Study On Vitrification Of Bovine Oocytes

This study mainly explored the cryopreservation method of vitrification and its effects on cytoskeleton, organelles, and subsequent developmental potential of bovine oocytes. The results are as follows:1. Mâ…¡o ocytes were froze with the vitrification solutions of groupâ… ,â…¡,â…¢,â…£,â…¤a ndâ…¥. After thawing the optimal datas of the six groups were selected to analyze significance of difference. Comparing with the cleavage rate ofâ…£3(24.6%)after thawing,â… 3 (2.2%),â…¡3(5.4%),â…¤3(5.6%) andâ…¥3 (7.2%)possessed significant differences (P <0.01), the difference withâ…¢3 (26.2%)was not significant(P>0.05), while the morphology normal rates ofâ…¢3 andâ…£3 were significantly different(P <0.05). Thus it was determined thatâ…£3(HM +20% EG +20% DMSO +0.5M sucrose)was the best combination of cryoprotectants.2. After thawing the survival rates and cleavage rates of GV and Mâ… oocytes were lower than that of Mâ…¡oocytes with significant differences(P<0.05) (P <0.01)respectively. This suggested that Mâ…¡s tage was the best time for cryopreservation.3. Use ordinary straws, OPS tubes, capillary tubes, and disposable culture dishes (35mm)as frozen carrier to freeze Mâ…¡oocytes. After thawing the survival rate and cleavage rate of oocytes on disposable culture dishes (35mm)were higher than those on straws and OPS tubes (50.5% VS 40.8%, 49.1%) (25.2% VS 17.1%, 20.5%). Furthermore, the differences were significant (P <0.05). Thus the disposable culture dish (35mm) was the most suitable frozen carrier.4. After thawing the morphology normal rate, survival rate and cleavage rate of the control group were higher than the freezing group with significant differences (P<0.05) (P<0.01) (P<0.01) respectively. Therefore, it was demonstrated that freezing had irreversible effects on developmental ability of oocytes.5. Immunofluorescence staining showed that after thawing microfilaments of GV, Mâ… , Mâ…¡stage oocytes reduced distribution in the cortex, and reduced or disappeared in the nuclear area. Besides, microtubules of Mâ… , Mâ…¡oocytes disintegrated, disappeared or partially lost, mitochondria of GV,Mâ… ,Mâ…¡oocytes decreased in number after thawing. These results indicated that freezing would produce injury to some structures and organelles of oocytes.