Effect of Small Molecules on Blastocyst Development and Outgrowth Establishment of Bovine Haploid Parthenogenetic Embryos

Haploid embryos constitute a valuable model for genetic and epigenetic studies; however, their developmental competence is reduced compared with diploid counterparts. This study evaluated whether supplementation of the culture medium with specific small molecules could improve developmental competence and outgrowth establishment of parthenogenetic haploid embryos. The effects of TGF-β inhibition (A83-01), WNT pathway modulation (CHIR99021 and IWR-1), and activin A (AA) supplementation were assessed from the morula stage onward under serum-free conditions. A83-01 treatment did not improve blastocyst formation or morphology and was associated with reduced total cell numbers relative to IVF controls. CHIR99021 supplementation increased the number of SOX2-positive cells compared with IWR-1 and vehicle-treated embryos, suggesting partial support of pluripotency; however, overall developmental progression remained inferior to diploid controls. In contrast, activin A significantly increased the proportion of haploid morulae developing into blastocysts and improved hatching rates. Nevertheless, AA supplementation did not restore CDX2-positive cell numbers or total cell counts to diploid levels. Furthermore, neither CHIR99021 nor AA affect DNA fragmentation levels, although a tendency toward increased TUNEL-positive cells was observed. Activin A treatment also failed to improve embryonic outgrowth formation. Collectively, these findings demonstrate that although activin A enhances blastocyst yield and hatching in bovine haploid embryos, modulation of TGF-β or WNT signaling alone is insufficient to restore diploid-like proliferative developmental competence.