Effects of ACTH, capture, and short term confinement on glucocorticoid concentrations in harlequin ducks (Histrionicus histrionicus).

Little is known about baseline concentrations of adrenal hormones and hormonal responses to stress in sea ducks, although significant population declines documented in several species suggest that sea ducks are exposed to increased levels of environmental stress. Such declines have been observed in geographically distinct harlequin duck populations. We performed an adrenocorticotropic hormone (ACTH) challenge to evaluate adrenal function and characterize corticosterone concentrations in captive harlequin ducks and investigated the effects of capture, surgery, and short term confinement on corticosterone concentrations in wild harlequin ducks. Harlequin ducks responded to the ACTH challenge with an average three-fold increase in serum corticosterone concentration approximately 90 min post injection, and a four- to five-fold increase in fecal glucocorticoid concentration 2 to 4 h post injection. Serum corticosterone concentrations in wild harlequin ducks increased within min of capture and elevated levels were found for several hours post capture, indicating that surgery and confinement maintain elevated corticosterone concentrations in this species. Mean corticosterone concentrations in wild harlequin ducks held in temporary captivity were similar to the maximum response levels during the ACTH challenge in captive birds. However, large variation among individuals was observed in responses of wild birds, and we found additional evidence suggesting that corticosterone responses varied between hatch year and after hatch year birds.

Variability in leptin and adrenal response in juvenile Steller sea lions (Eumetopias jubatus) to adrenocorticotropic hormone (ACTH) in different seasons.

Eight free-ranging juvenile Steller sea lions (SSL; 6 males, 2 females; 14-20 months) temporarily held under ambient conditions at the Alaska SeaLife Center were physiologically challenged through exogenous administration of adrenocorticotropic hormone (ACTH). Four individuals (3 males, 1 female) underwent ACTH challenge in each of two seasons, summer and winter. Following ACTH injection serial blood and fecal samples were collected for up to 3 and 96 h, respectively. A radioimmunoassay (RIA) was validated for leptin, and using a previously validated RIA for cortisol, collected sera were analyzed for both cortisol and leptin. ACTH injection resulted in a 2.9-fold increase (P=0.164) in leptin which preceded a 3.2-fold increase (P=0.0290) in cortisol by 105 min in summer. In winter, a 1.7-fold increase in leptin (P=0.020) preceded a 2.1-fold increase (P=0.001) in serum cortisol by 45 min. Mean fecal corticosteroid maxima were 10.4 and 16.7-fold above baseline 28 and 12 h post-injection and returned to baseline 52 and 32 h post-injection, in summer and winter, respectively. Data indicate acute activity in juvenile adrenal glands is detectable in feces approximately 12-24 h post-stimulus in either season, with a duration of approximately 40 h in summer and 20 h in winter. Changes in serum cortisol proved statistically significant both seasons and elevated concentrations were detected by 30 min post-stimulus (baseline 64.8+/-4.2; peak 209.5+/-18.3 ng/ml: summer; baseline 87.0+/-15.7; peak 237.6+/-10.0 ng/ml: winter), whereas the changes that occurred in serum leptin proved to be significant only in winter (baseline 6.4+/-0.6; peak 18.7+/-7.0 ng/ml: summer; baseline 4.2+/-0.5; peak 7.5+/-0.6 ng/ml: winter). Changes in fecal corticosteroids proved significant only in summer (baseline 117.8+/-36.7; peak 1219.3+/-298.4 ng/g, P=0.038: summer; baseline 71.8+/-13.7; peak 1198.6+/-369.9 ng/g, P=0.053: winter) due to a high degree of individual variability in winter months. The data indicate that ACTH stimulates leptin production earlier than cortisol in both summer and winter, and that while the leptin response appears most variable in summer, fecal corticosteroids are most variable in winter.

Seasonal and predator influences on adrenal function in adult Steller sea lions: gender matters.

Chronically heightened adrenal activity indexed by fecal corticosteroids has been shown to be a valid descriptor of stress in many species. As part of an ongoing investigation of adrenal activity in Steller sea lions (SSL), adrenocorticotropic hormone (ACTH) challenges were performed during the summer at the Alaska SeaLife Center (ASLC). These results were compared to earlier data from winter months. Additionally, adrenal response of free-ranging females SSL to a presumed in situ stressor, pup predation by killer whales (Orcinus orca), was evaluated as a field trial of developed methodologies. Summer ACTH results indicated that gender-dependent differences in baseline fecal corticosterone concentrations exist, with summer baseline fecal corticosterone concentrations higher in males than in females, based on season when compared with previously reported values in winter ACTH trials for this species. ACTH trials in the male in the summer resulted in 2468 ng/g basal to 10,937 ng/g maximal fecal corticosterone concentrations, a fourfold change. Female 1 exhibited a 30.5-fold increase 24 h post-ACTH stimulation (27.9-852.0 ng/g dry weight), with a return to just above baseline concentrations by hour 25. Additionally, female 2 exhibited a 64.4-fold increase at 25 h post-stimulation (31.7-2042.0 ng/g dry weight), with a return to just above baseline concentrations by hour 45. In situ female fecal corticosterone tripled 24h subsequent to orca predation on pups (54.6+/-18.5 ng/g mean pre-attack, 542.5+/-252.7 ng/g mean post-attack) during the field trial. Data from both the summer ACTH and subsequent field trial underscore the necessity of gender determination of collected scat source and knowledge of conditions at collection sites for proper interpretation of fecal corticosterone data, particularly in studies that focus on population-wide stress. With elimination of gender bias and delineation of acute response to natural stimuli, corticosterone data can now be evaluated within context and provide meaningful information about stress and potentially reproductive physiology in free-ranging Steller sea lions.

Postnatal ontogeny of erythropoietin and hematology in free-ranging Steller sea lions (Eumetopias jubatus).

The hormone erythropoietin (EPO) is responsible for the increased production of red blood cells (RBC) in response to tissue hypoxia. While the role of EPO in hematological development has been established in humans and terrestrial mammals, this relationship has never been examined in marine mammals that rely heavily on stored oxygen to maintain aerobic metabolism while diving. Since blood is the major oxygen storage site in marine mammals, it was hypothesized that EPO may have a significant influence on the development of hematology parameters associated with the expansion of blood oxygen stores during development. To explore this hypothesis, serum EPO concentrations were determined by radioimmunoassay in 235 free-ranging Steller sea lions (Eumetopias jubatus), throughout their Alaskan range. Hematocrit (Hct), hemoglobin (Hb), and red blood cell (RBC) counts were also measured, and mean corpuscular hemoglobin content (MCHC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCV) values determined. Erythropoietin and most hematological parameters varied with age. Hematocrit, Hb, RBC, and MCHC decreased after birth, reached their lowest values at two to three months of age, and then increased to values similar to those of adults by five months of age. Since changes in Hct and Hb account for the majority of the changes in blood oxygen stores and EPO was negatively correlated with both, it appears that EPO may play an important role in blood development of Steller sea lions, similar to previous studies on terrestrial mammals.

Evaluation of adrenal function in serum and feces of Steller sea lions (Eumetopias jubatus): influences of molt, gender, sample storage, and age on glucocorticoid metabolism.

Fecal corticosterone concentrations, measured via radioimmunoassay (RIA), were validated as a method to monitor adrenal function in Steller sea lion physiology. Quantification of adrenal response to an acute stressor and relevance of data produced by developed methodologies was determined through physiological challenge with exogenous administration of adrenocorticotropic hormone (ACTH) to captive adult, reproductively intact, Steller sea lions of both sexes (n=3, 1 male, 2 female) during seasonal molt. Following ACTH administration, serial blood and fecal samples were collected and analyzed by RIA to determine adrenal response. Storage regimens and weather exposure were examined to establish external impact on fecal corticosterone concentrations. High-pressure liquid chromatography (HPLC) of both serum and feces of Steller sea lions was employed to explore potential gender-based differences extant in either sample media. ACTH challenges produced >3-fold increases in serum cortisol concentrations which were reflected in >18-fold increases in fecal corticosterone concentrations post-injection at 3.25 and 32 h, respectively, and fecal corticosterone concentrations returned to baseline 52 h post-injection. Neither outdoor exposure to weather nor variation in duration and temperature of freezer storage impacted fecal corticosterone concentrations. HPLC of individual fecal samples produced eluate immunoreactivity profiles that differed consistently with both sex and age class. Techniques developed herein effectively detected physiologically relevant corticosterone data in Steller sea lion feces, unaffected by conditions likely to be encountered with field collection samples. Additionally, results quantify an acute response to ACTH and provide methodology for examining chronically heightened adrenal activity in Steller sea lions.