# Non-Invasive Assessment of Adrenal Activity in the Subterranean Rodent Ctenomys talarum in Field and Laboratory Conditions

## TL;DR

This study introduces a non-invasive method to measure stress hormones in a subterranean rodent, improving animal welfare and data collection in ecological research.

## Contribution

The paper validates a non-invasive fecal glucocorticoid metabolite assay for measuring stress in Ctenomys talarum.

## Key findings

- Blood sampling causes a temporary spike in stress hormones in Ctenomys talarum.
- Fecal glucocorticoid metabolite analysis is a reliable non-invasive method to assess adrenal activity.
- Stress hormone levels correlate with reproductive seasonality and handling stress.

## Abstract

In nature, animals experience stress in response to challenges such as harsh weather, predators or food shortages. Particularly in vertebrates, the response to these conditions includes the activation of the hypothalamic–pituitary–adrenal axis, releasing glucocorticoid hormones that quickly boost energy levels. Short bursts of this response help animals survive, but prolonged elevation of these hormones can weaken immunity, impede growth and even disrupt reproduction. Researchers studying wildlife often capture and handle animals through immobilization, blood sampling or temporary captivity, which triggers stress in itself. To understand these effects, we studied Ctenomys talarum, a subterranean rodent that lives in natural and human-altered grasslands. We found that standard blood collection causes a brief spike in stress hormones, which return to normal soon afterwards. We also validated a non-invasive assay to measure glucocorticoid metabolites in feces. This test detected increases in hormones after handling, captivity, immobilization and also during breeding seasons. Fecal hormone analysis is less invasive and improves animal welfare while still providing reliable data. Adopting these methods will enhance conservation efforts and help to balance scientific objectives with the well-being of wild animals.

The endocrine stress response is a valuable tool for evaluating how organisms cope with environmental challenges. However, selecting an appropriate matrix for measuring glucocorticoids (GCs) requires careful consideration of sample quality and accessibility. This study reveals that blood sampling affects plasma cortisol levels in the subterranean rodent Ctenomys talarum, with the effect being reversed shortly thereafter. To facilitate a non-invasive approach, an enzyme-linked immunosorbent assay (EIA) that had previously been validated for measuring plasma cortisol in C. talarum was evaluated to measure adrenocortical activity by analyzing fecal glucocorticoid metabolites (FGCs). Using this assay, we monitored the stress response during wild capture, transport to captivity, adrenocorticotropic hormone (ACTH) stimulation, and immobilization. This showed that FGC levels accurately reflect adrenal activation in these contexts. We also documented a relationship between reproductive seasonality and FGCs. Finally, we provide evidence for a relationship between adrenal activity and behavior. Our results suggest that when considering plasma GCs for the assessment of acute stress, it is crucial to understand the magnitude and timing of the effects of blood sampling on the stress state of organisms. The validation of FGC measurement in C. talarum provides a new option for advancing ecophysiological studies in both the wild and captivity.