Age-related changes in testis, epididymis and caudal epididymal sperm in dogs

Abstract

The present study aimed to investigate the effect of aging on sperm quality, testicular degeneration, interstitial fibrosis of dog’s testis and alteration of androgen receptor (AR) and proliferation using Ki-67 as a marker in testicular tissues. Fifty-five healthy medium-sized dogs were divided into 4 groups; young (1-3 y/o, n=14), adult (>3-6 y/o, n=12), old (>6-9 y/o, n=14) and senile (>9 y/o, n=15). Spermatozoa were flushed from epididymal tails for routine sperm evaluation. Testes, epididymides (head, body and tail) and vas deferens were collected. The degrees of testicular degeneration and fibrosis on cut surface area were subjectively evaluated. Later, collected tissue sections were stained with H&E and Masson-Trichrome staining for evaluation of testicular degeneration in the seminiferous tubule and the area proportion of fibrotic tissue accumulation in testis, respectively. Microscopically, the semi-quantitative severity scoring of seminiferous tubule degeneration and quantification of testicular cells for Spermatic index (SI) and Sertoli cell index (SEI) were performed. Accumulation of the connective tissue in testis was determined using image analysis software (PatternQuant, 3DHISTECH) and quantified as percent of fibrosis. Expression of AR and Ki-67 protein was investigated by immunohistochemistry and evaluated using image analysis software (NuclearQuant, 3DHISTECH). The results showed that significant lower percentages of sperm motility, progressive motility and viability were found in adult, old and senile dogs, compared to young dogs. Animal’s age negatively correlated with sperm motility, progressive motility and sperm viability. The primary, secondary, major and minor sperm defects were significantly higher in senile compared to young dogs. Severity of seminiferous tubule degeneration gradually increased with age (p<0.05), being highest in senile dogs (4.7±0.2); and lowest in young dogs (1±0). Age positively correlated with the severity of seminiferous tubule degeneration. The score of testicular degeneration and fibrosis on cut surface area of testis was higher in senile than other age groups (p<0.05). In addition, the percent of fibrosis was found to be higher in senile dogs (30.9±2.5) compared to other groups. Significant positive correlations between age and degrees of testicular degeneration and fibrosis on cut surface as well as age and the percent of fibrosis were also observed. In senile dogs, SI was the lowest when compared to other groups (p < 0.05). Conversely, senile dogs appeared to have the statistically significant highest SEI. Expression levels of AR did not differ among different age groups. However, a positive correlation was found between age and AR expression in the testis. The Ki-67 index was lower in senile dogs compared to young dogs. Negative correlations were found between age and Ki-67 index. In conclusion, the present study demonstrated that senescence was associated with poor sperm quality, germ cell depletion and interstitial fibrosis of the testis as well as tubular germ cell potential and the efficacy of the final maturation process and spermatogenesis. Nevertheless, aging also has a negative effect on AR and Ki-67 protein expression in testicular tissues which may affect the efficiency of spermatogenesis especially in dogs over 9 years old.