Studies on the Survival and In Vitro Fertilization Rates of Frozne-Thawed Porcine Embryos and Immature Oocytes

돼지 수정란 및 미성숙 난자의 동결융해 후의 생존율에 관한 연구

In order to determine suitable conditions for rapid freezing of porcine embryos, the kind and concentration of cryoprotectants, sucrose concentrations, equilibration time and thawing temperature in freezing medium were examined in relation to the survival of frozen-thawed oocyte and embryos. The results obtained are as follows : 1. The suitable concentrations of cryoprotoctant in the freezing medium which consisted of TCM-199+20% FCS were 1.5M for glycerol, 2.0M for DMSO, 2.5M for ethylene glycol, and 2.0M for propanediol. The sucrose concentration of 0.25M in the medium was found to optimal because the survival rate was markedly higher at this concentration when compared to the others. The survival rate was relatively high when the frozen embryos were thawed at 30$^{\circ}C$ in the freezing medium containing 2.5M cryoprotectants. The equilibration periods of 2.0 and 5.0 minutes revealed the higher survival in the media containing 1.5 or 2.1M glycerol when compared to 10 and 15 minutes. 2. The fertilization rates of frozen-thawed follicular oocytes which matured in vitro for 1, 12, 24 and 48 hours were 6.7~26.7% depending on the maturation time, and the rates were relatively high for those matured for a short period of time. The survival rates of frozen-thawed oocytes which matured in vitro for certain periods and fertilized were 10.0~30.0% depending on the maturation time.

본 연구는 돼지 수정란의 동결에 있어서 내동제의 종류, sucrose 농도, 평형시간 및 융해온도가 생존율에 미치는 영향과 미성숙 난자의 체외배양과 동결융해 후 생존율과 체외수정율을 조사하고저 수행하였다. 1. 수정란의 급속동결에 있어서 1.5 M glycerol, 2.0 M DMSO, 2.5 M ethyleneglycol 및 2.0 M propanediol 농도에서 생존율이 유의하게 높았으며, 내동제에 첨가된 sucrose농도는 0.25 M 첨가가 다른 농도에 비해 생존율이 높게 나타났다. 동결수정란은 3$0^{\circ}C$에서 융해시 생존율은 33.3~40.6%로서 $25^{\circ}C$ 및 37$^{\circ}C$의 융해온도에 비해 높게 나타났으며, 동결시 1.5 M 및 2.0 M glycerol＋0.25 M sucrose가 첨가된 내동액으로 짧은 평형시간(2.0~5.0분)이 긴 평형시간 (10~15분)보다 높은 생존율을 나타냈다. 2. 미성숙란을 1~8시간 체외배양시킨 다음 동결융해 후 체외수정시켰을 때 수정율은 6.7~26.7%로서 단시간의 체외성숙 배양이 높게 나타났으며, 또한 체외배양시 생존율은 10.0~30.0%를 나타냈다.