Measurement of oxidative response and protein carbonylation in uterine samples from mares with post-breeding endometritis treated with ozone therapy

• Grant number: 25/13480-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Eneiva Carla Carvalho Celeghini
• Grantee: Fernanda Lopes da Silva
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Infertility in mares has become a relevant issue in equine reproduction, especially when associated with inflammatory processes triggered by artificial insemination (AI). Post-breeding endometritis (PBIE) is often caused by an exaggerated inflammatory response to semen deposited in the female reproductive tract, resulting in an excessive influx of leukocytes, reactive oxygen species (ROS), and pro-inflammatory cytokines, thus creating an unfavorable environment for fertilization and embryonic development. Oxidative stress, which arises from the imbalance between ROS production and the organism's antioxidant defenses, may lead to significant cellular damage to the endometrial epithelium. Ozone therapy has been extensively investigated in animal reproduction as a therapeutic alternative for reproductive tract disorders in females due to its potential antimicrobial, immunomodulatory, and indirect antioxidant effects.The main objective of this study is to evaluate oxidative stress in uterine samples from mares submitted to AI with cryopreserved semen at different time points: before AI (T0), 48 hours after AI (T48), and 24 hours after intrauterine treatment (T72) with either lactated Ringer's solution (O¿-, control) or ozonized lactated Ringer's solution (O¿+). Uterine samples had been previously collected and stored at -80°C in triplicate from mares classified as negative (E-, control) or positive (E+) for PBIE. Oxidative markers in the uterine samples will be analyzed by measuring malondialdehyde (MDA), a biomarker of lipid peroxidation, using the thiobarbituric acid reactive substances (TBARS) assay, as well as by assessing protein oxidation through carbonyl group detection. The study will follow a 2 × 2 factorial design, considering endometritis status (E- vs. E+) and treatment (O¿- vs. O¿+), and the effects of time (T0, T48, and T72) will also be evaluated. Data will be analyzed by ANOVA, and group means will be compared using Tukey's test (parametric) or the Wilcoxon test (non-parametric), with statistical significance set at p ¿ 0.05. It is expected that this experimental model will help establish a relationship between ozone therapy and oxidative byproducts present in the uterine samples.