1 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CAMPUS DE JABOTICABAL COMPOSIÇÃO DAS PENAS E DA PULPA DE FRANGOS DE CORTE Bruno Balbino Leme Zootecnista 2020 2 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CAMPUS DE JABOTICABAL COMPOSIÇÃO DAS PENAS E DA PULPA DE FRANGOS DE CORTE Bruno Balbino Leme Orientadora: Profa. Dra. Nilva Kazue Sakomura Co-orientadora: Profa. Dra. Kênia Cardoso Bícego Co-orientador: Prof. Dr. Robert Mervyn Gous Dissertação apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Mestre em Zootecnia. 2020 3 Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). 1. Frango de corte. 2. Penas. 3. Aminoácidos. 4. Remiges. 5. Penas de contorno. I. Título. Leme, Bruno Balbino Composição das penas e da pulpa de frangos de corte / Bruno Balbino Leme. -- Jaboticabal, 2020 47 p. : tabs. Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientadora: Nilva Kazue Sakomura Coorientador: Robert Mervin Gous; Kênia Cardoso Bícego L551c 4 5 DADOS CURRICULARES DO AUTOR BRUNO BALBINO LEME, filho de Flávio Donizete Leme e Aparecida de Fátima Balbino Leme, nascido no dia 27 de novembro de 1995 em Mirassol, São Paulo. Ingressou no curso de Zootecnia na Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) – Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, em março de 2013. Em fevereiro de 2018 obteve o título de Zootecnista. Em março de 2018 ingressou no Programa de Pós-Graduação em Zootecnia da Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) – Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, sob orientação da Profª Drª Nilva Kazue Sakomura, submetendo-se à defesa da dissertação em fevereiro de 2020. 6 Dedico Aos meus pais Flávio e Aparecida, por todo amor, carinho e dedicação que sempre tiveram por mim, pelos esforços e incentivo durante toda minha trajetória. Amo cada um de vocês incondicionalmente. 7 AGRADECIMENTOS Primeiramente a Deus, pela força, apoio, sabedoria, coragem nesta caminhada, por ter guiado meus passos e atender todos meus pedidos. A minha orientadora, Prof.ª Dr.ª Nilva Kazue Sakomura, pela confiança em deixar aos meus cuidados este estudo, por todo apoio, dedicação e credibilidade. O meu muito obrigado! Aos meus pais, Aparecida e Flávio, por todo apoio, carinho, dedicação, amor e contribuição para poder ter chegado até aqui, minha irmã, Fabiana, por sempre estar ao meu lado, ajudando nas decisões a serem tomadas para meu bem, pelo carinho e amor ímpar e por nunca desistir de mim, meu cunhado, Fábio, pelo companheirismo e força para alcançar meu sonho almejado. A Letícia, pelo companheirismo, amor, carinho e muita paciência, principalmente nos momentos difíceis. A Carol, por toda contribuição para a realização desta dissertação, pela amizade e companheirismo. A todos os amigos, graduandos e pós-graduandos, e funcionários do aviário, em especial Larissa (Pioia), Thaísa (Lorota), Gabriel (Macaé), Bernardo (Murango), Letícia (Pomba), Mariana, Rafael (Kuki), Thaís, Palloma, Mirella, Jefferson, Felipe (Torcido), Rony, Freddy, Luís, Matheus, Guilherme, Marllon e Heloísa pela paciência e aprendizado e aos funcionários Robson, Izildo e Vicente pela experiência. Aos meus co-orientadores, Profa. Dra. Kênia e Prof. Dr. Gous, pela paciência, credibilidade e ensinamento, e em me dar todo suporte necessário para realizar este trabalho. Ao Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) pelo bolsa de estudo concedida. A Faculdade de Ciências Agrárias e Veterinárias, campus Jaboticabal, pelo acolhimento e estrutura concedida. A todos que contribuíram de forma direta ou indireta para que eu pudesse obter o título de Mestre. 8 Sumário RESUMO – ..................................................................................................................... 10 ABSTRACT – ................................................................................................................. 11 CAPÍTULO 1. Considerações gerais .............................................................................. 12 INTRODUÇÃO .......................................................................................................... 12 REVISÃO DE LITERATURA ................................................................................... 13 Anagênese ................................................................................................................ 13 Produção e composição da pulpa............................................................................. 15 Estrutura e queratinização das penas ....................................................................... 17 Composição das penas ............................................................................................. 18 Referencias .................................................................................................................. 20 CAPÍTULO 2. Composition of feathers and pulp of two broiler genotypes ................. 23 INTRODUCTION ....................................................................................................... 26 MATERIALS AND METHODS ................................................................................ 27 RESULTS.................................................................................................................... 28 DISCUSSION ............................................................................................................. 30 REFERENCES ............................................................................................................ 33 9 10 COMPOSIÇÃO DAS PENAS E DA PULPA DE FRANGOS DE CORTE RESUMO – A composição em aminoácidos das penas é utilizada como base para predição das exigências nutricionais para o seu crescimento. No entanto, durante o crescimento dos frangos de corte as penas sofrem mudas e consequentemente ocorrem alterações em sua composição, e até então este evento não foi contemplado nos modelos de predição de exigências nutricionais para aves. Neste contexto, esta dissertação foi realizada com o objetivo de: 1) descrever a composição das penas em diferentes tratos do corpo durante o crescimento das aves; 2) descrever a composição da pulpa presente nas penas em crescimento; 3) descrever a composição dos diferentes tipos de penas maduras. Para descrever a composição das penas em diferentes tratos e da pulpa durante o crescimento das aves, 200 frangos de corte de cada sexo (macho e fêmea) x linhagem (Cobb 500 MX e Ross 308 AP (AP95)) foram distribuídos aleatoriamente em dez boxes de 20 aves cada e alimentadas com quantidades adequadas de proteína dietética usando um programa alimentar de quatro fases. Uma ave por box (10 aves por genótipo) foi amostrada e eutanasiada aos 14, 28, 42, 56, 70, 84, 98 e 112 dias de idade. Todas as penas foram arrancadas de cada um dos sete tratos, sendo eles capital-cervical, dorsopélvico, interescapular, peitoral, femoral, humeral-alar e dorsocaudal. Das remiges (primárias e secundárias) foram coletadas toda a pulpa presente. As penas foram secas em estufa de ventilação forçada (55°C por 72 horas) e a pulpa foi seca por liofilização (-80°C por 72 horas), após esses procedimentos foram quantificados os teores de água e proteína. Os aminoácidos foram quantificados apenas nos frangos machos da linhagem cobb, nas idades de 1, 28 e 70 dias. Para descrever a composição das penas maduras, um sub-ensaio foi conduzido utilizando 20 aves de cada um dos genótipos avaliados, as aves foram alojadas individualmente em gaiolas e as penas perdidas naturalmente foram coletadas diariamente. Foram separadas as penugens, penas de contorno, remiges e retrices, analisado os teores de água e proteína. Novamente, apenas as penas de frangos Cobb macho foram quantificados o conteúdo de aminoácidos. Foi verificada uma pequena variação nos teores de proteína, em base de matéria seca, em todas as idades, linhagem e sexo. Os tratos dorsopélvico e interescapular apresentaram um aumento discreto nos teores de proteína com o avanço da idade. No conteúdo de água, uma redução foi observada com o aumento da idade para ambos sexo e linhagem, em todos os tratos. Nas penas em crescimento, os aminoácidos metionina, lisina e ácidos aspártico tiveram uma redução durante o crescimento das aves, já os aminoácidos cistina, valina, leucina e serina aumentaram. Em relação a pulpa, o teor de proteína foi menor apenas aos 112 dias, para água foi semelhante ao longo de todo período. As penas maduras apresentaram baixo teor de água, e para proteína os valores foram semelhantes ao das penas em crescimento. Os aminoácidos metionina, lisina e histidina foram consideravelmente maiores na pulpa do que nas penas maduras. Cistina e valina foram maiores nas penas maduras do que na pulpa. Diferenças que evidenciam o processo de queratinização das penas. Contudo, há mudanças na composição de aminoácidos durante o crescimento das penas de frangos de corte, e que comumente não são computadas em modelos de predição de exigências de aminoácidos. Palavras chave: Aminoácidos, tratos das penas, penas de contorno, remiges 11 FEATHER AND PULP COMPOSITION OF BROILERS ABSTRACT – The amino acid composition of feathers is used to predict the nutritional requirements for their growth. However, during the growth of broilers the moult is current and consequently changes in their composition occur, and at the present moment this event was not contemplated in the prediction models of nutritional requirements for birds. In this context, this dissertation aimed: 1) describe the composition of feathers in different body tracts during the growth of birds; 2) describe the composition of the pulp present in growing feathers; 3) describe the composition of the different types of mature feathers. In order to describe the composition of feathers in different tracts and pulp during poultry growth, 200 broilers of each sex (male and female) x strain (Cobb 500 MX and Ross 308 AP (AP95)) were randomly distributed in ten boxes with 20 birds each and fed adequate amounts of dietary protein using a four-phase feeding program. One bird per pen (10 birds per genotype) was sampled and euthanized at 14, 28, 42, 56, 70, 84, 98 and 112 days of age. All feathers were plucked from each of the seven tracts, namely capital-cervical, dorso-pelvic, interscapular, pectoral, femoral, humeral-alar and dorso-caudal. From the remige (primary and secondary) all the pulp present was collected. The feathers were dried in a forced ventilation oven (55 ° C for 72 hours) and the pulp dried by lyophilization (-80 ° C for 72 hours), after which the water and protein contents were quantified. Amino acids were quantified only in male cobb broilers at the ages of 1, 28 and 70 days. In order to describe the composition of mature feathers, a sub-trial was conducted using 20 birds from each of the genotypes, the birds were individually housed in cages and the feather loss was collected daily. Down feathers, contour feathers, remiges and rectrices were separated, analyzed for water and protein contents. The same procedure, only male cobb broiler feathers were quantified for amino acid content. The protein content, on the dry matter basis, remains relatively constant throughout growth, strain, and sex. The dorso-pelvic and interscapular tracts showed a slight increase in protein content over time. In water content, a decrease was observed with the age for both sex and strain in all treatments. In growing feathers, the amino acids methionine, lysine and aspartic acids decreased during the growth of birds, while the amino acids cystine, valine, leucine and serine increased. Regarding pulp, the protein content was lower only at 112 days, for water content was similar throughout the period. The mature feathers had low water content, and for protein the values were similar to the growing feathers. The amino acids methionine, lysine and histidine were considerably higher in pulp than in mature feathers. Cystine and valine were higher in mature feathers than in pulp. Differences that evidence the process of feather keratinization. Therefore, there are changes in amino acid composition during the growth of broiler feathers, which are not commonly computed in amino acid requirement prediction models. Keywords: amino acids, feather tracts, contour feathers, remiges 12 CAPÍTULO 1. Considerações gerais INTRODUÇÃO A automação do setor avícola e os avanços em pesquisa elevaram a cadeia produtiva de frangos de corte a um patamar de excelência e competitividade que permitiu ao longo de anos a conquista e manutenção de importantes mercados consumidores. É indubitável a influência da nutrição e ambiência na produtividade de frangos de corte. Todavia, vale salientar que tais fatores, quando fornecidos em condições exigidas e adequadas, são o gatilho para a expressão de um potencial genético pré-existente, e que tal potencial é o fator determinante nas exigências de aves por nutrientes (Emmans, 1981). O crescimento do corpo deve ser compreendido como a soma dos componentes químicos tais como proteínas, água, cinzas e lipídios, cujas taxas de deposições no corpo e nas penas variam em função da idade da ave até sua maturidade, momento no qual a mesma se encontra em equilíbrio e as taxas de deposição são nulas (Ferguson, 2006). Em função de diferenças nas taxas de deposição de nutrientes e na composição química, sobretudo no que tange a composição aminoacídica, o crescimento do corpo e das penas deve ser avaliado separadamente. Ao contrário do corpo o crescimento das penas é descontínuo, ou seja, novas gerações nascem após a perda de penas maduras (Macari et al., 2017). Sob a ótica da nutrição, a principal implicação do crescimento de novas penas é que ocorre aumento na demanda do organismo por nutrientes, sobretudo aminoácidos, e que esta demanda deve ser vista como processo dinâmico uma vez que em função do estágio de maturação, a pena apresenta diferenças quanto ao perfil de aminoácidos (Rivera-torres et al., 2011). 13 A descrição do crescimento de penas, assim como sua composição em aminoácidos é contemplada em modelos fatorais de predição de exigências de aves por aminoácidos propostos por Martin et al. (1994). Contudo, estes modelos não comtemplam as variações na composição em aminoácidos das penas durante o crescimento das aves. O entendimento do perfil de aminoácidos em função da idade poderia aumentar o refinamento com o qual os modelos fatorais acima citados predizem as exigências de aves por aminoácidos, o que consequentemente permitiria atender com maior precisão exigências nutricionais aumentando a eficiência de conversão de proteína dietética em proteína corporal. OBJETIVOS Descrever a composição das penas em diferentes tratos do corpo durante o crescimento das aves; Descrever a composição da pulpa presente nas penas em crescimento; Descrever a composição dos diferentes tipos de penas maduras. REVISÃO DE LITERATURA Anagênese O crescimento das penas é denominado como anagênese, e tem seu início ainda na fase embrionária em frangos de corte (Lucas e Stettenheim 1972; Macari et. al., 2017). O início do desenvolvimento folicular ocorre no quinto dia de incubação por meio da condensação das células mesenquimais na camada dérmica (Yu et al., 2004). Após essa condensação, os aglomerados de células formam linhas que definirão os tratos de penas no corpo da ave (Lucas e Stettenheim 1972). Entre os dias 6 e 7 de incubação é possível observar os aglomerados de células que delimitam os tratos femoral, humeral, peitoral, esternal, espinhal, capital e cranial. A organização 14 dos tratos ocorre de forma sincronizada que definirá também o padrão de muda das penas de 2ª geração em diante (Stuart e Moscona, 1967). Durante o desenvolvimento embrionário os folículos em desenvolvimento darão origem as penugens (1ª geração) e após sofrerem muda, a penas de contorno, remiges e retrices. Aos 9 dias de incubação é possível observar a olho nu os folículos em desenvolvimento no embrião (Leeson e Walsh, 2004). Entre o 5° e 10° dia de incubação a diferenciação celular nos folículos em desenvolvimento é intenso (Lucas e Stettenheim, 1972), uma vez que no 12° dia os folículos já estão formados e as penas começam a apresentar características adultas (Watterson, 1942). Durante esse período, as penas não crescem apenas para cima (Fig. 1), mas também para baixo dando continuidade no desenvolvimento do folículo da pena (Leeson e Walsh, 2004). Ainda, neste período, ocorre a diferenciação dos tipos de penas, onde ráquis e barbas começam se desenvolver de acordo com sua função (Lucas e Stettenheim, 1972). Entre os dias 13-19 de incubação as penugens sofrem o processo de queratinização, uma vez que neste período já emergiram do folículo na epiderme (Beckingham-Smith, 1973). Contudo, o processo de desenvolvimento folicular e formação das penas é um processo complexo, e ainda, caso haja algum trauma no desenvolvimento folicular, este irá prosseguir durante toda a vida da ave, apresentando deformações nas penas juvenis e adulta (Lucas e Stettenheim, 1972). 15 Figura 1. Diagrama do desenvolvimento de um folículo da pena. (A) Desenvolvimento do placode da pena epidérmica e condensação dérmica. (B) Desenvolvimento de uma papila de penas através da proliferação de células dérmicas. (C) Formação do folículo da pena pela invaginação de um cilindro de tecido epidérmico em torno da base da papila. (D) Corte transversal do folículo da pena (Prum e Williamson, 2001). Produção e composição da pulpa “A pulpa é um amplo retículo mesenquimal, cujos interstícios são ocupados principalmente por uma substância gelatinosa homogênea que confere a pulpa uma consistência firme, resistente e elástica” Lillie, 1940. Esta é a definição mais completa da pulpa presente na literatura, porém pouco foi estudado sobre sua produção e composição até o momento. 16 Segundo Smith e Bath (1995) o crescimento das penas necessita da síntese da pulpa que é responsável por todo aporte nutricional das penas. Portanto, a pulpa apresenta aminoácidos em sua composição e é muito rica em fluido sérico. Lillie (1940) relata em seu estudo que a produção de pulpa é em média três vezes maior que a quantidade de pena produzida, ou seja, a relação da quantidade de pulpa para produção de penas é de 3:1. A pulpa está presente no interior do cálamo e da ráquis da pena em desenvolvimento, ainda apresenta uma ligação intima com o folículo da pena aderido na derme por meio do colarinho da pena, assim vasos sanguíneos e artérias conseguem manter o tecido irrigado e nutrido (Lillie, 1940; Lucas e Stettenheim, 1972). Segundo Moran et al. (1966) na pulpa ocorre conversão de metionina para cistina assim como ocorre no fígado, mostrando que a pulpa é um tecido proteico dinâmico diferentemente das penas que uma vez sintetizada não há mais degradação. Smith e Bath (1995) avaliaram a composição de água e proteína presentes na pulpa de remiges de frangos pescoço pelado até as 15 semanas de idade. Verificaram que à medida que a pena cresce a pulpa regride para 1 a 2%, ficando difícil coletá-la nos estágios de crescimento final das penas. Aos 14 dias de idade, as aves apresentavam penas com aproximadamente 50% pena e 50% pulpa, e a pena apresentava cerca de 50% de matéria seca enquanto a pulpa de 13 a 15%. No final do estudo, na 15ª semana, algumas penas apresentavam teor de matéria de seca de 85% enquanto a pulpa era de 10%. Além disso, Smith e Bath (1995) verificaram que a pulpa apresentava um teor de proteína, em base de matéria seca, entre 75 e 80%, enquanto as penas tinham de 90 a 92%. 17 Entretanto, não há relatos da composição de aminoácidos presentes na pulpa dérmica das aves. Porém, essa informação é relevante quando se trata de exigências nutricionais, uma vez que, para a formação das proteínas presentes nas penas ou em qualquer outro tecido é necessária uma cadeia de aminoácidos. Estrutura e queratinização das penas A estrutura das penas é composta por dois principais segmentos dispostos em seu eixo longitudinal, denominados de cálamo e ráquis. O cálamo é representado por uma base curta e tubular inserida no folículo das penas, composto por uma abertura localizada na porção inferior. Enquanto a ráquis compreende o eixo central acima da pele. No eixo central, existem ramificações primárias chamadas de barbas, as quais são conhecidas coletivamente por vexilo (Fig. 2). As barbas apresentam ramificações secundárias suportadas por um ramo, nomeadas de bárbulas que possuem ramificações ainda menores denominadas de barbículas (Prum e Williamson, 2001; Macari et al., 2017). Figura 2. Estrutura das penas (Macari et al., 2017) A estrutura e o tamanho das penas são diversos. Entretanto, segundo Leeson e Walsh (2004) existem três principais tipos de penas em aves comerciais, sendo as penas de contorno, remiges (são as penas de voo presentes nas asas) e retrices (são as penas presentes na cauda). As penas de contorno estão presentes em todos os 18 tratos do corpo das aves e são responsáveis pela cobertura da pele protegendo-a de lesões e auxiliando no equilíbrio térmico da ave. Ainda, existem as penugens que estão presentes em sua maioria nos pintinhos de um dia, que conferem uma estrutura “fofa” evitando a perda de calor para o meio. A pena se desenvolve a partir da queratinização de suas estruturas, onde durante o seu crescimento a divisão celular ocorre no colarinho do folículo (Lucas e Stettenheim, 1972). Desta forma, as penas se desenvolvem por proliferação e diferenciação dos queratinócitos que produzem a queratina. À medida que ocorre a multiplicação das células mais jovens, os queratinócitos mais velhos morrem e são deslocados para fora do colarinho, deixando para trás uma matriz de queratina depositada que constitui a pena madura (Prum e Williamson, 2001). A queratina é uma escleroproteína que é muito resistente a degradação pela maioria das enzimas proteolíticas (Leeson e Walsh, 2004), sendo dividida em duas classes por Block (1935), as euqueratinas e pseudoqueratinas, porém as penas de aves domésticas apresentam em sua composição as euqueratinas. Segundo Leeson e Walsh (2004) a queratina representa 85% das proteínas presentes nas penas, e é caracterizada por um alto teor de enxofre, em sua maioria na forma do aminoácido cistina. Contudo a exigência deste aminoácido é maior em períodos de crescimento das penas. Composição das penas As penas são compostas por aproximadamente 90% de proteínas, 7,9% de umidade e 1,3% de lipídeos (Maccasland & Richardson, 1966; Leeson & Summers, 1997). A proteína está presente, em sua grande maioria, na forma de queratina, 19 substância constituída principalmente por aminoácidos sulfurados que confere rigidez às penas (Macari et al., 2017; Maciel et al., 2005). Entretanto, durante o crescimento das aves a composição das penas pode variar, uma vez que a pena não apresenta um crescimento contínuo, após atingir seu tamanho máximo a mesma é substituída por uma nova pena (Macari et al., 2017). Hancock et al. (1995) ao avaliarem a composição das penas de seis linhagens de frangos de corte durante o crescimento, verificaram que os teores de proteína na matéria seca permaneceram constante em todo tempo. Porém, o conteúdo de água diminuiu consideravelmente com o avançar da idade das aves. Martin et al. (1994) avaliaram a composição das penas de frangas em crescimento e observaram o mesmo comportamento para água, já para proteína houve um pequeno aumento durante o crescimento das aves. Em um estudo mais recente, Gonçalves (2017), avaliou a composição das penas de três linhagens de frangos de corte durante o crescimento, os teores de proteína encontrado em base de matéria seca foram muito semelhantes aos observados por Hancock et al. (1995), porém, os teores de água encontrados foram distintos, sendo abaixo em todas as idades. Segundo Emmans et al. (1989) a composição em aminoácidos do corpo e das penas são distintos. Ferket et. al., (1997) observaram que o perfil de aminoácidos do corpo depenado não parece mudar durante o crescimento da ave, porém o das penas apresenta diferença. De forma similar, Rivera-torres et al., (2011) também não verificaram diferenças na composição aminoacídica do corpo de perus ao decorrer do crescimento. Contudo, os mesmos autores observaram decréscimo no percentual de lisina, metionina, triptofano, histidina, tirosina, arginina, asparagina e glutamina nas penas das aves com o avançar da idade, enquanto os teores de treonina, cistina, 20 isoleucina, leucina, valina, glicina, serina, alanina e prolina aumentaram, e de fenilalanina se mantiveram constante. Estes resultados corroboram aqueles encontrados por Fisher et. al., (1981) com frangos de corte machos de 1 a 49 dias de idade. Dentre os aminoácidos que compõem as penas os aminoácidos sulfurados, metionina e cistina, são os mais exigidos para frangos de corte durante o crescimento das penas (Macari et al., 2017). Segundo Leeson e Summers, (2008) 25% da cistina e 2% da metionina dietética são utilizadas exclusivamente para a síntese da queratina. Ainda, vale salientar que a cistina participa diretamente deste processo enquanto a metionina atua na conversão para cistina (Moran et al., 1966). Outros aminoácidos de grande importância para a formação da queratina, e consequentemente para o crescimento das penas, são os aminoácidos de cadeia ramificada (valina, leucina e isoleucina). Estes, juntos, correspondem a mais de 18% da queratina (Macari et al., 2017). Contudo, como a composição de aminoácidos das penas são utilizadas como base para predição das exigências em aminoácidos para o crescimento das mesmas, é vantajoso conhecer o efeito da idade nestes valores (Fisher et al., 1981). REFERENCIAS BECKINGHAM-SMITH, K. 1973. “Proteins of the embryonic chick epidermis. I. During normal development in ovo”. In: Dev. Control of Nuclear Activiry. Ed. Goldstein. Publ. Prentice Hall N.J. USA. BLOCK, R.J. 1935. “Basic amino acids of human skin”. Experimental Biology and Medicine. 32(9), 1574-1575. 21 BLOCK, R.J., & BOLLING, D. 1939. “The amino acid composition of keratins the composition of gorgonin, spongin, turtle scutes, and other keratins”. Journal of Biological Chemistry, 127(3), 685-693. EMMANS, G. C. A model of the Growth and Feed Intake of Ad Libitum Fed Animals, Particularly Poultry. BSAP occasional publication, v. 5, p. 103-110, 1981. EMMANS, G. C. The growth of Turkeys. In: Recent Advances in Turkey Science. Ed. Butterwordths, Poultry Science Symposium, n. 21, p. 135-166, 1989. FERGUSON, N. S. Basic concepts describing animal growth and feed intake. Mechanistic modelling in pig and poultry production’. (Eds RM Gous, TR Morris, C Fisher) pp, p. 22-53, 2006. FERKET, P. R., CHEN F., and THOMAS L. N. Effect of age on carcass and feather amino acid profile in turkeys. Poultry Science, 76(Suppl.1):82, 1997. FISHER, M. L.; LEESON, S.; MORRISON, W. D.; SUMMERS, J. D. Feather growth and feather composition of broiler chickens. Canadian Journal of Animal Science, v. 61, p. 769-773, 1981. GONÇALVES, C.A. 2017. “Modelagem do crescimento, composição do corpo e das penas em frangos de corte”. PhD thesis, Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias (Jaboticabal). HANCOCK, C. E.; BRADFORD, G. D.; EMMANS, G. C.; GOUS, R. M. The evaluation of growth parameters of six strains of commercial broiler chickens. British Poultry Science, v. 36, p. 247-64, 1995. LEESON, S. and SUMMERS, J.D. 2008. Feeding programs for broilers chickens. In: Commercial poultry nutrition. Ed Leeson, S. and Summers, J.D. University Books. Guelph. Pp.297-342. LEESON, S. and WALSH, T. 2004. “Feathering in commercial poultry. I. Feather growth and composition”. World’s Poultry Science Journal 60: 42 – 51. LILLIE, F.R. 1940. “Physiology of development of the feather. III Growth of the mesodermal constituents and blood circulation in the pulp”. Physiological Zoology 13: 143 – 176. LUCAS, A. M.; STETTENHEIM P. R. Avian anatomy-integument. Washington, DC: U. S. Department of Agriculture, p. 235-276, 1972. MACARI, M.; FURLAN, R. L.; GONZALES, E. Fisiologia Aviária Aplicada a Frangos de Corte. Jaboticabal: Funep, 2017. MACCASLAND, E.; RICHARDSON, L. R. Methods for determining the nutritive value of feather meals. Poultry Science, v. 45, p. 1231-1236, 1966. 22 MACIEL, J.L. Produção de hidrolisados proteicos de penas de frango utilizando bactérias queratinolíticas. 2005. Tese (Mestrado em Medicina Veterinária) – Universidade Federal do Rio Grande do Sul, Porto Alegre, 2005. MARTIN, P. A.; BRADFORD, G.; GOUS, R. M. A formal method of determining the amino acid requirements of laying-type pulleys during their growing period. British Poultry Science, v. 35, p. 709-724, 1994. MORAN, E.T., SUMMERS, J.D. and SLINGER, S.J. 1966. “Keratin as a source of protein for the growing chick”. Poultry Science. 45: 1257-1266. PRUM, R. O.; WILLIAMSON, S. Theory of the growth and evolution of feather shape. Journal of Experimental Zoology, p. 31-57, 2001. RIVERA-TORRES, V. NOBLET, J. VAN MILGEN, J. Changes in chemical composition in male turkeys during growth. Poultry Science, v. 90, p. 68–74, 2011. SMITH, W.K. and BATH, H.M. 1995. “Growth and composition of feathers in male broilers”. 1995 Spring meeting of the WPSA (UK branch), British Poultry Science, 36:5, 833-881. STUART, E.S. and MOSCONA. A.A. (1967). Embryonic morphogenesis: Role of fibrous lattice in the development of feathers and feather patterns. Science. 157: 947- 948. WATTERSON, R.I. (1942). The morphogenesis of down feathers with special reference to the development history of melanophores. Physiological Zoology. 15 : 234. YU, M. et al. The developmental biology of feather follicles. The International Journal of Developmental Biology, 48 ed, p. 181-191, 2004. 23 CAPÍTULO 2 - Composition of feathers and pulp of two broiler genotypes Este capítulo é apresentado de acordo com as normas da British Poultry Science. 24 Composition of feathers and pulp of two broiler genotypes B.B. Lemea ; L. Vargasa; C.C.N. Nascimentoa; F.A.P. Antayhuaa; M. Macaria; R.M. Gousb and N.K. Sakomuraa* a School of Agricultural and Veterinarian Sciences, São Paulo State University (Unesp) Jaboticabal, São Paulo, Brazil; bSchool of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, South Africa nilva.sakomura@unesp.br *corresponding author 25 Abstract 1. Male and female Cobb and Ross broilers were used to measure the water, protein and amino acid composition of feathers in seven tracts at two-weekly intervals from 14 to 112 d of age and in moulted natal down, contour feathers, remiges and rectrices. In addition, the composition of feather pulp was measured in the remiges collected over the growing period. 2. 200 chicks of each sex and strain were assigned randomly to ten pens of 20 chicks each and fed adequate amounts of dietary protein ad libitum using a four-phase feeding program. Ten birds per genotype were sampled and euthanized at each age. All feathers were dry-plucked from each of the seven tracts and pulp was removed from primary and secondary remiges. 3. Daily losses of feathers were collected from 20 individually-caged broilers of each genotype. 4. Amino acid contents of feathers from the seven tracts were measured only in Cobb males on days 1, 28 and 70; for pulp on days 28 and 70 of age; and for the four types of moulted feathers. 5. Protein content on a dry matter basis remained relatively constant over all ages and tracts during growth. Water content decreased with age in both sexes and strains. Lysine and methionine content in feathers decreased with age while cystine, valine, leucine and serine increased. 6. Contents of lysine, methionine and histidine were considerably higher in pulp than in mature feathers whereas cystine and valine contents were higher in mature feathers than in pulp. 7. These results, together with information about moulting patterns in broilers, enable the effects of age of the bird and of the feather, and of the type of feather being considered, to be taken into account thereby more accurately calculating the amino acid content of feathers. Keywords: amino acids; chicken; feather tracts; natal down. 26 INTRODUCTION Feather protein consists mainly (850 g/kg) of the scleroprotein keratin (Leeson and Walsh, 2004). Keratins have been divided into two classes, eukeratins and pseudokeratins (Block, 1935). It was suggested that those keratins which are chemically similar to cattle horn be named eukeratins, which are defined as insoluble proteins, resistant to enzymatic digestion, and which yield histidine, lysine, and arginine in the molecular ratios of approximately 1: 4: 12 respectively. Feathers from the domestic fowl have been classified as eukeratins (Block and Bolling, 1938). But the amino acid composition of feathers, as reported by various authors, appears to vary considerably (Leeson and Walsh, 2004). This may be due, in part, to changes that occur in the chemical composition of feathers over time (Hancock et al., 1995; Stilborn et al., 1997), that feathers are not always harvested and handled under similar conditions (Leeson and Walsh, 2004) and because of between-laboratory differences in the analytical techniques used to measure amino acids in protein. The most notable change that occurs in feather composition during the broiler growing period is that the water content is reduced. Feather protein content, on a dry matter basis, remains relatively constant throughout growth (Martin et al., 1994; Hancock et al., 1995). Few studies have been made of the amino acid composition of feathers and whether this changes during the growing period. The most comprehensive are those by Fisher et al. (1981) and Stilborn et al. (1997), both of whom concluded that only minor changes occur in some of the amino acids, with the remainder retaining relative stability throughout growth. Also, little research has been done on the composition of the pulp that is responsible for nourishing the feather as it grows. The results reported here are from a larger study of feathering in broilers, where feathers were collected not only at different stages of growth to 112 d of age but also from different feather tracts during that period (Vargas et al., 2020). In addition to the results reported in that paper, pulp samples and moulted feathers were also collected and analysed. The objective of the study was to ascertain the extent to which the amino acid composition of feathers from the different tracts, and at different ages, remained constant. 27 MATERIALS AND METHODS The study was approved by The Ethics Committee on Animal Use of the São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil, under protocol number n° 015111/17. The experiment reported here formed part of a larger experiment conducted at the above research facility, and the experimental design, husbandry and feeds and feeding program used in the main trial, involving male and female broilers chicks of two slow-feathering genetic strains (Cobb 500 MX and Ross 308 AP (AP95)), have been comprehensively described elsewhere (Vargas et al., 2020). At day-old, five chickens were randomly sampled from each strain x sex combination to provide information on the weight and chemical composition of down feathers at that age. On eight further occasions, with an interval of 14 days, between 14 and 112 d of age, ten birds were selected from each strain and sex, euthanized using isoflurane inhalation, and then defeathered. All feathers were dry-plucked from each of seven tracts (capital-cervical, dorso- pelvic, interscapular, pectoral, femoral, humeral-alar and dorso-caudal tracts). The feathers of each tract and age were analysed for water and crude protein contents. Only those feathers collected from male Cobb 500 MX broilers at 1, 28 and 70 d of age were analyzed for amino acid content. Pulp was removed manually from the remiges (primary and secondary) of males and females of both strains sampled on the above eight occasions, with the aid of blade and forceps. The pulp was stored in a freezer at -20 ° C, after which the first drying in freeze-drying was carried out to quantify water and protein contents. At 28 and 70 d, a pool of the pulp samples from male Cobb 500 MX broilers was used to quantify the amino acid contents. In a sub-trial, in which 20 broilers of each genotype were evaluated, birds were housed individually in cages surrounded by netting, and the daily losses of feathers were collected. These feathers were separated into natal down, contour feathers, remiges and rectrices and then pooled for each feather type, sex and strain to quantify their water and protein contents. Only those feathers collected from male Cobb 500 MX broilers were analysed for amino acid content. Chemical analysis The feathers were dried in forced air ventilation (55° C for 72 h) whilst the pulp was freeze- dried at -80°C in a Thermo VLP200 apparatus. Dried samples were finely milled in a 28 multipurpose mill (Tecnal, TE 631/4) and analyzed for dry matter and N content according to AOAC methods 920.39 and 2001.11, respectively, the latter with the use of Foss Kjeltec 8400. Crude protein was calculated by multiplying N content by 6.25. Samples were analyzed for amino acids in Germany by Evonik Nutrition & Care GmbH using ion-exchange chromatography with post-column derivatization with ninhydrin (Commission Directive 1998). Amino acids were oxidized with performic acid which was neutralized with Na metabisulfite, liberated from the protein by hydrolysis with 6 N hydrochloric acid for 24 h at 110°C and then quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. The amino acid contents were calculated to a 100% dry matter basis and then expressed as a proportion (g/kg) of feather protein for statistical purposes. Statistical analysis Mean feather and pulp water and protein contents were calculated for each strain x sex combination. The data were analyzed by analysis of variance as a factorial arrangement of strain, sex, and age, using the ANOVA procedure in Genstat 18th edition (VSN International, 2016). Values were considered significant at P < 0.05. Regression analyses were conducted to determine the change in protein and water contents over time for the feathers from each of the seven tracts. The constant terms and slopes from each tract and over all tracts were compared using simple linear regression with groups in Genstat. RESULTS The mean protein and water contents of feathers from each of the seven tracts and at each sampling age are given in Table 1. There was a greater variation in protein weight within tracts at 28 d than at any other age, as evidenced by the generally higher standard error (SE) at that age over all tracts. There was no discernable pattern in SE of water contents over time. There were no distinct trends in protein and water content with strain or sex, although both factors had some influence in some cases. Of more importance was the trend over time in the mean protein and water content over strains and sexes combined for each tract. In Table 2 the protein (dry matter) and water contents of feathers in each of the tracts have been regressed against age, with data from both strains and sexes being averaged for each age. It is apparent from this table that the protein contents of feathers from both the dorso-pelvic tract, which was used as the reference tract, and the interscapular tract, increased significantly (0.78 ± 0.042 g/kg.d-1) over time, and more so than that of the other tracts whose regression 29 coefficients are all lower than that of the dorso-pelvic tract. These lower slopes were exacerbated by higher intercepts compared with the reference tract. In spite of the change in protein content over time it is instructive to view the mean protein content over the entire test period for each tract, and these values are given in Table 3. Unlike the protein contents, the feather water contents (Table 2) all decreased with time, the rate of decline being the same for the dorso-pelvic, interscapular, dorso-caudal and femoral tracts, although the total amount of water initially was higher in the interscapular and dorso- caudal tracts. The rate of decline was lower for the remaining three tracts. When the feather protein and water contents from each strain and sex were averaged over all tracts (Table 4) and regressed against age, protein content increased (922±1.23 + 0.467±0.017 g/kg.d-1), and the water content decreased (580±3.66 – 3.035±0.052 g/kg.d-1). In both cases that fit was improved by separating the strains and sexes using Linear regression with Groups in Genstat (Table 5). The increase with time of the protein content of feathers from Cobb females, used for reference, was lower than for the males of both strains, but higher than for Ross females. However, the decrease in water content over time for Cobb females was the same as for Cobb males, but greater than for Ross females and males. The amino acid composition of natal down at day-old and feathers from seven tracts of Cobb males at 28 and 70 d of age is given in Table 6. In only a few cases were differences apparent at these three ages: the lysine content of natal down was the same as that at 70 d, but at 28 d it was 34 % higher than at the other ages. Methionine showed a similar pattern, with the content being 30 % higher at 28 d. Arginine and phenylalanine contents were 14 % higher at day-old than at the two later ages, when they were the same. Histidine content decreased by 59 % from day-old to 28 d and then by a further 34 % at 70 d of age. In all other cases the difference between ages was less than about 7 %. The content of water and protein in the moulted feathers (natal down, contour, remiges and rectrices) and from pulp is presented in Table 7. Water content was lowest in natal down and highest in the remiges, and protein content was highest in the remiges. Water content in feather pulp exhibited an exponential increase over time, with significant differences between males and females, as indicated in the equation Y = A + B (RX). For males, water content of pulp was 900 (± 1.10) – 88.4 (± 11.7) x 0.9406 (± 0.0084)^Tt where T is age (d). For females, the A coefficient was 904. R2 for these equations was 50.3. The protein content of feather pulp exhibited a linear decline over time, with differences being evident between strains. The 30 equation for Cobb broilers was 887 (± 4.12) – 0.7136 (±0.0583) T, where T is age, d, and for Ross, 859 (± 5.83) – 0.5101 (± 0.0824) T. The R2 value for these equations was 44.5. The amino acid composition of the moulted feathers and pulp is given in Table 8. Contour feathers had a higher total amino acid content than the remiges or rectrices, due to higher concentrations of cysteine, threonine, arginine, isoleucine, serine and proline. However, the mean concentration of each amino acid over the three types of feathers measured was very similar to the mean of feathers at 70 d (Table 6). The amino acid content of feather pulp remained constant over the two periods of measurement, but these differed markedly from the amino acid content of feathers. DISCUSSION In this study, the protein content of feathers in the different feather tracts of both strains and sexes, on a dry matter basis, remained constant throughout the growth period. When averaged over the feather tracts these results were the same as those reported by Hancock et al. (1995) and Gonçalves (2017). Interestingly, Hancock et al. (1995) observed that at 28 days of age the variation in feather protein content was greater than at other ages, and the same was observed in the results presented here. In an earlier publication on this study (Vargas et al., 2020) it was reported that peak feather loss occurred between 4 and 5 weeks of age, which may explain the greater variation in protein content of feathers at 28 d. Unlike feather protein content, the water content of feathers in all tracts decreased over time. These results mirror those observed by Hancock et al. (1995) but not by Gonçalves (2017) who could not detect any pattern in water content over the growth period. The latter result is possibly related to the way in which the feathers were sampled, emphasising the importance of sampling all feathers on the body when evaluating the mean composition. The decrease in water content over time is likely to be related partly to the presence of pulp in feathers. During feather growth, the pulp is responsible for providing the necessary nutrients for this growth (Lillie, 1940), and as shown in Table 7, feather pulp has a considerably higher water content than do feathers themselves. Smith and Bath (1995) evaluated the water content of feathers from broiler chickens at different stages of maturation and reported that as the feather grows the pulp percentage decreases and dry matter content increases. The water content of feather pulp, as reported by these authors (850 to 870 g/kg) was similar to that reported here (863 to 909 g/kg) whilst the protein content reported by them (750 to 800 g/kg dry matter) was similar to that at 112 d of age in the present study (797 g/kg) but lower than from 14 to 98 d of age (816 to 874 31 g/kg). This difference could be related to the stage of feather maturation, since as the feathers mature less pulp is present to be irrigated by plasma (Lillie, 1940). Smith and Bath (1995) evaluated the composition of individual primary remiges and observed that when these feathers were mature they contained about 110 g of water/kg and 900 to 920 g protein/kg dry matter. This composition is similar to that of moulted, i.e. mature, remiges in our study for both sexes and strains. The higher water content and lower protein content in the rectrices and contours feathers (136 and 843 to 850 g/kg, respectively) when compared to remiges is possibly related to the higher proportion of calamus and rachis present in the remiges (Harrap and Woods, 1964), which also influenced the amino acid composition of these three feather types, as discussed below. The amino acid contents of feathers in male Cobb broilers are in general agreement with those reported by Stilborn et al. (1997) and by Rivera-Torres et al. (2011) who evaluated the amino acid composition of feathers of growing male turkeys. Some variation was apparent as the birds aged, but not in relation to the different tracts. Of particular interest in the present trial was the increase in both lysine and methionine content at 28 d compared with the levels in natal down and at 70 d, and the step-down in histidine content over the three periods measured. Stilborn et al. (1997) reported a continual decline in the contents of lysine, methionine, tryptophan, tyrosine and histidine over the period from 14 to 112 d. Fisher et al. (1981) reported decreasing methionine contents with age and increases in isoleucine, threonine and valine, which occurred in the study of Stilborn et al. (1997) and in the present study. They did not report the highly significant decline in histidine content which was found in both our study and that of Stilborn et al. (1997). These relatively consistent changes in amino acid content with age should be taken into account when calculating the daily amino acid requirements of a given broiler. The amino acid content of feather pulp differed markedly from that of feathers themselves. The contents of both lysine and methionine in pulp were higher than in feathers (38 vs. 12 and 11 vs. 3.2 g/kg protein, respectively), whereas cysteine (33 vs. 68 g/kg), valine (47 vs. 71 g/kg), isoleucine (33 vs. 46 g/kg) and histidine (11.8 vs. 4.2 g/kg) were all at a lower concentration in the pulp. The higher levels of lysine and methionine in pulp are probably due to the presence of muscle tissue, blood, veins and arteries in the pulp (Lillie, 1940; Lucas and Stettenheim, 1972; Yu et al., 2004) which contain high levels of these amino acids in their composition (Conde-Aguilera et al., 2013). According to Moran et al. (1966) methionine is converted to cystine in the pulp, as happens also in the liver, evidencing this higher concentration of methionine in the pulp. Pacheco et al. (2018) in a study using labelled amino acids, indicated 32 that the conversion of methionine to cystine in the body was 54%, corresponding closely with the ratio of pulp to feathers in our study of 52%. A possible reason for the higher levels of methionine, lysine and histidine at 28 d than at 70 d is that new feathers are emerging at 28d following a moult (Vargas et al., 2020) and, being new, they would contain more pulp, and hence more of these three amino acids, than the feathers at 70 d that had by then attained their mature condition (Vargas et al., 2020). Mature feathers are higher in cystine, valine, serine and leucine than pulp, and these amino acids are consequently present in higher proportions at 70 d than at 28 d. Cystine, in particular, with its disulfide bond (Moran et al., 1966) plays a key role in ensuring the rigidity of the feather through the process of keratinization. As mentioned above, the amino acid composition of the three different types of moulted feathers differed, with remiges containing lower contents of all amino acids other than leucine and glycine. Harrap and Woods (1964) reported a higher proportion of these two amino acids in the calamus and rachis, these being morphological portions of feathers that are present in larger concentrations in the remiges. In addition, the same authors reported higher contents of cystine in feather barbs, which would explain our findings of higher cystine concentrations in contour feathers, as these have a higher proportion of barbs than do other feathers. These results give us insight into the reason for the small differences in amino acid composition observed between the types of feathers present in broilers. If differences in feather amino acid content with age, reported here and by Fisher et al. (1981) and Stilborn et al. (1997), are ignored. a comparison can be made of the amino acid composition of feathers reported in this and in four previous studies (Table 9). Inevitably there will be some variation in the amino acid composition reported, depending on the age at sampling and the composition of the samples taken, making it difficult to decide upon a reliable set of definitive values to use to describe the amino acid composition of feathers. The results presented in this present exercise, combined with information about moulting patterns in broilers (Vargas et al., 2020) enable the effects of age of the bird and of the feather, and of the type of feather being considered, to be taken into account when describing the amino acid content of feathers. These results also suggest that in considering the amounts of each amino acid used in the production of feathers, due consideration should be given to the composition of feather pulp, bearing in mind that the protein present in pulp is dynamic, with synthesis and degradation 33 both occurring, whereas feathers once synthesized are not degraded (Smith and Bath 1995). Lillie (1940) calculated that 3 g of pulp was required to manufacture each g of feather. Thus, since the amino acid requirements for feather growth have always been based on the composition of keratinized tissue making up the feather, with only synthesis occurring, the cost of feather development may have been underestimated by 25-30% (Smith and Bath 1995). As a result, by knowing the composition of pulp, growing feathers and mature feathers, it would be possible to better estimate the amino acid requirements for feather growth in broilers. REFERENCES BLOCK, R.J. 1935. “Basic amino acids of human skin”. Experimental Biology and Medicine. 32(9), 1574-1575. BLOCK, R.J., & BOLLING, D. 1939. “The amino acid composition of keratins: The composition of gorgonin, spongin, turtle scutes, and other keratins”. Journal of Biological Chemistry, 127(3), 685-693. CONDE-AGUILERA, J.A., COBO-ORTEGA, C., TESSERAUD, S., LESSIRE, M., MERCIER, Y., AND VAN MILGEN, J. 2013. “Changes in body composition in broilers by a sulfur amino acid deficiency during growth”. Poultry Science, 92(5), 1266-1275. FISHER, M.L., LEESON, S., MORRISON, W.D. and SUMMERS, J.D. 1981. “Feather growth and feather composition of broiler chickens.” Canadian Journal of Animal Science 61: 769– 773. GONÇALVES, C.A. 2017. “Modelagem do crescimento, composição do corpo e das penas em frangos de corte”. PhD thesis, Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias (Jaboticabal). HANCOCK, C.E., BRADFORD, G.D., EMMANS, G.C. and GOUS, R.M. 1995. “The evaluation of the growth parameters of six breeds of commercial broiler chickens.” British Poultry Science 36: 247 - 264. HARRAP, B.S. and WOODS, E.F. 1964. “Soluble derivatives of feather keratin.” Biochemical Journal 49: 8 - 26. HURWITZ, S., PLAVNIC, I., BENGAL, I., TALPAZ, H. and BARTOV, I. 1983. “The amino acid requirements of growing turkeys. 2. Experimental validation of model-calculated requirements for sulfur amino acids and lysine.” Poultry Science 62: 2387 – 2393. 34 LEESON, S. and WALSH, T. 2004. “Feathering in commercial poultry. I. Feather growth and composition”. World’s Poultry Science Journal 60: 42 – 51. LILLIE, F.R. 1940. “Physiology of development of the feather. III Growth of the mesodermal constituents and blood circulation in the pulp”. Physiological Zoology 13: 143 – 176. LUCAS, A.M. and P.R. STETTENHEIM. 1972. “Avian anatomy-integument”. Washington: United States Department of Agriculture. MARTIN, P.A., BRADFORD, G.D. and GOUS, R.M. 1994. “A formal method of determining the dietary amino acid requirements of laying-type pullets during their growing period.” British Poultry Science 35: 709 - 724. MORAN, E.T., SUMMERS, J.D. and SLINGER, S.J. 1966. “Keratin as a source of protein for the growing chick”. Poultry Science. 45: 1257-1266. NITSAN, Z., DVORIN, A. and NIR, I. 1981. “Composition and amino acid content of carcass, skin and feathers of the growing gosling”. British Poultry Science 22: 79-84. PACHECO, L. G., SAKOMURA, N. K., SUZUKI, R. M., DORIGAM, J. C., VIANA, G. S., VAN MILGEN, J., & DENADAI, J. C. 2018. “Methionine to cystine ratio in the total sulfur amino acid requirements and sulfur amino acid metabolism using labelled amino acid approach for broilers”. BMC veterinary research, 14(1), 364. RIVERAS-TORRES, V., NOBLET, J., VAN MILGEN, J. 2011. “Changes in chemical composition in male turkeys during growth. Poultry Science. 90:68-74. SMITH, W.K. and BATH, H.M. 1995. “Growth and composition of feathers in male broilers”. 1995 Spring meeting of the WPSA (UK branch), British Poultry Science, 36:5, 833-881. STILBORN, H.L., MORAN, E.T., GOUS, R.M. and HARRISON, M.D. 1997. “Effect of age on feather amino acid content in two broiler strain crosses and sexes.” Journal of Applied Poultry Research 6: 205 - 209. VARGAS, L., SAKOMURA, N.K., LEME, B.B., ANTAYHUA, F.A.P., CAMPOS, D., GOUS, R.M. and FISHER, C. 2020. “A description of the growth and moulting of feathers in commercial broilers” British Poultry Science 61: 454 – 464. VSN INTERNATIONAL. 2016. GenStat. Release 18.1. Rothamsted Experimental Station. (Clarendon Press: Oxford, UK) 35 YU, M., YUE, Z., WU, P., WU, D.Y., MAYER, J.A., MEDINA, M., WIDELITZ, R.B., JIANG, T.X., CHUONG, C.M. 2004. “The developmental biology of feather follicles”. The International journal of developmental biology, 48, 181. 36 Table 1. Mean feather protein (dry matter) and water content (g/kg) of feathers from seven feather tracts of male and female Cobb and Ross broilers from 14 d, at 14-d intervals, to 112 d of age Protein content (g/kg) Water content (g/kg) Cobb Ross Cobb Ross 1Age Male Female Male Female Mean S.E.M2 Male Female Male Female Mean S.E.M Capital-cervical tract 14 913 949 944 947 938 2.55 433 392 488 529 461 19.3 28 904 910 924 966 926 8.64 570 523 564 508 542 9.48 42 975 975 955 958 966 4.56 451 423 471 450 449 11.9 56 957 958 953 961 957 7.39 332 350 383 394 365 15.3 70 959 949 936 952 949 4.84 395 349 379 332 364 17.4 84 968 960 965 974 967 4.60 382 317 407 311 354 16.1 98 978 958 972 971 970 5.09 324 256 315 271 292 16.3 112 965 955 973 970 966 5.04 245 194 275 187 228 8.70 Dorso-pelvic tract 14 871 908 840 932 887 - 436 610 440 532 505 - 28 888 902 909 953 913 7.88 626 544 636 545 587 9.26 42 973 968 947 957 961 5.40 517 426 545 439 482 10.4 37 56 953 952 972 966 961 5.66 344 371 387 387 373 14.1 70 946 947 949 955 948 9.23 350 340 325 334 338 14.0 84 981 958 980 979 974 5.90 301 292 348 305 312 13.2 98 981 969 973 972 974 4.98 309 255 313 248 282 10.5 112 973 956 978 978 971 4.85 241 194 268 185 222 11.0 Interscapular tract 14 902 902 908 934 912 -3 433 547 437 542 490 - 28 875 910 912 952 912 9.79 692 591 704 586 644 13.4 42 978 976 945 961 965 5.67 559 465 539 480 522 14.0 56 958 952 960 955 957 5.93 385 393 441 403 406 12.1 70 956 945 944 958 951 6.41 396 361 369 338 366 13.8 84 968 960 984 986 975 4.98 303 279 346 295 306 15.9 98 987 982 986 978 983 3.95 305 276 317 262 290 13.6 112 976 965 985 979 976 4.24 233 214 242 189 220 11.7 Pectoral tract 14 937 933 922 934 932 3.31 469 418 564 581 509 22.6 28 897 905 923 960 921 8.51 573 527 552 527 545 11.8 38 42 975 968 953 962 964 4.60 460 429 488 438 454 10.1 56 958 950 962 958 957 4.66 336 397 391 455 395 12.6 70 960 935 940 934 942 6.27 395 406 403 379 396 15.6 84 965 950 968 958 960 5.43 390 335 422 353 375 15.7 98 966 954 971 963 963 5.49 337 294 340 277 312 19.9 112 968 950 984 974 969 4.31 234 213 250 203 225 10.9 Femoral tract 14 905 941 933 941 930 1.91 504 499 536 569 527 27.7 28 910 905 930 961 926 6.18 555 536 555 530 545 9.47 42 974 966 947 959 962 5.38 499 476 513 492 495 12.4 56 952 947 957 956 953 4.78 349 364 403 399 379 12.2 70 957 937 949 953 949 5.61 338 307 363 284 323 23.9 84 974 958 978 974 971 3.76 285 273 307 279 287 12.9 98 975 968 980 972 974 4.23 275 260 283 249 268 13.3 112 971 964 984 979 975 3.92 227 217 244 192 220 10.8 Humeral-alar tract 14 946 957 939 954 949 2.82 468 450 518 510 487 5.60 39 28 925 899 936 973 934 8.23 585 502 579 488 539 8.00 42 976 978 968 975 974 4.92 435 426 461 424 437 5.12 56 966 965 975 967 968 5.07 309 345 327 399 345 6.78 70 965 955 967 966 963 6.65 288 371 294 363 329 7.78 84 977 964 968 981 972 4.31 297 367 349 370 346 7.15 98 974 972 978 979 976 5.05 354 299 377 299 332 7.31 112 975 965 986 981 977 4.08 294 207 335 217 263 9.53 Dorso-caudal tract 14 905 929 909 950 923 - 655 561 656 568 610 - 28 888 887 907 959 910 6.88 616 523 647 517 630 12.1 42 944 956 929 954 946 6.62 551 459 564 475 512 17.1 56 931 931 923 930 929 6.04 372 444 425 467 427 18.0 70 918 918 925 932 923 6.34 346 433 348 445 393 14.5 84 935 939 941 956 943 8.86 297 357 334 411 350 14.2 98 936 949 935 952 943 6.56 346 325 378 352 356 18.4 112 932 941 936 958 942 7.11 313 242 329 235 280 15.3 1Values correspond to the average of 10 individual measurements per strain and sex 2 Standard error of the mean 3 Samples were pooled due to Insufficient material for individual analysis, hence no mean square error 40 Table 2. Linear regression with groups of feather protein and water contents against age for each of the seven feather tracts1, averaged over both strains and sexes Tract Constant term s.e. Regression coefficient s.e. R2 Protein content Dorso-pelvic 900 2.98 +0.780 0.042 0.37 Interscapular +9.61 N.S.2 Dorso-caudal +17.8 -0.544 Femoral +24.5 -0.294 Pectoral +27.4 -0.400 Capital-cervical +33.3 -0.437 Humeral-alar +42.9 -0.439 Water content Dorso-pelvic 605 8.44 -3.444 0.119 0.70 Interscapular +33.9 N.S. Dorso-caudal +44.4 N.S. Femoral N.S. N.S. Pectoral -22.0 +0.566 Capital-cervical -53.1 +0.748 Humeral-alar -65.3 +0.985 1 Using dorso-pelvic tract as reference 2 Not significantly different from the reference tract 41 Table 3. Mean feather protein (dry matter) content (g/kg) over all ages, strains and sexes per feather tract Tract Protein Mean S.E.M Pectoral 951 5.52 Femoral 955 4.64 Dorso-pelvic 949 6.04 Capital-cervical 955 5.62 Dorso-caudal 932 6.52 Interscapular 954 5.73 Humeral-alar 964 5.37 42 Table 4. Mean feather protein (dry matter) and water content (g/kg) over all feather tracts, strains and sexes per age of sampling Age Protein Water Mean S.E.M Mean S.E.M 14 924 2.04 511 4.92 28 920 8.09 568 3.72 42 963 5.35 479 3.56 56 954 5.71 384 3.37 70 947 6.60 358 3.74 84 966 5.62 332 3.63 98 969 5.11 304 3.86 112 968 4.90 237 3.17 43 Table 5. Linear regression with groups of feather protein and water contents against age averaged over feather tracts, strains and sexes Factor Constant term s.e. Regression coefficient s.e. R2 Protein content Cobb females 923 2.36 0.369 0.033 0.30 Cobb males -13.8 +0.253 Ross females +21.5 -0.115 Ross males -12.5 +0.253 Water content Cobb females 584 5.20 -3.219 0.089 0.91 Cobb males N.S.1 -0.211 Ross females +15.4 N.S. Ross males +24.0 -0.206 1 Not significantly different from Cobb females 44 Table 6. Amino acid composition (g/kg feather protein) of natal down and feathers from seven tracts of Cobb males at 1, 28 and 70 d of age Amino acids ¹Feathers tracts Lys Met + cys Met Cys Thr Val Arg Ile Leu His Phe Gly Ser Pro Ala Glu acid Asp acid Total 1 d Natal down 11.4 66.8 2.9 64.0 39.6 62.0 73.0 41.7 70.6 15.2 50.4 67.5 105 90.8 30.3 84.3 64.1 872 28 d Capital-cervical 20.1 71.3 4.8 66.5 42.8 65.1 60.9 42.2 68.8 7.2 42.0 61.4 97.6 91.5 35.7 93.9 61.5 862 Interscapular 17.2 70.2 4.5 65.7 42.6 68.0 63.7 45.7 69.6 6.1 42.9 61.9 103 92.9 35.9 94.7 60.4 875 Dorso-pelvic 18.1 69.6 4.7 64.8 42.6 67.4 63.9 45.5 70.0 6.3 43.0 62.1 103 93.1 36.5 95.0 60.4 877 Pectoral 18.7 71.3 4.6 66.6 42.7 64.6 62.0 42.9 67.7 6.6 41.6 60.8 98.5 91.1 35.0 92.4 60.9 857 Femoral 18.5 72.4 4.7 67.6 43.0 67.2 63.6 45.2 69.8 6.6 43.1 62.4 102 94.5 35.7 95.2 61.5 881 Humeral-alar 17.6 71.9 4.3 67.6 42.9 67.6 62.2 41.4 73.4 5.5 43.4 66.1 102 93.6 41.9 94.0 62.8 886 Dorso-caudal 16.1 71.8 4.2 67.6 42.9 67.6 61.6 42.5 71.2 5.7 43.3 64.0 102 93.6 38.5 94.2 62.3 877 Mean 18.0 71.2 4.5 66.6 42.8 66.8 62.6 43.6 70.1 6.3 42.8 62.7 101 92.9 37.0 94.2 61.4 874 70 d 45 Capital-cervical 11.7 74.8 3.0 71.7 43.6 69.4 64.7 44.2 70.0 4.3 43.0 63.6 110 97.4 35.2 90.3 58.6 881 Interscapular 12.4 70.7 3.4 67.3 42.4 71.1 64.8 46.8 72.5 4.4 44.4 65.8 113 97.0 38.0 93.4 58.1 895 Dorso-pelvic 12.3 71.6 3.3 68.3 42.6 71.8 65.2 47.6 71.8 4.1 43.8 65.0 113 97.6 36.9 92.6 56.9 893 Pectoral 11.1 71.4 3.0 68.3 42.5 71.7 63.8 46.7 73.0 3.9 43.8 66.4 114 98.8 38.6 91.2 56.9 894 Femoral 13.2 68.2 3.5 64.7 42.0 72.1 63.5 48.9 71.5 4.5 43.7 64.5 112 97.8 37.2 93.3 56.2 889 Humeral-alar 9.9 71.8 2.7 69.1 42.5 72.5 62.1 41.5 79.4 3.4 45.3 72.9 113 98.7 47.6 91.1 61.2 913 Dorso-caudal 12.8 73.2 3.4 69.7 43.0 71.3 62.2 43.5 73.7 4.5 44.8 67.2 108 97.5 40.6 94.8 62.4 900 Mean 11.9 71.7 3.2 68.4 42.7 71.4 63.8 45.6 73.1 4.2 44.1 66.5 112 97.8 39.2 92.4 58.6 895 1Values correspond to the pool of 10 feather samples 46 Table 7. Mean protein (g/kg dry matter) and water content (g/kg) of feathers and pulp moulted by male and female Cobb and Ross broilers Cobb Ross Cobb Ross Male Female Male Female Male Female Male Female Protein (g/kg) Mean Water (g/kg) Mean S.E.M Type Moulted feathers Natal Down 638 664 648 648 650 127 123 157 132 135 - Contour 850 817 823 873 841 137 141 145 123 136 - Remiges 917 893 891 906 902 112 123 117 115 117 - Rectrices 842 857 864 851 853 139 131 135 139 136 - Mean 812 808 807 820 812 129 130 139 127 131 Age, d Pulp 14 863 864 852 874 863 861 866 861 864 863 1.20 28 922 902 838 832 874 887 899 881 897 891 1.32 42 826 813 809 834 821 894 894 895 894 894 1.19 56 807 845 819 859 833 904 895 893 876 892 2.78 70 903 821 823 840 847 916 903 894 899 903 1.61 47 84 837 837 806 830 828 882 898 881 904 891 2.13 98 818 821 821 803 816 901 912 906 918 909 1.36 112 804 788 797 797 797 - 912 913 903 899 906 1.59 48 Table 8. Amino acid composition (g/kg of feather protein) of moulted contour feathers, remiges, rectrices and pulp from Cobb males Amino acids ¹Feather types Lys Met + cys Met Cys Thr Val Arg Ile Leu His Phe Gly Ser Pro Ala Glu acid Asp acid Total Moulted feathers Contour 12.1 74.5 3.4 71.1 43.4 71.6 64.1 44.6 75.4 4.0 44.6 69.3 116 101 41.1 91.1 58.8 911 Remiges 10.5 67.4 3.0 64.4 38.9 68.6 56.4 35.2 79.1 3.4 42.9 75.1 105 91.6 52.5 86.6 60.7 874 Rectrices 12.7 67.1 3.8 63.3 39.7 66.5 57.5 37.5 74.6 4.1 42.1 68.6 102 88.0 45.4 88.1 60.1 854 Mean 11.8 69.7 3.4 66.3 40.7 68.9 59.3 39.1 76.4 3.8 43.2 71.0 107 93.5 46.3 88.6 59.9 880 2Age Pulp 28 d 37.0 47.4 10.4 37.0 35.9 49.7 53.9 34.4 61.7 11.7 34.0 50.0 61.1 55.3 38.8 94.9 62.4 728 70 d 37.9 40.4 11.4 29.0 32.8 43.3 52.3 31.2 57.5 11.9 31.4 53.1 52.1 50.5 39.5 93.2 61.3 688 1Values correspond to the pool of 20 feather samples 2Values correspond to the pool of 10 pulp samples 49 Table 9. The content of essential amino acids (g/kg feather protein) of feathers as reported by various authors Amino acid Nitsan1 1981 Hurwitz2 1983 Fisher3 1981 Stilborn4 1997 This study5 Arg 61 72 78 67 71 Cys 67 84 69 75 76 His 8 7 10 7 6 Ile 37 50 50 46 50 Leu 66 81 90 79 81 Lys 18 19 24 20 17 Met 5 7 6 7 4 Phe 40 51 54 47 49 Thr 42 46 53 48 48 Val 57 77 84 62 78 1 Nitsan et al. (1981) 2 Hurwitz et al. (1983) 3 Fisher et al. (1981) 4 Stilborn et al. (1997) mean of six observations spanning period from 14 to 112 d of age 5 Mean of eight observations spanning period from 14 to 112 d of age