Studies on development of a simplified rapid freezing method for preservation of farm and small animal eggs

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1. When pig oocytes at the germinal vesicle stage without or with 0.5 M sucrose were cooled at 0.5゜C/min to 0, 10, 20 and 30゜C (control), the proportion of oocytes matured to metaphase II during culture was 0, 0, 16-21 and 46-52%, respectively. After oocytes were cooled 3゜C/min to 20゜C, none reached metaphase II during culture. Addition of 0.5 M sucrose and cryoprotectants (DMSO, glycerol and ethylene glycol) had no apparent effect on survival of cooled oocytes. These results show that pig oocytes were all killed when cooled to temperatures of 10 or 0゜C. In conclusion, pig oocytes appear to be extremely sensitive to cooling and there are differences between species in the responses of oocytes to cooling.2. Mouse morulae were frozen rapidly to -196゜C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7゜C after seeding into liquid nitrogen vapour at -170--180゜C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M glycerol, 2 M propylene glycol, 2 M ethylene glycol; 5-30 min equilibration time at 0゜C; 3-60 holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. The present freezing procedure provides a quick and easily performed technique for preserving mouse embryos with a minimum of specialized equipment and makes it possible to freeze a large number of embryo samples in a short time. The present liquid nitrogen vapour method, therefore, may be applied to the embryo preservation of other species.