一种改进的小鼠无透明带胚胎培养方法

摘要：为了探索一种高效的无透明带胚胎培养方法，为手工核移植提供技术支持，本研究以小鼠胚胎为材料，对比了现有的3 种无透明带胚胎培养方法，对其中效果较好的WOW法进行改进，以海藻酸钙凝胶包埋小凹并且尝试与共培养技术相结合。结果显示，海藻酸钙凝胶包埋小凹法（AEW法）的囊胚发育率（54.7 %）和囊胚细胞平均数（54.3）显著高于微滴单卵培养法（MDS法）和琼脂柱包埋培养法（AE法）（p＜0.05），与WOW法相当（p>0.05）；使用AEW法进行培养时，最佳的海藻酸钠及钙离子浓度分别为0.7 %和 1.5 %；该法与颗粒细胞共培养相结合后，胚胎卵裂率（89.7 %）与囊胚发育率(67.9 %)显著高于对照组（p＜0.05）。实验结果表明，AEW法培养无透明带胚胎的效果优于其他几种方法；AEW法与颗粒细胞共培养相结合可以大大提高无透明带胚胎的胚胎卵裂率及囊胚发育率，便于无透明带重构胚的大批量培养。

An Improved Method for The Culture of Mouse Zona-Free Embryos

Abstract: To search for an effective culture method for zona-free embryos and provide technical support for handmade cloning, this study displayed three methods for the culture of zona-free embryos and improved the more effective well of the well (WOW) culture method that embedded the well with calcium alginate gel and tried to combine with the co-culture technology. The results indicated that the blastocyst development rate(54.7 %) and the average cell numbers of blastocysts (54.3) culturing with calcium alginate gel embedding well(AEW) culture method was significantly higher than culturing with the micro-drops single culture method and agarose-pillar embedding culture method ( P＜0.05) and there was no significant differences (P＞0.05) with the WOW culture method(P＞0.05); This study also found the cleavage rate and the blastocyst development rate culturing with this new culture method were no significant differences(P＞0.05) when the concentration of the sodium alginate was used between 0.5 %～1.0 % and the concentration of the calcium ion was used between 1.0 %～2.0 %, and the optimal concentration of the sodium alginate and calcium ion were 0.7 % and 1.5 %; The study also found the cleavage rate(89.7 %) and blastocyst development rate (67.9 %) culturing with the AEW culture method combining with co-culture technology was significantly higher than culturing with the AEW culture method alone (P＜0.05). The whole results indicated that the effect of AEW culture method on culturing zona-free mouse PN-stage embryos was better than the other culture methods; this method combining with granule cells co-culture technology could greatly raised the cleavage rate and blastocyst development rate for the culturing of zona-free embryos..In conclusion,this method is suitable for the batch zona-free cloned embryo production.