DOI: 10.5061/dryad.n8pk0p34f
title: Blood cortisol and fecal cortisol metabolite concentrations following an ACTH challenge in unanesthetized brown bears (Ursus arctos)

description: 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.
description: 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 <em>et al</em>., 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<sup>st</sup>). We
 challenged bears with 5μg/kg Cortrosyn (Sandoz Pharmaceuticals and
 Amphastar Pharmaceuticals) injected intravenously (Cattet <em>et
 al</em>., 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
 (&lt;12h) to avoid potential bias associated with exposure time
 (Dalerum <em>et al., </em>2020; Stetz <em>et
 al.,</em> 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. <strong><em>Fecal Hormone
 Extraction</em></strong> 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 <em>et al.,</em> 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. <strong><em>Cortisol and
 Cortisol Metabolite Assay</em></strong>
 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.
 <strong><em>Assay
 Validation</em></strong> 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 <em>et
 al.,</em> 2020). Dilutions were parallel to the standard curve (test
 of equal slopes, p&gt;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. <strong>Ethical
 Statement</strong> 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.
description: # Blood cortisol and fecal cortisol metabolite concentrations following an
 ACTH challenge in unanesthetized brown bears (*Ursus arctos*) We collected
 54 blood cortisol concentration samples across six sampling times
 (pre-injection, 3 hours post-injection, 6 hours post-injection, 24 hours
 post-injection, 48 hours post-injection, 72 hours post-injection) among
 nine bears. We also collected 205 fecal cortisol metabolites (FCMs)
 samples from 24 hours prior to injection through 72 hours post-injection.
 We conducted ANOVAs and TukeyHSD to determine when/if baseline (average of
 cortisol or FCMs concentrations prior to injection) concentrations
 differed at each time point and when concentrations returned back to
 baseline. ## Description of File Structure ####
 FecalCortisolMetabolites.csv refers to fecal cortisol metabolite data.
 Column A: "Bear" in column refers to an individual. In our
 study, each bear was given a name ID and that's how we refer to them.
 Column B: "Day" refers to whether it was the day prior to
 injection, the first day of bears receiving injection, and so forth.
 Column C: "Date" refers to actual date of data collection Column
 D: "Time Deposited" refers to the time in which a bear defecated
 that particular sample. Column E: "Time" refers to how many
 hours since injection. Negative times are representative of fecal samples
 deposited prior to injection, whereas positive times were deposited after
 injection. Column F: "Cort" refers to Fecal Cortisol Metabolite
 concentration which was shorted for ease and to avoid a long name that
 could lead to typing errors in R code (ng/g). Column G: "Sex"
 refers to the sex of an individual bear. Column H "Age" refers
 to our classification of age groups for our individuals. ####
 BloodAssay.csv describes data collected in regard to blood cortisol Column
 A: "Bear" in column refers to an individual. In our study, each
 bear was given a name ID and that's how we refer to them. Column B:
 "Time" refers to what time following injection that sample was
 taken. e.g., time 0 represents the sample taken before injection, time 3
 is 3 hours after injection, etc. Column C: "Cort" refers to
 blood cortisol concentration (ng/mL). Column D: "Sex" refers to
 the sex of an individual bear. Column E "Age" refers to our
 classification of age groups for our individuals. ## R Packages
 library(ggplot2) library(dplyr) library(magrittr) library(tidyr)
 library(tidyverse)