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Male and female bovine embryos show several differences as early as the blastocyst stage. For example, differences are observed in metabolism, developmental kinetics, or gene expression that can lead to a shift in the sex ratio. Interestingly, the culture medium differentially affects male and female embryos. We previously showed that male Day 7 blastocysts present lower apoptotic rates than females (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The objective of the present study was to determine if such difference might be related to a differential sensitivity to oxidative stress, known to increase apoptosis in bovine blastocysts. In vitro-produced embryos were cultured in a SOF-based medium containing 0.4% BSA. At Day 5 post-insemination (pi) all the embryos were transferred in drops containing the same culture medium supplemented or not with 1 mM 2,2′-azobis(2-amidinopropane) dihydrochloride, an inducer of reactive oxygen species. Blastocysts were collected at Day 7, and apoptosis was evaluated by an immunofluorescent staining of cleaved caspase-3 (8 replicates, n = 175). Total and apoptotic cells were counted using an epifluorescence microscope. As expected, embryos cultured under stress conditions from Day 5 pi presented a lower blastocyst rate at Day 7 (10.9 ± 1.0% v. 23.1 ± 1.9% for the control group; standard least squares, P < 0.0001). The stressed blastocysts also showed fewer cells (113 ± 3 v. 139 ± 4; P < 0.0001) and higher apoptotic rates (15.3 ± 0.9% v. 9.4 ± 0.6%; P < 0.0001). As previously observed, the mean total cell number of the blastocysts was higher for males than females, whatever the culture condition (stress: males: 119 ± 4, females: 108 ± 4; control: males: 144 ± 5, females: 131 ± 6; sex effect: P = 0.005; interaction of sex × condition: P = 0.9). Interestingly, the sex ratio of the blastocysts was significantly different between control and stress conditions (χ2, P = 0.02); whereas a deviation in favour of the male embryos was observed in the control group (males: n = 57, 61%, females: n = 37, 39%; P = 0.04), it disappeared when embryos were submitted to oxidative stress (males: n = 35, 43%, females: n = 46, 57%; P = 0.22). However, oxidative stress had a similar impact on male and female blastocysts regarding the apoptotic rates (stress: males: 15.3 ± 1.3%, females: 15.4 ± 1.3%; control: males: 8.9 ± 0.7%, females: 10.2 ± 1.1%; standard least squares, sex effect: P = 0.99; interaction of sex × condition: P = 0.3). In conclusion, female embryos seem more resistant to oxidative stress than male ones when the stress is induced from Day 5 pi. Oxidative stress has a similar impact on the apoptotic rates in male and female blastocysts. The higher rate of apoptosis previously observed in female blastocysts can thus not be explained by a higher sensitivity of female embryos to oxidative stress. This is in accordance with the higher level of expression of several X-linked genes related to antioxidant pathways in female blastocysts.

Several studies have demonstrated differences in developmental rates and metabolism between bovine female and male embryos after IVF. Such differences seem related to the activity of both X chromosomes in female embryos up to the blastocyst stage and can lead to a shift in sex ratio. Developmental differences between male and female embryos are influenced by culture conditions. The objective of this study was to evaluate developmental and apoptotic rates of male and female bovine embryos in 2 SOF-based culture media, one with 5% FCS and the other with 4 mg mL–1 of BSA. Sex-sorted semen of one bull was used to produce cohorts of embryos of the desired sex. In preliminary experiments, IVF procedures were adapted to the use of sexed semen, and the purity of the sexed semen was verified through embryo sexing. The levels of apoptosis were assessed in Day-7 blastocysts using 2 techniques on the same embryos: TUNEL and detection of cleaved caspase-3 by immunostaining (caspase staining). Analysis by confocal microscopy and subsequent 3-D reconstruction allowed a precise cell count. A higher blastocyst rate on cleaved embryos was observed at Day 8 post-insemination for male than for female embryos in both media (BSA: male: 36.7 ± 4.0%, female: 28.6 ± 3.7%; FCS: male: 41.7 ± 2.9%, female: 31.7 ± 4.7%; ANOVA 3, P = 0.01). No significant difference in cell number was observed between male and female blastocysts (BSA: male: 188 ± 9, female: 170 ± 9; FCS: male: 186 ± 6, female: 177 ± 7; ANOVA 3, P = 0.14). In both media a higher proportion of cells showing caspase staining was observed in female than in male embryos (BSA: male: 7.3 ± 1.3%, female: 9.4 ± 2.1%; FCS: male: 9.2 ± 0.6%, female: 14.2 ± 1%; ANOVA 3, P = 0.01), whereas the proportion of stained cells was higher in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.02). The same tendency, although not significant, was obtained for the proportion of cells showing TUNEL staining with higher values in female than in male embryos (BSA: male: 9.3±2.1%, female: 10.5 ± 2.6%; FCS: male: 13.1 ± 0.9%, female: 16.5 ± 1.1%; ANOVA 3, P = 0.07) and higher values in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.05). A tendency for a higher proportion of double-stained cells (TUNEL and caspase-positive) was also observed in female embryos whatever the medium (BSA: male: 3.8 ± 1.1%, female: 5 ± 1.6%; SOF: male: 4.8 ± 0.5%, female: 7.0 ± 0.7%; ANOVA 3, P = 0.08). Intriguingly, only about half of the stained cells showed the double staining (TUNEL and caspase). This could be explained by the fact that caspase-3 activation can appear before DNA fragmentation during the apoptosis process and caspase staining disappear when TUNEL staining is still visible. But both caspase-3 activation and DNA fragmentation can also occur independently of apoptosis. In conclusion, male embryos seem to show a higher developmental rate in both media and could be less affected by apoptosis than female ones, particularly when cultured with FCS. Those experiments have to be repeated with the sexed sperm of another bull to draw final conclusions.