Data and code for: Previous assessments of faecal glucocorticoid metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed

Britnell, Jake 1 ; Palme, Rupert2; Kerley, Graham3; Jackson, John4; Shultz, Susanne 1

Research facility: University of Manchester

Published Jul 09, 2024 on Dryad. https://doi.org/10.5061/dryad.vq83bk42d

Abstract

Steroid hormones, especially glucocorticoids (GCs), are widely used to assess physiological responses to stressors. As steroid hormones are heavily metabolised prior to excretion, it is essential to validate enzyme immunoassays (EIAs) for measuring faecal glucocorticoid metabolites (fGCMs). Although problems with unvalidated assays have been raised repeatedly, their use persists widely. Lea et al. (2017) used an unvalidated corticosterone assay (CJM006) to relate fGCM concentrations to habitat quality, demography, and population performance in the Cape mountain zebra (Equus zebra zebra). Here, we revisit their findings and evaluate the validity of their conclusions using a validated EIA. First, we evaluate the biological sensitivity of six EIAs (three group-specific metabolite assays and three corticosterone assays, including CJM006) through a biological validation experiment (translocation) for two sub-species of mountain zebra, Cape mountain and Hartmann’s mountain zebra (E. z. hartmannae). Second, we reanalyse the faecal extracts from Lea et al. (2017) using a validated EIA. fGCM concentrations consistently increased following translocation, when using two 11-oxoaetiocholanolone (lab codes: 72T and 72a) and an 11ß-hydroxyaetiocholanolone (69a) EIA, but did not with three different corticosterone EIAs. All corticosterone EIAs (including CJM006) failed to detect an increase in fGCMs within the critical 48–72-hour period post translocation. Therefore, the CJM006 EIA utilised in Lea et al. (2017) does not sensitively measure hypothalamic-pituitary-adrenal (HPA) axis activity in CMZ faeces.

Using a validated assay (72T), fGCM concentrations were no longer associated with adult sex ratio or habitat quality (measured by grassiness) and these variables were dropped from predictive models. fGCM concentrations now varied between seasons and were negatively associated with female fecundity (foal:mare ratio). Consequently, we can conclude that the results of the previous study are unreliable. We introduce the terms “insensitive” and “sub-optimal” to categorise assays that are tested but fail validation, and assays that are comparatively poor at detecting relevant hormone changes, respectively. We discuss how both “insensitive” and “sub-optimal” assays could lead to incorrect inferences about population stressors and counterproductive conservation recommendations.

In this dataset, we provide all R code scripts, raw data and data to reproducible all results and figures in Previous assessments of faecal glucocorticoid metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed. We have structured the project as an internally consistent directory with files corresponding to code, data, raw lab_ouputs, and figures.

https://doi.org/10.5061/dryad.vq83bk42d

Description of the data and file structure

GENERAL INFORMATION

1. Title of Dataset: Data and analysis code for: A grazer’s niche edge is associated with increasing diet diversity and poor population performance

2. Author Information

Corresponding Investigator and Lead Supervisor:

	Name: Professor Susanne Shultz

	Institution: Department of Earth and Environmental Sciences, University of Manchester, United Kingdom

	Email: Susanne.shultz@manchester.ac.uk

Lead Author and investigator

	Name: Dr Jake Alan Britnell

	Institution during work: Department of Earth and Environmental Sciences, University of Manchester, United Kingdom

	Current institution: Faculty of Life Sciences, University of Bristol, United Kingdom

	Email: jake.britnell@bristol.ac.uk

Co-investigator 1:

	Name: Professor Rupert Palme

	Institution: Department of Biomedical Sciences, University of Veterinary Medicine, 1210 Vienna, Austria 

Co-investigator 2

	Name:  Professor Graham Kelley

	Institution: Centre for African Conservation Ecology, Nelson Mandela University, Gqeberha, 6031, South Africa

Co-investigator 3

	Name:  Dr John Jackson 

	Institution during work: Department of Biosciences, University of Sheffield, Sheffield, S10 2TN, UK

	Current institution: Estación Biológica de Doñana

3. Brief summary of work:

Lea et al. (2017) used an unvalidated corticosterone assay (CJM006) to relate fGCM concentrations to habitat quality, demography and population performance in the Cape mountain zebra (Equus zebra zebra). Here, we revisit their findings and evaluate the validity of their conclusions using a validated EIA.

Here, we evaluate the biological sensitivity of six EIAs (three group-specific metabolite assays and three corticosterone assays, including CJM006) through a biological validation experiment (translocation) for two sub-species of mountain zebra, Cape mountain and Hartmann’s mountain zebra (E. z. hartmannae). Second, we reanalyse the faecal extracts from Lea et al. (2017) using a validated EIA.

We found that GCM concentrations consistently increased following translocation, when using two 11-oxoaetiocholanolone (lab codes: 72T and 72a) and an 11ß-hydroxyaetiocholanolone (69a) EIA, but did not with three different corticosterone EIAs. All corticosterone EIAs (including CJM006) failed to detect an increase in fGCMs within the critical 48–72-hour period post translocation. Therefore, the CJM006 EIA utilised in Lea et al. (2017) does not sensitively measure hypothalamic-pituitary-adrenal (HPA) axis activity in CMZ faeces.

4. Date of data collection:

In this paper, we used three different sample sets of Mountain zebra faeces. These were collected at the respective time-periods.

1.	Hartmann's mountain zebra (Equus zebra hartmannae) captive zoo population translocation samples:  Faecal samples were collected January 2019 and January 2020

2.	Cape Mountain zebra (Equus zebra zebra) wild zoo population translocation samples:  Faecal samples were collected January 2019 and January 2020

3.	Cape Mountain zebra macroecological sampling across populations:  Faecal samples were collected January 2019 and January 2020

5. Description of sample sizes:

1. 4 Hartmann's mountain zebra (Equus zebra hartmannae) (1 male and 3 females)

2. 5 Cape mountain zebra (Equus zebra zebra) (1 male and 4 females)

3. 365 Cape mountain zebra (Equus zebra zebra) faecal samples from 163 individuals across seven populations - Collected by Lea et al 2017

6. Geographic location of data collection:

1. Captive Hartmann’s mountain zebra (Equus zebra hartmannae) population - Tierpark Berlin, Germany

2. Translocation Cape Mountain zebra (Equus zebra zebra) - population: Sanbona Wildlife Reserve, Barrydale 6720, South Africa

3. Wild Cape mountain zebra populations — populations: Bakkrans Nature Reserve,Camdeboo National Park, De Hoop Nature Reserve, Gamkaberg Nature Reserve, Mount Camdeboo Private Game Reserve, Swartberg Private Game Reserve and Welgevonden Game Farm. All in South Africa

PROJECT DIRECTORY FORMAT

We have arranged our project into an internally consistent project directory folder. Once Dryad is downloaded, the scripts in the code will generate all outputs such as figures. The lab_outputs have been processed separately such Below we describe the name of each file and what it contains

•	code:  This folder contains all the scripts to reproduce all the analysis and figures produced in the manuscript

•	data:  This folder contains all the data sheets used for the analysis and figures produced in the manuscript. Please NOTE THAT WE STRONGLY ENCOURAGE USERS TO NOT MANUALLY EDITED FILES WITHIN THE data FILE. OUR SCRIPTS DO NOT EDIT THESE FILES INSTEAD THEY PRODUCE OUTPUTS SO THAT ANALYSES ARE REPRODUCIBLY EDITED IN R

•	figures: This folder contains copies of all figures in the manuscript. These are saved separately for publication

•	lab_outputs: This folder contains all raw datafiles that where produced during laboratory work. These have been consolidated into data file for analysis but for transparency we include all data

•	README.

DATA FILES OVERVIEW

cartridge_correlations.csv

•	Description: Contains correlation data for various extraction methods (Methanol dried and Cartridge) across different enzyme immunoassays (EIA).

Format: CSV. No special formatting. 

Sheets: One sheet

•	Role in Analysis: Used to assess the correlation between different extraction methods for glucocorticoid metabolites, aiding in the evaluation of their reliability and consistency.

NAs: NAs occur in this csv. We evaluated the loss of metabolite concentrations using two different protocols (C8 cartridge extractions and methanol drying extraction). We ran two sets of experiments. One of which was a smaller subset containing all the enzyme immunoassays while the other contained 72a alone. Hence, there are some NA gaps.

Header	Meaning	Units
EIA_72a_Methanol_dried	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The Methanol_dried denotes the extraction protocol and storage technique used to extract the faecal glucocorticoid and store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.
EIA_72a_Cartridge	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The _Cartridge denotes storage technique used to store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.
EIA_72T_Methanol_dried	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The Methanol_dried denotes the extraction protocol and storage technique used to extract the faecal glucocorticoid and store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.
EIA_72T_Cartridge	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The _Cartridge denotes storage technique used to store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.
EIA_69a_Methanol_dried	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The Methanol_dried denotes the extraction protocol and storage technique used to extract the faecal glucocorticoid and store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.
EIA_69a_Cartridge	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The _Cartridge denotes storage technique used to store them during the experiment.	ng/g (concentration of metabolite in faeces). Numeric format.

cartridge_loss_test.xlsx

•	Description: Details the loss of glucocorticoid metabolites during the extraction process using different cartridges. Includes columns for sample identifiers and metabolite measurements before and after extraction.

Format: XLSX. No special formatting. 

Sheets: Two sheets. Named Cart_raw and Cart_longformat. These are equivalent datasheets but in two different formats. The formats are needed for different analyses 

•	Role in Analysis: Utilized in the S5_supp_cartridge_loss.R script to compare and quantify the loss of metabolites across different extraction cartridges.

NAs: There are no NAs within either sheet.

Header	Meaning	Units
Sheet 1 - Cart_raw	The following correspond to Sheet 1 in the .xlsx
Sample	Sample ID	NA. Character string.
11'17DOA_lab	The concentrations of 11'17 DOA from the laboratory extraction and storage. 11' 17 DOA originate from the 72a enzyme immunoassay. The laboratory extraction and storage are the 80% methanol extraction and dried storage at 50-60 degrees celcius	ng/g (concentration of metabolite in faeces). Numeric format.
11'17DOA_cart	The concentrations of 11'17 DOA from the cartridge extraction and storage. 11' 17 DOA originate from the 72a enzyme immunoassay. The cartridge extraction and storage are described in the manuscript.	ng/g (concentration of metabolite in faeces). Numeric format.
Sheet 1 - Cart_longformat	The following correspond to Sheet 2 in the .xlsx
Sample	Sample ID	NA. Character string.
Category	Whether the row corresponds to the "Laboratory" or "Cartridge" treatment.	NA. Character string.
ng/g	nanograms per gram of faeces for metabolite (concentration of metabolite in faeces).	ng/g (concentration of metabolite in faeces). Numeric format.

cmz_cjm006_parallelism.csv

•	Description: Contains data on the parallelism of CMZ samples using the CJM006 assay. Includes columns for sample identifiers, B/B0 ratios, and concentration values.

Format: CSV. No special formatting. 

Sheets: One sheet. 

•	Role in Analysis: Used in the S5_supp_parallelism.R script to validate the parallelism of the CJM006 assay across different samples.

NAs:There are no NAs within this csv.

Header	Meaning	Units
Sample	Sample ID	NA. Character string.
B/B0	Binding of metabolite within the enzyme immunoassay (EIA) at the specified concentration divided by 100% binding within the same EIA	Proportion of binding against absolute binding. Numeric format. (Proportion)
concentration	nanograms per gram of faeces for metabolite (concentration of metabolite in faeces).	ng/g (concentration of metabolite in faeces). Numeric format.

cmz_fgcm_fm_ratio.csv

•	Description: Provides the fecal glucocorticoid metabolite (FGCM) to fecal matter (FM) ratio for various reserves. Includes columns for reserve names, FGCM measurements, and FM measurements.

Format:CSV. No special formatting.

Sheets:One sheet. 

•	Role in Analysis: Employed in the S3_fgcm_fm_ratio.R script to calculate and analyze the FGCM/FM ratios, helping to understand the variability in metabolite concentrations.

NAs: There are no NAs within this csv.

Header	Meaning	Units
Reserve	The reserve initials. Intials: BKR: Bakkrans, CNP: Camdeboo National Park, DHNR_1, De Hoop Nature Reserve, GNR: Gamkaberg Nature Reserve, MCPR: Mount Camdeboo Private Reserve, SWT: Swartberg, WGN: Welgevonden Game Farm	NA. Character string.
GC_munro	Concentration of faecal glucocorticoids detecting using the CJM006 "munro" corticosterone enzyme immunoassay.	ng/g (concentration of metabolite in faeces). Numeric format.
GC_munro_se	Standard error of the faecal glucocorticoid metabolites analysed using the CJM006 "munro" corticosterone enzyme immunoassay.	Dimensionless standard deviation. Numeric format.
X72T	Concentration of faecal glucocorticoids detecting using the 72T metabolite specific enzyme immunoassay.	ng/g (concentration of metabolite in faeces). Numeric format.
X72T_se	Standard error of the faecal glucocorticoid metabolites analysed using the 72T enzyme immunoassay.	Dimensionless standard error. Numeric format.
pg_all	Population growth rate of the population.	Rate of growth of population (lamdba). Numeric format.
fm	Foals per mare in the population	ratio of the number of foals in the populations per mare in the population. Numeric format.
density	Density of Cape mountain zebra within the population	individuals per km squared. Numeric format.

cmz_fgcm_macroecology_reanalysis.csv

•	Description: Contains extensive data on FGCM measurements along with associated macroecological variables such as reserve, season, individual IDs, and environmental conditions.

Format: CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Used in the S4_reanalysis_models_cjm006_72t.R script to perform reanalysis of FGCM data, considering various ecological and biological factors.

NAs: NAs are denoted with NA. NAs occur when information was either not collected or is unavailable.

Header	Meaning	Units
Sample	Sample Number	NA. Numeric integer.
Code	Sample ID	NA. Character string.
Reserve	The reserve initials. Intials: BKR: Bakkrans, CNP: Camdeboo National Park, DHNR_1, De Hoop Nature Reserve, GNR: Gamkaberg Nature Reserve, MCPR: Mount Camdeboo Private Reserve, SWT: Swartberg, WGN: Welgevonden Game Farm	NA. Character string.
Season	Season that the sample was collected in during the first trip	NA. Character string.
Season2	Season that the sample was collected in during the second trip	NA. Character string.
VI	Vegetation index (estimate of the grassiness of the habitat/reserve)	Standardized unitless metric. Numeric format.
Frozen	Whether the same was frozen or not.	NA. Boolean Y/N
Dry weight	Weight of the faecal sample after full evaporation of water in the faeces and constant weight	grams (g). Numeric format
Date	Date/Month/ Year that the sample was collected	D/MM/YY. Date format
Day	Day of sample collection starting from first day of sampling	NA. Numeric integer.
Time	Time of day that the sample was collected.	Hour.minute. Numeric integer.
Group	Group that the Cape mountain zebra sampled belonged to.	Group number. Numeric integer/
Individual	Details on the individual that the faecal samples were collected from	NA. Character string.
Group_Size	Number of individuals within the same group of the individual that the faecal sample was collected from.	Individuals. Numeric integer.
Sex	Sex of the individual	Sex of the individual. M/F.
Details	Details on the social position of the individual.	NA. Character string.
Trip	Which sampling trip the faecal sample was collected within.	Trip 1 or Trip 2.
ID	Unique identifier for the individual that the faecal sample was collected from	NA. Character string.
GC_munro	Concentration of faecal glucocorticoids detecting using the CJM006 "munro" corticosterone enzyme immunoassay.	ng/g (concentration of metabolite in faeces). Numeric format.
GC_munro_cor	Concentration of faecal glucocorticoids detecting using the CJM006 "munro" corticosterone enzyme immunoassay. Corrected for corticosterone estimate.	ng/g (concentration of metabolite in faeces). Numeric format.
GC_munro_DM	Concentration of faecal glucocorticoids detecting using the CJM006 "munro" corticosterone enzyme immunoassay corrected for Dry Matter in the faeces.	ng/g (concentration of metabolite in faeces). Numeric format.
72t Merl 1:10 10¬µl	Concentration of faecal glucocorticoids detecting using the 72T metabolite specific enzyme immunoassay at a 1:10 dilution.	ng/g (concentration of metabolite in faeces). Numeric format.
72T	Concentration of faecal glucocorticoids detecting using the 72T metabolite specific enzyme immunoassay. Corrected for dilution factor	ng/g (concentration of metabolite in faeces). Numeric format.
T3	Concentration of faecal thyroid hormone (T3) levels detecting during the Lea et al 2017 study.	ng/g (concentration of metabolite in faeces). Numeric format.
T3_cor	Concentration of faecal thyroid hormone (T3) levels detecting during the Lea et al 2017 study. Corrected for dilution factor	ng/g (concentration of metabolite in faeces). Numeric format.
pg_all	Population growth rate of the population.	Rate of growth of population (lamdba). Numeric format.
fm	Foals per mare in the population	ratio of the number of foals in the populations per mare in the population. Numeric format.
density	Density of Cape mountain zebra within the population	individuals per km squared. Numeric format.
sex_ratio	Number of Males to females in the population	Ratio. Numeric format.
max_temp	Maximum temperature from remote sensing for interval of sampling.	Degrees Celsius. Numeric format.
min_temp	Minimum temperature from remote sensing for interval of sampling.	Degrees Celsius. Numeric format.
av_temp	Minimum temperature from remote sensing for interval of sampling.	Degrees Celsius. Numeric format.
precip	Amount of precipitation calculated from remote sensing for interval of sampling.	millimetres (mm). Numeric format.
precip3	Amount of precipitation in three months prior to sampling calculated from remote sensing	millimetres (mm). Numeric format.
net_con	Network connectivity within social network of the population.	Unitless. Numeric format.
gr_var	grassiness variation.	Unitless. Numeric format.
prop_bat	Proportion of batchelors within the population	Ratio of bachelor males to stallions
min_temp2	Minimum temperature in 2 month around sampling from remote sensing.	Degrees Celsius. Numeric format.
precip2	Amount of precipitation in two months prior to sampling calculated from remote sensing	millimetres (mm). Numeric format.
Grassiness	Equivalent to Vegetation index. Estimated proportion of estimated grass available within habitat	Standardized unitless metric. Numeric format.
Season3	Duplicated season.	NA. Season
Rainfall	Whether rainfall in the habitat is seasonal or aseasonal	NA. Character string. Seasonal or aseasonal.
Growth	Population growth rate of the population.	Rate of growth of population (lamdba). Numeric format.
Day_of_year	Day of the year	Day. Integer
Month	Month of the year	Month. Integer
Dekad	Dekad of the year	Dekad. Integer

cmz_fgcm_translocation.csv

•	Description: Includes FGCM measurements related to translocation events. Contains columns for sample identifiers, assay types, pre/post-translocation statuses, and metabolite concentrations.

Format: CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Utilized in the S5_supp_cartridge_loss.R script to assess the impact of translocation on FGCM levels.

NAs: There are no NAs within this csv.

Header	Meaning	Units
Sample	Sample ID	NA. Character string.
Assay	The Enzyme immunoassay that used to generate concentrations of metabolites	Character string.
pre_post	Whether the sample was collected Pre or Post translocation.	NA. Character string.
ng/g	nanograms per gram of faeces for metabolite (concentration of metabolite in faeces).	ng/g (concentration of metabolite in faeces). Numeric format.
percentage_meanbaseline	Percentage of change in metabolite concentration from mean baseline of all samples (i.e. average of all samples)	Percentage. Numeric format.
percentage_from_pre	Percentage of change in metabolite concentration from pre baseline of all samples (i.e. average of all pre samples only)	Percentage. Numeric format.
percentage_meanbaseline_stand	Standardized Percentage of change in metabolite concentration from mean baseline of all samples (i.e. average of all samples)	Percentage. Numeric format.

cmz_macroecology_72t_cjm006_comparsion.csv

•	Description: Provides a comparison of FGCM concentrations using the 72T and CJM006 assays across different sampling trips, reserves, and seasons.

Format:CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Used in the S5_supp_reanalysis_sex_res_season_cjm006_72t.R script to compare and analyze the performance of the two assays under various ecological conditions.

NAs: NAs occur in this document. NAs here are all in the androgen metabolite concentration columns. Lea et al 2017 did not test for androgen metabolite in female faeces.

Header	Meaning	Units
Sampling trip	Which sampling trip the faecal sample was collected within.	Trip 1 or Trip 2.
Sample no. (Trip	Which sampling trip the faecal sample was collected within.	Trip 1 or Trip 2.
sample	Sample ID	NA. Character string.
Reserve	The reserve initials. Intials: BKR: Bakkrans, CNP: Camdeboo National Park, DHNR_1, De Hoop Nature Reserve, GNR: Gamkaberg Nature Reserve, MCPR: Mount Camdeboo Private Reserve, SWT: Swartberg, WGN: Welgevonden Game Farm	NA. Character string.
Season	Season that the sample was collected	NA. Character string.
Rainfall seasonality	Whether rainfall in the habitat is seasonal or aseasonal	NA. Character string. Seasonal or aseasonal.
Rainfall_2seasonality_2	Whether rainfall in the habitat is seasonal or aseasonal	NA. Character string. Seasonal or aseasonal.
Grass abundance	Vegetation index (estimate of the grassiness of the habitat/reserve)	Standardized unitless metric. Numeric format.
Individual ID code	Unique identifier for the individual that the faecal sample was collected from.	NA. Character string.
Sex	Sex of the individual	Sex of the individual. M/F.
Details	Details on the social position of the individual.	NA. Character string.
Sex ratio (M:F)	Number of Males to females in the population	Ratio. Numeric format.
Group size	Number of individuals within the same group of the individual that the faecal sample was collected from.	Individuals. Numeric integer.
Glucocorticoid metabolite concentration (ng/g)	Concentration of faecal glucocorticoids detecting using the CJM006 "munro" corticosterone enzyme immunoassay. Corrected for corticosterone estimate.	ng/g (concentration of metabolite in faeces). Numeric format.
Androgen metabolite concentration (ng/g)	Concentration of faecal androgen hormone (Testosterone) levels detecting during the Lea et al 2017 study.	ng/g (concentration of metabolite in faeces). Numeric format.
72t metabolite concentration (ng/g)	Concentration of faecal glucocorticoids detecting using the 72T metabolite specific enzyme immunoassay. Corrected for dilution factor	ng/g (concentration of metabolite in faeces). Numeric format.
Population growth rate	Population growth rate of the population.	Rate of growth of population (lamdba). Numeric format.
Mean foal:mare ratio	Foals per mare in the population	ratio of the number of foals in the populations per mare in the population. Numeric format.

cmz_translocation_corrplot.csv

•	Description: Contains correlation data for FGCM measurements using different extraction methods (Methanol and Diethylether) and assays (72a, 72T, 69a, Arbor Corticosterone, CJM006 Corticosterone).

Format:CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Used in the S5_supp_corrplots.R script to generate correlation plots, helping to visualize the relationships between different extraction methods and assays.

NAs: No NAs occur in this csv.

Header	Meaning	Units
72a Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
72a Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
72T Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
72T Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
69a Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
69a Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
Arbor Corticosterone	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), Arbor Cortiocosterone .	ng/g (concentration of metabolite in faeces). Numeric format.
CJM006 Corticosterone	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), CJM006 Cortiocosterone.	ng/g (concentration of metabolite in faeces). Numeric format.

cort_parallelism_and_validation.csv

•	Description: Provides data for validating the parallelism of corticosterone assays. Includes columns for sample identifiers, optical density (OD), B/B0 ratios, and concentration values.

Format:CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Employed in the S5_supp_parallelism.R script to validate the consistency and reliability of corticosterone assays across different samples.

NAs: No NAs occur in this csv.

Header	Meaning	Units
Sample	Sample ID	NA. Character string.
OD	Optimal density reading from Enzyme immunoassay	Unitless. Numeric format.
B/B0	Binding of metabolite within the enzyme immunoassay (EIA) at the specified concentration divided by 100% binding within the same EIA	Proportion of binding against absolute binding. Numeric format. (Proportion)
logOD	Log Optimal density reading	Unitless.Numeric format.
concentration	nanograms per gram of faeces for sample.	ng/g (concentration of sample). Numeric format.
log_concentration	Log concentration	log ng/g (concentration of sample). Numeric format.
Binding	Binding of metabolite within the enzyme immunoassay (EIA) at the specified concentration binding within the same EIA	Proportion of binding . Numeric format.
log_binding	Binding of metabolite within the enzyme immunoassay (EIA) at the specified concentration divided by 100% binding within the same EIA	Log Proportion of binding. Numeric format.

mz_longitudual_fgcm_zoo_samples.csv

•	Description: Contains longitudinal FGCM data for zoo samples, including individual identifiers, extraction methods, storage conditions, assay types, and metabolite concentrations.

Format:CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Utilized in the S1_fgcm_zoo_movement.R script to analyze the movement patterns of zoo animals and their FGCM levels over time.

NAs: NAs are denoted by NA. NAs occur in this document as some samples were too low to be above the detection limit for the Enzyme immunoassay or because the percentage baselines were calculated from that sample.

Header	Meaning	Units
Individual	Individual ID	NA. Character string.
Extraction	The extraction technique used to extract the faecal glucocortiocoids	NA. Character string.
Storage	The storage technique used to store the faecal glucocorticoids	NA. Character string.
Date	The day in order that the sample was collected	NA. Numeric Integer
Pre/post	Whether the sample was collected Pre or Post translocation.	NA. Character string.
Metabolite	The metabolite that the Enzyme immunoassay targets to generate concentrations of metabolites	Character string.
Assay	The Enzyme immunoassay that used to generate concentrations of metabolites	Character string.
Assay_cartridge	The Enzyme immunoassay and whether storage technique combined that used to generate concentrations of metabolites	Character string.
ng/g	nanograms per gram of faeces for metabolite (concentration of metabolite in faeces).	ng/g (concentration of metabolite in faeces). Numeric format.
percentage_initial_sample	Percentage of change in metabolite concentration from first of all samples (	Percentage. Numeric format.
percentage_mean_pre	Percentage of change in metabolite concentration from pre baseline of all samples (i.e. average of all pre samples only)	Percentage. Numeric format.
percentage_mean_baseline	Standardized Percentage of change in metabolite concentration from mean baseline of all samples (i.e. average of all samples)	Percentage. Numeric format.

zoo_validation_corrplot.csv

•	Description: Contains correlation data for FGCM measurements using different extraction methods (Methanol and Diethylether) and assays (72a, 72T, 69a, Arbor Corticosterone, CJM006 Corticosterone) for Hartmann's zebra zoo translocation samples

Format:CSV. No special formatting.

Sheets:One sheet.

•	Role in Analysis: Used in the S5_supp_corrplots.R script to generate correlation plots, helping to visualize the relationships between different extraction methods and assays.

NAs: There are no NAs within this csv.

Header	Meaning	Units
72a Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
72a Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72a. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
72T Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
72T Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 72T. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
69a Methanol	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The Methanol denotes the extraction protocol used to extract the faecal glucocorticoids.	ng/g (concentration of metabolite in faeces). Numeric format.
69a Diethylether	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), 69a. The Diethylether denotes the extraction protocol used to extract the faecal glucocorticoids	ng/g (concentration of metabolite in faeces). Numeric format.
Arbor Corticosterone	Concentration of faecal glucocorticoids detecting using the enzyme immunoassay (EIA), Arbor Cortiocosterone .	ng/g (concentration of metabolite in faeces). Numeric format.

Laboratory outputs

Laboratory outputs are additional csv and xlsx files that contain all raw, unprocessed data that were outputted from laboratory equipment. These have already been collated into the data files for analysis but for transparency and reproducibility we include them all here. These are usually XLSX documents with multiple sheets. They are NOT a traditional datasheet format and we strongly encourage users not attempt to use them in analysis directly. Below we describe each laboratory output with a table of what is contained within each sheet and explain the formatting.

LAB FILES OVERVIEW

There are five specially formatted outputs in Excel format, each related to different aspects of corticosterone and metabolite validation studies. Below is a detailed description of each file, including their structure and key columns.

Files and Descriptions

arbor_cort_cmz_translocation.xlsx

Description: This file contains raw data on corticosterone measurements in a study involving CMZ translocation.

Sheet	Description	Format explanation	Key Features
Results_validation	Datasheet for the metabolite concentrations from the Cape mountain zebra translation	This sheet is formatted like a traditional spreadsheet	`Sample`: Identifier for each sample.`individual`: Identifier for each individual in the study.`avgconc`: Average concentration of corticosterone.`pre/post`: Indicates if the measurement was taken before (pre) or after (post) translocation.`logconc`: Logarithm of the concentration.`extractvol`: Volume of the extract used.`df`: Dilution factor.`faecalweight`: Weight of the faecal sample.`samplevolume`: Volume of the sample.`ng/g`: Nanograms per gram of faeces.`logng_g`: Logarithm of ng/g values.

parl_omit_standards_nonlinearpt	Datasheet for the metabolite concentrations from the Cape mountain zebra parallelism	This sheet is formatted like a traditional spreadsheet	`Sample`: Identifier for each sample.`OD`: Optimal Density measurement.`logOD`: Logarithm of the OD values.`concentration`: Concentration of the measured substance.`log_concentration`: Logarithm of the concentration values.`Binding`: Binding measurement.`log_binding`: Logarithm of the binding values.

Plate map	This is the plate map used for the Enzyme immunoassay. This is plate 1 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
Parallelism plate 10_09_20	This is the Raw output from the Enzyme immunoassay. This is display the raw optical density values per cell of the enzyme immunoassay	This is a special format where the raw optimal density values of a 96 well enzyme immunoassay is displayed.	NA
myassays_10_09_20	This is the Raw output from my assays analyses. My assays is a online software for processing raw optimal density outputs.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Plate Map validation	This is the plate map used for the Enzyme immunoassay. This is plate used for validation layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
Validation plate 15_02_09	This is the Raw output from the Enzyme immunoassay. This is display the raw optical density values per cell of the enzyme immunoassay	This is a special format where the raw optimal density values of a 96 well enzyme immunoassay is displayed.	NA
Validation plate myassay	This is the Raw output from my assays analyses. My assays is a online software for processing raw optimal density outputs.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.

arbor_cort_validation_mz_zoo.xlsx

Description: This file includes validation data for corticosterone measurements in a zoo setting.

Sheet	Description	Format explanation	Key Features
CV <15	This sheet displays all samples that have a coefficient of variation less than 15. In the field, a CV greater than 15 is must be reanalysed as there is little certainty that the concentration is reproducible.	This sheet is formatted like a traditional spreadsheet	`Sample`: Identifier for each sample.`individual`: Identifier for each individual in the study.`avgconc`: Average concentration of corticosterone.`pre/post`: Indicates if the measurement was taken before (pre) or after (post) translocation.`logconc`: Logarithm of the concentration.`extractvol`: Volume of the extract used.`df`: Dilution factor.`faecalweight`: Weight of the faecal sample.`samplevolume`: Volume of the sample.`ng/g`: Nanograms per gram of faeces
all_Data	This sheet displays all samples.	This sheet is formatted like a traditional spreadsheet	`Sample`: Identifier for each sample.`individual`: Identifier for each individual in the study.`avgconc`: Average concentration of corticosterone.`pre/post`: Indicates if the measurement was taken before (pre) or after (post) translocation.`logconc`: Logarithm of the concentration.`extractvol`: Volume of the extract used.`df`: Dilution factor.`faecalweight`: Weight of the faecal sample.`samplevolume`: Volume of the sample.`ng/g`: Nanograms per gram of faeces
Plate map plate1	This is the plate map used for the Enzyme immunoassay. This is plate 1 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
RAW readings plate 1	This is the Raw output from the Enzyme immunoassay. This is display the raw optical density values per cell of the enzyme immunoassay	This is a special format where the raw optimal density values of a 96 well enzyme immunoassay is displayed.	NA
Stan curve 1	This is the Raw output from my assays analyses. My assays is a online software for processing raw optimal density outputs.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Plate map Plate 2	This is the plate map used for the Enzyme immunoassay. This is plate 2 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
RAW readings plate 2	This is the Raw output from the Enzyme immunoassay. This is display the raw optical density values per cell of the enzyme immunoassay	This is a special format where the raw optimal density values of a 96 well enzyme immunoassay is displayed.	NA
Standard curve P2	This is the Raw output from my assays analyses. My assays is a online software for processing raw optimal density outputs.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Plate map3	This is the plate map used for the Enzyme immunoassay. This is plate 3 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
RAW readings plate 3	This is the Raw output from the Enzyme immunoassay. This is display the raw optical density values per cell of the enzyme immunoassay	This is a special format where the raw optimal density values of a 96 well enzyme immunoassay is displayed.	NA
Standard Curve P3	This is the Raw output from my assays analyses. My assays is a online software for processing raw optimal density outputs.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.

cjm006_validation_both.xlsx

Description: This file contains validation data for CJM006 in both zoo and other contexts.

Sheet	Description	Format explanation	Key Features
Plate map 1	This is the plate map used for the Enzyme immunoassay. This is plate 1 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
Plate 1 results	This is the processed from analysis of optimal density analysis. In this spreadsheet, we generated our own conversion and standard curve estimates to calculate concentration.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Plate map 2	This is the plate map used for the Enzyme immunoassay. This is plate 2 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
Plate 2 results	This is the processed from analysis of optimal density analysis. In this spreadsheet, we generated our own conversion and standard curve estimates to calculate concentration.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Plate map 3	This is the plate map used for the Enzyme immunoassay. This is plate3 layout for this experiment.	This is a special format where the layout of a 96 well enzyme immunoassay is displayed.	NA
Plate 3 results	This is the processed from analysis of optimal density analysis. In this spreadsheet, we generated our own conversion and standard curve estimates to calculate concentration.	This is a special format where the standard curve for the enzyme immunoassay is calculated to correct and estimate concentraton of the metabolite measured for each sample.	A standard curve is displayed at the top left of the document. Corrected estimates of concentration from optimal density markers are below.
Inter-assay variation	In this sheet we calculate inter-assay variation from our pooled samples across assays.	This is not formatted to any standard. It is simply the concentrations of the assay in the top left and then the calculation furth below	NA
Parallelism	This sheet displays metabolite concentrations for the parallelism samples. This is the pooled sample and the samples.	This sheet is formatted like a traditional spreadsheet.	`Sample`: Identifier for each sample. B/b0: Binding measurement of sample divided by total binding. concentration: Nanograms per gram of faeces
Results	This sheet displays metabolite concentrations all samples.	This sheet is formatted like a traditional spreadsheet.	`Sample`: Identifier for each sample. cv: coefficient of variation of the sample. concentration4pl: Nanograms per gram of faeces of the first sample. concentration4pl2: Nanograms per gram of faeces of the sample if it needed to be replicated.concentration**_avg average Nanograms per gram of faeces of the sample.

metabolite_validation_cmz_translocation.xlsx

Description: This file includes validation data for metabolites in the context of CMZ translocation.

Sheet	Description	Format explanation	Key Features
Sheet1	This sheet displays metabolite concentrations all samples.	This sheet is formatted like a traditional spreadsheet but headings are formatted slightly differently as colours correspond to the different metabolites used.	`No`: Sample number.`Experiment`: Type of experiment.`Reserve`: Reserve identifier.`Sample ID`: Identifier for each sample.`Tube ID`: Tube identifier.`Date`: Date of sample collection.`Time`: Time of sample collection.`Perturbation`: Perturbation details.`ID`: Individual identifier.`Band`: Band identifier.`Sex`: Sex of the individual.`age`: Age of the individual.Multiple columns for `pg/50µl`, `ng/g`, and `%`: Concentration metrics and percentages for different substances.

metabolite_validation_mz_zoo.xlsx

Description: This file includes validation data for metabolites in a zoo setting.

Sheet	Description	Format explanation	Key Features
Sheet1	This sheet displays metabolite concentrations all samples.	This sheet is formatted like a traditional spreadsheet but headings are formatted slightly differently as colours correspond to the different metabolites used.	`No`: Sample number.`Experiment`: Type of experiment. Extraction: Extraction technique.`Individual` : Individual that the sample was collected from.`Date`: Date of sample collection.Multiple columns for `pg/50µl`, `ng/g`, and `%`: Concentration metrics and percentages for different substances.

Sharing/Access information

This is a section for linking to other ways to access the data, and for linking to sources the data is derived from, if any.

This project builds on the original dataset within:

DOI: 10.1111/1365-2435.13000

Code/Software

All analyses were preformed in R. We demonstrated reproducibility of our results on Monday 24th July 2024 on R version 4.3.3 (2024-02-29) - "Angel Food Cake". Platform: aarch64-apple-darwin20.

Importantly, for use, we encourage users to edit the SET DIRECTORIES section at the top of each script to the directory in which they are keeping the data files. All scripts are heavily annotated and we have attempted to give all functions calls a namespace:: call. This will inform users which package we are using for each function call. Packages are also called at the top of each script users the traditional library() call.

CODE OVERVIEW

There are multiple scripts and we encourage users to run in the following order set:

All scripts are described in using both annotations in the scripts as well as briefly here.

Scripts should be run using R either on a computer or on a cluster.

1.	S1_fgcm_zoo_movement

2.	S2_fgcm_cmz_validation

3.	S3_fgcm_fm_ratio

4.	S4_reanalysis_models_cjm006_72t

All scripts that begin with S5... denote scripts to generate all information within the supplementary material of the article.

CODE DESCRIPTIONS

1. S1_fgcm_zoo_movement.R

This script analyzes the movement patterns of zoo animals and their fecal glucocorticoid metabolite (FGCM) levels. It includes data cleaning, transformation, and visualization steps to explore the relationships between animal movements and FGCM levels.

2. S2_fgcm_cmz_validation.R

This script performs validation of the FGCM data for different animal populations. It includes statistical analyses and model fitting to ensure the reliability of the FGCM measurements. The script also generates visualizations to compare observed and predicted FGCM levels.

3. S3_fgcm_fm_ratio.R

This script calculates the fecal glucocorticoid metabolite (FGCM) to fecal matter (FM) ratio. It includes data processing steps to compute the ratios and performs statistical analyses to assess the significance of the findings. The script also includes plots to visualize the FGCM/FM ratios across different samples.

4. S4_reanalysis_models_cjm006_72t.R

This script reanalyzes data using various statistical models, focusing on the CJM006 and 72T assays. It includes model fitting, comparison of different models using Akaike Information Criterion (AIC), and generation of summary statistics. The script also includes visualizations to illustrate the model results and the relationships between variables.

5. S5_supp_cartridge_comparsions_zoo.R

This script performs supplementary analyses to compare different assay cartridges used in zoo studies. It includes statistical comparisons of assay performance, visualization of assay results, and interpretation of the findings. The script generates plots to illustrate differences in assay performance and reliability across different cartridges.

6. S5_supp_cartridge_loss.R

This script evaluates the loss of glucocorticoid metabolites during the extraction process using different cartridges. It includes data processing, statistical comparisons, and visualizations to assess and compare the performance of various cartridges in minimizing metabolite loss.

7. S5_supp_cmz_validation_diethylether.R

This script validates the use of diethylether as a solvent for glucocorticoid metabolite extraction in CMZ samples. It involves statistical analyses and comparisons with other solvents to determine the efficacy and reliability of diethylether in the extraction process. The script also generates plots to illustrate the validation results.

8. S5_supp_corrplots.R

This script generates correlation plots to visualize the relationships between different variables in the dataset. It includes data preparation, calculation of correlation coefficients, and creation of graphical representations to highlight significant correlations among variables.

9. S5_supp_diethylether_cmz_validation.R

Similar to S5_supp_cmz_validation_diethylether.R, this script focuses on validating the use of diethylether for extracting glucocorticoid metabolites, specifically for CMZ samples. It includes detailed statistical analyses, model fitting, and visualizations to support the validation process.

10. S5_supp_diethylether_extraction_comparsions.R

This script compares the efficiency and reliability of diethylether extraction against other extraction methods. It involves processing experimental data, performing statistical tests, and generating comparative plots to evaluate the performance of diethylether extraction in glucocorticoid metabolite analysis.

11. S5_supp_parallelism.R

This script assesses the parallelism of different glucocorticoid metabolite assays. It includes statistical analyses to determine the consistency and reliability of assay results across different samples. The script also generates plots to visualize the parallelism and ensure the assays' validity.

12. S5_supp_reanalysis_sex_res_season_cjm006_72t.R

This script reanalyzes the data focusing on the effects of sex, reserve, and season on glucocorticoid metabolite concentrations using the CJM006 and 72T assays. It involves advanced statistical modeling, comparison of different models, and generation of visualizations to illustrate the findings and their implications.

Data and code for: Previous assessments of faecal glucocorticoid metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed

Data files

Abstract

README: Data and code for: Previous assessments of faecal glucocorticoid metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed.

Description of the data and file structure

Laboratory outputs

Files and Descriptions

Sharing/Access information

Code/Software

Methods

Works referencing this dataset