Dietary Patterns Modulate Ozone-Induced Reproductive Injury in C57BL/6J Mice

Dietary patterns orchestrate systemic metabolism and reproductive health, yet whether specific dietary patterns modulate susceptibility to ozone-induced male reproductive toxicity and its intergenerational consequences remains unclear. Here, male C57BL/6J mice were fed ad libitum (AL), a 30% calorie-restricted diet (CR), or a 45% high-fat diet (HFD), followed by ozone exposure (1 ppm, 4 h/day, 5 days/week for 3 months) to recapitulate real-world condition. We found that ozone exposure exacerbated reproductive injury under both CR and HFD, each diet perturbing metabolism through distinct pathways. Under CR, ozone further suppressed glycolysis, the TCA cycle, and amino acid metabolism, resulting in severe testicular energy depletion and impaired spermatogenic differentiation. In contrast, HFD amplified ozone-induced metabolic overload, inflammatory activation, and mitochondrial stress, accompanied by dysregulated lipid metabolism. Integrated metabolomic analyses identified disruption of the glucose-lactate-TCA axis as a central mechanism underlying testicular energy failure. Furthermore, exposed males (F0) were subsequently mated with unexposed females to generate offspring (F1 and F2) for assessment of next-generation health risks. The results showed that paternal dietary regimen and ozone exposure exclusively perturbed metabolic homeostasis in F1 offspring, indicating a reversible metabolic reprogramming rather than stable transgenerational inheritance. Collectively, these findings establish systemic metabolic status as a critical determinant of ozone-induced reproductive toxicity and reveal that paternal nutritional and environmental challenges jointly prime metabolic susceptibility in the next generation (F1).