Maximum Corticosteroid Binding Capacity Research Articles

Effects of capture on stress-axis measures in endangered little brown bats (Myotis lucifugus)

Abstract Little brown bats (Myotis lucifugus) are a widely distributed species in North America that have been decimated by the fungal disease white-nose syndrome. As such, little brown bats are the focus of monitoring and research initiatives that often include capturing and handling free-ranging individuals. We examined the stress response of 198 adult female little brown bats after being captured from three bat houses, during the summer. Our objective was to inform best practices to researchers capturing and handling bats in the wild. We compared the stress response among bats held for <3 min (baseline), 15–30 min, or >30 min, and then among bats held alone or in a group with conspecifics. We measured the levels of plasma total and free cortisol, maximum corticosteroid binding capacity (MCBC), and blood glucose. Relative to baseline, total and free cortisol levels were significantly higher in bats held for 15–30 min and higher still in those held for > 30 min. Blood glucose levels were elevated after >30 min of holding. MCBC levels showed no differences among holding times. We detected a weak effect of social holding condition, with solitary-held bats having lower total cortisol levels than group-held bats, but MCBC, free cortisol, and blood glucose levels showed no effect of social holding condition. Our findings demonstrate that capture time should be minimized and suggest that little brown bats should be handled and released within 30 min of capture as means of reducing stress. Further, solitary holding did not appear to increase stress measures, which supports holding bats individually after capture, instead of in groups, to reduce risk of pathogen and parasite transmission.

The stress of Arctic warming on polar bears.

Arctic ecosystems are changing rapidly in response to climate warming. While Arctic mammals are highly evolved to these extreme environments, particularly with respect to their stress axis, some species may have limited capacity to adapt to this change. We examined changes in key components of the stress axis (cortisol and its carrier protein-corticosteroid binding globulin [CBG]) in polar bears (Ursus maritimus) from western Hudson Bay (N=300) over a 33year period (1983-2015) during which time the ice-free period was increasing. Changing sea ice phenology limits spring hunting opportunities and extends the period of onshore fasting. We assessed the response of polar bears to a standardized stressor (helicopter pursuit, darting, and immobilization) during their onshore fasting period (late summer-autumn) and quantified the serum levels of the maximum corticosteroid binding capacity (MCBC) of CBG, the serum protein that binds cortisol strongly, and free cortisol (FC). We quantified bear condition (age, sex, female with cubs or not, fat condition), sea ice (breakup in spring-summer, 1year lagged freeze-up in autumn), and duration of fasting until sample collection as well as cumulative impacts of the latter environmental traits from the previous year. Data were separated into "good" years (1983-1990) when conditions were thought to be optimal and "poor" years (1991-2015) when sea ice conditions deteriorated and fasting on land was extended. MCBC explained 39.4% of the variation in the good years, but only 28.1% in the poor ones, using both biological and environmental variables. MCBC levels decreased with age. Changes in FC were complex, but more poorly explained. Counterintuitively, MCBC levels increased with increased time onshore, 1year lag effects, and in poor ice years. We conclude that MCBC is a biomarker of stress in polar bears and that the changes we document are a consequence of climate warming.

Measurement of free glucocorticoids: quantifying corticosteroid binding capacity and its variation within and among mammal and bird species.

Plasma glucocorticoid (CORT) levels are one measure of stress in wildlife and give us insight into natural processes relevant to conservation issues. Many studies use total CORT concentrations to draw conclusions about animals’ stress state and response to their environment. However, the blood of tetrapods contains corticosteroid-binding globulin (CBG), which strongly binds most circulating CORT. Only free CORT (CORT not bound by CBG) leaves the circulation and exerts biological effects on CORT-sensitive tissues. Measuring free CORT concentrations provides insight to an animal’s stress response that cannot be revealed by simply measuring total CORT. To calculate free CORT concentrations in plasma or serum samples, one needs three measurements: the binding affinity of CBG for CORT (which varies by species), the total CORT concentration in the sample and the maximum corticosteroid binding capacity (MCBC) of CBG in the sample. Here, we detail the measurement of CBG binding capacity. We compare and contrast the three main methods to measure MCBC: charcoal, cell harvester and dialysis. Each is defined by the means by which free and bound CORT are separated. We weigh the relative merits and challenges of each. We conclude that sample volume, species and taxon binding specificity, and availability of equipment are the primary considerations in selecting the appropriate separation method. For most mammals, the charcoal method is recommended. For birds, the harvester method has critical advantages over the charcoal method. The dialysis method is widely regarded as the gold standard and has lower equipment costs but is more time-intensive and costly in terms of radioactive isotope needed and is less suited to processing large numbers of samples. The binding capacity of CBG varies tremendously within and among the bird and marine mammal species studied, and we discuss the implication of this variation for understanding the role of stress in wildlife.

Reproduction at All Costs: The Adaptive Stress Response of Male Arctic Ground Squirrels

We tested the hypothesis that adult male arctic ground squirrels (Spermophilus parryii plesius) exhibit an adaptive stress response during the mating period that may compromise their survival, whereas males at other times (nonreproductive adult males and juvenile males) have a normal functional stress response. We assessed the physiological responsiveness of the stress axis, of energy mobilization, and of the immune response by subjecting adult breeding males, adult nonbreeding males, and juvenile males to a hormonal challenge and an immunocompetence challenge. At the onset of the breeding season in spring, only 25–30% of the population were males, and of those present during the mating period, half disappeared soon thereafter, and 82% were not replaced by immigrants. Adult breeding males had the highest levels of free cortisol, the lowest maximum corticosteroid-binding capacity, slight dexamethasone resistance, the lowest hematocrit, the lowest number of white blood cells, the highest number of eosinophils, and the poorest ability to respond to the foreign antigen challenge in comparison with the other two male classes. All of these characteristics were indicative of chronic stress in breeding males that may directly compromise their survival. Juvenile males in mid-August also showed many, but not all of these characteristics, indicative of a prolonged period of stress, presumably associated with the period of dispersal. Testosterone levels remained high irrespective of age or breeding condition, decreased when dexamethasone was injected, and increased when ACTH was injected. These latter results are unique in mammals. High testosterone levels and their augmentation with stressors may play a key role in maintenance of aggressive behavior. We conclude that breeding male arctic ground squirrels exhibit an adaptive stress response in which they trade off survival for reproduction. We hypothesize that similar stress responses may have evolved in other species with comparable life histories.

Seasonal changes in glucocorticoid and testosterone concentrations in free-living arctic ground squirrels from the boreal forest of the Yukon

We examined how glucocorticoid and testosterone concentrations changed from spring to summer by livetrapping free-living populations of arctic ground squirrels (Spermophilus parryii). The primary glucocorticoid was found to be cortisol, with corticosterone below measurable concentrations in most individuals. Livetrapping elicited a strong stress response in both sexes: breeding males and females trapped in spring had free cortisol concentrations 4 and 34 times, respectively, those of base-line animals. The maximum corticosteroid-binding capacity (MCBC) was unaffected by trapping and was about 3 times higher in breeding females than in breeding males. Over the active season, MCBC values were lowest in all male classes (juveniles, nonreproductive adults, and reproductive adults), being less than half those in all female classes; pregnant females had values approximately twice those of juvenile females. However, free cortisol concentrations were similar in all female classes and in juvenile males and about half those in adult males. Livetrapping increased testosterone concentrations in males over those found in samples from base-line males, and testosterone concentrations did not affect MCBC values. Testosterone concentrations in livetrapped animals differed significantly among male classes, with nonreproductive males maintaining concentrations 64% of those in breeding males and 10 times those in juveniles.

Seasonal changes in glucocorticoid and testosterone concentrations in free-living arctic ground squirrels from the boreal forest of the Yukon

We examined how glucocorticoid and testosterone concentrations changed from spring to summer by livetrapping free-living populations of arctic ground squirrels (Spermophilus parryii). The primary glucocorticoid was found to be cortisol, with corticosterone below measurable concentrations in most individuals. Livetrapping elicited a strong stress response in both sexes: breeding males and females trapped in spring had free cortisol concentrations 4 and 34 times, respectively, those of base-line animals. The maximum corticosteroid-binding capacity (MCBC) was unaffected by trapping and was about 3 times higher in breeding females than in breeding males. Over the active season, MCBC values were lowest in all male classes (juveniles, nonreproductive adults, and reproductive adults), being less than half those in all female classes; pregnant females had values approximately twice those of juvenile females. However, free cortisol concentrations were similar in all female classes and in juvenile males and about half those in adult males. Livetrapping increased testosterone concentrations in males over those found in samples from base-line males, and testosterone concentrations did not affect MCBC values. Testosterone concentrations in livetrapped animals differed significantly among male classes, with nonreproductive males maintaining concentrations 64% of those in breeding males and 10 times those in juveniles.

Contrasting stress response of male Arctic ground squirrels and red squirrels

A hormonal-challenge protocol was used to compare the stress response of males of Arctic ground squirrels and red squirrels during the breeding season (May). These squirrels live in the same boreal forest of the Yukon, but have very different life histories and utilize the forest in markedly different ways. Red squirrels had levels of total cortisol, maximum corticosteroid-binding capacity, and free cortisol that were 5, 7, and 2 times, respectively, those of Arctic ground squirrels. Red squirrels were resistant to suppression by an artificial glucocorticoid, dexamethasone (DEX); Arctic ground squirrels were not. Cortisol levels in red squirrels responded slowly but continuously to the ACTH injection; Arctic ground squirrels responded rapidly and then stabilized. Testosterone levels in red squirrels were extremely sensitive to the challenge, being suppressed by both DEX and ACTH; levels in Arctic ground squirrels were resistant to the challenge, being modestly suppressed by DEX and stimulated by ACTH. Energy mobilization, as measured by glucose and free fatty acid responses, was not affected. Red squirrels had four times the levels of white blood cells and higher proportions of lymphocytes and lower proportions of eosinophils than Arctic ground squirrels, indicating that the latter were in worse condition immunologically. Our evidence suggests that the functions associated with the hypothalamic-pituitary-adrenal axis are compromised in breeding male Arctic ground squirrels, but not in red squirrels. We propose that in male red squirrels this axis has evolved in the context of a stable social system based on long-lived animals with individual territories which are needed to deal with unpredictable winter food supplies. In contrast, Arctic ground squirrels escape the rigors of winter by hibernation and this hormonal axis has evolved in short-lived males in the context of intense intra-sexual competition in a social system based on female kin groups and regular male dispersal to avoid inbreeding. J. Exp. Zool. 286:390-404, 2000.

The Impact of Predator-Induced Stress on the Snowshoe Hare Cycle

The sublethal effects of high predation risk on both prey behavior and physiology may have long-term consequences for prey population dynamics. We tested the hypothesis that snowshoe hares during the population decline are chronically stressed because of high predation risk whereas those during the population low are not, and that this has negative effects on both their physiology and demography. Snowshoe hares exhibit 10-yr population cycles; during declines, virtually every hare that dies is killed by a predator. We assessed the physiological responsiveness of the stress axis and of energy mobilization by subjecting hares during the population decline and low to a hormonal-challenge protocol. We monitored the population demography through live-trapping and assessed reproduction through a maternal-cage technique. During the 1990s' decline in the Yukon, Canada, hares were chronically stressed—as indicated by higher levels of free cortisol, reduced maximum corticosteroid-binding capacity, reduced testosterone response, reduced index of body condition, reduced leucocyte counts, increased overwinter body-mass loss, and increased glucose mobilization, relative to hares during the population low. This evidence is consistent with the explanation that predation risk, not high hare density or poor nutritional condition, accounted for the chronic stress and for the marked deterioration of reproduction during the decline. Reproduction and indices of stress physiology did not improve until predation risk declined. These findings may also account for the lag in recovery of hare reproduction after predator densities have declined and thus may implicate the long-term consequences of predation risk on prey populations beyond the immediate effects of predators on prey behavior and physiology.

Population Declines in the Snowshoe Hare and the Role of Stress

Every 10 years snowshoe hare populations across the boreal forest of North America go through a population cycle, culminating in a decline lasting 4 or more years. We tested the hypothesis that snowshoe hares during the decline are in poor condition and less able to respond to challenges in their environment by examining the stress response of male hares. Three groups from February and May, 1991 (the second year of the hare decline in the Yukon), were compared: baseline hares were collected to obtain resting hormone levels; control hares were wild animals caught at randomly placed sites: and fed hares were wild animals caught on supplementary fed areas. The latter two groups were sequentially bled to examine their response to dexamethasone (DEX) followed by adrenocorticotropic hormone (ACTH). Trapping and handling were stressful to the experimental hares as the initial blood levels of total and free cortisol levels were higher (especially in controls), testosterone levels were lower, and glucose levels were higher in experimental hares than in baseline hares. Control and fed hares showed similar total and free cortisol responses, falling to low levels after the DEX injection and increasing rapidly in response to the ACTH injection. However, control hares were in worse condition than fed hares as indicated by the higher free cortisol levels and lower maximum corticosteroid-binding capacity (MCBC) in control hares. In addition, though testosterone levels fell in both groups in response to DEX, only the fed hares showed a large, transitory increase 30 min after the ACTH injection. An unexpected finding was a dramatic increase in MCBC levels 30 min after the ACTH injection in both experimental groups, but it was more pronounced in the fed group. We conclude that the pituitary-adrenocortical feedback system in hares from declining populations is operating normally anti that they should be able to cope with acute, short-term stressors, but that they are in poor condition and are exposed to higher levels of free cortisol than fed hares in good condition.

Reproduction in Sminthopsis-Macroura (Marsupialia, Dasyuridae) .2. The Male

The breeding season of S. macroura extends from June to February, and individual males (both wild- caught and laboratory-reared) are capable of breeding over extended periods during the breeding season, and for up to three seasons, in the laboratory. Gross and histological changes in the reproductive organs and endocrine changes in relation to reproductive activity have been investigated. Males do not appear to reach sexual maturity until the season following that in which they were born, although spermatorrhoea may commence in the season of birth. Testis and epididymis weight of these males, which commence spermatorrhoea late in the season, approximates that of sexually mature males early in the season but androgen levels and the weight of the accessory glands are low in all males except during the early months of the season. The age at which spermatorrhoea commences ranges from 141 to 350 days. The minimum scrota1 width at which it commences is 7.9 mm and the minimum body weight, 14.0 g. The onset of spermatorrhoea is not a function of age or season and in S. macroura should be used with caution as an indicator of impending sexual maturity. Maximum corticosteroid- binding capacity (MCBC) generally exceeded corticosteroid concentration and no androgen-related fall in MCBC was evident.

The Effect of Mating and Agonistic Experience on Adrenal Function and Mortality of Male Antechinus stuartii (Marsupialia)

The effects of agonistic and mating behavior on androgen and total plasma corticosteroid concentrations and the maximum corticosteroid binding capacity (MCBC) were investigated in male Antechinus stuartii. Males were subjected to three experimental conditions: isolated from all conspecifics, in daily physical contact with conspecific males, and in intermittent contact with conspecific estrous females. There were no differences among groups in either androgen concentrations or MCBC. Males in contact with other males and therefore subjected to daily aggressive encounters showed greater mortality and significantly higher total plasma corticosteroid levels in mid-August at the end of the breeding season. Results suggest that interactions with estrous females may lower total corticosteroid concentrations and have an ameliorating effect on the stress caused by competition with other males.

Effects of Corticotropin and Heat on Corticosteroid-Binding Capacity and Serum Corticosteroid in White Rock Chickens

The effects of the administration of corticotropin (ACTH) or high environmental temperature on serum maximum corticosteroid-binding capacity (MCBC) and serum corticosteroid concentrations were studied in 6- to 8-wk-old White Rock chickens. Highly significant increases in serum corticosteroids produced by either intramuscular or intravenous injections of ACTH were followed by significant reductions in MCBC within 24 hr. A single high-temperature episode (46 C) caused a significant increase in serum corticosteroids when blood was sampled immediately following exposure to heat; however, serum corticosteroids were not significantly increased following two heat episodes spaced 11 hr apart. Exposure to high temperature did not alter MCBC levels.

The effects of individual and group penning of pigs on total and free plasma corticosteroids and the maximum corticosteroid binding capacity

Total and free plasma corticosteroid concentrations and the maximum corticosteroid binding capacity (MCBC) were measured twice a day in pigs penned individually or in groups following a period of individual penning. Moving animals into groups after 5 weeks in individual pens resulted in a short-term stress response as evidenced by an increased corticosteroid concentration measured 5 1 2 hr later, but within 24 hr the hormonal pattern established was of higher levels in the morning than in the afternoon. In group-penned pigs the total corticosteroid concentration was generally well below that of the MCBC. In contrast, in individually penned pigs the MCBC was lower and the afternoon corticosteroid concentration was elevated to levels similar to the morning values. Both morning and after-noon mean corticosteroid values approached the mean MCBC. It was concluded that individual penning resulted in a chronic stress response.

Effects of photoperiod and feeding on plasma corticosteroid concentrations and maximum corticosteroid-binding capacity in pigs.

The effects of time of feeding and photoperiod on the circadian rhythm of total and free plasma corticosteroid concentrations and the maximum corticosteroid-binding capacity (MCBC) were measured in pigs. There were four photoperiod treatments: natural long and short daylengths (light:dark, 14:10; 101/2:131/2, respectively), artificial short daylength and reverse artificial short daylength (light:dark, 9:15). Photoperiod had a significant effect on corticosteroid concentrations. The corticosteroid concentrations on long daylength showed a major increase in the morning (from an average of 8 ng ml-1 at 0700 h to a peak level of 53.5 ng ml-1 at 0910 h). The amplitude of the morning peak was lower in the other treatments. Apart from this increase, corticosteroid levels at other times of day for pigs in all treatments were generally low. The MCBC varied greatly between animals (less than 25 - greater than 50 ng ml-1) but showed no circadian rhythm. There was no change in corticosteroid levels directly attributable to feeding. It was concluded that, in experiments where the effect of acute stressors on corticosteroid levels is examined, blood sampling should be performed in the afternoon-evening period because the between-animal variation in hormone levels is less and the hormonal levels are more stable, and also that time of feeding did not affect the rhythm of corticosteroids. A blood-sampling regime to define the circadian rhythm in corticosteroid concentration and to determine the MCBC is described.

Relationship between plasma corticosteroids and weight change in recently parous lactating and dry sheep

Twin-bearing ewes were assigned to three treatments at lambing. Treatment 1 was the adult with both lambs retained; treatment 2 was the adult with one lamb retained; and treatment 3 was the adult alone. All sheep were fed on an amount calculated to maintain an adult and one lamb, and consequently animals in treatments 1, 2 and 3 lost, maintained and gained weight respectively. Chronic under-nutrition of the lactating ewe (treatment 1) resulted in a small increase in the maximum corticosteroid binding capacity and a large increase in total corticosteroid levels in peripheral plasma, resulting in a significant increase in biologically active (free) corticosteroid concentrations. In dry sheep (treatment 3) no such increase in free hormone concentration occurred. There was a significant negative relationship between free corticosteroid concentrations and weight change during both the 2-week intervals and the entire 6-week experimental period.