Fecal cortisol metabolites (FCMs) are increasingly used to index physiological stress in wildlife. Cortisol and other stress hormones act to mobilize glucose, providing energy for the organism to respond to environmental perturbations. Cortisol, the predominant glucocorticoid (GC) in most mammals, is metabolized by the liver and excreted as FCMs. For FCMs to serve as a meaningful physiological index of stress in brown bears (Ursus arctos), we sought to quantify the relationship between blood cortisol and FCM concentrations. Consequently, we conducted an adrenocorticotropic hormone (ACTH) challenge on nine unanesthetized captive brown bears at the Washington State University Bear Research, Education, and Conservation Center. We collected 10mL of blood at 0, 3, 6, 24, 48, and 72 hours post-injection to measure changes in blood cortisol concentrations. Fecal samples were collected between 7:00 am to 8:00 pm from 24 hours prior to injection through 72 hours post-ACTH challenge. We found that the peak blood cortisol concentrations occurred between 3 to 6 hours following an ACTH challenge whereas FCMs peaked between 10 and 27 hours after injection.

We conducted this study in June 2021 using nine captive brown bears (five females, four males) ranging in age from six to 20 years. Bears were housed at the Washington State University Bear Research, Education, and Conservation Center. For the duration of the experiment, bears were housed individually with indoor (3m x 3m x 2.5m) and outdoor (3m x 5m x 5m) access. The study bears had been trained previously to enter a holding crate and present a rear leg through the bars for blood collection. All bears were trained via positive reinforcement using dilute honey (in water), a method shown to not influence plasma cortisol levels (Joyce-Zuniga et al., 2016). Bears were fed a commercial bear diet in the form of kibble from Mazuri (Wild Carnivore Bear Plus), apples, and a small amount of meat (e.g., chicken, beef, or wild game) at 7:20 am and 3:00 pm during the active season (Mid-March through November 1^st).

We challenged bears with 5μg/kg Cortrosyn (Sandoz Pharmaceuticals and Amphastar Pharmaceuticals) injected intravenously (Cattet et al., 2021). Bears received Cortrosyn injections between 8:40 am to 9:29 am and we collected 10mL of blood from the metatarsal or lateral saphenous vein beginning at approximately 8:00 am (0h) and then at 3h, 6h, 24h, 48h, and 72h following injection to measure changes in serum cortisol concentrations. Once collected, the blood was centrifuged, and the serum was stored at -80ºC until analyzed. Fecal samples were collected between 7:00 am to 8:00 pm from 24h pre-ACTH challenge through 72h post-ACTH challenge and placed in a -20°C freezer until shipped overnight on dry ice to Northern Michigan University where samples were stored in a -80°C freezer until analyzed. Bears were under 24-hour video monitoring, individuals could be identified, and thus the time and source of each fecal deposition could be identified. Fecal samples deposited between 7:00 am to 8:00 pm were collected as soon as possible, usually within 6 hours, while samples deposited overnight were collected the following morning (<12h) to avoid potential bias associated with exposure time (Dalerum et al., 2020; Stetz et al., 2013). Baseline serum cortisol levels were calculated as the average cortisol concentrations of plasma drawn at 0h for each bear. FCM baselines were calculated as the average FCM concentration of samples deposited prior to the ACTH challenge (time 0). Peak blood cortisol and peak FCM concentrations were identified as the sample with the largest concentration of cortisol or cortisol metabolites following the ACTH challenge.

Fecal Hormone Extraction

Fecal samples were thawed at room temperature for 30 minutes prior to FCM extraction. We weighed 0.5±0.01g of wet feces and placed the feces in a 15mL centrifuge tube with 5mL of 80% methanol (Palme et al., 2013). Samples were vortexed for one minute and then centrifuged at 2500g for 15 minutes. After being centrifuged, the supernatant was analyzed immediately via an ELISA kit. The final concentration was then converted from ng/mL into ng/g.

Cortisol and Cortisol Metabolite Assay

Serum cortisol concentrations and FCM concentrations were determined in duplicate using commercially available cortisol ELISA kits (Cortisol ELISA K003, ArborAssay, Ann Arbor, MI 48108, USA). This kit binds to immunoreactive cortisol metabolites indiscriminately and the upper and lower detection limits of the assay were 45.4 and 27.6 pg/mL, respectively. Serum cortisol samples were brought to room temperature prior to assay, following the manufacturer's protocol. For FCM samples, we modified the manufacturer’s protocol by extending the time samples were on the plate shaker to an hour and a half to increase the time for FCMs to bind to the ELISA antibodies.

Assay Validation

Fecal extracts were tested for parallelism by diluting high FCM concentration samples (one for each sex) from 1:20 to 1:2.5 with assay buffer (Hein et al., 2020). Dilutions were parallel to the standard curve (test of equal slopes, p>0.30), indicating no additional substances in the extract were cross-reacting with the antibody.

All statistical analyses were conducted in R (version 4.2.2, R Core Team 2022) using the packages ggplot2, dplyr, magrittrm tidyr, and tidyverse. Alpha was set at 0.05 and all tests were two-tailed. Samples were included in the final analysis if the intra-assay %CV was below 10 and the inter-assay %CV was below 15. For both FCM and serum cortisol concentrations, we evaluated the change from baseline through four days following the ACTH challenge with a repeated measure analysis of variance (ANOVA). Additionally, we performed a two-way repeated measures ANOVA to determine the influence of sex (male, female) and age (young, old) and the day of feces collection/plasma collection before and after injection, in addition to the interaction between our variables (Sex:Age, Sex:Time, Age:Time). We considered young individuals as bears six years old and younger, and old bears to be older than six years old. Next, we performed a post-hoc Tukey’s test to determine which days of fecal collection were significantly different from one another.

To assess whether blood cortisol concentrations and FCMs concentrations were correlated, we added individual lag time between peak blood cortisol and peak FCM to each blood cortisol time point for all bears. Blood cortisol concentrations were then paired with the corresponding fecal samples deposited within an hour of the adjusted time. When fecal samples did not fall within an hour of the adjusted time, FCMs concentration of the prior and following samples of that time point were averaged. We then plotted and conducted a linear regression for each individual bear to determine if there was a correlation between blood cortisol concentrations and FCM concentrations.

Ethical Statement

All procedures were approved by Washington State University Institutional Animal Care and Use Committee (IACUC) and we confirm that all procedures were performed in accordance with the approved IACUC ASAF #6874.