Use of non-conventional sample matrices for endocrine studies of pit vipers: assay validation and applications

Understanding a species’ physiological state is important for advancing animal ecology and conservation. Endocrine responses to reproduction, stress, and nutritional status are commonly assessed through gonadal, adrenal, and thyroid hormones, respectively. Using non-conventional samples for endocrinological evaluation is an increasingly utilized method but remains uncommon for snakes. In this study, we assessed whether feces, urine, or shed skin from two Neotropical pit vipers (Bothrops jararaca and B. jararacussu) contain detectable testosterone (T), progesterone (P4), 17β-estradiol (E2), corticosterone (CORT), and triiodothyronine (T3) using enzyme immunoassay (EIA). We collected samples from 23 individuals, 10 B. jararaca and 13 B. jararacussu, and assessed detectability of hormones and/or immunoreactive hormone metabolites (IHM). We used tests of parallelism and accuracy to validate assays. Triiodothyronine was not detected in urine of either species; all other hormones were detected in all matrices. Testosterone and T3 showed good parallelism for all matrices tested. Parallelism tests for E2 (urine, both species), CORT (urine, B. jararaca, and shed skin, B. jararacussu), and P4 (urine, B. jararaca), showed marginal results. Most accuracy validations were successful, except for T3 in shed skin extract (B. jararacussu) and P4 in urine extract (B. jararaca). This study demonstrates the applicability of non-conventional samples for hormone and IHM detection and quantification, offering valuable tools to monitor the endocrinological status of both free-ranging and confined snakes.