Majelantle, Tshepiso LesediBennett, Nigel CharlesGanswindt, Stefanie BirgitHart, Daniel WilliamGanswindt, Andre2023-09-272023-09-272023-04Majelantle, T.L.; Bennett, N.C.; Ganswindt, S.B.; Hart, D.W.; Ganswindt, A. Validation of Enzyme Immunoassays via an Adrenocorticotrophic Stimulation Test for the Non-Invasive Quantification of Stress-Related Hormone Metabolites in Naked Mole-Rats. Animals 2023, 13, 1424. https://doi.org/10.3390/ani13081424.2076-2615 (online)10.3390/ani13081424http://hdl.handle.net/2263/92430DATA AVAILABILITY: Data supporting the reported results will be sent by the corresponding author upon request.SUPPLEMENTARY MATERIALS : TABLE S1: Summary of saline volume and synthetic adrenocorticotropic hormone (Synacthen® depot, Novartis, South Africa (Pty) Ltd.) administered to each individual during the ACTH challenge; TABLE S2: Summary of EIA sensitivities and intra-assay and inter-assay coefficients of variation in high- and low-value quality controls for all EIAs applied to measure immunoreactive urine glucocorticoid metabolite (uGCM) and fecal glucocorticoid metabolite in naked mole-rats.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.en© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).Adrenocorticotrophic challengeCoristolDose-dependent responseGlucocorticoidsUrineFecesAfrican mole-rats (Bathyergidae)Naked mole-rat (Heterocephalus glaber)Enzyme immunoassay (EIA)SDG-03: Good health and well-beingNaked mole-rat (NMR)Adrenocorticotropic hormone (ACTH)Glucocorticoid metabolitesArticle