Optimization of One-Step Dilution Method of Vitrified Bovine IVM/IVF/IVC Blastocysts

초자화 동결된 체외생산 소 배반포기배의 1 단계 융해 방법의 적정화

This study was to establish an effective dilution technique in a vitrification of bovine blastocysts for the field trial. For vitrification, blastocysts were exposed in glycerol (G) and ethylene glycol (EG) mixture in m-DPBS supplemented with 10% FBS. Blastocysts were first exposed to 10% (v/v) G for 5 min, and subsequently were transferred to 10% G plus 20% EG (v/v) for 5 min. Finally, embryos were transferred to 25% G plus 25% EG (v/v) for 30 see and were placed in nitrogen vapor for 3 min, and then were plunged into L$N_2$. At thawing, the straw containing blastocysts was placed in air for 10 sec, and then plunged into a water bath at $25^{\circ}C$ until all ice had disappeared. They were placed in $25^{\circ}C$ and 36$^{\circ}C$ water according to treatment group for different time. Also, in vitro survival was assessed by the re-expansion and hatched rates at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) In the survival rates of vitrified bovine blastocysts according to different dilution time at thawing, the data of 1 min group (86.6, 56.6%) were higher than those of other treatment groups (2 min; 93.5, 35.4%, 2.5 min; 76.9, 30.7%, 3 min; 88.8, 36.1% and 3.5 min; 83.7, 8.1%). 2) When the in vitro survival of vitrified groups according to different developmental stage was examined at 48 h after thawing using 1 min dilution method, the hatching rates of fast developed embryos (expanded blastocyst: 81.3%: early hatching blastocyst: 86.2%) were higher than that of delayed developed one (early blastocyst: 46.6%). 3) In addition, when the in vitro survival of vitrified groups according to different embryo age was compared, the hatched rates at 48 h after thawing of Day 7 (66.6%) and Day 8 embryos (60.0%) were significantly higher than that of Day 9 embryos (22.7%) (P＜0.05). These results demonstrate that vitrified bovine IVM/IVF/IVC blastocysts can be successfully survived in vitro using one-step dilution (1 min) method.

본 실험은 초자화동결된 소 배반포기배를 실험현장에서 효율적으로 융해할 수 있는 기술을 찾고자 실시하였다. 초자화동결은 glycerol (G)과 ethylene glycol (EG) 그리고 10% FBS가 들어있는 m-DPBS를 이용하였으며, 배반포기배는 3단계로 초자화동결 되었는데, 10% G에 5분간 평형, 10% G와 20% EG에 5분간 평형, 그리고 25% G와 25% EG에 30초간 노출하였다. 질소 증기를 3분간 씌고 액화질소에 침지하였다. 융해는 straw 를 공기 중에서 10초간 노출시키고, $25^{\circ}C$ 물에서 빙정이 없어질 때까지 녹인 후 $25^{\circ}C$ 와 36$^{\circ}C$ 에 각각 시간차에 따라 처리군을 나누었다. 초자화동결된 배반포기배를 융해시 시간차에 따라 체외생존능은 융해 24시간과 48시간 후 재팽창과 완전탈출 배반포기배로 평가하였다. 그 결과를 요약하면 다음과 같다. 1) 초자화 동결된 배반포기를 융해시 시간차에 따라 체외생존농을 보았을 때, 1분으로 융해한 군이(86.6, 56.6%) 다른 처리군들보다 (2분 : 93.5, 35.4% ; 2.5분 : 76.9, 30.7% ; 3분 : 88.8, 36.1%; 3.5분 : 83.7, 8.1%) 체외생존능이 높게 나타났다. 2) 1분 융해방법으로 배반포기배의 발달단계에 따라 생존능을 조사하였을 때, 융해 48시간 후 빠르게 발달된 배반포기배의 부화율 (팽윤 : 93.8, 56.3% : 부화초기 : 86.2, 58.6%)은 느리게 발달하는 난자군의 부화율 (초기 : 83.3, 36.6%) 보다 높은 체외생존능을 나타내었다. 3) 또한, 1분 융해방법으로 배반포기배가 생산된 나이에 따라 체외생존능을 조사하였을 때, 융해 48시간 후, 7일 (66.6%) 과 8일 (60.0%)에 생산된 배반포기배가 9일 (22.7%)에 생산된 완전탈출 배반포기배율 보다 유의하게 높은 체외생존율을 나타내었다 (P＜0.05). 그러므로 초자화동결된 배반포기배를 1분 융해방법으로 융해하였을 때 빠르고 효율적으로 체외생존능을 얻을 수 있음을 알 수 있었다.