DOI: 10.5061/dryad.vq83bk42d
title: Data and code for: Previous assessments of faecal glucocorticoid metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed

description: 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. 
description: The faecal samples were collected from Cape Mountain zebra
 <em>(Equus zebra zebra) </em>Hartmann's Mountain zebra
 <em>(Equus zebra hartmannae) </em>in South Africa and from
 European zoos respectively. We calculated faecal metabolite concentrations
 using 6 different glucocorticoid assays. All raw data generated is
 available as laboratory outputs as supplementary files. We used a mixed of
 custom and my assay software to convert raw optimal density estimates to
 metabolite concentration. All data files have been provided and all
 processing steps can be seen in the laboratory outputs supplementary xlsx
 files. 
description: # Data and code for: Previous assessments of faecal glucocorticoid
 metabolites in Cape Mountain zebra (Equus zebra zebra) were flawed.
 [https://doi.org/10.5061/dryad.vq83bk42d](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. 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. 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 &lt;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. concentration*4pl: 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.