Freezing of stallion semen with modified INRA-82 extender with two combinations of glycerol and dimethylformamide

The individual or combined use of cryoprotectants in the diluent for the freezing of equine semen is a determining factor in the post-thawing viability of spermatozoa. The objective of this investigation was to evaluate the effect of the modified INRA-82 diluent, supplemented with two combinations of cryoprotectant glycerol and dimethylformamide, on the post-thaw quality of equine semen. The semen was obtained from stallions of the following breeds; Argentinean Sport horses, Belgian horses, American Quarter Horses, Percheron, American Paint Draft horses, Zangersheide and Selle Francais breed located in the breeding facilities of the National Colombian Police (Facatativá-Cundinamarca). Each semen sample was divided into two equal parts that were afterwards diluted in BotuSemen® at 37 ° C. After two hours, each sample was centrifuged at 800g for 12 minutes and the pellet was resuspended in the modified INRA-82® diluent previously supplemented according to the T1 treatments: 2.5% dimethylformamide (DMF) and 2.5% glycerol (GLY) or T2: 4% DMF and 1% GLY, both at a concentration of 200 x 1 06 cells/mL. Samples were packed in 0.5 mL straws, which were then refrigerated at 5°C for 80 minutes followed by exposure to liquid nitrogen vapors for 15 min. The samples were stored in liquid nitrogen until thawed in water at 37°C. Post-thawing evaluation of sperm motility was performed through the Sperm Class Analyzer® computerized system; the vitality and morphology of spermatozoa were evaluated through Eosin-Nigrosin staining; the functional integrity of the membrane was measured by the HOST hyposmotic test and the structural integrity of the membrane was analyzed using fluorescent SYBR14 / IP probes. The statistical analysis was performed using generalized linear models and the comparison of means by the Tukey test. A higher proportion of morphologically normal sperm was observed for T1 (70.7%) in comparison to T2 (67.0%) (p