Evaluation of therapeutic effects of D‐limonene following orchiopexy in the rat model of cryptorchidism

BACKGROUND: Cryptorchidism impairs spermatogenesis, causes infertility, and increases malignancy risk. Orchiopexy, the standard treatment, often fails to fully restore testicular function. This study evaluated the adjunctive effects of D-limonene in prepubertal rats with bilateral cryptorchidism. METHODS: Twenty-four male rats were allocated into four groups: sham-operated (SH), cryptorchid control with orchiopexy (CT), and two orchiopexy groups receiving low-dose (50 mg/kg, Lim-50) or high-dose (100 mg/kg, Lim-100) D-limonene. Assessments included oxidative stress markers in testicular tissue (total antioxidant capacity [TAC], malondialdehyde [MDA], superoxide dismutase [SOD], and glutathione peroxidase [GPx]), macroscopic indices (testicular index [TI], absolute testicular weight [TW], and testicular weight index [TWI]), histopathological parameters (Johnsen score [JS], epithelial height [EHS], and seminiferous tubule diameter [STD]), apoptosis-related proteins (Bax, Bcl-2, Caspase-3, and TNF-α), serum testosterone, and sperm parameters. RESULTS: D-limonene improved several indices of testicular injury following orchiopexy in cryptorchid rats. Both treatment doses increased TAC, while the higher dose more effectively reduced MDA and improved SOD activity. Testicular morphometric and histopathological parameters, including TW, TI, TWI, JS, EHS, and STD, were significantly improved. D-Limonene also decreased pro-apoptotic markers (Bax and Cleaved Caspase-3), increased anti-apoptotic markers (Bcl-2 and Pro-Caspase-3), reduced TNF-α expression and apoptotic cell counts, and enhanced serum testosterone and sperm quality. However, full normalization of all parameters was not achieved. CONCLUSION: Adjunctive D-limonene administration following orchiopexy attenuated oxidative stress, inflammation, and apoptosis, and partially restored testicular structure and function in experimental cryptorchidism. These findings support D-limonene as a promising supplementary strategy with partial but meaningful protective effects.