243 THE SEX OF THE BOVINE EMBRYO AFFECTS APOPTOTIC RATES AT THE BLASTOCYST STAGE

Abstract

Male and female pre-implantation bovine embryos may differ in several aspects such as kinetics of development, metabolism, or gene expression. These differences vary between culture conditions and may lead to shifts in sex ratio. In a previous study, we showed that female Day 7 blastocysts display a higher apoptotic rate than male ones when cultured in the presence of 5% FCS (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The difference was less important in the presence of BSA. This previous study was performed on in vitro-produced embryos produced with sexed semen and analysed using confocal microscopy. The objective of the present work is to confirm these differences using a) first, the unsexed semen of the same bull (bull 1) as the one used in the previous study in order to exclude any potential bias induced by the use of sexed semen and b) second, the unsexed semen of another bull (bull 2) in order to generalise our findings to the Bos taurus species. Levels of apoptosis were assessed in Day 7 blastocysts using immunohistochemical staining of cleaved caspase-3 and detection of fragmented DNA by TUNEL reaction on Day-7 blastocysts cultured with 5% FCS. Quantification of the number of stained cells was achieved with a fluorescence microscope. After cell counting, embryos were recovered and sexed by PCR. In both experiments, a higher proportion of cells showing caspase staining was observed in female (n = 145) than in male (n = 215) embryos (Bull 1: male: 11.8 ± 0.6%; female: 17.6 ± 1.1%, 2-way ANOVA, P ≤ 0.0001; bull 2: male: 9.5 ± 0.4%; female: 13.3 ± 0.6%, P ≤ 0.0001), whereas the proportion of TUNEL-stained cells was only significantly higher in female embryos produced with the semen of bull 2 (bull 1: male: 10.8 ± 0.4%; female: 11.4 ± 0.6%, P = 0.12; bull 2: male: 7.2 ± 0.3%; female: 9.1 ± 0.4%, P = 0.0009). A significant difference in cell number was observed between male and female blastocysts produced with the semen of bull 2 (male: 172 ± 5; female: 154 ± 5, P = 0.02) and the same tendency was observed for embryos generated with the semen of bull 1 (male: 143 ± 4; female: 132 ± 4, P = 0.07). In conclusion, our study demonstrated that the use of sexed semen does not interfere with the pattern of caspase and TUNEL staining previously observed. Moreover, a similar pattern was observed with 2 different bulls. We can thus conclude that the level of apoptosis of bovine Day 7 blastocysts produced in the presence of FCS is higher in female than male embryos. This could be related to the tendency towards a lower cell number in female blastocysts and to the shift in sex ratio in favour of male embryos often observed in the presence of serum.