Divergent molecular strategies underlie bovine blastocyst hatching and cryopreservation stress adaptation revealed by integrated multi-omics.

Cryopreservation tolerance and blastocyst hatching are two major challenges for successful embryo transfer. Whether embryos employ distinct molecular strategies to navigate programmed developmental transitions versus acute environmental stress remains unclear. To address this, we performed an integrated multi-omics analysis of bovine early (E_B), hatched (H_B), and vitrified-warmed hatched (V_B) blastocysts using Trio-seq2. This approach enabled simultaneous transcriptome and methylome profiling from the same embryo, allowing us to examine dynamics in gene expression, transcription factors (TFs), alternative splicing (AS), transposon mobility, and DNA methylation. Our results reveal a fundamental difference: blastocyst hatching entails global transcriptomic and epigenomic reprogramming, shifting from a proliferation-centered state toward metabolic activation. In contrast, cryopreservation induced minimal global gene expression changes but triggered widespread alterations in AS patterns and targeted DNA methylation adjustments, all while preserving core developmental programs. Collectively, these findings suggest that embryos may adopt distinct molecular regulatory modes-extensive epigenetic reprogramming for developmental progression and targeted post-transcriptional/epigenetic modulation for cryostress adaptation. The alternative splicing events and correlated methylation-expression relationships uncovered here could serve as potential molecular markers for evaluating bovine embryonic developmental potential and cryotolerance, and may offer clues for refining in vitro culture and cryopreservation protocols.