EFFECT OF ANTIOXIDANTS (SODIUM PYRUVATE AND CATALASE) ON POST THAW MOTILITY, VIABILITY AND DNA INTEGRITY OF RAM SPERMATOZOA

Freezing of the semen in liquid nitrogen is the only method for long term storage of fertile spermatozoa. The procedure involves different steps which are harmful to spermatozoa and consequently reduce their quality and fertility. In many animal species the addition of antioxidants to semen before freezing was found to have positive effect on the quality and fertility of frozen/thawed (F/T) spermatozoa. As in rams there are contradictory results in the literature concerning the influence of specific antioxidants on sperm quality of F/T spermatozoa, the present study was performed to evaluate the effects of two different antioxidants on post thaw motility, viability and DNA integrity of F/T ram spermatozoa. Ejaculates from six crossbreed rams were frozen according to the standard procedure after two step dilution with modified Tris-egg yolk extender. The second extender, which contained 14% of glycerol, was added to the semen at 5 degrees C. Ejaculates were divided into three parts and extended with diluents that were either supplemented with 5 mM sodium pyruvate (group 1), 20IU/ml catalase (group 2), both antioxidants together (group 3) or contained no antioxidants (control group). Diluted samples were loaded into 0.5 ml straws and frozen in nitrogen vapour, 4 cm above the liquid nitrogen. Frozen samples were kept in liquid nitrogen for at least two month before thawing and analysis. Frozen straws were thawed in water bath at 37 degrees C for 17s. Motility and viability (Viadent (R)) of the F/T samples were analysed with Hamilton Thorne Biosciences, Version 12.3, after incubation in water bath at 37 degrees C for 10 min, 6, 12 and 24 h. The percentages of sperm showing a high DNA fragmentation (DFI%) of F/T spermatozoa was analysed 5 min and 3 h after thawing by using the flow cytometric sperm chromatin structure assay (SCSA (TM)). Statistical analyses were performed with Sigma Stat (Version 3.10). Statistical difference between the groups was tested with Kruskal-Wallis One Way Analysis of Variance on Ranks. Values were considered to be statistically significant when p < 0.05. There were no differences in percentages of motile (74.0 +/- 23.6% vs. 73.6 +/- 23.8 vs. 73.6 +/- 23.7% vs. 73.3 +/- 22.9% and 58.4 +/- 25.4% vs. 55.6 +/- 24.3% vs. 60.2 +/- 25.1 vs. 57.7 +/- 26.8% and 45.5 +/- 25.6% vs. 45.2 +/- 23.1% vs. 45.7 +/- 22.4 vs. 45.6 +/- 24.3% and 10.0 +/- 9.4% vs. 13.3 +/- 11.5% vs. 13.3 +/- 12.1 vs. 11.9 +/- 11.7% for group 1 vs. 2 vs. 3 vs. control respectively for 0, 6, 12 and 24 h after F/T) and viable spermatozoa (81.0 +/- 16.3% vs. 82.5 +/- 13.5% vs. 82.7 +/- 13.2 vs. 80.7 +/- 17.0% and 78.7 +/- 15.0% vs. 79.8 +/- 13.9% vs. 79.7 +/- 14.8 vs. 75.8 +/- 18.9% and 79.0 +/- 13.0% vs. 77.9 +/- 14.2% vs. 80.1 +/- 12.2 vs. 77.6 +/- 15.1% and 67.6 +/- 26.2% vs. 71.4 +/- 19.9% vs. 70.5 +/- 23.4 vs. 66.4 +/- 27.2% for group 1 vs. 2 vs. 3 vs. control respectively for 0, 6, 12 and 24 h after F/T) in groups with antioxidants in comparison to control group at 0, 6, 12 and 24 h (P > 0.44). Analysis of SCSA (TM) also revealed no difference (1.7 +/- 1.2% vs. 1.6 +/- 0.9% vs. 1.7 +/- 1.0 vs. 1.6 +/- 1.1% and 1.8 +/- 1.3% vs. 1.7 +/- 1.0% vs. 1.9 +/- 1.2 vs. 1.7 +/- 1.3% for group 1 vs. 2 vs. 3 vs. control respectively for 0 and 3 h after F/T) (p > 0.06) the p-value is rather low and indicates that there is somewhere a difference by trend (0.05 < p < 0. 10) n DFI%-values between groups with antioxidants compared to control group 0 and 3h after thawing. These results indicate no positive effect of pyruvate and catalase on motility, viability and chromosome integrity of F/T ram spermatozoa.