OPS两步法玻璃化冷冻小鼠8-细胞胚胎生存发育率的研究

Survival of Two-step Open-pulled Straw Vitrification of Mouse Eight-cell Embryos

目的为实验动物、家畜和人类胚胎的冷冻保存探索简易、高效的玻璃化冷冻程序。方法以小鼠8-细胞胚胎为材料,在25℃室温和37℃恒温台条件下,利用玻璃化冷冻溶液EDFS30, 对小鼠8-细胞胚胎进行玻璃化冷冻保存,解冻后培养60 h后的囊胚发育率为其体外发育能力的考核指标,同时对解冻后培养1-3 h的胚胎进行移植以判定其体内发育潜力。OPS(Open-pulled straw)两步法冷冻保存,即胚胎首先移入预处理液(10％EG+10％DMSO)中平衡30 s,再移入玻璃化溶液中吸入OPS平衡15、25、35、45和60 s连同胚胎直接投入液氮中冷冻保存。结果小鼠胚胎8-细胞冷冻后囊胚发育率最佳组高达95．6％,与对照组(96．8％)差异无统计学意义(P> 0．05)。利用最佳冷冻组获得的165枚胚胎移植于11只假孕63-67 h的受体母鼠,结果有4只妊娠,产仔23只,妊娠产仔率为38．3％(23/60),与对照组37．9％(22/58)差异无统计学意义(P>0．05)。结论 EDFS33为玻璃化冷冻液,OPS两步法能有效地冷冻保存小鼠8-细胞胚胎。

Objective To establish potentially an effective and easy method for the vitrification of embryos from laboratory animals, domestic animals and human. Methods The experiments were conducted at 37℃ hot plate and 25℃ room temperature to cryopreserve mouse eight-cell embryos with EDFS30 （an ethylene glycol-based vitrification solution） in Open-pulled Straw （OPS）. In the two-step OPS method, the superovulated embryos were first pretreated with 10% EG＋10% DMSO for 30s, then exposed to EDFS30 for 15s, 25s, 35s, 45s or 60s and then immersed in liquid nitrogen. After warming, the survival of embryos was assessed by their development to blastocysts or to term after transfer. Results The higher blastocyst rates of vitrified embryos were 95.6%, which were similar （P〉0.05） to that （93.0%） of control. The embryos derived from the best vitrified group or fresh eight-cell embryos （used as control） after culture for 1 to 3h were transferred to 63 to 67h pseudo-pregnant female mice. Twenty-three fetuses were obtained from 4 of 11 recipients which had received 165 vitrified-warmed embryos. In the pregnant recipients, the percentage of transferred embryos developed to young in the treated group （38.3%） and control （37.9%） showed no significant difference （P〉0.05）. Conclusion The mouse eight-cell embryos could survive cryopreservation by vitrification with EDFS30 in OPS.