Studies On Cryo-preservation Of Bovine Ooctyes

The objective of this study is to provide an effective method to cryopreserve bovine GV oocytes for research and practice of gamete bank, in vitro fertilization, nuclear transfer and cell cloning. The study compared the effects of cryopreservation reagent , freezing method, the developmental stages of oocytes and equilibration method in bovine. The results as follows:Studies on cryoprotectants toxicity for bovine oocytes. Bovine GV oocytes were treated with four 1.5-7.2 mol/L cryoprotectants (DMSO, PROH, EG, GL) at 25 and 39 , then diluted cryoprotectants and compared the ratio of the maturation in vitro. The results demonstrated that the four cryoprotectants' toxicity for bovine oocytes were low and increased depend on the concentration at 25 ; And those toxicity were evident at 39 , but EG's is lowest. The toxicity from the high to low was GL>PROH>DMSO>EG. Using the programmed freezing method to freeze the bovine oocytes with 1.5 mol/L DMSCK PROH, EG, GL, the effects of EG and PROH were better than GL, DMSO.Using programmed freezing method, Straw vitrification, open-pulled-straw (OPS), glass micropipette (GMP) to freeze the bovine GV oocytes, Freezed and warmed oocytes were matrured and fertilized in vitro . The cleavage ratio was 3.33% and 8.33% respectively in programmed freezing method and Straw vitrification, and the latter was higher by 5.1% than the former, but the results was not different (P>0.05 ); The cleavage ratio using OPS and GMP was similar, 36.84% and 36.67% respectively, and the results was not different (P>0.05) ,but was significant different with programmed freezing method and Straw vitrification( PO.01 ) .This demonstrated that vitrification was better than programmed freezing method,that GMP and OPS was better than Straw vitrification, that the effect of GMP and OPS was similar.We study the effects of the developmental stages of oocytes. GV oocytes and ooctyes cultured 6h, 18h, 22h in vitro were vitrification using GMP. the cleavage ratio of warmed oocytes cultured 24h, 18h, 6h and 2h and fertilized in vitro was compared .The cleavage ratio of warmed GV oocytes was 36.67%, 40.98% and 41.27% after culture 6h and 18h. The three was not different (P>0.05).However, this was significant different compared with oocytes cultured 22h (52.38%) and the control (68.42%) (P>0.05) . The cleavage ratio of IVM freezed and thawed oocytes was 52.38%, and it was not different with the control (68.42%) (P>0.05).This results indicated the cyroperservation effectiveness of oocytes cultured 22h was better than that of GV and those oocytes cultured 6h and 18h. The more maturation of bovine oocytes were, the more cyroperservation effectiveness was.We study the effects of equilibration for vitrification. Prior to vitrification of bovine oocytes in the vilification solution, the oocytes were exposed to equilibration solution (1%EG, 3%EG, 6%EG, 10%EG, 1/2 VS solution) for 1-1 5min, the results indicated that effectiveness of 3%EG is best than others, whose cleavages and maturation ratio in vitro were high.This suggested the diluted solution equilibrium can improve the cyroperservation effectiveness before vitrification.The conclusions proved that GMP vitrification procedure can be used to cyro-perserve bovine GV oocytes.