Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

Abstract

The duration of sperm–oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm–oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1–3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P < 0.05), and the highest rate of normal fertilization was observed for 5 h culture (73.4%; P < 0.05). The sex ratio was significantly ( P < 0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1 h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P < 0.05) and decreased the proportion of HOST positive sperm (65.8–48.6%; P < 0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5–10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1 h (treatment 5) or 5 h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P < 0.05), and the 1 h co-incubation significantly skewed ( P < 0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5 h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P < 0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly ( P < 0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus–sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.