180 SPERM SELECTION OF STALLION PONIES THROUGH GLASS WOOL

Abstract

Two techniques of sperm concentration (centrifugation or filtering) and sperm separation technique with glass wool were applied to the sperm samples collected from 3 pony stallions (6 ejaculates; 2 from each stallion). Ejaculates were extended to a final concentration of 50 × 106 spermatozoa mL–1 using a nonfat dry milk-based extender and evaluations occurred at 24, 48, and 72 h after immediate ejaculate dilution and cooling. Each stallion was considered as a block, and semen from each stallion was assigned to 4 treatments: Group A (control): extended semen alone; Group B: extended-centrifuged semen; Group C: extended-sperm filtered semen; Group D: extended-glass wool-separated semen. All groups were tested for pH, osmolarity, motility, morphology, membrane functionality (hyposmotic), and cell viability (MTT assay). The experimental design was performed using a split-plot model. Data analysis at the level of 5% was performed using ANOVA and Bonferroni as post-hoc test. Data are presented as mean ± standard error. Group D had the highest rate of viable cells (P < 0.05) after the separation procedure (Table 1). Group B had a higher percentage of cells with tail defects after processing compared with the controls and Groups A, C, and D (P < 0.05). More than 60% of the cells retained on the filter showed defects (P < 0.001). Progressive motility was greater in group D at 0, 24, and 48 h (P < 0.05). Seventy-two hours after cooling, motility in groups A and B was lower than in Group D (P < 0.01). Group D showed a higher number of cells with mitochondrial activity during the cooling period. In conclusion, the technique of sperm selection by gravity using a glass wool filter resulted in an increased number of viable sperms after cooling pony semen for 24, 48, and 72 h. Table 1.Effect of sperm concentration and separation techniques on mean ± standard error percent of intact sperm from 3 stallions ponies (2 ejaculates/pony) stained with eosin-nigrosin