89 Influence of sperm preincubation on development and sex ratio of in vitro-produced bovine embryos

The sex ratio of female and male in vivo-derived bovine embryos and newborn calves is nearly 1:1. However, in vitro-produced embryos tend to shift towards more males. The aim of the present study was to investigate whether a bull-specific sperm preincubation before in vitro fertilisation may influence embryonic development and embryonic sex ratio. Cumulus–oocyte complexes (COC) from abattoir-derived ovaries were in vitro-matured for 24 h. For each of the three bulls (bull A, B, C) used for IVF, capacitation time was assessed by chlortetracycline staining. Therefore, 90 (bull A) or 180 (bull B, C) min before IVF, cryopreserved/thawed sperm was prepared using a standard protocol and preincubated in FertTALP. This preincubation step was omitted for sperm of all three bulls serving as control. After 19 h of IVF, the fertilisation rate was analysed via Hoechst staining. Presumptive zygotes were denuded and placed in synthetic oviductal fluid with amino acids (SOFaa) for in vitro culture up to Day 8 (IVF = Day 0). At Days 7 and 8, cleavage and developmental rates were recorded, and non-expanded blastocysts (BL) and expanded blastocysts (ExB) were collected for sexing via PCR. Live and dead cells of Day 8 embryos (BL + ExB) were counted after Hoechst 33342 and ethidium homodimer staining. Data were analysed by ANOVA followed by a Tukey test (sperm parameter), a t-test (IVP data), or chi-squared test (sex ratio). A mean of 1335 COC were used per treatment for each bull. Preincubated sperm showed a decreased motility (28.3%, 29.0%, 28.0%) compared to the control for that bull (63.3%, 65.0%, 64.1%) and increased capacitation patterns (31.9%, 33.3%, 35.2% vs. 24.8%, 25.6%, 25.4%; P < 0.001). Fertilisation rate (70.0%, 72.5%, 68.0% vs. 70.0%, 72.5%, 66.0%), developmental rate (28.9%, 25.6%, 24.2% vs. 27.1%, 27.5%, 27.0%) and ratio of live/dead cells in Day 8 BL and ExB (22.0, 19.0, 20.0 vs. 22.2, 21.8, 23.0) were not affected by sperm preincubation within bull (P > 0.05). For bull A, cleavage rate was higher after IVF with preincubated sperm than in control (82.6%, vs. 77.8%; P < 0.05) and, at Day 7, more hatching blastocysts were detected in the preincubated group (2.4% vs. 0.0%, P < 0.05). The sex ratios for bull A did not differ from the control (60.0% vs. 61.1% male embryos). For bull B, the overall sex ratio was skewed towards male in the preincubated treatment (91.7% vs. 66.7% for control; P < 0.05). For bull C, interestingly, the overall sex ratio did not differ from control (54.5% vs. 48.0% males); however, the sex ratio of the different subgroups was 50.0%, 58.3%, 50.0%, and 57.1% for Day 7 embryos (BL + ExB), Day 8 (BL + ExB), BL (Day 7 + Day 8), and ExB (Day 7 + Day 8), respectively, compared to 33.3%, 90.9%, 90.0%, and 37.5 respectively for control. The sex ratios of bull C Day 8 embryos and of BL differed significantly between treatments (P < 0.05). These data show that preincubation of sperm might have a bull-dependent effect on cleavage rate, proportion of hatching blastocysts at Day 7, and sex ratio of the resulting embryos.