecian siglos.
—¡Piensa en mí! ¡Piensa en quién eres! ¡Piensa en D.
Rodrigo Venegas! ¡Piensa en el Niño Jesus!—murmuró D.
Trinidad, levantando hácia el jóven las abiertas manos, en
ademan de plegaria.
Manuel tembló de piés á cabeza, como si, al renunciar á
su última y suprema arrogancia, renunciase tambien á la
vida, y, quitándose respetuosamente el sombrero, saludó al
hombre á quien habia jurado matar.
Arregui se descubrió casi al mismo tiempo, respondiendo
hidalga y afectuosamente á aquel saludo.
Una salva de aplausos estalló entónces entre el gentío,
miéntras que mil y mil voces ensordecian el aire gritando:
—¡Viva Manuel Venegas! ¡Viva Antonio Arregui! ¡Viva D.
Trinidad Muley! ¡Viva el Niño Jesus!
Manuel habia metido espuelas entre tanto, y
desaparecido como una exhalacion, sin que la Volanta, que
corrió detras de él, consiguiera darle alcance, ni detenerlo
con sus descompasados gritos.
 EPÍLOGO.

I.
LLEGADA DE DESAIX Á MARENGO.

De buena gana hubiéramos terminado esta obra con el

capítulo anterior...—Nada habria perdido en ello la dignidad
del género humano (en cuanto puedan representarla
personajes tan imperfectos y oscuros como Manuel Venegas
y la Dolorosa), y mucho nos lo hubiesen agradecido
nuestros lectores predilectos..., que, si no son los más
sabidos y leidos, tampoco son los de peor alma.
Pero hoy no tenemos la libertad discrecional del
novelista: hoy somos esclavos de unos hechos
desgraciadamente reales y positivos, y, por lo tanto, nos
vemos en la dura obligacion de referir aquí el trágico suceso
que llenó de luto la Ciudad aquel inolvidable dia, y que
sobrepujó á los deseos del mismo Vitriolo y á las aficiones
románticas de la forastera.
No creais, sin embargo, que la indicada catástrofe
contradijo en el fondo (ya que sí en apariencia) el saludable
concepto final que, á nuestro juicio, se desprende de lo que
llevamos narrado hasta ahora. Ántes bien le sirvió de
comprobacion inmediata, demostrando cuán en lo cierto
estuvo don Trinidad Muley al decir á Manuel Venegas, luégo
que se enteró de que habia perdido la fe religiosa (cuya
restauracion por el sentimiento apénas se habia iniciado
despues en su pobre alma):—«¡Ya serás del último que
llegue!...» esto es: ya no tendrá para tí más autoridad el
Bien que el Mal: ya elegirás entre ellos segun tus aficiones,
ó segun el estado de lucidez de tu conciencia: ya no
regulará tus actos otra Ley que la que dicten tus propios
afectos: ya no servirá de límite á tu soberbio albedrío el
angosto cauce de la obediencia: ya caerás en todos los
abismos que te atraigan...
Pero dejémonos nosotros de estas filosofías ó teologías,
cuyo esclarecimiento no nos incumbe; y, reduciéndonos al
humilde oficio de narradores de hechos consumados,
volvamos á aquella plaza de la ciudad moruna, de donde
acababa de salir para su voluntario destierro nuestro inculto
y apasionado protagonista.
Poquísima gente quedaba ya en ella. Antonio Arregui,
cuya austeridad de carácter conocemos, no habia tardado
en alejarse de aquel sitio, rehuyendo conversaciones
ociosas ó dañinas. D. Trinidad Muley habia hecho lo propio,
anunciando que iba á meterse en la cama, pues con tantas
fatigas y emociones, aumentadas por el dolor de ver partir
para siempre á su adorado Manuel, sentíase muy mal y
creia que estaba amenazado de un tabardillo. El
septuagenario Capitan le dió el brazo y se marchó con él,
jurando no volver más á la puerta de la Botica... Y, con todo
esto, se disolvió el concurso, y cada cual tornó á sus
quehaceres ordinarios, despidiéndose unos de otros «hasta
la tarde, en la Rifa», no obstante el escaso interes que ya
les ofrecia la fiesta.
 En cuanto á Vitriolo, cualquiera habria dicho que una
especie de vértigo le dominaba, pues no hacía más que dar
vueltas y vueltas en la trasbotica, mirando al suelo, como si
invocase al infierno, miéntras que sus labios proferian
imprecaciones tan espantosas y repugnantes contra
Soledad, contra Antonio, contra Manuel, contra el Capitan y
contra el Cura, que, de todos sus discípulos, solamente uno
le seguia fiel y le acompañaba.—Los demas se habian
marchado en pos del ideólogo Paco Antúnez, proclamando
que no querian servir de juguete á viles pasiones; que ellos
eran incrédulos, pero no criminales, y que harto claro veian
que el desalmado farmacéutico, más que adversario de la fe
en Dios, era enemigo de la especie humana, y muy
particularmente de aquellos individuos que se interponian
entre él y la Dolorosa, por la cual continuaba sintiendo
todos los furores del amor, de la desesperacion y de la
impotencia.
Al único discípulo que permanecia fiel á Vitriolo lo
conocemos ya moralmente, por un conato de fechoría que
estorbó la tarde ántes el Capitan retirado, echándole mano
al pescuezo en la calle de Santa Luparia.—«Filemon» se
llamaba aquel celoso voluntario de la maldad, cuyo nombre
ha conservado la Historia por el odioso papel que al cabo
logró representar este otro dia, no habiendo conservado
tambien su apellido, como el de Drouet, por la sencillísima
razon de que era expósito.
—¡Cálmate, Vitriolo! (decia Filemon á su maestro.) ¡Yo
no te abandonaré jamás, como esos traidores que se han
ido con Paco Antúnez! ¡Yo tengo tambien en el alma mucha
amargura que escupir al mundo, y te seré fiel hasta la
muerte!
—¿Qué me importa? (chilló el miserable, llorando... no
lágrimas, sino verdadero vitriolo.) ¿Crees que mi furor es
porque esos necios me han abandonado? ¿De qué me
estarian sirviendo ahora? ¿De qué puede servirme ya nadie?
¿De qué me sirve la vida?
En este momento llamaron al mostrador.
Filemon se asomó á ver quién era, y dijo á Vitriolo:
—Sal á despachar.
—¡No despacho!—respondió el farmacéutico.
—¡Mira que es la Volanta!...
—¡Ah! ¡la Volanta! ¡Que éntre! ¡Que éntre!—¡Es el último
recurso que me queda!
La bruja entró jadeante, sin aliento, bañada en sudor, y
se dejó caer en una silla. En sus verdes ojos relucia tanta
perversidad en accion, que Vitriolo columbró un rayo de
esperanza.—Dióle, pues, á falta de aguardiente, un poco de
espíritu de vino con agua y jarabe, y le dijo, en són y estilo
de cómitre:
—¡Vamos pronto! ¡Desembucha!—¡Tú tienes algo que
contarme!
La Volanta miró á Filemon, como si le estorbase su
presencia.
—¡Descuida! (añadió Vitriolo.) Este es de los buenos, y
podrá ayudarnos, si hay algo que hacer.—Conque ¡habla!
—¡Deja que pueda respirar!... (resolló al fin la vieja.)—
Vengo reventada de correr detras de ese demonio..., y es lo
peor que no he conseguido que oiga mis gritos.
—¿De quién se trata?
 —¿De quién se ha de tratar?—¡Del Niño de la Bola!
—¡Cómo! ¿Tú deseabas hablarle? ¿Tenías acaso algo
que decirle? ¿De parte de quién?
—¡Conque no has observado nada! ¡Conque no me viste
cuando me acerqué á él y se atravesó el Cura!...—¡Me
alegro! ¡Así te cojo más de nuevas, y me pagarás mejor mi
secreto!
—¿Qué secreto?—¡Dímelo pronto, ruin hechicera, ó te
estrujo hasta sacártelo!
—¡Así me gusta á mí la gente! ¡Con entrañas!—Dáme
otro poco de esa bebida, que está buena...—Pues, señor,
recordarás que esta madrugada me fuí de acá cerca de las
cuatro (despues de referirte lo que ocurria en casa de
Manuel), á contárselo á Soledad, que me aguardaba para
salir de dudas acerca de si se iba ó no se iba hoy del pueblo
su antiguo amante, y á enterar de camino á Antonio Arregui
(por consejo tuyo) de que su suegra y su hijo estaban
pasando la noche en casa de Manuel Venegas...
—Bien ¿y qué?—¡No me desesperes!
—¡Vamos despacio; que no soy costal!—Llegué á casa
de la Dolorosa, que lo tenía todo preparado para que me
abrieran la puerta sin que lo notase su marido...—(¡Una vez
dentro, no habia cuidado; pues, como duermo allí muchas
noches, mi presencia en la casa no podia chocar á nadie!)—
El bueno de Antonio no se habia desnudado, y estaba
abajo, en su despacho, paseándose como un basilisco, á
causa de haber recibido á prima noche contestaciones muy
agrias de su mujer (que, como sabes, lo domina
completamente) sobre si ésta habia llorado ó no habia
llorado en la Procesion...—Es decir, que, por medio de
aquella pelea, habia conseguido la muy pícara lo que
deseaba, que era desterrar al pobre marido de la cama de
matrimonio, á fin de esperarme sola...,—y, con este mismo
objeto, habia hecho que la madre se llevase á su casa el
niño, diciéndole que aquel era el mejor modo de
destetarlo...
—¡Acaba, con cinco mil demonios!
—¡Allá voy, hombre! ¡allá voy!—Pues, señor: encontré á
doña Dulcinea metida en la cama, con muchos encajes y
moños, como de costumbre (pues es presumida y orgullosa
hasta cuando duerme), y con dos ojos abiertos como los de
una lechuza, aguardando las noticias que yo debia darle
sobre su adorado tormento.—¡Siempre te dije que la
Dolorosa no habia nacido para mujer de bien!—¡Es hija de
Caifás, y basta!—¡La triste comida que me da, en cambio de
las fincas que me robó su padre, tengo que tragármela
revuelta con mil burlas é insultos por mi aficion á beber una
gota de lo blanco, y, desde que no vive con su madre, la
mayor parte de los domingos se queda sin misa!...
—¡Lo mismo haces tú, y las dos haceis bien! (exclamó
Vitriolo.)—¡Vamos adelante; que estoy consumiéndome de
impaciencia!
—Pues atiende, que ahora entra lo bueno.—«¡Ay, Lucía!
¡cuánto has tardado! (me dijo al verme.) ¿Se va el pobre
Manuel? ¿Lo ha convencido el Cura?»—Ahora mismo acaba
de convencerlo... (le respondí), y creo que se marchará hoy
por la mañana.—«¡Hoy por la mañana! (gritó, hecha una
loca.) ¡Eso no puede ser!... ¡Tú no sabes lo que te dices»!...
—Contéle entónces todo lo que habia presenciado en casa
del Chantre, y, segun yo le iba hablando, ella se ponia unas