Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology.

To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4 days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7 h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2 days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2 days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8-12 h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8-12 h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive field studies of stress in hares.

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Live‐capture is a necessary component for the scientific study and management of most mammals, but it may negatively affect their health and physiology. We compared blood parameters related to the stress response (nominal base levels) from red squirrels Tamiasciurus hudsonicus after capture of up to 4.5 h in five different live trap models (Hava‐hart, Sherman, Tomahawk 102, Tomahawk 103 and ‘Special Squirrel’ trap) with true base levels (obtained in less than three minutes). In addition, we evaluated the capture rate in the five trap models. We found that (1) prolonged time in live traps altered stress hormone concentrations compared with true base levels, but maximum corticosteroid‐binding capacity was unaffected; (2) squirrels captured in a trap model with reduced visibility (a roof cover – Hava‐hart) had significantly lower (c. 50%) mean free cortisol levels compared with those captured in a trap model with full visibility (Tomahawk 102), but all other blood parameters were similar; (3) cortisol levels and white blood cell counts (mainly neutrophil counts) were positively related to duration of capture; (4) a non‐covered trap (Tomahawk 102) was most effective and fully covered trap (Sherman) was least effective at capturing squirrels. We discuss the use of effective, yet less stress‐inducing trap models to mitigate the stress caused by live‐capture on these animals. We conclude that covered traps such as the Hava‐hart may reduce trap‐induced stress in red squirrels, but at the same time also reduces their capture rates.

Arctic ecosystems are changing rapidly in response to climate warming. While Arctic mammals are highly evolved to these extreme environments, particularly with respect to their stress axis, some species may have limited capacity to adapt to this change. We examined changes in key components of the stress axis (cortisol and its carrier protein—corticosteroid binding globulin [CBG]) in polar bears (Ursus maritimus) from western Hudson Bay (N = 300) over a 33 year period (1983–2015) during which time the ice‐free period was increasing. Changing sea ice phenology limits spring hunting opportunities and extends the period of onshore fasting. We assessed the response of polar bears to a standardized stressor (helicopter pursuit, darting, and immobilization) during their onshore fasting period (late summer–autumn) and quantified the serum levels of the maximum corticosteroid binding capacity (MCBC) of CBG, the serum protein that binds cortisol strongly, and free cortisol (FC). We quantified bear condition (age, sex, female with cubs or not, fat condition), sea ice (breakup in spring–summer, 1 year lagged freeze‐up in autumn), and duration of fasting until sample collection as well as cumulative impacts of the latter environmental traits from the previous year. Data were separated into “good” years (1983–1990) when conditions were thought to be optimal and “poor” years (1991–2015) when sea ice conditions deteriorated and fasting on land was extended. MCBC explained 39.4% of the variation in the good years, but only 28.1% in the poor ones, using both biological and environmental variables. MCBC levels decreased with age. Changes in FC were complex, but more poorly explained. Counterintuitively, MCBC levels increased with increased time onshore, 1 year lag effects, and in poor ice years. We conclude that MCBC is a biomarker of stress in polar bears and that the changes we document are a consequence of climate warming.

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Interest in the ecology of stress in wild populations has triggered the development of noninvasive methods for quantifying stress hormones. Measurement of fecal corticosteroid metabolites (FCMs) is one such method, but it is still unclear whether FCMs can be a reliable proxy of free plasma glucocorticoids. To assess the validity of this assumption, we carried out a robust assessment on brown lemmings (Lemmus trimucronatus) from Bylot Island, Nunavut, Canada, that were hand captured and anesthetized and related plasma glucocorticoid levels to fecal metabolite glucocorticoid levels. We examined endogenous factors that could explain interindividual variability. Blood corticosterone was measured from samples obtained on capture and 30 min later, and FCM levels were measured from animals kept in captivity for 72 h. Plasma free corticosterone increased 135-fold over baseline values 30 min after capture, which confirmed that initial handling was perceived as a stressor. We found that FCM levels were highly related with free (marginal [Formula: see text] = 0.53) but not with total ([Formula: see text] = 0.02) corticosterone levels, regardless of age, sex, and reproductive condition. FCM levels started increasing 2 h after capture and reached maximum levels 4 h after capture. No circadian rhythm in FCMs was found. Plasma total corticosterone levels were much higher in adult females compared with adult males, but this difference was much smaller when measuring free corticosterone levels and FCM levels. Our results suggest that FCM levels are good measures of stress by being closely related to plasma free corticosterone levels in brown lemmings.

Social interactions among conspecifics can have marked effects on individual physiology, especially through its modulation of the stress axis by affecting the production of adrenal glucocorticoids (GCs). Previous research has focused on how individual GC levels may be influenced by social status, but few studies have considered how the balance between positive (e.g. cooperation) and negative (e.g. competition) social interactions shape individual GC levels. A lack of association between individual GC levels and social factors may be confounded by opposite effects of social competition on the one hand, and social cooperation on the other. We tested for these effects in the Columbian ground squirrel (Urocitellus columbianus), a colonial rodent. During the breeding season, females are exposed to territorial unrelated neighbors and to territorial, but more tolerant, close kin. On the one hand, territoriality and competition for resources led us to predict a positive association between local colony density and female GC levels. On the other hand, higher tolerance of philopatric kin females and known fitness benefits led us to predict a negative association between kin numbers and female GC levels. We compared levels of fecal cortisol metabolites (FCMs) in females at two different spatial scales during lactation: local (a female's core territory during lactation, 30m-radius about her nest burrow) and colony-wide. At the local scale, female FCM levels were neither related to colony density nor to the number of co-breeding female kin, but FCM levels increased with age. At the colony scale, female FCM levels varied in a quadratic fashion with female kin numbers. FCM levels decreased from 0 to 1 co-breeding kin present and increased with >1 kin present. Among females that had only one co-breeding kin present, daughters (and littermate sisters and mothers, but not significantly) led to a 14% reduction in FCM levels compared with females that had no kin. Our results reject the idea that local colony density is associated with increased GC levels this species, but indicate subtle (positive and negative) effects of kin on individual GC secretion. They further call into question the importance of the nature of social relationships in modulating the stress experienced by individuals.