The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO₂ in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O₂, 5% CO₂ at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.