Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm

Abstract

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22–24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 μg/ml and three sperm concentrations: 0.5×10 6, 1.5×10 6, and 4.5×10 6 ml −1; 10 μg/ml of heparin and 1.5×10 6 ml −1 of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52–54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18–20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased ( P<0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm ( P<0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5×10 6 ml −1) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 μg/ml) and sperm concentrations (4.5×10 6 ml −1). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.