Vitrificação de ovócitos imaturos de bovinos e seu efeito na taxa de maturação "in vitro"

• Autor(a) principal: Martins, Rodrigo Duarte
• Data de Publicação: 2004
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5036
• topic: Bovino; Oócito; Criopreservação; Bovine; Oocytes; Cryopreservation; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL

The aim of this study was to evaluate the vitrification of immature bovine oocytes, using differents equilibrium times and differents concentrations of ethylene glicol (EG) during equilibrium period, with a vitrification solution containing EG associated with two disaccharides. It was evaluated the influence of each treatment on the oocytes maturation rates, after thawing. The experiment was conducted in the LRA-DVT-UFV. It was tested three equilibrium solutions (ES), containing 3, 20 or 40% of EG, three equilibrium times (ET), 0,5, 5 and 15 minutes, and two vitrification solutions (VS), containing 40% of EG + 1,0 mol L-1 of trehalose or 40% of EG + 1,0 mol L-1 of sucrose. The three ES, the three ET and the two VS were combined among each other, completing 18 treatments. The controlled treatment had fresh oocytes non vitrified maturated in vitro. It was used 2.103 immature oocytes, distributed in 19 treatments. The selected oocytes were kept in the ES (basemedium with 3, 20 or 40% of EG) for a determined ET (0,5, 5 or 15 minutes). After the end of the ET, the oocytes were transfered to the VS (base-medium with 40% of EG + 1,0 mol L-1 of trehalose or 40% of EG + 1,0 mol L-1 of sucrose), for one minute. During this time, the oocytes were loaded in 0,25 mL straw and dipped directly in liquid nitrogen. The oocytes were thawed by exposing the straw for five seconds to air, followed by immersion in water bath at 37 °C for 30 to 45 seconds, and were gradually rehydrated in trehalose solutions (for treatments 1 to 9) or sucrose solutions (for treatments 10 to 18). After rehydratation, the recovery rate and morfology of the oocytes were evaluated. The oocytes were cultured for 22 to 24 hours. The recovery rate was not affected by ES, ET and VS, but the morfology of the oocytes was. The ES containing 40% of EG showed the highest rate of normal oocytes (NORM) (76,94%). The ET of 15 minutes showed the highest rate of NORM (80,58%) and lowest rate of oocytes with citoplasmic retraction (CITRET) (16,02%). Vitrifications solutions containing trehalose also showed the highest rate of NORM (76,80%) and the lowest rate of CITRET (19,93%). The combinations ET for 15 minutes and ES containing 3, 20 ou 40% of EG showed the lowest rates of CITRET (17,40; 20,78 and 9,88%, respectively). The combination (treatment 14) VS with sucrose, ET for five minutes, ES with 20% of EG showed the highest rate of MII (metaphase II) (44,55%). The lowest rate of MII in the treatments that used sucrose, were found in the combinations ES with 40% of EG and ET for 15 and five minutes (0,95 and 0,00%, respectively). The highest rate of MII in the treatments that used trehalose was 5,32%. The highest rates of CC (cromatin condensation) were found in the treatments that used trehalose. It was observed that the rate of MII of treatment 14 was significally different from the controlled treatment (44,55% and 74,95%, respectively). The results point out that morfologic evaluation of immature oocytes after thawing and rehydratation, based on citoplasmic retraction, do not reflect the potencial of the oocytes fo in vitro maturation. The use of trehalose in the VS influenced negatively the oocyte maturation. The use of sucrose in the VS influenced positively the oocyte maturation. Elevated concentrations of EG (40%) associated with a long ET (five and 15 minutes) did not favor the oocyte development. The protocol using 20% of EG in the ES, with ET of five minutes and the VS containing 40% of EG + 1,0 mol L-1 of sucrose, showed good rates of in vitro maturation, for immature bovine oocyte.