Motility and CASA parameters of thawed sperm of the Tobet canine breed after vitrification

Dogs of the unique and endangered indigenous national breed Tobet are on the verge of extinction. The cryopreservation of sperm via slow freezing and vitrification may be an effective approach to save these dogs. In this study, the effectiveness of different freezing rates for spermatozoa cryopreservation was examined. Ejaculates were obtained from 16 dogs of the Tobet breed. One part was frozen via slow freezing in nitrogen vapor at heights of 4 cm, 5 cm, and 6 cm above the liquid nitrogen level for 5-6 minutes, and the other part was subjected to programmed freezing in Slow Freezing Rate (SFR) and Fast Freezing Rate (FFR) modes. Vitrification was carried out by adding sucrose at different volumes and BSA to the vitrification solution. The results showed that during slow freezing, the most optimal cryoprotectant/freezing rate combination in nitrogen vapor was freezing 5 cm above the liquid nitrogen level, but the most optimal method was the use of a programmed freezer with SFR and FFR. Additionally, using a vitrification solution (sucrose 0.2 M + 1% BSA) without adding penetrating cryoprotectants also allowed for effective preservation of important sperm parameters and the successful cryopreservation of sperm from dogs of the Tobet breed.