Abstract 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.
