Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress

Abstract

Despite being undomesticated, African Grey Parrots (Psittacus erithacus) are commonly found in captivity, in zoos or as pets. Captivity can be an ongoing stressor. Non-invasive glucocorticoid metabolites (GCM) measurements from bird droppings are of interest for assessing stress but require careful evaluation in each newly studied species. This study describes the assessment of such methods for Psittacus erithacus to provide tools for evaluating stress and monitoring welfare. We evaluated 12 method combinations of GCM extraction and enzyme immunoassay (EIA) from a pool of African Grey Parrot droppings, through the validation of several analytical parameters. Then, Heterophil: Lymphocyte Ratios (HLR), another reliable stress marker, were determined and correlated to individual dropping GCM concentrations for 29 birds to determine whether the method is biologically relevant. We found that the best procedure to measure GCM in African Grey Parrot droppings is a combination of 60% methanol extraction measured using a Corticosterone EIA kit (Cayman Chemical Company) from fresh or dry droppings. The establishment of a significant correlation (Pearson coefficient correlation = 0.48; p = 0.0082) between HLR and GCM in the studied population confirmed the method biological relevance. This method can thus be applied to assess stress in Psittacus erithacus and support welfare monitoring in zoo populations.

Keywords: Fecal Glucorticoid Metabolites; Heterophil:Lymphocyte Ratio; analytical validation; captivity; enzyme immunoassay; non-invasive stress measurement; parrot; welfare monitoring.

Figure 1

Test for parallelism. The supernatants of three extracts of African Grey Parrots’ droppings (SPL1, SPL2, SPL3) were serially diluted and compared to the standard curve (STD) of the CCC EIA kit. X-axis shows the concentration of GCM obtained from the kit in pg/mL. Y-axis is the percent of binding (B)/total binding (Bo).

Figure 2

Global precision profile established from the measurements of samples used during the procedure selection and analytical validation of the CCC EIA kit (n = 57) showing the obtained intra-assay %CV in function of the GCM concentrations in the EIA wells (pg/mL). The dotted line stands for the 20% limit of acceptable CV%.

Figure 3

Scatter plot displaying the relationship between individual droppings GCM concentrations (ng/g) and HLR in the studied populations of African Grey Parrots (n = 29). The dotted line and the solid line stands for the 70% and 80% prediction ellipses respectively.