Ethics statement: procedures involving field sampling and animal subjects have been approved by the School of Animal, Rural and Environmental Science (ARES) at Nottingham Trent University.

1. Collection of Fecal Samples

NOTE: Gloves should be worn when handling fecal samples and methanol. If there is a strong suspicion that an animal could be suffering from a zoonotic disease, protective clothing such as a lab coat should also be worn.

1. Collect the fecal samples as soon as possible (within min to a few hr) following defecation and place them into a sample bag. Take three to five subsamples of any defecated sample with a total weight of approximately 10 g. Label the bag with the time and date of collection, individual ID, and freeze the sample at -20 °C until analysis.
2. To minimize error, freeze all samples at -20 °C until the study is complete then gather the samples together for analysis as a single batch.

2. Fecal Hormone Extraction

1. Leave the fecal samples to thaw at RT.
2. Ensure that the samples are homogenized by manually mixing each sample in its own sample bag.
3. For each sample weigh 0.50 (± 0.003 g) of fecal material on a microbalance in a weigh boat then transfer the feces to a 8 ml glass vial. Use a new weigh boat for each sample and clean the tweezers with 30% methanol between each sample.
4. Combine the 0.5 g of fecal material with 5 ml of 90% methanol: water, and shake O/N on an orbital shaker at RT.
5. The following morning, vortex the sample and then centrifuge for 20 min at 600 g.
6. Decant the methanol fraction into a 16 x 125 mm² glass tube and place in a rack in a water bath set to a temperature of 37 °C. Evaporate to dryness using air in a fume cupboard. Rinse the extraction vial and place the remaining fecal pellet into a clinical waste bag for disposal.
7. Re-suspend the fecal extracts in 100%, 1 ml methanol and transfer to a 12 x 75 mm² polypropylene tube. Immediately cap and store at -20 °C until analysis.

3. Hormone Analysis

1. Carry out the analysis with an enzyme linked-immunoassay (EIA) using polyclonal corticosterone CJM006 antiserum.
   NOTE: A second EIA was tested (Cortisol R4866 antiserum, but this failed to achieve the biochemical validation step (See Section 4.1). The Polyclonal corticosterone CJM006 antiserum and Cortisol R4866 antiserum and the respective HRP's are supplied by CJ Munro, University of California, Davis, CA.
2. Dilute the antiserum at 1:15,000 in coating buffer (0.05 M NaHCO3, pH 9.6) using a repetitive pipette and a 50 µl pipette tip, load a 96-well microtitre plate with 50 µl/well. Cover the plate with a plate sealer and incubate O/N at 4 °C. Leave two wells uncoated to represent the non-specific binding wells (NSB).
3. Immediately before use wash the microtitre plates five times using an automated microtitre plate washer (wash solution; 0.15 M NaCl, 0.05% Tween 20).
4. Load the plates with 50 µl per well for the NSB's and 'zero' wells [EIA buffer (0.1 M NaPO₄, 0.149 M NaCl, 0.1% bovine serum albumin, pH 7.0) only], standards (corticosterone, 3.9 – 1,000 pg/well) or fecal extract samples (appropriately diluted at 1:20 EIA buffer, see section 4.1). Standards and zeros should be run in triplicate and quadruplicate, respectively. Diluted fecal extract samples and NSB's should be run in duplicate.
5. Follow this immediately with addition of 50 µl/well of the label horseradish peroxidase conjugate^ᵻ diluted in EIA buffer to 1:70,000.
6. Incubate the microtitre plates in the dark for 2 hr at RT.
7. Wash the microtitre plates with wash solution 5 times and incubate the plates at RT in the dark with 100 µl/well of RT substrate [0.4 mM 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt, 1.6 mM H₂O₂, 0.05 M citrate, pH 4.0]. Prepare the substrate immediately prior to use.
8. Use a spectrophometer at a wavelength of 405 nm to read the microtitre plates. Incubation of plates is considered complete when the optical density of the zero wells reaches 0.8 to 1.0.
9. To insure repeatability, use controls [EIA standards or a biological sample diluted in EIA buffer to bind at approximately 30% (EIA standard), 50% (a biological sample e.g., pool of domestic horse fecal extract) and 70% (EIA standard)] to demonstrate an intra- and inter-assay coefficients of variation of < 10% and < 15% respectively.

4. Enzyme Linked-immunoassay: Validation for the Study Species and Sex

1. Biochemical Validation (Parallelism)
   1. Using EIA buffer, perform a serial dilution on pooled fecal extract, ideally taken from suspected high and low hormone concentration samples from several individuals (range: neat, 1:2 to 1:8192) and run on the EIA as described in section 3.
      NOTE: A parallelism is considered achieved when the serial dilutions of fecal extracts yield a displacement curve parallel to the standard curve and linear regression results demonstrate R2 > 0.90, a slope > 0.50 and p < 0.05. The serial diluted fecal extract that binds around 50% on the standard curve should be considered the ideal dilution to run all fecal extracts (e.g., 1:20). There is no interference considered on the assay when the linear regression results demonstrate R2 > 0.90 and p < 0.05.
2. Biochemical Validation (Interference)
   1. Spike 200 µl of serially diluted standards (zero, 3.9 to 1000 ng/well corticosterone) with 200 µl of the appropriately diluted pooled fecal extract [diluted at the 50% binding determined by parallelism (1:20)] and run on the EIA as in section 3. Following EIA analysis plot the observed concentration minus the background (concentration of the zero sample) versus the expected concentration.
      NOTE: If the addition of diluted pooled fecal extract to standards does not significantly alter the amount expected and linear regression results yields R² > 0.90, a slope close to 1.0 and p < 0.05. No interference with the EIA has been achieved.
3. Biological Validation:
   NOTE: A demonstration that the chosen EIA has the ability to measure biologically relevant changes of the hormone of interest [e.g., the rise in glucocorticoid concentrations following a pharmacological (ACTH is often a licenced procedure) or biological challenge (e.g., psychological and/or environmental; more likely to be an opportunistic unregulated event)].
   1. Select, extract and analyse fecal samples for glucocorticoid metabolite concentrations using the process detailed in sections 1 – 3 before and after a challenging event.
      NOTE: A challenging event will be species specific. An example method is provided below for the domestic horse. Full examples of challenging events for horses have been published previously including the use of management⁹ and husbandry procedures¹⁶. A significant rise in glucocorticoid metabolite concentrations following the challenging event must be observed and confirmed using statistical analysis. This must incorporate where appropriate, the effect of repeated measures, sample date and individual. Biological validation is then considered achieved.