Abstract
Various cryoprotectans and freeze-thawing procedures were examined for the preservation of rab-bit embryos. Rabbit morulae were recovered 60-65h after mating from females which had been in-duced to superovulate by injections of FSH and hCG. The morulae were suspended in 0.2ml PBS (supplemented with 20% rabbit serum) containing 1.5M-DMSO, -ethylene glycol, -glycerol or -ery-thritol in a glass test-tube at room temperature. The samples were cooled at 1C/min to -4C and were seeded. Tubes were then cooled either (a) slowly to -75C at 1C/min and then plunged into liquid nitrogen, or (b) rapidly by transfer into -20C ethanol, kept for 10min, suspended in nitrogen vapor (-100C) for 10min, and then plunged into liquid nitrogen. After storage for 10-150 days, these samples were thawed either slowly at room temperature or rapidly in warm water at 30-35C. The cryoprotectants were removed by transferring the embryos into PBS containing the cryoprote-ctant and 0.5M-sucrose, PBS with 0.5M-sucrose, and finally into fresh PBS. The embryos were cul-tured for 48h in a modified Tyrode solution containing 20% rabbit serum to determined their survival rate, which was assessed by the ability to develop to expanded blastocysts. The results obtained are as follows.
In the freeze-thawing procedures examined, the highest survival rates were obtained when embryos were frozen in the presence of DMSO, followed by ethylene glycol, and the third glycerol. Slow thawing of slowly frozen samples gave good survival rates of 70% in DMSO, 38% in ethylene glycol and 24% in glycerol. When slowly frozen samples were thawed rapidly, survival rates of embryos stored with DMSO and glycerol decreased. Survival rates of embryos frozen-thawed rapidly were lower than those of slowly frozen embryos.
When erythritol was used as a cryoprotectant, morphologically normal embryos were not obtained after freeze-thawing except for the case of rapid freezing, and no embryos developed in culture.
