小鼠休眠胚胎与正常胚胎冻融后体内外发育潜力的比较

Comparison of the in vivo and in vitro developmental potentiality of dormant and normal mouse embryos before and after cryopreservation

目的检验小鼠休眠胚胎冻融后的质量及体内外发育潜力,为胚胎休眠技术的生产应用提供必要的参考。方法采用常规冷冻方法将正常孵化期胚胎和休眠胚胎进行冷冻,之后分别进行体外复苏培养实验和胚胎移植实验。随后利用双重荧光染色的方法分别对冻融前后的小鼠休眠胚胎与正常孵化期胚胎进行细胞计数,观察两种胚胎冻融前后的质量变化。结果休眠胚胎的冷冻解冻回收率、发育率均极显著高于孵化期胚胎(72.1%vs 50.2%,P<0.01;94.2%vs 73.9%,P<0.01)。休眠胚胎的移植妊娠率显著高于孵化期胚胎(40.8%vs 30.1%,P<0.05)。休眠胚胎的内细胞团细胞数显著高于孵化期胚胎(27.83 vs 19.53,P<0.05),滋养层细胞数差异不显著。冻融培养后休眠胚胎的内细胞团数,滋养层细胞数均显著高于孵化期胚胎(25.18 vs 14.68,P<0.05;114.09 vs 73.88,P<0.05)。结论小鼠休眠胚胎冻融后胚胎质量及体内外发育潜力均优于小鼠正常孵化期胚胎。

Objective To verify the quality as well as in vitro and in vivo development potentiality of mouse dormant embryos after freezing and thawing, and provide a reference for the production and application of embryonic dormancy technology. Methods Normally incubated and dormant embryos were frozen by conventional freezing method, followed by in vitro resuscitation culture and embryo transfer experiments. Subsequently, the cells of dormant and normally hatched embryos before and after freezing and thawing were counted by double immunofluorescence staining, and the qualities of the two embryo types before and after freezing and thawing were observed. Results The recovery rate from freezing and thawing and the development rate of dormant embryos were significantly higher than those of hatched embryos (72.1% vs 50.2%, P < 0.01;94.2% vs 73.9%, P < 0.01). The pregnancy rate of dormant embryos was significantly higher than that of hatched embryos (40.8% vs 30.1%, P < 0.05). The number of cells in the inner cell mass of the dormant embryo was significantly higher than that in the hatched stage (27.83 vs 19.53, P < 0.05), while the difference in the number of trophoblasts was not significant. The number of inner cell clusters in the dormant embryos after freezing and thawing was significantly higher than that in the hatched embryos (25.18 vs 14.68, P < 0.05;114.09 vs 73.88, P < 0.05). Conclusions The embryo quality as well as in vitro and in vivo development potential of mouse dormant embryos after freezing and thawing are better than those of normally hatched embryos.