Noninvasive methods for assessing animal hormonal status are increasingly applied in ecophysiological studies, which is particularly important for rare and vulnerable species. In vertebrates, the hypothalamic–pituitary–adrenal (HPA) axis and its effector hormones, glucocorticoids, play a central role in regulating physiology and behavior in response to environmental challenges, including various stressors. This study aimed to validate a noninvasive method for assessing HPA axis activity by measuring corticosterone metabolites in the droppings of two migratory passerine bird species, the European robin (Erithacus rubecula) and the garden warbler (Sylvia borin), using EIA-4164 kits (DRG International Inc., USA). Physiological validation using an ACTH challenge induced a significant increase in plasma corticosterone concentrations, followed by an increase in corticosterone metabolite levels in droppings. In both species, metabolite levels peaked within three hours after ACTH injection, consistent with gut passage time. Experimental procedures (blood sampling and ACTH injection) temporarily suppressed feeding activity in some birds, leading to a negative correlation between droppings mass and corticosterone metabolite concentrations, i.e., metabolite concentrations were higher in samples with lower droppings mass. Given the considerable variation in feeding intensity among experimental birds, expressing results as the absolute amounts of corticosterone metabolites excreted over fixed time intervals (excretion rate) reduced bias associated with variation in droppings mass and provided a more reliable assessment of hormonal status than concentration-based measures. This study validates the EIA-4164 kit for noninvasive monitoring of HPA axis activity in two small migratory passerine species and highlights the importance of accounting for the mass of droppings to improve the accuracy of hormonal status assessments.
Tsvey et al. (2026) studied this question.