Validation of enzyme-linked immunosorbent assay for measurement of faecal cortisol in fish

Quantification of glucocorticoid (GC) levels in faeces has become an established method for the non-invasive assessment of adrenocortical activity. These hormones are frequently determined in plasma samples as parameters of adrenal activity and response to stress. Because GCs are metabolized and excreted with both intact hormone and their metabolites present in faeces, the concentration of GCs can be measured in excreta. Faecal samples present the advantages of easy collection, no stress to the animal and elimination of the issue of potentially misleading acute GC spikes. The aim of this study was to determine if an enzyme-linked immunosorbent assay (ELISA) for cortisol was appropriate for monitoring adrenocortical activity in faecal casts of fishes. Performance of the cortisol ELISA was validated by comparison to high-performance liquid chromatography, which is an established method for measuring free GCs and GC metabolites in faeces. Parallelism and sample extraction efficiency were compared for the two methods. Pearson's correlation across samples for these methods was 0.996. Results demonstrated that the ELISA was an efficient, sensitive and reliable method for cortisol measurement in faecal extracts, which should permit integration of non-invasive stress monitoring into studies of fish behaviour and physiology.