OPS法玻璃化冷冻牛卵母细胞和囊胚

OpenPulledStraw (OPS) vitrification of bovine oocytes and blastocysts derived from invitro production

本文报道了利用拉细开口型细管 (Open Pulled Straw,OPS)方法进行玻璃化冷冻保存体外成熟(IVM)的牛卵母细胞及体外生产 (IVP)的牛囊胚的实验结果。实验 1的牛卵母细胞冷冻处理分 2组 ,组 1(G1 )是将卵母细胞在 IVM2 1 h时去除部分卵丘细胞后马上进行冷冻处理。组 2 (G2 )卵母细胞在 IVM6h时去除部分卵丘细胞 ,然后继续培养至 2 2 h冷冻 ;冷冻后的卵母细胞在液氮中保存 0 .5～ 2 h后解冻再继续 IVM1 h后与正常 IVM2 4h的对照组卵母细胞一起进行体外受精 (IVF)处理。实验 2则冷冻了来自IVP的 6、 7日龄的牛囊胚。结果显示 ,G1的卵母细胞 IVF后 8d的囊胚率 (2 2 .8% ,2 6/1 1 4)和孵化囊胚率 (1 4.9% ,1 7/1 1 4)都极显著地高于 G2 (分别为 2 .9% ,5 /1 71和 0 .5 % ,1 /1 71 ,P<0 .0 0 1 ) ;但两处理组的受精卵裂率差别不大 (分别为 48.2 %和 41 .5 % ,P>0 .3 )。冷冻第 6d和第 7d的体外囊胚 ,其冻后存活率和囊胚孵化率分别为 92 .7%、 89.2 %和 83 .6%、 66.7%。结果表明 ,利用

In 2 experiments using invitro produced bovine ova,285 oocytes and 148 blastocysts were vitrified in heatsoftened and pulled straws by the OpenPulledStraw (OPS) method The methods for vitrification and for invitro production (IVP) were performed as described earlier. In Experiment 1,the oocytes were partially denuded of cumulus cells at 6 h (Group 2, n=171 ) or21 h (Group 1, n =114) after the onset of invitro maturation (IVM) by careful pipetting All oocytes were then vitrified at 21~ 22 h after IVM,stored in liquid nitrogen for 1/2 to 2 h,and then warmed and matured for one additional hour before being invitro fertilized and cultured for 8 d The cleavage rates of vitrified oocytes from Group 1 and Group 2 were 48 2% and 41 5%, respectively,which was significantly lower than for nonvitrified control oocytes (88 1%, n=285, P <0 001) Of the vitrified oocytes from Group 1,22 8% developed to blastocysts on Day 8 after fertilization and 14 9% to hatched blastocysts This was significantly higher compared to Group 2 (2 9% and 0 5%,respectively, P< 0 001) In Experiment 2,Day6 ( n =55) and Day7 ( n =93) IVP blastocysts were vitrified,stored in liquid nitrogen for about one hour,and then warmed before continuing invitro culture The respective reexpansion/hatching rates were 92 7%/83 6% for the Day6 and 89 2%/66 7% for the Day-7 blastocysts These results indicate that bovine blastocysts derived from IVP can be cryopreserved without the loss of viability,and that bovine oocytes matured invitro can also be successfully vitrified by the simple OPS method