Freezing Morula Stage Rabbit Embryos

Abstract

Optimization procedures were utilized to determine the levels of 5 factors that maximized survival and development of frozen and thawed morulae collected from superovulated Dutch-Belted rabbits. The factors were dimethylsulfoxide (DMSO) concentration, equilibration time to the cryoprotective agent, cooling rate, temperature at which slow cooling was terminated and warming rate. DMSO concentration and equilibration time were the most critical factors, and cooling rate, the least important. After 10 experiments, which utilized 192 samples and 2164 embryos, the optimal values were estimated to be: 2.0 M DMSO; 10.7 min equilibration time; 1.1.degree. C per min cooling rate; -100.degree. C to stop slow cooling and 17.degree. C per min warming rate. Through use of these values, 83% of frozen and thawed morulae developed into blastocysts in vitro. To test survival in vivo of frozen and thawed New Zealand White and Dutch-Belted morulae, embryos subjected to these optimal conditions were transferred to 1 horn of Dutch-Belted and New Zealand White recipients, while the contralateral horn received nonfrozen morulae of the opposite strain. After freezing the percentage of embryos that implanted and the overall pregnancy rate were significantly (P < 0.05) reduced. New Zealand White recipients had higher pregnancy and implantation rates than the Dutch-Belted recipients, but embryo genetics had little apparent effect on these parameters. Twenty-six percent of the frozen and thawed Dutch-Belted morulae developed into viable fetuses, with no apparent developmental aberrations, when New Zealand White recipients were used.