小鼠-猪种间核移植胚胎玻璃化冷冻保存

Vitrification of embryos derived from mouse-pig interspecies nuclear transfer

以小鼠卵丘细胞为供核细胞,猪体外成熟去核卵母细胞为受体,构建小鼠-猪种间体细胞核移植(iSCNT)胚胎,并以猪体细胞核移植(pSCNT)胚胎为对照,采用开放式拉长细管(OPS)法对所获iSCNT胚胎进行冷冻保存,研究不同冷冻保护液(ES、EFS和EDFS)及胚胎不同发育阶段(2-、4-、8-细胞)对冷冻效果的影响。结果显示:小鼠-猪iSCNT胚胎卵裂率与猪pSCNT胚胎相比无显著差异(P>0.05),但其8-细胞发育率则明显较低(28.1%对56.8%,P<0.05)。采用ES液作为冷冻保护液,iSCNT胚胎冻后存活率与EDFS组相似,但显著高于EFS组(28.6%和22.7%对9.5%,P<0.05);与其他发育阶段相比,2-细胞期小鼠-猪iSCNT胚胎可获得最高的冻后存活率;小鼠-猪iSCNT各期胚胎的冻后存活率均低于pSCNT组,但无统计学差异(P>0.05)。结果表明,以ES液为冷冻保护液,选择2-细胞期小鼠-猪iSCNT胚胎进行OPS法冷冻保存,可获得相对较高的冻后存活率,但iSCNT胚胎的冻后存活率和体外发育能力均较pSCNT胚胎低。

The purpose of this experiment was to investigate the effects of different cryoprotectant（ES,EFS and EDFS）and developmental stages（2-,4-,8-cell）on viability of mouse-pig interspecies somatic cell nuclear transfer（iSCNT）embryos using open-pulled straws（OPS method）.Mouse cumulus cells were used for nuclear donor cells,and the in vitro maturated porcine oocytes were used as recipients after enucleation.Mouse-pig iSCNT embryos were produced then and compared with the control of porcine somatic cell nuclear transfer（pSCNT）embryos.The results showed that mouse-pig iSCNT embryos had similar cleavage rate but a significant lower developmental rate to 8-cell stage（28.1% vs 56.8%,P0.05）compared with the control.When the ES solution was used as cryoprotectant,the survival of iSCNT embryos after warming was much similar with that from EDFS group but both were significantly higher than that of EFS group（28.6% and 22.7% vs 9.5%,respectively,P0.05）.As compared to other developmental stages,the re-warming survival rate of mouse-pig iSCNT embryos at 2-cell stage were the highest.The iSCNT embryos from every group displayed a relative lower survival rate as compared to the pSCNT groups,but there were no statistical differences（P0.05）.This study revealed that the ES was the optimal vitrification solution and 2-cell stage is suitable for the vitrification of iSCNT embryos.However,the iSCNT embryos exhibited a relative lower developmental competence as well as a lower survival rate when compared with pSCNT embryos.