ZP-free enucleation for efficient haploid androgenetic embryo production and the identification of an activation-dependent barrier to hemiclone development in bovine

Abstract Purpose This study aimed to establish a simplified and efficient zona pellucida (ZP)-free protocol for bovine haploid androgenetic embryo (hAE) production and to identify molecular barriers during the embryonic genome activation (EGA) window that limit the development of reconstructed hemiclones. Methods hAE were generated by ZP-free enucleation of zygotes following short in vitro fertilization (IVF). Haploidy was confirmed by chromosome analysis and commercial microsatellite profiling. Hemiclones were produced by fusing hAE blastomeres with mature oocytes followed by activation (FA) or with pre-activated oocytes (AFA). Gene expression of markers regulating chromatin status, RNA processing, metabolic activity, and pluripotency was evaluated by qRT-PCR at the 8–16 cell and blastocyst stages. Results ZP-free enucleation simplified nuclear identification by removing adherent sperm, yielding 76.9% haploid blastomeres. Microsatellite analysis provided complete monoallelic profiles in blastocysts, validating the platform for pre-zygotic genetic screening (PZGS) using standard market tools. Hemiclone blastocyst development was markedly lower in both FA (4.6%) and AFA (1.2%) groups compared to parthenogenetic (PA) control (23.4%). Molecular analysis at the 8–16 cell stage revealed that artificial activation (FA and PA) disrupts EGA programming and metabolic status compared to naturally activated groups (hAE and IVF). Conclusion ZP-free enucleation provides a scalable and efficient platform for bovine hAE production and practical PZGS. While the altered gene expression resulting from artificial activation may have detrimental consequences for development, the marked developmental disparity between FA and PA embryos suggests that inherent constraints of the hemiclone condition represent the primary barrier to developmental competence.