自制载体冷冻小鼠原核期胚胎效果分析

Vitrification of Mouse Pronuclear Stage Embryos: Effect of a Newly Self-Made,Economical Loading Device

目的探讨自制冷冻载体冷冻保存昆明小鼠体内原核期胚胎的可行性。方法首先,比较了两种流行的商业化载体:开放式拉长麦管(open pulled straw,OPS)和冷冻帽(cryotop)开展小鼠原核胚玻璃化冷冻保存效果。其次,以cryotop为对照,利用自制简易载体(cryotip)开展小鼠原核期胚胎的玻璃化冷冻保存。之后,利用ANOVA对各组胚胎在复苏后的体外培养卵裂率、囊胚率进行统计分析。结果 OPS和cryotop两组之间,胚胎在玻璃化冷冻/复苏后发育的2-细胞率、4-细胞率和囊胚率差异均无显著性(P>0.05),但cryotop冷冻效果更接近对照组;cryotip玻璃化冷冻载体与cryotop相比,胚胎复苏后各组差异均无显著性(P>0.05),数值上除了2-细胞发育率外,cryotip其他几项结果都稍微高于cryotop组。结论 OPS,cryotop,cryotip冷冻保存昆明小鼠体内原核期胚胎均是可行的;cryotop在冷冻效果上要优于OPS,笔者自制的cryotip因其成本低,制作简单,操作安全可靠,在实验中替代昂贵的商业化载体OPS和cryotop是可行的。

Objective To explore the possibility of using a newly self-made loading device to replace expensive commercial carriers to vitrify Kunming white mouse pronuclear stage embryos derived by in vivo fertilization. Methods Firstly,the effect of two popular and commercial carriers： open pulled straw （OPS） and Cryotop,on in vitro development of mouse in vivo derived pronuclear embryos,subjected to vitrification and thawing,was examined. Secondly,with the Cryotop as control,the feasibility of using self-made loading carrier （ Cryotip） to vitrify mouse pronuclear embryos was evaluated. Data of cleavage and blastocyst formation were analyzed by ANOVA. Results The results showed that the rates of 2-cell embryo,4-cell embryo,and blastocyst in both OPS and Cryotop groups were similar （ P〉0. 05）,but the rates of cleavage and blastocyst formation in the Cryotop group were likely to be closer to that in the control group. The rates of each group were not significantly different between Cryotip and Cryotop groups （ P〉0. 05）,although Cryotop tends to have a higher rate in 2-cell embryo rate. Conclusion It is feasible to cryopreserve Kunming white mouse pronuclear stage embryos derived from in vivo fertilization with OPS,Cryotop,and Cryotip. The results of cryopreservation by Cryotop are likely better than that by OPS. In particular,our self-made novel loading device – Cryotip,is of good quality and lower cost, easier to make,may replace the expensive commercial carriers such as OPS and Cryotop,widely used in mouse embryo or oocyte vitrification practice.