小鼠胚胎的玻璃化冷冻技术研究
摘要
本试验探讨了冷冻保护剂基础液、冷冻载体的选择及不同玻璃化冷冻方法对小鼠3.5d～4d桑椹胚和囊胚的冷冻保存效果。（1）玻璃化液分别使用TCM-199和D-PBS为基础液，在基础液中添加10% EG+10% DMSO为玻璃化1液（VS1），添加20% EG+20%DMSO为玻璃化2液（VS2），冷冻小鼠桑椹胚和解冻后获得的发育率分别为70.00%和72.34%，二者差异不显著(p > 0.05)；（2）使用玻璃管和塑料管作为胚胎的承载材料进行玻璃化冷冻效果比较，玻璃材质的OPS管玻璃化冷冻桑椹胚，解冻后形态正常率和发育率分别为95.23%和87.57%，而使用塑料OPS管玻璃化冷冻桑椹胚解冻后形态正常率和发育率分别为89.66%和86.40%；玻璃材质的OPS管玻璃化冷冻囊胚，解冻后形态正常率和发育率分别为92.00%和85.83%，而使用塑料OPS管玻璃化冷冻桑椹胚解冻后形态正常率和发育率分别为86.84%和77.14%，使用玻璃OPS管和使用塑料OPS管在胚胎解冻后形态正常率和发育率上差异均不显著(p > 0.05)；（3）采用程序化冷冻与OPS玻璃化冷冻法进行对比，结果使用玻璃化冷冻法和程序化冷冻法对小鼠胚胎进行冷冻保存都可以取得很好的结果。程序化冷冻在形态正常率桑椹胚97.78%，高于玻璃化冷冻94.64%，囊胚为95.12%，低于玻璃化冷冻100%。在发育率方面，程序化冷冻桑椹胚解冻后发育率为95.45%，玻璃化冷冻法为88.94%，差异显著(p < 0.05)；程序化冷冻囊胚解冻后发育率为86.16%，玻璃化法为83.33%，差异不显著(p > 0.05)；(4) 使用OPS细管进行玻璃化冷冻桑椹胚，解冻后形态正常率和发育率分别为97.01%和86.91%，0.25ml细管冷冻桑椹胚，解冻后胚胎形态正常率和发育率分别为91.84%和75.51%，差异不显著(p > 0.05)；使用OPS细管进行玻璃化冷冻囊胚，解冻后形态正常率和发育率为88.46%和83.51%，0.25ml细管冷冻囊胚，解冻后胚胎形态正常率和发育率分别为75.47%和68.89%，差异不显著(p > 0.05)。得出结论，使用玻璃化冷冻法也可以获得与程序化冷冻相同的良好结果。
In the present study, mouse embryos (Day 3.5 – 4) collected from superovulated mice were frozen by open pulled straw (OPS) vitrification and conventionally frozen before in vitro culture. The objective was to assess embryo survival rate and process of embryo handling during commercial embryo transfer (ET) using the conventional freezing method as a comparison. The results as following: (1) During the vitrification of using different medium, the developed morulae were reached by 70.00% for medium TCM-199, and 72.34% for medium D-PBS. There were no significant difference between 2 mediums (p > 0.05); (2) The morphologically normal rates of thawed morulae and blastocysts with glass straw and open pulled straw as a carrier were 95.23% vs. 89.66% and 92.00% vs. 86.84%. Developing rates of thawed morulae and blastocysts were 87.57% vs. 86.40% and 85.83% vs. 77.14% (p > 0.05) respectively; (3) Morphologically normal rates of embryos vitrified or conventionally frozen were 97.78% vs. 94.64% at the morula stage, and 95.12% vs. 100%. The developing rates of embryos vitrified or conventionally frozen were 95.45% vs. 88.94% at the morulae stage, and 86.16% vs. 83.33% at the blastocysts stage respectively. No significant difference was found at the blastocysts stage between vitrified or conventionally frozen (p > 0.05), although significant difference was found at the morulae stage between vitrified or conventionally frozen (p < 0.05); (4) The morphologically normal rates of thawed embryos between OPS straw or 0.25ml straw at the stage of morulae were97.01% and 91.84%, 88.46% and 75.47% at the blastocysts stage. The developing rates of embryos vitrified with OPS and 0.25ml straw at the morula were 86.91% vs. 75.51%, at the blastocyst were 83.51% vs. 68.89% (p > 0.05) respectively. The study demonstrated about the same survival rates for vitrified and conventionally cryopreserved embryos of morulae and blastocysts stages during in vitro embryo culture. OPS vitrification is an effective and rapid method of cryopreservation of mouse embryos.
In the present study, mouse embryos (Day 3.5 – 4) collected from superovulated mice were frozen by open pulled straw (OPS) vitrification and conventionally frozen before in vitro culture. The objective was to assess embryo survival rate and process of embryo handling during commercial embryo transfer (ET) using the conventional freezing method as a comparison. The results as following: (1) During the vitrification of using different medium, the developed morulae were reached by 70.00% for medium TCM-199, and 72.34% for medium D-PBS. There were no significant difference between 2 mediums (p > 0.05); (2) The morphologically normal rates of thawed morulae and blastocysts with glass straw and open pulled straw as a carrier were 95.23% vs. 89.66% and 92.00% vs. 86.84%. Developing rates of thawed morulae and blastocysts were 87.57% vs. 86.40% and 85.83% vs. 77.14% (p > 0.05) respectively; (3) Morphologically normal rates of embryos vitrified or conventionally frozen were 97.78% vs. 94.64% at the morula stage, and 95.12% vs. 100%. The developing rates of embryos vitrified or conventionally frozen were 95.45% vs. 88.94% at the morulae stage, and 86.16% vs. 83.33% at the blastocysts stage respectively. No significant difference was found at the blastocysts stage between vitrified or conventionally frozen (p > 0.05), although significant difference was found at the morulae stage between vitrified or conventionally frozen (p < 0.05); (4) The morphologically normal rates of thawed embryos between OPS straw or 0.25ml straw at the stage of morulae were97.01% and 91.84%, 88.46% and 75.47% at the blastocysts stage. The developing rates of embryos vitrified with OPS and 0.25ml straw at the morula were 86.91% vs. 75.51%, at the blastocyst were 83.51% vs. 68.89% (p > 0.05) respectively. The study demonstrated about the same survival rates for vitrified and conventionally cryopreserved embryos of morulae and blastocysts stages during in vitro embryo culture. OPS vitrification is an effective and rapid method of cryopreservation of mouse embryos.
引文
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