Optimization of canine sperm cryopreservation by focusing on glycerol concentration and freezing rate

The purpose of this study was to improve the quality of frozen-thawed canine spermatozoa through the optimization of glycerol concentration (GC) and freezing rate in the semen freezing protocol. Ejaculates from nine dogs were diluted with an extender containing 0%, 1.5%, 3%, 6%, or 9% glycerol. The suspensions were loaded into 0.25 ml straws, frozen in nitrogen vapor in a closed box, and immersed in liquid nitrogen (LN₂). The freezing rate was controlled by setting the distance from the LN₂ surface to the straws as 1, 4, 7, or 10 cm. Firstly, freezing curves for each GC and freezing rate were analyzed. The analysis showed that the temperature of ice nucleation, freezing point, and immersion were changed with a certain trend depending on the GCs and freezing rates. Secondly, the sperm motility index (MI), viability and mitochondrial (MT) activity were evaluated. At 0 h after thawing, the MI was higher in the 3% and 6% GCs than the 0% GCs (P < 0.05). At 24 h, the 3% GC with 1 cm LN₂ distance (1 cm-3%) and the 7 cm-6% showed higher viability than the other conditions (P < 0.05), and the highest MT activity was obtained in the 1 cm-3%, which was higher than the other conditions (P < 0.05). The present findings indicate that the rapid freezing rate at 1 cm (average - 31 °C/min) with 3% GC provided the optimal condition in this study; use of this condition should reduce the detrimental damage to dog spermatozoa caused by ice crystal formation during freezing.