Effect of Single layer centrifugation using Bovipure on frozen-thawed bovine sperm during a 6 hours survival test

Aim of the study: to evaluate the functional parameters of bovine spermatozoa before and after separation by single layer centrifugation using Bovipure gradient. Material and Methods: Frozen semen from 8 bulls was used in this study. Thawed pellets were subjected to the separation process through gradient media according to the manufacturer’s instructions. Qualitative assessment of semen was performed at initial time (T0) and during a 6h survival test (T6) before and after gradient centrifugation preparation. Beside conventional examination, the functional parameters of sperm such as acrosome integrity (using Spermac Stain), membrane potential (JC 1 fluorescent dye) and DNA fragmentation (HalomaxBos Taurus) were evaluated. Results: An extremely high variability in percentages of sperm obtained after separation of thawed pellets was noted, the average sperm recovery (± SD) from thawed pellets after separation being 29.6 ± 13.9% of original sperm count. Sperm preparation by SLC selected functionally and morphologically high quality sperm compared to the control samples, the overall advantages of SLCselected samples over the control group being particularly evident during the 6 h survival test. Conclusions: This procedure selected spermatozoa that were motile, possessed good membrane integrity, high respiratory activity and additionally, maintain these characteristics during storage significantly better than control samples, prolonging its longevity.