Decoding canine male reproductive dysfunction: a multi-level assessment of sperm quality in dogs from the ‘Land of Fires’ (Campania, Italy)

Male canine infertility is increasingly recognized in clinical practice, yet diagnostic workups are often limited to conventional semen parameters. Building on our previous characterization of testicular lesions in stray dogs from the ‘Land of Fires’ (Campania Region, Italy), this study extends the phenotyping of male reproductive dysfunction by integrating testicular tissue remodeling and redox balance with sperm output and nuclear quality. Testes from 40 stray male dogs were classified into four groups: Group A: testes with normal spermatogenesis; Group B: testes showing mild degeneration; Group C: testes showing moderate to severe degeneration; Group D: neoplastic testes. Testicular remodeling was assessed by immunofluorescence for vimentin and α-smooth muscle actin (α-SMA), while testicular oxidative status was examined by measuring hydrogen peroxide (H₂O₂) production and superoxide dismutase (SOD) activity. Subsequently, epididymal sperm concentration (ESC) was quantified, and Toluidine Blue (TB) staining was performed to evaluate sperm chromatin condensation through an automated whole-slide image analysis pipeline. Finally, sperm nuclear basic proteins (SNBPs) were profiled by AU–PAGE to evaluate protamine representation. Overall, the histopathological lesions were found to be associated with both a reduction in epididymal sperm output and a deterioration in sperm quality, as evidenced by the presence of nuclear and biochemical abnormalities. These results support a multi-level model linking testicular pathology to impaired spermatogenic function in dogs.