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

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.