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

## TL;DR

This study shows that non-invasive methods can accurately measure steroid hormones in desert spadefoot toads, reducing stress during research.

## Contribution

The paper validates non-invasive and minimally invasive hormone measurement methods in a desert-adapted amphibian species.

## Key findings

- Corticosterone, estradiol, and testosterone can be reliably measured in water samples from Couch’s spadefoots.
- Pharmacological challenges can be detected in waterborne corticosterone in both male and female frogs.
- Plasma corticosterone correlates with both waterborne and salivary corticosterone levels.

## Abstract

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, E2; testosterone, TST) in Couch’s spadefoots (Scaphiopus couchii)—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 E2 in male frogs, but not consistently for TST or E2 in female frogs. Lastly, plasma hormone concentrations were consistently correlated with their waterborne complements for CORT (both sexes), as well as TST and E2 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.