# Punica granatum L. Modulates Antioxidant Activity in Vitrified Bovine Ovarian Tissue

## TL;DR

This study shows that an extract from pomegranate can help preserve bovine ovarian tissue during freezing and thawing by reducing oxidative stress and maintaining tissue structure.

## Contribution

The novel finding is that pomegranate extract improves vitrification outcomes in bovine ovarian tissue by modulating antioxidant activity and preserving morphology.

## Key findings

- EE-PG at 10 and 100 µg/mL preserved follicular morphology and mitigated vitrification-induced damage.
- EE-PG increased SOD activity and reduced MDA levels, indicating improved redox balance.
- Despite downregulation of antioxidant genes, EE-PG supported tissue metabolism and structure.

## Abstract

This study aimed to evaluate the effects of an ethanolic extract from Punica granatum L. (EE-PG) on bovine ovarian tissue vitrification, focusing on follicular morphology, ultrastructure, stromal cell density, collagen distribution, redox status, and mRNA expression of antioxidant-related genes. Bovine ovarian cortex fragments were divided into a fresh control group for in vivo tissue evaluation or vitrified either with the base vitrification solution (αMEM) alone or supplemented with different concentrations of EE-PG (10, 50, and 100 µg/mL), and subsequently stored in liquid nitrogen for 5 days. After warming, fragments were allocated for morphological and oxidative stress analyses or incubated for 24 h to resumption of cellular metabolism. The concentrations of 10 and 100 µg/mL preserved follicular morphology immediately after warming, and were therefore selected for ultrastructural evaluation. Both concentrations mitigated vitrification-induced damage. Gene expression analysis showed decreased levels of catalase (cat), Glutathione Peroxidase 1 (gpx1), and Nuclear Factor Erythroid 2-Related Factor 2 (nrf2) compared with the fresh control, whereas Superoxide Dismutase (SOD) enzymatic activity increased after incubation with 10 µg/mL EE-PG compared with all experimental groups. Moreover, Malondialdehyde (MDA) levels in tissues treated with 10 or 100 µg/mL were comparable to fresh controls after incubation. Overall, EE-PG at 10 or 100 µg/mL in the vitrification solution supported the maintenance of tissue morphology, redox balance—despite the downregulation of essential antioxidant genes, which may be associated with a reduced demand for enzymatic antioxidant defense—and cellular metabolism, indicating potential for improving bovine ovarian tissue vitrification outcomes.