Zinc chelation and supplementation to enhance artificial oocyte activation efficiency in the bovine

Zinc homeostasis is essential for oocyte maturation, activation, and genomic stability. Oocyte activation is physiologically triggered by calcium oscillations and a rapid exocytotic zinc release that coordinates meiotic exit and early embryogenesis. Although zinc chelation has been shown to induce oocyte activation in other mammalian species, its efficacy, optimal conditions, and developmental consequences in bovine oocytes remain poorly defined. Moreover, the interaction between zinc chelation-based activation and zinc availability during in vitro maturation (IVM) has not been systematically evaluated in cattle. This study aimed to optimize artificial oocyte activation (AOA) via zinc chelation using 1,10-phenanthroline (PHEN) and to evaluate the impact of zinc sulfate (ZnSO₄) supplementation during IVM on parthenogenetic embryo development. Bovine cumulus-oocyte complexes were matured with different concentrations of ZnSO₄ and activated using ionomycin or PHEN. Intracellular zinc levels in oocytes were measured, and blastocyst diameter and DNA fragmentation levels were assessed. Zinc supplementation significantly increased intracellular Zn²⁺ levels at 1.5 μg/mL ZnSO₄ but did not improve cleavage or blastocyst rates. PHEN at 250 μM for 30 min induced zinc depletion and meiotic resumption yet produced lower developmental rates than ionomycin, whereas 250 μM for 60 min achieved comparable cleavage and blastocyst rates. Ionomycin combined with 1.0 or 1.5 μg/mL ZnSO₄ significantly reduced blastocyst DNA fragmentation, while PHEN activated embryos maintained consistently low fragmentation levels. Collectively, these findings demonstrate that optimized zinc chelation can support bovine embryo development comparable to calcium-based activation, while zinc supplementation enhances intracellular zinc levels and genomic integrity rather than developmental yield.