DOI: 10.5061/dryad.7h44j0zvk
title: Quantification of faecal glucocorticoid metabolites as a measure of stress in the rock hyrax (Procavia capensis) living in an urban green space

description: Despite the abundance of rock hyrax (Procavia capensis) within South
 Africa's urban areas, there is not much information available about
 the effect of anthropogenic activities on rock hyrax wellbeing. To
 determine the potential impact of anthropogenic disturbance on
 adrenocortical activity, we conducted an ACTH challenge to identify a
 suitable enzyme-immunoassay (EIA) for measuring faecal glucocorticoid
 metabolite (fGCM) concentrations in the rock hyrax. This study identified
 an 11β-hydroxyaetiocholanolone EIA as the most suitable assay in this
 regard. The fGCM levels measured, indicate the physiological stress
 response in different rock hyrax populations, living in an area with
 varying degrees of anthropogenic activity (low, medium, high) within the
 National Botanical Garden of Pretoria, South Africa. The species’
 habituation to human numbers (weekly mean number of people) was examined
 by determining individual flight initiation distance (FID). Seasonally,
 there were overall higher fGCM concentrations in late spring compared to
 winter. The fGCM concentrations, although not significantly
 different but possibly biologically relevant, in the section with the
 lowest anthropogenic disturbance were ~10% higher compared to those in the
 section with medium disturbance, and ~20% higher compared to those in the
 section with the highest disturbance. Animal FID did not differ
 significantly between seasons but they did differ significantly between
 sections, and decreased in accordance with fGCM concentrations. The
 non-invasive approach established in this study provides a foundation for
 assessing rock hyrax wellbeing, and can help better understand how
 anthropogenic presence is perceived as a stressor in this species.
description: For this study, we conducted an ACTH challenge test at the SANBI
 Mokopane Biodiversity Conservation Centre on 3 animals (two females and
 one male) to collect serum cortisol data and fGCM concentrations pre and
 post ACTH test. To confirm effective ACTH
 administration, we collected four blood samples (0.25-0.4 ml)
 from the brachiocephalic vein from
 each individual; one immediately before and three at 15, 30, 45 min
 post-injection. We also collected faeces for 46 h after the ACTH test, to
 measure fGCM concentrations. All the samples were collected using clean,
 sterile latex gloves, stored in single labelled tubes, and immediately
 frozen at –20°C, until transferred for analysis to
 the Endocrine Research Laboratory (ERL) at the University of
 Pretoria. To determine alteration in fGCM concentrations
 post-defecation, in order to get an indication of suitable collection
 intervals in the field without compromising adrenocortical activity
 analysis reliability, fresh faeces were collected on one day from a
 communal latrine at the SANBI Mokopane Biodiversity Conservation Centre.
 Faeces were collected using sterile gloves, homogenized into one single
 sample, subsequently divided into 21 sub-samples, and stored outside (20 -
 27°C, 40 - 50% humidity, no precipitation). Following the methodology
 explained by Webber et al. (2018), sub-sample triplicates were frozen at
 –20°C at 0 h, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. All frozen samples were
 sent to the ERL for enzyme immunoassay analysis.  For
 the second part of the research, we collected faeces, using the same
 methodology, from a colony of free roaming <em>Procavia capensis
 </em>at the Pretoria National Botanical Garden (PBG). All samples
 were collected over 35 days in winter (July-August 2019) and
 over 25 days in late spring (October-November 2019), from 07:00 to 08:00. 
 The intent was to monitor whether there was a correlation between human
 presence and procavia's fGCM concentrations, thus, as visitor numbers
 were not evenly distributed, due to various activities offered in distinct
 areas of the park, we divided PBG into three different sections (1,2,3)
 based on human activity present (low, medium, high). To evaluate
 the assumption of different levels of human
 numbers in different sections of the PBG, we used digital motion activated
 cameras (Cuddeback digital C.5.1- 1347 - Wisconsin, USA), with a trigger
 time of 0.25 seconds, and a detection zone of 20 m, to quantify human
 activity in the respective areas. Cameras were set up following Miller and
 colleagues (2017). Pictures were taken from 06:00 – 18:00 over a total of
 50 days, split into two sampling periods (Period 1: 35 days in winter,
 July-August, and Period 2: 25 days in late spring, October-November
 2019). Following Chapman and colleagues 2012, during the same study period
 of camera traping and faecal sampling (35 days in winter, July-August, and
 25 days in late spring, October-November 2019), we also took FID
 measurements from animals living in each section, using a laser range
 finder (Nikon Forestry Pro laser range finder, accuracy: +18.2 mm,
 distance: 10-500 m). A total of 132 individual total
 measurements for colony one, 123 for colony two and 115 for colony three
 were collected, and daily median were calculated (n= 15) for each colony
 during both study periods. Blood samples analysis: the
 ERL analysed 15 serum samples using a
 Cortisol EIA, originally described by Palme and Möstl (1997)
 and following procedures previously described (Ganswindt et al. 2012).
 Palme and Möstl (1997) provide detailed assay characteristics, including
 antibody cross-reactivities. Faeces analysis: Frozen
 faeces were lyophilized, and the resulting dry faeces pulverized and
 sifted with a wire filter to remove any undigested material (Ganswindt et
 al. 2002). Steroid extraction was performed by dissolving 0.050 - 0.055 g
 of faecal powder in 3.0 ml of 80% ethanol, with each sample being
 subsequently vortexed for 15 min and then centrifuged for 10 min at 1500 g
 (Ganswindt et al. 2002). Lab technicians decanted supernatants into 1.5 ml
 safe-lock microcentrifuge tubes, labelled, and frozen at -20°C until
 further analysis. To determine the suitability of the EIAs for fGCM
 quantification, a subset of the ACTH challenge faecal
 extracts (n = 13 per animal), were measured for
 immunoreactive fGCM concentrations using five different
 enzyme-immunoassays (EIA): (i) a 11-Oxoaetiocholanolone I EIA (detecting
 11,17 dioxoandrostanes) (lab code 72a), (ii) a 11-Oxoaetiocholanolone II
 EIA (detecting fGCMs with a 5β-3α-ol-11-one structure) (lab code 72T),
 (iii) a 5α-pregnane-3β,11β,21-triol-20-one EIA (detecting
 fGCMs with a 5α-3β,11β-diol structure) (lab code 37e), (iv) a Cortisol
 EIA (lab code CSL), and (v) a 11β-hydroxyaetiocholanolone EIA
 (detecting fGCMs with a 5β,3α,11β-diol structure)
 (lab code 69a).
 Detailed assay characteristics, including cross-reactivities,
 can be found in Palme and Möstl (1997) for the 11-oxoetiocholanolone I and
 cortisol EIAs, Möstl et al. (2002) for the 11-oxoetiocholanolone II EIA,
 Touma et al. 2003 for 5α-pregnane-3β,11β,21-triol-20-one EIA and Frigerio
 (2004) for the 11β-hydroxyaetiocholanolone EIA. The
 sensitivity of the EIAs used were 1.2 ng/g dry weight (DW)
 (11-oxoaetiocholanolone I, 11-oxoaetiocholanolone II and cortisol), 2.4
 ng/g DW (11β-hydroxyaetiocholanolone), and 4.8 ng/g DW
 (5α-pregnane-3β,11β,21-triol-20-one). The cortisol
 and 11β-hydroxyaetiocholanolone EIA) for analysing the steroid extracts
 from the fGCM stability post-defecation test. Serial dilutions of faecal
 extracts gave displacement curves that were parallel to the respective
 standard curves (relative variation (%) of the slopes of respective trend
 lines &lt;5%) for both assays. Subsequently, faecal samples collected
 at the PBG were analysed by using only the 11β-hydroxyaetiocholanolone
 EIA. All hormone analyses were
 conducted at the ERL. Statistcal
 analysis: We used descriptic statistics to analyse the pattern in serum GC
 and fGCM concentrations following ACTH administrations and to describe
 alterations in fGCM concentations post defecation. Changes in fGCM
 concentrations post-defecation, determined with the cortisol and 11β-hydroxyaetiocholanolone EIA, were further analysed using One-way Analysis of Variance (ANOVA), followed by Holm-Sidak’s t-test as <i>post hoc</i>. Individual serum GC concentrations, collected at the point of ACTH injection (time 0h), and individual median fGCM concentrations determined from samples collected up to 26 h pre-ACTH administration, were set as baseline (100%). Subsequently, we calculated relative changes (%) in serum GC concentrations for individual samples collected at 15-, 30-, and 45-min post-injection. Differences in peak fGCM concentration 15-22 h post-ACTH administration were identified by comparing individual peak values with individual median pre-injection (baseline) values and normalised those by expressing these differences in percent. Peak values with an increase of &gt;100% above baseline were deemed considerable (Young et al. 2017). Variations in human numbers and FID for each study period were determined by using a generalised linear model (GLM), using the weekly mean number of people and, calculating daily median FID for each colony, as a response variable and section and season as predictor variables. An interaction between season and section was included to the model. The GLM analyses were followed by a Tukey’s <i>post hoc </i>analysis, conducted at 95% family wise confidence levels. A GLM was also used to calculate whether the covariates section (1, 2, 3) and season (late spring and winter) significantly influenced variations in fGCM concentrations. Subsequently, the use of a Tukey’s <i>post hoc</i> test, conducted at 95% family wise confidence levels, followed by Bonferroni adjustment, helped to determine whether significant differences in seasonal fGCM concentrations for each section occurred. Data are presented as mean ± standard deviation (SD), and significance was taken at 0.05. Data were statistically analysed using R Studio software (R Core Team 2016, 4.0.2 version).
description: Please note that animals ID are different in the dataset compared
 to those used in the manuscript: Animal A5-1 in the
 dataset corresponds to Female F1 in the manuscript
 Animal A5-2 in the dataset corresponds to Male M1 in the
 manuscript Animal A5-3 in the dataset corresponds to
 Female F2 in the manuscript