In vitro embryo production alters spatial organization of mesodermal and endodermal markers in elongating bovine...

In brief: Embryo production technologies can affect early developmental processes critical for pregnancy establishment in cattle. This study reveals that in vitro-derived embryos exhibit impaired elongation and altered lineage patterning compared to those derived from artificial insemination. Abstract: This study aimed to characterize and compare cell lineage specification in the embryonic disc (ED) of elongating bovine conceptuses derived from artificial insemination or from in vitro-produced embryos, including fresh and cryopreserved blastocysts. Conceptuses were recovered on day 16 of gestation, measured, and EDs were dissected for whole-mount immunofluorescence using markers of epiblast, mesoderm, and endoderm lineages. Image analysis was performed to assess disc morphometry, epiblast cell number, and spatial organization of lineage-specific markers. Conceptuses derived from artificial insemination were longer and exhibited larger EDs compared to those derived following transfer of in vitro-produced embryos, particularly vitrified embryos, which showed reduced elongation and altered disc morphology. Epiblast cell numbers were lower in vitrified conceptuses but scaled proportionally with disc size across all groups. The initiation of gastrulation and hypoblast differentiation had occurred in most conceptuses, regardless of origin; however, in vitro-produced conceptuses displayed altered spatial organization of mesodermal and endodermal markers, with less defined patterning compared to artificial insemination-derived conceptuses. These findings indicate that while key developmental processes are preserved, in vitro embryo production, especially when combined with cryopreservation, is associated with impaired elongation and disrupted spatial organization of cell lineages during early development. Such alterations may contribute to reduced developmental competence and increased embryonic loss.