Measuring Faecal Glucocorticoid Metabolites to Assess Adrenocortical Activity in Reindeer

Abstract

Simple SummaryWe validated an 11-oxoaetiocholanolone enzyme immunoassay for measuring faecal cortisol metabolites (FCMs) in reindeer. Samples were collected from eight male reindeer following adrenocorticotrophic hormone (ACTH) stimulation and from another group of reindeer during handling and calf marking. The overall FCM levels peaked after seven to eight hours in both locations, proving that the assay is suited to evaluate the adrenocortical activity in reindeer.AbstractSeveral non-invasive methods for assessing stress responses have been developed and validated for many animal species. Due to species-specific differences in metabolism and excretion of stress hormones, methods should be validated for each species. The aim of this study was to conduct a physiological validation of an 11-oxoaetiocholanolone enzyme immunoassay (EIA) for measuring faecal cortisol metabolites (FCMs) in male reindeer by administration of adrenocorticotrophic hormone (ACTH; intramuscular, 0.25 mg per animal). A total of 317 samples were collected from eight male reindeer over a 44 h period at Tverrvatnet in Norway in mid-winter. In addition, 114 samples were collected from a group of reindeer during normal handling and calf marking at Stjernevatn in Norway. Following ACTH injection, FCM levels (median and range) were 568 (268–2415) ng/g after two hours, 2718 (414–8550) ng/g after seven hours and 918 (500–6931) ng/g after 24 h. Levels were significantly higher from seven hours onwards compared to earlier hours (p < 0.001). The FCM levels at Stjernevatn were significantly (p < 0.001) different before (samples collected zero to two hours; median: 479 ng/g) and after calf marking (eight to ten hours; median: 1469 ng/g). Identification of the faecal samples belonging to individual animals was conducted using DNA analysis across time. This study reports a successful validation of a non-invasive technique for measuring stress in reindeer, which can be applied in future studies in the fields of biology, ethology, ecology, animal conservation and welfare.

Fecal glucocorticoid metabolites reflect hypothalamic–pituitary–adrenal axis activity in muskoxen (Ovibos moschatus)

“…During both the winter and summer challenges, there was a high variability among individuals in the presence and magnitude of their response to a single dose of ACTH, as well as in the timing of the peak and duration of the increase in FGMs post-injection. Similar inter-individual variations have been reported in other ungulates (e.g., Rocky mountain goats ( Oreamnos americanus ) [ 29 ], reindeer ( Rangifer tarandus tarandus ) [ 30 ]; S1 Table ). These could be due to a variety of factors, including sex, age, reproductive status, health condition, and/or “individual” characteristics, such as genetic background or past and recent experiences, which may influence GC levels, the responsiveness of the HPA axis, and the metabolism and excretion of FGMs [ 19 , 31 ].…”

“…The 2,814% increase observed in MX-1169 during the summer challenge sits at the high end of the ranges of FGM peak magnitudes. The summer FGM peak timing of 7 h with a return to time 0 levels by 22 h post-injection would be closest to the responses measured in reindeer [ 30 ], caribou ( Rangifer tarandus granti ) [ 21 ], cattle ( Bos taurus ) [ 10 , 35 ], and sheep ( Ovis aries ) [ 35 ]. The later peaks and long responses observed during the winter challenge (i.e., peak in FGMs at 47 h with a decrease by 55 h in MX-283 and increase after 25 h followed by a return to time 0 levels by 95 h in MX-1189) are at the high end of the ranges measured in other even-toed ungulate species, but such responses have been observed in reindeer [ 21 ] and dromedary camels ( Camelus dromedaries ) [ 36 ].…”

“…The few studies that have compared seasonal responses of FGMs to ACTH administration have had differing results depending on the species. A study of reindeer in Norway in which FGMs were measured following a pharmacological challenge in the winter and a stressful event (i.e., calf marking) in the summer found no major differences in the timing of the FGM elevation [ 30 ]. A study in white-tailed deer ( Odocoileus virginianus ) detected earlier peaks in FGMs following a pharmacological challenge in the winter (10–13 h post-ACTH injection) compared to the fall (20–24 h) [ 38 ].…”

“…Due to species-specific differences in hormone metabolism during transit through the gut, the excreted metabolites vary in structure and proportion, and consequently, will only be effectively detected using an antibody that cross-reacts with the specific structures present ( S1 Table ). For example, a study in giraffes ( Giraffa camelopardalis ) found variations both between the animals and among the six EIAs used to quantify FGMs following an ACTH injection [ 50 ], and the contrasting results of two pharmacological challenges done in reindeer may have been due to the use of different EIAs (see S1 Table for study details) [ 21 , 30 ]. While the corticosterone EIA detected responses in several muskoxen, an EIA more specifically targeting the metabolites excreted by this species may have allowed us to more reliably detect changes in FGMs with greater consistency across animals.…”