Ultrastructural Characteristics of Bovine Embryos Produced In Vitro And Vitrified Using the Cryotop Method

BACKGROUND: Despite advancements in bovine embryos cryopreservation techniques, challenges remain, warranting further investigation into their impact on embryo morphology and viability so that outcomes can be improved. OBJECTIVE: To analyze, through transmission electron microscopy (TEM), in vitro-produced bovine embryos vitrified using the Cryotop method. MATERIALS AND METHODS: Groups of embryos were transferred to a stabilization solution (SS) containing 7.5% EG, 7.5% DMSO in maintenance medium (TCM-199 supplemented with 20% FBS) for 2 min, and then transferred to a vitrification solution (VS) containing 15% EG, 15% DMSO, and 0.5 M sucrose in maintenance medium. Warming was performed in five stages with decreasing concentrations of sucrose. After warming, the blastocysts were cultured for 24 h for subsequent survival analysis and ultrastructural evaluation. In vitro-produced bovine embryos that did not undergo the vitrification process were used as a fresh control. RESULTS: Blastocoel reestablishment was observed in 52.3% (66/126) of vitrified embryos 24 h after warming, demonstrating the method's effectiveness in postcryopreservation survival. Ultrastructural analysis of embryos from the fresh control group showed flattened trophoectodermal cells with prominent nuclei, well-preserved mitochondria, and Golgi complexes were also evident. Microvilli were observed in some regions near the zona pellucida. Embryos vitrified using the Cryotop method exhibited lesions consistent with the cryopreservation process, such as intracellular disorganization, mitochondrial injuries, and dispersion of microvilli. CONCLUSIONS: Ultrastructural evaluation of in vitro-produced bovine embryos vitrified using the Cryotop method is an effective tool for increased understanding of the injuries caused to embryonic cells during the cryopreservation process.