Validation of Enzyme Immunoassays via an Adrenocorticotrophic Stimulation Test for the Non-Invasive Quantification of Stress-Related Hormone Metabolites in Naked Mole-Rats

SIMPLE SUMMARY: Naked mole-rats (Heterocephalus glaber) have unique physiological and behavioral characteristics that make them a species of interest in biomedical research. However, due to their small size, frequent blood sampling to monitor the animals is not possible. Thus, we aimed to validate enzyme immunoassays, tools which detect the quantity of hormone molecules, for monitoring stress-related hormones, glucocorticoids, and/or their metabolites, in naked mole-rats using urine and feces. We administered a high and low dose of adrenocorticotrophic hormone, which causes the animal to physiologically respond to stress, and a saline administration as a control, which should not elicit a physiological stress response. The results revealed two enzyme immunoassays, namely, a 5α-pregnane-3β,11β,21-triol-20-one detecting glucocorticoid metabolites with a 5α-3β-11β-diol structure and an 11-oxoaetiocholanolone detecting glucocorticoid metabolites with a 5β-3α-ol-11-one structure, for measuring stress-related hormones in male and female urine, respectively. Furthermore, one enzyme immunoassay, namely, 11-oxoaetiocholanolone detecting 11,17 dioxoandrostanes for measuring stress-related hormones in feces of both sexes. There were sex-related differences in how the animals responded to the high and low doses of the adrenocorticotrophic hormone. We recommend using feces to monitor stress-related hormones in naked mole-rats, which will be valuable when investigating housing conditions and other animal welfare aspects. ABSTRACT: Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat (Heterocephalus glaber, NMR) using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3β,11β,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3β-11β-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5β-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.