Study On In Vitro Maturation And Cyopreservation Of Donkey Germinal Vesicle Oocytes

This study mainly explored that three different methods were evaluated for efficiency of donkey oocyte collection and the effect of different maturation media by culturing donkey oocyte.in addition,GV oocyte were froze with vitrification method and it’s microstructure were observed,The results are as follows:1. incision and combination method were significant higher than aspiration method for efficiency of donkey oocyte collection (P<0.05)2. GV oocytes were cultured in different concentrations of FSH to mature, and the optimal concentration of FSH was analyzed based on maturing rate finally.The maturation rate of donkey oocytes for adding 100 μg/mL FSH (51.77%) was significantly higher than those of for adding 60、80μg/mL FSH(21.43%、38.98% respectively)(P<0.05), but not significantly difference with 120μg/mL(47.29%) (P >0.05).3. GV stage oocytes were cultured in different gradient time to mature. The results show that maturation rate gradually increases along with in vitro culture time,and the maturation rates are stable after being cultured for 30～32 h.4. GV donkey oocytes were frozed with four kinds of refrigerant (Ⅰ、Ⅱ、Ⅲ、 Ⅳ), finally the best refrigerant was definited according to the normal morphology rates and maturing rates. The results showed that the normal morphology rates of group Ⅰ、Ⅱ、Ⅲ、Ⅳ were 84.62%、87.50%、71.43% and 68.63%,the maturation rate of group Ⅱ (28.13%) was significantly higher than other three groups, (P< 0.05),but significantly lower than normal group. Thus it was determined that group Ⅱ (20%DMSO+20%EG+60%M199+0.25M Sucrose) was the best combination of cryoprotectants.5. Use ordinary straws, OPS tubes and GMP tubes as frozen carrier to freeze GV donkey oocytes, finally the best refrigerant was definited according to the normal morphology rates and maturing rates. The results showed that the normal morphology rates of group ordinary straws, OPS tubes and GMP tubes were 81.67% 87.50%、 76.47%, The maturation rate of using OPS tubes (28.13%) was significantly higher than ordinary straws(11.67%) (P< 0.05) but not significantly difference with GMP tubes(23.53%)(P>0.05).6. Immunofluorescence staining showed that after thawing microfilaments of GV stage oocytes reduced distribution in the cortex and a part of microfilaments in the cytoplasm of central. mitochondria of GV stage oocytes decreased in number after thawing. These results indicated that freezing would be produce injury oocytes of microstructure.