초자화 동결법을 이용한 생쥐배아 발달단계별동결성적의 비교
Comparative study on the development of frozen-thawed mouse embryo using vitrification at each developmental stage
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목적 : 생쥐배아를 각 발달 단계별로 각각 초자화 동결과 완만 동결을 시행하여 융해 후 생존율과 배반포 발달율을 비교하여 가장 적절한 동결 시기와 방법을 알아보고자 하였다.연구 방법 : 총 1,437개의 생쥐 배아 (초자화 동결군 743, 완만 동결군 694)를 0.4% BSA를 포함한 T6 배지에서 각 발달 단계별 (1-, 2-, 4-, 8-세포, 상실기, 배반포)로 배양하여, 초자화 동결과 완만 동결을 시행하였다. 동결융해 후, 회수율, 생존율 및 배반포 발달율을 분석했다. 두가지 다른 동결법에서 단계별로 비교하였다. 통계분석은 Student's T-test를 이용하였다.결과 : 해동 후 두 동결군의 배아 회수율에는 큰 차이가 없었으나, 생존율 및 배반포 발달율은 초자화 동결군에서 의미있게 높았다. 초자화 동결군에서 생존율과 배반포 발달율은 상실배에서 각각 98.4% 및 86.4%로 가장 높았다. 완만 동결군에서 배아의 생존율은 상실배에서 87.2%로 가장 높았으나, 배반포 발달율은 8세포기 배아에서 78.1%로 가장 높았다. 이상의 연구결과로 배아 동결 보관시 두 동결군 모두에서 상실기 배아의 동결융해가 가장 적절한 것으로 나타났다. 8세포기에서의 배반포 발달율을 제외하고 모든 발달 단계에서 생존율과 배반포 발달율은 초자화 동결군이 완만 동결군에 비해 높음을 알 수 있었다.
Abstract
Objective : To find out the optimal freezing time in terms of developmental stage of mouse embryo and the effectiveness of vitrification method for freezing them. Methods : Superovulation induction was performed using PMSG and hCG. Total 1,437 mouse embryos (vitrification group: 743, slow-freezing group: 694) were obtained and cultured with the T6 containing 0.4% BSA medium. Each developmental stage of embryos (1-, 2-, 4-, 8-cell, morula & blastocyst) were cryopreserved by vitrification and also by slow freezing method for comparison of the results. After thawing, the recovery rate, the survival rate and the blastocyst developmental rate were analysed and compared in two different settings. Student's t-test was used for statistical analysis. Results : The survival and developmental rates at all subgroups of vitrification method are significantly higher than those of slow-freezing groups, but not in the recovery rates. In vitrification group, the survival rate and the blastocyst developmental rate are highest when frozen at morula stage, 98.4% and 86.4%, respectively. In slow-freezing group, the survival rate is also highest when frozen at morula stage, 87.2%, and the blastocyst developmental rate is highest when frozen at 8-cell stage, 78.1%. Conclusion : The vitrification method is more efficient for mouse embryo freezing compared with slow freezing one. Among various developmental stages of mouse embryos, morula stage seems to be the optimal stage for cryopreservation, whatever the freezing method applied. Therefore, we recommend embryo freezing at morula stage by vitrification method.
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