Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa

The objective of this study was to evaluate post-thaw quality of frozen dog semen processed with diluents containing different antioxidants. Ejaculates were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF movement), viability, acrosomal integrity and by the hypo-osmotic swelling test. Also, superoxide production, hydroxyl radicals and total reactive oxygen species (tROS) were determined. The pool was divided in seven aliquots, for control and test conditions, which were processed for cryopreservation. The sperm pellets were diluted to a final concentration of 200 × 106 sperm/ml with TRIS-glucose-egg yolk extender containing one of the following supplements: vitamin C (1.5 mM), NAC (N-acetyl-l-cysteine; 1.5 mM), taurine (0.6 mM), catalase (300 U/ml), vitamin E (0.3 mM) and B16 [5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid; 0.3 mM]. Post-thaw semen evaluation showed that mean (±S.E.M.) motility was increased (p < 0.001) after addition of catalase (49.75 ± 3.63 versus 39.00 ± 2.90 in controls), whereas more spermatozoa with RSF movement were observed (p < 0.001) after the catalase, NAC and vitamin E treatments (31.75 ± 3.46, 28.00 ± 3.27, 26.75 ± 3.15, respectively, versus 17.00 ± 2.26 in controls). Viability was increased (p < 0.001) after addition of catalase, taurine, NAC and tocopherol (66.00 ± 3.03, 61.90 ± 2.48, 60.60 ± 1.93 and 60.50 ± 4.12, respectively, versus 51.70 ± 2.81 in controls). The percentage of swollen spermatozoa was increased after addition of catalase and taurine (61.75 ± 1.61 and 61.25 ± 1.49, respectively, versus 55.65 ± 1.64 in controls). Acrosomal integrity was not influenced in any case. B16 addition had adverse effects on all parameters evaluated. None of the reactive oxygen species were significantly reduced post-thaw in antioxidant treated semen. The results suggest that catalase had the most pronounced effect in improving post-thaw quality of canine spermatozoa.