생쥐 수정란의 동결보존법을 개선하고자 유리화 동결과정에서 제1보존액에서의 평형시간, 제2보존액에서의 전냉각 여부 및 straw loading 방법에 따른 동결보존 후 수정란의 생존율을 조사하고, 또한 유리화 동결보존 과정에서 수정란의 부분적인 손상 여부를 확인하기 위하여 동결보존 후 배양하여 수정란의 할구수를 Hoechst 33342로 염색하여 조사하였던 바 그 결과는 다음과 같다. 1. 동결융해 후 생존율은 Medium-1에서 10분간 평형시간을 준 경우(81.0%)는 5분간(40.0%) 및 15분간(74.1%)의 평형시간을 준 경우보다 유의적(P<0.05)으로 높았다. 2. Double Medium-2 column방법으로 얻은 생존율(81.0%)은 single Medium-2 column방법으로 얻은 생존율(62.8%)보다 유의적(P<0.01)으로 높았다. 그리고 Double Medium-2 column방법에서는 유리화 용액이 희석액에 오염되지 않아 순수하게 투명한 유리화를 이룰 수 있었다. 3. 전냉각을 않은 경우에 비하여 전냉각한 Medium-2로 동결한 수정란은 생존율에 있어서 다소 높은 성적을 보였으나 유의적인 효과는 없었다. 4. 상실배기의 수정란을 24~28시간동안 배양하여 얻은 후기 배반포를 Hoechst 33342로 염색하여 할구수를 조사하였던 바 신선란(53.3${pm}$1.6)보다 동결란(41.4${pm}$1.5)에서 유의적(P<0.05)으로할구수가 적었다. 이는 동결융해 과정에서의 부분적인 손상에 의하였던 것으로 사료된다.
This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${pm}$1.6 vs 41.4${pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.
