Characterization of Longitudinal Testosterone, Cortisol, and Musth in Male Asian Elephants (Elephas maximus), Effects of Aging, and Adrenal Responses to Social Changes and Health Events.

The conservation of endangered species and sustainability of managed populations requires considerations to ensure the health and welfare of individuals. Male elephants experience a biological phenomenon called "musth", which is characterized by increased testosterone production, temporal gland secretion and urine dribbling, heightened aggression and sexual behavior, and therefore can pose unique challenges for human safety and animal welfare. This study characterized longitudinal (9 to 22 years) patterns of circulating testosterone and cortisol in relation to musth in four adult Asian elephant bulls spanning ages from 12 to 54 years. Age-related effects on musth activity and adrenal responses to social changes and clinical health events were also examined. All bulls exhibited regular annual musth cycles. Circulating cortisol covaried positively with testosterone and musth, highlighting intrinsic patterns that should be considered when evaluating the impact of social, health, and environmental changes on adrenal glucocorticoid activity. Except for an end-of-life cortisol increase in one bull, there was no clear evidence of chronically elevated cortisol secretion outside of musth in any individual. Testosterone decreased with age in sexually mature bulls, whereas age-related changes in cortisol varied across individuals, with the three older bulls showing the greatest rate of change during musth versus inter-musth periods. In contrast to physiological factors, there was no evidence of social factors, such as addition of a new male and death of male herdmates, impacting adrenal glucocorticoid activity in these bulls in the short term. Changes in cortisol were associated with treatment for Mycobacterium tuberculosis (M. tb) in two bulls, increasing after start of treatment and decreasing with cessation of treatment, but were not clearly associated with activation of disease. This study highlights the importance of longitudinal hormone monitoring to track changes in physiological function and responses to social, health, and environmental change in elephant bulls, which is important for making more informed decisions on how to manage male elephants under varying degrees of human care to ensure welfare and safety.

Serum Health Biomarkers in African and Asian Elephants: Value Ranges and Clinical Values Indicative of the Immune Response.

Serum biomarkers indicative of inflammation and disease can provide useful information regarding host immune processes, responses to treatment and prognosis. The aims of this study were to assess the use of commercially available anti-equine reagents for the quantification of cytokines (tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL) 2, 6, and 10) in African (Loxodonta africana, n = 125) and Asian (Elephas maximus, n = 104) elephants, and alongside previously validated anti-human reagents for acute-phase proteins (serum amyloid A and haptoglobin), calculate species-specific biomarker value ranges. In addition, we used opportunistically collected samples to investigate the concentrations of each biomarker during identified clinical cases of illness or injury, as a first step to understanding what biomarkers may be useful to managing elephant health. Immune biomarkers were each elevated above the calculated species-specific value ranges in at least one clinical case, but due to variability in both clinical and non-clinical samples, only serum amyloid A was significantly higher in clinical compared to non-clinical paired samples, with tendencies for higher TNF-α and IL-10. We also detected increased secretion of serum amyloid A and all five cytokines following routine vaccination of a single Asian elephant, indicating that these biomarkers can be beneficial for studying normal immune processes as well as pathology. This study indicates that assays developed with commercial reagents can be used to quantify health biomarkers in wildlife species and identifies several that warrant further investigation to elucidate immune responses to various pathologies.

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.