Development in vitro of rabbit embryos after freezing by two-step or ultra-rapid cooling methods

The effects of rapid freezing by two-step and ultra-rapid cooling methods on 2-cell, 8 to 16-cell, compacted morula and early blastocyst stages of rabbit embryos were examined. Dimethylsulfoxide (DMSO, 3.5 mol/l) combined with sucrose (0.25 mol/l) was used as the freezing medium. The embryos were loaded into plastic straws with the freezing medium, held during 2.5 min and then were directly plunged into liquid nitrogen (ultra-rapid cooling) or after a time from 30 to 45 min held at -27 degrees C (two-step cooling). After rapid thawing embryo development was evaluated by in vitro developmental capacity shown by the embryos and was compared to control. All studied embryonic stages developed in vitro after two-step and ultra-rapid cooling procedures. However, higher developmental rates were obtained when the two-step cooling method was used. Using the two-step cooling method, best results were obtained at compacted morula and blastocyst stages, and no significant differences were shown when compared with control embryos at blastocyst stage. All embryonic stages to which the ultra-rapid cooling method was applied showed lower developmental rates than control. The results show that a high proportion of rabbit embryos can develop in vitro after freezing by two-step cooling method.