Alteration of sex ratio and DNA integrity of bovine sperm centrifuged in continuous density gradients

Abstract

The objective of the present study was to verify the sex ratio and presence of DNA fragmentation by TUNEL technique (In situ terminal deoxinucleotidyl transferase mediated dUTP nick end labeling assay) in bovine spermatozoa centrifuged in density gradients of Percoll or OptiPrep during the sperm separation. Approximately 40 million of frozen/thawed bovine spermatozoa were deposited on each density gradient composed of Percoll or OptiPrep with three layers ranging from 1.110g/mL to 1.123g/mL in polystyrene tubes of 15mL. The tubes were kept at 4°C for 24h before the deposition of sperm. Centrifugation of the tubes was performed at 500xg for 15min at 22°C. Supernatants were aspirated and the sediment recovered for verification of DNA fragmentation by TUNEL technique. A deviation of in vitro produced embryos for females in Percoll gradient (62% females) was obtained in relation to OptiPrep and Control groups, (47.1 and 48.7% of females, respectively). No sperm DNA fragmentation was detect in centrifuged samples (Percoll or OptiPrep gradients). Thus, it was possible to sexing sperm, obtaining a higher percentage of X sperm than the control group, using a methodology that is simple and without damage to the sperm DNA.