METHODS: Validating an immunoassay to measure fecal glucocorticoid metabolites in yellow-bellied marmots.

Quantifying physiological stress in wild animals is essential for understanding their health, reproductive success, and survival in a variable environment. The yellow-bellied marmot (Marmota flaviventer) study at the Rocky Mountain Biological Laboratory near Crested Butte, Colorado, USA is the world's second longest study of free-living mammals. Historically, we used a validated corticosterone radioimmunoassay (RIA) to measure fecal glucocorticoid metabolites (FGMs) as a proxy for physiological stress. However, the costs and risks associated with working with radioisotopes drove us to consider a more sustainable method. Here we evaluate the suitability of two competitive corticosterone enzyme assays (EIA), one from Cayman Chemical Company (CCC) and one from Arbor Assays (AA), to measure marmot FGMs via their cross-reaction. The findings revealed that the AA EIA better matched the RIA in terms of accuracy across high and low FGM concentrations, had superior assay parameters, showed the highest correlations with RIA results and effectively captured the annual variations in FGM concentrations, thus demonstrating its reliability for use in longitudinal studies. We further analytically validated the AA EIA for FGMs and confirmed its efficacy and lack of matrix effects, thus establishing its suitability for ongoing and future studies of FGMs in marmots. The transition to the AA EIA from the RIA ensures continued data integrity while enhancing safety and environmental sustainability.