Epididymal lithiasis, characterized by the formation of stones in the epididymis, has been associated with a decline in fertility in roosters. This study aimed to investigate the reproductive performance, ultrastructural characteristics, and expression of aromatase cytochrome P450 (CYP19) and aquaporin 9 (AQP9) in aged broiler breeder roosters affected by epididymal lithiasis. X-ray analysis confirmed the presence of genital stones in both the epididymis and testicular tissue regions. While there was a significant decrease in sperm concentration in the affected roosters compared to non-affected roosters, no significant differences were observed in total and progressive sperm motility between the two groups. Furthermore, the affected roosters exhibited significant abnormalities in semen parameters, except for sperm concentration and morphology. Transmission electron microscopy (TEM) revealed the depletion and deciliation of ciliated cells in the distal efferent ductules of the epididymis in affected roosters. Additionally, the expression of CYP19 and AQP9 was found to be increased in the epididymal region of affected roosters. Notably, we report the presence of testicular stones for the first time in this study, in addition to epididymal stones. Considering the male reproductive tract lesions observed, we propose the term “genital stones” to describe these conditions. Moreover, our findings suggest that the overexpression of AQP9, which is associated with a high copy number of the CYP19 gene in the epididymal region of affected aged roosters, may contribute to the formation of genital stones by promoting increased reabsorption of fluids in the epididymis. The condensation of epididymal duct contents and reduction in the population of ciliated cells further impairs semen movement and can lead to the blockage of extra-testicular ducts, resulting in the low fertility syndrome observed in aged roosters.
