不同温度条件下小鼠囊胚OPS法玻璃化冷冻保存技术的研究

本实验采用OPS法在不同温度条件下对小鼠囊胚实施冷冻保存,研究用EDFS和EFS溶液冷冻保存囊胚的效率和提供不同温度下筛选玻璃化溶液的依据,为家畜和人类胚胎的冷冻保存建立模型。25℃室温和37℃恒温台条件下OPS一步法冷冻保存小鼠囊胚,EFS40和EDFS40冷冻组扩张囊胚率(92.31%,92.30%)与对照(97.26%)均无显著差异(P>0.05),但EDFS40孵化囊胚率(59.62%)显著低于对照组(83.56%)(P<0.05);二步法冷冻结果显示,采用EDFS30和EFS40均能高效保存小鼠囊胚,解冻后扩张囊胚率(95.69%和95.05%)和孵化率(80.48%和78.95%)与对照无显著差异(P>0.05)。当改为25℃室温不使用恒温台条件下,一步法冷冻的胚胎解冻后,仅EDFS40冷冻组扩张囊胚率和孵化囊胚率(85.96%和75.44%)与对照(96.05%和82.89%)无显著性差异(P>0.05);实施二步法冷冻的胚胎,解冻后EDFS30,EDFS40和EFS40组均获得理想效果,扩张囊胚率(92.03%-95.31%)及孵化囊胚率(67.19%-76.76%)与对照均无显著差异(96.05%和82.89%)(P>0.05)。据体外发育结果,选择最佳冷冻组胚胎移植给假孕4d的受体母鼠,其妊娠率和产仔率(90.90%和37.33%)与新鲜胚对照组(91.67%和42.33%)无显著差异(P>0.05)。结果证实,EDFS30、EDFS40和EFS40三种冷冻液在不同的温度条件和采用不同冷冻程序,均能成功保存小鼠囊胚。

VITRIFICATION OF MOUSE BLASTOCYST BY OPEN PULLED STRAW (OPS) METHOD AT DIFFERENT TEMPERATURE

To examine the efficiency of EDFS and EFS for blastocyst cryopreservation and to provide information for preparation of vitrification medium at different temperature, the present investigation was conducted to establish a model for vitrification of domestic animal's blastocyst and human blastocyst using mouse blastocyst as a model and OPS (open pulled straw) as the container. When ambient temperature was 25 degrees C and heating plate temperature was 37 degrees C, mouse blastocysts were vitrified using 1-step OPS method, after thawing, the expanded blastocyst (EB) rates of EDFS40 and EFS40 group (92.31%, 92.30%) were not different from that of the control (97.26%) (P > 0.05), however, the hatched blastocyst (HB) rate of EDFS40 group (59.62%) was significantly lower than that of the control (83.56%); When 2-step method was adopted at the same temperature, mouse blastocyst was successfully cryopreserved with EDFS30 and EFS40, the post-thawing EB rate (95.69%, 95.05%) and HB rate(80.48%, 78.95%) of both EDFS30 and EFS40 groups did not differ from those of the control (P > 0.05). When ambient temperature was 25 degrees C and heating plate was free, the EB rate (85.96%) and HB (75.44%) of embryos vitrified with EDFS40 using 1-step OPS method were not statistically different from those of the control (96.05% and 82.89%, respectively) (P > 0.05); the EB and HB rates (92.03%-95.31%, 67.19%-76.76%) of embryos vitrified with EDFS30, EDFS40 and EFS40 using 2-step OPS method were similar to those of the control (P > 0.05). According to in vitro development of vitrified embryos, the promising group was chosen for embryo transfer to examine in vivo development of vitrified embryos. When vitrified blastocysts were transferred into uterus of pseudopregnant mouse, the pregnancy rate of receipts (90.90%) and birth rate of foster mother (37.33%) were similar to those of fresh embryos (91.67% and 42.33%) (P > 0.05). All data demonstrated that mouse blastocyst was successfully cryopreserved with EDFS30, EDFS40 and EFS40 at different temperature and using different procedures.