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Universidade Estadual Do Ceará Faculdade De Veterinária Programa De Pós-Graduação Em Ciências Veterinárias Mestrado Acadêmico Em Ciências Vaterinárias

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Universidade Estadual Do Ceará Faculdade De Veterinária Programa De Pós-Graduação Em Ciências Veterinárias Mestrado Acadêmico Em Ciências Vaterinárias 0 UNIVERSIDADE ESTADUAL DO CEARÁ FACULDADE DE VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS MESTRADO ACADÊMICO EM CIÊNCIAS VATERINÁRIAS YASMIM MAIA FERREIRA EFEITO DA OSMOLALIDADE DE DIFERENTES SOLUÇÕES ATIVADORAS SOBRE A CINÉTICA ESPERMÁTICA DO SÊMEN FRESCO, RESFRIADO E DESCONGELADO DE Prochilodus brevis (ACTINOPTERYGII:PROCHILODONTIDAE) FORTALEZA – CEARÁ 2019 1 YASMIM MAIA FERREIRA EFEITO DA OSMOLALIDADE DE DIFERENTES SOLUÇÕES ATIVADORAS SOBRE A CINÉTICA ESPERMÁTICA DO SÊMEN FRESCO, RESFRIADO E DESCONGELADO DE Prochilodus brevis (ACTINOPTERYGII:PROCHILODONTIDAE) Dissertação apresentada ao Curso de Mestrado Acadêmico em Ciências Veterinárias do Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para à obtenção do título de Mestre em Ciências Veterinárias. Área de concentração: Reprodução e Sanidade Animal. Orientador: Dr. José Ferreira Nunes Coorientadora: Prof.ª Dr.ª Carminda Sandra Brito Salmito Vanderley FORTALEZA – CEARÁ 2019 2 3 YASMIM MAIA FERREIRA EFEITO DA OSMOLALIDADE DE DIFERENTES SOLUÇÕES ATIVADORAS SOBRE A CINÉTICA ESPERMÁTICA DO SÊMEN FRESCO, RESFRIADO E DESCONGELADO DE Prochilodus brevis (ACTINOPTERYGII:PROCHILODONTIDAE) Dissertação apresentada ao Curso de Mestrado Acadêmico em Ciências Veterinárias do Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para à obtenção do título de Mestre em Ciências Veterinárias. Área de concentração: Reprodução e Sanidade Animal. Aprovada em: 10 de dezembro de 2019. 4 Dedico este trabalho aos meus pais, meus queridos irmãos e ao meu eterno e amado avô, Valdir Maia. 5 AGRADECIMENTOS À Deus, por me permitir realizar mais este sonho e por ter concedido luz e sabedoria para a conclusão desta etapa. Ao meu avô, Valdir Maia, que viverá eternamente em meu coração. Aos meus pais Valdilene Maia e Francisco Fernandes e irmãos Ingrid Maia, Mayara Maia e Marcus Wilson, por todo o amor, carinho, proteção, paciência e apoio nestes anos dedicados à vida acadêmica. Aos meus amores, André Siqueira, Vitório e Django por todo cuidado, amor e parceria. À Universidade Estadual do Ceará (UECE), por todo o suporte durante minha caminhada acadêmica, e ao Programa de Pós-Graduação em Ciências Veterinárias (PPGCV), pelo ensino de excelência. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo suporte financeiro. Ao meu orientador, Dr. José Ferreira Nunes e coorientadora Dra. Carminda Sandra Brito Salmito Vanderley, pela oportunidade e confiança na realização deste trabalho. Aos membros da banca Dra. Caroline Vieira Feitosa, Dr. César Carneiro Linhares Fernandes e Dra. Cristiane Clemente de Mello Salgueiro pela disposição em contribuir com este trabalho. Ao Laboratório de Biotecnologia da Reprodução de Peixes (LBRP) e ao Núcleo Integrado de Biotecnologia (NIB) pela oportunidade de ensino e aprendizado. A todos os amigos de laboratório: Júlia Trugilio, Mayara Setúbal, Larissa Teixeira, Renata Vieira, Vanessa Alves, Priscila Almeida, Edna Raquel, Pedro Emídio Leite, Thais Maia, Hiago Gadelha, Jessica Lobato, Yara Silvino, Levi Kalil, Guilherme Melo, Carla Pamela, Carla Tatiana, Samuel Victor, Vitória Correia e Karen Karoline. Agradeço à todos pela grande parceria, disponibilidade em ajudar, companhia e amizade. Aos amigos queridos Pedro Arruda, Jonas Felipe, Kalil Mubarac, Matheus Magalhães e Lucas Abreu, por todos os momentos descontraídos e de felicidade que tornaram a vida, acadêmica e pessoal, mais leve e feliz. 6 “São as nossas escolhas que revelam o que realmente somos, muito mais do que as nossas qualidades.” (Albus Dumbledore) 7 RESUMO Prochilodus brevis é uma espécie de peixe reofílica de importância econômica e ecológica no nordeste brasileiro. No entanto, ações antrópicas tornaram sua população vulnerável, sendo necessário realizar estudos sobre sua biologia reprodutiva para aperfeiçoar sua reprodução em cativeiro. Sendo assim, objetivo deste estudo foi avaliar os efeitos das soluções ativadoras ACP-104, Frutose e NaCl em três diferentes osmolalidades (100, 160 e 220 mOsm Kg-1) na cinética espermática e na duração da motilidade em sêmen de P. brevis in natura, resfriado e criopreservado. Os machos receberam duas doses de extrato hipofisário de carpa (0,4 e 4 mg kg-1 peso corporal) com um intervalo de 9 horas entre as aplicações. Após 14 h, os animais foram sedados, tiveram o sêmen coletado e foram formados oito pools de sêmen. Amostras de sêmen in natura foram ativadas e analisadas; posteriormente, foram resfriadas e criopreservadas, com base em metodologia padronizada para esta espécie. ACP-104 e NaCl (P> 0,05) a 100 e 160 mOsm Kg-1 registraram resultados cinéticos satisfatórios para o sêmen fresco, enquanto o ativador NaCl obteve resultados cinéticos satisfatórios para espermatozoides resfriados nas três concentrações de osmolalidade (P <0,05). NaCl a 220 mOsm Kg-1 obteve os melhores resultados para espermatozoides descongelados (P <0,05). O ACP-104 a 100 mOsm Kg-1 manteve durações de motilidade espermática mais longas no sêmen submetido às três condições experimentais adotadas no presente estudo. Sendo assim, é possível concluir que o sêmen fresco de P. brevis ativado com ACP-104 ou Frutose em baixos níveis de osmolalidade permite altas taxas de motilidade espermática. Espermatozoides resfriados e descongelados podem ser ativados com NaCl em níveis mais altos de osmolalidade. Para todas as condições seminais, indica-se ACP-104 a 100 mOsm kg-1, por proporcionar uma maior duração da motilidade. Palavras-chave: Characiformes. CASA. Criopreservação seminal. ACP-104. Ativadores espermáticos. 8 ABSTRACT Prochilodus brevis is a rheophilic fish species of economic and ecological importance. However, anthropic actions have made its population vulnerable; therefore, it is necessary conducting studies about its reproductive biology to help improving its reproduction in captivity. The aim of the current study is to evaluate osmolality effects (100, 160 and 220 mOsm Kg-1) of activated solutions such as ACP-104, Fructose and NaCl on sperm kinetics and motility duration in fresh, cooled and thawed P. brevis semen. Fresh sperm samples were analyzed; subsequently, they were cooled and cryopreserved, based on standardized methodology for this species. ACP-104 and NaCl (P >0.05) at 100 and 160 mOsm Kg-1 recorded satisfactory kinetic results for fresh semen, whereas NaCl recorded satisfactory kinetic results for cooled sperm at all three osmolality levels (P <0, 05). NaCl at 220 mOsm Kg-1 recorded the best results for thawed sperm (P <0.05). ACP-104 at 100 mOsm Kg-1 maintained longer sperm motility durations in semen subjected to the three experimental conditions adopted in the current study. It is possible concluding that fresh P. brevis semen activated with ACP-104 or Fructose at low osmolality levels enables high sperm mobility rates. Cooled and thawed sperm can be activated with NaCl at higher osmolality levels. In addition, ACP-104 at 100 mOsm Kg-1 enables longer sperm mobility duration in fresh, cooled and cryopreserved semen. Palavras-chave: Characiformes. SCA. Sperm cryopreservation. ACP-104. Sperm activators. 9 LISTA DE FIGURAS CAPÍTULO 1 - OSMOLALITY OF DIFFERENT ACTIVATING SOLUTIONS INFLUENCES THE MOTILITY DURATION AND SPERMATIC KINETICS OF Prochilodus brevis FRESH, COOLED AND CRYOPRESERVED SPERM. Figura 1 - The percentage of motile Prochilodus brevis fresh sperm activated in ACP- 104, Fructose and NaCl at different osmolalities. Different letters indicate significant differences (P < 0.05) between solutions and osmolalities......................................................................................................................47 Figura 2 - The duration of sperm motility in P. brevis fresh sperm activated in ACP-104, Fructose and NaCl. Different letters indicate significant differences (P < 0.05) between solutions………………………………….......................................................................48 10 LISTA DE TABELAS CAPÍTULO 1 - OSMOLALITY OF DIFFERENT ACTIVATING SOLUTIONS INFLUENCES THE MOTILITY DURATION AND SPERMATIC KINETICS OF Prochilodus brevis FRESH, COOLED AND CRYOPRESERVED SPERM. Tabela 1 - Motility rate and velocities (curvilinear = VCL, straight line = VSL, average path = VAP) of fresh sperm of P. brevis activated with ACP-104, Fructose and NaCl, with osmolalities of 100, 160 and 220 mOsm Kg-1. Data are expressed as mean ± standard deviation…………………………………………..........................................................49 Tabela 2 - Motility rate, velocities (curvilinear = VCL, straight line = VSL, average path = VAP) and duration of motility of cooled sperm of P. brevis activated with ACP-104, Fructose and NaCl, with osmolalities of 100, 160 and 220 mOsm Kg-1. Data are expressed as mean ± standard deviation.……………………..........................................................50 Tabela 3 - Motility rate, velocities (curvilinear = VCL, straight line = VSL, average path = VAP) and duration of motility of frozen sperm of P. brevis activated with ACP-104, Fructose and NaCl, with osmolalities of 100, 160 and 220 mOsm Kg-1. Data are expressed as mean ± standard deviation. .…………………….........................................................51 11 LISTA DE ABREVIATURAS E SIGLAS ACP Água de coco em pó AIA Ácido indol acético AMPc Monofosfato cíclico de adenosina ANOVA Análise de variância CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CASA Computer assisted sperm
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