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PALOMA ROCHA ARAKAKI Evaluation of Two Extenders For PALOMA ROCHA ARAKAKI Evaluation of two extenders for cryopreservation of semen from golden-headed lion tamarin (Leontopithecus chrysomelas) São Paulo 2017 PALOMA ROCHA ARAKAKI Evaluation of two extenders for cryopreservation of semen from golden-headed lion tamarin (Leontopithecus chrysomelas) Thesis submitted to the Postgraduate Program in Animal Reproduction of the School of Veterinary Medicine and Animal Science of the University of São Paulo to obtain the Doctor’s degree in Sciences Department: Animal Reproduction Area: Animal Reproduction Advisor: Prof. André Furugen Cesar de Andrade, Ph.D. In agreement: _________________________ Advisor São Paulo 2017 Note: The original version is available at FMVZ/USP Library Autorizo a reprodução parcial ou total desta obra, para fins acadêmicos, desde que citada a fonte. DADOS INTERNACIONAIS DE CATALOGAÇÃO NA PUBLICAÇÃO (Biblioteca Virginie Buff D’Ápice da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo) T. 3569 Arakaki, Paloma Rocha FMVZ Evaluation of two extenders for cryopreservation of semen from golden-headed lion tamarin ( Leontopithecus chrysomelas ) / Paloma Rocha Arakaki. -- 2017. 101 f. : il. Título traduzido: Avaliação de dois diluidores para a criopreservação do sêmen de mico-leão-de-cara-dourada ( Leontopithecus chrysomelas ). Tese (Doutorado) - Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal, São Paulo, 2017. Programa de Pós-Graduação: Reprodução Animal. Área de concentração: Reprodução Animal. Orientador: Prof. Dr. André Furugen Cesar de Andrade. 1. Conservação de primatas. 2. Primatas neotropicais. 3. Vibroestimulação peniana . 4. Análise seminal. 5. Teste de ligação de espermatozoides. I. Título. Ficha catalográfica elaborada por Camila Molgara Gamba, CRB 7070-8. EVALUATION FORM Author: ARAKAKI, Paloma Rocha Title: Evaluation of two extenders for cryopreservation of semen from golden- headed lion tamarin (Leontopithecus chrysomelas) Thesis submitted to the Postgraduate Program in Animal Reproduction of the School of Veterinary Medicine and Animal Science of the University of São Paulo to obtain the Doctor’s degree in Sciences Date: _____/_____/_____ Committee Members Prof. _____________________________________________________________ Institution:_____________________________ Decision:____________________ Prof. _____________________________________________________________ Institution:_____________________________ Decision:____________________ Prof. _____________________________________________________________ Institution:_____________________________ Decision:____________________ Prof. _____________________________________________________________ Institution:_____________________________ Decision:____________________ Prof. _____________________________________________________________ Institution:_____________________________ Decision:____________________ ACKNOWLEDGEMENTS I thank The Lord, because without Him I am nothing. “For from Him and through Him and for Him are all things. To Him be the glory forever. Amen”. Rom. 11.36. My family, especially my parents Nelson and Raquel, for all support during my whole life, for guiding me and teaching me the values that north who I am. My sisters Kelly and Barbara, for sharing the best moments until now, my brother-in-law Matthew, for friendship, my brother Rafael, his wife Camila and my nephews Joaquim and Heloísa, for bringing joy to my life. My fiancé Leonardo, for the love, joy and hope shared; for helping me with every sort of problems, for cheering me up, and for saying that we would make it. Prof. Marcelo Alcindo de Barros Vaz Guimarães (in memoriam), for being an excellent and real Professor, an example of professional and human being. Prof. Marcílio Nichi, for helping me in a moment when I most needed during my academic life, guiding me through the experiments and kindly answering all of my questions. Prof. Ricardo José Garcia Pereira, for all the moments of talks, discussions and guidance, helping me find solid alternatives whenever something went wrong. Prof. Mario Binelli, for receiving me in a delicate moment, for the encouragement, support, and even help in supplying materials when I needed. Prof. Rodrigo del Rio do Valle, for introducing me to the beautiful field of primate reproduction, and for the help during my first steps in research. Prof André Furugen Cesar de Andrade, for all the help on the final stretch of doctorate. Dr Cristiane Schilbach Pizzutto, for being willing to help me in the most diverse aspects. The professors from Postgraduate Program in Animal Reproduction of the School of Veterinary Medicine and Animal Science of the University of São Paulo, for the precious teachings and contributions to the project and also to my formation during graduation. Mrs. Harumi Doi Shiraishi for all the help and friendship developed over the past 6 years. My colleagues from the Postgraduate Program in Animal Reproduction, especially João Diego de Agostini Losano, for the immense help, for teaching me the sperm- binding assay, for the statistical analysis, and for sharing his knowledge always with patience and kindness; Bruno Rogério Rui and Marcel Henrique Blank, for giving their time to teach me techniques of sperm staining and Fernanda Maria de Carvalho, for donation of TYB extender and support. Dr Paulo Magalhães Bressan and Dr João Batista da Cruz, for the opportunity to develop this research at São Paulo Zoological Park Foundation (FPZSP). Dr Patrícia Locosque Ramos, for supporting my research, making available material resources, and helping me with the obstacles that arose during the study. Paula Andrea Borges Salgado and Débora Rodrigues Gonçalves, for everyday help in the lab, the conversations, discussions, and for the friendship and good times shared. São Paulo Zoological Park Foundation staff, especially Cauê Monticelli, Luan Henrique Morais, Amanda Alves de Moraes, Fabrício Braga Rassy, Luis Barbosa de Miranda Filho, Karina Craveiro dos Santos, Mara Cristina Marques, Paulo Gil Proença Soares, Denise Martins Sanches, and all interns and trainees, for the essential help in this project. Chico Mendes Institute for Biodiversity Conservation (ICMBio), from Ministry of Environment (MMA), for authorization to collect biological samples from primates. FINANTIAL SUPPORT Doctoral Fellowship from Coordination for the Improvement of Higher Education Personnel (CAPES). RESUMO ARAKAKI, P. R. Avaliação de dois diluidores para a criopreservação do sêmen de mico-leão-de-cara-dourada (Leontopithecus chrysomelas). [Evaluation of two extenders for cryopreservation of semen from golden-headed lion tamarin (Leontopithecus chrysomelas)]. 2017. 101 f. Tese (Doutorado em Ciências) – Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2017. Muitas espécies de primatas neotropicais são ameaçadas de extinção. As biotecnologias da reprodução podem contribuir consistentemente para a conservação das espécies, estando entre elas os bancos de recurso genético. Entretanto, o sucesso no uso de tais tecnologias depende de avanços no conhecimento da biologia reprodutiva básica de uma certa espécie. Durante esse processo, a avaliação da qualidade espermática é de suprema importância. Nós concentramos nosso trabalho em micos-leões-de-cara-dourada (Leontopithecus chrysomelas), mantidos na Fundação Parque Zoológico de São Paulo. Leontopithecus sp. é um alvo importante para o desenvolvimento de técnicas reprodutivas com o propósito da conservação, sendo que todas as espécies do gênero são classificadas como ameaçadas ou criticamente ameaçadas de extinção pela IUCN. Para a avaliação da qualidade espermática em L. chrysomelas, técnicas como o teste de ligação do espermatozoide, e as colorações não fluorescentes para a avaliação de integridade de acrossomo – fast-green/rosa bengala, integridade de membrana plasmática – eosina-nigrosina e atividade mitocondrial – 3,3’ diaminobenzidina – foram validados para sêmen fresco. Nós também validamos uma técnica de avalição de integridade de acrossomo – corante azul de coomassie, para amostras descongeladas devido a inadequação do método utilizado em sêmen fresco. Algumas espécies de primatas neotropicais apresentam variação na qualidade seminal ao longo do ano. Para avaliar a qualidade do sêmen para essa espécies e para verificar se mico-leão-de-cara-dourada possui essa variação, nós colhemos sêmen nas estações de seca e chuva, durante diferentes períodos da estação reprodutiva – no intervalo dos períodos conceptivos e no final do segundo período conceptivo. As medições dos testículos foram realizadas previamente a cada colheita de sêmen, e uma diferença significativa foi encontrada no volume testicular total entre as estações, com maiores volumes encontrados durante a estação de seca (p = 0,0011). As amostras colhidas durante a estação de chuva mostraram uma maior porcentagem de motilidade total (x̅ = 93,85) e integridade de membrana plasmática (x̅ = 95,54) (p = 0,0149 e p = 0,0279, respectivamente), em comparação com a estação seca (motilidade total x̅ = 90,96 e integridade de membrana plasmática x̅ = 92,46). Mesmo com essas diferenças, sêmen de ambas as estações apresentaram boa qualidade. A taxa de sucesso das colheitas com a técnica de vibroestimulação peniana foi de 100%, e todas as amostras eram constituídas de somente uma fração coagulada. Apesar de não obtermos a dissolução completa do coágulo, nós pudemos acessar um grande número de espermatozóides pela adição do meio
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