Simplified method to measure glucocorticoid metabolites in faeces of horses
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Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3α,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3α,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.
KeywordsACTH challenge Enzyme immunoassay Stress behaviour Cortisol
We thank Jürgen Heinze for helpful suggestions, Sandra Hake-Paulus, Petra Kölle, Anika Pachten, Bettina Wollanke (LMU Munich) and Hans-Peter Remler (LVFZ Haupt- und Landgestüt Schwaiganger Pferdehaltung) for assistance with conducting the experiment, Alexandra Kuchar and Andreas Trindl for technical assistance in the laboratory, and Henning Thies for language corrections. Horses have been provided by the LMU Munich and the LVFZ Haupt- und Landgestüt Schwaiganger Pferdehaltung. The study was supported by an Excellence grant of the Universität Bayern e.V. and an HWP II grant of the University of Regensburg.
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