UNIVERSIDADE ESTADUAL PAULISTA “JULIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ALTERAÇÕES METABÓLICAS E MORFOMÉTRICAS INDUZIDAS POR HIPÓXIA EM Gallus gallus Paula Andrea Toro Velasquez Zootecnista JABOTICABAL – SÃO PAULO – BRASIL FEVEREIRO DE 2014 UNIVERSIDADE ESTADUAL PAULISTA “JULIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ALTERAÇÕES METABÓLICAS E MORFOMÉTRICAS INDUZIDAS POR HIPÓXIA EM Gallus gallus Paula Andrea Toro Velasquez Orientadora: Profa. Dra. Kênia Bicego Co-orientador: Prof. Dr.Marcos Macari Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Doutora em Zootecnia. JABOTICABAL – SÃO PAULO – BRASIL Fevereiro de 2014 Toro-Velasquez, Paula Andrea T689a Alterações metabólicas e morfométricas induzidas por hipóxia em Gallus gallus / Paula Andrea Toro-Velasquez. – – Jaboticabal, 2014 v, 127 p. ; 28 cm Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2014 Orientadora: Kênia Cardoso Bicego Co-orientador: Marcos Macari Banca examinadora: Isabel Cristina Boleli, Luciano Hauschild, Jose Eduardo Pereira Wilken Bicudo, Denis Otavio Vieira de Andrade Bibliografia 1. Incubação. 2. Taxa metabólica. 3. Intestino. 4. Células calicifomes. 5. Respostas ventilatorias. I. Título. II. Jaboticabal- Faculdade de Ciências Agrárias e Veterinárias. CDU 636.5:636.082.47 Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação – Serviço Técnico de Biblioteca e Documentação - UNESP, Câmpus de Jaboticabal. DADOS CURRICULARES DA AUTORA PAULA ANDREA TORO VELASQUEZ – natural de Medellín – Antioquia, Colombia, nasceu no dia 27 de julho de 1976. Em fevereiro de 1994 ingressou no curso de graduação em Zootecnia, na Faculdade de Ciências Agrárias, da Universidade De Antioquia da Colômbia em Medellín, formando-se em julho de 2000. Em agosto de 2004 iniciou o curso de Mestrado em Zootecnia (Área de concentração: Nutrição e Alimentação Animal, Linha de pesquisa: Avaliação de Alimentos para Animais) na Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Câmpus de Jaboticabal e concluiu em junho de 2006. Na mesma instituição, em março de 2010 iniciou o curso de Doutorado em Zootecnia (Área de concentração: Bioquímica e Fisiologia Animal, Linha de pesquisa: Biologia de Desenvolvimento Animal) concluindo em fevereiro de 2014. Realizou doutorado sanduiche no Laboratório do Professor Jacopo Mortola na Área de Fisiologia Respiratória, do Departamento de Fisiologia, da Universidade de McGill, em Montreal de outubro de 2012 a outubro de 2103, Quebec, Canada. Foi bolsista do Programa Convênio de Pós-Graduação (PEC-PEG) da CAPES durante março de 2010 até junho de 2011, Bolsista FAPESP de julho de 2011 a setembro de 2012 e bolsista BEPE durante o doutorado sanduiche. “I have only made this letter longer because I have not had the time to make it shorter.” “I would prefer an intelligent hell to a stupid paradise.” “Nature is an infinite sphere of which the center is everywhere and the circumference nowhere.” “We arrive at truth, not by reason only, but also by the heart.” Blaise Pascal “The true sign of intelligence is not knowledge but imagination.” “Insanity: doing the same thing over and over again and expecting different results.” “A person who never made a mistake, never tried anything new.” Albert Einstein “The greatest enemy of knowledge is not ignorance; it is the illusion of knowledge.” Stephen Hawking “Education is the most powerful weapon which you can use to change the world.” “I learned that courage was not the absence of fear. But the triumph over it. The brave man is not he who does not feel afraid, but he who conquers that fear.” “It always seems impossible until it’s done.” Nelson Mandela “In the depth of winter I finally learned that there was in me an invincible summer.” Albert Camus “What matters in life is not what happens to you but what you remember and how you remember it.” “Nobody deserves your tears, but whoever deserves them will not make you cry.” Gabriel García Márquez “Friendship is the source of the greatest pleasures, and without friends even the most agreeable pursuits become tedious.” Thomas Aquinas Aos meus queridos pais; Martha Lucia e Manuel Antonio À minha irmã, Lina Marcela E a minha família em especial Ana Josefa, Anita, Catalina,Juan David ,Juan Camilo, Juliana, Odilia e Rosalba, Por seu apoio, orações e incentivo incondicional o tempo tudo, que a pesar da distância sempre estiveram comigo. Obrigada por todo o amor e ensinamentos no caminhar juntos, porque vocês fizeram, fazem e farão sempre parte da minha história! Vocês são maravilhosos. Amo vocês. Minha dedicação especial À Profa Dra. Kênia C. Bicego, Pela orientação, paciência, ensinamentos, amizade e por acreditar em mim! Ao Prof. Dr. Marcos Macari Pela orientação, aceitação, incentivo, escuta e concelho durante todo meu doutorado. Ao Prof. Dr. Jacopo Mortola, Pelos ensinamentos, formação, paciência, oportunidade e bons momentos em Montreal. Meus agradecimentos especiais AGRADECIMENTOS A Deus pela vida. Antes de começar a falar das pessoas, quero dizer que Deus coloca as pessoas certas, no momento certo de sua vida e que você pode chama-las de amigos, mas também pode chama-las de anjos. Agradeço a todos esses anjos que cruzaram o meu caminho. À Profa. Dra. Luciane H. Gargaglioni pelos concelhos e amizade. À Carol Scarpellini e à Marcia Fernandes, porque sem elas a respirometria não seria uma realidade, só elas sabem como foi difícil o começo do caminhar! Além dos bons momentos compartilhados. À Lilian Arantes, colega e amiga da pós-graduação, pela ajuda nas análises histológicas e a companhia no caminhar da pós. À Lara pelo apoio na respirometria! À banca examinadora da tese pelas sugestões importantes para o aprimoramento da mesma: Profa Dra. Isabel Cristina Boleli, Prof. Dr. Luciano Hauschild, Prof. Dr. Jose Eduardo Pereira Wilken Bicudo e Prof. Dr. Denis Otavio Vieira de Andrade. Aos grandes amigos de pós-graduação e da cidade, Miryelle Freire Sarcinelli, Katiani Silva Venturini, Viviane Morita, Daniella Donato, Joy Sato, Helena Espitia, Marilia Petruz, Vania, Vitor e Kris pelos momentos de descontração. Aos colegas e amigos do Laboratório de Fisiologia pelo convívio, risadas e ensinamentos, em especial Livia Espinha, Vivian Biancardi, Lucas Zena, Luis Gustavo, Debora, Mariane, Victor, Caroline, Camila, Luana, Aretuza, Danuzia, Bruno, Valter, Elisa, Gabriela, Jolene, e Carol! Aos colegas de pós-graduação, Fabricio, Marcos, Wedson, Fernando, Raquel, Miguel Daniel e Paulo, pela valiosa ajuda na execução na fase de campo e momentos vividos. Aos funcionários da FCAV/UNESP, Jaboticabal, Euclides, Orandi, William e Damares (Departamento de Morfologia e Fisiologia Animal), Robson e Izildo (Setor de Avicultura). A todos, muito obrigada pela valiosa colaboração na realização deste trabalho. Ao Anselmo pelo apoio e compreensão durante este tempo. Aos amigos Sunitha, Angie, Kelly, François, Flavia, Carlos, Carolina e Marie-Claude que fizeram o Canadá ser inesquecível. E, a todos que de uma forma ou outra, fizeram parte deste caminhar de quatro anos aqui no Brasil ou no Canada, muitíssimo obrigada. Ao programa PEC-PG pela concessão da bolsa de estudos incial. À Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, pela concessão da bolsa de estudos no país, no exterior e o suporte financeiro do projeto. i SUMÁRIO Página CERTIFICADO DA COMISSÃO DE ÉTICA NO USO DE ANIMAIS........... iii RESUMO...................................................................................................... iv ABSTRACT.................................................................................................. v CAPÍTULO 1 – CONSIDERAÇÕES GERAIS............................................. 1 1. INTRODUÇÃO E REVISÃO DE LITERATURA..................................... 1 1.1. Modelos Experimentais: Frangos de corte e galinhas poedeiras......... 1 1.2. Desenvolvimento pré-natal em Gallus.................................................. 3 1.3. Hipoxia................................................................................................. 5 1.3.1. Hipóxia aguda............................................................................. 6 1.3.2. Hipóxia crônica perinatal............................................................. 7 2. OBJETIVO............................................................................................... 10 3. REFERÊNCIAS BIBLIOGRÁFICAS....................................................... 10 CAPÍTULO 2 – EFFECTS OF HYPOXIA DURING INCUBATION ON THE DEVELOPMENT OF THE INTESTINE IN CHICKEN HATCHLINGS......... 16 ABSTRACT................................................................................................. 17 1. INTRODUCTION...................................................................................... 18 2. METHODS............................................................................................... 20 3. RESULTS................................................................................................. 23 4. DISCUSSION .......................................................................................... 25 5. REFERENCES......................................................................................... 29 CAPÌTULO 3 – METABOLIC RESPONSES TO HYPOXIA IN CHICKS WITH DIFFERENT GROWTH RATES……….............................................. 41 ABSTRACT................................................................................................. 42 1. INTRODUCTION...................................................................................... 43 2. METHODS............................................................................................... 45 ii 3. RESULTS................................................................................................. 47 4. DISCUSSION .......................................................................................... 50 5. REFERENCES......................................................................................... 52 CAPÍTULO 4 – VENTILATORY RESPONSE TO HYPOXIA OF THE 1- DAY OLD CHICKEN HATCHLING AFTER PRENATAL COLD-INDUCED HYPOMETABOLISM................................................................................... 64 ABSTRACT................................................................................................. 64 1. INTRODUCTION...................................................................................... 64 2. METHODS............................................................................................... 64 3. RESULTS.AND DISCUSSION................................................................ 65 4. REFERENCES......................................................................................... 66 APÊNDICES APÊNDICES 1 - THE MOTILITY OF THE CHICKEN EMBRYO: ENERGETIC COST AND EFFECTS OF HYPOXIA………………………… 68 APÊNDICES 2 - BREATHING PATTERN AND VENTILATORY CHEMOSENSITIVITY OF THE 1-DAY OLD MUSCOVY DUCK (Cairina moschata) IN RELATION TO ITS METABOLIC DEMANDS……………….. 76 APÊNDICES 3 - THE THERMAL PREFERENCE OF THE CHICKEN HATCHLING: BELOW THERMONEUTRALITY……………………………... 81 APÊNDICES 4 - THERMOGENESIS, VOCALIZATION AND TEMPERATURE PREFERENCE OF 1-DAY OLD CHICKEN HATCHLINGS AFTER COLD-EXPOSURE IN LATE EMBRYOGENESIS.. 104 iii iv ALTERAÇÕES METABÓLICAS E MORFOMÉTRICAS INDUZIDAS POR HIPÓXIA EM Gallus gallus RESUMO- Baixa pressão parcial de O2 durante o desenvolvimento embrionário e/ou fetal pode diminuir a taxa metabólica e a taxa de crescimento do embrião, podendo levar a alterações morfofisiológicas após o nascimento, além de atenuar a resposta ventilatória hipóxica no recém-eclodido de galinhas. No presente estudo, várias incubações foram realizadas para avaliar o efeito da hipóxia pre-natal sobre a taxa metabólica e a resposta à hipóxia aguda em diferentes linhagens e idades de Gallus gallus. Além disso, foi verificada a possibilidade de que a redução da taxa metabólica por si e não especificamente induzida por hipóxia prolongada durante desenvolvimento pré-natal seria um fator contribuinte sobre a diminuição da taxa metabólica em neonatos. Para isso embriões de galinha foram incubados a 35 ◦C (grupo de frio), que é conhecido por diminuir o consumo de oxigênio embrionário por ~30% ao longo de incubação, e aumenta o período de incubação em ~2 dias. Quanto a aves precoces, nenhum estudo havia abordado a influência da hipóxia em diferentes fases de incubação no desenvolvimento morfométrico do intestino, importante não somente para os processos digestivos e de absorção, mas também para a proteção contra patógenos; por isto avaliamos as características morfométricas e a quantidade de células caliciformes por vilo das regiões do intestino delgado em pintainhos recém- eclodidos de frango de corte. Palavras-chave: incubação, taxa metabólica, intestino, células calciformes, respostas ventiltorias v METABOLIC AND MORPHOMETRIC RESPONSES INDUCED BY HYPOXIA IN Gallus gallus ABSTRACT- Low O2 partial pressure during embryonic and fetal development can decrease the growth and metabolic rate of the embryo, leading to morphophysiological changes after birth, and blunt the hypoxic ventilatory response in newly hatched chicken. In the present study, different conditions during incubation (cold and hypoxia) were performed to evaluate the effect of prenatal hypoxia on metabolic rate, and the response to acute hypoxia in different strains and ages of Gallus gallus. We also considered the possibility that prolonged metabolic depression per se, but not specifically induced by hypoxia, during incubation would be a contributing factor to decreased metabolic rate in neonate. Thus chicken embryos were incubated at 35◦C (cold group), which is known to decrease the embryonic oxygen consumption (VO2) by ~ 30% throughout the incubation period and increases the incubation time in ~2 days. Finally, in precocial birds, no study has approached the influence of hypoxia at different stages of incubation in the morphometric development of the intestine, not only important for the digestive processes and absorption, but also for protection against pathogens, so we evaluated the morphometric characteristics and quantity of goblet cells per villi of the small intestine regions in newly hatched broiler chicks . Keywords: incubation, metabolic rate, intestine, calciform cells, ventilatory response 1 CAPÍTULO 1 - Considerações Gerais 1. Introdução e revisão de literatura Nos últimos anos, observa-se um crescente interesse da comunidade científica e dos produtores nas consequências pós-natais de mudanças ambientais (estresses nutricionais, térmicos, gasosos, dentre outros) durante o desenvolvimento pré e/ou perinatal, podendo ser resultado de adaptações epigenéticas, ou seja, de mudanças na expressão gênica (Hala et al., 2012; Jiménez-Chillarón et al., 2012) com possíveis alterações fenotípicas. Do ponto de vista zootécnico, a aquisição de tais conhecimentos visa melhorar o entendimento desses processos e os aspectos relativos à produção como, por exemplo, tolerância a estresses ambientais, prevenção de doenças, redução da janela de eclosão, dentre outros. No presente estudo, o estímulo estressor abordado foi a hipóxia, queda da pressão parcial de oxigênio, durante a incubação de frangos de corte e/ou galinhas. Foram investigadas as influências da hipóxia pré-natal sobre o desenvolvimento morfométrico do intestino delgado e as características gerais dos neonatos de frangos de corte e as respostas metabólicas frente a uma nova exposição aguda à hipóxia na fase pós-natal (1 e 10 dias) de frangos de corte e galinhas. Também foi verificado se a redução metabólica induzida pelo frio durante a fase final da incubação teria o mesmo efeito que da hipóxia pré-natal sobre as respostas ventilatórias à hipóxia aguda de neonatos de galinhas. 1.1. Modelos Experimentais: Frangos de corte e galinhas poedeiras A produção de frangos de corte e ovos de galinha em países de clima tropical e subtropical aumentou de forma marcante nos últimos 40 anos, sendo este um grande desafio para o setor devido à baixa tolerância ao frio na fase inicial e, principalmente para os frangos de corte, à baixa tolerância ao calor na fase final de criação. Nesse intervalo de tempo, o Brasil foi o país de maior destaque no cenário 2 avícola mundial, sendo o principal exportador e o terceiro produtor de carnes de frango (UBABEF, 2013). Segundo a USDA (United States Department of Agriculture) a tendência de crescimento do setor avícola do ano 2013 para 2014 é 2,7% para produção, 2,45% para o consumo, 3,46% para a exportação (todos maiores quando comparados aos das carnes bovina e suína) e 1,93% para importação (AVISITE, 2013). Apenas cinco países produzem 55% do total de ovos no mundo, o equivalente a 64 milhões de toneladas do produto por ano. Hoje, no mundo, há cerca de 6,5 bilhões de poedeiras em produção, garantindo mais de 90% da produção global de ovos. A China, que é o maior produtor mundial de ovos, produziu em 2011 27,9 milhões de toneladas do produto. Atrás da China, seguem os Estados Unidos, a Índia, o Japão e o México no ranking dos maiores produtores de ovos. O Brasil aparece em sexto lugar (OVOSITE, 2013). Na década de 2002-2012 a produção brasileira de ovos aumentou perto de 35% (AVISITE, 2013). As pesquisas nacionais contribuíram sobremaneira para o avanço da produção de carne e ovo nas condições tropicais. Aproximadamente desde a década de 1950 o melhoramento genético, praticado nas linhagens de frango de corte e galinhas de postura, levou a um grande progresso na produção de carne e ovos. As aves comerciais de hoje são híbridos produzidos por meio de cruzamentos entre linhagens intensivamente selecionadas para crescimento rápido, eficiência alimentar e rendimento de carne (frangos de corte) e produção de ovos (galinhas poedeiras), entre outras (Harvestein et al., 1994a, 2003). O direcionamento da seleção artificial de frangos de corte tem se baseado em uma alta taxa de ganho de massa corporal em um intervalo curto de tempo (Havenstein et al. 1994a,b); o melhoramento da composição corporal (alto rendimento de tecido muscular magro e baixo teor de gordura abdominal) também tem sido um importante critério de seleção (Rance et al., 2002). 3 1.2. Desenvolvimento pré-natal em Gallus O desenvolvimento embrionário começa aproximadamente 3 horas após a fecundação, a qual ocorre na porção superior do infundíbulo do oviduto. Esse desenvolvimento continua progredindo paralelamente à formação do ovo no interior do oviduto da ave. Como a duração da formação do ovo é de ± 26 horas, o desenvolvimento embrionário no interior do organismo da ave tem aproximadamente 22 horas (Gilbert 2000). Depois do inicio da incubação o evento mais importante para a clivagem do embrião é o estabelecimento de eixos de polaridade. Primeiro, os lados dorsal e ventral tornarão aparentes (o eixo dorso-ventral) e, em seguida, o ântero-posterior (o eixo crânio-caudal) (Bellairs & Osmond, 2005). Pelas 22 horas de incubação, a maioria das células presumíveis da endoderme estão no interior do embrião, embora as células presumíveis mesodérmicas continuam a migrar para dentro de um tempo mais longo. No segundo dia de incubação a partir dos três folhetos embrionários (ectoderme, mesoderme e endorderme), começam a desenvolver os esboços primários dos órgãos: um sistema vascular primitivo surge para suprir as necessidades nutritivas do embrião e um coração tubular surge a partir deste sistema de vasos sanguíneos. Dois distintos sistemas circulatórios se estabelecem: uma circulação intra- embrionária e uma circulação extra-embrionária que se estende pelo saco vitelino. O Sistema Nervoso se diferencia a partir do ectoderme e pelo processo de Neurulação, um tubo cilíndrico se destaca das demais células ectodérmicas. A modelagem do corpo do embrião inicia-se mediante dobra cefálica, na região anterior, dobra caudal, na região posterior, e dobras laterais. Ao final do terceiro dia de incubação, as vesículas encefálicas e o aparelho ocular estão visíveis, o bico começa a se desenvolver e esboços das asas e pés estão presentes (Hamburger & Hamilton 1951). No quarto dia de incubação uma torção e flexão do corpo do embrião estão visíveis e o embrião gira 90º para a direita e repousa com seu lado esquerdo sobre o vitelo. Com a flexão, a cabeça e a cauda se aproximam e o embrião tem a forma de 4 um “C”. A boca e a língua e as fendas nasais desenvolvem-se como partes dos sistemas digestório e respiratório, respectivamente. O coração tubular transforma-se em uma estrutura com quatro câmaras e pode ser visto batendo, se o ovo for aberto nesta etapa do desenvolvimento. No final do quarto dia de incubação, o embrião tem todos os órgãos necessários para sustentar a sua vida até a eclosão e inclusive para ser distinguido dos mamíferos. O embrião cresce e se desenvolve rapidamente. Pelo sétimo dia as papilas das penas aparecem nas asas e pés, o coração está completamente dentro da cavidade torácica e o embrião já tem muitas características de ave, assim ele passa a ser referido como feto. Após o décimo dia as plumas são visíveis e o bico endurece (Hambruger & Hamilton, 1951). No décimo quarto dia as garras são formadas e o feto se move para a posição de eclosão. Após vinte dias, o pintinho está em posição de eclosão, o bico perfura a câmera de ar e a respiração pulmonar começa (internal pipping). Após 21 dias de incubação, o pintainho finalmente começa a sair da casca (external pipping) e eclode (Hambruger & Hamilton, 1951). Em aves, a região das futuras divisões do intestino pode ser reconhecida pelo dia embrionário seis (E6). Uma pequena dobra do duodeno, que se encontra sob o lobo direito do fígado, é seguido pela curvatura do duodeno-jejuno e mais uma dobra que o liga ao saco vitelínico. O endoderme forma as camadas epiteliais do intestino e os ductos das glândulas mucosas, enquanto o mesoderme dá origem à parede muscular e a estruturas associadas (Bellairs & Osmond 2005). O peso do intestino, como peso relativo do embrião passa de 1% no dia 17 de incubação (E17) a 3,5% no momento de eclosão (Uni, 2006). Para ajustar-se à rápida transição de fontes de nutrientes internos para fontes externas, o intestino delgado da ave passa por alterações morfológicas, moleculares e celulares no final da incubação. Dois dias antes da eclosão, o intestino delgado de frangos tem a estrutura das vilosidades e um potencial de digestão e absorção de carbohidratos (Uni et al., 2003a). Embora a capacidade digestiva comesse a se desenvolver poucos dias antes de eclosão, a maior parte do desenvolvimento ocorre após o nascimento, quando o pintainho passa a consumir ração (Uni, 2006). Assim, a importância do desenvolvimento 5 precoce do intestino delgado (antes da eclosão e durante a primeira semana de vida) está relacionada ao próprio desenvolvimento geral do animal, tendo influências sobre o crescimento posterior e a sua saúde (Michell & Moretto, 2006). A superfície do intestino é coberta por uma camada de muco produzido pelas células caliciformes, o que é importante para a prevenção de patologias gastrointestinais e desempenha um papel na digestão e absorção de nutrientes (Forstner et al., 1995). A camada de muco atua tanto como um meio para proteção da borda em escova contra danos causados por produtos químicos ou microorganismos e ajuda no transporte entre o conteúdo luminal e a borda em escova (Forstner & Forstner, 1994). Recentemente também foi demonstrada uma importante função das células caliciformes na resposta imunológica inata, absorvendo as imunoglogulinas maternas e as secretando juntamente com o muco na superfície intestinal, mantendo essa primeira linha de defesa dos pintainhos até que eles produzam suas próprias imunoglobulinas (Bar-Shiba et al., 2014). O desenvolvimento das pequenas células secretoras de muco intestinal em frangos ocorre na fase tardia de incubação e na fase imediatamente após o nascimento (Uni et al., 2003b). 1.3. Hipóxia A redução da pressão parcial de O2, ou seja, a hipóxia pode ser encontrada em situações fisiológicas e patológicas. Situações fisiológicas resultam de ambientes hipóxicos como altas altitudes, tocas e habitats aquáticos privados de oxigênio, enquanto que condições patológicas podem ser apneia obstrutiva do sono, doença pulmonar obstrutiva crônica, hipoxemia associada com distúrbios circulatórios, especialmente os relacionados com a septicemia ou choque endotoxêmico (revisado por Bicego et al., 2007). As respostas fisiológicas à hipóxia podem ser diferentes considerando se é uma exposição curta ou mais prolongada ou ainda dependendo da fase de desenvolvimento em que o animal se encontra, sendo críticas as fases pré e/ou perinatal para influenciar processos fisiológicos mais tarde na vida desse animal. Essas considerações das respostas à hipóxia no domínio do tempo são abordadas 6 abaixo. 1.3.1. Hipóxia aguda Hipoxia gera respostas ventilatórias e metabólicas como aumento na ventilação, queda na taxa metabólica e queda na temperatura corporal (Tc) em recém-nascidos e adultos de diferentes espécies de aves e mamíferos (Gautier, 1996, Mortola 2009, Bicego et al., 2007) que tendem a reduzir a demanda por O2 e facilitar a sua captação. As respostas ventilatórias à hipóxia em aves e mamíferos são predominantemente ativadas por ação de quimiorreceptores chamados arteriais ou periféricos. Tais quimiorreceptores são sensíveis a mudanças das pressões parciais arteriais de oxigênio (PaO2) e CO2 (PaCO2) e do pH arterial (pHa), estando, portanto envolvidos também nas respostas ventilatórias à hipercapnia e/ou à acidemia (Powell, 2000). Os mais importantes desses quimiorreceptores são os corpos carótideos (1-mm de diâmetro) localizados bilateralmente entre a artéria carótida e o gânglio nodoso do nervo vago (Adamson, 1958). Eles são ricamente perfundidos por um ramo da artéria carótida, e inervados por um ramo do vago. Os corpos carotídeos estão perto das glândulas paratireoide e ultimobranquial em aves e são envolvidos dentro da glândula paratireoide em algumas espécies (Yamatsu & Kameda, 1995), não sendo ainda bem conhecido o papel fisiológico dessas interações. A queda da PaO2 promove ativação das células quimiosensíveis (células glomus) dos corpos carotídeos, sendo esta informação enviada ao sistema nervoso central via aferencias vagais. É bem descrita a queda da Tc frente à hipóxia em recém-nascidos e adultos de muitas espécies (revisado por Bícego et al., 2007; Mortola 2009), bem como a redução da temperatura crítica inferior especialmente em mamíferos (Frappell et al, 1992; Mortola & Matsuoka, 1993; Tattersall et al, 2002). Essa queda da Tc frente à hipóxia não é uma simples hipotermia, constituindo um mecanismo regulado resultante da inibição da produção de calor e estimulação da perda de calor (Gautier 7 et al., 1987; Barros et al., 2001; Tattersall & Milson, 2003), além da seleção comportamental por ambientes mais frios (Hicks & Wood, 1985; Gordon & Fogelson, 1991; Malvin & Wood, 1992). Trata-se, portanto, de reduções da taxa metabólica e da Tc ativamente induzidas pelo organismo. Pelo menos em mamíferos, a hipóxia parece atuar nos neurônios sensíveis ao calor localizados na área pré-óptica do hipotálamo, principal região do encéfalo envolvida na termorregulação, para induzir essas alterações termoefetoras (Branco et al., 2006; Scarpellini et al., 2009; Tattersall & Milson, 2009). Para aves, o cenário do processamento central das informações térmicas é menos conhecido e parece apresentar variações quanto às regiões termosensíveis e termointegradoras no hipotálamo e fora dele (Bícego et al., 2007). A sensibilidade do sistema nervoso central (SNC) frente à hipoxia tanto no que se refere às lesões deste e possíveis alterações no limiar termogênico e na termossensibilidade tem sido investigada em mamíferos (Vannucci & Hagberg, 2004; Tattersall & Milsom, 2009). Os trabalhos têm mostrado que o encéfalo “imaturo”, isto é, durante o desenvolvimento embrionário ou fetal, parece ser resistente a condições de hipóxia ou hipóxia isquêmica. Fato este que não ocorre após a maturação do SNC. No feto, uma redução aguda da PaO2 leva a respostas cardiovasculares envolvendo uma elevação da pressão arterial e redistribuição do débito cardíaco em favor de órgãos vitais (Ruijtenbeek et al., 2002). Em embriões de aves o aumento das concentrações de catecolaminas circulantes participa desta resposta (Mulder et al., 2000 e 2001). 1.3.2. Hipóxia crônica perinatal Diversas alterações metabólicas e morfométricas foram demonstradas em aves submetidas à hipóxia durante o desenvolvimento pré-natal, tais como incremento na mortalidade, diminuição na massa corporal, anormalidades no desenvolvimento cardiovascular e de outros sistemas e até mesmo na embriogênese, como resultado da oferta restrita de oxigênio aos tecidos (Dzialowski et al., 2002; Chan & Burggren, 2005; Ghatpande et al., 2008). Em geral, o efeito 8 hipóxico depressor da taxa metabólica manifesta-se como redução da taxa de crescimento durante a embriogênese (Mortola & Cooney, 2008; Mortola & Awan, 2010) podendo levar à imaturidade de órgãos e sistemas no neonato. Além disso, também foi demonstrado que a exposição à hipóxia a partir do quinto dia de incubação de ovos de galinha altera a preferência térmica de pintainhos normóxicos recém-eclodidos, sendo que estes selecionam Tas menores do que os controles, diferença esta não observada naqueles com mais de 8 horas pós-eclosão (Azzan et al., 2007). Existe uma interação multivariada entre o estágio de desenvolvimento, o ambiente, o tempo de exposição a um estímulo ambiental e o fenótipo do adulto, e quanto tempo vai levar para alcançar esse fenótipo. Além disso, durante o desenvolvimento pré e perinatal existem fases que apresentam maior sensibilidade a mudanças ambientais, sendo chamadas de “janelas críticas” (Burggren, 1998). Cada órgão, cada sistema orgânico e cada organismo tem suas próprias janelas críticas, ou seja, fases de maior suscetibilidade a apresentarem um fenótipo alterado devido à exposição a uma variação ambiental. Entender como processos fisiológicos derivam de estruturas anatômicas é particularmente importante na compreensão de como relações de forma e função surgem no embrião em desenvolvimento e, sobretudo, como essas relações mudam durante o desenvolvimento posterior do animal. Assim, o estresse hipóxico pode gerar diferentes efeitos no desenvolvimento (estruturais e fisiológicos), dependendo da fase e do intervalo de tempo de exposição à hipóxia (Burggren, 1998; Dzialowski et al., 2002; Chan & Burggren, 2005; Ghatpande et al., 2008). Neonatos de galinha que foram expostos à hipóxia (crônica) durante a incubação apresentam reduzidas respostas metabólicas e ventilatórias a uma nova exposição hipóxica (aguda). Tal fato tem sido atribuído a possíveis alterações no SNC em regiões envolvidas no controle metabólico e ventilatório e a uma ação negativa sobre o desenvolvimento dos quimiorreceptores periféricos, os quais se tornam funcionais na última fase da incubação (Mortola, 2009). 9 Desta forma, tem sido sugerigo que a hipóxia crônica em recém-nascidos tem um efeito depressor a longo prazosobre a resposta ventilatória quando os animais são expostos a um novo episodio de hipóxia aguda na vida juvenil/adulta. Este fenômeno, tanto em mamíferos e aves, tem sido atribuído a um desarranjo do desenvolvimento normal dos quimiorreceptores (revisado em Carroll, 2003; Mortola, 2009). No entanto, em embriões de galinha, hipóxia crônica durante toda a incubação ou apenas no último terço, resultou em uma resposta ventilatória hipóxica diminuída (RVH) do recém-eclodido (Szdzuy & Mortola, 2007; Ferner & Mortola, 2009). Enquanto a hipóxia não carregava consequências nas RVH do recém- eclodido, se a hipóxia ocorre apenas em estágios anteriores embrionários (Ferner & Mortola, 2009). O fato de que a hipercapnia embrionária (Szdzuy & Mortola, 2008) e a hiperóxia (Bavis & Simons, 2008; Mortola, 2011a) pós-natal causou alguma diminuição das RVH foi considerado compatível com a ideia de que uma estimulação crônica dos quimiorreceptores pode interferir com o seu desenvolvimento pré-natal normal. Hipóxia pré-natal reduz a taxa metabólica e impede o crescimento de muitos órgãos, incluindo os pulmões (Mortola, 2009). A possibilidade do que o baixo peso ao nascer, por si só e independentemente da hipóxia pré-natal, pode contribuir para a diminuição das RVH foi testado no recém-nascido experimentalmente e descartado (Mortola, 2010). Da mesma forma, a possibilidade de que a hipóxia pré- natal pode resultar em um incremento da impedância mecânica do sistema respiratório do recém-nascido também foi rejeitado (Mortola, 2011b). Ainda resta testar se a condição do hipometabolismo pré-natal, por si só independentemente da hipóxia, pode contribuir para alguma depressão das RVH neonatais. É interessante notar que os trabalhos citados acima demonstrando as influências da hipóxia durante a incubação somente abordaram possíveis alterações morfofisiológicas durante a fase pré-natal (embrião e/ou feto) e/ou no primeiro dia pós-eclosão. Além disso, a grande maioria dos estudos foi realizada com linhagem de galinhas poedeiras, sendo que as alterações morfológicas avaliadas restringiram- se a possíveis mudanças de massa dos órgãos. 10 2. Objetivo Diante do que foi apresentado acima, o presente estudo teve como objetivo investigar: a) o efeito da hipóxia na primeira ou na última semana da incubação sobre características morfofisiológicas do intestino delgado (morfometria de vilos e quantidade de células caliciformes) e características gerais do recém-nascido de frango de corte (Capítulo 2). b) as possíveis diferenças entre as respostas metabólicas à hipóxia aguda de pintinhos de frangos de corte (selecionado para crescimento rápido) e de galinha poedeira (crescimento lento) de 1 e 10 dias pós-eclosão e qual a interferência da hipóxia durante a fase final da incubação sobre essas respostas (Capítulo 3). c) se a depressão metabólica fetal (dias 18-20 de incubação) pelo frio pode exercer a mesma influência que aquela pela hipóxia sobre a resposta ventilatória hipóxica no recém-eclodido de galinha poedeira (Capítulo 4). 3. Referências Bibliográficas ADAMSON, T. P. ‘‘The ComparativeMorphology of the Carotid Body and Carotid Sinus.’’ Chas. C. Thomas, Springfield, 1958. AVISITE, 2013, disponível em: http://www.avisite.com.br/noticias/index.php?codnoticia=14664. Acesso em 15 de novembro de 2013. AZZAM, M. A.; SZDZUY, K.; MORTOLA, J. P.; Hypoxic incubation blunts the development of thermogenesis in chicken embryos and hatchlings. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, 292: R2373–R2379, 2007. BAR-SHIRA, E.; COHEN, I.; ELAD, O.; FRIEDMAN, A. Role of goblet cells and mucin layer in protecting maternal IgA in precocious birds. Developmental & Comparative Immunology, 44: 186-194. 2014. BARROS, R. C.; ZIMMER M. E.; BRANCO L. G.; MILSOM W. K. Hypoxic metabolic 11 response of the golden-mantled ground squirrel. Journal of Applied Physiology, 91(2): 603-12, 2001. BAVIS, R. W.; SIMONS, J. C. Developmental hyperoxia attenuates the hypoxic ventilator response in Japanese quail (Coturnix japonica). Respiratory Physiology & Neurobiology, 164: 411-418, 2008. BELLAIRS, R.; OSMOND, M.; The atlas of Chick development. Elsevier Academic Press, p. 470 (ISBN 0-12-084791), 2005. BRANCO, L. G.; GARGAGLIONIB L. H.; BARROS R. C. H.; Review Anapyrexia during hypoxia. Journal of Thermal Biology, 31: 82–89, 2006. BICEGO, K. C.; BARROS R. C. H.; BRANCO, L. G. S. Review Physiology of temperature regulation: Comparative aspects. Comparative Biochemistry and Physiology A, 147: 616–639, 2007. BURGGREN, W. W. Studying physiological development: pas, present and future. Biological Bulletin of National Taiwan Normal University, 33(2): 71-84, 1998. CARROLL, J. L. Plasticity in respiratory motor control. Invited review: developmental plasticity in respiratory control. Journal of Applied Physiology, 94: 375-389, 2003. CHAN, T.; BURGGREN, W. Hypoxic incubation creates differential morphological effects during specific developmental critical windows in the embryo of the chicken (Gallus gallus). Respiratory Physiology & Neurobiology, 145: 251- 263, 2005. DZIALOWSKI, E. M.; PLETTENBERG, D. V.; ELMONOUFY, N. A.; BURGGREN, W. W. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comparative Biochemistry and Physiology A, 131: 713-724, 2002. FERNER, K.; MORTOLA, J. P. Ventilatory response to hypoxia in chicken hatchlings: a developmental window of sensitivity to embryonic hypoxia. Respiratory Physiology & Neurobiology, 165: 49-53, 2009. FRAPPELL P.; LANTHIER C.; BAUDINETTE R. V.; MORTOLA J. P. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian 12 species. American Journal of Physiology, 262(6 Pt 2):R1040-6, 1992. FORSTNER, J. F.; FORSTNER, G. G. Gastrointestina mucus. In: Physiology of the Gastrointestinal Tract. 3rd ed. P. Leonard and R. Johnson, ed. Raven Press, New York, p. 1255–1283, 1994. FORSTNER, J. F.; OLIVER, M. G.; SYLVESTER, F. A. Production, structure and biologic relevance of gastrointestinal mucins. In: Infections of the Gastrointestinal Tract. R. L. Guerrant, ed. Raven Press, New York, NY, p. 71– 88, 1995. GAUTIER, H. Interactions among metabolic rate, hypoxia, and control of breathing. Journal of Applied Physiology, 81: 521-527, 1996. GAUTIER, H.; BONORA, M.; SCHULTZ, S. A.; REMMERS, J. E. Hypoxia-induced changes in shivering and body temperature. Journal of Applied Physiology, 62(6): 2477-84, 1987. GHATPANDE, S.K.; BILLINGTON, J. Jr.; RIVKEES, S. A.; WENDLER, C.C. Hypoxia Induces Cardiac Malformations via A1 Adenosine Receptor Activation in Chicken Embryos. Birth Defects Ressearch. Part A, Clinical and Molecular Teratology, 82(3): 121–130, 2008. GILBERT, S. F. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; Early Development in Birds. Disponível em: http://www.ncbi.nlm.nih.gov/books/NBK10070/ Acesso em 24 de janeiro de 2014, 2000. GORDON, C. J.; FOGELSON, L. Comparative effects of hypoxia on behavioral thermoregulation in rats, hamsters, and mice. American Journal of Physiology, 260: R120-5, 1991. HALA, D.; HUGGETT, D. B.; BURGGREN, W. W. Environmental stressors and the epigenome, Drug Discovery Today: Technologies, 2012. Disponível em: http://dx.doi.org/10.1016/ j.ddtec.2012.05.004. HAMBURGER, V.; HAMILTON, H. A series of normal stages in the development of the chick embryo. Journal of Morphology, 88: 49-92, 1951. HAVENSTEIN, G. B.; FERKET, P. R.; QURESHI, M. A. Growth, liveability and feed conversion of 1957 vs 2001 broilers when fed representative 1957 and 2001 13 broiler diets. Poultry Science, 82: 1500-1508, 2003. HAVENSTEIN, G. B.; FERKET, P. R.; SCHEIDELER, S. E.; LARSON, T. B. Growth, liveability and feed conversion of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poultry Science, 73: 1785–1794, 1994a. HAVENSTEIN, G. B.; FERKET, P. R.; SCHEIDELER, S. E.; RIVES, D. B. Carcass composition and yield of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poultry Science, 73: 1795–1804, 1994b. HICKS, J. W.; WOOD S. C. Temperature regulation in lizards: effects of hypoxia. American Journal of Physiology, 248: R595-600, 1985. JIMENEZ-CHILLARON, J. C.; DIAZ, R.; MARTINEZ, D.; PENTINAT, T.; RAMON- KRAUEL, M.; RIBO, S.; PLOSCH, T. the role of nutrition on epigenetic modifications and their implications on health. Biochimie, 94: 2242-2263, 2012. MALVIN G. M.; WOOD S. C. Behavioral Hypothermia and Survival of Hypoxic Protozoans Paramecium caudatum. Science, 13; 255(5050):1423-5, 1992. MITCHELL, M.A.; MORETÓ, M. Absorptive function of the small intestine: adaptations meeting demand. In: Avian Gut Function in Health and Disease, G.C. Perry (Ed), CAB International, [ISBN: 1-84593-1807], p 43-64, 2006. MORTOLA, J. P. Metabolic and ventilatory sensitivity to hypoxia in avian embryos. Respiratory Physiology & Neurobiology, 178: 352–356, 2011a. MORTOLA, J. P. Respiratory mechanics in 1-day old chicken hatchlings and effects of prenatal hypoxia. Respiratory Physiology & Neurobiology, 175: 357–364, 2011b. MORTOLA, J. P. Small birth weight does not compromise ventilator chemosensitivity in the 1-day old hatchlings. Respiratory Physiology & Neurobiology, 172: 206–209, 2010. MORTOLA, J.P., Review Gas exchange in avian embryos and hatchlings. Comparative Biochemistry and Physiology, A, 153: 359–377, 2009. MORTOLA, J. P.; AWAM, K. A., Growth of the chicken embryo: Implications of egg size. Comparative Biochemistry and Physiology, A, 156: 373–379, 2010. MORTOLA, J. P.; COONEY E. Cost of growth and maintenance in chicken embryos during normoxic or hypoxic conditions. Respiratory Physiology & 14 Neurobiology, 162: 223–229, 2008. MORTOLA, J. P.; MATSUOKA, T. Interaction between CO2 production and ventilation in the hypoxic kitten. Journal of Applied Physiology, 74: 905–910, 1993. MULDER, A. L. M.; VAN GOLDE, J. M. C. G.; VAN GOOR, A. A. C.; GIUSSANI, D. A.; BLANCO, C. E. Developmental changes in plasma catecholamine concentrations during normoxia and acute hypoxia in the chick embryo. Journal of Physiology, 527: 593–599, 2000. MULDER, A. L. M.; VAN GOOR, C. A.; GIUSSANI, D. A.; BLANCO, C. E. Adrenergic contribution to the cardiovascular response to acute hypoxia in the chick embryo. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, 281: R2004–R2010, 2001. OVOSITE, 2013, disponível em: http://ovosite.com.br/noticias/index.php?codnoticia=13537. Acesso em 15 de novembro de 2013. POWELL, F. L. Respirtion. In: Sturkie's Avian Physiology, 5th Edition, Whittow G. (Ed). ISBN [9780127476056], p. 233-264, 2000. RANCE, K. A.; McENTEE, G. M.; McDEVITT, R. M. Genetc and phenotypic relationships between and within support and demand tissues in a single line of broiler chicken. British Poultry Science, 43: 518-527, 2002. RUIJTENBEEK, K.; KESSELS, C. G. A.; VILLAMOR, E.; BLANCO, C. E.; DE MEY J. G. R. Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, 283: R331-R338, 2002. SCARPELLINI C.S., GARGAGLIONI, L. H. BRANCO L.G.S., BÍCEGO K.C. Research Report Role of preoptic opioid receptors in the body temperature reduction during hypoxia. Brain Research,1286: 66 – 74, 2009. SZDZUY, K,; MORTOLA, J. P. Ventilatory chemosensitivity and thermogenesis of the chicken hatchling after embryonic hypercapnia. Respiratory Physiology & Neurobiology, 162: 55-62, 2008. SZDZUY, K,; MORTOLA, J. P. Ventilatory chemosensitivity of the 1-day old chicken 15 hatchling after embryonic hypoxia. American Journal of Physiology 293: R1640-R1649, 2007. TATTERSALL, G.; MILSOM W. K. Hypothermia-induced respiratory arrest and recovery in neonatal rats. Respiratory Physiology & Neurobiology, 137: 29- 40, 2003. TATTERSALL, G. J.; BLANK, J. L.; WOOD, S. C. Ventilatory and metabolic responses to hypoxia in the smallest simian primate, the pygmy marmoset. Journal of Applied Physiology, 92: 202-10, 2002. TATTERSALL, G.; MILSOM W. K. Hypothermia-induced respiratory arrest and recovery in neonatal rats. Respiratory Physiology & Neurobiology, 137: 29- 40, 2003. TATTERSALL, G. J.; MILSOM, W. K. Hypoxia reduces the hypothalamic thermogenic threshold and thermosensitivity. Journal Physiology, 587.21: 5259–5274, 2009. UBABEF Uniao Brasileira de avicultura. 2013. Disponível em: http://www.ubabef.com.br/estatisticas/frango/producao_mundial_carne_frango_ 2012. Acesso em:14 de novembro de 2013. UNI, Z. Early Development of Small Intestinal Function. In: Avian Gut Function in Health and Disease, G.C. Perry (Ed), CAB International, [ISBN: 1-84593-1807], p. 29-42, 2006. UNI, Z.; SMIRNOV, A.; SKLAN, D. Pre- and posthatch development of goblet cells in the broiler small intestine: effect of delayed access to feed. Poultry Science, 82: 320-7, 2003a. UNI, Z.; TAKO, E.; GAL-GARBER, O.; SKAN, D. Morphological, molecular and functional changes in chicken small intestine of late-term embryo. Poultry Science, 82: 1747-1754, 2003b. VANNUCCI S. J.; HAGBERG H. Review Hypoxia–ischemia in the immature brain. The Journal of Experimental Biology, 207: 3149-3154, 2004. YAMATSU, Y.; KAMEDA, Y. Accessory carotid body within the parathyroid gland III of the chicken. Histochemistry, 103: 197–204, 1995. 16 CAPÍTULO 2 - Effects of hypoxia during incubation on the development of the intestines in chicken hatchlings Toro-Velasquez, Paula A.1,2; Souza, Lilian F.A.1; Gargaglioni, Luciane H. 1,2; Macari, Marcos1; *Bícego, Kênia C.1,2 1Department of Animal Morphology and Physiology, College of Agricultural and Veterinarian Sciences, São Paulo State University, Jaboticabal, São Paulo, 14884-900, Brazil. 2National Institute of Science and Technology on Comparative Physiology (INCT- Fisiologia Comparada) Running title: Late hypoxia incubation decreases goblet cell quantity in hatchlings *Corresponding Author: Via de acesso Paulo Donato Castellane s/n, 14884-900, Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita Filho, Jaboticabal, SP, Brasil. Telephone: 55 16 32092656. Telefax: 55 16 32024275. E-mail: keniacb@yahoo.com.br; keniacb@fcav.unesp.br. 17 Abstract Hypoxia during pre-natal development can affect body growth differently depending on the phase of incubation during which it is applied. Regarding precocious birds, no study assessed the influence of hypoxia during different phases of incubation on the morphometric development of the gut. The gut is important not only for digestion and absorption, but also for protection against exogenous pathogens. In this case, goblet cells play a significant role in gastrointestinal surface protection because of mucus secretion. We investigated the effect of 15% O2 in air during the first (HxE) or the last (HxL) week of incubation on the morphometric characteristics and quantity of goblet cells per villus in regions of the small intestine in chicken hatchlings. Neither HxE nor HxL changed the villus height or surface in the duodenum, jejunum or ileum. The number of goblet cells per villus was lower in the duodenum of the HxL compared to the HxE and normoxia groups. Hypoxia at the beginning or the end phase of incubation did not affect body or intestinal mass. HxE, but not HxL, induced a higher quantity of immature neonates, characterized mainly by the presence of remaining membrane in the navel area, incomplete closure of the navel area and weak activity. The results indicate that hypoxia in the last week of incubation, but not in the first, seems to affect intestinal morphophysiological development, especially regarding the reduced quantity of goblet cells in the duodenum, which may lead to reduced protection of the brush border and poor immune defense and/or digestive function. Key words: Goblet cells, villus, duodenum, jejunum, ileum 18 1. Introduction Hypoxia is an important challenge during prenatal development and is reported to decrease body growth in avian embryos (Dzialowsky et al., 2002; Azzan and Mortola, 2007; Zhang and Burgreen, 2012) since primary energy saving during a low oxygen supply probably originates from the blunting of body growth (Mortola, 2009 for review). Thus, hatchlings may present anomalies that can influence performance or even survival, which may be related to the decrease in growth rate during embryonic and/or fetal development. Evidence exists that many of the abnormalities seen in late fetuses or hatchlings have their origins in mishaps at early stages, which are considered to be the most vulnerable phases to general disturbances (Bellairs and Osmond, 2005). However, chicken hatchlings subjected to hypoxia at the third, but not at the first or the second, week of incubation exhibit reduced body mass after 21 days of incubation (Dzialowsky et al., 2002). These results indicate that the embryo/fetus seems to be able to gain weight during the subsequent normoxia, compensating for the slower hypoxic growth rate (Dzialowsky et al., 2002). Except for the chorioallantoic membrane, which develops out of proportion, there is a correlated reduction of body mass and the masses of several organs, such as the lungs, heart and intestine, in chicken hatchlings subjected to hypoxia during incubation (Azzan and Mortola, 2007). Regarding the intestine, its full development is associated with the maintenance of appropriate digestive and absorptive functions for the supply of nutrients, and also protection, since its enormous mucosal surface is associated with lymphoid tissue (Kasahara et al., 1993; Beal et al., 2006) and is covered by a mucus layer (Forstner et al., 1995). The mucus, produced by goblet cells, is important for preventing gastrointestinal pathologies, and also plays a role in nutrient digestion and absorption (Forstner et al., 1995). 19 The mucus layer acts as a medium for protection of the brush border against damage by chemicals and microorganisms, lubrication and also influences transport between the luminal contents and the brush border (Forstner and Forstner, 1994). The region of future divisions of intestine in the chicken can be recognized by embryonic day 6 (E6) (Bellairs and Osmond, 2005). Intestinal mass, as a proportion of embryonic mass, increases from approximately 1% at 17 days of incubation (E17) to 3.5% at hatching (Uni, 2006). To accommodate the rapid transition from internal to external nutrient sources, the chicken small intestine goes through morphological, cellular and molecular changes toward the end of incubation. Two days prior to hatching, the small intestine has a villus structure and the potential for carbohydrate digestion and absorption (Uni et al., 2003a). Although the digestive capacity begins to develop a few days before hatching, its full development occurs post-hatch when the neonatal chick begins consuming food (Uni, 2006). Thus, the importance of early development of the small intestine is associated with influences on later growth, development and health of the animal (Michell and Moretto, 2006). Even though there are many studies showing the effect of hypoxia during chicken embryo and fetus development on organ mass, including the intestines, (Dzialowsky et al., 2002; Azzan and Mortola, 2007; Zhang and Burgreen, 2012), no one has characterized this effect on the morphometric characteristics of the intestine, specifically the duodenum, jejunum and ileum, or the quantity of goblet cells. Therefore, in the present study we investigated the effect of hypoxia during the first or last week of incubation on the morphometric characteristics of segments of the small intestine and the quantity of goblet cells, as well as general physical characteristics, in chicken hatchlings. 20 2. Methods Experiments were conducted on chicken hatchlings (broilers, Cobb-500® strain). Fertile eggs were obtained from a local supplier and placed in incubators (Premium Ecológica, Belo Horizonte, MG, Brazil) set at a temperature of 37.5°C and 60% relative humidity, with automatic egg rotation every hour. The eggs were distributed in three incubators as follows: 1) normoxia for the whole incubation (21% O2; Nx); 2) hypoxia for the first week and normoxia during the rest of incubation (15% O2 from day 0 to day 7; HxE); and 3) normoxia during the first two weeks and hypoxia for the third week of incubation (15% O2 from day 12 to day 19; HxL). Hypoxia was induced by leaking a small stream of N2 into the incubator under the control of a flowmeter (AFSG 165; 0.4-5 Lpm +-3%fe; White Martins, Brazil). The O2 concentration in the incubator was continuously sampled by an O2 analyzer (Sensepoint XCD, Honeywell, USA). The study wasconducted with the approval of the local Animal Care and Use Committee (protocol number 024166/13). 2.1. Internal pipping, external pipping and spread of hatching At day 19 of incubation, all the eggs were candled, and those with evidence of a living fetus were transferred from turning trays to hatchery baskets and placed individually in a net sac (15 x 10 cm). Between 462 and 522 h of incubation, the transferred eggs were frequently checked (every 6 h), candled and verified for internal pipping, external pipping and hatching. The hatchlings were individually labeled and weighed. After 522 h of incubation (21.75 days), the hatching process was ended in all groups. For morphometric analyses of the intestine and the counting of gobletcells, chicks that hatched at around 504 h were used for all experimental groups. 21 2.2. General characteristics of hatchlings All hatchlings were examined and scored for general characteristics as previously described by Tona et al., (2003). Briefly, physical parameters were scored according to their importance for the survival of the chick and the severity of possible anomalies. These included activity (good: 6; weak: 0), feathering (clean and dry: 10; wet: 8; dirty and wet: 0), eye condition (opened and bright: 16; opened and not bright: 8; closed eyes: 0), conformation of legs (normal legs and toes: 16; one infected leg: 8; two infected legs: 0), navel area condition (completely closed and clean: 12; not completely closed and not discolored: 6; not closed and discolored 0), yolk retraction (body with normal swallowed yolk: 12; body with swallowed large yolk and rather hard to the touch: 0), and status of the remaining membrane (no membrane: 12; small membrane: 8; large membrane: 4; very large membrane: 0) and remaining yolk (no yolk: 16; small yolk: 12; large yolk: 8; very large yolk: 0). Each chick was classified by its total score (maximum 100) resulting from the sum of the scores for all characteristics. For the morphometric analyses of the intestine and the counting of goblet cells, chicks with a score of 100 were used for all experimental groups (n= 6/group). The remaining hatchlings were used in other two studies. 2.3. Body and intestinal mass Hatchlings were weighed and six were selected according to the average body mass of animals in each treatment (Nx, HxE or HxL) and euthanatized by lethal dose of ketamine (1.15 g/kg BW). The yolk sac and total intestine (small + large) were excised and weighed. The duodenum, jejunum and ileum were separated (see next section 2.4) and weighed. Body 22 and yolk sac masses were also determined in six other 19-day-old fetuses (E19; at this age, the yolk sac is not incorporated into the body yet). 2.4. Tissue sampling The intestine samples consisted of pieces of about 2 cm: duodenum (from the pylorus to the distal duodenal loop); jejunum (from the distal duodenal loop to Meckel’s diverticulum); and ileum (starting from Meckel’s diverticulum to the ileocecal junction). The samples were washed in saline solution, fixed in Bouin, and then dehydrated in a series of increasing alcohol concentrations, diaphanized in xylene, and embedded in paraffin (Luna, 1968) for analysis with light microscopy. 2.5. Intestinal morphometry and goblet cell counting Semi-serial 5-�m-thick cross-sections of the duodenum, jejunum and ileum were prepared and stained with hematoxylin-eosin for morphometry and periodic acid-Schiff reagent for the counting of goblet cells (Luna, 1968). Images were captured using a light microscope (Leica Microsystems Inc., USA) and were examined using the Image J® computerized analyzer (Rasband, 2004). Measurements of the villus height were taken from the basal region, which coincides with the upper portion of the crypts to the apex, and the crypts were measured from the base to the crypt-villus transition region. The villus surface area was calculated using the following formula: 2 x width / 2 x villus height (Sakamoto et al., 2000; Sohail et al., 2012), where width was measured at the middle part of the villus. The number of goblet cells per villus in the duodenum, jejunum and ileum was determined for a 200 m extension in the middle of the villus. A total of 30 villi and 30 crypts were considered per segment/bird (1 bird/replicate) for all the analyses. 23 2.6. Statistical analyses Values are expressed as mean ± SEM. The data for incubation duration, body mass, yolk sac mass, organ mass, and intestinal parameters were processed with an analysis of variance using the General Linear Models procedure of SAS (SAS Institute Inc., 2002). Means were compared by the Tukey´s test and significance was based on p < 0.05. Data for chick scores and spread of hatching was analyzed with a two-tailed test for comparison of variance. 3. Results 3.1. Effect of early or late hypoxia on incubation duration and spread of hatching in broiler hatchlings Hypoxia during the first or the last week of incubation did not affect internal pipping, external pipping or incubation duration (Table 1). Figure 1 depicts the percentage of hatching during a 36-hour interval at the end of incubation (from 486 to 522 h). In normoxia, the peak of hatching occurred between 504 and 506 h corresponding to 40% of hatching. Late hypoxia induced a slightly lower peak of 30% hatching at the same time as normoxia. However, early hypoxia increased the spread of hatching, as demonstrated by a lower and larger peak (between 498 and 510 h) and a larger base of the hatching curve (Fig.1). 3.2. Effect of early or late hypoxia on general characteristics and body mass of hatchlings 24 The average scores of hatchlings were not different among groups (Table 2). For the normoxia group, 40.3% of animals had a score of 100 and 29% of them presented a score less than 88. These proportions were not different from those obtained in the late hypoxia group. In contrast, hypoxia during the first week of incubation induced a smaller proportion of hatchlings with a score of 100 (24.2%) and a higher proportion of chicks with a score less than 88 (46.8%) compared to normoxia and late hypoxia groups. The main problems observed in early hypoxia chicks were incomplete closure of the navel area, weak activity and the presence of remaining membrane in the navel area. No differences among groups were observed in the body or yolk sac mass of 19-day- old fetuses or the body mass of hatchlings, while yolk sac mass of hatchlings was higher in the late hypoxia group compared with normoxia and early hypoxia (Table 3). 3.3. Effect of early or late hypoxia on intestinal development in broiler hatchlings There was no effect of different incubation conditions on the absolute and relative masses of total intestine (small + large) or the duodenum, jejunum and ileum segments (Table 4). In Table 5, it can be observed that there were no significant differences in villus height, crypt depth and villus area of the duodenum, jejunum and ileum among normoxia, early and late hypoxia groups. The number of goblet cells in each villus in the duodenum was lower in late hypoxia, but not early hypoxia, compared to the normoxia group. For the other segments, jejunum and ileum, there were no significant differences among groups (Fig. 2). 25 4. Discussion The main finding of the present study is that hypoxia during the last, but not the first, week of incubation interferes with the development of the small intestine, decreasing the number of goblet cells per villus in the duodenum of chicken hatchlings. As previously suggested (Dzialowsky et al, 2002; Azzan and Mortola, 2007; Zhang and Burggren, 2012), the present results reinforce the idea that critical windows for pre-natal development exist in chickens. Thus, hypoxia exerts general influences when applied during the first week of incubation (embryonic development) increasing the proportion of immature hatchlings, and affects maturation of systems, the digestive system in this case, when applied during the last week of incubation (fetus development). Early hypoxia (HxE) affects the spread of hatching and general characteristics of hatchlings, but not the morphometry of the intestines Despite the lack of treatment effects on duration of incubation, HxE increased the spread of hatching, inducing a larger base and lower peak of the curve (Fig. 1). This result seems to be related to the lower chick scores observed in the HxE hatchlings (Table 2). In fact, the main problems observed in the HxEchicks were the presence of remaining membrane in the navel area, incomplete closure of the navel area and weak activity, indicating hatchlings were in incomplete stages of development. In agreement with this assumption, acute hypoxia (2, 4 or 6 h) on embryonic day 2, 4 or 6 was reported to affect embryo survival and cause developmental abnormalities (Altimiras and Phu, 2000). In addition, incubation at an altitude of 2,000 m (corresponding to an approximate fraction of 17% O2) during the first 10 days (Bahadoran et al., 2010) or during the entire incubation (Hassanzadeh et al., 2004) induced 26 hatching earlier than development at sea level. It is interesting to note that in both studies, the authors observed increased T4, T3 and corticosterone levels in embryos at days 10 (Bahadoran et al., 2010) and 19 (Hassanzadeh et al., 2004; Bahadoran et al., 2010), which may be a factor that induced early hatching in those studies and probably in our chicks as well. Thus, the imbalance in embryonic neurochemical regulations induced by hypoxia early in incubation may affect the proper time for total development before hatching, since a high percentage of our HxE hatchlings were immature. None of the hypoxic conditions affected the body masses of fetuses (19 days) or hatchlings (Table 3). Bahadoran et al. (2010) also did not observe any difference in absolute or relative embryo body mass after incubation from 1-10 days at high altitude (1,800 m). Other studies also showed that hypoxia during incubation decreases growth rate (Azzan and Mortola 2007; Mortola, 2009), but after removal of hypoxia, chicken embryos are able to recover body mass (Dzialowski et al., 2002). On the other hand, Chan and Burggren (2005) found no differences in embryo and yolk mass at day 18 of incubation from early, late and continuous hypoxia, suggesting that embryos are able to recover from the deleterious effects of hypoxia on growth rate. In the present study, HxL hatchlings, but not HxE, presented bigger yolk sacs than the Nx group (Table 3). This result agrees with a hypoxia-induced reduction in metabolic rate (Mortola and Labbè, 2005) and, consequently, less yolk consumption during the late phase of incubation, while metabolic recovery possibly occurred in HxE hatchlings. 27 Late hypoxia (HxL) affects the number of goblet cells per villus in the duodenum, but not the spread of hatching and general characteristics of hatchlings Both the absolute and relative masses of the intestine and its segments (duodenum, jejunum and ileum) were not affected in hatchlings by any incubation with hypoxia (Table 4). Azzam&Mortola (2007) demonstrated that at the same age, the intestines presented a lower specific weight in embryos incubated with hypoxia compared to a higher specific weigh of the brain and heart. However, the authors attributed this effect to the generalized blunted growth, as differences in organ weight between hypoxia and normoxia disappeared when organ weight was compared as a function of body mass. Thus, it appears that hypoxia has minimal selective effects on the growth of specific organs. Furthermore, we showed that neither HxE nor HxL caused changes in morphometric parameters of the duodenum, jejunum and ileum, such as villus height, crypt depth and villus surface area (Table 5). Pre-villus ridges appear in the chicken embryo duodenum on the 7th or 8th day and increase in numbers thereafter; at 18 or 19 days they elongate rapidly (Coulombre&Coulombre, 1958). Despite the fact hypoxia did not change intestinal morphometry, the number of goblet cells per villus in the duodenum was reduced in the HxL group. It is known that the development of these cells occurs in the late embryonic and immediate post-hatch phase (Uni et al., 2003b). Goblet cells arise by mitosis from pluripotential stem cells at the base of the crypt (Cheng and Leblond, 1974) or from poorly differentiated cells in the lower crypt, referred to as oligomucous cells (Cheng, 1974). These cells migrate from the crypt toward the villus tip where they are sloughed into the lumen, a process that takes 2 to 3 days (Geyra et al., 2001). The goblet cells secret mucin, which is the main component of the mucus gel layer covering the surface of the gastrointestinal tract (Allen and Flemström, 2005; Uni, 2006). In 28 general, the mucus gel layer acts as a lubricant, improving the propulsion of chyme; protects the intestinal epithelium from enteric pathogens, modulating their adherence to the epithelium and acting as a physical barrier; acts as a stable and adherent layer where secreted bicarbonate creates a pH gradient that protects against luminal acid in the stomach and duodenum; protects the intestinal wall against proteolytic digestion by luminal enzymes (Allen and Flemström, 2005; Uni, 2006). At least in rats, acute intestinal ischemia induces mucin breakdown, which is accompanied by epithelial cell disruption and increased non-selective intestinal permeability (Chang et al., 2012). Interestingly, a new protective function of goblet cells was recently suggested, as they are thought to aid in the persistence of maternal IgA (important for immune defense at the perinatal period and first weeks post-hatch) in the chicken gut (Bar-Shira et al., 2014). These authors provided evidence that goblet cells serve as a reservoir for maternal IgA, as they seem to uptake this IgA (supplied by the egg) and release it along with mucin secretions, which are confined to the enterocyte surface, protecting this area and limiting IgA loss due to intestinal flushing. In our chicks, the hypoxia- induced ~20% reduction of goblet cells, especially in the duodenum (Fig. 2), may result in a poorly protected intestinal epithelium, affecting the integrity of this surface and, consequently, the general health of the animal, at least in the early phase post-hatch when hatchlings start to consume external food and water. In summary, hypoxia during the late, but not the early, phase of incubation reduced the number of goblet cells per villus in the duodenum, which may affect protective and digestive/absorptive functions of the intestine in chicken hatchlings. 29 Acknowledgement: The presentstudywassupportedby Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (10/20285-1). P. A. T. Velasquez was the recipient of a FAPESP (11/07509-0) PhD scholarship. References Allen, A., Flemström, G. 2005. Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin. Am. J. Physiol. Cell. Physiol. 288, C1–C19. Altimiras, J., Phu, L. 2000. Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia. J. Exp. Zool. 286(5), 450-6. Azzan, M., Mortola, J. P., 2007.Organ growth in chicken embryos during hypoxia: Implications on organ “sparing” and “catch-up growth”. Resp. Physiol. Neuro, 159, 155-162. Bahadoran, S., Hassanzadeh, M., Zamanimoghaddam, A. K., 2010. Effect of chronic hypoxia during the early stage of incubation on prenatal and postnatal parameters related to ascites syndrome in broiler chickens. Iran JVetRes, 11, 64-71. Bar-Shira, E., Cohen, I., Elad, O., Friedman, A. 2014.Role of goblet cells and mucin layer in protecting maternal IgA in precocious birds.Dev. Comp. Immunol. 44, 186-194. Beal, R. K., Powers, C., Davison, T.F., Smith, A.L., 2006. Immunological Development of the Avian Gut.In: Avian Gut Function in Health and Disease, G.C. Perry (Ed), CAB International, [ISBN: 1-84593-1807], pp 85-106. Bellairs R., Osmond, M., 2005.The atlas of Chick development. Elsevier Academic Press, p. 470 [ISBN 0-12-084791]. Cheng, H. 1974. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. II. Mucous cells. Am. J. Anat. 141, 481–501. 30 Chang, M., Alsaigh, T., Kistler, E.B., Schmid-Schönbein, G.W. 2012.Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine.PLoS ONE 7(6): 1-12. Chan, T., Burggren W., 2005. Hypoxic incubation creates differential morphological effects during specific developmental critical windows in the embryo of the chicken (Gallus gallus). Resp. Physiol. Neuro. 145, 251-263. Cheng, H., Leblond, C. P. 1974. Origin, differentiation and renewal of the four main epithelial cells in the mouse small intestine. IV. Unitarian theory of the origin of the four epithelialcell types. Am. J. Anat. 141, 537–561. Coulombre, A. J., Coulombre, J. L. 1958. Intestinal development. I. Morphogenesis of the villi and musculature. J. Embryol. Exp. Morphol. 6 (3), 403-11. Dzialowski, E. M., Plettenberg, D. V., Elmonoufy, N. A., Burggren, W. W., 2002. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp. Biochem. Physiol. A, 131, 713-724. Forstner, J. F., Forstner G. G. 1994. Gastrointestina mucus.Pages 1255–1283 in Physiology of the Gastrointestinal Tract.3rd ed. P. Leonard and R. Johnson, ed. Raven Press, NewYork. Forstner, J. F., Oliver, M. G., Sylvester, F. A. 1995. Production, structure and biologic relevance of gastrointestinal mucins. In: Infections of the gastrointestinal tract. Guerrant, R.L. (Ed) Raven Press, New York, NY.Pages 71–88. Geyra, A., Uni, Z., Sklan, D. 2001. The effect of fasting atdifferent ages on growth and tissue dynamics in the small intestine of the young chick. Br. J. Nutr. 86, 53–61. Hassanzadeh, M., Fard, M.H.B., Buyse, J., Bruggeman, V., Decuypere, E., 2004. Effect of chronic hypoxia during embryonic development on physiological functioning and on hatching 31 and post-hatching parameters related to ascites syndrome in broiler chickens, Avian Pathol. 33, 558-564. Kasahara, Y., Chen, C.L., Gobel, T.W.F., Bucy, R.P., Cooper, M.D., 1993. Intraepithelial lymphocytes in birds. In: Mucosal Immunology: Intraepithelial Lymphocytes. Kiyono, H., McGhee, J.R. (Eds) Raven Press, New York. Luna, L. G. 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology.McGraw-Hill, New York, NY. Mitchell, M.A., Moretó, M., 2006. Absorptive function of the small intestine: adaptations meeting demand. In: Avian Gut Function in Health and Disease, G.C. Perry (Ed), CAB International, [ISBN: 1-84593-1807], pp 43-64. Mortola, J.P., 2009. Gas exchange in avian embryos and hatchlings. Comp. Biochem. Physiol. A, 153, 359-377. Mortola, J.P., Labbè, K., 2005. Oxygen consumption of the chicken embryo: interaction between temperature and oxygenation. Respir. Physiol. Neurobiol. 146, 97-106. Sakamoto, K.,Hirose, H., Onizuka, A., Hayashi, M., Futamura, N. , Kawamura, Y., Ezaki, T. 2000.Quantitative Study of Changes in Intestinal Morphology and Mucus Gel on Total Parenteral Nutrition in Rats.J. Surg. 94, 99-106. Sas Institute. SAS®.2002 (Statistical Analyses System) User’s guide, Statistics, versão 8.1, v.2, 4ª. Ed. Cary. Sohail, M. U., Hume, M. E., Byrd, J. A., Nisbet, D. J., Ijaz, A., Sohail, A., Shabbir, M. Z., Rehman, H. 2012. Effect of supplementation of prebiotic mannan-oligosaccharides and probiotic mixture on growth performance of broilers subjected to chronic heat stress. Poultry Sci. 91, 2235–2240. 32 Tona, K., Bamelis, F., De Ketelaere, B., Bruggeman, V., Moraes, V.M.B., Buyse, J., Onagbesan, O., Decuypere, E. 2003. Effects of egg storage time on spread of hatch, chick quality, and chick juvenile growth. Poultry Sci. 82, 736-741. Uni, Z., 2006. Early Development of Small Intestinal Function. In: Avian Gut Function in Health and Disease, G.C. Perry (Ed), CAB International, [ISBN: 1-84593-1807], pp 29-42. Uni, Z., Tako, E., Gal-Garber, O., Skan, D. 2003a.Morphological, molecular and functional changes in chicken small intestine of late-term embryo. Poultry Sci. 82, 1747-1754. Uni, Z., Smirnov, A., Sklan, D., 2003b. Pre- and posthatch development of goblet cells in the broiler small intestine: effect of delayed access to feed. Poultry Sci. 82, 320-7. Zhang, H., Burggren, W.W., 2012. Hypoxic level and duration differentially affect embryonic organ system development of the chicken (Gallus gallus). Poultry Sci. 91, 3191–3201. 33 Table 1 – Internal pipping, external pipping and incubation duration (ID) from the normoxia (n=45), early hypoxia (n=52) and late hypoxia (n=48) incubations of broiler chicken Normoxia Early hypoxia Late hypoxia p value Internal pipping (h) 476.10 ± 4.26 480.93 ± 4.35 476.73 ± 4.76 0.0683 External pipping (h) 488.85 ± 3.42 489.00 ± 3.16 490.79 ± 3.71 0.6183 ID (h) 506.44 ± 2.63 505.18 ± 3.33 505.09 ± 3.37 0.7285 Data are expressed as mean ± SEM. 34 Table 2 – Hatchling general scores from the normoxia (n=62), early hypoxia (n=62) and late hypoxia (n=61) incubations of broiler chicken Normoxia Early hypoxia Late hypoxia % of chicks with score 100 40.32a 24.19b 40.98a % of chicks with score < 88 29.03a 46.77b 29.51a Average score of all chicks 91.60 87.56 91.90 Different letters (a, b) means significant difference among experimental groups (p< 0.05). � 35 Table 3 – Body mass (without yolk sac) and yolk sac mass of broiler 19-day fetus and hatchling of the normoxia, early hypoxia and late hypoxia incubations Normoxia Early hypoxia Late hypoxia p value Fetus Body mass (g) 33.45 ± 1.77 31.68 ± 0.76 29.56 ± 0.71 0.1006 Yolk sac mass (g) 12.42 ± 0.79 14.65 ± 0.57 14.54 ± 0.77 0.0769 Hatchling Body mass (g) 41.70 ± 0.56 41.22 ±0.55 40.41± 0.36 0.2117 Yolk sac mass (g) 4.13 ± 0.46 b 4.93 ± 0.56 b 7.22 ± 0.40 a 0.0011 ����������� �� ���� ���� ��������������������� �������� �� ������� ���� ������������� �� �� �������� 36 Table 4 – Total intestine (small + large), duodenum, jejunum and ileum absolute and relative mass (% of yolk free hatchling mass) of hatchlings from the normoxia, early hypoxia and late hypoxia incubations Normoxia Early hypoxia Late hypoxia p value Absolut Mass (g) Intestine 1.93 ± 0.14 1.87 ± 0.08 1.82 ± 0.07 0.7548 Duodenum 0.40 ± 0.03 0.42 ± 0.02 0.40 ± 0.02 0.7929 Jejunum 0.46 ± 0.05 0.44 ± 0.03 0.41 ± 0.02 0.6240 Ileum 0.37 ± 0.04 0.33 ± 0.01 0.35 ± 0.02 0.4862 Relative mass (%) Intestine 4.61 ± 0.29 4.54 ± 0.19 4.51 ± 0.18 0.9457 Duodenum 0.95 ± 0.06 1.00 ± 0.06 0.96 ± 0.04 0.7786 Jejunum 1.10 ± 0.11 1.07 ± 0.08 1.02 ± 0.05 0.7707 Ileum 0.88 ± 0.08 0.79 ± 0.03 0.86 ± 0.05 0.5228 ����������� �� ���� ���� ��������������� � � � 37 Table 5 –Duodenum, jejunum and ileum villus height, crypt depth and villus surface area of hatchlings from the normoxia, early hypoxia and late hypoxia incubations Normoxia Early hypoxia Late hypoxia p value Duodenum Villus height (μm) 825.4 ± 43.1 771.4 ± 41.6 790.6 ± 22.4 0.5887 Crypt depth (μm) 78.3 ± 3.5 74.7 ± 5.2 76.0 ± 5.4 0.8707 Villus surface area (mm2) 0.261 ± 0.020 0.242 ± 0.024 0.222 ± 0.011 0.3768 Jejunum Villus height (μm) 448.4 ±42 365.6 ±28 436.7 ±25 0.1871 Crypt depth (μm) 55.8 ±5.1 61.2 ±4.0 58.5 ±2.7 0.6473 Villus surface area (mm2) 0.108 ± 0.014 0.096 ± 0.007 0.096 ± 0.012 0.7124 Ileum Villus height (μm) 387.2 ±33 368.8 ±20 405.3 ±15 0.5627 Crypt depth (μm) 64.3 ±2.3 66.3 ±2.9 61.4 ±2.6 0.2972 Villus surface area (mm2) 0.088 ± 0.009 0.098 ± 0.007 0.079 ± 0.005 0.1852 Data are expressed as mean ± SEM. N= 6. � 38 ������� ��� �� � ������ �� � �����!�� ���� ��"� � ��#���#����#��$� ��� ���#�� �� ����� ���%��&�����'�#' ����� ��� �(��� �������#' ����� ���%)��� �*+��� ���������# �� ���� �� �������� �� � ������ �,� * �� �" +�������� �� *�+���-�(���.��� ���!���* � � ��#���* �� *�&�/�/* *��� �����*�� �� #���#�� " ��� ���#� �� ���&�����'�#' ����� �������#' ����� �*+��� ���������� � 39 � ������� � 40 � � ������� 41 CAPÍTULO 3 - Metabolic responses to hypoxia in chicks with different growth rates Toro-Velasquez, Paula A.1,2; Mortola, Jacopo3; Amaral, Lara1,2; Fernandes, Marcia1,2; Gargaglioni, Luciane H. 1,2; Macari, Marcos1; *Bícego, Kênia C.1,2 1Department of Animal Morphology and Physiology, College of Agricultural and Veterinarian Sciences, São Paulo State University, Jaboticabal, São Paulo, 14884-900, Brazil. 2National Institute of Science and Technology on Comparative Physiology (INCT- Fisiologia Comparada) 3Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada. *Corresponding Author: Via de acesso Paulo Donato Castellane s/n, 14884-900, Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita Filho, Jaboticabal, SP, Brasil. Telephone: 55 16 32092656. Telefax: 55 16 32024275. E-mail: keniacb@yahoo.com.br; keniacb@fcav.unesp.br. 42 Abstract For most endotherms, birds and mammals, the acute response to hypoxia includes decreases in metabolic rate and body temperature (Tb) and increase in ventilation, all together seems to minimize the imbalance between oxygen supply and demand. Therefore, in the present study we investigated what is/are the main factor(s) affecting metabolic depression by acute hypoxia in young chicks: age, rate of growth, body mass and/or previous exposure to hypoxia during incubation in the last week (late hypoxia: HxL). The resting metabolic rate (VO2) normalized by body weight increased with age independent on the incubation condition for fast and slow growth. The exposure to acute hypoxia decreased VO2 in all animals. Regardless incubation condition, the Fast H5 and Slow H10 chicks presented similar body weights but Fast H10 birds had higher resting metabolic rates. Fast H10 chicks presented the highest body weight and metabolic rate. Key words: oxygen consumption, incubation, fast growth, slow growth, 43 1. Introduction Energy metabolism in vertebrates is primarily based on aerobic processes. Thus, reduction of O2 partial pressure (hypoxia) can affect all of these processes influencing cellular and organ functions including cell membrane transports, muscle tone, maintenance, tissue growth, locomotion, states of alertness and sleep and general behavior (Mortola and Maskrey, 2011). Hypoxia can be found in both physiological and pathological conditions. Physiological situations usually result from hypoxic environments such as high altitude, burrows and oxygen-deprived water habitats; as to pathologies, it can be listed obstructive sleep apnea, chronic obstructive pulmonary disease and hypoxaemia associated with circulatory disorders including those related to septicemic or endotoxemic shock (Tsioutou et al., 2005; Bicego et al., 2007, Tattersall and Milsom, 2009). For most endotherms, birds and mammals, the acute response to hypoxia includes decreases in metabolic rate and body temperature (Tb) and increase in ventilation, all together seems to minimize the imbalance between oxygen supply and demand. These responses are observed in newborns and adults of several species (Gautier, 1996; Mortola, 2009; Bícego et al., 2007; Mortola and Maskrey, 2011) and the metabolic suppression is accompanied by increase in autonomic (Tattersall and Milsom, 2003) and behavioral (Mortola and Feher, 1998; Bícego et al., 2007; Mortola and Maskrey, 2011) heat loss responses. During prenatal stages, the hypoxic metabolic drop reflects mainly in depression of tissue growth because at this phase growth is the most energy-demanding function. In contrast, after birth, at least in mammals, thermogenesis becomes the principal source of energy expenditure, and its inhibition is the main factor involved in hypoxia-induced metabolic reduction (reviewed by Mortola and Maskrey, 2011). Moreover, there is a general consideration that the greater the 44 metabolic rate (thermogenesis), the larger the metabolic reduction during hypoxia. This reflects the fact that smaller, younger and exposed to cold mammals show a more pronounced reduction in metabolic rate during hypoxia (Mortola and Maskrey, 2011). To the best of our knowledge, no study compared metabolic effects of hypoxia in young birds of different ages and growth rates. In this context, chickens present interesting characteristics for this sort of investigation since they are precocial birds with detectible thermogenesis and independence from maternal care as early as at birth (Mortola, 2009, for review; Toro-Velasquez et al., unpublished). Besides acute hypoxia, chronic exposure to low levels of oxygen can induce long lasting morphophysiological changes, especially if the challenge occurred at critical moments of pre and/or perinatal phases (Mortola, 2009, Dzialowski et al., 2002). Several metabolic and morphometric changes were demonstrated in laying hen chicks submitted to hypoxia during prenatal development as a result of tight supply of oxygen to tissues (Mortola, 2009, Dzialowski et al,. 2002; Chan and Burggren, 2005; Ghatpande et al., 2008). In general, the hypoxic reduction in the growth rate during embryogenesis can lead to immaturity of organ systems in the neonate (Mortola and Cooney, 2008; Mortola and Awan, 2010). Furthermore, it was shown that exposure to hypoxia from the fifth day of incubation reduces the thermal preference of newly hatched normoxic chicks; they select smaller ambient temperatures than the controls (Azzan et al., 2007). Neonates also show less pronounced metabolic reductions to acute hypoxia if they are incubated in low oxygen atmosphere (Szdzuy and Mortola, 2007). Based on the considerations above, we investigated what is/are the main factor(s) affecting metabolic depression by acute hypoxia in young chicks: age, rate of growth, body mass and/or previous exposure to hypoxia during incubation. To this end, we used hatchlings 45 and 10 days old chicks of two strains of domestic chicken, a bred with a normal growth rate (Slow) and a bred which grows twice as quickly (Fast) (Gyles, 1989; Havenstein et al., 1994a,b). 2. Methods Experiments were conducted on chicks (Gallus gallus) of fast-growing (Fast; Cobb 500®) and slow-growing (Slow; White Leghorn) strains at two different ages, 15-18 hrs (hatchlings; H0) and 10 days post-hatching (H10). Experiments were also performed using 5 days old Fast chicks (H5; see explanation in Protocols section). Freshly laid fertilized eggs were obtained from local supplier. After noting the weight, the eggs were placed in incubators set at the temperature of 37.5°C and 60% relative humidity, with a 45° egg rotation at least four times a day. The start of incubation was denoted embryonic day 0 (E0). Incubation temperature and relative humidity were monitored by sensors inside the incubator. Half of the eggs at E12 was transferred to a different incubator with hypoxia 15% until E19. At the end of incubation (E20, internal pipping phase) all eggs were moved to a hatchery-incubator with no rotation; hatching day and time were noted. Chicks were maintained in temperature-controlled and ventilated chambers under a light:dark cycle of 14h:10h up to the day of the experiment (5th or 10th day) with free access to water and commercial starter diet. Ambient temperature was about 32oC (from day 1 to 6) and 30oC (from day 7 to 10). The protocols were conducted with the approval of the local Animal Care and Use Committee (CEUA- number 024166/13). 46 2.1. Oxygen consumption (VO2) VO2 was measured by an open-flow methodology (Szdzuy et al., 2008). The hatchlings (H0) of both strains were placed individually in a 200 mL container inside a temperature controlled chamber (BOD 347CDG, FANEM, Brazil) and a steady gas flow of 150 ml/min was continuously delivered through the respirometer. Regarding the H5 and H10 chicks, they were maintained in a 1000 ml container with a steady gas flow of 1500 ml/min continuously delivered through the respirometer. The outflow gas passed through a drying column (Drierite, Sigma) and the O2 and CO2 concentrations were recorded continuously by calibrated gas analyzers (Sable Systems International Fox, Henderson, NV). The inflow concentrations were monitored intermittently. After mathematical correction of the gas concentrations for a respiratory quotient different from unity (Depocas and Hart 1957; Mortola and Besterman 2007), VO2 was computed from the flow rate and the inflow-outflow gas concentration difference. 2.2. Protocols The same protocol was conducted on chicks of all ages, strains and different incubation conditions (Normoxia: Nx and Hypoxia: HxL). Each bird was left undisturbed in the respirometer for at least half an hour at ~35ºC (H0) or 30 ºC (H5 and H10). Following this time for habituation, the chick was maintained for 15 min in normoxia, 15 min in hypoxia 15%, 15 min in hypoxia 10%, and 20 min in normoxia again (recovery). The same animal was used at H1, H5 and H10. We used Fast H5 chicks in order to isolate age and body mass as a factor of differences between strains, since Fast H5 and Slow H10 had a similar weight (Table 1). 47 2.3. Data Analysis Data are presented as means ±1 SEM. Statistical comparisons among groups were done by one-way ANOVA (resting metabolic rates) and repeated measures two-way ANOVA (effect of acute hypoxia), with a Bonferroni’s post hoc test. It was applied linear regression for testing the relation between metabolic depression during hypoxia and resting metabolic rate in normoxia. The statistical analysis was performed using the software GraphPad Prism 5.0. Differences were considered statistically significant at P < 0.05. 3. Results The characteristics of the eggs and chicks (N=28) are in Table 1. The postnatal age was at least 10 hours at the time of the first measure of VO2 consumption. The temperature at the respirometry chamber changed with age, for H0 it was 35-37 °C, for H5 it was 31-32°C, and for H10 it was 30-31°C for both strains and incubation conditions. The egg of various groups had statistically similar weight. Also the incubation age at hatching, the body temperature at H0 and H5 and the body weight at H5 were not different among strains and incubation condition. Fast H0 and H10 chicks were heavier than Slow ones (p<0.0001). Body temperature of Fast animals from hypoxic incubation was lower than the other groups (p<0.01). 3.1 Effect of hypoxia on metabolic rate of slow-growing (Slow) chicks incubated in normoxia (Nx) and late hypoxia (HxL) The resting metabolic rate (VO2) normalized by body weight increased with age independent on the incubation condition (Nx or HxL) (p< 0.0001) (Fig. 1a). The VO2 of H0 48 were 18.77 ± 1.48and 23.02 ± 1.29 mL/kg.min, and of H10 were 49.2 ± 4.32 and 50.6 ± 3.18 mL/kg/min for the Nx and HxL incubations respectively. At the same age, no differences were observed in VO2 between the two incubation conditions. The exposure to acute hypoxia decreased VO2 in all animals (Fig. 1b, c and d). The H0 and H10 chicks of the Nx incubation had a reduction in VO2 at both hypoxic levels (p<0.0001), but this response was more pronounced in H10 submitted to 10% O2 (p< 0.001; Fig. 1b). The VO2 during recovery was higher in H10 at 10 min (p<0.01) compared to H0. For the HxL incubation group VO2 reduced at 15%O2 (p< 0.05) but did not decreased further at 10%O2 in both ages (Fig. 1c). At 10 min after hypoxia exposure H10 had a higher VO2 than H0 (p< 0.05). Comparing the two incubation conditions, it can be observed that the biggest difference among responses of the same age chicks was the much lower VO2 of H10-Nx than H10-HxL during exposure to 10%O2 (p<0.001; Fig. 1d). The figure 2 depicts the comparison between the resting VO2 at the beginning of the experiment and during the recovery phase at 10 min (Fig. 2a) and 20 min (Fig. 2b) after the end of 15 and 10%O2 exposure. At time 10 min values of H10 chicks of both Nx and HxL incubations were above the oblique line (line of identity: initial VO2 = recovery VO2), which means recovery VO2 is higher than initial metabolic rate. In the fig 2.b all the data points are on the line of identity. 3.2. Effect of hypoxia on metabolic rate of fast-growing (Fast) chicks incubated in normoxia (Nx) and late hypoxia (HxL) Regardless the incubation condition, the resting metabolic rate normalized by body weight increased with age (p<0.0001; Fig. 3a). The VO2 of H0 were 22.65±0.51and 49 26.30±1.8 mL/kg.min, while those of H5 were 35.93±2.1 and 39.02±2.42mL/kg/min and of H10 were 40.85±1.4 and 39.25±1.1mL/kg/min for the Nx and HxL incubations respectively. H0 chicks had lower VO2 than both H5 and H10 (p<0.0001) but no difference was observed between H5 and H10. In the Nx incubation, metabolic rate of H5 and H10, but not H0, decreased during hypoxia 10%O2. During the recovery time VO2 of H0, but not H5 and H10, increased at time 10 min (p< 0.0001), decreasing at 20 min but remaining higher than in the other ages (p< 0.05; Fig 3b). In the HxL incubation (Fig 3c) the hypoxic reduction of VO2 was similar in H5 and H10 while it was less pronounced in H0 during 10%O2 exposure (p< 0.05). After hypoxia, VO2 of H0 increased at 10 min and remained high at 20 min compared with the other chicks (p<0.001). Comparing Nx and HxL incubations, it can be observed that H5 as well as H10 chicks showed similar responses to hypoxia and recovery to normoxia, while H0-Nx presented a higher hypoxic VO2 (10%O2) than H0-HxL (p<0.0001; Fig. 3d). The figure 4 depicts the comparison between the resting VO2 at the beginning of the experiment and during the recovery phase at 10 min (Fig. 4a) and 20 min (Fig. 4b) after the end of 10%O2 exposure. At the time 10 min data values of H0 chicks of both Nx and HxL incubations were above the oblique line (line of identity: initial VO2 = recovery VO2), which means recovery VO2 is higher than initial metabolic rate. In the fig 4b all the data points are on the line of identity. 50 Comparisons of metabolic rates between fast- and slow-growing chicks incubated in normoxia (Nx) and late hypoxia (HxL) ������ ���� ���������������������������������� ����� ����� ������� ����������� O2 of all animals in normoxia. The Fast and Slow H0 chicks of both Nx and HxL incubations showed similar weights and resting normoxic VO2. Regardless incubation condition, the Fast H5 and Slow H10 chicks presented similar body weights but Fast H10 birds had higher resting metabolic rates. Fast H10 chicks presented the highest body weight and metabolic rate. As can be seen in Fig. 5b hypoxic metabolic rates of Fast and Slow H0 chicks from both incubation conditions were close to the 0 line, which indicates no difference between the hypoxic and normoxic VO2. Regardless incubation condition Slow H10 chicks had similar body weights as Fast H5; however the Slow H10 birds from Nx group showed further drop in hypoxic VO2 while HxL incubation caused a great variability of the responses, with values similar to Fast H5. Fast H10 chicks, on the other hand, presented higher body weight but similar reduction in hypoxic VO2 compared to Slow H10, independent on incubation condition. Regardless both incubation condition and chicken strain, the higher the resting VO2 (per unit weight) in normoxia the greater was its hypoxic drop (Fig. 6). The slopes of the linear regressions were not significantly different among groups. Discussion In the present study we demonstrate that the magnitude of drop in hypoxic VO2 is 51 dependent on the level of normoxic resting metabolic rate in precocial chicks, which is similar to the pattern observed in several species of mammals (Mortola and Maskrey, 2011). This conclusion is based on the fact that regardless all conditions analyzed, i.e., age, body mass, growth rate, strain or hypoxia exposure during the last phase of incubation, the greater the normoxic resting VO2 the larger the metabolic depression during acute hypoxia (Fig.6). However, differences are observed in the factors that contribute to the resting metabolic rate in these animals. Although hypoxia during incubation is known to reduce growth rate, body weight of both slow and fast growing H0 chicks were not significantly affected by hypoxia during incubation (Table 1). In previous studies (Dzialowski et al., 2002; Azzam et al., 2007) it was suggested that the hatchling's body weight may be almost normal because in the process of hatching the abdomen incorporates the remaining yolk not consumed during embryogenesis (Mortola, 2009). Slow H10 chicks had a higher metabolic rate normalized by body weight than Fast chicks. Konarzewski et al (2000) also studied two trains, Slow and Fast growing chicken and found that, despite growing six times faster, Fast chicks had lower resting VO2, a difference that disappeared after one week of life. Those differences between strains in growth rate during the first week after hatching were not reflected in similar differences in the relative masses of the heart, liver, and small intestine. However, Fast animals had heavier intestines once they reached a body mass of 80 g, but had relatively smaller brain. If we consider the major tasks for energy in the animal are biosynthesis, maintenance and external work, but the most expensive budge for an organism is maintenance, gain muscle is less expensive than organ activities. Although Slow H10 chicks were almost 2 times smaller than Fast H10, they 52 showed similar hypoxic metabolic depressions (Fig. 5b), which may be attributed to the relatively higher resting metabolic rate of Slow H10, even higher than the Fast H5 of similar body weight. Acknowledgment: The present study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (10/20285-1). P. A. T. Velasquez was the recipient of a FAPESP (11/07509-0) PhD scholarship. References Azzam, M. A., Szdzuy, K., Mortola, J. P. 2007. Hypoxic incubation blunts the development of thermogenesis in chicken embryos and hatchlings. Am J Physiol Regul Integr Comp Physiol. 292: R2373–R2379. Bicego, K.C., Barros R.C.H., Branco L.G.S. 2007. Review Physiology of temperature regulation: Comparative aspects. Comparative Biochemistry and Physiology, Part A 147: 616–639. Chan, T., Burggren W. 2005. Hypoxic incubation creates differential morphological effects during specific developmental critical windows in the embryo of the chicken (Gallus gallus). Resp. Physiol. Neuro. 145, 251-263. Dzialowski, E. M., Plettenberg, D. V., Elmonoufy, N. A., Burggren, W. W. 2002. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp. Biochem. Physiol. A, 131, 713-724. Depocas, F., Hart, J.S., 1957. Use of the Pauling oxygen analyzer for measurement of oxygen 53 consumption of animals in open-circuit systems and in a short-lag, closed-circuit apparatus. J. Appl. Physiol. 10, 388-392. Gautier, H. 1996. Interactions among metabolic rate, hypoxia, and control of breathing. J Appl Physiol 81, 521±527. Ghatpande, S.K., Billington, J. Jr., Rivkees, S.A., Wendler, C.C. 2008. Hypoxia Induces Cardiac Malformations via A1 Adenosine Receptor Activation in Chicken Embryos. Birth Defects Res A Clin Mol Teratol. 82(3): 121–130. Gyles, N.R. 1989. Poultry, people and progress. Poultry Science, 68: 1–8. Havenstein, G.B., Ferket, P.R., Scheideler, S.E., Larson, T.B. 1994a. Growth, liveability and feed conversion of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poultry Science, 73: 1785–1794. Havenstein, G.B., Ferket, P.R., Scheideler, S.E., Rives, D.B. 1994b. Carcass composition and yield of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poultry Science, 73: 1795–1804. Konarzewski, M., Gawin, A., McDevitt, R., Wallis, R. 2000. Metabolic and organ mass responses to selection for high growth rates in the domestic chicken (Gallus domesticus) Physiol Bioche Zoo, 73: 237-248. Mortola, J.P. 2009. Review Gas exchange in avian embryos and hatchlings. Comp. Biochem. Physiol. A 153: 359–377. Mortola, J.P., Awam, K.A. 2010. Growth of the chicken embryo: Implications of egg size. Comparative Biochemistry and Physiology, Part A 156, 373–379. 54 Mortola, J.P., Besterman, A.D., 2007. Gaseous metabolism of the chicken embryo and