Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species.

Assessment of luteinizing hormone and prolactin immunoactivity in Asian and African elephant urine using assays validated for serum.

Analysis of serum hormones is useful for timing artificial insemination (Luteinizing hormone) and diagnosing pregnancy (prolactin) in elephants. However, these tests require blood collection, which is not tolerated by all animals, and is impractical for field studies. Thus, developing a means to obtain these measures noninvasively could improve species management. Matched urine and serum was collected from Asian and African elephants daily throughout the follicular phase and after administration of a GnRH analogue for LH determination, and in pregnant and nonpregnant females for prolactin analyses using immunoassays validated for elephant serum. Despite identifying robust increases in circulating hormone concentrations, no concomitant changes in urinary LH or prolactin immunoactivity was detected. Concentration of samples by centrifugal filtration or ethanol precipitation did not increase the ability to measure biologically relevant changes in endogenous urinary LH or prolactin immunoactivity. Sample matrix interference was ruled out following sufficient recovery of exogenous LH or prolactin added to samples, except for samples concentrated >35-fold where some interference was suspected. These results suggest that elephants either do not excrete native LH or prolactin in urine, or concentrations are too low to be measured accurately by standard immunoassay techniques that are valid for serum analyses. Thus, it does not appear feasible or economically viable to use these noninvasive tests for ovulation detection or for pregnancy diagnosis in elephants.