Effects of Trehalose and Sucrose, Osmolality of the Freezing Medium, and Cooling Rate on Viability and Intactness of Bull Sperm After Freezing and Thawing

The influence of the presence of trehalose or sucrose, the osmolality of the freezing medium, and the cooling rate on success of cryopreservation of bull sperm were investigated. Bull semen was frozen at four different cooling rates varying from 40 to 300 degrees C/min in standard Tris-egg yolk medium and in hypertonic or isotonic media containing 0.2 M trehalose or sucrose. Sperm motility, H33258 exclusion, and acrosome morphology were evaluated. In the hypertonic media the motility was almost completely inhibited, but could partly be restored by returning to isotonic conditions. Medium composition and cooling rate had significant effects, with a strong interaction of medium x cooling rate. The optimal cooling rate was found to be between 76 and 140 degrees C/min. At the highest cooling rate, both the presence of the sugars and the high osmolality of the hypertonic media conferred significant protection against fast-cooling damage, sucrose being significantly better than trehalose. At all other cooling rates the medium differences were less pronounced, but the isotonic sugar media were generally superior to standard medium as to preservation of acrosome intactness and H33258 exclusion. This indicates that the isotonic sugar media could enable some improvement of cryopreservation of bull semen over the currently used Tris-egg yolk medium. Further improvement of sperm survival at the AI station should be possible by implementation of controlled rate freezing, using a cooling rate in the optimal range between 76 and 140 degrees C/min.