Freezing of stallion semen: I - In vitro evaluation of motility and acrosin activity in sperm cells cryopreserved under different glycerol concentrations

The aim was to verify the efficiency of different glycerol concentrations (1%, 3%, and 5%) for cryopreservation of stallion semen. After semen collection, sperm cells were evaluated for motility and acrosin activity. The semen was divided into three equal parts and resuspended in freezing diluent without glycerol in a 1:1 proportion and were incubated for 20 minutes at room temperature. Immediately after centrifugation for 10 minutes at 600g and discarding the supernatant, each pellet was diluted with the same diluent and further centrifuged for 10 minutes. After the second centrifugation, each pellet received freezing glycerol-containing diluent under concentrations of 1%, 3%, and 5% and was loaded in 4mL macro-tubes. These macro-tubes were placed in liquid nitrogen vapor for 15 minutes for immediate immersion in liquid nitrogen and storage at -196 degrees C. After thawing at 50 degrees C for 40 seconds, semen samples were initially evaluated for sperm motility and acrosin activity. Moreover, semen was also incubated at 37 degrees C and evaluated for sperm motility after 60 and 120 minutes in a thermoresistance test (TRT). Immediately after thawing and during the TRT, both progressive and total sperm motility was lower in 1% glycerol (P < 0.05) than those frozen with 3 and 5% of this cryoprotectant, while there was no difference (P > 0.05) between these two later concentrations. The acrosin activity in fresh semen was only greater (P < 0.05) than those frozen with 1% glycerol that was also lower than both semen with 3% and 5% of this cryoprotectant, although there was no difference (P > 0.05) between these later glycerol concentrations. In conclusion, based on sperm motility and acrosin activity, that 3% and 5% glycerol concentrations are suitable for stallion semen cryopreservation.