2.4. In Vitro Embryo Culture

At approximately 18 h after IVF, presumptive zygotes were washed four times and transferred to four-well plates containing 25 μL droplets of IVC medium: SOF supplemented with 3 mg/mL of bovine serum albumin (BSA) under 750 μL of mineral oil (Nidacon, Gothenburg, Sweden). Embryos were incubated at 38.5 °C in 5% CO₂, 5% O₂, and 90% N₂ air with high humidity for 8 days post-insemination (dpi). Cleavage rate was evaluated at 48 h post-insemination (hpi), and blastocyst rate was recorded on 6, 7, and 8 dpi. All expanded blastocysts were washed three times in PBS supplemented with 0.1% w/v PVA (PBS-PVA) and either snap-frozen and stored at −80 °C until mRNA analysis or fixed in a 100 µL drop of glutaraldehyde solution (4% w/v in PBS, pH 7.4) at room temperature for cell-number evaluation.