Ana Laura Reyes-Ábalos, Bruno Barcelona, Nélida Rodríguez-Osorio, Francisco Báez
The zona pellucida (ZP), a viscoelastic extracellular matrix surrounding oocytes and preimplantation embryos, plays essential roles in embryo protection, embryo-environment interactions, and blastocyst hatching. Although ZP function depends on its structural organization, its remodeling during bovine preimplantation development remains poorly characterized, particularly in embryos produced in vitro. This study characterized stage-dependent changes in ZP morphology throughout bovine preimplantation development using scanning electron microscopy (SEM), confocal microscopy, and digital image analysis. Embryos at the 2-4-cell, morula, early blastocyst, and expanded blastocyst stages were processed for SEM. Calibrated images were analyzed in FIJI to quantify pore number, area, diameter, porous surface coverage, and pore shape descriptors including roundness and circularity. Embryo diameter was measured from low-magnification SEM images, whereas expanded blastocysts were additionally evaluated for ZP thickness and hatching-associated discontinuities. Quantitative analyses revealed progressive changes in ZP architecture throughout development. Compared with earlier stages, expanded blastocysts exhibited fewer pores, smaller pore areas and diameters, lower porous surface coverage, and more circular pore shapes, consistent with a transition toward a less porous and more homogeneous ZP structure. Expanded blastocysts also displayed heterogeneous ZP thickness, ruptures and rounded openings associated with hatching, together with abundant vesicle-like spherical structures within the exposed perivitelline region adjacent to these openings. Overall, this study provides the first quantitative SEM-based characterization of ZP ultrastructural remodeling during bovine preimplantation development and establishes an objective framework for future studies investigating the relationship between ZP architecture, embryo development, and hatching.