玻璃化冷冻方法三种冷冻载体冻存卵裂期胚胎效果的比较

Comparison of the cryopreservation effects of cleavage stage embryos with vitrification in three frozen carriers in mice

目的应用冷冻环、冷冻膜和自制麦管片三种冷冻载体,采用玻璃化冷冻方法冻存昆明小鼠卵裂期胚胎,比较三种载体的冻存效果。方法采集通过体外受精在体外培养第2天得到的4-8细胞期的昆明小鼠胚胎247个,对其中177个按载体不同随机分为冷冻环组59个,冷冻膜组59个,自制麦管片组59个;剩余的新鲜胚胎70个作为对照组,比较冻融后鼠胚的复苏率和囊胚形成率。结果冷冻环组、冷冻膜组和自制麦管片组的胚胎复苏率分别为98.3%、98.3%和94.9%,三组比较差异无统计学意义(P>0.05);冷冻环组、冷冻膜组和自制麦管片组的囊胚形成率分别是84.5%、86.2%和82.1%,三组比较无统计学差异(P>0.05);新鲜对照组囊胚形成率为94.3%,与冷冻环组、冷冻膜组分别比较无统计学差异(P均>0.05),与自制麦管片组有统计学差异(P<0.05)。结论采用玻璃化冷冻方法冻存小鼠卵裂期胚胎,使用冷冻环和冷冻膜作为冷冻载体具有复苏率高、囊胚形成率高的优点,更适合应用于临床。

Objective To compare the cryopreservation effects of cleavage stage embryos with vitrification in three frozen carriers including frozen ring （Cryoloop） , frozen membrane（Cryotop） and self-made straw in mice. Methods A total of 247 embryonic cells of Kunming mice in 4 - 8 cell stages got by fertilization in vitro and culture in vitro in the second day were collected. According to different carriers, 177 of 247 embryonic cells were randomly divided into three groups （n =59 each） ： Cryoloop group, Cryotop group and self-made straw group, and other 70 fresh embryos were served as control. The recovery rate of mice embryos and blastocyst formation rate after frozenthawing in three carries were compared with control group. Results The embryo recovery rates in Cryoloop group, Cryotop group and self-made straw group were 98.3% , 98.3% and 94.9% , respectively, and there was no significant difference in the three groups （ P 〉 0.05 ）. The blastocyst formation rates in Cryoloop group, Cryotop group and self-made straw group were 84.5% , 86.2% and 82.1% , respectively, and there was no significant difference in the three groups （P 〉0.05）. The blastocyst formation rate in control group （94.3%） was similar with Cryoloop group and Cryotop group （ all P 〉 0.05 ） but higher than that in self-made straw group （ P 〈 0.05 ）. Conclusions As carries for cryopreservation of the cleavage embryo of mice, all the Cryoloop and Cryotop have higher recovery rate and higher blastoeyst formation rate, and their cryopreservation effects are all superior to self-made straw.