106 The sex ratio of bovine embryos using conventional semen

The objective of this study was to determine baseline sperm and embryo sex ratio in an in vitro embryo-production system using conventional semen. Cumulus-oocyte-complexes (COCs) were obtained from a local abattoir and matured in serum-free maturation medium for 22 h. Frozen spermatozoa from three Holstein bulls were used for this experiment. Once per bull, a semen sample was collected from the straw. Then, for each replicate of in vitro fertilisation, a post-density gradient sperm sample was collected for each bull to determine whether the gradient affected the sex ratio of the sperm used for fertilisation. All semen and sperm samples were washed three times using DPBS, and lysed using Lucigen lysis buffer and 500 µM DTT using the following protocol: 65°C for 1 h, 98°C for 2 min. For fertilisation, gametes were co-incubated for 18 h, then putative zygotes were denuded by vortexing. Putative zygotes were cultured using a two-step culture procedure, with a media exchange occurring 96 h post-fertilisation. On Day 8 post-fertilisation, twenty blastocysts, ranging from early to fully hatched, were individually collected per bull, in addition to twenty arrested embryos ranging from 3–16 cells. Each embryo was washed three times in DPBS-PVP, then lysed using Lucigen lysis buffer using the following protocol: 65°C for 15 min, 98°C for 2 min. This experiment was repeated three times. A dual priming oligonucleotide-based multiplex real-time TaqMan assay was utilised to identify the presence of the X and Y chromosomes in both sperm and embryo samples by amplifying X-specific PLP and Y-specific SRY. To determine the percentage of X and Y sperm, standard curves were created using male somatic cell DNA, and relative copy numbers for X and Y sperm were generated by calculating sample CT values from the standard curve. Data were analysed via one-way ANOVA using the PROC GLM procedure of Statistical Analysis Software v. 9.4. Differences in means were determined using the pdiff option of LSMEANS. All sex ratio results are presented as male:female. The pre-density gradient (n = 3) and post-density gradient mean sperm ratios (n = 9) were 1.7:1, and 2.3:1, respectively. There was no effect of the density gradient (P = 0.91) on the sex ratio of the sperm; however, there was a trend for the effect of bull (P = 0.0508). Day 8 arrested embryos (n = 157) were 1.6:1, and Day 8 blastocysts (n = 175) were 1.4:1. There was no effect of embryo stage (P = 0.78) or sire (P = 0.17) on embryo sex. In conclusion, for the three tested bulls, there was a bias toward male sperm in the semen straw, although statistically insignificant, and that bias was maintained throughout embryo culture. Therefore, the current media used for in vitro embryo production has no bias toward development of female or male embryos.