Parthenogenetic Activation And Cryopreservation Of Sheep Oocytes

The objective of this paper attempted to find an optimum cryopreseryation technique to provide enough oocytes for in vitro fertilization, nuclear transfer and genetic transfer. It was designed to carry out parthenogenetic activation and cryopreservation of sheep oocytes derived from IVM under various time of matuation culture, ethanol treatment, cryoprotectant reagent and methods. The results showed as follows:1.Oocytes cultivated to mature for 22h, 24h and 26h, the cleavage rate using ethanol activation were 60.7%,75.2% and 74.5% respectively and that cleavage rate matured for 24h was significantly higher than that of matured for 22h, but there was no significantly difference between group of 24h and 26h(75.2%vs74.5%, P>0.05). The morula rate of oocytes matured for 24h was significantly higher than that of oocytes matured for 26h(38.6%vs27.3%, P<0.05).2.The cleavage rate of oocytes co-cultured in 2mmol/L 6-DMAP for Oh, 2h was significantly lower than that of co-cultured in 2mmol/L 6-DMAP for 6h (66.4%, 70.3%vs 83.4%, P<0.05) and there was no significantly difference between group of 6h and 4h (83.4%vs79.4%, P<0.05).3.The cleavage rate was no significantly difference between activation time of 3min group, 5min group and 7min group(76.7%,81.4%,70.4%, respectly, P>0.05). The blastocyst rate of 3min group was significantly higher than that of 7min group(13.3%vs9.3%, PO.05).4.The cleavage rate of oocytes activated with 7% ethanol concentration was significantly higher than that of 5% ethanol concentration (85.5%vs72.2%, PO.05), but both of morula rate and blastocyst rate among three group were no significantly difference.5.The blastocyst rate using oviduct cell co-culture and cumulus cell co-culture were higher than that using CM embryo cultural solution(20.4%, 18.7% vs 10.1% respectively, PO.05). There were no significantly difference of cleavage rate among three embryo cultural methods, using CM embryo cultural solution, oviduct cell co-culture and cumulus cell co-culture (71.4%, 75.2% and 75.8% respectively, P>0.05), which was also found with the morula rate.6.Leaving the oocytes in different cryoprotectants reagents under 20%v/v for 120 seconds, sheep oocytes were actived and cultivated. The cleavage rate of DMSO group was 40.6%, which was lower than that of EG group and PROH group (P<0.01). The morula rate was 23.2%, which was also lower than that of EG group and PROH group (P<0.05). There was no significantly difference among blastocyst rate (P>0.05).7.After thawing, the normal morphological rate of oocytes frozen with Straw Vitrification and OPS method were lower than that of GMP method(9.2%, 15.7% vs 70.3% respectively, P<0.01). Oocytes using GMP method for frozen obtain higer cleavage rate, morula rate and blastocyst (26.5%, 11.9% and 3.2%) than those using Straw Vitrification and OPS methods(P>0.05).8. Usingâ‘ ,â‘¡andâ‘¢ represent the mixture vitrification solution of 20%EG + 20%DMSO, 20%PROH + 20%DMSO and 10%PROH + 10%DMSO + 20%EG respectively. After thawing, the normal morphological rate of oocytes using â‘  and â‘¢ group for frozen were higer than that of â‘¡ group (P<0.01).