UNIVERSIDADE ESTADUAL PAULISTA – UNESP CENTRO DE AQUICULTURA DA UNESP Grãos de milho de destilaria secos com solúveis em dietas para juvenis de Piaractus mesopotamicus (Holmberg 1987) Kátia Rodrigues Batista de Oliveira Jaboticabal, São Paulo 2016 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CENTRO DE AQUICULTURA DA UNESP Grãos de milho de destilaria secos com solúveis em dietas para juvenis de Piaractus mesopotamicus (Holmberg 1987) Kátia Rodrigues Batista de Oliveira Orientador: Dra Elisabete Maria Macedo Viegas Dissertação apresentada ao Programa de Pós-graduação em Aquicultura do Centro de Aquicultura da UNESP - CAUNESP, como parte dos requisitos para obtenção do título de Mestre. Jaboticabal, São Paulo 2016 Oliveira, Kátia Rodrigues Batista de O48g Grãos de milho de destilaria secos com solúveis em dietas para juvenis de Piaractus mesopotamicus (Holmberg 1987) / Kátia Rodrigues Batista de Oliveira. – – Jaboticabal, 2016 ii, 103 p. : il. ; 29 cm Dissertação (mestrado) - Universidade Estadual Paulista, Centro de Aquicultura, 2016 Orientadora: Elisabete Maria Macedo Viegas Banca examinadora: Giuliana Parisi, Leonardo Tachibana Bibliografia 1. DDGS. 2. Pacu. 3. Enzimas digestivas. 4. Peixes nativos. 5. Biocombustíveis. I. Título. II. Jaboticabal-Centro de Aquicultura. CDU 639.3.043 Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação – Serviço Técnico de Biblioteca e Documentação - UNESP, Câmpus de Jaboticabal. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP i Sumário Agradecimentos.................................................................................................... 1 Apoio Financeiro .................................................................................................. 3 Resumo ................................................................................................................. 4 Palavras-Chave .....................................................................................................8 Abstract .................................................................................................................9 Key-words ...........................................................................................................10 Introdução Geral ................................................................................................... 8 Manuscript 1. Effect of corn DDGS on growth performance, feed utilization and digestibility of Piaractus mesopotamicus juveniles ................................ 13 Abstract ............................................................................................................... 13 Key-words ........................................................................................................... 14 Introduction ......................................................................................................... 15 Material and Methods ......................................................................................... 16 DDGS Digestibility trial ......................................................................................... 16 Growth and Digestibility Trail ................................................................................ 17 Sampling ............................................................................................................... 19 Chemical Analyses ............................................................................................... 20 Economic Viability ................................................................................................. 20 Statistical Analyses ............................................................................................... 21 Results ................................................................................................................. 21 DDGS and Experimental Diets Digestibility .......................................................... 21 Growth and Economic Viability ............................................................................. 22 Discussion .......................................................................................................... 23 DDGS Digestibility ................................................................................................ 23 Growth and Economic Viability ............................................................................. 28 Conclusion .......................................................................................................... 30 References .......................................................................................................... 31 Tables Index ........................................................................................................ 42 Tables .................................................................................................................. 43 Manuscript 2. Effect of corn DDGS on digestive enzymes, oxidative stress, and intestine morphology of Piaractus mesopotamicus juveniles ................ 48 Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP ii Abstract ............................................................................................................... 48 Key Words ........................................................................................................... 49 Introduction ......................................................................................................... 50 Material and Methods ......................................................................................... 51 Experimental Trial ................................................................................................. 51 Sampling ............................................................................................................... 52 Digestive Enzymes Analyses ................................................................................ 53 Oxidative Stress Analyses .................................................................................... 56 Intestine Histology ................................................................................................ 57 Statistical Analyses ............................................................................................... 57 Results ................................................................................................................. 57 Intestine pH and digestive enzymes ..................................................................... 57 Oxidative Stress Enzymes .................................................................................... 60 Histology ............................................................................................................... 61 Discussion .......................................................................................................... 61 Intestinal pH profile ............................................................................................... 61 Digestive enzymes profile ..................................................................................... 62 Intestine Oxidative Status and Morphology .......................................................... 65 Conclusion .......................................................................................................... 69 References .......................................................................................................... 70 Tables Index ........................................................................................................ 85 Figures ................................................................................................................. 85 Tables .................................................................................................................. 86 Discussão Geral.................................................................................................. 92 Referências Complementares ........................................................................... 95 Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 1 Agradecimentos Ao Deus pai, sabedoria suprema de todas as coisas. A toda minha família, meu alicerce, minha base. Em especial àqueles já não mais presentes: minha mãe Eliete e minhas avós Gercy Lara e Dica. Ao meu pai José Maria pelo apoio e amor incondicional. Por ser sempre exemplo de força e luta, também tranquilidade, compaixão e humildade. Sempre mostrando o caminho do bem, quem me ensinou e ensina os grandes valores e ensinamentos da vida. À minha madrinha Marilene e À tia Direne pelo apoio, dedicação, paciência, carinho e amor. Por serem tão importantes e essenciais, grandes responsáveis pela pessoa em que me tornei e pelo o que tenho conquistado. Agradeço pelo exemplo de luta e perseverança. Por sempre ressaltarem o valor e a importância do estudo e o quão longe podemos chegar com ele. Às minhas irmãs Layra, Karina, Kênia e Stella (de coração) pela presença, companheirismo, parceria, carinho e amor, com quem sei poder contar sempre e a quem tanto amo. Ao Diego Zanetti, pelo carinho, amor, confiança, compreensão, paciência e dedicação. Obrigada por contribuir na realização deste trabalho e por participar e estar presente ao meu lado em todos os momentos. À Universidade Estadual Paulista “Júlio de mesquita Filho”, principalmente ao Centro de Aquicultura pelos ensinamentos e contribuição em minha formação acadêmica. À Faculdade de Zootecnia e Engenharia de Alimentos – FZEA/USP, principalmente ao Departamento de Zootecnia pelo suporte e acolhimento. Ao CNPq e FAPESP pela concessão da bolsa durante todo o curso de mestrado. À professora Elisabete Maria Macedo Viegas, pela orientação, apoio, ensinamentos e confiança. Obrigada pela oportunidade. À professora Ana Lúcia Salaro, que tanto contribui para minha formação profissional e pessoal, pela amizade e bons conselhos. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 2 Agradeço em especial, à professora Helena Peres, pelos ensinamentos, disposição, atenção e carinho. Muito obrigada por sua contribuição na realização deste trabalho. Ao laboratório de aquicultura da FZEA-USP e todos os seus membros. Aos técnicos José Apolináro e Daflin por toda a ajuda e amizade ao longo destes anos. Aos parceiros e amigos Adja, Júlio, Mariene (Bitoca), Rosa, Joana, Beatriz, Sheyla. Rachel, Francine e Thaisa. Obrigada pela amizade e excelente convivência. Especialmente àqueles que tanto me ajudaram na realização dos experimentos, até mesmo de madrugada! Ao laboratório Nutrition and Immunobiology Research Group (NUTRIMU) da Universidade do Porto e a todos os seus membros. Especialmente ao professor Aires Oliva-Teles pelos ensinamentos e contribuições no trabalho. Aos amigos Alexandre e Renan pela ajuda e amizade. Aos grandes amigos que fiz durante os períodos de intercâmbio. Aos membros e grandes irmãos das repúblicas Mara de Viçosa e Cortiço de Pirassununga A todos vocês que contribuíram para a concretização deste trabalho. Muito obrigada. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 3 Apoio Financeiro CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico– Bolsa de Mestrado, Processo 130664/2014-6, Período de Vigência: março/2014 a fevereiro/2015. FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo - Bolsa de Mestrado, Numero do Processo 2014/16685-5, Período de Vigência: março/2015 a junho/2016. FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo - Bolsa de Estágio no Exterior, Numero do Processo 2015/21245-7, Período de Vigência: novembro/2015 a fevereiro/2016. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 4 Resumo Devido ao maior interesse por biocombustíveis, indústrias brasileiras iniciaram, recentemente, a produção de etanol também a partir de grãos de milho, gerando um resíduo com potencial de uso como ingrediente em rações para animais, o DDGS (grãos secos de destilaria com solúveis). Por resultar de processos de fermentação de grãos de milho por leveduras e enzimas, este resíduo possui um elevado teor proteico e baixo teor de carboidratos solúveis, o que o torna boa fonte de proteína vegetal em rações para animais. Além do baixo custo, possíveis benefícios relacionados aos resíduos de leveduras e enzimas restantes da fermentação também contribuem para seu potencial de mercado. Desta forma, com este trabalho, objetivou-se avaliar a viabilidade de inclusão do DDGS do milho em dietas para juvenis de Piaractus mesopotamicus em substituição ao farelo de soja. Para tal, foram realizados três ensaios experimentais. No primeiro ensaio avaliaram-se os coeficientes de digestibilidade aparente (CDA) de nutrientes do DDGS para juvenis de P. mesopotamicus (13±0.3 gramas), distribuídos, em delineamento inteiramente casualizado (DIC), em seis tanques de fibra de vidro, na densidade de 35 peixes tanque -1 em sistema de recirculação contínuo de água. A coleta das fezes foi realizada em sistema de Guelph modificado. Após obtenção dos CDAs, foram formuladas dietas contendo cinco diferentes níveis de inclusão de DDGS (0, 10, 20, 30 e 40%) utilizadas nos ensaios posteriores. O segundo ensaio consistiu na avaliação dos CDAs dos nutrientes das dietas contendo 0, 10, 20, 30 e 40% DDGS, onde juvenis de P. mesopotamicus (27±1.4 gramas) foram distribuídos em cinco tanques de fibra de vidro na densidade de 30 peixes tanque -1 em sistema de recirculação de água. Utilizou-se delineamento em Quadrado Latino, 5x5 (05 dietas e 05 períodos). Concomitantemente ao segundo ensaio, e sob o mesmo sistema de recirculação, juvenis de P. mesopotamicus (21±0.2 gramas) foram distribuídos em 20 tanques de fibra de vidro, na densidade de 15 peixes tanque -1, em DIC, e alimentados com as dietas por 100 dias. Neste terceiro ensaio foram avaliados parâmetros de desempenho produtivo, viabilidade econômica, atividade das enzimas digestivas e de estresse oxidativo do intestino, bem como morfologia intestinal dos juvenis. Os dados obtidos de desempenho produtivo, estresse oxidativo e morfometria intestinal foram submetidos à one-way Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 5 ANOVA e em caso de significância (p < 0.05) foi realizado teste de Tukey adotando- se 5% como nível de probabilidade. Dados de enzimas digestivas foram submetidos a two-way ANOVA e em caso de significância para interação foi feita uma one-way ANOVA e teste Tukey a 5%. Os valores obtidos para os CDA do DDGS confirmaram seu potencial de uso como ingrediente proteico em dietas para P. mesopotamicus, assim como os resultados de desempenho produtivo, onde se obteve menor valor de conversão alimentar e melhor eficiência de retenção de proteína para a dieta contendo maior nível de inclusão de DDGS (40DDGS). Os demais parâmetros de desempenho não foram afetados significativamente. A atividade das enzimas digestivas foi reduzida da porção anterior do intestino para distal e para as dietas com níveis superiores a 10% de DDGS. A inclusão de DDGS levou a redução do status oxidativo do intestino e melhoras na morfometria intestinal. Sendo assim, é possível o uso de até 40% de DDGS do milho como ingrediente proteico em dietas para juvenis de P. mesopotamicus, substituindo em totalidade o farelo de soja, mantendo os valores de desempenho produtivo, melhorando a saúde intestinal dos peixes bem como a capacidade de absorção e aproveitamento dos nutrientes disponibilizados na dieta. Palavras-Chave Biocombustíveis, DDGS, enzimas intestinais, pacu, peixes nativos. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 6 Abstract Due to the increased interest in biofuels, Brazilian companies started recently, the production of ethanol from corn, generating a waste with potential for use as an ingredient in animal feed, the DDGS (dried distillers grain with soluble). As its processes results from fermentation of corn grain by yeast and enzymes, this residue has high protein and low soluble carbohydrates, which makes it good source of vegetable protein for animal feed. Besides the low cost, possible benefits related to yeast residues and other enzymes from fermentation may also contribute to DDGS market potential. Thus, this work aimed to evaluate the feasibility of inclusion of corn DDGS in diets for Piaractus mesopotamicus juveniles to replace soybean meal. To this end, there were three experimental runs. In the first assay we evaluated the apparent digestibility coefficients (ADC) of DDGS nutrients for P. mesopotamicus (13 ± 0.3 grams), distributed in a completely randomized design (CRD) in six fiberglass tanks, at density of 35 fish tank-1 in a continuous recirculating water system. The collection of feces was carried out in modified Guelph system. After obtaining the ADCs, diets were formulated with five different levels of DDGS inclusion (0, 10, 20, 30 and 40%) used in subsequent assays. The second test was the evaluation of ADCs of nutrient in the diets containing 0, 10, 20, 30 and 40% DDGS where P. mesopotamicus juvenile (27 ± 1.4 g) were distributed in five fiberglass tanks at density of 30 fish tank-1 in a recirculating water system. We used a Square Latino design, 5x5 (05 diets and 05 periods). Concomitantly to the second test, and under the same recirculation system, P. mesopotamicus juveniles (21 ± 0.2 grams) were divided into 20 fiberglass tanks, at density of 15 fish tank-1 in CRD, and fed diets for 100 days. In this third test were evaluated growth performance, economic viability, activity of digestive enzymes and oxidative stress of the intestine and intestinal morphology of juveniles. Data obtained for growth performance, oxidative stress and intestine morphology were subjected to one-way ANOVA and in case of significance (p <0.05) Tukey's test was carried out adopting a 5% probability level. Data from digestive enzymes were subjected to two-way ANOVA and in case of significance to interaction was made a one-way ANOVA and Tukey's test at 5%. The ADCs values obtained for the DDGS confirmed its potential use as a protein ingredient in diets for P. mesopotamicus, as well as the results of Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 7 productive performance, which showed lower value of feed conversion ratio and an improved on protein retention efficiency for the diet containing higher inclusion level of DDGS (40DDGS). The other performance parameters were not significantly affected. The activity of the digestive enzymes was reduced from anterior to distal portion of the intestine and for diets with levels above 10% DDGS. The DDGS inclusion led to reduction of oxidative status of the intestine and improvement in intestinal morphology. Thus, the use of up to 40% corn DDGS as a protein ingredient for P. mesopotamicus juvenile is possible, replacing in whole soybean meal, keeping the growth performance, improving fish gut health as well as the absorption and utilization of nutrients available in the diet. Key-words Biofuels, DDGS, intestinal enzymes, pacu, native fish Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 8 Introdução Geral A aquicultura mundial encontra-se em fase de intenso crescimento, com produção prevista de mais de 186 milhões de toneladas para o ano de 2030 (World Bank, 2013). Este aumento de produção resulta em maior demanda por ingredientes para fabricação de rações, como farinha e óleo de peixe. Contudo, a produção destes ingredientes não tem se mostrado suficiente para atender a demanda de mercado (FAO, 2012), elevando os preços de custo e consequentemente o valor a ser pago pelo consumidor. Dessa forma, torna-se necessário a busca por ingredientes alternativos sustentáveis, que apresentem maior disponibilidade e menor custo de produção, como é o caso de alguns produtos de origem vegetal (Ayadi, 2012). O principal ingrediente utilizado atualmente como substituto à farinha de peixe é o farelo de soja, por apresentar alto teor de proteína (48%), considerável balanço de aminoácidos (Gatlin et al., 2007), alta disponibilidade e preço razoável (Refstie et al., 2000; Thompson et al., 2008). Porém, este possui alguns fatores antinutricionais que podem prejudicar o consumo das rações e o ganho de peso dos animais (Francis et al., 2001). Existe um vasto grupo de ingredientes que possuem potencial para serem utilizados na fabricação de rações para animais como fontes proteicas alternativas (Ayadi, 2012). Os resíduos de grandes indústrias, por exemplo, tornam-se uma boa opção, visto que seu uso também permite a reutilização de produtos já descartados pelas indústrias e que possivelmente se tornariam um problema ambiental, como é o caso dos resíduos da indústria do etanol (Wyman, 1996). O milho e a cana de açúcar são as principais matérias primas utilizadas na fabricação de etanol devido ao elevado teor de sacarose e amido, sendo a cana de açúcar utilizada principalmente no Brasil e o milho nos Estados Unidos. No entanto, no período da entressafra da cana de açúcar no Brasil as destilarias ficam com a produção limitada devido à menor disponibilidade desta matéria prima (Duarte et al., 2012). Estima-se que a produção de álcool no Brasil alcance 64 bilhões de litros em 2017, podendo até mesmo superar os Estados Unidos em exportação de etanol (Carvalho, 2009). Dessa forma, a utilização de milho como matéria prima para a produção de etanol no Brasil aparece como uma provável opção para usinas, uma Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 9 vez que possibilitaria, principalmente, uma produção de etanol mais constante ao longo do ano, permitindo assim uma maior competitividade do etanol brasileiro no mercado externo. Quanto à produção de milho, mesmo sendo crescente e as exportações estarem em alta, ainda há uma perda de competitividade do milho brasileiro no mercado mundial devido, principalmente, às deficiências no transporte da produção até os portos de exportação (Duarte et al., 2012). Portanto, utilizar o milho como substrato para produção de etanol no Brasil torna-se uma alternativa, tanto por prover uma destinação viável ao excedente de produção, manter a produtividade das usinas na entressafra da cana, bem como por gerar resíduos com potencial de uso pelas fábricas de ração, como é o caso do DDGS. DDGS ou grãos secos de destilaria com solúveis resultam da fermentação de grãos de milho pela adição de leveduras e enzimas. Após a fermentação, o líquido produzido é destilado e destinado à produção do etanol, enquanto o restante segue para um conjunto de centrífugas, onde se separam a parte fina, que será reaproveitada no processo ou levada para evaporação, e a parte mais grosseira, que após centrifugação originará o DDG (Disttilers Dried Grains) (Wyman, 1996). Ao adicionar a parte fina ao DDG obtém-se o DDGS (Distillers Dried Grains with Soluble) que também pode ser utilizado na alimentação animal. Portanto, as características do produto final, DDG ou DDGS, dependerão das práticas de fabricação adotadas pelas usinas de etanol. Por perder grande parte dos carboidratos durante a fermentação, o DDGS torna-se um produto mais concentrado que o milho em proteína e baixa quantidade de amido (Jacques et al., 2003). Além de não possuir fatores antinutricionais (Lim et al., 2009), seu custo está bem abaixo quando comparado ao da soja (Ayadi, 2012), onde a tonelada de DDGS está cotada em R$ 500,00 (Fonte: Libra Etanol, 2016) e a da soja a R$ 1.060,00 (Fonte: Conab, 2016). Por ser um resíduo industrial sua composição nutricional está sujeita a grandes variações que podem estar relacionadas tanto aos métodos de produção do DDGS quanto à qualidade e composição do grão utilizado (Shurson e Alghamdi, 2008). Cromwell et al. (1993) avaliando nove diferentes fontes de DDGS, observaram diferenças na composição nutricional, química e até mesmo física das amostras. O mesmo foi observado por Spiehs et al. (2002) onde foram avaliadas 118 amostras Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 10 de DDGS oriundos de 10 diferentes usinas de etanol durante os três anos consecutivos 1997, 1998 e 1999 e por Belyea et al. (2004) analisando a composição de 235 amostras de DDGS, produtos de uma usina de etanol no estado de Minnesota, Estados Unidos. Liu (2008) também avaliou a composição nutricional em função da distribuição dos tamanhos de partículas contidos em 11 amostras de DDGS do milho. Dentre estes autores, maiores variações na composição química das amostras de DDGS foram obtidas por Cromwell et al. (1993) contrastando com Spiehs et al. (2002), Belyea et al. (2004) e Liu (2008). Essa diferença pode estra relacionada à maior padronização dos métodos de produção de DDGS pelas usinas ao longo dos anos, visto que esta é uma necessidade do ponto de vista nutricional, uma vez que permite a otimização da inclusão do DDGS na alimentação animal. Dentre os nutrientes contidos no DDGS a proteína apresenta variações mais bruscas, o que pode estar relacionado ao perfil de aminoácidos. Segundo Cromwell et al. (1993) e Spiehs et al. (2002) a lisina é o aminoácido que mais sofre variações dentre as diferentes amostras de DDGS, seguida pela metionina e triptofano. Durante seu processamento o DDGS passa por temperaturas muito elevadas, podendo chegar até 550°C. Estas elevadas temperaturas podem modificar a estrutura das proteínas contidas no DDGS bem como o perfil dos aminoácidos. A lisina, por exemplo, é altamente susceptível a altas temperaturas, estando sua concentração muitas vezes ligada aos tons de coloração do DDGS (Cromwell et al., 1993; Fastinger et al.,2006). Tons mais escuros indicam provável ocorrência de reação de Maillard (Stein et al., 2006) e portanto menor concentração de lisina, sendo o oposto observado para DDGS de coloração mais clara. Contudo alterações na coloração do DDGS também podem estar relacionadas a outros fatores como a cor do grão e quantidade de solúveis adicionados antes do processo de secagem final. Quanto à composição de minerais, o DDGS possui grande quantidade de fósforo na forma disponível, o que possibilita maior absorção deste mineral pelos peixes e minimiza impactos ambientais. Possui também um elevado teor de sódio, cálcio e enxofre, podendo ter uma concentração destes minerais de até três vezes do que o observado para grãos de milho (Liu e Han, 2006). Além disto, o DDGS possui relativa quantidade de leveduras, estimada em 3.9% (Ingledew et al., 1999), Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 11 o que pode trazer benefícios tanto para a saúde da microbiota intestinal (He et al., 2013) como para o sistema imune dos animais (Lim et al., 2009). Na aquicultura, o DDGS tem se apresentado como candidato à substituição da farinha de peixe quando utilizado em conjunto com outra fonte proteica animal (Coyle et al., 2004) e também quando em substituição ao farelo de soja (Øverland et al., 2013; Zhou et al., 2010). Para bagre do canal (Ictalurus punctatus) resultados positivos foram obtidos para níveis de até 30% de inclusão de DDGS (Tidwell, 1990; Webster et al., 1993; Robinson e Lee, 2008; Zhou et al., 2010). A inclusão de DDGS em dietas para tilápia do Nilo (Oreochromis niloticus) na faixa de 20 a 30% pode ser realizada sem causar alterações deletérias no crescimento e ou saúde do animal (Schaeffer et al., 2009). Já para truta do arco-íris (Oncorhynchus mykiss) é possível à substituição de até 50% da farinha de peixe nas dietas (Cheng e Hardy 2004a), sendo que 15% de DDGS foi o nível máximo incorporado com sucesso para esta espécie. Pode ser uma eficiente fonte de proteína principalmente para espécies onívoras, uma vez que as exigências por dietas com altos níveis proteicos não são tão acentuadas como para as espécies de hábito alimentar carnívoro (Boscolo et al., 2011; Gatlin et al., 2007; Hardy, 2010). Além disso, peixes onívoros podem tolerar até 9% de fibra bruta na dieta (Rodrigues et al., 2010), minimizando as limitações ao uso do DDGS pelo seu alto teor de fibras. Portanto, a determinação dos níveis de inclusão de DDGS em dietas para animais aquáticos vai depender, dentre outros fatores, do hábito alimentar da espécie e demanda por nutrientes. Piaractus mesopotamicus é uma espécie de peixe tropical, endêmico das planícies alagadas da região Centro-Oeste do Brasil. Pertencente à família Myleinae é um peixe de hábito alimentar onívoro-frutívoro, alimentando-se principalmente de folhas, caules, flores, frutos e sementes. Comumente conhecido como pacu ou pacu-caranha, esta espécie apresenta relativa facilidade de cultivo em cativeiro devido a sua rusticidade, boa adaptabilidade e capacidade de ganho de peso (Nunes, 2006). Apreciado pela culinária devido à boa qualidade e sabor da carne, também é explorado na pesca esportiva (Jomori et al., 2003). Pesquisas relacionadas à nutrição do pacu vêm sendo conduzidas visando melhorar a eficiência de criação desta espécie e alguns dados já se encontram disponíveis, como exigência proteica (Batista et al., 2000;. Abimorad et al., 2008), exigência de Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 12 lisina (Bicudo et al., 2009) e coeficientes de digestibilidade aparente para alguns ingredientes (Abimorad et al., 2008). Tendo em vista a importância do pacu na aquicultura brasileira, faz-se necessário maior incentivo e investimento nas pesquisas utilizando ingredientes proteicos alternativos à farinha de peixe e farelo de soja, gerando informações que visem melhorias na eficiência de criação desta espécie. Dessa forma objetivou-se avaliar o valor nutritivo do DDGS do milho e a influência de seu uso em substituição ao farelo de soja, em dietas para juvenis de pacu (Piaractus mesopotamicus) sobre os parâmetros de desempenho produtivo, atividades das enzimas digestivas e de estresse oxidativo do intestino, microbiologia intestinal e viabilidade econômica. Para fins didáticos, este projeto foi dividido em dois capítulos, ambos escritos em formato de artigos para serem prontamente submetidos à publicação científica. O primeiro capítulo “Effect of corn DDGS on growth performance, feed utilization and digestibility of Piaractus mesopotamicus juveniles” refere-se a uma avaliação prévia do potencial de uso do DDGS do milho em dietas para o pacu, através da avaliação dos parametros de digestibilidade, desempenho produtivo e viabilidade econômica. O segundo capítulo “Effect of corn DDGS on digestive enzymes, oxidative stress and intestine morphology of Piaractus mesopotamicus juveniles” trata-se de uma avaliação a nível fisiológico dos possíves efeitos provocados pela inclusão de DDGS do milho nas dietas para o pacu em remoção total ao farelo de soja, através da determinação da atividade das enzimas digestivas, status oxidativo e morfologia intestinal. Ao final, correlacionado os dois capítulos apresentados, torna-se possível uma análise mais ampla e completa do potencial de uso do DDGS do milho em dietas para pacu, como produtividade, viabilidade econômica e ambiental, bem como possíveis efeitos na saúde intestinal dos peixes. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 13 Manuscript 01: Effect of corn DDGS on growth performance, feed utilization and digestibility of Piaractus mesopotamicus juveniles Oliveira, K.R.B*; Viegas, E.M.M. * Universidade Estadual Paulista “Júlio de Mesquita Filho”- Via Professor AC Paulo D Castellane - s/n Km 5, Jaboticabal – SP Scientific Journal Target of Publication: Aquaculture Nutrition, ISSN: 1365-2095, Impact Factor: 1.395 Abstract DDGS (Distiller’s Dried Grains with Soluble) is a potential ingredient for soybean meal replacement in animal diets due to reductions on production costs. The aim of this study was to evaluate the use of DDGS as a substitute of soybean meal in diets for P. mesopotamicus juveniles. Three experimental trails were performed, being the digestibility of the ingredient (DDGS) the first one, with 210 P. mesopotamicus juveniles (13±0.3 grams) randomly allocated in six tanks (35 fish tank-1), fed a diet containing 30% DDGS, 69.5% referential diet and 0.5% chromic oxide, for seven days. Fecal samples were collected and analyzed due to estimate Apparent Digestibility Coefficient (ADC) for protein, energy, dry matter, lipid and phosphorus. After obtaining data, diets were formulated to contain different levels of DDGS inclusion (0, 10, 20, 30 and 40%). The second trial consisted in determine the ADCs for the diets mentioned above. Thus, P. mesopotamicus juveniles (27±1.4grams) were stocked in five tanks (30 fish tank-1) in Latin square design 5 x 5 (five treatments and five experimental periods). During each period, fish were fed with experimental diets, added 0.5% chromic oxide, for seven days and feces were collected. The third experiment was a growth trial with 300 P. mesopotamicus juveniles (21±0.2 grams) randomly distributed in 20 fiber glass tanks, divided in five treatments (0, 10, 20, 30 and 40% DDGS) and four replicates, feeding experimental diets for 100 days. Growth performance, economic viability, feed utilization and Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 14 digestibility were evaluated. All data were subjected to a one-way ANOVA analysis of variance to determine significant (p ≤ 0.05) differences among treatments and subsequently Tukey’s test. The lower ADC was obtained for dry matter (62.7 %) and the higher for protein (94.8 %) and phosphorus (91 %). The ADCs for dry matter and energy reduced significantly (p < 0.05) with DDGS inclusion. The opposite was true for lipid that decreased from 94.1% for 40DDGS diet to 87.8 % for the control without DDGS inclusion. Also, it was observed a decrease (p < 0.05) in total phosphorus release in the water with increasing levels of DDGS in the diets. Feed conversion ratio and efficiency protein ratio were positively affected by DDGS inclusion (p < 0.05). The other growth parameters did not differ (p > 0.05) between treatments. Dietary costs of weight gain were reduced (p < 0.05) in 26% with DDGS inclusion and soybean meal replacement. Thus, it is possible the inclusion of 40% of corn DDGS as a plant protein source in diets for P. mesopotamicus in total replacement of soybean meal without provide negative effects on fish production. Key-words DDGS, industry residues, native fish, pacu, plant protein, phosphorus availability Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 15 Introduction Residues represent a challenge for industries since they have not more propose in the productive chain, generating difficulties especially related with storage and discard. In the United States of America, where the corn is the major source of starch for ethanol production, the amount of product and residues remained are relatively close. Each ton of corn grain produces 465 liters of ethanol and 300 kg of residue (DDGS) (Wyman, 1996). To evaluate the potential reuse of residues from industries is an alternative that would benefit both, industries and environment. The evaluation of the viability of these residues as ingredients in animal feed appears to be an alternative solution. Distiller’s dried grain with soluble is the residue derived from the fermentation of grains by the action of enzymes and yeasts on ethanol production (Wyman, 1996). Its chemical composition may vary according to the grain source and methods of fermentation (Liu, 2009; Lim et al., 2011), but basically consists of 28 - 33% of crude protein, 3.5- 12.8% of lipid, 5.4 to 10.6% of crude fiber, 2.8 to 9.8% of ash, 0.5-1.1% of lysine and 0.5-0.8% of methionine (Ayadi et al., 2012). DDGS apparent digestibility coefficients have being established for vary terrestrial animals species with commercial interest as broilers (Liu, 2011), swine (Urriola and Stein, 2010; Yang et al., 2010; Pedersen et al., 2007) and rabbits (Youssef et al., 2012). Nevertheless, some aquatic animals are also being focus of the studies regardless DDGS digestibility like Pacific white shrimp, Liponeaus vannamei (Lemos et al., 2008), channel catfish, Ictalurus punctatus (Li et al., 2011), meagre, Argyrosomus regius and European seabass, Dicentrarchus labrax (Magalhaes et al., 2015) and sunshine bass, Morone chrysops x M. saxatilis (Thompson et al., 2008). In aquaculture, the use of corn DDGS has been focus of research since the 80s (Lovell, 1980). For catfish (Ictalurus punctatus) studies have shown that DDGS can replace soybean meal up to 35% without the addition of lysine (Webster et al., 1991; Webster et al, 1992; Webster et al, 1993) and up to 70 % with lysine inclusion (Webster et al.1991). Recent studies reaffirm the potential of DDGS uses in diets for several species with importance in the aquaculture such as Ictalurus punctatus Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 16 (Li et al, 2011.), Oreochromis niloticus (Suprayudi et al., 2015) and Oncorhynchus mykiss (Welker et al., 2014; Øverland et al., 2013). Piaractus mesopotamicus is a native Characid from rivers and floodplains of the Brazilian Midwest, commonly known as pacu or pacu-caranha. It is one of the most important native species for national aquaculture, characterized by omnivorous food habit, high growth rates, good meat quality and good acceptance by the consumer (Urbinati & Gonçalves 2005). Given Brazil's potential in grain production, the growing interest in biofuel and the benefits of using DDGS in animal feed, it is necessary to develop studies that evaluate the use of the DDGS, produced in Brazil, in diets for native fish species with commercial interest. Thus, the objective of this study was to evaluate the inclusion of different levels of corn DDGS replacing soybean meal in diets for juvenile P. mesopotamicus on growth performance, economic viability, feed efficiency and digestibility. Material and Methods 1. DDGS Digestibility trial A reference diet was formulated to contain 32% of crude protein, 17.6 kj g-1 of crude energy and 0.5% of chromic oxide III. The test diet was formulated containing 70% of the reference diet and 30% of DDGS. Corn DDGS was supplied by Libra Etanol LTDA, São Jose do Rio Claro, Mato Grosso, Brazil. All the ingredients used to compound experimental diets were grounded (1mm) and mixed on their respectively proportions. The mixtures underwent to extrusion process (Extrutech, 2mm) at Fish Nutrition Laboratory from Aquaculture Center of Sao Paulo State University (CAUNESP), Jaboticabal, Sao Paulo, Brazil. Diets were dried at 50°C for 12 hours and stocked at -20°C. Diets proximate composition is shown in Table 1. The digestibility trial was performed at Aquaculture Nutrition Laboratory from College of Food Engineer and Animal Science of Sao Paulo University, Pirassununga, Sao Paulo, Brazil. Apparent digestibility coefficients of DDGS nutrients were measured by the indirect method, following methodology describe Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 17 on NRC, 2011. For that, 210 P. mesopotamicus juveniles (13.6±0.3 grams mean weight) were distributed in six rectangular tanks (50 L), at density of 35 fish tank-1. Tanks were disposed in a recirculated system supplied with aeration and temperature kept constant by heathers (26°C). Each tank was considered as an experimental unit, arranged in a completely randomized design. Water quality was maintained by the use of supplemental aeration (central line and air diffusers), mechanical and biological filtration. Fish were fed during seven days, twice daily (09:00 and 17:00 hours) with experimental diets and feces were collected using Guelph modified system. After being collected, feces passed through centrifugation process (1800 xg, 10 min), -20°C storage and afterward, freezing dryer prior to chemical analysis. Apparent digestibly coefficients (ADCs) of protein, dry matter, energy, lipid and phosphorus of the experimental diets were calculated, according to NRC, 2011, as follows: � = × ( − � × � � � ) (1) The apparent digestibility coefficients of protein, dry matter, energy, lipid and phosphorus of the test ingredient (DDGS) were calculated according to NRC, 2011: � = + [ – ( .7 . � � )] (2) Where ti = test ingredient; td = test diet; rd = reference diet; D ref. is the % nutrient (or kj g-1) of reference diet (dry matter basis) and D test ingredient is the % nutrient (or kj g-1) of test ingredient (dry matter basis). 2. Growth and Digestibility Trail After obtaining the apparent digestibility coefficients of DDGS nutrients, five diets with increasing levels of DDGS inclusion (0, 10, 20, 30 e 40%) were formulated to be, on digestible basis, isoproteic (29% of digestible protein) and isoenergetic (13.4 kj g-1 of digestible energy). Due to formulate the experimental diets, it was used the apparent digestibility coefficients of soybean meal, fishmeal, wheat meal, Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 18 corn, rice bran, poultry meal (Abimorad & Carneiro 2004) and corn gluten meal (Fabregat et al., 2008) previously obtained for P. mesopotamicus. A mixture based on soybean meal, soybean oil and corn was formulated to attain DDGS protein and energy contents. As DDGS was being included on the diet, the mixture was being replaced, until reach 40% of DDGS inclusion, where all soybean content was removed. All the ingredients used to compound the experimental diets were grounded (1mm), mixed on its respectively proportions and the mixture extruded (Extrutech, 2mm) at Fish Nutrition Laboratory at Sao Paulo State University (ESALQ), Piracicaba, Sao Paulo, Brazil. Diets were dried at 50°C during 12 hours and then stocked at -20°C. The proximate composition of the ingredients and experimental diets are shown in Table 2 and Table 3, respectively. The growth trial was conducted at Aquaculture Nutrition Laboratory of College of Food Engineer and Animal Science from Sao Paulo University, Pirassununga, Sao Paulo, Brazil. P. mesopotamicus juveniles, coming from commercial fish farming, were stocked and acclimatized for 30 days, feeding a commercial diet (Pira 32, Guabi 32% of crude protein). To set up the trail, fish were weighed (mean weight 21±0.2 grams) and redistributed in 20 fiber glass tanks (100 L), at density of 15 fish tank-1. Tanks were disposed in a recirculated system supplied with aeration and temperature kept constant by heathers. Each tank was considered as an experimental unit, arranged in a completely randomized design with five treatments (0, 10, 20, 30 and 40% of DDGS) and four replicates. Tanks were covered with netting to prevent fish from jumping out. Also, a plastic hideout was set up on the border of the tank due to reduce stress stemming from feedlot. Water quality was maintained by the use of supplemental aeration (central line and air diffusers), mechanical and biological filtration. Water temperature was controlled with a heat exchanger (26°C) and measured twice daily. Dissolved oxygen and pH were monitored daily using a multiparameter Horiba (model U – 10). Total ammonia and nitrate by the use of commercial kits (Labcon Test Fresh Water Toxic Ammonia and Labcon Test Nitrito NO2-). Fish were fed with experimental diets during 100 days, twice daily (9:00 and 17:00), until apparent satiation. At the same time that it was being performed the growth trial, another experimental trial was being conducted to determinate the digestibility of DDGS Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 19 diets, with the same P. mesopotamicus livestock and at the same recirculated system. However, it was added to the experimental diets 0.5% of chromic oxide. The apparent digestibility coefficients for protein, energy, lipid, dry matter and phosphorus of the experimental diets were measured by the indirect method, following methodology describe on NRC, 2011. 150 P. mesopotamicus juveniles (27.09 ± 1.4 grams mean weight) were distributed in five fiber glass tanks (100 L), at density of 30 fish tank-1. Each tank was considered an experimental unit and the experiment was lain out in Latin square design 5x5 (five treatments and five experimental periods). Fish were fed during seven days, twice daily (09:00 and 17:00 hours) with the experimental diets. Feces were collected using Guelph modified system. After collection, feces passed through centrifugation process (1800 xg, 10 min), -20°C storage and afterward, lyophilized prior to chemical analysis. The apparent digestibly coefficients (ADCs) of the experimental diets were calculated as exemplified in Formula 1. Right before and immediately after each feces collection period, it was taken a sample of 200 ml of water due to analyze dissolved and total phosphorus released in the water by fish excretions. Dissolved and total phosphorus were determinate according to methodology described on AOAC (2000). 3. Sampling At the end of the growth trial, fish were fasted for 24 hours, anesthetized in benzocaine (50 mg L-1) and slaughtered through spinal cord section. After that, each fish was weighted and gutted to obtaining the following parameters: � ℎ �� = � � � ℎ − � �� � ℎ (3) � � ℎ � = [ log − log / � � ] × (4) � � ℎ = + / (5) � � � � = � � � ℎ / ∗ (6) Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 20 � � � � = �� � ℎ / ∗ (7) � � �� � = � � � ℎ / ∗ (8) � = � � � (9) � � � = � / � ℎ �� (10) � � � � � = � ℎ �� / � � (11) 4. Chemical Analyses Chemical analyses of ingredients, diets and feces were conducted at Aquaculture Laboratory from Animal Science and Food Engineer Faculty of Sao Paulo University, Sao Paulo, Brazil, following AOAC (2000) methodology. The ingredients and diets were first of all, plated in a forced air drying oven (105°C) to dry matter determination and feces were lyophilized. Samples were analyzed for crude protein (N x 6.25) by Kjeldahl method; ash by incineration in muffle (450°C for 16h) and crude lipid through extraction with petroleum ether using a Soxtec system. Chromium oxide and phosphorus were determined by absorbance in spectrophotometer (770 and 350nm, respectively), after acid digestion process. Gross energy was estimated by the use of the Atwater general factor system (FAO, 2012; NRC, 2011) based on the heats of protein, lipid and carbohydrate (including fiber) combustion. 5. Economic Viability The evaluation of economic viability of the use of corn DDGS in diets for P. mesopotamicus juveniles was analyzed following Hoffman (2006) methodology and Gameiro (2009) suggestions. Data about the costs of the dietary ingredients was collected from the last 10 years (2005 to 2015) from the Instituto de Economia Agricola (IEA – APTA, Brazil) due to determinate the total cost of the experimental diets, using the following formulas: � � � = % � � � ∗ � / (12) � ℎ �� = ∗ (13) Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 21 6. Statistical Analyses All data were subjected to a one-way analysis of variance to determine significant (p ≤ 0.05) differences among the treatment means. Tukey’s test was used when appropriate to distinguish significant differences between treatment means. All statistical analyses were conducted using SPSS 22.0 software package for Windows. Results DDGS and Experimental Diets Digestibility The apparent digestibility coefficients (ADC) of corn DDGS nutrients and experimental diets containing the different levels of DDGS inclusion are shown in Table 4. The ADC of the test ingredient (DDGS) was higher for protein (94.8%), phosphorus (91.0%) and lipid (88.8%), followed by energy and dry matter that presented the lowest value of ADC, 66.3 and 62.7% respectively. The different levels of DDGS included in the experimental diets for P. mesopotamicus juveniles caused modifications on the ADC of the most nutrients evaluated, excepted for protein. The values of ADC found for dry matter was significantly (p < 0.05) lower for diet 30DDGS comparing to the other treatments reaching 62.9 %. The diet with 40 % of DDGS inclusion (40DDGS) presented values of ADC for dry matter similar to the 30DDGS. The same ongoing was observed for energy, where the ADC was reduced (p < 0.05) from 79.1 % to control and to 69.4 % for the 30DDGS diet. Energy ADC increased (p < 0.1) in 40DDGS diet, however the values obtained were not different (p > 0.05) from the control, 10DDGS and 20DDGS diets. The lipid ADC ranged from 87.8 for the control to 94.1% for the 40DDGS diet, being the last one the highest value of ADC obtained among all treatments. Even though no differences (p > 0.05) were obtained between the other treatments, all diets with DDGS, independent of the level of inclusion, presented ADC for lipid statistically different from the control (p < 0.05). Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 22 The values of dissolved and total phosphorus released in the water during the feces collection period, are shown in Table 5. The amount of total phosphorus varies (p < 0.05) among treatments. It was observed that the value of total phosphorus decreased as DDGS was included in the diets, ranging from 0.61% for control to 0.37% for the 40DDGS diet. Dissolved phosphorus did not differ significantly (p > 0.05) between treatments. Growth and Economic Viability Fish accepted the diets without difficulty and also there was no evidence in all treatments of disease or mortality during the trial. Growth parameters and feed utilization efficiency of the fish are presented in Table 6. There were variation (p < 0.05) between treatments for feed conversion ratio (FCR) and protein efficiency ratio (PER). The inclusion of DDGS in the diets caused a slighted decrease (p < 0.05) on FCR, turning 1.14 from control to 1.00 for 30DDGS diet and 1.02 for 40DDGS. Diets 30DDGS and 40DDGS were not different between them. The PER of 30DDGS was significantly higher (p < 0.05) than control and 20DDGS diets. The fish that received 10DDGS and 40DDGS diets did not show difference on PER (p > 0.05). Results obtained for specific growth rate (SGR), viscero somatic (VSI), hepato somatic (HSI) indexes and carcass yield (CY) did not vary (p > 0.05) between the treatments. The same was true for weight gain (WG) that did not vary significantly (p >0.05) between treatments. Dietary costs are presented in Table 7. It was observed a significant reduction (p < 0.05) on the cost of weight gain (R$ kg -1 of weight gain) as DDGS was being included in the diets and soybean meal being replaced. Diets have the cost of weight gain varying from R$2.21 for control to R$1.64 for the 40DDGS diet, without in soybean meal, what represents a reduction of 26% of costs. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 23 Discussion DDGS Digestibility The success of fish rearing mostly depends on quality of the food supplied in fish farms. Thus, exhibit nutrients availability of dietary ingredients appears as an important practice to allow the maximum expression of animal productive performance and reduce nutrients waste through fish excretions (Lee, 2002). The availability of nutrients can be obtained through determination of ingredients apparent digestibility coefficients (Smith et al 1995; NRC, 2011), practice that is recognized as the first step to evaluate the potential use of ingredients in animal feed production (Allan et al., 2000). Thus, corn DDGS apparent digestibility coefficients (ADC) were firstly determined to pacu (Piaractus mesopotamicus) juveniles in the present study. The ADC of protein found for DDGS (94.8%) remained higher than the obtained for some ingredients majority used as protein source in P. mesopotamicus diets such as poultry meal (83.4%), soybean meal (81.1%) (Abimorad, 2004), corn gluten meal (78.6%) (Fabregat et al., 2008), fish meal (84.6%) and yeast extract (81.5%) (Abimorad et al., 2008). Also, the ADC of protein contained in DDGS for pacu was higher than the value of 86.2% reported for channel catfish, (Li et al., 2011) and 64.2% for sunshine bass (Thompson et al., 2008). Therefore it is possible to emphasize the potentiality of DDGS uses as protein source in P. mesopotamicus diets. However, even though DDGS has shown great digestible protein value, it is required more detailed studies evaluating DDGS amino acids profile and digestibility for pacu. Magalhaes et al. (2015) found that the ADCs of amino acids in diets with DDGS inclusion trend to be lower than those obtained for fishmeal- based diet for meagre (Argyrosomus regius) and seabass (Dicentrarchus labrax), what is probably related with the lower availability of amino acids in DDGS when compared to fishmeal. Although high contents of fiber and nitrogen free extract in diets can lead in a decrease of protein digestibility (Lech and Reich, 2012), Magalhaes et al. (2015) also did not find differences between protein digestibility coefficients of diets with Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 24 DDGS inclusion and control, for both fish species, with ADCs ranging from 89 to 91% for meagre and 91.9 to 92.9% for seabass. Results obtained by Magalhaes et al. (2015) for protein digestibility of DDGS diets were similar to those obtained in the present study, whereas increasing levels of DDGS inclusion also did not reflect in modifications on diets protein ADCs for P. mesopotamicus. Furthermore, the digestibility of protein obtained for pacu juveniles in diets with DDGS, independent of inclusion level, were higher than the observed for other animals feeding DDGS diets such as pigs (Ren et al., 2011) and broilers (Liu, 2011). Ren et al. (2011) evaluating diets with three different sources of corn DDGS from ethanol production observed values of 72.9, 50.0 and 51.4% for protein apparent ileal digestibility for growing pigs, while Liu (2011) observed values of 85% for total tract and 74% for ileal apparent digestibility coefficients in broilers feeding diets with 20% of corn DDGS without xylanase addition. Trough dry matter ADCs investigation it is possible to get a general estimative about the digestibility of a particular ingredient by indicating the amount of non-digestible nutrients presents in it. As well obtained for P. mesopotamicus, low dry matter digestibility in corn DDGS was reported for the majorly of the aquatic species studied until present moment (Magalhaes et al., 2015; Seo et al., 2011; Li, 2011; Chan et al., 2004). Farther, Cheng and Hardy (2004) and Li (2011), corroborating with the present study, observed a reduction on the digestibility of dry matter and energy of diets with increasing levels of DDGS inclusion. Authors correlated the results obtained with the high amount of fiber present in DDGS, what was confirmed through the analysis of neutral detergent fiber (NDF) promoted in this study. Krogdahl et al. (2005) also mentioned high amounts of cellulose and hemicellulose, such arabinoxylans, as an adverse feature of DDGS used in animal feed. Among factors capable for induce changes on dietary nutrients digestibility, fiber is the biggest issue when talking about DDGS. In fish, there are two ways of accessing some of the dietary fiber, by acid hydrolysis in the stomach or through microbial enzymes activity in intestine (Kaushik, 2001). High fiber content may provide modifications in nutrients metabolism reducing the availability of dietary nutrients in many ways, either by changing the ADCs of other dietary ingredients Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 25 (Liu, 2011) or inducing endogenous losses (Back Knudsen and Hansen, 1991; Noblet and Perez, 1993). Previous studies have been reported low digestibility coefficients of diets, not only for dry matter but also for other dietary nutrients, with increasing levels of fiber such those obtained for rainbow trout (Hilton et al., 1983), common carp (Kirchgessner et al., 1986), red drum (McGoogan and Reigh, 1996) and rockfish (Lee, 2002). The use of large amounts of fiber in diets would leads to reduction on gut retention time and digestive enzyme contact with intracellular contents (Vanderoof, 1998) which impairs the capability of absorbing nutrients available from dietary ingredients (Enes et al., 2011; Fontoulaki et al., 2005; Stone et al., 2003). Additionally, high fiber can lead in an increase of microbial activity and substrates from fermentation, what would end up in endogenous losses. All energy released by DDGS comes, basically, from lipids since starch is degraded in the fermentation process (Han and Liu, 2010). Nevertheless, in the present study, the source of corn DDGS incorporated in the diets had lipids level (4.0 %DM) not as expressive as the reported by literature that shown values ranging from 9.0 to 12.0% (Feedstuff, 2016; USGC, 2012). During the ethanol production, the oil restrained in the wet cake can be extracted due to generate corn oil as another ethanol co-product. So, variations in lipid present in DDGS will be dependent of the process adopted by ethanol plants behind DDGS production. Adding to that, the starch remained in the DDGS used in this experiment (5.3 % DM) does not have a remarkable contribution as energy source. Therefore, most of the energy coming from the DDGS used in the present study is probably arising from the others nutrients like fiber and protein. Due to that, the low value of apparent digestibility coefficient obtained for energy for P. mespotamicus either in DDGS (66.3%) or in the experimental diets with different levels of DDGS inclusion may be explained by over quantity of nutrients with low energy digestibility such as fiber components (neutral detergent fiber), what contrast with others nutrients with higher digestible value like starch and lipid (Stein and Bohlke, 2007; NRC, 2011). Thereby, even though P. mesopotamicus is a species that tolerate high levels of fiber in the diet (Rodrigues et al., 2010) there was not effectiveness in the use of the energy coming from the fiber content in DDGS. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 26 The ADC of DDGS obtained for lipid in this study (88.8%) was lower than the ADCs found for corn (91.1%), soybean meal (93.1%) and fish meal (94.1%) for Oreochromis niloticus juveniles (Furuya et al., 2001), an omnivorous fish species with similar food habit than P. mesopotamicus. Also, pacu showed a lower ADC for lipids in DDGS in the present study than the 93.8 % obtained for channel catfish (Li, 2011), however higher than the 68.7% found for sunshine bass (Thompson et al., 2008). Although, lipid content in the sources of DDGS used were higher for the studies cited above, 13 % (dry matter basis) for sunshine bass and 7 % (dry matter basis) for catfish than the 4% of lipids of the DDGS source used in the present study for pacu. Increasing amounts of lipid content in diets may lead in higher digestibility of this nutrient, due to delay in release gastric fluids making available more time for dietary lipids digestion (Quigley & Meschan, 1941; Windell et al., 1969). As obtained in the present study, increase in lipids digestibility of diets with rising lipid content is also reported for Oncorhynchus mykiss (Takeuchi et al., 1978), Oreochromis niloticus x Oreochromis mossambicus (De Silva et al., 1991), Dicentrarchus labrax (Peres and Oliva-Teles, 1999) and Seabastes schlegeli (Lee, 2002). Corn oil, as the lipid source in DDGS, has on its composition higher amounts of unsaturated fatty acids than saturated (NRC, 2011). Adding the fact that unsaturated fatty acids are better digested than saturated (Ng, 2003; Bahurmiz and Ng, 2007), it is possible to relate the increasing digestibility of lipid in DDGS diets with fatty acids profile of the experimental diets. As soybean oil and corn oil have similar saturated (SFA) and unsaturated (UFA) percentages (14 – 16% of SFA and 86 – 84% of UFA) (NRC, 2011; Martin et al., 2008) modifications on the amount of those two ingredients in experimental diets did not bring any damage on lipids digestibility due to changing SFA/UFA ratio. Thus, increasing values of lipid digestibility of diets with higher levels of DDGS inclusion may be related with the equally increase in dietary lipids content. High amounts of fiber can also cause decrease on lipid digestibility by endogenous losses (Back Knudsen and Hansen, 1991; Noblet and Perez, 1993), however, the increasing on fiber content in DDGS diets in this study was not achievable of damage lipid diets digestibility. Otherwise, low ADCs of lipids were reported for rainbow trout (Chan et al., 2004), sunshine bass (Thompson et al., Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 27 2008), meagre and seabass (Magalhaes et al., 2015) feeding diets with corn DDGS inclusion. In the present study, diets were extruded due to follow fish feed commercial production process, allowing to get results as closest to real fish culture as possible. The influence of feed processing methods on ingredients digestibility was reported by Wilson and Poe (1985) for corn grains. They found higher corn energy ADC for extruded diets than pelleted, but no differences were found for soybean meal and wheat meal. However, Allan and Booth (2004) observed higher dry matter, protein and energy digestibility of soybean meal in extruded diets than pelleted diets, and the opposite for canola meal. Thus, the possibility of changings on nutrients digestibility of dietary ingredients caused by the type of processing method used in feed production is not completely discarded. Digestible phosphorus refers to the portion absorbed by the gastrointestinal tract. This portion can be compounded by non-phytic or phytic phosphorus, being the last one available after hydrolysis process by intrinsic phytase (Bünzen, 2008; Cao et al., 2007). Thus, the high digestible phosphorus content found in the DDGS may be explained by its high bioavailability. Fermentation processes and heating, which is subjected DDGS, may have resulted in an increase of the hydrolysis of phytate molecules (Kim et al., 2008), component non digestible for animals. The high available phosphorus content in DDGS can also be observed through measurements of body retention and the amount of phosphorus release in the water. Cheng & Hardy (2004) and Overland et al. (2013) could observed that increase amounts of DDGS in diets for Nile tilapia and rainbow trout, respectively, ended up in higher phosphorus retention in the body comparing to control diets without DDGS inclusion. Increase in nutrient retention leads in smaller quantity of nutrient released in water as waste (Bureau & Hua, 2010; Prachom et al., 2013). In the present study, it was evaluated the discharge of phosphorus in the water, and results obtained are in agreement with Cheng & Hardy (2004) and Overland et al. (2013). This reaffirm that the inclusion of DDGS in fish diets provides higher amounts of digestible phosphorus leading in better body retention and less release of this mineral. Also, according to Conama 357/2005, only the experimental diets with DDGS inclusion Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 28 were in the range of phosphorus concentration recommended for aquaculture water that accepts values above 0.05 mgP L-1. Apparent phosphorus digestibility contained in the DDGS was considerably high (91%) when compared to ingredients such as soybean meal (35.1%), fishmeal (27.1%), bone meal (54.6%), corn meal (7.3% ) and wheat bran (30.5%), obtained for tilapia (Oreochromis niloticus) fingerlings (Miranda et al., 2000). Furuya et al. (2001) also obtained ADCs for corn (45.1%), wheat bran (29.5%), soybean meal (47.1%) and fishmeal (49.8%) for tilapia below values found in the present study. Furthermore, phosphorus ADC for DDGS obtained in the present study was even higher than that observed for catfish (Li et al., 2015) and pigs (Almeida et al., 2012; Rojas et al., 2013). Whereby corn DDGS has higher phosphorus content availability compared to other ingredients commonly used in diets for aquaculture, its use is also recommended as a strategy for the development of a complete and appropriate aqua feed, allowing the reduction of phosphorus excretion to the environment. It is important to emphasize that possible variations on the quality and nutrient composition of the DDGS, between different facilities or even the same facility (Spiehs et al., 2002; Kleinschmitt et al. 2007) may occur, basically, by differences between plants and process methods (Ortin and Yu, 2009; Liu, 2011). Therefore, the success of DDGS inclusion on fish diets will be dependent of the protein and amino acid requirements of the fish, the type of feed ingredient being replaced and the source and composition of the DDGS. Growth and Economic Viability Changes on dietary protein source can interfere on fish growth performance due to variations on nutritional values, nutrients digestibility and amino acid composition (Anderson et al., 1992). Thus, to obtain a considerable growth performance it become necessary to check some nutritional aspects of the new ingredient being added, mainly when changing a protein source in fish diets. In agreement with the present experiment, a decreased on feed conversion ratio (FCR) was also observed for rainbow trout (143 grams mean initial weight) feeding diets with 50% of DDGS inclusion as substitute of a mixture with plant protein ingredients (Øverland et al., 2013) without significant effects on weight gain Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 29 and feed intake. Distinctly, it was reported improvements on weight gain for hybrid catfish feeding diets with DDGS inclusion (Zhou et al., 2010). Robinson & Li (2008) observed that the lower FCR found for channel catfish feeding 30% DDGS diet was not due to improvements on feed intake but by an increase in fish weight gain. Li et al. (2010) suggested that the high lipid level of the diet containing 30% DDGS was partially responsible for the increase in feed efficiency ratio in catfish juveniles. Also, improvements on feed utilization were reported for catfish feeding diets with DDGS, DS (distillers’ solubles) and EDS (distillers’ solubles from corn endosperm) when compared with HPDDG (high protein distillers dried grain with solubles) diet, probably due to the absence of the soluble portion in HPDDG (Li et al., 2010). Thus, it is possible to affirm that DDGS may have some components that facilitated the digestibility of the diet or nutrient absorbance. Some studies related with the inclusion of brewer’s yeast in fish diets shown improvements on feed efficiency ratio for hybrid striped bass, Morone chrysops x Morone saxatilis (Li and Gatlin, 2006) and sea bass, Dicentrarchus labrax (Oliva- Teles and Gonçalves, 2001). That may be related with the nucleotides, β glucans and oligosaccharides present in yeast cells that can cause modifications on intestine morphometry of some fish species, increasing the area of nutrients absorption (Burrells et al., 2001; Santin et al., 2001; Yang et al., 2007; Dimitroglou et al., 2009) and consequently the use of the dietary nutrients. As DDGS is composed by considerable amounts of yeast cells (Ingledew, 1999; Zohu et al. 2010) it is possible that its components had acted modifying P. mesopotamicus intestine, improving the absorption of nutrients present in the experimental diets. The inclusion 50% of dried brewer’s yeast (Saccharomyces cerevisiae) in replacement of fishmeal resulted in improvements on feed efficiency, growth performance and reduction on nitrogen release for pacu, P. mesopotamicus (initial mean weight 26.6 ± 1.7 grams) (Ozorio et al., 2010). Adding to that, Ozorio et al. (2010) observed high digestibility of nutrients and amino acids in experimental diets, suggesting high digestibility of this ingredient for pacu and its benefic interference on the digestibility of other dietary ingredients. Thus, considering results obtained in the present study it is possible to affirm that yeast present in DDGS may have exerted positive influence on fish growth performance, feed utilization and nutrients digestibility. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 30 Instead of Shelby et al. (2008) did not find differences for weight gain, feed intake and feed efficiency ratio on Nile tilapia fed 30 or 60% DDGS diets with lysine addiction, compared to the control. The same behavior was described for catfish feeding diets with 40% of DDGS inclusion (Lim et al., 2009). No differences in body weight gain was observed for yellow perch, a carnivorous specie, feeding diets with increased DDGS levels (Schaeffer et al., 2011) however weight gain and FCR showed better results for the fishmeal based diet than the others with DDGS inclusion. Furthermore, the success of DDGS use in fish diets may be dependent on the combination of dietary ingredients and fish food habit. Improvements on feed utilization in the present study reflected on economic viability, where inclusion of DDGS promotes reduction of 26% on dietary cost per weight gain for P. mesopotamicus fed 40% DDGS diets. Once feed contributes with 60 to 80% of total production costs (Campos et al., 2007; Rola and Hasan, 2007) turns priority the use of low-cost ingredients in aqua feed, as long as do not cause losses on animal production. Conclusion This is the first work that investigated the inclusion of corn DDGS in total soybean meal replacement in diets for P. mesopotamicus, and the data obtained indicate that, the use of 40% DDGS in the diet did not provide negative consequences for growth and digestibility of the juveniles, although improved feed utilization and reduced dietary costs and phosphorus release in culture water. Thus it is possible the inclusion of corn DDGS in levels up to 40% in the diets for P. mesopotamicus juveniles in total replacement of soybean meal. Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 31 References Abimorad, E.; & Carneiro, D. J. 2004. 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Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 42 Tables Index Table 1: Composition and proximate analyses of reference and test diets .......... 43 Table 2: Proximate analyses of the experimental dietary ingredients .................. 44 Table 3: Composition and proximate analyses of experimental diets with increasing levels of DDGS inclusion ...................................................................................... 45 Table 4: Apparent digestibility coefficients (%) of corn DDGS and experimental diets nutrients for P. mesopotamicus juveniles ..................................................... 46 Table 5: Dissolved Phosphorus (DP) and Total Phosphorus (TP) present in the water during feces collection of P. mesopotamicus fed diets with distinct levels of corn DDGS inclusion ............................................................................................ 46 Table 6: Growth performance and feed utilization efficiency of P. mesopotamicus juveniles fed experimental diets ............................................................................ 47 Table 7: Dietary costs and costs of weight gain of diets with increasing levels of corn DDGS in soybean meal replacement for P. mesopotamicus juveniles ......... 47 Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 43 Tables Table 1: Chemical and percentage composition of reference and test diets Reference Diet Test Diet Ingredients (%) DDGS - 30.0 Poultry meal 14.5 10.1 Fishmeal 6.0 4.2 Soybean meal 30.0 20.9 Wheat meal 24.0 16.7 Corn 15.0 10.5 Rice bran 9.0 6.3 BHT1 0.1 0.0 Chromium oxide III 0.5 0.5 Vit. And Min. Premix2 1.0 0.7 Total 100.0 100.0 Chemical Composition Dry Matter 93.5 94.5 Crude Protein (% DM) 36.2 35.5 Gross Energy (kj g-1) 18.8 19.2 Crude Lipid (% DM) 3.4 4.5 Phosphorus (% DM) 1.0 0.8 Digestible Protein (% DM) 33.0 30.9 Digestible Energy (kj g-1) 14.5 14.0 1 BHT (Butyl hydroxytoluene); 2 Vitamin and Mineral Premix: vitamin A - 500.000 UI; vitamin D3 - 250.000 UI; vitamin E - 5.000 mg; vitamin K3 - 500 mg; vitamin B1 - 1.500 mg; vitamin B2 - 1.500 mg; vitamin B6 - 1.500 mg; vitamin B12 - 4.000 mg; folic acid - 500 mg; pantothenate Ca - 4.000 mg; vitamin C - 10.000 mg; biotin - 10 mg; Inositol - 1.000; nicotinamide - 7.000; choline - 10.000 mg; Co - 10 mg; Cu - 1.000 mg; Fe - 5.000 mg; I - 200 mg; Mn - 1500 mg; Se - 30 mg; Zn - 9.000 mg3. (Agromix LTDA, Sao Paulo, Brazil) Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 44 Table 2: Proximate analyses of the experimental dietary ingredients1 DDGS SBM CM WM RB CG FM PM Chemical Composition Dry Matter 92.9 88.9 89.3 90.3 86.9 92.7 97.7 97.3 Crude Protein (% DM) 33.6 44.8 8.4 14.8 13.2 71.5 58.0 48.9 Gross Energy (MJ kg-1) 20.9 17.1 16.2 16.7 19.4 21.1 14.6 13.1 Crude Fiber (% DM)* - 7.6 2.2 9.8 9.0 2.2 1.5 1.5 Lipid (% DM) 4.0 2.0 4.0 2.9 15.2 1.9 3.4 15.2 Ash (% DM) 2.0 7.8 1.3 5.5 10.2 2.2 27.5 15.0 Phosphorus (% DM) 0.2 0.6 0.3 0.9 1.6 0.4 3.0 2.7 Starch (%DM) 5.3 - - - - - - - 1 DDGS: Corn Distiller’s Dried Grains with Soluble (Libra Etanol, Mato Grosso, Brazil); SB: Soybean meal, CM: Corn Meal, WM: Wheat Meal, RB: Rice Bran (Cargill, Sao Paulo, Brazil); CG: Corn Gluten, FM: Fish meal (In Vivo, Sao Paulo, Brazil); PM: Poultry Meal (Agromix, Sao Paulo, Brazil) * DDGS: neutral detergent fiber (NDF) = 56.4% (DM basis); Mestranda - Kátia Rodrigues Batista de Oliveira Orientadora - Dra Elisabete Maria Macedo Viegas CAUNESP 45 Table 3: Composition and proximate analyses of experimental diets with increasing levels of DDGS inclusion Control 10 DDGS 20 DDGS 30 DDGS 40 DDGS Ingredients DDGS 0.0 10.0 20.0 30.0 40.0 Soybean meal 23.1 17.3 11.5 5.8 0.0 Corn 25.4 21.8 18.1 14.5 10.8 Soybean oil 3.4 2.8 2.3 1.7 1.1 Poultry meal 6.6 6.6 6.6 6.6 6.6 Fish meal 11.0 11.0 11.0 11.0 11.0 Corn Gluten 8.3 8.3 8.3 8.3 8.3 Wheat meal 13.0 13.0 13.0 13.0 13.0 Rice bran 7.7 7.7 7.7 7.7 7.7 Lysine1 0.5 0.5 0.5 0.5 0.5 BHT2 0.1 0.1 0.1 0.1 0.1 Vit. and Min. Premix3 1.0 1.0 1.0 1.0 1.0 Total 100.0 100.0 100.0 100.0 100.0 Chemical Composition Dry Matter 93.1