不同的胚胎密度、培养液体积及微孔培养对小鼠早期胚胎发育的影响

摘要

ObjectiveTo optimize culture system of early embryonic development and improve development rate and quality, the effects of different embryo densities, culture medium volumes and well of the well (WOW) on early mouse embryonic development were discussed.Methods (1) 5, 10, 20 and 40 2-cell embryos were cultured in 20μl culture droplets covered by paraffin oil and 8-cell embryo, blastocyst and hatched blastocyst rates were counted. (2) 15 2-cell embryos were cultured in 5, 10, 20, 50, 100μl culture droplets and 500μl WOW system (one embryo was placed in one WOW) and 8-cell embryo, blastocyst and hatched blastocyst rates were counted. (3) Resultant blastocysts were stained by differential dyeing and were counted.ResultsCompared to other groups, 10 2-cell embryos cultured in 20μl droplet got higher 8-cell embryo, blastocyst and hatched blastocyst rate, and the resultant blastocysts had more mean cell number of ICM, TE and ICM+TE than 5 or 40 embryo group. In different medium volume and WOW system, 5μl medium volume got lower developmentrates than other groups in each embryonic stages and the resultant blastocysts had lower mean cell number of ICM, TE and ICM+TE. There was no significant difference between 500μl WOW culture system and 20μl or 50μl medium groups which got high developmental rates while the blastocysts of 500μl WOW system and 20μl medium group got higher mean cell number of ICM, TE and ICM+TE.Conclusion10 embryos cultured in 20μl medium had higher developmental rates compared to others and the resultant embryos had better quality. Less culture volume was not conducive to embryonic development and most appropriate volume of liquid droplets was 20-50μl. Meanwhile, WOW culture system under our experimental conditions got good embryonic development rates.%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。