Selection for Developmental Speed Skews Sex Ratios and Metabolic Profiles in Bovine IVP Embryos

The developmental pace and sex ratio of preimplantation embryos have major implications for pregnancy outcome and herd replacement. We asked whether selecting embryos that develop faster preferentially captures distinct molecular and metabolic states and inadvertently affects the sex ratio in bovine in vitro produced (IVP) blastocysts. Hatching-stage blastocysts (pool of blastocysts undergoing and having completed the hatching process) developed on Day 7 and Day 8 of in vitro culture (IVC) were analyzed by RNA-sequencing, alongside assessments of lipid content, mitochondrial activity, and reactive oxygen species (ROS). Sexing was performed via Y-chromosome multiplex PCR, and candidate genes were quantified by qPCR in individually sexed hatching-stage embryos. RNA-sequencing identified 192 differentially expressed genes. Notably, significantly higher expression of pregnancy recognition factors (IFNT2, IFNT3) and the trophoblast marker TKDP1, along with glycolytic, lipid transport genes was observed on Day 8. In contrast, hatching-stage blastocysts from Day 7 displayed upregulation of oxidative phosphorylation genes. Mitochondrial activity and ROS levels were comparable between groups, but Day 7 embryos contained significantly more lipids. Interestingly, sexing analysis revealed a significant male bias among these blastocysts on Day 7 (66.7%) and Day 8 (58.5%). Candidate gene expression analysis revealed sexually dimorphic regulation of UQCRC2 and developmental day-dependent regulation of ENO1, UQCRC2, HSP90B1, and APOA1. Collectively, these findings indicate that hatching-stage blastocysts developed on Day 7 and Day 8 of IVC represent distinct physiological states and demonstrate that selecting embryos for rapid development can inadvertently skew sex ratios and molecular profiles in bovine IVP embryos.