Abstract
The present study was designed to examine the effects of several factors on the survival of mouse embryos frozen rapidly by liquid nitrogen vapour based upon the two-step procedure. The mouse morulae in 2 M glycerol were frozen by direct transfer into liquid nitrogen vapour at -170-180°C after seeding at -7°C and then being plunged into liquid nitrogen 1015 min later. Frozen samples were thawed rapidly by agitating the tubes in a 3840°C water bath. The frozen-thawed embryos were placed in PBS containing 2 M glycerol plus 0.5 M sucrose and then placed in PBS containing 0.5 M sucrose each for 3 min at room temperature, followed by two washes in fresh PBS. The morulae were then cultured for 4045 h to assess their ability to develop into blastocysts. 1. Glycerol (survival rate: 78%), propylene glycol (70%) and ethylene glycol (67%) were effective cryoprotectants and some pro-tection was also afforded by erythritol (53%). In contrast, the degree of protection given by DMSO was very low and no embryos survived freezing in the presence of methanol or sucrose. 2. A higher survival rate (81%) was found after exposure to glycerol at 0°C for 10 min prior to freezing than after exposure at 20 (41%) or 38°C (19%). 3. Exposure of embryos to 0.5 M sucrose in PBS for 5 min at room temperature prior to freezing that makes partial dehydration of blastomeres osmotically prior to freezing did not improve the survival of frozen embryos as compared to the survival of embryos un-pretreated with sucrose. 4. There was no difference in the survival rate between embryos where ice-seeding was induced at -7°C and embryos where ice-seeding was omitted. 5. The temperature (0, 20 and 38°C) at which glycerol was diluted with sucrose out of frozen-thawed embryos did not affected the survival rate. 6. Glycerol was diluted out of the frozen-thawed embryos by three methods at room temperature. The survival rate improved after glycerol-dilution with sucrose described previously (74%) as compared to the stepwise dilution (addition of 0.2, 0.2 and 0.4 ml PBS at 1-min intervals) (51%) or the direct dilution (direct transfer of embryos into PBS) (8%). 7. Although the survival rate (76%) of embryos frozen in glycerol rapidly by liquid nitrogen vapour appeared a little lower than that (89%) of embryos frozen slowly, the difference was not significant. In conclusion, a relatively high proportion of mouse embryos survived rapid freezing by direct transfor from 0 (without ice-seeding) or -7°C (with ice-seeding) to liquid nitrogen vapour at -170180°C before plunging into liquid nitro-gen. Further experiments are needed to determine the optimum conditions for freezing mouse embryos by using liquid nitrogen vapour.
