Michael J. Sheriff

Measuring stress in wildlife: techniques for quantifying glucocorticoids

Stress responses play a key role in allowing animals to cope with change and challenge in the face of both environmental certainty and uncertainty. Measurement of glucocorticoid levels, key elements in the neuroendocrine stress axis, can give insight into an animal’s well-being and can aid understanding ecological and evolutionary processes as well as conservation and management issues. We give an overview of the four main biological samples that have been utilized [blood, saliva, excreta (feces and urine), and integumentary structures (hair and feathers)], their advantages and disadvantages for use with wildlife, and some of the background and pitfalls that users must consider in interpreting their results. The matrix of choice will depend on the nature of the study and of the species, on whether one is examining the impact of acute versus chronic stressors, and on the degree of invasiveness that is possible or desirable. In some cases, more than one matrix can be measured to achieve the same ends. All require a significant degree of expertise, sometimes in obtaining the sample and always in extracting and analyzing the glucocorticoid or its metabolites. Glucocorticoid measurement is proving to be a powerful integrator of environmental stressors and of an animal’s condition.

The sensitive hare: sublethal effects of predator stress on reproduction in snowshoe hares

1. Prey responses to high predation risk can be morphological or behavioural and ultimately come at the cost of survival, growth, body condition, or reproduction. These sub-lethal predator effects have been shown to be mediated by physiological stress. We tested the hypothesis that elevated glucocorticoid concentrations directly cause a decline in reproduction in individual free-ranging female snowshoe hares, Lepus americanus. We measured the cortisol concentration from each dam (using a faecal analysis enzyme immunoassay) and her reproductive output (litter size, offspring birth mass, offspring right hind foot (RHF) length) 30 h after birth. 2. In a natural monitoring study, we monitored hares during the first and second litter from the population peak (2006) to the second year of the decline (2008). We found that faecal cortisol metabolite (FCM) concentration in dams decreased 52% from the first to the second litter. From the first to the second litter, litter size increased 122%, offspring body mass increased 130%, and offspring RHF length increased 112%. Dam FCM concentrations were inversely related to litter size (r(2) = 0.19), to offspring birth mass (r(2) = 0.32), and to offspring RHF length (r(2) = 0.64). 3. In an experimental manipulation, we assigned wild-caught, pregnant hares to a control and a stressed group and held them in pens. Hares in the stressed group were exposed to a dog 1-2 min every other day before parturition to simulate high predation risk. At parturition, unsuccessful-stressed dams (those that failed to give birth to live young) and stressed dams had 837% and 214%, respectively, higher FCM concentrations than control dams. Of those females that gave birth, litter size was similar between control and stressed dams. However, offspring from stressed dams were 37% lighter and 16% smaller than offspring from control dams. Increasing FCM concentration in dams caused the decline of offspring body mass (r(2) = 0.57) and RHF (r(2) = 0.52). 4. This is the first study in a free-ranging population of mammals to show that elevated, predator-induced, glucocorticoid concentrations in individual dams caused a decline in their reproductive output measured both by number and quality of offspring. Thus, we provide evidence that any stressor, not just predation, which increases glucocorticoid concentrations will result in a decrease in reproductive output.

The ghosts of predators past: population cycles and the role of maternal programming under fluctuating predation risk.

Maternal effects may be a major factor influencing the demography of populations. In mammals, the transmission of stress hormones between mother and offspring may play an important role in these effects. Laboratory studies have shown that stressors during pregnancy and lactation result in lifelong programming of the offspring phenotype. However, the relevance of these studies to free-living mammals is unclear. The 10-year snowshoe hare (Lepus americanus) cycle is intimately linked to fluctuating predation pressure and predation risk. The enigma of these cycles is the lack of population growth following the decline phase, when the predators have virtually all disappeared and the food supply is ample. We have shown that a predator-induced increase in maternal stress hormone levels resulted in a decline in reproduction. Here we examine population and hormone changes over a four-year period from the increase (2005) to the decline (2008). We report (1) that an index of maternal stress (fecal corticosteroid metabolite [FCM] concentrations) fluctuates in synchrony with predator density during the breeding season; (2) that maternal FCM levels are echoed in their offspring, and this occurs at a population-wide level; and (3) that higher maternal FCM levels at birth are correlated with an increased responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis in their progeny. Our results show an intergenerational inheritance of stress hormones in a free-ranging population of mammals. We propose that the lack of recovery of reproductive rates during the early low phase of the hare cycle may be the result of the impacts of intergenerational, maternally inherited stress hormones caused by high predation risk during the decline phase.

Assessing stress in animal populations: Do fecal and plasma glucocorticoids tell the same story?

Many studies have recently focused on stress as a marker of an animals well being. Since animals respond to a stressor by increasing their glucocorticoid (GC) levels there has been much interest in measuring these hormones. Fecal GC analyses have been used in a wide range of studies as they are an easily obtained, non-invasive measure of these stress hormones. However, these analyses rest on two major assumptions. First, they assume that fecal GC metabolites reflect free, biologically active levels of GCs in the plasma. Second, they assume that differences in fecal GC metabolite levels among animals are an accurate reflection of their physiological state and thus of their ability to respond to a stressor. We tested these assumptions in a population of free-ranging snowshoe hares (Lepus americanus) in the southwestern Yukon, from 2006 to 2008. Both assumptions were verified. Plasma free cortisol levels mirrored bile and fecal cortisol metabolite (FCM) levels, but plasma total cortisol levels did not. Differences in FCM concentrations among hares robustly predicted their response to a hormonal challenge. Hares with higher FCM concentrations showed a greater resistance to the suppression of their free plasma cortisol following a dexamethasone injection and a more marked increase of free plasma cortisol following an ACTH injection. Furthermore, we found that changes in FCM concentrations in autumn and winter over two years reliably tracked changes in plasma free cortisol levels obtained from the hormonal challenge test. These results indicate that both fecal and plasma measures of an animals stress physiology are concordant: they tell the same story.

From process to pattern: how fluctuating predation risk impacts the stress axis of snowshoe hares during the 10-year cycle.

Predation is a central organizing process affecting populations and communities. Traditionally, ecologists have focused on the direct effects of predation—the killing of prey. However, predators also have significant sublethal effects on prey populations. We investigated how fluctuating predation risk affected the stress physiology of a cyclic population of snowshoe hares (Lepus americanus) in the Yukon, finding that they are extremely sensitive to the fluctuating risk of predation. In years of high predator numbers, hares had greater plasma cortisol levels at capture, greater fecal cortisol metabolite levels, a greater plasma cortisol response to a hormone challenge, a greater ability to mobilize energy and poorer body condition. These indices of stress had the same pattern within years, during the winter and over the breeding season when the hare:lynx ratio was lowest and the food availability the worst. Previously we have shown that predator-induced maternal stress lowers reproduction and compromises offspring’s stress axis. We propose that predator-induced changes in hare stress physiology affect their demography through negative impacts on reproduction and that the low phase of cyclic populations may be the result of predator-induced maternal stress reducing the fitness of progeny. The hare population cycle has far reaching ramifications on predators, alternate prey, and vegetation. Thus, predation is the predominant organizing process for much of the North American boreal forest community, with its indirect signature—stress in hares—producing a pattern of hormonal changes that provides a sensitive reflection of fluctuating predator pressure that may have long-term demographic consequences.

A non-invasive technique for analyzing fecal cortisol metabolites in snowshoe hares (Lepus americanus)

To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4xa0days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7xa0h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2xa0days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2xa0days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8–12xa0h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8–12xa0h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive field studies of stress in hares.

Seasonal Metabolic Acclimatization in a Northern Population of Free-Ranging Snowshoe Hares, Lepus americanus

Abstract Seasonal acclimatization in high-latitude endotherms may involve increases or reductions in body size and metabolic rate to, respectively, augment thermoregulatory capacity or reduce energy requirements. We investigated seasonal acclimatization in a northern population of wild snowshoe hares (Lepus americanus) that is exposed to low food availability and extremely cold temperatures in winter. Snowshoe hares were livetrapped and transported to a nearby mobile laboratory. Hares were placed in a metabolic chamber and oxygen consumption was measured for 55 min at each of the following temperatures: 10°C, 0°C, −10°C, −15°C, and −20°C. Hair length and density were measured on a sample of collected hares. Snowshoe hares maintained similar body mass and body temperature between the seasons, but average resting metabolic rate and thermal conductance were, respectively, 20% and 32% lower in winter than in autumn. The lower critical temperature was −10°C to −15°C in winter and 0°C to −10°C in autumn. Guard hairs were 36% longer and 148% denser in winter than autumn, whereas downy hairs were the same length but 128% denser in winter than autumn. Collectively, these results suggest that resource constraints associated with a herbivorous diet in regions and seasons of poor forage quality favors an energetically conservative approach to winter acclimatization.

The cold shoulder: free-ranging snowshoe hares maintain a low cost of living in cold climates

The hypothesis that cold air temperatures (Ta) constrain the metabolic diversity of high-latitude endotherms is based on the observation among birds and mammals that mean field metabolic rate (FMR) increases, whereas the variability of FMR decreases, from the warm tropics to the cold poles. However, there is a paucity of FMR measurements from above 60° latitude and below 0 °C. We measured the daily energy expenditure of a high-latitude population of free-ranging snowshoe hares (Lepus americanus Erxleben, 1777) in Yukon, Canada, in winter (Ta-mean = –16.4 °C) and in autumn (Ta-mean = 0.5 °C). Doubly labelled water measures of FMR were approximately 20% lower in winter than in autumn, and were a similar, low multiple of resting metabolic rate in both seasons (2.04 and 1.94, respectively). The mass-corrected FMR of snowshoe hares in winter was only half the value predicted by extrapolating the relationship between FMR and Ta > 0 to –16.4 °C. These results contribute to an emerging pattern of a reversal in the relationship between FMR and Ta in free-ranging mammals from negative above 0 °C to positive below 0 °C. We refer to the positive, low Ta portion of this relationship as the cold shoulder, and suggest that it may reflect the general necessity for free-ranging mammals to use behavioural and (or) physiological means to conserve energy during long winters when cold conditions coincide with resource scarcity.