Study On Vitrification Preservation Technology Of Mouse Morula And Early Blastocyst

In this study, mouse morula and blastocysts were vitrified using Straw Method and OPS Method, after frozen for 1-6d, embryos were thawed using DPBS contained 0.5M sucrose. Embryos were cultured in TCM199 for 24h and development in vitro and the pregnant rate and litter rate after transferred were observed to evaluate the best method and stage for vitrifying mouse morulas and blastocysts. The results indicated as below:1,When embryos were balanced in EFS40 for 0.5 minutes using the One Step Method, then cultured in TCM199 for 24 hours after vitrification and thawed, up to 70.59% morulas developed to blastocysts while only 41.94% blastocysts hatched. When embryos were balanced in EFS40 for 1.0 minutes, the development rates of morulas and blastocysts after thawed were low(52.27%,50.00%, respectively). When embryos were balanced in EFS20 for 2.0 minutes and EFS40 for 0.5 minutes using the Tow Step Method, 81.13% morulas developed to blastocysts after 24h culture, winch is higher than expand rate of blastocysts (48.94%)very significantly(P＜0.01). When embryos were balanced in EFS40 for 1.0 minutes using Tow Step Method, the development rate of morulas and blastocysts were 75.00% and 45.00%, respectively.2,Fresh morulas and morulas using Tow Step Method obtained 42.86% and 28.57% pregnant rates after transfered to receptor. When embryos were balanced in EFS20 for 2.0 minutes and EFS40 for 1.0 minutes, neither morulas nor blastocysts obtained pregnant receptor. When embryos were balanced in EFS20 for 2.0 minutes and EFS40 for 0.5 minutes, the development rate of morulas(11.11%) was higher than the hatch rate of blastocysts(4.11%) significantly(P＜0.05). After transferred the pregnant rate and litter rate of morulas using Tow Step Method(28.57%,11.11%) were higher than using One Step Method (14.29%,3.80%)(P＜0.05). 3,When embryos were balanced in EFS40 for 0.5 minutes using the One Step Method, then cultured in TCM199 for 24 hours after treated, 72.31% morulas developed to blastocysts. This was significantly higher than the one balanced in EFS40 for 1.0 minute(47.62%)(P＜0.05), while no significantly different to fresh embryos(81.54%)(P＞0.05). When embryos were balanced in EFS40 for 0.5 minutes using the One Step Method, then cultured in TCM199 for 24 hours after treated, the expand rate of blastocysts(25.00%) was very significantly lower than the development rate of morulas (47.62%) and fresh embryos(81.54%)(P＜0.01). When embryos were balanced in EFS20 for 2 minutes and EFS40 for 0.5 minutes using the Tow Step Method, the development rate of morulas (81.43%) was very significantly higher than the expand rate of blastocysts(46.15%)(P＜0.01).4,Morulas vitrified with One Step OPS Method obtained 96.83% development rate, which was no significant difference with One Step Staw Method(P＜0.05). Both development rate of One Step Staw(90.16%) and OPS(96.83%) Method vitrified morulas was higher then expand rate of blastocysts(77.36%,56.36%)(P＜0.05 or P＜0.01). Blastocysts vitrified with Tow Step OPS Method Obtained higher expand rate (71.15%) than Staw(P＜0.05). Morulas vitrified with Tow Step Staw and OPS Method obtained 93.44% and 96.72% development rate, not significantly(P＞0.05).5,When embryos were balanced in EFS40 for 0.5 minutes using the Tow Step OPS Method, then cultured in TCM199 for 24 hours, the pregnant rate(42.86%) and litter rate(12.35%) of morulas were significantly higher than One Step Method(2.53%) and morulas balanced inEFS40 for 1.0 minutes (3.57%)(P＜0.01). When embryos were balanced in EFS40 for 0.5 minutes using the Tow Step Method, the pregnant rate (28.57%) and litter rate(5.48%) of blastocysts were significantly lower than morulas (42.86%, 12.53 % )(P＜0.05).