Freezing Morula Stage Rabbit Embryos

Abstract

Optimization procedures were utilized to determine the levels of five factors that maximized survival and development of frozen and thawed morulae collected from superovulated Dutch-Belted rabbits. The factors were 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 found to be 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° C/min cooling rate; −100° C to stop slow cooling and 17° C/min warming rate. Through use of these values, 83 percent of frozen and thawed morulae developed into blastocysts in vitro.