Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Development of Effective Cryopreservation Method for Mammalian Embryo

포유류 초기 배아의 효율적인 동결 보존 방법에 관한 연구

  • Kim, Hyun (Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo) ;
  • Cho, Young Moo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Sung Woo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Seong, Hwan-Hoo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Yamanouchi, Keitaro (Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo)
  • 김현 (일본 동경대학교 수의과학대학 수의생리학 교실) ;
  • 조영무 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 야마노우치 케이타로 (일본 동경대학교 수의과학대학 수의생리학 교실)
  • Received : 2014.09.16
  • Accepted : 2014.09.26
  • Published : 2014.09.30
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Abstract

This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p<0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p<0.01, p<0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

Keywords

References

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