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目的通过比较不同胚胎密度和培养液体积及微孔(WOW)培养系统对小鼠早期胚胎发育的影响,以期优化早期胚胎发育的培养条件和环境,提高胚胎的发育率和质量。方法 (1)在液状石蜡覆盖的20μl的发育液小滴中培养5、10、20和40枚2-细胞胚胎,观察并统计8-细胞率、囊胚率和孵化囊胚率。(2)15枚2-细胞胚胎放入在5、10、20、50、100μl的发育液小滴和500μl WOW系统(每个WOW中放一枚胚胎)中,观察并统计8-细胞率、囊胚率和孵化囊胚率。(3)对各组得到的囊胚进行差异染色并计数。结果在不同胚胎密度的比较中,20μl的培养液小滴中培养10枚2-细胞胚胎得到了较高的8-细胞胚胎率、囊胚率和孵化囊胚率,而且得到囊胚的ICM、TE和ICM+TE的平均细胞数显著高于5枚和40枚胚胎组。在不同培养液体积和WOW培养系统中,5μl培养液组在各阶段胚胎发育率和得到囊胚的ICM、TE和ICM+TE的平均细胞数方面显著低于其他各组。500μl WOW培养系统组在各阶段胚胎发育率方面与得到较高发育率的20μl和50μl培养组均没有统计学差异,同时500μl WOW培养系统和20μl培养液组在孵化囊胚率和得到囊胚的ICM、TE和ICM+TE平均细胞数方面显著高于100μl培养液组。结论 20μl的培养液小滴培养10枚胚胎可以得到较高的胚胎发育率,且得到的胚胎质量较好。较少的培养液体积不利于胚胎发育,较适宜的培养液小滴体积为20~50μl。同时,WOW培养系统在本实验条件下取得了较好的胚胎发育效果。
OBJECTIVE: To compare the effects of different embryonic densities, culture volume and micropore (WOW) culture system on early embryo development in mice, in order to optimize the culture conditions and environment for early embryo development and improve the embryo development rate and quality. Methods (1) 5, 10, 20, and 40 2-cell embryos were cultured in liquid paraffin-covered 20 μl droplet of developmental fluid and the 8-cell ratio, blastocyst rate and hatched blastocyst rate were observed and counted. (2) Fifteen 2-cell embryos were placed in 5, 10, 20, 50, 100 microliters of developing liquid droplets and 500 microliters of WOW system (one embryo per WOW), 8-cell ratio was observed and counted, Blastocyst rate and hatched blastocyst rate. (3) The blastocysts obtained from each group were differentially stained and counted. Results In comparison of different embryo densities, 10 2-cell embryos cultured in 20 μl culture broth gave higher 8-cell embryo rate, blastocyst rate and hatching blastocyst rate, and the blastocyst ICM, The mean cell numbers of TE and ICM + TE were significantly higher than those of 5 and 40 embryos. In different culture medium volume and WOW culture system, 5μl culture medium group was significantly lower than other groups in terms of embryonic development rate and mean cell number of ICM, TE and ICM + TE for blastocysts. There was no significant difference in the rate of embryo development between the 500 μl WOW culture system group and the 20 μl and 50 μl culture groups that obtained the higher developmental rates, while the blastocyst rate of 500 μl WOW culture medium and 20 μl of the culture medium group were not significantly different ICM, TE, and ICM + TE were significantly higher in cell numbers than 100 μl culture medium. Conclusion 20 l of culture broth droplet culture of 10 embryos can get a higher rate of embryonic development, and get a better embryo quality. Less volume of culture medium is not conducive to embryonic development, the more appropriate culture medium droplets volume of 20 ~ 50μl. At the same time, WOW training system achieved better embryo development under the experimental conditions.