奶牛体外生产胚胎玻璃化冷冻技术研究

Cryopreservation Technique of Bovine Embryos produced in vitro by Vitrification

利用乙二醇(EG)及二甲基亚砜(DMSO)作为主要冷冻保护剂,对牛体外生产囊胚进行了冷冻保存试验,探讨了不同冷冻剂组合、平衡时间、蔗糖浓度、冷冻方法对牛胚玻璃化冷冻的影响,结果显示:牛胚置于30%DMSO、15%DMSO+15%EG和30%EG等3种不同冷冻保护剂,其解冻后发育率分别为75.3%、81.8%及71.4%(P>0.05),差异不显著;含有10%EG与10%DMSO冷冻保护剂平衡30 s、45 s及60 s,其解冻后发育率分别为54.5%、75%和44.4%,45 s显著较30 s及60 s者高(P<0.05);以15%DMSO+15%EG冷冻保护剂中添加0.25、0.5及1.0 M的蔗糖浓度,其解冻后牛胚后续发育率分别为50%、75.0%及33.3%,蔗糖添加浓度为0.5 M者效果显著较佳(P<0.05);利用GMP、MDS及Cryoloop3种不同冷冻方式进行冷冻,其解冻后发育率分别为66.7%、71.1%及80.0%,三者间并无显著差异(P>0.05)。

Cryopreservation experiment was carried out for preserving Bovine Embryos produced in vitro with Dimethyl sulfoxide （DMSO） and Ethylene glycol （EG） as main cryoprotectant solutions. Vitrification efficiency of different factors, including combination of cryoprotectants, equilibration time in vitrification solution, sucrose concentration, and freezing methods were evaluated. The results showed that： （1） when 30% DMSO, 15% DMSO＋15% EG, and 30% EG were used, frozen-thawing development rates （75.3%, 81.8%, and 71.4%, respectively） were not significantly different. （2） The frozen-thawing development rate （75.0%） after exposed to cryoprotectant solution of 10% EG ＋10% DMSO and equilibrated for 45 s time was significantly higher than those for 30 s （54.5%） and 60 s （44.4%）. （3） Different concentrations of sucrose （0.25 M, 0.5 M and 1.0 M） were added to 15% DMSO＋15% EG solution, the frozen-thawing development rates were 50.0%, 75.0% and 33.3%, respectively, which indicated that addition of 0.5 M sucrose was suitable. （4） The development rates to blastoeyst stage for the bovine embryos vitrified by GMP method, MDS method, and cryoloop were 66.7%, 71.1% and 80%, respectively, with no significant difference （P〉0.05）.