7 EFFECT OF DENSITY GRADIENT CENTRIFUGATION WITH TRYPSIN ON THE FERTILIZING CAPABILITY OF BOVINE SPERM

The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.