Abstract
Global warming-induced thermal stress is an escalating threat to livestock fertility, perturbing ovarian function, oocyte maturation, and preimplantation embryo development through excessive accumulation of reactive oxygen species (ROS), which drive follicular oxidative damage. Although embryo transfer technologies offer a practical abatement strategy for mitigating such implications caused by HS, in vitro systems lack the endogenous antioxidant defenses present in vivo, leaving follicular cells, gametes, and embryos particularly vulnerable. Here, we aimed to investigate whether antioxidants, including quercetin (QUE), carnosol (CAR), and sulforaphane (SFN), mitigate HS-induced follicular oxidative damage in bovine granulosa cells (GCs), oocytes, and embryos. For this, antioxidant supplementation, either individually or in combination, was performed during in vitro GC culture and oocyte maturation under normothermic (NT) or HS conditions. Across all models, QUE and SFN supplementation activated nuclear NRF2, reduced ROS accumulation, and restored mitochondrial function and apoptosis levels under conditions of HS. In oocytes exposed to thermal stress, QUE and SFN supplementation also led to increased blastocyst rates and total cell numbers. Single-embryo metabolic profiling revealed reduced oxygen consumption (OCR) and extracellular acidification (ECAR) rates in blastocysts derived from antioxidant-treated oocytes, indicative of enhanced metabolic efficiency. Moreover, quantitative analysis of recently defined embryo competence-associated genes demonstrated a restoration of the embryo competence index (ECI) following QUE and SFN supplementation. In conclusion, antioxidant supplementation during GC culture and oocyte maturation alleviates HS-induced reproductive dysfunction by restoring redox homeostasis, preserving metabolic efficiency, and re-establishing embryo competence, thereby providing a mechanistically grounded strategy to mitigate climate-driven fertility decline in cattle.