Ethanolic Extract of Pomegranate (Punica granatum L.) Prevents Oxidative Stress and Preserves the Morphology of Preantral Follicles Included in Bovine Ovarian Tissue Cultured In Vitro

Oxidative stress compromises follicle survival during in vitro culture. Natural antioxidants may reduce cellular damage and preserve tissue integrity. This study evaluated the ethanolic extract from <i>Punica granatum</i> L. (EE-PG) on bovine ovarian tissue cultured in vitro. Bovine ovarian (<i>n</i> = 24) fragments were cultured for 6 days in αMEM⁺ medium with or without EE-PG at 10, 50, or 100 µg/mL. At the end of the in vitro culture, the medium was used to evaluate antioxidant capacity (DPPH and ABTS assays), while the fragments were collected for morphological and biochemical analyses. HPLC-UV-Vis confirmed the presence of α-punicalagin in the pure EE-PG. At 100 µg/mL, EE-PG showed the strongest effects: it had higher antioxidant capacity, preserved follicle morphology and ultrastructure, and promoted follicle activation. At 50 and 100 µg/mL, the extract also reduced malondialdehyde (MDA) and increased thiol levels, indicating protection against lipid peroxidation. In contrast, 10 µg/mL had little effect. Follicle and oocyte diameters were not significantly altered, but the collagen I/III ratio increased at higher concentrations, suggesting extracellular matrix remodeling. Together, these findings demonstrate that EE-PG protects bovine preantral follicles from oxidative stress, maintains redox balance, and preserves tissue integrity. These results reinforce the potential of <i>Punica granatum</i> L. extract as a natural antioxidant in reproductive biotechnologies and fertility preservation.