Extracellular Vesicles From Heat- and Oxidative-Stressed Granulosa Cells Improve Redox Balance and Embryo Development in Bovine Oocytes.

In vitro culture (IVC) exposes oocytes to suboptimal conditions that may disrupt redox homeostasis and compromise developmental competence. Extracellular vesicles (EVs) mediate intercellular communication, and their cargo reflects the physiological state of donor cells. We hypothesized that EVs derived from heat-stressed (HS) or oxidative-stressed (OS) granulosa cells (GCs) differentially modulate oocyte redox status and embryo development. GCs were exposed to 42°C for 24 h or to 5 μM H2O2 for 40 min, and EVs were subsequently isolated from conditioned media. GC-EVs (1 × 109 particles/mL) were supplemented during in vitro maturation (IVM). Redox parameters were assessed using triple fluorescence staining for reactive oxygen species (ROS), glutathione (GSH) and mitochondrial activity, combined with 3D confocal segmentation. EV-treated oocytes showed reduced ROS/GSH ratios compared with controls, indicating an improved redox balance. HS-EVs specifically enhanced mitochondrial activity. Cleavage rates were unaffected, whereas OS-EVs increased Day 7 blastocyst rates and HS-EVs improved hatching rates. These findings demonstrate that stress-conditioned GCs-EVs differentially regulate oocyte redox status, mitochondrial activity and developmental competence, supporting their potential to optimize IVC systems.