Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32/46) in Bull A and 54.2% (58/107) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 °C; (II) 30 to 80%, 10 °C; (III) 30 to 80% 20 °C; (IV) 30 to 90%, 20 °C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV)_66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.