Preliminary studies for elaborating a semen cryopreservation protocol of the Hungarian Giant rabbit

It is well established that semen freezability varies considerably among rabbit breeds and lines, therefore, breed-specific cryopreservation protocols are required. In the study, three experiments were conducted. Semen quality was assessed based on sperm motility, the proportion of live/dead cells and morphological abnormalities. In Experiment 1, modified Lake's, BotuCrio and INRAFreeze extenders were compared using a simple static cryopreservation protocol. Post-thaw sperm motility was significantly higher with modified Lake's and BotuCrio than with INRAFreeze. However, no significant differences were found in the proportion of live, intact cells, therefore, modified Lake's extender was selected for further studies. In Experiment 2, the timing of cryoprotectant addition was evaluated. In half of the samples, 8% dimethyl sulfoxide (DMSO) was added at the beginning of the 45-minute equilibration period, while in the other half it was added only during the final 5 min. Both the percentage of live, intact cells and total motility were significantly higher when sperm were exposed to the cryoprotectant for only 5 min. In Experiment 3, modified Lake's extender supplemented with 1% glycerol and 4% dimethylformamide (DMF) was compared with BotuCrio using a rapid freezing protocol. Sperm motility and the proportion of live, intact cells were significantly higher with the modified Lake's containing glycerol and DMF than with the BotuCrio. In conclusion, the most effective method identified in this study for rabbit semen cryopreservation was the use of modified Lake's extender supplemented with 1% glycerol and 4% DMF.