Received October 14, 2023
Accepted April 19, 2024

Abstract Methotrexate (MTX) is widely administered for the treatment of various cancers. However, MTX induces male reproductive toxicity. In the current study, the effect of ozone therapy (OT) on reducing the toxic effects of MTX in the mouse testicles has been investigated. Twenty-four mice were divided into four groups: control, OT (4 mg/kg Ozone), MTX (20 mg/kg), and MTX + OT. Testosterone levels, histological changes, and oxidative stress biomarkers were assessed to evaluate the protective effects of OT. The results demonstrated that MTX disrupted germinal epithelium, reduced serum testosterone levels, and enhanced oxidative stress in testicular tissue. However, treatment with OT attenuated these adverse effects. OT effectively restored the levels of antioxidant enzymes, such as catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD). OT reduced lipid peroxidation, as indicated by decreased malondialdehyde (MDA) levels. OT preserved normal spermatogenesis, improved morphometric parameters, and reduced histological changes by MTX. Moreover, OT effectively restored testosterone levels. In conclusion, OT protects against MTX-induced testicular damage by suppressing oxidative stress.