水牛徒手克隆胚胎培养及冷冻条件的优化

摘要

This study was conducted to optimize the culture and cryopreservation conditions for buffalo hand-made clone (HMO embryos. Microdrops (MD) , well of the wells (WOW) , flat surface (FS) were adopted to culture HMC reconstruc ted embryos;and 40% EG, 25% EG (ethylene glycol, EG) + 25% DMSO (dimethylsulphoxide, DMSO) and 20% EG + 20% DMSO + 0.5 mol/L sucrose were adopted to vitrify HMC blastocysts. Furthermore, the efficiency of HMC and traditional somatic cell nuclear transfer (SCNT) were also compared. The results showed that the cleavage rate of embryos cultured in WOW was higher than those cultured on FS (P<0. 05) and in MD(P<0. 01) ;the blastocyst rate of embryos derived from WOW system (40.0%) was also higher than those derived from FS (19.8%) and MD (8. 3%) systems (P<0. 01);the cryo-survival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0. 5 mol/L sucrose was higher than those vitrified with 40% EG (P<0. 01) ;both the fusion rate and blasotycst rate of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos (P<0. 05;P<0. 01) , while the cryotolerances of HMC and SCNT blastocysts were not different from each other. These results indicated that HMC could be an alternative to traditional SCNT in buffalo, WOW was most suitable for culture of HMC embryos, and vitrification of HMC blastocysts with 20% EG+20% DMSO + 0. 5 mol/L sucrose could get a reasonable cryosurvival rate.%本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养；采用了40％EG(ethylene glycol,EG)、25％EG+ 25％DMSO(dimethylsulphoxide,DMSO)和20％ EG+20％ DMSO+0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻；并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率.结果表明,微穴系统的卵裂率要显著高于平板系统(P＜0.05),极显著高于微滴系统(P＜0.01)；且微穴系统的囊胚率(40.0％)极显著高于平板(19.8％)和微滴系统(8.3％)(P＜0.01).采用20％ EG+20％ DMSO+0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40％ EG(P＜0.01)；HMC重组胚的融合率和囊胚率均高于传统核移植法(P＜0.05;P＜0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异.以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20％ EG+20％ DMSO+0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果.