Vitrificação de ovócitos imaturos de bovinos

• Ano de defesa: 2011
• Autor(a) principal: Santos, Giancarlo Magalhães dos
• Orientador(a): Costa, Eduardo Paulino da
• Banca de defesa: Espeschit, Claudio José Borela , Benjamin, Laércio dos Anjos , Figueiró, Giuliano Moraes
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Medicina Veterinária
• Departamento: Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de
• País: BR
• Palavras-chave em Português: Ovócitos; Bovinos; Vitrificação
• Palavras-chave em Inglês: Oocytes; Bovine; Vitrification
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1449

The aim was to investigate the effects of vitrification solutions and vitrification of bovine immature oocytes. The oocytes were obtained from ovaries collected from bovine females immediately after slaughter, morphologically selected and distributed according to the treatments. The experimental procedure was divided into two phases, in the first phase was tested the toxicity of vitrification solutions and in the second phase was tested the effect of vitrification. In Experiment 1, the oocytes were matured in vitro after maintenance in differents vitrification solutions (61 treatments). Solutions of equilibrium (SE) of 3, 8, 16, 20, 32 and 40% ethylene glycol (EG) were tested to equilibration time (ET) of 1, 2, 4, 5, 6, 8 and 10 minutes and then maintained by 1 minute in the vitrification solution (VS) containing 40% EG and 1.0 mol L-1 sucrose. The same procedure was performed in other tratament, but using the vitrification solution containing 25% EG, 25% DMSO and 1.0 mol L-1 sucrose. After these procedures, the oocytes were rehydrated and subjected to cultivation in vitro for 24 hours. Nuclear maturation was observed in most treatments except in SE combinations with 32 and 40% EG at different ET. Likewise, the oocytes exposed to SV 25% EG + 25% DMSO + 1 mol L-1 sucrose did not allow maturation. The best results were obtained with SE solutions containing 3% and 16% exposed f or five minutes and 20% EG exposed for 10 minutes (76.7, 57.1 and 66.7% of oocytes in metaphase II, respectively). All treatments groups were exposed to the end of the equilibrium to the SV, containing 40% EG + 1.0 mol L -1 sucrose. In Experiment 2, oocytes were submitted to the procedures described in the Experiment 1. Were tested SE containing 3% EG (for 1, 5, 6, 8, and 10 minutes), 8% EG (for 1, 5 and 10 minutes), 16 and 20% EG (for 5 and 8 minutes). Subsequently, the oocytes were maintained for 1 minute at SV containing 40% EG and 1.0 mol L-1 sucrose. Subsequently, the oocytes were packed into 0.25 mL straws, vitrified (immersed in liquid nitrogen) and subsequently desvitrificated, rehydrated and submitted to maturation in vitro for 24 hours. It was observed that the SE combination with 3% of EG, in ET of 1, 5, 6, 8 and 10 minutes provided the metaphase II rates of 10.4, 19.3, 10.0, 7.6 and 14 3% respectively. The combinations SE with 8% EG with ET 1, 5 and 10 minutes and SE with 16% EG, 5 minutes with ET and SE of 20% EG with ET 8 minutes provided rates of metaphase II 12.9, 6.9, 2.4, 23.1 and 2.0% respectively, all of which differ (P <0.05) than the control group (73.7%). Oocytes were processed for the evaluation of the ultrastructure and gene expression. The ultrastructure of oocytes indicated that all treatments (except control group) had degenerated organelles. Additionally, it not observed cytoplasmic distribution of mitochondria typical of a mature oocyte. In the gene expression was observed that oocytes exposed to the equilibrium solution of 16% EG in five minutes have a higher amount of transcripts related to oxidative stress (HSP 70.1). However, the transcripts of the maternalzygotic transcription (MATER and ZAR1) were under-regulated (P <0.05). We conclude that the procedures of dehydration tested for cryopreservation of oocytes, except for combinations of SE with 32% EG and 40 in different TE, cannot compromise the rate of nuclear maturation after maturation "in vitro". However, ultrastructural compromise the integrity, citoplasmatic maturation and gene expression.