Validation of non-invasive methods for the measurement of gonadal and inter-renal steroid hormones in a desert-adapted amphibian ().

For aquatic and semi-aquatic vertebrates like amphibians, it is possible to estimate excreted hormone levels using non-invasive methods such as waterborne and salivary sampling. These techniques allow monitoring of endocrine activity over varying, repeated and simultaneous integration periods while minimizing handling-related stress that can 'contaminate' hormone estimates, including estimates of baseline glucocorticoids. Here we have validated the extraction and quantification of three steroid hormones (corticosterone, CORT; 17-b estradiol, E; testosterone, TST) in Couch's spadefoots ()-a desert-adapted anuran of special interest for physiology, evolution and conservation-using non-invasive waterborne and minimally invasive salivary hormone methods. We combined extraction and enzyme immunoassay methods to conduct conventional technical validations of parallelism, recovery and time-course. Next, we carried out biological validations by testing the correlation between excreted and circulating concentrations and conducting pharmacological challenges. We found that all three hormones can be precisely estimated from 60-min water baths, exhibit robust parallelism, and have high recoveries. Further, we demonstrated that secretory responses to pharmacological challenges can be detected in waterborne CORT in male and female frogs; in TST and E in male frogs, but not consistently for TST or E in female frogs. Lastly, plasma hormone concentrations were consistently correlated with their waterborne complements for CORT (both sexes), as well as TST and E in males (but not females). Plasma CORT was also positively correlated with salivary CORT. Together, our findings suggest that sampling waterborne and salivary hormones offers a minimally invasive method that field endocrinologists and conservation physiologists can use to obtain biologically informative endocrine estimates from desert-adapted amphibians.