Simple SummaryThis study aimed to investigate the changes in sexual hormones around puberty in dogs by analyzing the concentrations of testosterone (T), 17β-estradiol (E2), and progesterone (P4) in hair and nails. Puberty in dogs is a complex process with various factors involved, making it challenging to study. Traditional methods using blood samples are not feasible for long-term studies. However, hair and nails have been identified as suitable biological specimens for retrospectively evaluating hormone accumulation. The study included five male and five female dogs. The results showed a significant increase in T levels in hair and nails of male dogs just before puberty. In females, there was a significant increase in E2 levels at puberty and a marked increase in P4 levels after puberty in both biological specimens. Sex-related differences were observed only in T concentrations in hair, but considering sex and sampling time together, T concentrations in hair and nails, as well as P4 concentrations in nails, were able to differentiate between male and female dogs at specific sampling times. This study demonstrates the usefulness of hair and nails as biological specimens for studying the changes in T, E2, and P4 during the peripubertal period in dogs.AbstractStudies about puberty in dogs are few, probably because many factors are involved in the delicate process of puberty onset, leading to difficulties in the proper enrollment of subjects. Moreover, the use of blood for monitoring hormonal changes can be problematic, and not feasible for long-term studies. Hair and nails proved to be suitable matrices for the retrospective evaluation of hormones’ long-term accumulation. This study was performed using hair and nails for the evaluation of testosterone (T), 17β-estradiol (E2) and progesterone (P4) concentrations to assess possible sexual steroid changes during the peripubertal period in dogs. The results, obtained on five males and five females, showed a significant increase in T in hair and nails of males immediately before puberty. In females, a significant increase in E2 at puberty and a marked increase in P4 after puberty was found in both biological specimens. Sex-related differences were detected only for T hair concentrations, but when the sex and sampling time were considered together, hair and nails T and nails P4 concentrations allowed us to discern between male and female dogs at specific sampling times. The results from this study showed that hair and nails are useful biological specimens for the retrospective evaluation of changes in T, E2 and P4 concentrations in peripubertal dogs.