Study On Cryopreservation Of Bovine Follicular Oocytes

Cumulus oocytes complexes(COCs) were recovered from Φ2-8 mm superficial follicles of slaughter discarded ovaries. After that, experiment I was conducted to optimize conditions of cryopreservation of COCs, subsequently, experiment II was carried out to analyze the effect of cryopreservation on COCs meiotic division progression.1 Factors affecting cryopreservation of bovine COCsAfter facilitating practice of in vitro maturation, factors like cryoprotectants, equilibrium temperatures, pre-equilibrium methods, thawing methods and carriers including conventional glass micropipette(GMP), pulled glass micropipette(pulled GMP) and solid surface(SS), were studied. Results showed as following: (l)choice of 26.2mM NaCHO₃ as buffer of maturation solution and classification of ovaries as growth stage and non-growth stage could improve maturation rate; (2)EG as permeable cryoprotectant was positively compared to GL in both procedure freezing and vitrification; (3)instead of 0.3M, addition of 0.1 M sucrose in procedure freezing solution could improve COCs maturation rate after thawing; (4)as equilibrium emperature, 38.5℃ and 25℃ were similarly more appropriate to 4℃ during vitrification; (5)among pre-equilibrium concentrations of 10%,5%,l%, 5% was sufficient to satisfy vitrification; (6)when thawing, multiple-step dilution was advised; (7)finally, different carriers of COCs were compared and results showed that pulled GMP was the best.2 Effect of freezing on COCs meiotic division progression using fluorescent stainingFirstly, fluorescent staining was used to establish the morphological standard of chromosomes, subsequently, based on the established standard, effect of freezing on meiotic division was analyzed. Results indicated: at the beginning of fresh COCs in vitro maturation, 70% COCs was found at GV atage,12.5% at GVBD stage,7.5% at CONDENSED stage, which demonstrated COCs from slaughterhouse were greatly heterogeneous; after 8 hours of maturation, frozen COCs at MI stage was significantly less than fresh COCs(p<0.01),while COCs at other stage were not significantly different between two groups; after 15 hours of maturation, 16.67% frozen COCs reached MI stage, which was significantly less than 38.89% in fresh COCs(p<0.05), furthermore, 19.44% and 16.67% fresh COCs reached AI stage and TI stage, respectively, while only 6.67% frozen COCs did so; after 22 hours of maturation, 55.56% fresh COCs reached MII stage, whichwas significantly higher than 27.03% frozen COCs(p<0.01),and significantly higher rate of frozen COCs(24.32%) was found arrested at CONDENSED stage(p<0.05),while only 5.56% fresh COCs did so. Collectively, this paper found most frozen COCs couldn't finish CONDENSED→MI transition, and which perhaps accounted for lower nuclear maturation rate of frozen COCs, therefore this paper concluded that injury of freezing of COCs occurred at CONDENSED→MI transition.