Poultry Science: The Many Faces of Chemistry in Poultry Production and Processing 9783110683714, 9783110683912, 9783110684094

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Katarzyna Stadnicka, Aleksandra Dunisławska and Bartosz Tylkowski (Eds.) Poultry Science

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Food Contamination by Packaging Migration of Chemicals from Food Contact Materials Ana Rodríguez Bernaldo de Quirós, Antía Lestido Cardama, Raquel Sendón and Verónica García Ibarra,  ISBN ----, e-ISBN ----

Poultry Science The Many Faces of Chemistry in Poultry Production and Processing Edited by Katarzyna Stadnicka, Aleksandra Dunisławska and Bartosz Tylkowski

Editors Dr. Katarzyna Stadnicka Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń Faculty of Health Sciences Łukasiewicza 1 85-821 Bydgoszcz Poland [email protected] Dr. Aleksandra Dunisławska UTP University of Science and Technolog Faculty of Animal Breeding and Biology Dpt of Animal Biotechnology and Genetics Mazowiecka Str 28 85-084 Bydgoszcz Poland [email protected] Dr. Bartosz Tylkowski Eurecat, Centre Tecnològic de Catalunya Campus Sescelades Carrer de Marcel-li Domingo 2 43007 Tarragona Spain [email protected]

ISBN 978-3-11-068371-4 e-ISBN (PDF) 978-3-11-068391-2 e-ISBN (EPUB) 978-3-11-068409-4 Library of Congress Control Number: 2023932791 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the internet at http://dnb.dnb.de. © 2023 Walter de Gruyter GmbH, Berlin/Boston Cover image: intararit / iStock / Getty Images Plus Typesetting: TNQ Technologies Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Preface According to global surveys, the poultry products (meat and eggs) are heading to become top animal protein sources, globally. This book has been prepared by invited, acknowledged experts from leading academic institutions and the industry, providing a holistic, interdisciplinary overview of the selected key aspects related to poultry science. The authors intention was to focus on the current, practical issues related to poultry production and meat processing, and provide the opinions on advancements in scientifically based solutions. Contributors to this book represent several disciplines (health sciences, zootechnics and fisheries, veterinary sciences, chemical sciences, biological sciences, food technology and human nutrition), as well as the industry sector. Therefore, it is a unique compilation of carefully arranged, original chapters that may serve as a guide to the current challenges that food producers must face in the context of globalization of production, Green Deal strategy and health and welfare aspects. The first chapter provides the readers with a comprehensive understanding of poultry nutrition, the chemistry of poultry feed and practical aspects related with feed formulation. Chapter no. 2 focuses on the key challenges on use of antibiotics in avian care, when it comes to face up the antimicrobial resistance and use of veterinary medicinal products in the light of regulations aiming to protect the human (and animal) health. Chapter no. 3 gives an outlook on poultry health in the context of the One Health concept. It is a follow up to discussion on antibiotics, featuring the foremost elements that make up the immunological status of the avian flock and shape the so called resilient phenotype of the animal in a production environment. It also identifies major poultry pathogens that may cause zoonoses, as well as introduces to natural bioasecuration measures to oppose these threats. Due to the intensification of food production methods, an increase in the importance of analytical chemistry can be observed. Therefore, the authors of Chapter 4 propose an overview of analytical chemistry in food production and processing. In Chapter 5, the authors with expertise in genetics, give their standpoint on ecological footprint of poultry production and effect of environment on poultry genes (therefore, introducing to the epigenetics science). In Chapter no. 6, an introduction to the prospects of genetically edited poultry is offered. The genetically engineered chickens have not only be adopted as a model for numerous study fields, but bring a promise as bioreactors producing therapeutic proteins to unsupported rare diseases and those molecules, that are too large for synthesis capacity in conventional cell bioreactors. The next, Chapter no. 7 is an attempt of a brief review on emerging, industrially applicable so called in ovo techniques. It provides an insight to various innovative solutions from sexing in ovo to in ovo stimulation, vaccination, and feeding. In the second part, the use of embryonated egg as a preclinical model is highlighted, describing development of xenografts of cancer to embryo towards personalized medicine, and utilization of CAM toxicological

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tests. The final Chapter no. 8 defines major problems related with meat (and product) quality in contemporary poultry production. It is an educational and opinion giving material, with original figures from the expert in food product assessment. We would like to express our gratitude and appreciation to the contributing authors, who allowed this project become a success. We particularly appreciate the exceptional support from Stella Müller DeGruyter Publisher, Germany, for Her assistance and editorial patience in this project. We dedicate the book to scientific Mentors, who teach the art of Collaboration, and the quality of science, with no borders. We believe, that the book will satisfy You, the Reader, as a source of knowledge and educational material on “multi faces” of poultry science. Katarzyna Stadnicka Aleksandra Dunisławska Bartosz Tylkowski

Contents Preface V List of contributing authors

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Marcin Barszcz, Anna Tuśnio and Marcin Taciak 1 1 Poultry nutrition 1 1.1 Introduction 3 1.2 Gastrointestinal tract physiology in poultry 1.3 Composition and activity of intestinal microbiota in poultry 10 1.4 Nutrient requirements in poultry 10 1.4.1 Broiler chickens 13 1.4.2 Laying hens 14 1.4.3 Turkeys 15 1.4.4 Geese 17 1.4.5 Ducks 18 1.5 Feedstuffs for poultry 18 1.5.1 Cereals 21 1.5.2 Legumes 26 1.5.3 By-products 28 1.5.4 Feed additives 30 1.5.5 Genetically modified organisms 30 References

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Adam Lepczyński, Agnieszka Herosimczyk, Mateusz Bucław and Michalina Adaszyńska-Skwirzyńska 2 Antibiotics in avian care and husbandry-status and alternative 41 antimicrobials 41 2.1 From antibiotics to antibiotic growth promoters 42 2.1.1 Mechanism of AGP action 43 2.1.2 Selected antibiotic stimulants applied in poultry production 44 2.1.3 Phenomenon of bacterial antibiotic resistance 45 2.1.4 Introduction of AGP ban the current global status 2.1.5 Impact of AGP ban and problems associated with antibiotics used in 46 animal production 47 2.2 Probiotics 47 2.2.1 General definition and characteristics 2.2.2 Intestinal mucosa colonization ability and modulation of intestinal 48 microbiota composition by probiotic bacteria 2.2.3 Modification of organic acid concentration in the poultry digestive tract 49 content

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2.2.4 2.2.5 2.2.6 2.2.7 2.3 2.3.1 2.3.2

2.3.3 2.4 2.4.1 2.4.2 2.4.3 2.5

Contents

Effect of probiotics on gastrointestinal tract architecture, digestibility of 50 feed components and their bioavailability 53 Probiotics and intestinal barrier impermeability 54 Impact of probiotics on production traits 56 Effect of probiotics on birds immune status 56 Prebiotics General definition and characteristics of prebiotics commonly used in 56 poultry feed Effect of dietary non-digestible oligosaccharides (NDOs) on productive performance of broiler chicken, nutrients digestibility and meat quality 59 traits 70 Effects of dietary NDOs on overall gut health of broiler chickens 74 Phytobiotics 74 General description 75 Antimicrobial and antiparasitic activity 78 Antioxidant activity 79 Conclusions 80 References

Aleksandra Dunislawska, Elżbieta Pietrzak, Aleksandra Bełdowska and Maria Siwek 3 Health in poultry- immunity and microbiome with regard to a concept of one 95 health 95 3.1 OneHealth concept 3.2 The microbiome of gastrointestinal tract – how it is developed and its 96 role 100 3.3 Immune status of the flock and ways to control it 104 3.4 Overview of immune system in poultry species 106 3.5 Major poultry pathogens that may because zoonoses 107 3.6 Chlamydia psittaci 108 3.7 Salmonella spp. 108 3.8 Campylobacter jejuni 109 3.9 Yersinia spp 109 3.10 Escherichia coli 109 3.11 Erysipelothrix rhusiopathiae 109 3.12 Listeria monocytogenes 110 References Przemysław Kosobucki, Waldemar Studziński and Sanling Zuo 115 4 The role of analytical chemistry in poultry science 120 4.1 Physicochemical analysis 120 4.2 Pollution and residues tests

Contents

4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12

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Residues of veterinary medicines 121 121 Residues of plant protection products 122 Dioxins and polychlorinated biphenyls 122 Mycotoxins 123 Polyaromatic hydrocarbons 123 Heavy metals 123 Nuturion determination 124 Vitamins 124 Feed additives 124 Final remarks 125 References

Ramesha Wishna Kadawarage, Aleksandra Dunislawska and Maria Siwek 5 Ecological footprint of poultry production and effect of environment on 127 poultry genes 127 5.1 Introduction 128 5.1.1 Environmental effects on poultry 129 5.2 Pre-hatch environmental effects 129 5.2.1 Effects of maternal environment 132 5.2.2 Effects of incubator environment 133 5.3 Post-hatch environmental effects 135 5.4 Ecological perspective and pro-ecological measures 144 References Mariam Ibrahim and Katarzyna Stadnicka 151 6 The science of genetically modified poultry 151 6.1 Introduction 152 6.2 Genome editing and genetically modified poultry 153 6.3 Avian bioreactors 157 6.4 Genome engineering of chicken primordial germ cells 159 6.5 Programmable genome editing systems 161 6.6 Delivery approaches for therapeutic genome editing 163 6.7 Future outlook 164 References Akhavan Niloofar, Bednarczyk Marek, Krajewski Krzysztof and Stadnicka Katarzyna 7 Emerging in ovo technologies in poultry production and the re-discovered 169 chicken model in preclinical research 7.1 Emerging industrial in ovo applications during embryonic development 170 and outlook on their implementation 172 7.1.1 In ovo technologies to determine the sex pre-hatch

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7.1.2 7.1.3 7.1.4 7.2 7.3 7.3.1 7.3.2 7.3.3

Contents

In ovo vaccination 173 175 In ovo feeding 175 In ovo modulation of immunity through gut health stimulation 177 Microbiome and microbiota in the chicken embryo 178 Chicken model in human studies Embryonic chorioallantoic membrane (CAM) to asses pharmacokinetics, 179 pharmacodynamics and systemic toxicity Chicken CAM as onco-immunological embryonic model and towards 179 personalized oncology 180 Germline cells, and reproductive tissue in cancer research 182 References

Joanna Bogucka and Katarzyna Stadnicka 8 Quality of poultry meat- the practical issues and knowledge based 185 solutions 8.1 Major issues related with poultry meat quality, including myopathies and 186 other defects 196 8.2 Research and innovations to improve poultry meat quality 8.3 The quality aspects of raw materials and processed meat, including the chemical additives incorporated to the meat. Opinion – what needs to be 197 done? 201 References Index

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List of contributing authors Michalina Adaszyńska-Skwirzyńska Department of Monogastric Animal Sciences West Pomeranian University of Technology Szczecin Poland

Mateusz Bucław Department of Monogastric Animal Sciences West Pomeranian University of Technology Szczecin Poland

Niloofar Akhavan Nicolaus Copernicus University in Toruń Faculty of Biology and Veterinary Sciences, Department of Microbiology Lwowska 1 87-100 Toruń Poland Email: [email protected]

Aleksandra Dunislawska Department of Animal Biotechnology and Genetics Bydgoszcz University of Science and Technology Mazowiecka 28 85-084 Bydgoszcz Poland Email: [email protected]

Marcin Barszcz Department of Animal Nutrition The Kielanowski Institute of Animal Physiology and Nutrition Polish Academy of Sciences Instytucka 3 05-110 Jabłonna Poland Email: [email protected]

Agnieszka Herosimczyk Department of Physiology Cytobiology and Proteomics West Pomeranian University of Technology Szczecin Poland

Marek Bednarczyk Bydgoszcz University of Science and Technology Faculty of Animal Breeding and Biology Mazowiecka 28 85-084 Bydgoszcz Poland Email: [email protected]

Mariam Ibrahim PBS University of Science and Technology Department of Animal Biotechnology and Genetics Mazowiecka 28 85-084 Bydgoszcz Poland Email: [email protected]

Aleksandra Bełdowska Department of Animal Biotechnology and Genetics Bydgoszcz University of Science and Technology Mazowiecka 28 85-084 Bydgoszcz Poland

Przemysław Kosobucki Department of Food Analysis and Environmental Protection Faculty of Chemical Technology and Engineering Bydgoszcz University of Science and Technology 3 Seminaryjna Street 85-326 Bydgoszcz Poland Email: [email protected]

Joanna Bogucka The Independent Research Laboratory STANLAB LLC Nakło nad Notecią Poland Email: [email protected]

Krzysztof Krajewski Vetdiagnostica Sp. z o.o Otorowo 30 86-050 Makowiska Poland Email: [email protected]

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List of contributing authors

Adam Lepczyński Department of Physiology Cytobiology and Proteomics West Pomeranian University of Technology Szczecin, Poland Email: [email protected] Elżbieta Pietrzak Department of Animal Biotechnology and Genetics Bydgoszcz University of Science and Technology Mazowiecka 28 85-084 Bydgoszcz Poland Maria Siwek Department of Animal Biotechnology and Genetics Bydgoszcz University of Science and Technology Mazowiecka 28 85-084 Bydgoszcz Poland Katarzyna Stadnicka Department of Oncology Collegium Medicum Nicolaus Copernicus University 85-821 Bydgoszcz Poland Email: [email protected] Waldemar Studziński Department of Food Analysis and Environmental Protection Faculty of Chemical Technology and Engineering Bydgoszcz University of Science and Technology 3 Seminaryjna Street 85-326 Bydgoszcz Poland

Marcin Taciak Department of Animal Nutrition The Kielanowski Institute of Animal Physiology and Nutrition Polish Academy of Sciences Instytucka 3 05-110 Jabłonna Poland Anna Tuśnio Department of Animal Nutrition The Kielanowski Institute of Animal Physiology and Nutrition Polish Academy of Sciences Instytucka 3 05-110 Jabłonna Poland Ramesha Wishna Kadawarage Department of Animal Biotechnology and Genetics Bydgoszcz University of Science and Technology Mazowiecka 28 85-084 Bydgoszcz Poland Sanling Zuo Department of Oncology Faculty of Health Sciences Collegium Medicum Nicolaus Copernicus University Łukasiewicza 1 85-821 Bydgoszcz Poland

Marcin Barszcz*, Anna Tuśnio and Marcin Taciak

1 Poultry nutrition

Abstract: Nutrition is the most important environmental factor affecting development, health status, growth performance and profitability of poultry production. Feeds for poultry constitute up to 70–75% of total production costs. Poultry nutrition differs considerably from that of other livestock, which is determined by the specific anatomy of the gastrointestinal tract. Protein, energy, fat, fiber, minerals, vitamins, and water are of basic importance for poultry nutrition and their content in feeds must cover the requirement that differ depending on the bird’s age and species. In general, feed protein must be of good value including the content of essential amino acids. Among them lysine, methionine, cysteine, threonine and tryptophan are the limiting ones. The main ingredient of poultry feeds are cereal grains, i.e. wheat and maize, which predominantly constitute an energy source because their protein content is insufficient for birds. Because of that cereals cannot be the only feed for poultry and must be combined with protein sources such as soybean or rapeseed meal, legume seeds or protein concentrates. Despite birds’ requirement for nutrients and chemical composition of feeds are well known, nutrition must face many problems. One of the most important issues is to find alternatives to antibiotic growth promoters. Keywords: feedstuffs; gastrointestinal tract; microbiota; nutrient requirements.

1.1 Introduction Nutrition is the most important environmental factor affecting development, health status, growth performance and profitability of poultry production. Feeds for poultry constitute up to 70–75% of total production costs [1]. Poultry nutrition differs considerably from that of other livestock, which is determined by the specific anatomy of the gastrointestinal tract [2]. The digestive system of birds is short which causes that an average daily feed intake is low. Therefore, the feed must be varied and contain all the necessary nutrients. Even a short-term feeding a diet that does not cover nutrient requirements impairs growth and development of young birds [3], while in older birds decreases or inhibits production [4, 5] and contributes to worsening of the health status [6–11].

*Corresponding author: Marcin Barszcz, Department of Animal Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland, E-mail: [email protected] Anna Tuśnio and Marcin Taciak, Department of Animal Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Barszcz, A. Tuśnio and M. Taciak “Poultry nutrition” Physical Sciences Reviews [Online] 2022. DOI: 10.1515/psr-2021-0122 | https://doi.org/10.1515/9783110683912-001

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Protein, energy, fat, fiber, minerals, vitamins, and water are of basic importance for poultry nutrition and their content in feeds must cover the requirement that differ depending on the bird’s age and species. In general, feed protein must be of good value including the content of essential amino acids. Among them lysine, methionine, cysteine, threonine, and tryptophan are the limiting ones. Nowadays, plant feeds supplemented with crystalline amino acids are the main source of amino acids. In poultry diets, the metabolizable energy to protein ratio is very important. An appropriate energy concentration in diets is ensured by carbohydrates, fat, and protein. Dietary fiber is a feed constituent which importance in poultry nutrition has increased considerably in recent years [12]. The reason for that is the increasing knowledge about the composition and activity of the intestinal microbiota which produces numerous bioactive compounds during fiber fermentation [13–18]. Ducks and geese better utilize dietary fiber than chickens, and geese better than ducks. This is because of that geese have an efficient and powerful proventriculus and gizzard and an effective microbial fermentation occur in their large intestine [19]. Minerals and vitamins are necessary for the regulation of metabolism and ensure a proper development of young organisms and birds’ productivity [20, 21]. Water is another essential constituent in poultry nutrition. Requirement for water depends on: species, body weight, age, physiological state, productivity, external temperature, and rearing system [22]. The main ingredient of poultry feeds are cereal grains, i.e. wheat and maize, which predominantly constitute an energy source because their protein content is insufficient for birds. Because of that cereals cannot be the only feed for poultry and must be combined with protein sources such as soybean or rapeseed meal, legume seeds or protein concentrates. Animal protein sources are also available for poultry nutrition and include fish meal, blood meal or feather meal. Forages are mainly used in ducks and geese feeding but their use depend on the production system [23, 24]. Poultry feeds also contain fat sources such as plant oils or animal fat, mineral–vitamin mixes, and a wide range of feed additives including enzymes, pre- and probiotics, antioxidants, etc. Usually, all these ingredients are included in one balanced feed, which composition is adjusted to the bird species, type of production, growth phase, metabolic rate, as well as anatomy and physiology of the digestive tract [25–29]. This is the most convenient method of poultry feeding ensuring the coverage of nutrient requirement and high productivity. Despite birds’ requirement for nutrients and chemical composition of feeds are well known, nutrition must face many problems. One of the most important issue is to find alternatives to antibiotic growth promoters which will ensure intestinal microbiota balance without the risk of development of antibiotic resistance in bacteria species [30]. Another one is the decreasing consumer acceptance for products originating from birds fed diets containing the genetically modified crops. Owing to consumer concerns as well as increasing prices of soybean meal, alternative protein sources must be taken into consideration [27, 31, 32]. However, the use

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of diets without or with reduced content of soybean meal requires studies on the effect on growth performance, gastrointestinal tract physiology and production profitability. Modern poultry nutrition also must ensure birds’ welfare as well as reduction of the negative impact of production on the environment. Improvement of nitrogen and phosphorus utilization by birds and reduction of emission of ammonia, hydrogen sulfide, and other odorous compounds are of particular importance [33–35]. Nowadays, climate warming and the resulting heat stress are becoming a growing problem [28]. Therefore, modern approach in poultry nutrition must include solutions for alleviation of the negative impact of the heat stress on birds’ physiology and productivity. There are many effective feed additives that may counteract and prevent some of these problems [36]. Also, plant breeders have obtained modern cultivars of legumes that may partially replace soybean meal in poultry diets. In this chapter, anatomical and physiological bases of poultry nutrition, recent advances in the field of intestinal microbiota research, feed characteristics, and the most important directions of feeding of different bird species are given.

1.2 Gastrointestinal tract physiology in poultry The digestive tract of poultry is important in converting the feed that is eaten into the nutrients which are necessary for growth, maintenance, and reproduction. Feed is broken in the body by mechanical and chemical processes. In many animals, the mechanical action involves chewing but due to the fact that birds do not have teeth, their bodies use other mechanical action. The chemical action involves digestive enzymes and fluids which are released from various parts of the digestive system. After being released from feed during digestion, nutrients are absorbed and distributed throughout the bird’s body. The digestive tract of chickens and most other birds consists of several sections which differ in morphological structure and function. The first part is the mouth, which, as compared to the mammalian organ, has no lips, cheeks, teeth, masseter muscles, and jaws but there is an organ called the beak. The hen’s beak is slightly curved and pointed at the apex of its upper part. It is a prehensile organ. The hen’s palate is the arch of the beak cavity and is not completely separate from the nasal cavity. The gap in it ensures communication between the beak and the nasal cavity. The beak cavity in the back goes directly into the esophagus. Chickens and most birds have no soft palate. Both the palate and the entire oral cavity are lined by a multilayered flat epithelium, in which branched tubular glands are located. The lack of complete separation of the oral cavity from the nasal cavity, as well as the lack of a soft palate, causes chickens and other species of birds to pour liquids collected into the mouth and into the esophagus by lifting the head up. The hen’s tongue is weakly muscled; its shape follows the shape of the beak, in which it fits completely. The front part is covered with horny epithelium, the middle part is covered with

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transversely arranged lingual papillae, and behind them lies the mucosa equipped with numerous salivary glands. The well-developed salivary glands of the chicken form an almost continuous layer in the wall of the mouth and pharynx. There are maxillary, palatine, rostral lingual, and rostral submandibular glands that open into the mouth cavity of a chicken. The mouth contains salivary glands that add a watery discharge containing mucus to moisten the feed to make it easier to swallow. Mucus is secreted throughout the entire gastrointestinal tract, it contains complex carbohydrates. In addition, in the saliva, enzymes such as amylase are released which starts the digestive process [37]. An adult hen produces between 16 and 30 mL of saliva per day. There is also evidence of antimicrobial activity in this part of the gastrointestinal tract, since the expression of antimicrobial peptides was detected on the tongue, including β-defensin 3 in chicken (AvBD3, gallinacin-3; [38]) and β-defensin 6 in duck end chicken (AvBD6, gallinacin-6; [39]). Additionally, Langerhans cells are present on the chicken tongue, which take a part in antigen presenting process [40]. The hen’s throat is wide enough to facilitate taking larger bites of feed. In the pharyngeal mucosa, there are the salivary glands but there are no lymphatic structures in the form of lymph nodes and tonsils, which can be found in the throat of mammals. The esophagus is the second segment of the bird’s digestive tract. Its length corresponds to the length of the neck. However, in a hen, it is a little shorter than the neck. It begins with the pharyngeal threshold, expanding in the lower section, forming a crop. Behind the crop, the esophagus passes over the heart and between the lungs, and ends at the entrance to the proventriculus. The esophagus is composed of the muscular, submucosal and mucosal layers. During embryonic development the mucosa of the esophagus consists of ciliated epithelium which is replaced by stratified epithelium after hatching [41]. In the layer of these cells also Langerhans cells are present [40]. During embryonic development, endocrine cells showing immunoreactivity against chromogranin A, chromogranin B, neurotensin, bombesin, and serotonin are observed in the esophagus. It is not known to what extent or if these compounds are secreted but it is suspected that they may play a role in development. These cells disappear after hatching [42]. Esophagus secrets mucus in which antimicrobial peptides (gallinacin-3 and gallinacin-6), produced by the chicken esophageal mucosa, are present [38, 43]. Esophagus shows peristaltic movement resulting from muscular contractions of the circular smooth muscles, which are stimulated by the parasympathetic nervous system. Contraction of the smooth muscle of the esophagus is also controlled by neuropeptides such as ghrelin, which is known to stimulate contractions of this part of the gastrointestinal tract in chicken and Japanese quail [44, 45]. The crop is in the form of a distensible sac. Main function of this organ is fermentation rather than digestion but some digestive enzymes are also present. During the time when ingesta are stored in the crop, digestion of specific dietary

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components can occur, mainly due to dietary supplements, such as amylase, protease, phytase, and xylanase. In addition, some endogenous enzyme activities may take place such as β-glucanase originating from the barley based feeds [46]. The rate of passage of ingesta through the gastrointestinal tract is strongly related to crop function [47]. Particle size of the diet affects the entrance of ingesta into the crop. Generally larger particles are favored in entering the crop [48]. The chemical composition of the diet also affects the rate of crop emptying. Feeding of phenylalanine or tyrosine over supplemented diets delayed or increased the rate of crop emptying, respectively [49]. Dietary medium-chain triacylglycerols delay feed passage from the crop as compared to long-chain triacylglycerols [50]. Crop emptying is also delayed by the ketone bodies [51]. Evidence for specificity of crop emptying as a function of dietary variability is supported by a soybean trypsin inhibitor that has no effect on crop emptying [52]. There is evidence that gut peptides and nerves are influencing crop emptying. Crop emptying is reduced after peripheral administration of neuromedin C and bombesin but not of neuromedin B [53]. It is not known if these effects are mediated by gastrin-releasing peptide or bombesin receptors. Similarly, ghrelin stimulates crop contraction [45]. Lactic acid is the main product of fermentation in the chicken’s [54, 55] and the turkey’s crop [56]. Fermentation in the crop leads also to production of acetic, propionic and butyric acids [57]. These carboxylic acids lower pH of the crop ingesta. There are speculations [58] that short-chain fatty acids such as acetate and butyrate in the crop increase growth rate of the small intestine, although the mechanism for this is still not understood. During the night, when ingesta are stored in the crop, the pH declines but when feed is withdrawn, the pH of the crop contents increases [57, 59]. In pigeons during the breeding season, the epithelium cells of the crop in both females and males fill with fat and degenerate. After falling off, they mix with the feed in the crop, creating the so-called milk-like mixture that parents use to feed their hatchlings. The stomach of the birds consists of two clearly demarcated parts. The front part, lying just behind the esophagus, is called the glandular stomach (proventriculus), while the posterior part, separated from the anterior stomach, is called the muscular stomach (gizzard). The proventriculus is baggy. Its thickened wall contains two kinds of glands in its mucosa. The larger ones, having a tubular structure, composed by oxyntic peptic cells, secrete gastric juice, while the smaller ones secrete mucus. The common orifice of enzyme-secreting ducts is a characteristic feature of birds that distinguish them from mammals, which have three types of secretory glands in the stomach. Additionally, surface cells of the proventriculus mucosa in the chicken express spasmolytic polypeptide [60], which is proposed to support repair of mucosal epithelium [61]. The major function of the proventriculus is to secrete gastric acid which is added to stomach

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lumen. The gastric acid consists of hydrochloric acid and pepsinogen, which is activated to pepsin responsible for protein digestion. Gizzard is larger and more powerful organ than proventriculus. It has dense muscular walls that reflect the organ’s responsibility for breaking up and grinding ingesta mechanically. The wall of the gizzard consists of 4 layers. First is the mucous membrane, where cuticle-producing glands are clustered in groups of 10–30. Second is the submucosa, consisting of connective tissue to create a strong connection between the mucosa and underlying tissues in order to allow effective grinding. Third is the muscle tunic, composed of 2 pairs of opposing circular muscles and fourth is the serosa. The interior surface of the gizzard is covered by a greenish or brownish carbohydrate/protein layer (koilin), which serves as a protection from the acid and proteolytic enzymes secreted by the proventriculus, and from injury during grinding of hard food items. This cuticula is secreted by the ventricular and pyloric glands. Its color, however, derives from the reflux of bile pigments from the duodenum. The koilin is continuously worn away and replaced. The pyloric region, about 0.5 cm long, connects the gizzard to the duodenum and its mucosal glands secrete mucous instead of a cuticle [62]. Besides the functions mentioned above, the poultry stomach (proventriculus/gizzard) promotes feed intake regulation through: (1) induction of satiety by vagal signals as a result of stretching and muscular activity; (2) humoral signals, by hormones like ghrelin, gastrin, and cholecystokinin, and (3) limiting feed passage from the gizzard by the pylorus [63]. Due to the nature of the hydrochloric acid and pepsinogen, enzymes secreted in the proventriculus, and their optimal activity values, pH values of broiler chicken stomach range somewhere between 1.6 and 4.5. When chickens are fed common pelleted diets, pH values range between 3 and 4 [63]. It is proved that stomach pH values depend on the physical (i.e. mash vs. pellets) and chemical characteristics of the feed (i.e. pH of feed), on the amount of feed in the organ (i.e. feed intake), and on the feed retention time [62]. The intestine of birds is divided into several sections, both morphologically and functionally different. The small intestine, the caeca and the colon are distinguished. The length of the intestine depends on the species, in hens it varies between 160 and 170 cm, in geese up to 350 cm. The chicken’s small intestine consists of three sections: duodenum, jejunum and ileum. In the intestine cross-section, one can distinguish the mucous layer consisting of villi and crypts, and the muscle layer consisting of four layers: outer longitudinal, outer circular, inner circular, and inner longitudinal [64]. Multiple digestive enzymes are produced in the small intestine i.e. sucraseisomaltase, amino-peptidase, lipase, amylase, maltase, and trypsin [65]. Mucus is also produced in this part of the digestive tract. For instance, goblet cell secretes the threonine- and proline-rich mucin 2. The addition of threonine to the diet results in increased expression of this glycoprotein in the jejunum and ileum [66].

1.2 Gastrointestinal tract physiology in poultry

7

Pancreatic fluid, which consists of multiple digestive enzymes and zymogens (amylase, chymotrypsinogen, procarboxypeptidase, lipase, and members of cationic and anionic trypsin sub-families – trypsinogen I and trypsinogen II, respectively), is secreted to the lumen of the duodenum [67, 68]. Bile produced in the liver is secreted to the duodenum via two ducts, i.e. from the left lobe directly into the duodenum via the hepatic duct, and from the right lobe to the gall bladder and then to the duodenum via cystic duct [65]. Bile contains bile acids in the form of salts (facilitating digestion of fat), bile pigments and several proteins such as IgA [65]. In chicken, two types of bile acid can be distinguished: dihydroxycholanic acid and trihydroxycholanic acid. In laying hens daily release of bile is about 190 mL. Studies on bile acid have shown its almost complete reabsorption [69]. Absorption of sugars into the enterocyte occurs via Na+-dependent glucose transporters, SGLT1 and SGLT5 for glucose and GLUT 5 for fructose. Out of enterocyte, glucose is transported by GLUT 2 [65]. In chicken, the expression of glucose transporters has been demonstrated in all three sections of the small intestine [70]. In case of fatty acids, most of them, are absorbed in the jejunum, however absorption of linoleic, stearic, and palmitic acid in the ileum is also possible [69]. Proteins undergo enzymatic digestion in the small intestine by the enzymes: trypsin, chymotrypsin, and aminopeptidase. First two must be activated from trypsinogen and chymotrypsinogen by proteolytic cleavage [65]. Proteins are absorbed in the small intestine in the form of amino acids, di- and tripeptides [71]. Based on studies performed on chickens, main site of absorption is jejunum [72], however, later studies shown relatively little difference in the expression of the peptide transporter 1 and amino acid transporters, i.e. ATBo,+, rBAT, bo,+AT, CAT1, CAT2, y+LAT2, BoAT, LAT1, y+LAT1, and EAAT3 for duodenum, jejunum and ileum [70]. The small intestine is equipped with an innate and adaptive immune system. Action of the innate immune system leads to the production of antimicrobial peptides such as β-defensins: AvBD8, AvBD10, and AvBD13 (in chicken, [73]; in ducks, [39]). Moreover, in chickens with necrotic enteritis, increased expression of cytokine TNFSF15 has been observed [73]. Gut associated lymphoid tissues (proximal and distal Payer’s patches) consist of B cells expressing IgM, IgG, or IgA [74]. Moreover, a pattern recognition receptor that recognizes bacteria lipoproteins (TLR2b) and pro-inflammatory cytokine, i.e. interleukin-6, are expressed in the ileum [75]. Similarly to mammals, there is a turnover of intestinal cells. The mucosal cells migrate from the crypt along the villus up to its top, where they die. After excretion to the intestinal lumen cells are digested. Estimated turnover times for mucosal cells in the chicken small intestine are: 111 h in duodenum, 55 h in jejunum, and 55 h in ileum [65]. In poultry, a phenomena of single peristaltic contraction occurs [76], which cause retrograde movements (reflux) of ingesta mixed with bile salts, lipase and trypsin, from the duodenum into gizzard.

8

1 Poultry nutrition

Ceca are two finger-like shape sacs in the proximal part of the colon, in adult chicken about 20 cm long [77]. Only part of the ingesta is able to enter ceca either from the ileum or from the colon, by retrograde transport. Particle size and solubility determine the possibility of passage into the ceca [48]. It is caused by the presence of villi in the proximal part of these organs. The villi decrease in height to finally disappear with the distance from ileocecal valves. Main functions of ceca are absorption of sodium, chloride and water [78], secretion/excretion of uric acid [77], and fermentation leading to formation of volatile fatty acids and vitamins [79]. There are two types of contraction of the ceca, low amplitude and high amplitude leading to mixing and evacuation of ingesta, respectively. Low amplitude contractions may be coordinated with contractions of the ileum and colon [80]. Similarly to the small intestine, also ceca is equipped with an innate and adaptive immune system. Last part of the digestive tract is a colon; its overall structure is similar to that of small intestine. Colon has many functions, one of them is water absorption. Colostomized chickens have high water intake needs [81]. Colostomy also decreases nitrogen utilization in adult male chickens on low protein diet, suggesting its important role in nitrogen metabolism [82]. In addition, sodium, chloride, potassium, and folic acid are absorbed in the colon [78, 83, 84]. The rate of passage of ingesta through the gastrointestinal tract can be measured by addition of markers to the diet which are not digestible and absorbable. In chickens and turkeys, markers first appear in the excreta 2–2.5 h after meal and most of the marker can be recovered within 24 h [85, 86]. There are many factors that can affect the passage time, for example, plant polysaccharides such as mannan and xylan which increase the viscosity of the ingesta and thereby slow transit. Supplementation of β-mannanase [87] and xylanase [88] to the diet decrease both ingesta viscosity in the small intestine and passage time.

1.3 Composition and activity of intestinal microbiota in poultry Composition of intestinal microbiota in poultry plays a role in health, development, and responses to a diet. The ceca are found to be the most important microbial ecosystem in the gastrointestinal tract. They contain one of the most diverse and abundant bacterial populations including pathogens such as Salmonella enterica and Campylobacter jejuni [13]. There are many techniques of detection and identification of intestinal microbiota but recently next generation sequencing (NGS) has been the most popular and used to determine changes in microbial ecology in response to a diet. Nowadays, Illumina sequencing predominates and is applied in research related to complex bacterial populations. In these techniques fluorescently tagged nucleotides are

1.3 Composition and activity of intestinal microbiota in poultry

9

added one by one to a DNA template. Individual templates are captured by a solid glass surface and amplified via bridge PCR [89–91]. The NGS technique coupled with an amplification of 16S rRNA gene is particularly useful and substantially contributed to the increase of knowledge in the field of intestinal microbiota. This gene contains alternating regions of sequence that are highly conserved across bacterial species [92], which enables designing PCR primers specific for these sites. This allows the amplification of regions of variability and further identification of wide range of bacteria on every taxonomic level [92–95]. The diversity of chicken gut microbiota differs depending on bird’s age, part of the gastrointestinal tract, and diet [14]. Each segment of the gastrointestinal tract has specific metabolic functions that affect the microbiota population. In the crop, bacteria content approximates 108–109 cfu/g and Lactobacillus genus predominates [14]. In the gizzard, there is a similar content of bacteria. However, the intensity of fermentation is low because of the low pH. The gizzard microbiota is mainly composed of Lactobacillus spp., Enterococcus sp., lactose-negative enterobacteria, and coliform bacteria [96]. In the duodenum, there are mainly species belonging to Clostridum, Streptococcus, Enterobacteriaceae, and Lactobacillus genera. Short digesta passage and bile secretion contributes to the lowest bacterial density in the duodenum as compared with other segments of the small intestine. According to Lu et al. [97] ileal microbiota population consist of Lactobacillus members (70%), Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). Cecal microbiota is more diverse, rich and stable than ileal [14]. During first 6 weeks of chickens life, richness and diversity increase, and shifts from Proteobacteria, Bacteroides, and Firmicutes to almost entirely Firmicutes by the third week of age [98, 99]. Within Firmicutes, Lachnospiraceae and Ruminococcaceae were found to be dominant families, while phylum Bacteroidetes was represented by Rikenellaceae, Bacteroidaceae, Porphyromonadaceae, and Prevotellaceae [16]. In turkeys, irrespective of the segment of the gastrointestinal tract and bird age, the dominating phylum was Firmicutes (ca. 84%), which was followed by Bacteroidetes (9.3%), Actinobacteria (4.1%), and Proteobacteria (1.5%). Lactobacillus, Streptococcus, and Clostridium XI predominated on a family/genus level. The cecal microbiota diversity was the highest in turkeys of all ages, i.e. from 6 to 16 week of age. The abundance of Alistipes, Anaerovorax, Bacteroides, Barnesiella, Blautia, Butyricicoccus, Campylobacter, Clostridium XIVb, Hallela, Paraprevotella, Phascolarctobacterium, Pseudoflavonifractor, Roseburia, Ruminococcus, Slackia, Subdoligranulum, Syntrophococcus, and unclassified bacteria was higher in the cecum than in the small intestine and colon [100]. Cecal microbiota of one-day old pekin ducks is dominated by the phylum Proteobacteria (77–99%) but within the next few days a shift to phylum Firmicutes occurs. This dominance extends through the rest of rearing period [101]. During the transition from Proteobacteria to Firmicutes from third to 10th day of age, there is an increase of the classes Bacilli, Clostridia, and Erysipelotrichi. In this period,

10

1 Poultry nutrition

Clostridia reach 45–78% of bacterial population. Within Clostridia, the dominant taxa were members of Lachnospiraceae and genus Blautia, Clostridiaceae and genus Clostridium and an uncharacterized genus in this family; Ruminococcaceae and genera Oscillospira and Butyricicoccus [101]. Application of the NGS in studies on the cecal microbiota in geese revealed that 80% of the sequences belonged to Bacteroidetes and Firmicutes phyla. The dominant species belonged to the taxa Bacteroides, Ruminococcaceae (uncultured), the Prevotellaceae Ga6A1 group, Faecalibacterium, Desulfovibrio, and Alistipes [17]. Intestinal microbiota interacts with the host and plays an important role in bird physiology [14]. Commensal bacteria prevent colonization of the intestinal epithelium by pathogenic bacteria through a competition for binding sites and produce variety of compounds, which are crucial for health, such as: vitamin K, vitamins B, short-chain fatty acids (SCFA), lactic acid, and bacteriocins. SCFA such as acetate, propionate, and butyrate are one of the most important bacterial metabolites of pleiotropic effect on the host. These compounds reduce the growth of pathogens by lowering digesta pH, serve as energy source for the host and regulate its metabolism, stimulate epithelial cell proliferation and blood flow, exert a trophic effect on the epithelium, increase absorption of water and sodium. Moreover, butyrate affects neutrophil function and migration, increases expression of tight junction proteins and mucins, and exerts anti-inflammatory effect [14, 102, 103]. Microbiota participates in metabolism of choline, bile acids, and nitrogenous compounds [14, 103]. Proteolytic fermentation leads to production of branched-chain fatty acids, ammonia, amines, phenolic compounds, and hydrogen sulfide. Most of these compounds are detrimental for the host [104]. On the other hand, ammonia released during proteolysis or uric acid degradation may be absorbed and used for the synthesis of amino acids, e.g. glutamine [14]. Bacteria also contribute to an improvement of blood lipid profile and induce non-pathogenic immune response, which is crucial for a proper development of the immune system. Stimulation of the immune system development includes both the intestinal epithelium and gut-associated lymphoid tissue which form a barrier between the host and microorganisms. However, microbiota competes with the host for nutrients and increase bird requirements for energy and protein necessary for mucus and secretory immunoglobulin A production. Therefore, commensal microbiota may affect growth performance of birds [14].

1.4 Nutrient requirements in poultry 1.4.1 Broiler chickens Broilers are meat type chickens that appropriate feeding enables optimal growth rate and obtaining a carcass of desirable consumer value [105]. The specificity of

1.4 Nutrient requirements in poultry

11

broiler feeding results from: a very intensive growth, high metabolic rate, relatively short gastrointestinal tract of a small volume, and short digesta passage rate. Throughout the whole rearing period broilers are fed ad libitum with a balanced feed, which ingredients should be characterized by a good value and high nutrient concentrations. Appropriate feed utilization and production profitability depends on covering the nutrient requirements which vary between breeds, lines, body weight, age, physiological state, environmental conditions, etc. Energy demands of broiler chickens depend on body weight and area, growth rate, productivity, housing temperature, and degree of feathering [106]. Feed mixture for broilers must be characterized by an appropriate ratio of metabolizable energy concentration to crude protein content because feed intake is strongly negatively correlated with the energetic value of feed. Too high energy concentration in feed may cause lower feed intake and nutrient deficiencies, particularly protein and minerals, in intensively growing chickens. On the other hand, lower caloric value of a diet increases feed intake and worsens feed utilization ratio. Relatively small volume of the digestive tract limits possibilities of increasing the feed intake, which would ensure the increase of growth rate, while the increase of caloric value of a diet may cause carcass fatness, particularly when oil or lard are used as an additional fat source in the feed mixture. Dietary fat addition increases the energetic value of feed and provides essential polyunsaturated fatty acids (PUFA), if plant oils are used as a fat source. However, the use of oil requires dietary supplementation with lecithin, vitamin E, and selenium [107–110]. Protein requirements result from a very intensive growth of chickens. Precise balancing of feed mixtures for poultry requires covering the need for all essential amino acids. The best results can be obtained by balancing the digestible amino acids [111]. Broiler requirement for amino acids is expressed as a ratio of particular amino acid to lysine which is the limiting amino acid [112]. Feeds for broilers are often supplemented with crystalline methionine, lysine, and threonine. Appropriate amino acid balance in feed is of particular importance in the intensive production system as it contributes to reduction of nitrogen emission to the environment [113]. Protein quality and quantity also affects meat characteristics [114, 115]. Dietary protein level necessary for maximum gain of meat protein in broilers is much higher than protein level ensuring maximum body weight gain. The increase of protein content in a diet to 27% may efficiently reduce fat gain [116]. Thus, feed conversion ratio and body fatness depend on both energy and protein content in feed mixture. Broiler chickens have limited ability to digest dietary fiber because of a small number of cellulolytic bacteria species in the caeca and colon. Crude fiber content in a diet must be limited but is necessary for a proper gut function and intestinal microbiota physiology. Crude fiber content between 2 and 3% improves growth parameters of broiler chickens fed low-fiber diets [117].

12

1 Poultry nutrition

The appropriate level of minerals and vitamins in a diet is necessary for growth, development, and health of broilers. Sodium and potassium requirements do not change with birds’ age, while demands for calcium, phosphorus, and trace elements is strongly associated with growth and productivity of birds. Mineral balancing in feed must include their availability and maintaining proper ratios between minerals because excessive levels of some elements may reduce availability of others. Calcium to available phosphorus ratio in broiler diet should equal 2.2:1 [118]. Mineral constituents from plants are often of poor availability; therefore, feed mixtures are supplemented with salts, phosphorus compounds, chalk, and mineral–vitamin premixes [111]. The traditional intensive rearing system includes three periods of broiler chicken feeding with diets of starter (1–14 day of life), grower (15–35 day), and finisher (36–42 day) type [119]. This ensures the adjustment of energy and protein level to bird’s requirements changing during growth and development. These changes are characterized by growing demands for metabolizable energy (ME) and decreasing protein requirement. The energy to protein ratio increases linearly from 130 in few-days old chicks to ca. 170 in six week-old birds. In the last phase of rearing feed conversion ratio is worse because of intensive deposition of both meat and adipose tissue. Therefore, finisher diet is of lower crude protein (CP) content in comparison with starter and grower diets. Additionally, it is devoid of coccidiostats due to a required waiting period. The ME concentration and CP content in these diets are as follows [119]: – starter: 13.1 MJ/kg ME and 21.3% CP, – grower: 13.4 MJ/kg ME and 20.2% CP, – finisher: 13.6 MJ/kg ME and 17.7% CP. However, in the modern programs of broiler production, there are three, four, or five feeding phases depending on the target live weight, i.e. 1.70–2.40 kg, 2.50–3.00 kg, or 3.10–3.50 kg [25]. In all programs, the first phase of feeding a starter type diet lasts from 0 to 10 day of bird’s age. Then, a grower type diet is given until 24 day of age. Subsequently, one (from 25 day to slaughter), two (from 25 to 39 day and from 40 to slaughter) or three (from 25 to 39 day, from 40 to 46 day, and from 47 day until slaughter) finisher diets are given [25]. It is also recommended to use a prestarter diet during first five days after hatching. Such diet should be of lower metabolizable energy concentration, as well as of lower fat and fiber content than a starter diet to facilitate a yolk sac resorption [120]. Components of the prestarter diet should be characterized by high nutrient digestibility, particularly amino acids. It is advantageous to supplement the prestarter with prebiotics, probiotics, and phytobiotics [112]. ME and CP contents in diets used in the five-phase feeding program are shown in Table 1.1.

1.4 Nutrient requirements in poultry

13

Table .: Metabolizable energy and crude protein content in diets for broiler chickens in the fivephase feeding program; target live weight .–. kg []. Ingredients

ME, MJ/kg CP, %

Age of birds, days –

–

–

–

>

. 

. .

. .

. .

. .

ME, metabolizable energy; CP, crude protein.

1.4.2 Laying hens Feeding of laying hens includes rearing phase (growth of hens) and laying period. During the rearing, it is of particular importance to cover protein requirements with regards to its quantity and high biological value. Feeding of growing hens is not aimed at an increase of growth rate but at reaching sexual maturity at an appropriate body weight and age planned in advance for particular laying breed, and at minimum feed intake [28]. Too intensive feeding of chickens with high protein diet accelerates sexual maturation and, in consequence, may lead to laying small eggs and low immunity of hens to disease. The excess of energy in a diet leads to fattening of chickens and delay of laying. From hatching until the 4th – 5th week of life birds are fed ad libitum a starter diet, then a grower-type diet until 10th week of life. From 11–16 weeks of age, chickens are given ad libitum a diet of reduced nutrient content but assuring appropriate development of the organism (developmental diet). From the 17th week of life hens are fed a diet with increased calcium content and slightly higher protein and amino acid levels. This diet is offered for ca. 10 days, until the flock reaches 2% laying (Table 1.2). The aim of this period is to stimulate the hen’s body via bone enhancement and calcium accumulation to prevent osteoporosis development at later age [28, 121]. Laying hens are fed ad libitum because they can adjust feed intake to energetic demands [28]. Nutrient requirements during the laying period are high due to a high

Table .: Basic nutrient requirements during rearing of laying hens []. Ingredients

ME, MJ/kg CP, g/kg Calcium, g/kg Available phosphorus, g/kg

Age of birds, weeks –

–

–

–% lay

.–.  .–. .–.

.–.  .–. .–.

.–.  .–. .–.

.–.  .–. .–.

ME, metabolizable energy; CP, crude protein.

14

1 Poultry nutrition

metabolic rate and intensive egg production. During the laying period three types of diet are used, depending on the laying efficiency that determines requirements for energy and nutrients, including calcium (Table 1.3). Table .: Recommended nutrient content for laying hens during egg production []. Ingredients

ME, MJ/kg CP, g/kg Calcium, g/kg Available phosphorus, g/kg

Laying efficiency, % >%

–%

26.7 °C), higher RH may have adverse effects on feed conversion, weight gain, feathering and pigmentation [64]. Since birds don’t have sweat glands, they mainly dissipate heat via panting [51]. However, when the environmental RH is associated with higher temperature, the efficiency of evaporative heat dissipation is reduced, and birds fail to regulate their body temperature [65] and can eventually cause death due to heat prostration [66]. Apart from this, higher RH can also cause wet litter problems affecting the health of the birds [67]. Ventilation is extremely crucial to avoid the implications of higher environmental temperature and RH. Natural or forced ventilation systems can facilitate continuous replacing of hot air with cool air to help with the heat dissipation of chickens [68]. It also facilitates removal of ammonia generated by poultry litter [69] and thus good ventilation help to prevent respiratory diseases in poultry [70]. Managing a proper ventilation system in poultry system is therefore imperative for the optimum physiology of the birds. Lighting is another important environmental effect. Major aspects of light are the day light length (duration of artificial lighting), intensity and the color (wavelength). Many researchers have checked different lighting schedules with different intensities and color of lights to assess their effects on poultry. Typically, different colors of light showed different behavioral effects in chickens such as calming effect (blue), reduction of feather pecking (red), reproduction (orange + red) and growth (green + blue) [71–73]. Apart from that light has numerous effects on health and immunity, growth, muscle development, bone health, reproduction enhancement, carcass characteristics etc (as reviewed by Abo-Al-Ela et al. [74]). Intermittent lighting showed relieving effect of the heat stress as well as growth enhancement in chickens [75]. Light is also very important

5.4 Ecological perspective and pro-ecological measures

135

for the circadian rhythm of birds and as a result it may influence the egg production in layers [76]. Therefore, light is a highly influencing and complex environmental factor for poultry production and therefore, should be carefully managed to optimize the production.

5.4 Ecological perspective and pro-ecological measures The ecological footprint measures how much nature we use comparing to how much nature we have. This approach is investigating how much biologically productive land and water area is used by each individual, population or activity to produce all necessary resources and also to absorb its waste. The ecological footprint in agriculture determines how much resources (water, nutrients, fuel, electricity) is used to obtain of a certain amount of product and how much waste generated waste is utilized. The term “ecological footprint” was developed by Mathis Wackernagel and William Rees at the University of British Columbia in 1990. The growing human population requires larger resources what leads to increased food demand. To meet the global food demand by 2050, agricultural production must be increased by 60%. The meat production needs to increase by 196 million tonnes, to reach the level of 455 million tonnes [77]. The meat products are supplied by the limited number of animal species. Cattle, poultry and pigs supply 88% of world meat production. Laying hens supply 92% egg production. The poultry production is growing rapidly past years. It accounted for 122 Mt and 37% of global meat production in 2017. It is expected to increase by 40 Mt till 2028. The egg production coming from laying hens accounted for 87 Mt in 2017 [78]. The poultry production as part of the agriculture and livestock is facing two challenges: supplying increasing food demand yet developing into direction of environmentally sustainable system. Over the past decades, due to the technological development, poultry production has reached high performance levels. Nevertheless the sustainability of this production system still needs a lot of attention [79]. However, it should be noted that amongst livestock systems, poultry production is considered to be relatively environmentally friendly, e.g. their greenhouse gas emission is at relatively low level. The negative environmental impact of the poultry production is due to nitrogen emission in form of ammonia, nitrous oxide emission and nitrate leaching. Hence, the improvement of the environmental sustainability should be considered in larger perspective than the direct farm activities such as: reduction of the emissions to the environment, reduction of the energy at the farm. The factors of direct and indirect environmental impact might have confounding effects, i.e. improvement at one part of the production chain might have harmful effect in the other. To be able to account for all possible interactions between direct and indirect factors influencing the

136

5 Ecological footprint on poultry production and environmental effects

environment a quantitative tool was developed called: life cycle assessment (LCA). LCA is used to evaluate an environmental impact in livestock production. It does take into account the entire production chain from the beginning until the end product. There are three sources of the poultry production impact on the environment: 1. Feed production, 2. Direct farm energy use, 3. Emission from housing and manure management [80]. Feeding is considered to be the major factor impacting the environment accounting for up to 82% of the overall impact on climate change [79]. Poultry production requires external feed production, especially imported protein sources which is most of the cases originated from soya. The negative impact related to fodder ingredients such as soya or palm oil is due to the fact they are produced on the land which has been converted from natural vegetation to agricultural use. Another indirect factor related to negative environmental impact of feed production is the use of fossil energy in the process of fertilizers production, field operations, and transportation [80]. The second source, the direct farm energy use is often considered as the primary target to address in the course of reducing the negative impact of the poultry production on the environment. In the poultry production the energy generated from electricity, oil or gas is used at hatcheries, breeder farms, egg production, and broiler production. The environmental impact is strictly depended on the production system. For example, the conventional indoor broiler system, using liquid propane gas for heating chicken houses has an impact on global warming potential below 15%. Global warming potential accounts for greenhouse gas emission to the atmosphere. The third source, which is emission from housing and manure management influenced euthrophication and acidification potential regardless of the production system. Applying LCA tool for poultry production showed that the feed production, including processing and transporting has the highest negative impact on the environment especially the global warming potential [80]. However the consumers and society preferences go for less intense poultry systems, i.e. free range, organic broiler production and barn, free range organic egg production, these systems have more negative environmental impact. It is due to the efficiency and the duration of the production cycle [80]. Looking 50 years back and applying an LCA tool for the retrospective analysis showed that an overall trend in term of the environmental impact of the poultry production is decreasing. It is due to improvement in animal performance by better genetics, breeding, feeding, management, housing technologies and in background process increased in the yield of feed production, better energy efficiency, lower emission related to transportation [78] (Table 5.1). Yet, there is still a room for an improvement. The mitigation interventions related to feed production has to focus on protein source and diet formulation. There are trends to replace soybean in broiler and laying hens diet with other protein crops such as: bean, pea, or sunflower. However such a replacement and formulation the diet based on other crops requires greater inclusion in the diet synthetic amino acids or vegetable

Reference Species

In-ovo CORT treatment (low dose) In-ovo CORT treatment (high dose)

.

High incubation temperature

High and low incubation temperature

High incubation temperature Low incubation temperature

.

.

.

Incubator environmental effects

.

Unpredictable ligh-dark rhythm for parents Unpredictable light rhythm for parents

.

Hypothalamus

Brain

Hypothalamus or pitutary

Organ/site

[] Chicken (cobb broiler)

[] Chicken (cobb broiler) Breast muscle

Yolk sac tissue

[] Chickens Heart (Punjab broiler ) Liver and spleen

[] Chicken (commercial layers – Hyline W) [] Chicken

[] Chicken (white leghorn)

Parent’s environmental effects and simulations

No. Environmental factor

Table .: Environmental effects at the genomic level in poultry.

HSP alpha (over-expressed) HSP alpha, HSP (underexpressed) Dynamics of PEPT, APOA, DIO, GYS, LRP, FBP, DIO genes throughout incubation period MYH, MYL, MYOG (over-expressed) and more genes affected SMAD, RUNX, MED (underexpressed) and more genes affected

-HTRA, MAO-A (over-expressed) TPH (under-expressed) GR and CRH gene promoters methylated

CRH, AVT (under-expressed)

RLX, FBXO, BDNF, MRPL, MMP, GABRR (over-expressed) and more genes over and under-expressed CQC, PER, CIRBP, CRIP, MAPKIP (over-expressed) SOUL (underexpressed)

Genomic changes

Increase of muscle growth and physiology Growth inhibition, alteration of global transcription regulation,

Variations in the yolk utilization in the embryo

Increase in heat tolerance capacity

Aggressive behavior

Reduced spatial learning ability and more competitiveness in offsprings from stressed parents Ability to cope with unpredictable environment

Main effects

5.4 Ecological perspective and pro-ecological measures

137

High incubation temperature

High incubation temperature High incubation temperature High incubation temperature

.

.

High incubation temperature . High incubation temperature . Monochromatic green light during first  days of incubation

.

.

.

High incubation temperature

.

No. Environmental factor

Table .: (continued)

Pectoral muscle

MYOD, MYOG, PAX, PCNA, IGF-, GH (over-expressed) INS, IRAK, PIKCG (over-expressed) HDAC, NFKBIA (under-expressed) FGA, FGB, FGG (over-expressed) ALB, AMBP, APOH, TRR, CELA, CLPS, CPA, CPA, CPA, CPB, CTRB, CTRC, CTRL (under-expressed) DHTKD, GOLPHL, INS, MAGI, mir, PDIA, SEMAD, SYTL (overexpressed) APOB, APOC, CQTNF, CD, CXCL, ENPP, GC, HADH,

MyoD, MyoG, Pax, PCNA, IGF-I, GH (over-expressed) myostatin (underexpressed) VEGFA, MyoG, IGF-I (over-expressed)

HKME and HKME post translational modifications in many genes

Genomic changes

Main effects

Cell and organ morphology and digestive system development and function

Improved muscle growth in post hatch broilers Immune response and energy metabolism Stimulate innate immunity upon vaccination

Improved breast muscle growth

Improved muscle growth

Neurodevelopmental, metabolic and gene regulation functions related to environmental memory Liver/heart/leg DNMT, DNMTA, DNMTB, MBD, Epigenetic regulation of embryo and breast muscle MBD genes differentially expressed in development a timely fashion Pectoral and thigh HSP, HSP, HSF- and HSF- Improved thermotolerance muscle tissue (over-expressed) Liver and blood FAS, ACC, ELOVL (over-expressed) Improved lipid metabolism in liver

Hypothalamus

Organ/site

[] Chicken (cobb Breast muscle and ross broilers) [] Chicken Pectoral muscles (cobb broiler) [] Chicken (layers) Spleen

[] Chicken (cobb broiler)

[] Chicken (ross broiler) [] Pecking ducks

[] Pecking ducks

[] Chicken (cobb broiler)

Reference Species

138 5 Ecological footprint on poultry production and environmental effects

[] Turkey (black bronze)

Reference Species

Chronic heat stress

Acute heat stress

.

.

.

Short term heat stress Long term heat stress High ambient temperature

.

[] Chicken (cobb broiler) [] Chicken (cobb broiler)

[] Chicken (hi-line layers)

[] Chicken (layer)

Post hatch environmental effects

. Lighting during first  days of incubation (red and blue)

No. Environmental factor

Table .: (continued) Genomic changes

Main effects

Pectoralis major

Illeum

Hypothalamus, duodenum and jejunum Jejunal mucosa

Pre-ovlulatory follicles

SGLT, GLUT, FABP (underexpressed) FABP, FATP, SGLT, PEPT (underexpressed) FABP, PEPT (over-expressed) FATP, SGLT (under-expressed)

GHRL and CART (over-expressed) CCK (under-expressed)

CYPA, CYPA (under-expressed)

CYPA (under-expressed)

Tissue specific changes to nutrient transport and utilization

Reduction in nutrient transportation

Reduction in feed intake

Decline of reproductive activity of ovary

INSIG, LAPTMB, PID, IVA, SYTL (under-expressed) CRik, CELA, CELAA, CLPS, Carbohydrate, lipid and protein CPA, CPA, CPA, CPB, CTRB, CTRC, metabolism CTRL, HSST, RBPJL (over-expressed) AHSG, AMBP, APOH, CRABP, FGA, RBP, SERPINC, SFTPA, SFTPA, TTR (under-expressed) Pectoralis muscle MyoG, MyoD (red light: OverEnhanced muscle growth expressed) FGF (blue light: Over-expressed)

Organ/site

5.4 Ecological perspective and pro-ecological measures

139

Chronic heat stress (continuous) Chronic heat stress (cyclic) Acute heat stress (AHS)

.

.

Acute heat stress

Chronic heat stress (CHS)

Acute heat stress

.

.

Cold stress

.

Chronic heat stress

No. Environmental factor

Table .: (continued)

Jejunal mucosa

Pectoralis major

Illeum

Organ/site

[] Chicken (cobb broiler)

Main effects

HSP (over-expressed) HSP (underexpressed) HSP (over-expressed)

SOD and HSP (over-expressed) GH (under-expressed) HS, HSF, HSF, HSF (overexpressed) HSF, HSF, HSP (over-expressed)

Organ damage via inflammation

Tissue specific antioxidation mechanisms induced by chronic and acute heat stress

Reduction in growth

FATP, GLUT, PEPT (over-expressed) FABP, PEPT, GLUT (underexpressed) GLUT, GLUT, PEPT (overexpressed) FABP, PEPT (underexpressed) OCLN, ZO-, HSF-, iNOS, AMPKα Increased energy metabolism and (over-expressed) barrier function possibly induced by the stress avUCP (under-expressed) Stimulate mitochondrial superoxide production SOD and HSP (over-expressed) Activation of antioxidant mechanisms

Genomic changes

Liver (CHS) Muscles (CHS) Liver, spleen, kid- TLR (over-expressed) ney, small intestine and heart

[] Chicken (female Heart (AHS) broiler breeders – Arbor acres) Liver (AHS) Muscle (AHS) Heart (CHS)

[] Chicken (cobb Skeletal muscle broiler) tissue [] Chicken (cobb Liver and ross broilers)

[] Chicken (arbor acres broiler)

Reference Species

140 5 Ecological footprint on poultry production and environmental effects

. Blue and green monochromatic light . Different intensities of light . Green light

. Blue color cool light

No. Environmental factor

Table .: (continued) Organ/site

[] Chicken (cobb broiler) [] Chicken (arbor acre broiler)

TPH and TH (under-expressed) CLOCK, BMAL and CRY (overexpressed)

Hypothalamus

ERα, ERβ and PR (over-expressed)

TLR and IL- (over-expressed)

Genomic changes

Brain

[] Chicken (fayoumi Spleen layers) [] Chicken (layers) Ovarian follicles

Reference Species

Regulation of the circadian rhythm

Behavioral modifications

Enhanced reproductive activity

Enhanced immunity

Main effects

5.4 Ecological perspective and pro-ecological measures

141

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5 Ecological footprint on poultry production and environmental effects

Table .: Protein content in various alterative products. Adapted from ref. []. Type

Product

Insects

Black soldier fly larvae Oxya fuscoviatta Acrida exaltata Housefly larvae Mealworm Locust Chlorella vulgaris Dunaliella salina Spirulina platensis

Algae

Protein content (%) – . . . . . –  –

oils. There are other alternative protein sources such as e.g. microalgae, worm or insects. Hence the use of these products in poultry feeding is mostly limited by current regulations [78]. Several examples of these alternatives are presented in Table 5.2. The modern poultry breeds require highly absorbable nutrients, especially highly digestible protein. Properly balanced animal feed performs building function, directly supports host intestinal microbiota and indirectly host immune system [102]. The most commonly used soybean meal has several advantages for poultry production. It has high (40%) protein content and well balanced amino acid profile. In poultry feeding several protein feeds are used: non – GMO extruded full – fat soybean, non-GMO raw full-fat soybean, extruded loose, and distiller’s dried grains. The major disadvantage is the necessity of importing of large quantities of soy feed from Brazil (57%) and USA (22%) to Europe. It does have a negative impact on the environment and it’s cost inefficient. There are attempts to replace soybean products with field peas. The advantages of such a solution are: good source of protein and starch, cost reduction, availability within EU. The disadvantage is lower crude protein and lysine content in peas comparing to soybean. It also does contain antinutritional factors like: Trypsin inhibitors, lectins, and phytate which have a negative impact on the poultry production results. The positively tested solution to decrease the level of the antinutritional ingredients is heat treatment [102]. As presented in Table 5.2, insects might serve as valuable alternative source of protein in animal diet. This protein has a nutritive value comparative to soybean meal. Insects might be grown on low value by products or organic waste from agriculture or food industry. Further on, the waste from insects rearing might be used as an organic fertilizer [103]. Insects have a higher conversion rate and are reared on much smaller surface comparing to livestock species. On the top of that their emission of GHG and ammonia per kg meat is lower comparing to most of the livestock species [103, 104]. It has been showed that replacing of soybean meal with feed based on insects has some advantages in quality of poultry products. In egg production, insects based feed

5.4 Ecological perspective and pro-ecological measures

143

increased weight and eggshell thickness. In poultry meat production, fodder based on grasshoppers improved meat taste [103]. Yet another alternative source of proteins are microalgae. The protein content of some strains of microalgae is up to 70%, and the essential amino acid profile is very similar to the one of soya bean. Microalgae contain also valuabe bioactive peptides with valuable properties such as: antioxidative, antihypertensive, anticoagulative, antitumor, and immune – stimulative. They are able to boost host immune system what might eventually support reduction of the antibiotic use in the poultry production. Hence, the microalgae have large potential also due to the number of beneficial biomolecules (vitamins, lipids, minerals). Several species: Chlorella sp., Tetraslemis sp., Dunaliella slina have confirmed prebiotic properties [105]. It has been shown that laying hens fed with fodder containing Nanochloropsis gaditana produced eggs with higher amount of ω-3 FA in the yolk, due to the high level of these fatty acids in the microalgae. The future of poultry production requires addressing two, rather opposite, demands: global need of poultry products and society awareness of animal welfare and ecological footprint. As presented in this chapter, the analysis of the restrospective data shows that an overall trend of poultry production is towards more sustainable systems. There is a general understating that global efforts must take into account improvement at each stage of the poultry breeding and poultry production. The further reduction of the ecological footprint requires engagement of the most advanced achievements in term of poultry genetics, nutrition, and health and management strategies. As presented in this chapter, the technological progress allows for better understanding of the complexity of biological processes of the chicken organism and its interaction with the environment. The detailed information about the chicken genome allows for deciphering the genetic background of the most complex traits. The epigenetic analysis grant to understand the impact of the environment on the DNA sequence. Poultry being an oviparous species give the opportunity of a controlled environmental intervention at the very early stage of embryo development. There is the well advanced in ovo technology giving the opportunities for hatching already more sustainable chickens. Some solutions for further decreasing the ecological footpring of poultry production has been already validated at the experimental scale (e.g. alternative protein sources), other solutions are to be developed. However the successful application of these developments requires wide acceptance among agriculture sector stakeholders, citizens, and farmers. These technological solutions have to be both: Societally acceptable and economically viable for poultry producers.

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Mariam Ibrahim* and Katarzyna Stadnicka

6 The science of genetically modified poultry Abstract: The exuberant development of targeted genome editing has revolutionized research on the chicken genome, generating chickens with beneficial parameters. The chicken model is a crucial experimental tool that can be utilized for drug manufacture, preclinical research, pathological observation, and other applications. In essence, tweaking the chicken’s genome has enabled the poultry industry to get more done with less, generating genetically modified chickens that lay eggs containing large amounts of lifesaving humanized drugs. The transition of gene editing from concept to practical application has been dramatically hastened by the development of programmable nucleases, bringing scientists closer than ever to the efficient producers of tomorrow’s medicines. Combining the developmental and physiological characteristics of the chicken with cutting-edge genome editing, the chicken furnishes a potent frontier that is foreseen to be actively pursued in the future. Herein we review the current and future prospects of gene editing in chickens and the contributions to the development of humanized pharmaceuticals. Keywords: bioreactor; chicken; genome editing; pharmaceuticals; primordial germ cells.

6.1 Introduction Chickens have been a focus of vertebrate embryology research for several decades as their embryos are internally fertilized and then meticulously loaded into eggs, providing easy access to developmental stages for leading manipulative experiments [1]. Thanks to various emerging technologies like in vivo electroporation, stem cell research tools, innovative methods for transgenic animals, and accessible genomic sequence resources, as well as traditional approaches like grafting and lineage tracking, the chick embryo has now grown even more robust, adding to its extensive and notable history as an important scientific model system [2]. In the last 30 years, diverse research entities have opened the doors to the development of genetically engineered chickens [3]. Adopting genetically engineered chickens as a model for numerous study fields, including behavior, developmental biology, immunology, physiology, and neurology, has lately gained prominence in the avian research community, contributing to our

*Corresponding author: Mariam Ibrahim, Department of Animal Biotechnology and Genetics, PBS University of Science and Technology, 85-084 Bydgoszcz, Poland, E-mail: [email protected]. https://orcid.org/0000-0001-6471-0237 Katarzyna Stadnicka, Department of Oncology, Collegium Medicum Nicolaus Copernicus University, 85-821 Bydgoszcz, Poland As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Ibrahim and K. Stadnicka “The science of genetically modified poultry” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/ psr-2022-0352 | https://doi.org/10.1515/9783110683912-006

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understanding of the different aspects of these sciences [2, 3]. With the advent of genome editing tools, there has been a growing scope for developing genetically engineered poultry that are impervious to certain infectious diseases [3]. Loads of research have shown that genetically modified chicken genomes hold an immense promise for boosting poultry production, providing a formidable engine for industrial protein genesis [4]. A bird that has been genetically altered is also referred to as a transgenic bird. A transgenic organism is one that has undergone genetic engineering techniques to incorporate a portion of the DNA of another species into its own. Reportedly, transgenic methods have been developed for producing transgenic birds that express recombinant human proteins in eggs, and tissue-specific genes as an avian model [4]. Implementation of effective approaches for genetically modified chicks is believed to contribute to industrial applications in livestock farming as well as to expanding our knowledge of avian biology [4]. The development of breed-specific traits in the chicken genome appears to have been heavily influenced by artificial selection [5]. Artificial selective breeding, whereby natural superior variations are chosen to generate subsequent generations of organisms with the goal of transferring the superiority to descendants, is likely to be the oldest known form of genome modification that became genetically governed after Mendel’s discovery of the law of heredity [5]. It is a testament to the effectiveness of classical artificial selection that several breeds with distinctive, stable traits have been developed and produced all over the world [5]. Selective breeding has altered the chicken’s genetic make-up for favorable phenotypes and biological features, increasing its ability to adapt to specific production environments [6]. Advances in breeding and biotechnology have fueled the efforts of research and industry to enhance animal welfare and productivity [6]. As germ line cell culture, genome editing, and transgenic technologies have flourished, it has become easier and more practical to alter the chicken genome. This has led to a recent upturn in the use of different methods for genome editing in chickens that can generate deletions, insertions, and base substitutions. For the purpose of studying the genetic circuitry, it will be extremely advantageous to create conveniently accessible genome-engineered resource lines. This review intends to outline the different facets of genetically modified poultry and how it is used to govern the synthesis of recombinant therapeutic proteins.

6.2 Genome editing and genetically modified poultry Inspired by both evolutionary and health research, the chicken was the second species of vertebrates after Homo sapiens to have its genome fully sequenced, which enabled molecular genetic research using the full power of modern genomics [7]. Genome editing is a technique by which precise alterations into an organism’s genome are introduced, in order to boost livestock production or disease resistance without introducing any undesirable traits [8, 9]. Genome editing tools include transcription activator-like

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effector nucleases (TALENs) [10], meganucleases [11], zinc finger nucleases (ZNFs) [12], and clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) [13], which are programmable site-specific nucleases. All of these genome editing tools have the ability to effectively generate precise genetic alterations at specified genomic loci via double-stranded breaks (DSBs) [14]. DSBs are naturally occurring detrimental DNA lesions that can be repaired by two common cellular machinery routes: the non-homologous end-joining (NHEJ) and the homology-directed repair (HDR) mechanisms [14]. NHEJ is the primary method by which cells mend DSBs through direct rejoining of exposed DNA ends [15]. However, this reparatory pathway is error-prone as it is frequently linked with disruptive insertion-deletion (indels) of nucleotides [15]. The HDR, on the other hand, employs homologous DNA sequences spanning the DSB as repair templates. By providing an exogenous repair template, this route can be used to accurately edit genomic sequences or incorporate foreign DNA [8, 15]. HDR process can precisely produce specified minor genetic indels as well as exact insertion of large foreign transgenes into the host cellular genome in a footprintless manner, as it generates site-specific genetic changes identical to those created by natural genetic alterations [8]. HDR has boosted transgenic research, through generating nonhuman models with more focused and exact genome modifications. All in all, unlike traditional selective breeding, which takes a long time to produce desired features as it depends on naturally existing genetic variants, genome editing technologies can swiftly insert favorable genetic changes in target genes [9]. Thus, genome editing provides a powerful and secure technique that works well in conjunction with conventional breeding to elevate production, immunity and disease resistance, develop animal models for conducting human health research, protect native species, minimize vector-borne diseases and plausibly bring extinct species back to life [16]. The advent of genome sequencing technologies and the availability of widely accessible chicken genome databases (Table 6.1) have accelerated the growth of poultry breeding through the ability to edit genetic markers linked to productivity by different genome editing systems.

6.3 Avian bioreactors Recombinant proteins has become increasingly popular in recent years for usage in industrial, medicinal, and scientific fields [17]. Cultured cells have often served as hosts for recombinant protein synthesis through ectopic expression of foreign genes [17]. Howbeit, this kind of large-scale recombinant protein generation demands costly and complicated mechanical bioreactors giving the opportunity for tailoring livestock animals as alternative bioreactors due to their potential benefits of low cost and high production volume [18]. Animal bioreactors are genetically altered animal systems that have the potential to lower manufacturing costs and increase the productivity of recombinant proteins with therapeutic relevance to conquer diseases and enhance the quality of man’s life, taking advantages of developments in medicine and

Aim Gives access to various databases, genetic information network tools, discussion forums, and other features. Enables access to comparative genomics, sequence variations, variant effect prediction, gene annotation and others. Presents an overview of the chicken genome sequencing projects. Provides a genome browser of different species including chicken enabling graphical interface and a user-friendly access method for sophisticated queries. Provides a way to access genomic data, including reference genomes. Includes BLAST facilities, gene ontology-searching, single nucleotide polymorphism (SNP) variants, RNA interference (RNAi) design tool, and other features. Contains primer-pair kits for microsatellites covering the chick linkage map for quantitative trait loci (QTL) mapping and other applications. Gives a comprehensive stock and curator list of accessible avian research stocks. Provides an SNP detector and online visualization tool for chicken research groups. Provides a public source for genomic mapping data from livestock including chicken and other animals. Integrates both NCBI and ensemble gene models and connects them to experimental gene expression data and QTL information. Brings together multidisciplinary educators and researchers who collaborate with commercial poultry farmers and small flock owners

Database ChickNET – The chicken genome information network Ensembl project Wash U. genome project UCSC chick genome browser

NCBI BBSRC ChickEST database

BACPAC genomics UCDAVIS Chicken SNP database ARKdb genome database Chickspress genome browser MSU extension poultry

Resource URL

http://www.chicken-genome.org/

http://www.ensembl.org/Gallus_gallus

http://genome.wustl.edu/projects/chicken/ http://www.genome.ucsc.edu/cgi-bin/hgGateway

http://www.ncbi.nlm.nih.gov/genome/guide/chicken http://www.chick.umist.ac.uk/

https://bacpacresources.org/

http://animalscience.ucdavis.edu/AvianResources/

https://ngdc.cncb.ac.cn/chickensd/

http://thearkdb.org/

http://geneatlas.arl.arizona.edu/

https://www.canr.msu.edu/poultry/

Table .: Some chicken genome resources tools.

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biotechnologies [19]. Many researchers have used genetically modified animal models including mammals and avians to produce desired proteins into milk, egg whites, blood, or other body fluids [19, 20]. FDA approval for the first transgenic animal-produced therapeutic protein came in 2009 [21]. Transgenic goats that produce anti-thrombin in their milk were created by a bio-therapeutics company using a mammary specific promoter [21]. A transgenic rabbit model that secretes a drug-based protein for the treatment of hereditary angioedema in its milk was the second transgenic animal bioreactor system that the FDA had approved [22]. However, utilizing avians as bioreactor systems has a number of benefits over mammals that has to do with simplicity in setup and scaling, reduced costs, and high production yields [19, 20]. The cost of keeping chickens is quite low, and small flocks of founder birds may quickly grow into big flocks [23]. Moreover, avian embryos grow in a maternal effect-free system, making different in ovo experimentations possible while allowing for the reliable identification of reactions to chemical or physical stress interventions [20]. Besides, it is possible to do numerous sorts of investigations on living cells through stage-specific embryonic manipulation, such as isolation, transplanting, and gene transfer [20]. Despite the fact that the human genome is twice the size of the chicken genome, it is realized that there are around the same number of genes in chickens as there are in man [20]. Egg laying, weight gain, and lipid metabolism are examples of well-developed, commercially significant features in birds that are controlled by genetic pathways that might be exploited in human research to, among other things; uncover the etiology of human diseases including obesity and cardiovascular anomalies [20, 24]. When it comes to researching human ovarian cancer, birds are more useful than rodents or humans because they lay a significant number of eggs for their lifespan and have a short ovulation cycle, which makes them more likely to acquire ovarian cancer than other models [25]. Consequently, it may be able to develop avian models of human ovarian cancer and, using gene editing technologies, to elucidate the genetic pathways underlying the pathogenesis of ovarian cancer [25]. The ample protein synthesis capability of the laying hen—a typical 60 g egg contains about 3.5 g of protein in the egg white, and each hen may lay over 300 eggs per year—has sparked additional interest in using hens as bioreactors [23]. Additionally, the abundance of biologically active nutrients and the structural simplicity of the egg proteins render the chicken an ideal bioreactor system [26]. The comparatively simple nature of egg white proteins makes it simple to purify recombinant proteins from egg white proteins. The fact that a number of the oligosaccharides bonded to the polypeptides in chicken proteins are more comparable to those of humans than those of mammals might be a benefit of adapting chickens as a bioreactor, despite the dearth of information about glycosylation modification in birds [17, 26]. Hence, recombinant proteins with human-like glycosylation can be produced in avian bioreactors [17]. In contrast to the majority of other mammals, humans do not typically have certain protein glycosylation. Mammals, apart from human and primates, have α1-3-galactose (α1,3-Gal) biomarkers on their tissues

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and glycoproteins. The enzyme alpha-1,3-galactosyltransferase is non-functional in humans, and around 1% of effective B-lymphocytes release anti-α-Gal antibodies in reaction to enteric bacteria, which commit to the rejection of transplanted xenografts and pose complications if individuals are exposed to bodily fluids from mammals [26]. Contrarily, chickens do not synthesize this enzyme, hence there is a reduced possibility of an immunological reaction to pharmaceutical proteins produced in eggs [26]. Evidently, the use of the egg as a bioreactor has expanded owing to developments in genetic manipulation by DNA microinjection, gene transfer via retroviral and lentiviral vectors, and the creation of chimaeras utilizing chick embryonic stem cells or primordial germ cells (PGCs), enabling the production of stable and well-characterized biopharmaceuticals with high-yield in egg white [27]. During the process of egg laying, each ovulated yolk gains layers of egg white at first, then shell membranes, and finally a shell as it travels down the adult oviduct, which ranges in length between 50 and 70 cm [28]. Typically, the white part constitutes 60% of the total egg weight with albumen protein constituting the primary component of egg white. The genes that code for egg white proteins are translated in the secretory cells of the magnum of the laying hen’s oviduct [28]. As the yolk descends the oviduct, secretion is triggered. Transgenic production of therapeutic proteins in egg white can be conveyed by driving expression of a sequence encoding a therapeutic protein through the regulatory sequences of the genes encoding egg white proteins [29]. Ovalbumin and lysozyme, two egg white protein genes whose regulatory sequences have been thoroughly described at the molecular level, are the two most prominent candidate genes to edit in order to tailor expression to the oviduct. Ovalbumin gene encodes a protein that makes up more than half of the egg’s white and is expressed only in the tubular gland cells of the oviduct [28]. Lillico et al. described the development of transgenic hens that produce functional recombinant pharmaceutical protein in high amount specifically in the oviduct of laying hens as a component of egg white with no indication of any transgene silencing following germ-line transmission [28]. Lysozyme, on the other hand, is expressed in chicken macrophages and the oviduct [30]. Several studies have revealed the development of chimeric genes composed of chicken lysozyme gene regulatory sequences and sequences encoding foreign pharmaceutical proteins that could be introduced randomly into the chicken genome and maintain adequate expression patterns [30, 31]. Recently, several human recombinant proteins entailing human interferon alpha-2b [32] and beta-1a [28] as well as human granulocyte-colony stimulating factor [33] have been successfully synthesized in chicken bioreactors. Anti-cancer therapeutic antibodies with improved Fc effector capabilities were also reported to be produced by transgenic chicken bioreactor systems [33]. Furthermore, lysosomal acid lipase was recently synthesized and purified from egg white of transgenic hens using the distinct N-glycosylation pattern of egg white protein [34]. Interestingly, Kanuma, the first pharmaceutical protein was recently licensed for the treatment of lysosomal acid lipase

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insufficiency [35]. Likewise, human flu vaccine was also produced by avian bioreactors [34]. Noteworthy, the purification of human monoclonal antibodies from chicken appears to be an effective application that has been reviewed elsewhere [36]. Currently, the OmniChicken, developed by Ligand Pharmaceuticals Inc., is a worldwide unique platform that uses transgenic chicken to generate human monoclonal antibodies. Different studies have shown that egg white produces tissue plasminogen activator for anti-thrombotic medicines [37], monoclonal antibodies for breast cancer therapy [38], epitope peptides for pollen [39], and antimicrobial peptides [40]. Additionally, human erythropoietin [41], parathyroid hormone [42], and other bio-products were also made available by chicken bioreactors. It is foreseen that numerous options for implementing the production of therapeutic proteins in chicken eggs will become available owing to genome editing versatility.

6.4 Genome engineering of chicken primordial germ cells The effectiveness of genome editing to create potent chicken bioreactors has recently increased owing to a number of technological developments, which has led to the development of genetically modified chicken lines. These developments encompass improved chicken PGCs culture conditions [43], the generation of sterile chicken surrogate hosts [44], and novel techniques for efficient and focused gene editing [44]. PGCs are unipotent stem cells that eventually differentiate into gametes, spermatogonia, or oocytes, which have the ability to transfer genetic information to subsequent generations [45]. During embryonic development, PGCs reveal special relocating and migrating abilities, after which they populate the forming embryo’s urogenital ridges [45]. Avian PGCs can be isolated at different developmental stages, mainly from vascular system of HH stage 14–15 embryos as circulating PGCs (cPGCs), and from gonadal ridge of HH 29–31 embryos as gonadal PGCs (gPGCs). Cell-surface antibodymediated techniques, density gradient centrifugation, and size-dependent separation techniques can all be used to identify and purify avian cPGCs and gPGCs. Interestingly, the chicken is one of the few species that allows PGCs to be easily replicated in vitro to increase cell abundance when cultured in a specific medium, where an in vitro PGCs growth from a single chicken embryo may reach over 100,000 cells after around four weeks [46]. In birds, transgenesis and genome editing are based on a characteristic germline transmission mechanism involving PGCs. Cultured PGCs can be altered in the lab by transgenically inserting a foreign gene or making particular genome editing modifications and then microinjected into blood arteries of recipient embryos in windowed eggs after about two days of incubation where they migrate to the developing gonads [47]. The recipient embryos are sealed and incubated until they hatch and

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Figure 6.1: An approach for developing chickens with altered genomes. Genome-edited birds can be generated by micro-injecting genetically modified PGCs cultured in vitro into the recipient embryos’ blood vasculature. Bioreactor models, in addition to other avian models, can be created using avian genome editing methods.

then hatched chickens are reared to sexual maturity and crossed with wild-type chickens. Using genome editing techniques to modify the germ cell lineage, the animal’s transformed genetic makeup can be passed down to its descendants [47]. The offspring of such a cross are then screened to identify those derived from the transgenic PGCs. Such a strategy for the production of genetically edited birds enable the production of bioreactor models as well as other avian models (Figure 6.1). Using this method will result in descendants that inherit half of their chromosomes from the donor PGCs [44]. The fact that injected donor PGCs and endogenous host PGCs coexist in host embryonic gonads poses a drawback to this method by lowering the likelihood that offspring from successive breeding will be produced from donor PGCs [48]. Thus, other initiatives aim to construct germline chimera more effectively by diminishing or depleting the endogenous PGCs in the host embryo using a variety of techniques, including gamma radiation treatment, busulfan injection to the embryo, and blood evacuation from recipient embryos during HH stages 14–15 [48]. According to Nakamura et al., chickens treated with busulfan had a much higher germline transfer efficiency (∼99%) compared to controls (∼6%) [49]. Prior to incubation, they first administered a dosage of busulfan to chicken embryos and then the busulfan-treated embryos were subsequently injected with donor PGCs, creating chimeric chickens that gave rise to progeny derived from donor PGCs [49]. Direct breeding of sire and dam surrogates can produce pure genetically edited eggs and therefore chicks (Figure 6.2).

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Figure 6.2: An approach for developing germline chimera more efficiently using surrogate recipient embryo. PGCs from a pure breed of chicken are isolated and cultured in vitro. Both the male and female PGCs are injected into surrogate host embryos following genome editing and clonal isolation. The embryos are developed into sexually mature adults and then mated. The intended genome modification is present in all of the laid eggs and hatched progeny that are from the donor breed of interest.

6.5 Programmable genome editing systems Using programmable genome editing technologies, the genome can be modified in a variety of ways and to varying degrees while also being effective and accurate. Biological characteristics, disease resistance, and the creation of bio-functional proteins are all benefitted by the novel breeding methods that these technologies have brought about [5]. Genome editing is used to add the desired genes and DNA fragments, after which animals are bred to generate stable lines. In order to provide a thorough view of the current genome alteration research in birds, it would be helpful to look at the different genome editing technologies employed in avian species. ZNFs were the first programmable genome-editing tools, which were later joined by TALENs and CRISPR-Cas9 technology [9]. ZNFs are programmable DNA-binding proteins linked to custom DNA endonucleases. Virtually any sequence of interest can be targeted by engineered ZNFs. ZNFs can be used to edit cultured cells, encompassing stem cells, which is very important for developing approaches for avian genome editing [5]. There have not been many studies on avian ZNF genome editing published yet, despite prior publications employing ZNFs to target interesting genes [5]. Fan et al. have attempted to build a dimeric ZNF that recognizes an 18-nucleotide sequence in the 3′-untranslated region of chicken ovalbumin [50]. Even though the assembly seemed to have optimal characteristics, it failed to exhibit any nuclease activity [50]. Indeed, ZNF technology

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have smoothed the path for more accurate gene editing platforms, however the difficulty and expensive cost of building protein domains specifically for each genomic locus as well as the possibility of erroneous target DNA cleavage caused by single nucleotide alterations or improper domain interactions have built significant obstacles into this technology [5]. Transcription activator-like effectors (TALEs) are proteins constitutionally excreted by plant pathogenic bacteria of the genus Xanthomonas featuring DNA-binding domains consisting of repeated modules 33–35 amino acids in length, with each unit recognizing a single DNA base pair, based on a highly variable dipeptide at amino acids 12 and 13 known as repeat-variable di-residues (RVD) that determines the nucleotide-binding specificity of each repeat [51]. TALENs are created upon fusing the non-specific DNA-cleavage domain of FokI endonucleases onto the specific DNA binding domain TALEs enabling the generation of DSB upon dimerization (Figure 6.3A) [51]. Park et al. established the first gene knockout chickens using TALEN-mediated gene targeted knockout of the ovalbumin gene in the PGCs, and ovalbumin gene mutant progeny were obtained using test-cross analysis, suggesting that the TALEN approach employed in the chicken PGCs line is a potent strategy for creating particular genome editing in chicken safely for potential implementation [52]. In 2017, Taylor et al. successfully targeted DDX4 locus knockout utilizing TALEN in conjunction with a targeting vector and reported a germline transmission from the founder birds [53]. Currently, the most accurate, practical, and effective genome editing approaches are CRISPR and its accompanying Cas9 protein. The CRISPR-Cas9 system consists of two essential elements: a DSB-inducing endonuclease, Cas9, and a short single guide RNA (sgRNA) complementary to a specific area of the genome sequence (Figure 6.3B) [54]. Specific recognition is tightly dependent on the existence of a short protospacer adjacent motif (PAM) on each side of the target site [54]. Following target recognition, R-loop formation and strand scission are triggered by matching of complementary bases between the sgRNA and target DNA, Cas9 endonuclease-DNA interactions, and related conformational changes [54]. The CRISPR-Cas9 system has been documented to be applied to avian somatic cells and tissues [55, 56]. Targeting PGCs by CRISPR-Cas9 and exploiting these modified cells to create genetically edited chickens by introducing specific genetic alterations into the genome has also been reported [57]. The DSBs caused by genome editing tools are repaired by host-mediated DNA repair mechanisms (Figure 6.3C). The common error-prone NHEJ process is triggered in the absence of a repair template, leading to sporadic indels or even substitutions at the DSB site that typically affect gene function [51, 58]. The error-free HDR pathway can be activated in the presence of a donor template having a targeted sequence flanked by homology arms to cause intended variation by homologous recombination [51, 58]. This lays the groundwork for carrying out precise gene alteration, often including gene knock-in, deletion, correction or mutagenesis, empowering the efficient establishment of pharmaceutical producingchicken bioreactors and other avian models (Figure 6.3D).

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Figure 6.3: Schematic illustration of TALEN and CRISPR-Cas9 systems. (A) TALENs are made up of repeating modules that are fused to non-specific FokI cleavage domain. Each repeating unit differs at amino acids 12 and 13 which are known as repeat-variable di-residues (RVD) that defines the nucleotide-binding specificity. (B) A 20-nucleotide guide RNA (gRNA) in the CRISPR/Cas9 complex directs Cas9 to the target DNA. An immediate protospacer adjacent motif (PAM) sequence downstream of the target DNA is basic for Cas9 nuclease activation. (C) Double-stranded breaks induced by TALENs and CRISPR-Cas9 are then repaired by NHEJ or HDR pathway. (D) Avian bioreactors are created by inducing genome modification.

6.6 Delivery approaches for therapeutic genome editing Gene-editing tools offer the potential to enable remarkable flexibility in modifying the genome at specific sites for gene knockdown or reinstatement, insertion of a therapeutic transgene, or repair of mutations linked with genetic conditions. The lack of safe and efficient techniques for administration of gene-editing reagents remains a significant obstacle for therapeutic gene editing. Recombinant DNA technology-based transgenesis has produced novel pharmaceuticals, and offered new prospects for livestock enrichment [59]. Table 6.2 provides a summary of the key milestones made in the process of creating genetically edited chickens. Among the main goals of transgenesis is to create bioreactors or organisms with profitable features that could not be achieved using conventional methods [5]. Notably, transgene incorporation into the host genome in eukaryotic cells is necessary for stable transmission and expression of transgenes. Eukaryotic cells can be transfected with exogenous recombinant DNA in a number of methods. Exogenous DNA has been successfully delivered into cultured animal cells using liposome transfection; hence, endeavors have been undertaken to introduce

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Table .: Some of the main milestones in the development of genetically modified chickens. Avian genetic modification innovation

year

Reference

The first genetically modified chicken using retroviral vectors inserted into the chicken germ line The first culture system for chick embryos The first transgenic chickens by DNA microinjection Production of germline transgenic chickens using lentiviral vectors The first description of long term in vitro culture of PGCs followed by transfection and generation of transgenic chickens Generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in eggs Generation of transgenic chickens that express anti-avian influenza virus replication inhibitors PGC gene editing and the creation of the first gene knockout chickens In vivo transfection of PGCs generating transgenic offspring using lipofectamine  complexed with Tol transposon and transposase plasmids. The first transgenic chickens with fluorescent proteins expressed in specific immune cells Development of defined culture conditions for chicken PGCs Cryopreservation of specialized chicken lines using cultured PGCs CRISPR-mediated homology directed repair targeting the chicken immunoglobulin heavy chain locus in PGCs to produce transgenic progeny. Efficient TALEN-mediated gene targeting a reporter construct to the DDX (vasa) locus in chicken PGCs Restoration of roosters fertility by transplanting genetically modified PGCs Genetically engineered sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs



[]

   

[] [] [] []



[]



[]

 

[] []



[]

  

[] [] []



[]

 

[] []

recombinant DNA to chicken embryos via liposome-based techniques [60] or polyethylenimine encapsulation techniques [61]. Electroporation is another method for introducing recombinant DNA into chicken embryos [62]. These techniques, albeit useful for short-term gene expression studies, are less effective for directly delivering recombinant DNA to chicken embryos in order to produce animals with heritable transgenes. Deployment of a plasmid encoding the Cas9 protein and sgRNA is a common strategy. Lipofection, polyethyleneimine, and electroporation were used to introduce plasmids containing the Cas9 protein and the sgRNA sequence [58]. In the first work employing CRISPR-Cas9 system in chickens by Véron et al., embryos were electroporated with plasmids expressing Cas9 and sgRNAs directed against the transcription factor PAX7 [63]. Another practical method for manipulating the avian genome appears to be spermmediated DNA delivery [56, 64]. Cooper et al. applied artificial insemination to deliver gene editing vectors via transfected sperms that were obtained after introduction of Cas9 mRNA and sgRNA into cells using lipofection [65]. Thus far, viral vectors have demonstrated to be remarkably efficient at delivering transgenes [64]. Viral vectors are created

6.7 Future outlook

163

by modifying viral genomes, which include key components required for the delivery of genetic materials into cells [64]. Elements that are not requisite for incorporation are substituted by transgenes during vector construction [64]. When the viral genome invades the host cell during infection, the viral genes begin to be expressed [64]. Lentiviral, adenoviral, and adeno-associated virus (AAV) vectors are the three main kinds of viral vectors used in transgenesis [64]. Numerous investigations have shown that CRISPR-Cas9 is effectively delivered by adenoviruses for editing. However, due to the limitations associated with their immunogenicity, adenoviruses are currently only used in research settings [66]. The most commonly used approach for CRISPR-Cas9 mediated gene targeting comprises a dual AAV system to deliver Cas9, sgRNA, and donor template [66]. AAV-mediated CRISPR-Cas9 delivery has been used successfully in numerous preclinical investigations to modify genes [66]. Being replication-defective, viral vectors are safe and the majority of them are readily available in commerce [67]. Owing to the accessibility of well-established methodologies and high transduction efficacy, viral delivery vectors have been utilized to transport nucleic acids into cells and are the preferred delivery technique for CRISPR-Cas9 [66]. Lentiviral vectors were successfully used by Kwon et al. and other research bodies to transduce chicken embryonic cells, resulting in the effective creation of transgenic lines [67, 68]. An alternative form of gene delivery technology that is frequently utilized in transgenic research is transposon-based vectors including PiggyBac vectors that are characterized by the simplicity of manufacturing and the substantial cargo capacity [69]. PiggyBac vectors have been shown to effectively transpose into the genomes of both chicken embryos [70] and PGCs [71]. A further popular non-viral transposon-based technique for transgene delivery to increase the efficiency of producing stable germline transgenic birds is the Tol2 vector [60, 72]. Direct infusion of Tol2 transposon vectors into early embryos’ vasculature to target germ cells as they migrate through the circulation is one way to produce genetically engineered birds that express a reporter gene carried in the transposon [73, 74]. The use of Tol2 transposon vectors has been shown by Macdonald et al. to significantly improve the effectiveness of stable genetic modification of PGCs, enabling the application of genetic modifications of PGCs as a strategy for the production of transgenic chickens [60].

6.7 Future outlook To meet humanitarian demands, the biopharmaceuticals sector has altered its traditional modes of operation. Given the effectiveness of their end-products, transgenic flocks that may be employed as natural bio-manufacturing facilities for the pharmaceutical sector has gained strong attention especially with the continuous growth of the technical knowhow and the ability to produce large amounts of protein in egg white continuously and non-invasively. The use of the chicken as a bioreactor may have a significant impact on human health by offering alternative therapeutic options. Poultry bio-pharm initiatives are now needed to transform the chicken into a large scale pharmaceutical

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bioreactor platforms that can address the expanding demand for human therapeutics based on recombinant proteins. The ability to genetically modify animals through the use of advanced genetic technologies opens up new opportunities for breeding programs to effectively produce the animals of the future, including chickens that work as mini-pharmaceutical factories. Genome engineering, in conjunction with PGCs and transgenic technologies, offers the potential to improve breeding programs focused on increasing production efficiency and developing new animal-based products. Transgenic technologies have the potential to align the transgenic chicken model with, and even surpass, the more widely-used transgenic mammalian models. The chicken has now grown as an important model platform for pharmacology, and other domains of agriculture and biology. The FDA had already approved genetically edited chicken in 2015 as a bioreactor for the manufacture of protein-based medicines in eggs. Technically, the primary obstacles to fully utilizing these technologies are the lack of housing options for experimental birds and the difficulty of maintaining valuable, gene-edited lines for experimental embryo production over time. A comprehensive program for the production of transgenic poultry necessitates a sizeable investment in animal and lab infrastructure for breeding after the generation of founder birds and maintenance of transgenic lines. Future research is anticipated to lead to the creation of cutting-edge transgenic technologies that fully utilize and improve the merits of avian models for fundamental and applied research. Targeting PGCs with programmable genome editing systems have a lot to do for avian models. Humans will have far more options and benefits if gene editing technology is extensively exploited on bird species and adopted in husbandry industry.

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35. Sheridan C. FDA approves ‘farmaceutical’ drug from transgenic chickens. Nat Biotechnol 2016;34:117–9. 36. Flemming A. Human antibodies from chicken eggs. Nat Rev Drug Discov 2005;4:884–5. 37. Kaleri HA, Xiang L, Aniwashi J, Xu S. Oviduct-specific expression of tissue plasminogen activator in laying hens. Genet Mol Biol 2011;34:231–6. 38. Oishi I, Kim S, Yoshii K, Esteban CR, Izpisua Belmonte JC. Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells. BMC Biotechnol 2011;11:5. 39. Kawabe Y, Hayashida Y, Numata K, Harada S, Hayashida Y, Ito A, et al. Oral immunotherapy for pollen allergy using T-cell epitope-containing egg white derived from genetically manipulated chickens. PLoS One 2012;7:e48512. 40. Liu T, Wu H, Cao D, Li Q, Zhang Y, Li N, et al. Oviduct-specific expression of human neutrophil defensin 4 in lentivirally generated transgenic chickens. PLoS One 2015;10:e0127922. 41. Koo BC, Kwon MS, Lee H, Kim M, Kim D, Roh JY, et al. Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens. Transgenic Res 2010;19:437–47. 42. Lee SH, Gupta MK, Han DW, Han SY, Uhm SJ, Kim T, et al. Development of transgenic chickens expressing human parathormone under the control of a ubiquitous promoter by using a retrovirus vector system. Poultry Sci 2007;86:2221–7. 43. Whyte J, Glover JD, Woodcock M, Brzeszczynska J, Taylor L, Sherman A, et al. FGF, insulin, and SMAD signaling cooperate for avian primordial germ cell self-renewal. Stem Cell Rep 2015;5:1171–82. 44. Ballantyne M, Woodcock M, Doddamani D, Hu T, Taylor L, Hawken RJ, et al. Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating. Nat Commun 2021;12:659. 45. Ballantyne M, Taylor L, Hu T, Meunier D, Nandi S, Sherman A, et al. Avian primordial germ cells are bipotent for male or female gametogenesis. Front Cell Dev Biol 2021;9:1–9. 46. Woodcock ME, Gheyas AA, Mason AS, Nandi S, Taylor L, Sherman A, et al. Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds. Proc Natl Acad Sci USA 2019;116:20930–7. 47. Davey MG, Balic A, Rainger J, Sang HM, McGrew MJ. Illuminating the chicken model through genetic modification. Int J Dev Biol 2018;62:257–64. 48. Han JY, Park YH. Primordial germ cell-mediated transgenesis and GE in birds. J Anim Sci Biotechnol 2018;9:19. 49. Nakamura Y, Usui F, Ono T, Takeda K, Nirasawa K, Kagami H, et al. Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken. Biol Reprod 2010;83:130–7. 50. Fan B, Huang P, Zheng S, Sun Y, Fang C, Sun Z. Assembly and in vitro functional analysis of zinc finger nuclease specific to the 3′ untranslated region of chicken ovalbumin gene. Anim Biotechnol 2011;22:211–22. 51. Woodcock ME, Idoko-Akoh A, McGrew MJ. Gene editing in birds takes flight. Mamm Genome 2017;28: 315–23. 52. Park TS, Lee HJ, Kim KH, Kim J-S, Han JY. Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci USA 2014;111:12716–21. 53. Taylor L, Carlson DF, Nandi S, Sherman A, Fahrenkrug SC, McGrew MJ. Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development 2017;144:928–34. 54. Jiang F, Doudna JA. CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys 2017;46:505–29. 55. Kobayashi K, Fujii T, Asada R, Ooka M, Hirota K. Development of a targeted flip-in system in avian DT40 cells. PLoS One 2015;10:e0122006. 56. Collares T, Campos VF, De Leon PM, Cavalcanti PV, Amaral MG, Dellagostin OA, et al. Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide. J Biosci 2011;36:613–20. 57. Dimitrov L, Pedersen D, Ching KH, Yi H, Collarini EJ, Izquierdo S, et al. Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells. PLoS One 2016;11: e0154303. 58. Chojnacka-Puchta L, Sawicka D. CRISPR/Cas9 gene editing in a chicken model: current approaches and applications. J Appl Genet 2020;61:221–9.

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Akhavan Niloofar, Bednarczyk Marek, Krajewski Krzysztof and Stadnicka Katarzyna*

7 Emerging in ovo technologies in poultry production and the re-discovered chicken model in preclinical research Abstract: Prenatal programming is a concept based on assumptions that the events occurring in critical points of embryonic development may pose epigenetic changes resulting from chemical rearrangements on the DNA structure. Epigenetic changes may pose life lasting phenotypic effects in the animal, or can be heritable, like gene silencing associated with methylation in gene promoters regions. The technical advancements in biotechnology, bioinformatics, molecular techniques and robotization have brought to new technological applications in poultry production. Intentional stimulation of embryonic development and determination of the future health of the hatched organism is possible by in ovo application of natural antioxidants and prebiotics, gut stabilizers like probiotics and other immunological enhancers including vaccines. In parallel, the finetuned and generally accessible techniques of chicken embryo incubation along with the novel tissue engineering tools have led to focus the attention of scientists on chicken embryo as the alternative animal model for some pre-clinical approaches, in the context of reducing and replacing the experiments on animals. In this chapter, some key highlights are provided on current achievements in poultry embryonic applications, with the attention put to the emerging in ovo technologies (in ovo feeding, immunological stimulation and in ovo oncological tools), that address the societal challenges in food production and health management. Keywords: chicken embryo; in ovo feeding; in ovo technology; poultry; prenatal.

The scientific input is financed by: 1/ National Science Centre Poland grant no. 2019/35/B/NZ9/03186 (OVOBIOM), 2/ Inkubator Innowacyjności 4.0, Ministry of Education and Science Poland. *Corresponding author: Stadnicka Katarzyna, Oncology Department, Collegium Medicum of Ludwik Rydygier in Bydgoszcz, Nicolaus Copernicus University, Bydgoszcz, Poland, E-mail: [email protected] Akhavan Niloofar, Department of Microbiology, Faculty of Biology and Veterinary Science, Nicolaus Copernicus University, Toruń, Poland; and Oncology Department, Collegium Medicum of Ludwik Rydygier in Bydgoszcz, Nicolaus Copernicus University, Bydgoszcz, Poland, E-mail: [email protected] Bednarczyk Marek, Department of Animal Biotechnology and Genetics, Bydgoszcz University of Science and Technology, Bydgoszcz, Poland, E-mail: [email protected] Krajewski Krzysztof, Vetdiagnostica Sp. z o.o., Makowiska, Poland, E-mail: [email protected] As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: A. Niloofar, B. Marek, K. Krzysztof and S. Katarzyna “Emerging in ovo technologies in poultry production and the re-discovered chicken model in preclinical research” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2021-0130 | https://doi.org/10.1515/ 9783110683912-007

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Implementation of precise solutions to develop natural and sustainable immunity in the animal is an element of systemic approach defined by the World Health Organization, and referred to as the OneHealth. The goal is to optimize field solutions and take measures for implementations in a careful holistic manner that would balance the health of humans, animals and environment. Stakeholders of poultry production seek solutions to eliminate and/or control the pathogens transmitted with food and animals. The need for efficient prophylactic and controlling strategies is high, primarily due to a large scale of poultry sector and its importance in diets. According to the current public surveillance, the majority of EU poultry meat production comes from: Poland (16.8%), the United Kingdom (12.9%), France (11.4%), Spain (10.7%), Germany (10.4%) and Italy (8.5%). Despite a general trend to shift human diets to plant sources and reduce the meat consumption, still the OECD/FAO [1] foresee, that in the context of Zero Hunger initiative, the consumption of poultry meat would increase globally to 154 Mt by 2031 (which is 27% higher than the growth in consumption predicted for pork and twice as higher as for beef) [1]. With growing awareness on health challenges, climate challenges and demands to meet the highest animal welfare standards, the societal expectations and new legislations influence the strategic planning in poultry industry.

7.1 Emerging industrial in ovo applications during embryonic development and outlook on their implementation Concept of in ovo technology was initiated in United States, in Michigan, with a fundamental achievement of Sharma and Burmester [2]. They were first to vaccinate chickens with herpesvirus of turkey (HVT) on day 18 of egg incubation (EID). The vaccinated embryos were viremic with HVT at hatching, and showed to protect the in ovo immunized chickens better than the post-hatch vaccinated chickens. The research of Sharma and Burmester has paved all the further way leading to development of industrial techniques for massive application, in general referred to as “in ovo technologies”. The current major challenge of poultry sector is not “how to produce more and cheaper” anymore, but “how to produce efficiently, healthy and regeneratively (by means of sustainability)”. Problems and dilemmas of poultry research community and of poultry producers are therefore focused on 1/ delivering high quality products i.e. free of pathogens, free of antibiotics and with eliminated myopathies in meat; 2/ evolving farming systems to meet the highest welfare standards; 3/ implementing regenerative production systems, with naturally gained resistance, and (ideally) energetically independent. In ovo technologies, some of them already in use and the other just about to enter the production scale, are fully addressing the above needs, and are briefly presented below with schematic overview in Figure 7.1.

Figure 7.1: Overview of target timepoints for in ovo applications, situated on chicken embryo development axis. Major bacteria phyla naturally found in chicken embryo [3], are indicated. EID- embryo incubation day.

7.1 Emerging industrial in ovo applications during embryonic development

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7.1.1 In ovo technologies to determine the sex pre-hatch 7.1.1.1 In ovo hormonal and spectral assays For the last couple of years the layer type hens production industry have been struggling with a problem of elimination of newly hatched male chickens. The male hatchlings have been considered as the unwanted byproduct of egg production. The culling of one day old chickens is going to be eliminated as a common industrial practice in the hatcheries. A high ethical and social pressure is pushing the new regulations. Germany, followed by France, are the first countries that put a ban to killing of day-old male chicks by implementing innovative technical solutions in cooperation with research universities. The German provider SELEGGT developed a safe and non-invasive gender detection system to be applied as early as in nine-day old incubated embryos. The sample of allantoic fluid is sucked out from incubated egg by automated laser device and the patented chemical endocrinological assay detects the female gender hormone, estrone sulphate in allantoic fluid, therefore allowing to immediately discard the male embryos from incubated population. In parallel, the Dutch provider called In Ovo, developed and an automated technology called Meet Ella, based on a similar principle to sample the eggs and spectrally detect the marker indicating sex in nine-day old embryos. With increasing availability of magnetic resonance (MRI) spectroscopic systems, two other physiochemical approaches have been under development. Recently, Fioranelli et al. [4] proposed a noninvasive method to determine the gender by virus medical imaging technique, based on the knowledge about communication of the virus outside the shell. The principle of action is that virus acts like the receiver of signals from the organism inside the egg. It interestingly happens that viruses exchange two different electromagnetic waves with males and females, thus allowing for gender distinction and even the status of growth. Another potential solution is to use a non-invasive protocol that implies Raman spectroscopy to identify sex-specific spectra of blood circulating in the extra-embryonic vessels, as early as on day 3.5 od embryonic incubation [5]. These technologies need further development and refinement towards affordability and accuracy. 7.1.1.2 Gene editing for gender determination The Israeli eggXYt team has led to development and commercialization of a novel CRISPR/Cas technology. A breakthrough gene-edited chicken line was produced, in which the sex can be determined as soon as the egg is laid [6]. To understand the public acceptance for such a solution, researchers conducted the national online survey with over 1000 participants, asking about their attitude to accept a genetic modification to the poultry, should that change possibly lead to elimination of day old chicks’ culling. Among the egg consumers, the public acceptance to use a gene editing technique that enables an

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early gender determination in embryos was found to be as high as 60%, showing a high support of innovation for major ethical issues [7]. Animal welfare in production is a highly important social dilemma, and it is rated that 80% of European citizens would in general support changes in relevant legislations towards improvements in welfare [8]. Regardless of scientific ambitions in the field of in ovo research to determine the embryonic gender maximally early (at a moment of egg lay in genetically edited chicken line, or using the hormonal allantois tests on nine day of egg incubation), the major driver of innovations and investments to this approach are the legislative and social expectations related to animal health and welfare. The hormonal tests seem to be the most reliable and socially acceptable for realization in hatcheries at the moment. As these solutions are just about to enter the markets, no information is yet available regarding the implementation issues in the hatcheries, likely to be an extra time and labor resources related with additional handling. It can be expected that the legislative framework for “animal production and use”, will be gradually more and more rigorous for practitioners. The regulation of EC Council No 1099/2009 of 24 September 2009 on the protection of animals at the time of killing, is working in compatibility with horizontal legislation on the protection of animals used for scientific purposes stipulated in Directive 2010/63/EU, and amended by Regulation (EU) 2019/1010. The power of law shows that the amendments are usually applied with no hesitations, as soon as the new research results on animal behavior, perception of pain and health and welfare issues are arriving. Zumbrink et al. [9], suggested the need to develop new anesthetic methods in compliance with the latest scientific data on the onset of pain perception, in the second period of chicken egg incubation, which is between the day 8 and 15 of egg incubation. Therefore, even provided that the males can be hormonally detected very early with 100% certainty rate, the methods to eventually eliminate those embryos from further process will have to be ethically and societally accepted.

7.1.2 In ovo vaccination Vaccination of the incubated embryos is a precise immunization strategy applied to massive populations of industrially hatched chickens in USA (90% of hatcheries), Brazil, Spain, Japan, and over last several years, vastly expanding globally to Europe, Asia and Africa. The aim is to maximize chicken resistance and performance in field, but also to eliminate the transmission of infection in the environment, including the transmission to human population. The fast expansion of embryo immunizations and stimulations with bioactive ingredients, is pushed forward to novel implementations, driven by several key factors: 1/ new knowledge and growing awareness about the early developmental events that

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influence development of immune system, 2/ knowledge on the role of epigenetic factors that affect the organism prenatally, but pose life-lasting and/or transgenerational effects [10]; 3/ the latest lessons from global COVID-19 pandemics and awareness of increasing antibiotic resistance; 4/ awareness of a role of avian species (domesticated, intensively reared and wild, migrating birds) in transmission of disease agents, with avian influenza on top of the threats; 5/ continuous improvements to the existing vaccine formulations and development of novel in ovo vaccines against the major infectious diseases caused by viruses: Bursa of Fabricius disease (Gumboro), Infectious Newcastle disease, Infectious laryngotracheitis, Avian influenza (Bird flu). Adding to those, a novel anti-parasitic vaccine against the Eimeria caused coccidiosis have been developed [11], 6/ legislations on use of veterinary medicinal products and medicated feed [12, 13]; 7/ finally, the technical improvements that fine-tune the operating capacities of vaccinating machines in an automated environment, and increasing competitiveness of the machines producers, make the technology available to growing number of hatcheries, especially to smaller operators, that could automatically vaccinate relatively lower number of eggs amounting to 20,000–150,000 daily. Although the World Health Organization assesses infections with avian influenza to human at low risk, the ongoing research, especially in Asia, highlights the potential emerging risks to human populations and keeps the alert high [14, 15]. In all operational hatcheries, the key elements that determine success of immunization are the level of sanitation and organizational capacities on site, including the monitoring of vaccine preparations, and the correct diagnosis of embryo age and egg size in order to reach the correct vaccinations compartments in the egg (amniotic fluid, being the optimal for efficacy). Vaccinating machines are constantly modernized and operating with increasing accuracy and efficiency. Several leading systems implemented in the hatcheries are known to be: Egginject – based on a dual pressure injection system and enabling individual adaptation of the injection depth to single embryos, with injecting capacity of 60,000 eggs/h; Vinovo (Viscon Hatchery Automation), a machine that selectively injects vaccine only in eggs that contain viable embryos, with a heartbeat detection technology and egg positioning. The idea behind this solution is to minimize vaccine wastage and reduce potential contamination from nonviable embryos. Finally the Smart Vac system (Royal Pas Reform) is designated for in-ovo vaccination and nutrition, and allows automatic and individual adaptation of the injection depth to each embryo. Efficacies of vaccinations may vary and should be considered individually for each population, depending on the embryonic stage at vaccination and delivery route (amniotic, intramuscular or subcutaneous), dose and environmental challenge encountered [16]. Efficacy of immunization is highly determined by the compartment in the egg that was injected and vaccinated, with the highest serological responsiveness by 94.4% observed after a direct, amniotic injection [17].

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7.1.3 In ovo feeding In ovo feeding is an early life strategy originally developed by the team of researchers Uni and Ferket [18]. It involves injection of various nutrients (amino acids, carbohydrates) or vitamins directly to the amnion between days 17.5 and 19.2 of embryonic development, in order to accelerate the onset of feeding and maturation process of the newly hatched chicken. The mostly observed phenotypic results of in ovo feeding are related to an increased body weight gain with even up to 5–6% higher mass for newly hatched chickens, over the controls. Some exemplary effects are reported by Saeed et al. [19], after delivery of various compounds directly to the amnion. Typically, an increased utilization of amino acids and the improved feed efficiency are reported after amniotic injections of Threonine and Arginine. Whereas, injection of carbohydrates caused accelerated development of gut by means of broadened villi surface. The other, considered range of biologics as candidates for in ovo route are hormones, e.g. insulin growth factor to stimulate muscle tissue development, and novel immunostimulants based on nucleic acids particles. Oligodeoxynucleotides containing CpG motifs (agonists of TLR9) showed ability to activate lymphocytes B and mitigate the Salmonella infection challenge [20].

7.1.4 In ovo modulation of immunity through gut health stimulation 7.1.4.1 Readiness for applications With the same technique as for in ovo feeding on 18.5 EID (Embryo Incubation Day) or applied into the egg air cell on 12 EID, the bioactive formulations being probiotics, prebiotics and synbiotics and other immune enhancers, can be delivered through automatic injections. Lately, such applications are also considered as vaccine adjuvants [21]. The major economical and practical rationales to develop and approve novel formulations specifically for in ovo delivery, are listed below: 1. a commercial hatchery falling into medium or big enterprise category produces ab. 3,000,000 chicks/month. 2. producer of broiler chickens that uses a complete feed mix supplemented with a commercial bioactive substance to stimulate the immune system of the animals (e.g. probiotic), has to include an average dose of 0.85 kg/tone of feed, whereas the price of the supplement is about to cost 0.8 EUR/100 kg (calculated as average values that may vary in case of different feed supplements currently available on the market). Whilst, the dose of a bioactive formulation applied in ovo (e.g. synbiotic) is amounting maximally to 3.5 mg per egg, in 0.2 mL solution injected on 12 EID, or 0.05 (vaccine) to 0.7 mL of solution (nutrients and other biostimulants) at late stage of egg incubation.

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3. the expected effect of a bioactive formulation administered in ovo as a single dose, would result in at least the same or better achievement in terms of growth and health performance, compared to the efficacy of a similar compound applied in the feed. 4. the in ovo embryo stimulations are highly compliant with the strategy of European Green Deal, and the circular economy. The strategies are focused on supporting production models in agriculture (including animal sectors), with plans for energy regeneration and the approach to produce less, but with maintained or higher efficiency; in other words feeding and consuming less, but with a better quality. Therefore, the in ovo supplementation of the embryo provides, in perspective, a substantial cost benefit to production and the “health and life quality” factor. Although the use of probiotics in feed has been applied for several decades, the cost of supplementation makes the zootechnical additives affordable to less than 50% of farmers in Europe. The scientific survey performed in Poland, Spain, the Netherlands and Denmark showed that in central Europe the use of probiotics is considered as relatively expensive, irrespective of efficacy [22]. The in ovo feeding strategy is not a new concept, and the technology to feed in ovo in the hatcheries is already in place, but it is too early to report on the scale of effects. According to the knowledge available, no nutrients or other formulations have been announced commercially for in ovo feeding so far. The requirement to submit a detailed dossier for EFSA (European Food Safety Authority) verification and approval substantially prolongs the way to implement novel zootechnical feed additives, even with existing technology for massive treatments. If the formulations that are already approved for use in feed are a promising ingredient for prenatal application, they still would undergo assessment for new uses in EU/EFSA, i.e. for in ovo and peri-hatch feeding. The category of feed additives called zootechnical additives is new point on the EFSA’s agenda, and includes: digestibility enhancers (enzymes), gut flora stabilizers (typically probiotics), environmental enhancers (unused yet), Other (e.g. immune enhancement) and animal welfare category (new, pending to add to EFSA registration lists) [23]. 7.1.4.2 In ovo embryo stimulation on 12 EID (embryo incubation day) Delivery of probiotics, prebiotics and synbiotics to the egg air cell on 12 day of egg incubation underwent a proof of concept and showed to: – stimulate the beneficial microbiome, physiological processes and growth performance [24] – stimulate development of immune system on the level of immune organs and gut associated lymphoid tissue [25] – may be considered as the epigenetic impact [10] – increase the functional properties of meat by increasing the amount of polyunsaturated fatty acids and reducing myopathies in breast muscle [26], possibly through angiogenic mechanisms [27]

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The air cell develops in minutes after the egg is laid by hen. The mode of delivery of a prebiotic on 12 EID embryo incubation was first optimized by researchers Bednarczyk and Gulewicz almost 20 years ago, and described by Villaluenga et al. [28]. Later, a range of prebiotics (lupin raffinose family oligosaccharides, algae derived extracts, galactooligosaccharides, astragalus polysaccharides), probiotics (e.g. Lactobacillus plantarum and Bifidobacterium lactis strains) have been 1/ formulated in vitro in a stepwise manner using microbiological and cell culture methodologies, 2/ tested to optimize the doses and safety for in vivo treatments as the novel synbiotics (combinations of the above), 3/ optimized for in ovo inoculations and verified in field. The various combinations of those stimulants and broad analytical panels (from microbiota determination to transcriptomic analyses and changes in methylome), have been applied in numerous, applicative international field studies over the last years. Most of the developmental works have been based on EU funds and industrial cooperations using the in-house developed prototypes of multiple injection systems: THRIVE RITE, EU/FP7 http://www.thriverite.eu/, ECO FCE, EU/FP7 http://www.eco-fce.eu/, MonoGutHealth, H2020 https://monoguthealth.eu/, and regional support for research actvities, to name the recent ones: Ovobiom project from Polish National Science Centre, or Inkubator Innowacyjności 4.0. The application of 12 EID has been leveraged to further developments on industrial level, and owing to a fact that the in ovo interventions (such as gender determination) are to be massively applied in hatcheries as early as on nine day of eggs incubation, the implementation of embryo stimulation with immune enhancements and gut stabilizers on 12 EID might be considered a matter of time.

7.2 Microbiome and microbiota in the chicken embryo Microbiome refers to genetic content of microorganisms. Through improving digestive efficiency and preventing pathogen colonization, the optimally balanced microbiome is a key factor to foster immune system development and immune homeostasis in the animal. It is still a domain of investigation as to how the microbiome develops and is passed down from hen to the avian embryo. With sequencing tools, NGS (next-generation sequencing), it is feasible to identify microbial communities and provide profiles of the chicken embryonic and gastrointestinal microbiota at various embryonic developmental stages. Ding et al. [3] performed a comparative work in 4- and 19-day-old chicken embryos, providing the first insight into the gut colonization process during egg incubation. Whole embryo and intestine samples were subject to DNA sequencing. The microbial classifications showed 28 phyla, 162 genera and 76 microbiota species. In the four-day old embryos, the Proteobacteria phylum was the most prevalent (86%), followed by Firmicutes (5%), Bacteroidetes (4%), and Actinobacteria (3%). Among Proteobacteria the two most prevalent microbial genera were Halomonas (79%) and Ochrobactrum (5%). During the development, a decline in the proportion of Proteobacteria and a striking upward

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trend in Firmicutes, Actinobacteria, and Bacteroidetes representations was revealed. Ding et al. [29] did a follow up on that trial and performed metagenomic analysis on days 7, 11, 15 and 19 of embryo incubation. In total, 3836 operational taxonomical units (OTUs) were identified in gut and yolk in the analyzed embryos. Proteobacteria, Firmicutes, Bacteroidetes, Verrucomicrobia, Fusobacteria, Actinobacteria, and Tenericutes were the microorganisms with the highest abundances found at the phylum level; at the genus level, Pelomonas, Ralstonia, Aquabacterium, Faecalibacterium, Pseudomonadaceae, Asticcacausia and Roseburia were the most abundant in yolk and gut. The main activities of the gut microbiota include nutrition exchange, and as the effect of gut stabilization, the pathogen exclusion and immune system modulation are managed. The bulk of dietary carbohydrates is digested and absorbed in the proximal gut, whereas the remaining carbohydrates are fermented and broken down by bacterial populations in the distal gut. As a result of fermentation of the proteins and carbohydrates, a diverse range of metabolites is excreted, including SCFAs (short chain fatty acids). Due to their dual role: as signaling molecules and energy providers, SCFAs are the key influencers of the health of the host.

7.3 Chicken model in human studies The avian embryo is the only higher vertebrate accessible to research manipulations and analyses, prenatally at various developmental stages. It is easy and relatively inexpensive to implement in laboratory condition. Moreover, during the early incubation days of chicken embryo (by 6 EID) it is possible to perform cellular manipulations and monitor physiological effects in vivo without any detectable discomfort applied to the organism, thus in a full compliance with ethical restrictions. Therefore, the chicken model is gaining increasing appreciation as the alternative pre-clinical model and less sensitive to rodent species. Using the embryonated eggs for prenatal pharmaceutical research, at least for the front line screening trials, save lives of females (which is impossible for prenatal trials in rodents) and ensures the full control over developmental stages of individual fetuses. A limitation of the chicken embryonic model is the lack of relationship of embryo with mother organism, so that the information on metabolism of drug affecting the embryo cannot be provided, whilst it is the case for placental routes in mammals [30]. Nevertheless, a majority of rationales related to use of chicken is fully compliant with the concept of 3R rule to reduce, replace and refine the research methodologies. Regarding the genetics; since the genome of an ancestor of domestic chicken, a female Jungle Fowl (Gallus gallus gallus) was sequenced for the first time, the comparisons between gene maps of human (ca. 20,000 genes) and chicken (ab. 20–23,000 genes) continuously reveal a high representation of conservation synteny, more than between gene maps of mouse and human [31]. Gene orthologs are these genes in different species, which have evolved from the common ancestral sequence by speciation events and retain the same biological functions in the organism [32]. With advancements in

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genomics and bioinformatics, the potential of utilizing chicken as a model organism not only finds momentum for evolutionary and basic research, but the chicken animal model has also been leveraged to pre-clinical studies, which is no longer questioned [33]. Below, several medically relevant research areas are briefly highlighted, in which the chicken model has already been advanced, or emerges as a promising alternative tool:

7.3.1 Embryonic chorioallantoic membrane (CAM) to asses pharmacokinetics, pharmacodynamics and systemic toxicity Assays with chorioallantoic membrane are well established and the most explored in ovo and ex ovo (where embryos are deprived of shells, cultivated in plastic cups) simulation models of intraoral and intravascular administration routes to explore the pharmacodynamics and toxicity of drugs, potentially toxic chemicals including endocrine disrupting chemicals (EDCs), nanoparticles and novel biomaterials [34]. The ex ovo models have been optimized to reach survival rate of treated embryos as high as over 80% and allow for an excellent visualization of implanted biomaterials along with continuous observation of vascularization process during the incorporation and naturalization of the graft [35]. As for toxicological assays of EDCs, various categories of chemicals that pose concerns for human upon exposures are tested, among them dioxins, bisphenols and pesticides. The assays are performed at various developmental timepoints. To provide examples, bisphenol was studied between 4 EID and 18.5 EID, dimethyl sulfoxide between 15 EID and 18.5 EID, perfluorooctanoic acid was studied prior to incubation and on 3 EID, aroclors (chlorinated biphenyls) on day 18 EID, 17β-estradiol hormone on 7 EID. Worth noticing, the lack of blood–brain barrier (BBB) in early stage embryos facilitates toxicological tests on potential prenatal exposure to certain metabolites or nanoparticles [30].

7.3.2 Chicken CAM as onco-immunological embryonic model and towards personalized oncology Chorioallantoic membrane model has been recently optimized for numerous cancer cell lines. What seems to be the most relevant, CAM model has recently been commercialized under the EU project by the research INOVOTION team (https://www.inovotion.com/). Analyzing the progress and targets as they are claimed in the published research, the CAM xenograft models are undergoing standardization to become clinical tools in personalized oncology. The idea is to standardize CAM and other in ovo assays as so called patient derived chicken egg model (PDcE model), proposed already several decades ago [36]. In one of the recent research, Sokolenko et al. [37] grafted to chicken CAM several cell lines of uveal melanoma (UM), belonging to the most common intraocular malignant tumors. The studied UM cell lines were: UM92.1, UPMD2 and UPMM3 and Mel 270 cell

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lines, and the grafts were performed on 11 EID and 17 EID (Embryo Incubation Day). The attachment rate of spheroid tumor was the most effective with the matrigel co-applied, at a rate of 90%. However, the authors also highlighted key issues with high mortality rates of the model embryos or rejections of tumors, which need to be overcome. Vu et al. [38] grafted surgical sample of ovarian tumor from ovarian cancer patient to test the candidate drug efficiency. The obtained results were repeatable, and the rate of successful transplants onto CAM was 4/5, with a rapid tumor formation. A year later, Komatsu et al. [39] applied ovarian cell line OVCAR8 and lung cancer cell line A549 onto chicken CAM. They described in details formation of tumors, and the observations allowed hypothesizing as to how the communication of cancer cells with extracellular environment occurs to recruit the factors required for growth and angiogenesis. An important study was carried out by Harper et al. [40] to understand how the phenomenon of hypoxia condition, which is necessary for expansion of range of tumors, takes place on CAM. Interestingly, the hypoxic zones required by tumors were effectively self-formed upon grafting and incorporation of cancer cells. Several cell lines were used in the study: HT-1080 fibrosarcoma, PANC-1 pancreatic, HCT 116 colorectal, Caki-1 renal, and A549 lung cancer, but also the tumor tissue from renal cancer patients. In any case, the results cannot be directly extrapolated to the tumor behavior in the individual patients. Therefore, a step wise and careful standardization of the PDcE for personalized oncology, should involve -omic methods and sequencing platforms combined with careful immunochemical and histopathological assessments.

7.3.3 Germline cells, and reproductive tissue in cancer research Germ cell tumors (GCTs) occur in most cases in testes and ovaries and may be considered understudied and underestimated due to their embryonal origin. They are developed in young population [41]. GTCs represent the majority of solid malignancies (seminomas) in young men, with increasing incidence. Those tumors express markers of pluripotency, including OCT3/4, NANOG, LIN28 and SOX17. Ontogeny of seminomas is suspected to be preceded by neoplasia in germ cells, and therefore attention is put to histological and gene expression patterns in embryonic primordial germ cells [42]. Based on available genomic assays and recent sequencing, the point mutations causing pediatric cancers were found to be enriched in genes that encode epigenetic mechanisms [43]. Primordial germ cells are precursors of gametes that carry parental genetic material, and colonize the gonads in developing embryo to differentiate in functional oogonia and spermatogonia. PGCs are also undergoing demethylation and de novo methylation, which are the “repair” mechanisms to avoid potentially unfavourable genetic transmissions. It is suggested that migrating PGCs could provide key information to understand the origin of extragonadal tumors, as the PGCs that extravasate during migration can become tumorigenic [44].

Figure 7.2: A, B- Colonies of chicken embryonic oviductal epithelial cells, visualized at magnifications ×5 and ×20. The embryonic oviduct (C) was isolated on day 16 of embryo incubation.

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There is lack of in vitro models to study PGCs migration. A recent study of Saito et al. [45], includes important observations to potentially support tracking of the metastasis of migrating cancer cells and extravasation mechanics through PGCs model. It was visually demonstrated as to how the migrating PGCs may become arrested in vascular capillaries by occlusion. With regard to genetic similarities, middle developmental stages of embryonic reproductive tissue in chicken are similar to humans (differentiation of oogonia, formation of primary and secondary follicles). Recently, over 20 chicken (hen) genes related with development of oocytes and meiosis were found convergent with human [46]. Hen oviduct has become an accepted model to study the origin, potential biomarkers and drugs to combat the ovarian cancer in adult female reproductive tissue. Hen is the only higher vertebrate among non-human model species, in which ovarian cancer develops spontaneously. Sahin et al. [47, 48] studied in hens with ovarian cancer, the effect of daily intake of curcumin as the potential chemopreventive agent, and in curcumin fed animals they revealed less frequent KRAS and HRAS mutations in ovarian tumors and significant reductions in ovarian tumors incidence. We have previously developed successful methods to cultivate chicken (and quail) oviduct cells and these can also serve as supporting, in vitro models (Figure 7.2). As for the promising novel candidate biomarkers, the attention has been brought to miRNAs and specifically to miR-200 gene expression regulator, which is an informative blood biomarker for both human and laying hen ovarian cancer [49]. Summarizing, several key emerging areas for implementation of the in ovo techniques were highlighted in this chapter, showing an unlimited potential for interdisciplinary cooperations, including animal science, mechanical engineering, microbiology, medicine, chemical engineering and specializations thereof, to the real life applications.

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27. Bogucka J, Dankowiakowska A, Stanek M, Stadnicka K, Kirkiłło-Stacewicz K. Effect of synbiotics administered in ovo on microvascularization and histopathological changes in pectoral muscle and the biochemical profile of broiler chicken blood. Poultry Sci 2022;101:101628. 28. Villaluenga CM, Wardeńska M, Pilarski R, Bednarczyk M, Gulewicz K. Utilization of the chicken embryo model for assessment of biological activity of different oligosaccharides. Folia Biol (Krakow) 2004;52: 135–42. 29. Ding P, Liu H, Tong Y, He X, Yin X, Yin Y, et al. Developmental change of yolk microbiota and its role on early colonization of intestinal microbiota in chicken embryo. Animals 2022;12:16. 30. Ghimire S, Zhang X, Zhang J, Wu C. Use of chicken embryo model in toxicity studies of endocrine-disrupting chemicals and nanoparticles. Chem Res Toxicol 2022;35:550–68. 31. Burt DW. The chicken genome. Genome Dyn 2006;2:123–37. 32. Fang G, Bhardwaj N, Robilotto R, Gerstein MB. Getting started in gene orthology and functional analysis. PLoS Comput Biol 2010;6:e1000703. 33. Beacon TH, Davie JR. The chicken model organism for epigenomic research. Genome 2021;64:476–89. 34. Ribeiro LNM, Schlemper AE, Da Silva MV, Fonseca BB. Chicken embryo: a useful animal model for drug testing? Eur Rev Med Pharmacol Sci 2022;26:4828–39. 35. Chen L, Wang S, Feng Y, Zhang J, Du Y, Zhang J, et al. Utilisation of chick embryo chorioallantoic membrane as a model platform for imaging-navigated biomedical research. Cells 2021;10:463. 36. Dagg CP, Karnofsky DA, Roddy J. Growth of transplantable human tumors in the chick embryo and hatched chick. Cancer Res 1956;16:589–94. 37. Sokolenko EA, Berchner-Pfannschmidt U, Ting SC, Schmid KW, Bechrakis NE, Seitz B, et al. Optimisation of the chicken chorioallantoic membrane assay in uveal melanoma research. Pharmaceutics 2022;14:13. 38. Vu BT, Shahin SA, Croissant J, Fatieiev Y, Matsumoto K, Le-Hoang Doa T, et al. Chick chorioallantoic membrane assay as an in vivo model to study the effect of nanoparticle-based anticancer drugs in ovarian cancer. Sci Rep 2018;8:8524. 39. Komatsu A, Matsumoto K, Saito T, Muto M, Tamanoi F. Patient derived chicken egg tumor model (PDcE model): current status and critical issues. Cells 2019;8:440. 40. Harper K, Yatsyna A, Charbonneau M, Brochu-Gaudreau K, Perreault A, Jeldres C, et al. The chicken chorioallantoic membrane tumor assay as a relevant in vivo model to study the impact of hypoxia on tumor progression and metastasis. Cancers 2021;13:1093. 41. Vishnoi V, Liebenberg P, Reid F, Ward A, Draganic B. A primary germ cell tumour in the gastrointestinal tract: a caecal lesion of yolk-sac morphology in a young patient. J Surg Case Rep 2018;11:291. 42. Oing C, Skowron MA, Bokemeyer C, Nettersheim D. Epigenetic treatment combinations to effectively target cisplatin-resistant germ cell tumors: past, present, and future considerations. Andrology 2019;7:487–97. 43. Barbet V, Broutier L. Future match making: when pediatric oncology meets organoid technology. Front Cell Dev Biol 2021;9:674219. 44. Roelen BAJ, Chuva de Sousa Lopes SM. Stay on the road: from germ cell specification to gonadal colonization in mammals. Philos Trans R Soc B 2022;377:20210259. 45. Saito D, Tadokoro R, Nagasaka A, Yoshino D, Teramoto T, Mizumoto K, et al. Stiffness of primordial germ cells is required for their extravasation in avian embryos. iSciene 2022;25:105629. 46. Rengaraj D, Han JY. Female germ cell development in chickens and humans: the chicken oocyte enriched genes convergent and divergent with the human oocyte. Int J Mol Sci 2022;23:11412. 47. Kazim S, Cemal O, Mehmet T, Nurhan S, Hakkı T, İbrahim HÖ, et al. Chemopreventive and antitumor efficacy of curcumin in a spontaneously developing hen ovarian cancer model. Cancer Prev Res (Phila) 2018;11:59–67. 48. Sahin K, Yenice E, Tuzcu M, Orhan C, Mizrak C, Ozercan IH, et al. Lycopene protects against spontaneous ovarian cancer formation in laying hens. J Cancer Prev 2018;23:25–36. 49. Choi PW, Bahrampour A, Ng SK, Liu SK, Qiu W, Xie F, et al. Characterization of miR-200 family members as blood biomarkers for human and laying hen ovarian cancer. Sci Rep 2020;10:20071.

Joanna Bogucka and Katarzyna Stadnicka*

8 Quality of poultry meat- the practical issues and knowledge based solutions Abstract: Animal protein is the most demanded and expensive source of nutritive protein, globally. Taking into account various types of poultry, the broiler (meat-type poultry) is widely accepted by various religious societies and relatively cheap amongst others animal protein sources. In particular, the chicken and turkey product is perceived to be healthier and of better quality due to a low content of fat, cholesterol and sodium compared to red meat. In order to maintain an unabated development and competitiveness of poultry industry, the priority is to focus on quality and safety of meat, during whole production and processing route. Consumers awareness of what should be considered a high quality product is constantly increasing, especially in the light of European and worldwide strategies to meet the common societal and environmental challenges, i.e. addressing the Zero Hunger goals, Green Deal and One Health concept. In this chapter, a common area of interest for a dialogue of poultry scientists and industrial practitioners is drawn from the background given on the consumer (demands and health)-centered issues. Keywords: meat quality; myopathies; One Health; poultry.

In recent decades, poultry meat production sector has reached peaks of productivity, by the highest rates of muscle tissue growth, as well as an increasing proportion of muscle tissue mass within the total body weight. However, this success in efficiency has some negative consequences. Firstly, the sensory, organoleptic and technological properties of meat quality have substantially changed. Genetically driven fast growth rates in fast-growing birds have resulted in certain negative health and welfare consequences that damagingly affected raw meat products, namely by causing morphological and biochemical modifications of muscle tissue [1]. In fast-growing hybrid chicken breeds, the idiopathic myopathies (pathological changes in muscles) have emerged and become and emergency issue. Among them, the myopathies that are presumed to be stress-driven, are of a priority significance for the deteriorated quality of meat. Myopathies usually affect the most valuable parts of carcass, which is

*Corresponding author: Katarzyna Stadnicka, Faculty of Health Sciences, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Lukasiewicza 1, 85-821, Bydgoszcz, CuyavianPomorenian, Poland, E-mail: [email protected]. https://orcid.org/0000-0002-2349-7203 Joanna Bogucka, The Independent Research Laboratory STANLAB LLC, Nakło nad Notecią, Poland, E-mail: [email protected] As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: J. Bogucka and K. Stadnicka “Quality of poultry meat- the practical issues and knowledge based solutions” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2021-0121 | https://doi.org/10.1515/9783110683912-008

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the breast muscle tissue (the pectoralis major muscle). Therefore, significant economical losses are the consequence of these pathological changes [2, 3].

8.1 Major issues related with poultry meat quality, including myopathies and other defects An increasing demand for poultry meat has led to a gradual improvement in genetic pressure to obtain fast growing broiler chickens. There are two major consequences of this selection: maximally increased body weight gain and a shortened fattening period. Over the last 60 years, the fattening phase of broilers has declined by almost half, from 72 days to about 40 days. Whilst, the body mass of a bird sent to a slaughter has nearly doubled, or in some breeds increased even by 3–4 times. Such intensification of production has some obvious, negative consequences. The rapid growth has been accompanied by increasing immunodeficiency and changes in musculature [4]. Broiler breeding programs are known to be primarily focused on reaching higher productivity and slaughter yield. In particular, the goal of breeders was to increase the mass of breast muscles. However, the genetic pressure has been contradicting the natural physiology of a bird, leading in the end to pathological changes known as myopathies. Muscle development is considered to be in the center of negatively affected processes. Myogenesis is a process of formation of new muscle cells (myocytes)/ muscle fibers, during embryogenesis and early postnatal period. The newly formed muscle mass is for the most part determined by the total number of muscle fibers (hyperplasia-increase in number of fibers) and increase of their thickness (hypertrophy-enlargement of the fiber’s diameter). Hyperplasia occurs during embryogenesis or early after hatching. The hypertrophy happens predominantly after hatch (Figure 8.1). An intensive gain in mass and size of breast muscles in broilers has caused an excessive enlargement of muscle fibers, and affected the structure of blood vessels of skeletal muscles, which may potentially worsen the quality of raw meat product. The major physiological causes are ischemia, anoxia and reduced removal of metabolic wastes from muscle cells [4, 5]. The defects in muscle structure, namely breast muscle myopathies (BMM) negatively affect the consumption and technological properties of meat [3, 6]. As a consequence, BMM negatively influence the consumers choices and cause serious economical loss for the producer. Basic differences in microstructure of breast muscle of a fast growing breed Ross 308 broilers compared to the slow growing, multi-purpose domestic Green-legged Partridge like hen, are presented in Figure 8.2. On 42 day of life, the breast muscles of Ross 308 broiler are characterized by less than half number, but much larger diameters of muscle fibers compared to the Green-legged Partridge like. A rapid increase of muscle mass in relatively short period causes spontaneous, idiopathic abnormalities in muscles accompanied by the greater susceptibility to

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Figure 8.1: Myogenesis and muscle growth in chickens.

stress-driven myopathies. The most frequent myopathies occurring in broiler chickens are: DPM (Deep Pectoral Myopathy) – degenerative muscle disease, myopathy of deep muscles known as Green Muscle Disease, WS (white striping), WB (wooden breast syndrom) and so called Spaghetti meat-myopathy related with immaturity of connective tissue, manifesting loss of integrity of the breast muscle, and mushy structure resembling “spaghetti”. All myopathies are a consequence of damages in muscle fibers, destruction of blood vessels or intramuscular connective tissue. The myopathic spectrum of syndromes lead to regressive changes including atrophy, tissue degeneration and necrosis. In animals, there are two major categories studied: stress-induced and nutritional myopathies. It was shown, that one of the basic mechanisms of development of myopathies are the following successive processes: damage in the myocyte (muscle cell), damage of the cell membrane and release of mitochondrial calcium, which in turn causes hypercontraction, dystrophic changes, atrophy and necrosis of muscle fibers [3]. The most frequent histopathological changes among the myopathies, are: 1. Degeneration – state, in which the substances that physiologically do not occur at all, or appear only in trace amounts, are beginning to emerge in cells, or extracellular matrix. For example, the hyaline degeneration is resulting from deposition of a protein (hyaline) among the muscle fibers and inside them. The accumulation of this protein inside the fiber leads to obliteration of the transverse striation. On the other hand, the hyaline deposits between muscle fibers may lead to pressure on the capillaries, resulting in reduced nutrients absorption and impairment of muscle gas exchange, and in the end, resulting in muscle atrophy or necrosis. In addition, hyaline is dependent upon calcium ions and such has affinity to Ca binding, contributing to the calcification of muscle tissue.

Figure 8.2: (Bogucka J.). Microscopic image of a slow- (A) – Green Legged Partridge, 42 d., BW = 446 g, HE. Mag. 200×) and fast-growing and (B) – Ross 308, 42 d., BW = 3130 g, HE. Mag. 200×) chicken’s muscle.

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Size heterogeneity – fiber atrophy and giant fibers. Atrophy is a gradual decrease in cell size due to several factors: e.g., hypoxia, denervation (loss of nerve supply), or due to other degenerative changes. On a contrary, giant fibers (hypercontracted fibers) are excessively increased in diameter. Giant fibers are usually easily recognized under microscopic picture, as they are round in shape and have a homogeneous cytoplasmic structure. On the longitudinal sections, no transverse striation is observed. Giant fibers are formed by excessive fiber activity. Many authors explain the occurrence of giant fibers by susceptibility of animals to stress; Necrosis with phagocytosis, are the states leading to sudden cell death. They are the consequence of degenerative processes and fiber destruction. The fiber breakdown is accompanied by the emergence of phagocytic cells. This condition is called necrosis with phagocytosis; Connective tissue hypertrophy, is a typical feature of the histopathological image of myopathy. Overgrowing connective tissue may compress capillaries in the muscle, which leads to hypoxia of the fibers and degenerative changes. The connective tissue hypertrophy is often accompanied by adipose tissue hypertrophy; Splitting, is initiated by fractures, that partially divide/split the fiber. In the next phase, one or more parts of the fiber are completely separated, followed by the ingrowth of connective tissue between the individual parts. Daughter fibers possess individual, separate sarcolemma’s membrane, but they share endomysia sheath. Usually, a split fiber is formed out of the two, up to five daughter fibers. The causes underlying the degenerative splitting are associated with increased stress of the animal, or a ‘functional overload’ in some of the hypertrophied fibers. The split fiber fragments may continue to degenerate or enlarge; Increasing number of fibers; Moreover, the large spaces between bundles of muscle fibers and leukocyte infiltrations are found in myopathies [4, 7].

In terms of economical loss and consumers’ (buyers) complaints, the most significant, emerging types of myopathies occurring in poultry, are briefly characterized below: 1. DPM (Deep Pectoral Myopathy); is a pathological change found in deep breast muscles (supracoracoideus or minor pectoral muscle) caused by necrosis and atrophy of the tenders. It is also referred to as Oregon disease or Green Muscle Disease. It was first described as a ‘degenerative myopathy’ in breeding turkeys in 1968, in Oregon, USA. DPM is observed in meat type breeding poultry, that were subject to a process of genetic selection for the high yield of breast muscles. The disease affects pectoralis minor muscle (located deep to the pectoralis major), which is responsible for lifting the wings. Anatomically, Pectoralis minor is enveloped with nonelastic fascia and a breastbone, which together restrain the physiological swelling of a muscle mass during movement (wings fluttering). As a consequence, the blood pressure increases and the muscle size may be enlarged by even 20%. The excessive tension leads to anoxia, ischemia and necrotic changes. Often, DPM is referred to as ischemic

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necrosis [8, 9]. Most probably, the transformation of myoglobin and hemoglobin in anaerobic condition are causing the green color of muscles (Figure 8.3). Muscles with DPM are characterized by change of color and tissue texture. The observed symptoms are petechial hemorrhaging, swelling, extravasation and change of color of the muscles. However the meat affected with DPM is considered as safe for the customers, the buyers would never select it. Such a meat is aesthetically undesirable and causes severe economic loss for poultry industry, especially that DPM affects the valued part of the carcass. The study results show that unfavorable changes occur both in major (breast fillet) and minor (tender) pectoral muscles with DPM, even if the changes would not be seemingly apparent in the major muscle. The digital image analysis shows the histological traits of muscle fibers with a broad spectrum of pathological changes in structure. Analysis of cross-sectional area of the smaller breast muscle with DPM, reveals many large spaces with muscle fibers’ bundles [2, 4, 10]. 2. White striping (WS) is an incidence of white stripes, mostly found in pectoralis major, in meat type poultry. White stripes are related with a higher fat and connective tissue contents in muscles, being the consequence of fibers’ necrosis. White striping is located parallel to the direction of fibers. In classification, the intensity of this myopathy is assessed visually, and categorized as: normal, moderate and severe [11]. This myopathy is a result of an extremely intensive feeding of the birds, leading to enlargement of the adipocytes (fat cells), loss of cross-striations and changes in size, or even to the lysis of fibers, infiltration of immune cells, lipidosis and overgrowth of the connective tissue component (fibrosis) [12] (Figure 8.4). The WS myopathy has become an issue to the global poultry market, due to several aspects: unfavorable look of the degenerated meat, unwillingness to purchase, mainly due to the excessive fat content, loss of a nutritive value (protein) due to the increased total collagen content [12, 13]. Paradoxically, although in WS meat the nutritional value is worsen due to an increased fat content, it is still considered rich in protein and low in fat compared to other meat sources [6]. Moreover, the consumers may be discouraged by the negative effect of WS on technological properties of poultry meat, related with a decreased water holding capacity and a softer texture compared to the normal meat [14]. 3. WB (wooden breast) is an essentially undesirable defect, due a low technological value of the meat, which is hard, pale and dry. The breast muscle gets stiff, most frequently in the proximal part of a fillet. The muscle tissue is assessed organoleptically (manually) by sensing the breast muscle tense and intensity of stiffness (‘woodiness’). Depending on the advancement of pathological changes, various features are observed: a lighter color, superficial bleeding, or presence of an exudate on the muscle’s surface [15]. The histological analysis shows an active degeneration and regeneration of the muscle fibers, and infiltration of the leucocytes, with an increased deposition of a fat and connective tissue. These features make the wooden breast being classified as a myodegeneration (muscle fibers’ necrosis), with fibrosis and

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infiltration of macrophages (Figure 8.5). Fibrosis is a result of a necrotic processes, and leads to the synthesis of an excessive connective tissue and to the replacement of proteins which are typical for the muscles with a rich collagen cross-linking, as the myopathy upregulates expression of genes related with collagen synthesis and crosslinking. The latter make the meat look hard as wood [16]. Moreover, the RNA sequencing revealed a range of unfavorable biochemical mechanisms on cellular level: a local hypoxia (lower oxygen concentration), oxidative stress, an increased cellular concentration of calcium, lower glycogen content and trends in changes of metabolic profile in muscle fibers, as the key defects found in WB [17, 18]. 4. „Spaghetti meat” is a quality defect found in poultry meat, which is related with a poor cohesion of a muscle, resulting from immaturity of the intramuscular connective tissue. Intramuscular connective tissue is composed of three sheaths endomysium, perimysium and epimysium, which envelop fibers, muscle fibers’ bundles and the entire muscle. In SM meat, the density of a connective tissue in endomysium i perimysium gradually decreases, and the perimysium sheaths get thinner. As a result, the bundles of muscle fibers fall apart or become ‘mushy’, resembling ‘pasta’ (spaghetti) [19, 20]. The meat with this myopathy is loose in structure and the bundles of muscle fibers can be easily separated with fingers (Figure 8.6). Physiologically, this type of myopathy is resulting from immaturity of a collagen, which is a major component of a normal, intramuscular connective tissue, forming the scaffold to maintain a structural integrity of skeletal muscles, and playing a major role in determination of hardness of the meat [21]. After cooking, the SM becomes very soft and disintegrated. Therefore, SM is eliminated from the raw meat market and can be used only in processed meat products. Such a limitation causes significant financial loss in poultry industry [22]. 5. Another significant pathological changes are found in a meat classified as PSE (Pale, Soft, Exudative) and DFD (Dark Firm Dry). Both defects are the consequence of abnormal energetic metabolism in muscles. In case of PSE syndrom, it is already during and immediately after slaughter of birds, that a sudden and rapid anaerobic glycogenolysis occurs, leading to a fast and excessive cumulation of a lactic acid in muscles, which in turn causes a sudden reduction of pH. Rapid and massive drop of pH together with an increased temperature, cause denaturation of proteins in muscles. As a consequence, the water holding capacity by proteins, is considerably reduced. The meat becomes watery and soft, with a pale color due to denaturation of myoglobin (the modified structure of muscle proteins leads to a decreased reflection of the light). The ethiology of DFD syndrome is the same as for PSE and it is also related with energetic organization in muscles. The major difference is related with time, in which a sudden breakdown of a muscle glycogen occurs. The damage occurs still in the living animal, prior to slaughter. The produced lactic acid is neutralized by the partially preserved blood circulation, however the glycogen is no more recuperated. Thus, at slaughter, the muscle tissue has low ATP, glycogen and lactic acid levels, whilst the pH is high. There is no post-slaughter acidification

Figure 8.3: (Bogucka J.). Macro- (A) and microscopic (B) image of a meat with deep pectoral myopathy (DPM). In the image B, there are pathological changes in muscle fibers’ structure (G – giant fibres, S – splitting) and numerous, large spaces among the fibers shown, as well as infiltration of leucocytes. HE staining, magn. 100×.

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Figure 8.4: (Bogucka J.). Macro- (A) and microscopic (B) images of a meat affected by white striping (WS). In the image B, there are visible, degenerative changes in muscle fibers’ structure (N – necrosis with phagocytosis, S – splitting). HE staining, mag. 100×.

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Figure 8.5: (Bogucka J.). Microscopic image of the wooden breast (WB) meat. There are various pathological changes observed: CTH – connective tissue hypertrophy, G – giant fibre, S – splitting, N – necrosis with phagocytosis, atrophy (arrows). HE staining, mag. 100×.

of the muscle tissue, and the meat has a high water holding capacity, causing the so called “closed” meat structure, tough in texture with a dry look. Too high pH prevents the activation of autolytic enzymes, especially of the proteolytic ones, thus the meat remains hard, and is not getting tenderized. Moreover, the high pH facilitates growth of putrefactive microflora. Both of the mentioned meat defects are caused by stress factors, genetic predisposition, nutrition and pre-slaughter handling. Nevertheless, the main cause of PSE and DFD meat is a genetic susceptibility of birds to stress. Therefore, in order to prevent the occurrence of meat quality defects, it should be recommended to genetically select the breeds for stress resistance, and not only for high yield [23]. Cases of DFD meat can be limited if one understands that these deficiencies are initiated by the muscles fatigue/exhausted energy supplies (glycogen) prior to slaughter. The animal keeping and handling ante-mortem, are key factors, which significantly affect the quality of poultry meat. That is why, the treatment of an animal prior to slaughter, including heat stress, caging, transportation and feeding, are important aspects to maintain not only the expected quality, but also welfare of the birds [24]. Incidence of PSE meat in poultry is resulting from complex factors and requires selection of birds that would be stress resistant [25–28].

Figure 8.6: (Bogucka J). Macro- (A) and microscopic (B) images of a spaghetti meat type (SM). In image B, there are degenerative changes of muscle fibers shown, varying sizes of the fibers and poorly packed, loose, immature fiber bundles. HE staining, mag. 100×.

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8.2 Research and innovations to improve poultry meat quality Research on improvement of poultry meat quality is mainly focused on preventive measures. Major factors that influence variation in meat quality, are (1) genetics: species, breed, age and sex, (2) the environmental components: rearing condition (access to the run space, size of the surface occupied per bird, layout of the keeping space). The organoleptic and nutritional values depend on a type of muscle, handling ante-mortem and carcass storage conditions. The fodder used for birds also influences the organoleptic, nutritional and technological properties of poultry products [29]. Knowing the underlying myopathies that worsen the meat quality (myopathies caused by stress, and nutritional myopathies), there are attempts to prevent their incidence. As for nutrition, the compound feeding stuffs can be modified (e.g., by adding dietary supplements, antioxidants, organic minerals, vitamins and aminoacids). However, according to researchers [30, 31], implementation of such nutritional strategies in commercial conditions can be problematic, because reduction of frequency of myopathies can be considered as an indirect consequence of a decreased finished weight and the size of breast muscles [32]. On the other hand, genetic selection for increased body weight gain, has reached the pivotal point and the further increase trend may be limited by the biological potential of muscles and the welfare aspects [33]. As Velleman suggests [34], it may be interesting to pay attention to the mechanisms of producing extra muscle fibers during embryogenesis (hyperplasia), instead of relying on the post-hatch selection only, which aims to achieve the increased muscle mass through increased thickness of muscle fibers (hypertrophy). Understanding molecular and histological basis of growth and development of muscle tissue that determine, to a vast extent, the produced mass of skeletal muscles, may be an efficient way to elaboration of new methodologies to improve the yield and meat quality. Especially, if the embryogenic developmental aspects of myogenesis and functions of satellite cells would be included and comprehended in the new strategies [35]. There is a novel study direction called nutrigenomics, aim of which is to elucidate the effect of nutritional ingredients on gene expression and meat quality. Some of the newly defined dietary components are adaptogens and regulators of muscle tissue development. Revealing functions of these components and their application, have a potential to increase the stability of muscle tissues and resistance to the environmental stress factors [3]. Also, the research on application of various bioactive ingredients, both prenatally (in ovo) and addition to feed and/or water shown that the natural bioactive compounds have potential to beneficially modulate the microstructure of breast muscle in poultry. Majority of this research has revealed a specific mode of action the studied components (prebiotics and synbiotics), as they improved the angiogenic process in muscle tissue, likewise a reduction of ischemic changes and myopathies was observed rather than influence on thickness or a number of fibers [36– 38].

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8.3 The quality aspects of raw materials and processed meat, including the chemical additives incorporated to the meat. Opinion – what needs to be done? Meat and meat products are an important source of nutrients like proteins, lipids, mineral ingredients, vitamins and others. According to the Regulation (EC) No 853/2004 on specific rules on the hygiene of food of animal origin for food business operators, the meat, by definition, means edible parts of the animals, including internal organs and blood. Meat is composed of approximately 72–75% water, 21% nitrogenous compounds (19% proteins and 1.5% nonprotein nitrogen compounds which include nucleotides, peptides, creatine and creatinine), 2.5–5% lipids, 1% non-nitrogenous compounds (vitamins) and carbohydrates (a very small amount of glycogen, transformed into lactic acid during postmortem period) and 1% ash (potassium, phosphorus, sodium, chlorine, magnesium, calcium and iron). The composition of meat is complex, dependent on numerous factors, like species, breed, age, physical activity/fitness and nutrition. On the other hand, the meat preparations, by definition, are food including meat with addition of foodstuffs, seasonings or additives added to it, but with retained internal muscle fibre structure of the meat. Meat has been processed into various products due to a need for a long term storage, to maximally utilize the carcass, but also to improve the taste, increase diversity of food and for convenience. Composition of meat preparations differs depending on the type of raw materials and the methods applied for preservation (pickling, salting, drying, heating, etc.), which in turn define the qualitative and sensory attributes of meat. Novel strategies that have been applied and emerged from scientific knowledge, are essentially based on health promoting, functional products (e.g., prebiotics, probiotics, CLA-Conjugated linolenic acid, fiber, herbs), that through their mechanisms of action, lead to reduction of some components that may cause pathological changes [39, 40]. Changes in meat, that are related with various processing technologies and preservation methods, can be systemized as: physical and chemical. Physical changes are modifications in tissue structure, that affect certain sensory traits, like size, look, color, texture, flavor and aroma. Chemical changes in meat are part of concerns associated with food safety, and are caused by molecular interactions occurring during the thermal processing, addition of food additives, or a prolonged storage. There is a need for technologies that would ensure food safety whilst satisfying the consumers’ demands without compromising the nutritional value of traditional meat products [41, 42]. One of the precise methods to evaluate the meat preparations, is the histological assessment (Figure 8.7). The histological examination enables a direct identification of different tissue types that happen to be present in meat products. It is a complementary

Figure 8.7: (Bogucka J.). Histological methods of meat product assements, (A) skeletal muscle with connective tissue; Calleja’s staining. Mag. 100×, (B) defragmentation of muscle fibers; Calleja’s staining Mag. 100×, (C) minced meat; Calleja’s staining. Mag. 100×, and (D) the meat filling; Calleja’s staining. Mag. 100×.

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test, and often a verification tool for other assessment protocols. The histometric analysis is, next to the chemical analysis, a valuable source of information on composition and quality of the product [43, 44]. In meat industry, there are various functional additives applied, apart from the meat and animal fat material. Supplementary and additional chemical substances are intended for obtaining desired features of the meat products (more attractive sensory characteristic and increased availability: shelf life, easiness in portioning, short preparation time), improvement of technological processing, increased consumption safety. The regulations regarding the use of supplementary substances as well as the list of allowed additives is included in the Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. The use of food additives in food processing needs to be technologically justified, and the consumer has a right to access all the information on the additives used (name, E number, technological function), displayed on the product label. The basic food additives in meat processing are: – Pigments (nutritive or aromatic colors, natural or synthetic, e.g., capsanthin-paprika extract, betanin-beetroot red, curcumin, β-carotene, carmines) – Preservatives-use of thereof allows to prolong the shelf-life of foods by protecting them against deterioration caused by micro-organisms, and reduction of unfavorable chemical processes (e.g., nitritates and nitrates) – Acidity regulators-to control the acidity or alkalinity of a foodstuff; the regulators influence the taste through a change of pH, favorably affect the microbiological stability of a product, and may accelerate or slow down the enzymatic processes (e.g., lactates, acetates, citric acid, citrates) – Antioxidants-protect against deterioration caused by oxidation; reduce the lipid oxidation (both naturally occurring in meat, likewise of the added fat); prevent from the change of color (pigment oxidation) and flavor; some of acidity regulators are also antioxidants (e.g., ascorbic acid) – Stabilizers and emulsifiers-stabilizers maintain the physico-chemical state of a foodstuff through preventing the uncontrolled and undesirable changes during production and storage (e.g., the phosphoric acid, phosphates); emulsifiers maintain a homogenous mixture of two or more immiscible phases such as oil and water in a foodstuff; especially to improve the texture of a meat filling and homogeneous distribution of grease drops (protein preparations) – Flavour enhancers-enhance the existing taste and/or odour of a foodstuff (e.g., herbs, and mixes or standardized mixes of spices and extracts) – Firming and gelling agents-influence the texture through formation of a gel; these substances increase water holding capacity, texture, portioning and strengthens (firm) the slices; improve the juiciness, lubricity, prevent the thermal drip and fat loss from the final product (e.g., caragana preparations, locust bean gum, guar gum, xanthine gum, konjac).

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Apart from the permitted substances, there are ingredients added to the food, in processing of meat. There is no need to include the E number of the labels, and their use in technological processes should be performed with regard to the good manufacturing practice. The mentioned ingredients are, e.g.,: salt, plant protein preparations (mainly soy proteins) and animal proteins (milk proteins, collagen, gelatin, blood plasma), carbohydrate ingredients, e.g., starch (potato, corn) and sugar (saccharose, glucose), natural spices or spice mixes (naturally occurring flavourings, and smoke flavourings identical to natural ones), as well as the enrichment ingredients, e.g., bioactive compounds [45]. A raw material used in meat based preparations is the mechanically separated meat (MSM), which is obtained by removing meat from flesh-bearing bones after boning or from poultry carcases. It is done by mechanical means resulting in the loss or modification of the muscle fibre structure. For this reason, MSM is excluded from the definition of meat according to the Regulation of EC no. 853/2004. Thus, it is required to clearly indicate if a product belongs to MSM, and indicate the animal species from which it is originating (poultry or porcine). There are several methods of assessment of MSM in meat preparations, some of them described by Wilhelm et al. [46]. Figure 8.8 shows the microscopic image of a bone part detected in the product, stained with Alizarin red.

Figure 8.8: (Bogucka J.). A microscopic image of a bone part, stained with Alizarin red stain, in MSM-mechanically separated meat product; Mag. 100×.

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Summarizing, meat and meat preparations are an important part of a human diet, and a key protein source, especially during the growth of young organisms. For several years, an increasing trend of meat consumption has been constantly recorded. Meat is a source of energy and highly nutritional ingredients, including essential amino acids, proteins of high biological value, minerals (iron, zinc, selenium, manganese and vitamins from group B, especially B12). On the other hand, a high consumption of processed meat may be associated with an increased risk of certain diseases [40]. Therefore, the aim of researchers and producers should be collaborative engagement not only to elaborate novel methods of healthy and sustainable production, but in parallel, development of reliable, knowledge-based methods of the quality assessment.

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Index absorption 8 absorption surface 54 acetate 49 acidifier 28 acidity regulators 15 adeno-associated virus 163 adenovirus 163 adherens junctions 53 agricultural production 107 albumen 156 alfalfa 26 alkaloid 24 allantoic fluid 172 alternative source 143 amino acid composition 26 amino acid transporter 7 ammonia 3 amnion 174 analytical chemistry 115, 117, 120, 124, 125 anas platyrhynchos 17 animal growth 100 animal product 30 antibiotic 41, 79, 143, 174 antibiotic growth promoters 42, 79 antibiotic resistance 2, 44, 100 antibiotic-resistant bacteria 44 antifungal activity 77 antimicrobial peptide 4 antinutritional effect 23 antinutritional factor 23 antioxidant properties 78 antiparasitic activity 78 artificial insemination 162 artificial selection 152 atrophy 5 avian embryo 155, 177 avian influenza 174 avian model 152, 155, 158, 160, 164 bacillus thuringiensis 30 bacteria 2, 98, 99, 107, 108 bacteriocins 48 bacteroidetes 9 barley 19, 21 beak 3

https://doi.org/10.1515/9783110683912-009

beta-glucan 21, 58 bifidobacterium spp. 48 bile 7 bioavailability of feed components 51 biological value 13 biologically active substances 75 biology 130 bio-pharm 163 biopharmaceutical 156, 163 bioreactor 153, 155–158, 160, 161, 163, 164 biosecurity 100 body temperature 108 bombesin 5 bone 14 breast muscle 12, 30 breeding 136, 152, 153, 158, 159, 164 broiler 2, 175 broiler chickens 21 broiler production 136 butyrate 10, 49 caecum 6 cairina moschata 17 campylobacter 8 carbohydrate 25 carcass 14, 16 cell line 180 chemical composition 18 chemometry 119 chemopreventive agent 182 chicken genome database 153 chicken model 178 chimaera 156 chimeric gene 156 chorioallantoic membrane:CAM 179 chromatographic techniques 124 claudins 53 clostridium 10, 9 clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 153 coccidiostat 28 cold stress 134 colon 6 CRISPR-Cas9 153, 159, 160, 162, 163

206

Index

crop 4 crude fibre 11 cysteine 2, 7 dark firm dry meat 7 day-old male chicks 172 dehulling 25 development 97 digestibility 22–24, 51 digestion 109 digestive tract 3, 20, 98 dioxins 116, 120, 122 dioxins and polychlorinated biphenyls 122 disease 109, 174 DNA sequence 143 DPM 6 ecological footpring 135, 143 ecosystem 8 EFSA 176 egg 18, 105, 172 egg protein 155 eggshell 18 egg white 155, 156, 163 EID 176 eimeria 29 electroporation 162 embryo 105, 169 embryo incubation day 176 emission 136 endocrine disrupting chemical:EDC 179 energy value 22 environment 128 environmental factor 1, 132 epigenetic regulation 131 epigenetics 174 esophagus 3 essential oils 74 EU Regulation 1831/2003 45 European green deal 176 exogenous DNA 161 extracts 74 extravasation 182 extrusion 25 fattening 16 feather 16 feathering 17 feed additives 116

feed production 136 feeding 136 fermentation 10 fiber 2 fibrosis 7 finisher 12 firmicutes 9 flavonoid 29 flavour enhancers 15 food additive 13 food production 115, 116, 120 food safety 13 foodstuffs 13 forage 15 fructooligosaccharide 29, 56, 58 GALT 56 gastrointestinal tract 98 gelling agent 15 gender 172 gene editing 172 genetically engineered poultry 152 genetically modified chicken 152, 157 genetics 136 genome editing 152, 153, 157–160, 164 genome modification 152, 153 germ 22 germ cell tumor:GCT 180 germline chimera 158 ghrelin 4 giant fiber 5 gizzard 6 global warming 136 glucose transporter 7 gluten 19 glycoalkaloid 26 glycogen 7 gosling 15 graft 180 green muscle disease 5 grower 11, 12 growth performance 21 gut 96, 176 gut barrier 73 gut microbiome 70, 98, 128 gut stabilizer 177 hatchery 173 HDR 160

Index

health 1, 95, 97, 143, 169, 170 heat stress 104, 128, 134 heavy metals 116, 120, 121, 123, 126 hen 13, 182 herb 29 herd immunity 103 histological examination 13 homology-directed repair 153 host 109 host organism 109 humidity 132 hydrogen sulfide 3 hydrophobicity 76 hyperplasia 12 hypertrophy 12 immune response 131 immune status 10, 96, 104, 106, 143 immunity 75, 106, 110, 170 immunoglobulins 56 immunomodulation 106 immunostimulant 175 immunostimulatory properties 75 immunosuppression 104 in ovo 130, 169 in ovo experimentation 155 in ovo feeding 175 in ovo injection 103 incubation 132, 170 incubator temperature 132 infection 108, 110 infiltration 6 inflammation 105 ingredient 18 injection 174 innate immunity 105 intestinal bariere 53 intestinal mucosa 106 intestinal structural integrity 73 intestinal villi 100 intestine 109 inulin 29 inulin type fructans 57 isomaltooligosaccharides 58 lactic acid 5, 49 lactobacillus 9 lactobacillus spp 48 lactulose 56

lectin 24 legume 22 lentiviral vector 163 light program 15 liposome transfection 161 livestock 1, 103, 135 lupin 22 lymphoid tissue 10 lysine 19 lysozyme 156 maize 21 management, 136 mannan oligosaccharide 57 maternal stress 131 meat 2, 10 meat structure 10 mechanically separated meat:MSM 16 mechanisms of antibacterial action 42 meganucleases 153 metabolism 2 methylation 131 microbiome 96, 100 microbiota 54, 97, 177 microclimate 128 micronization 25 migration 180 monogastric animal 19 mucus 4 mulard 17 muscle 3, 132 mycotoxins 122 myodegeneration 6 myopathy 1, 6 natural antibodies 105 necrosis 3 NHEJ 160 nitrogen conversion 128 non-digestible oligosaccharides 70 non-homologous end-joining 153 non-protein nitrogen 27 nutrient utilization 21 nutrition 104, 123, 130 nutritional myopathy 12 nutritional value 22, 25, 26 oat 19 occludins 53

207

208

Index

offspring 129 of total antioxidant status 79 onehealth 95 osteoporosis 13 ovalbumin 156, 159, 160 oviduct 156 oviduct cell 182 pale, soft, exudative:PSE 7 pea 22 pectoralis minor 5 pellet 14 pesticide 120, 121 PGC 157, 158, 160, 163, 164 pharmaceutical 156, 157, 160, 161, 163, 164 phosphorus 12, 28 phytobiotics 74, 79 piggybac vector 163 plant breeder 3 plant protection products 121 plasmid 162 pollution 128 pollution and residues 120 polyaromatic hydrocarbons 123 polycyclic aromatic hydrocarbons 120, 121, 125 polyethylenimine 162 polysaccharide 25 polyunsaturated fatty acids 11, 78 potassium 12 potato protein 26 poultry 1, 96, 128, 169 poultry production 99, 135 poultry science 115 pre-clinical 169 pre-clinical study 179 prebiotics 13, 56, 29, 59 precise gene alteration 160 prelay diet 18 preservation methods 13 primordial germ cell 156 primordial germ cell:PGC 180 probiotics 2, 47, 79, 177 probiotic microorganisms 48 processed meat product 7 productivity of animals 54 propionate 49 protein 11 proteolysis 10

proventriculus 5 pyrimidine glycoside 25 quail 133 recombinant protein 153, 155, 156, 164 reflux 7 relative humidity 134 requirement 14 residues 116, 120, 121 resistance 79, 110 resting period 15 rye 20 saccharomyces 48 salivary gland 4 salmonella 8, 118 SCFA 10 SCFA:short chain fatty acid 178 secretion 8 seed coat 22 sequencing 8 sexual dimorphism 17 sexual maturation 13 short chain fatty acids 70, 73, 48, 49 sm meat 7 small intestine 6 sodium 12 solubility 14 soybean 25 spaghetti meat 7 spirulina 29 split fiber 5 stabilizers 15 starch 21 starter 12 stem cell 156, 157 stomach 5 supplementation 176 surrogate host 157 TALEN 153, 159, 160 tannin 24 technological process 27 temperature 132 the action of AGP 42 thermal stress 134 threonine 2

Index

TJ proteins 53 Tol2 vector 163 tonsil 4 transcription activator-like effector nuclease 153 transgene 30 transgenesis 157, 161, 163 transgenic bird 152, 163 transgenic hen 156 transmission 108 triticale 19 trypsin inhibitor 24 tryptophan 21 tubular gland 3 tumor 180 vaccination 173 validation 118 veterinary medicines 121

viral vector 162 vitamins 28, 124, 126 wattle 15 welfare 173 wheat 19, 20, 21 white striping 6 WS 6 xenobiotics 123 xylooligosaccharides 58 yeast 27 zinc finger nuclease 153 ZNF 153, 159 zonulin-1 53 zoonotic disease 107

209