Corridor-based approach with spatial cross-validation reveals scale-dependent effects of geographic distance, human footprint and canopy cover on grizzly bear genetic connectivity.

Understanding how human infrastructure and other landscape attributes affect genetic differentiation in animals is an important step for identifying and maintaining dispersal corridors for these species. We built upon recent advances in the field of landscape genetics by using an individual-based and multiscale approach to predict landscape-level genetic connectivity for grizzly bears (Ursus arctos) across ~100,000 km2 in Canada's southern Rocky Mountains. We used a genetic dataset with 1156 unique individuals genotyped at nine microsatellite loci to identify landscape characteristics that influence grizzly bear gene flow at multiple spatial scales and map predicted genetic connectivity through a matrix of rugged terrain, large protected areas, highways and a growing human footprint. Our corridor-based modelling approach used a machine learning algorithm that objectively parameterized landscape resistance, incorporated spatial cross validation and variable selection and explicitly accounted for isolation by distance. This approach avoided overfitting, discarded variables that did not improve model performance across withheld test datasets and spatial predictive capacity compared to random cross-validation. We found that across all spatial scales, geographic distance explained more variation in genetic differentiation in grizzly bears than landscape variables. Human footprint inhibited connectivity across all spatial scales, while open canopies inhibited connectivity at the broadest spatial scale. Our results highlight the negative effect of human footprint on genetic connectivity, provide strong evidence for using spatial cross-validation in landscape genetics analyses and show that multiscale analyses provide additional information on how landscape variables affect genetic differentiation.

Quantifying energetic costs and defining energy landscapes experienced by grizzly bears.

Animal movements are major determinants of energy expenditure and ultimately the cost-benefit of landscape use. Thus, we sought to understand those costs and how grizzly bears (Ursus arctos) move in mountainous landscapes. We trained captive grizzly bears to walk on a horizontal treadmill and up and down 10% and 20% slopes. The cost of moving upslope increased linearly with speed and slope angle, and this was more costly than moving horizontally. The cost of downslope travel at slower speeds was greater than the cost of traveling horizontally but appeared to decrease at higher speeds. The most efficient walking speed that minimized cost per unit distance was 1.19±0.11 m s-1 However, grizzly bears fitted with GPS collars in the Greater Yellowstone Ecosystem moved at an average velocity of 0.61±0.28 m s-1 and preferred to travel on near-horizontal slopes at twice their occurrence. When traveling uphill or downhill, grizzly bears chose paths across all slopes that were ∼54% less steep and costly than the maximum available slope. The net costs (J kg-1 m-1) of moving horizontally and uphill were the same for grizzly bears, humans and digitigrade carnivores, but those costs were 46% higher than movement costs for ungulates. These movement costs and characteristics of landscape use determined using captive and wild grizzly bears were used to understand the strategies that grizzly bears use for preying on large ungulates and the similarities in travel between people and grizzly bears that might affect the risk of encountering each other on shared landscapes.

Response of grizzly bears (Ursus arctos) to pipelines in Alberta.

This research provides the first in-depth analysis of fine-scale grizzly bear habitat selection and movement patterns in response to the linear footprints cleared for below-ground pipelines in Alberta. Using an extensive set of GPS location data from collared grizzly bears, we were able to determine that grizzly bears selected for younger pipelines (mean age since last construction~6.5 years), which are known to have a greater abundance of important bear foods. Bears also selected for wider corridors that were disturbed for construction more than once. During the spring season, sex/age class was an important predictor of grizzly bear use of pipelines, with adult female bears more likely to use these features than other sex/age classes. Examination of movement patterns revealed that pipeline density influenced grizzly bears' movement rates and path straightness, particularly in the spring, when bears moved more slowly and movement paths were more tortuous in areas with higher pipeline densities. These movement patterns are consistent with foraging behavior and further indicate that bears are not exhibiting avoidance behaviors or displacement by pipeline features, and pipelines may be functioning as seasonally important foraging areas for grizzly bears in Alberta.

Changing spring snow cover dynamics and early season forage availability affect the behavior of a large carnivore.

Changing climates are altering wildlife habitats and wildlife behavior in complex ways. Here, we examine how changing spring snow cover dynamics and early season forage availability are altering grizzly bear (Ursus arctos) behavior postden emergence. Telemetry data were used to identify spring activity dates for 48 individuals in the Yellowhead region of Alberta, Canada. Spring activity date was related to snow cover dynamics using a daily percent snow cover dataset. Snow melt end date, melt rate, and melt consistency explained 45% of the variation in spring activity date. We applied this activity date model across the entire Yellowhead region from 2000 to 2016 using simulated grizzly bear home ranges. Predicted spring activity date was then compared with a daily spring forage availability date dataset, resulting in "wait time" estimates for four key early season forage species. Temporal changes in both spring activity date and early season forage "wait times" were assessed using non-parametric regression. Grizzly bear activity date was found to have either remained constant (95%) or become earlier (5%) across the study area; virtually no areas with significantly later spring activity dates were detected. Similarly, the majority of "wait times" did not change (85%); however, the majority of significant changes in "wait times" for the four early season forage species indicated that "wait times" were lessening where changes were detected. Our results show that spring activity date is largely dictated by snow melt characteristics and that changing snow melt conditions may result in earlier spring activity. However, early season food stress conditions are likely to remain unchanged or improve as vegetation phenology also becomes earlier. Our findings extend the recent work examining animal movement in response to changing phenology from migratory birds and ungulates to an apex predator, further demonstrating the potential effects of changing climates on wildlife species.

Recognizing the importance of an all-inclusive approach to brown bear conservation now and into the future.

In their critique of our recent article in Oecologia (Pigeon et al. Oecologia 181:1101-1116, 2016a) investigating the influence of ambient temperature on brown bear habitat selection, Ordiz et al. (2017, current issue) argue that we downplay the role of human disturbance on bear behavior, and that we wrongly report on the findings of Ordiz et al. (Oecologia 166:59-67, 2011). We argue that our previous article in Oecologia (Pigeon et al. 2016a) by no means downplays the influence of human factors on bear behavior, and that we correctly stated that Ordiz et al. (2011) did not adequately consider the potential influence of temperature on their findings. Finally, we stress the relevance of considering all-inclusive approaches to the common goal of successful wildlife conservation.

The bear circadian clock doesn't 'sleep' during winter dormancy.

BACKGROUND: Most biological functions are synchronized to the environmental light:dark cycle via a circadian timekeeping system. Bears exhibit shallow torpor combined with metabolic suppression during winter dormancy. We sought to confirm that free-running circadian rhythms of body temperature (Tb) and activity were expressed in torpid grizzly (brown) bears and that they were functionally responsive to environmental light. We also measured activity and ambient light exposures in denning wild bears to determine if rhythms were evident and what the photic conditions of their natural dens were. Lastly, we used cultured skin fibroblasts obtained from captive torpid bears to assess molecular clock operation in peripheral tissues. Circadian parameters were estimated using robust wavelet transforms and maximum entropy spectral analyses. RESULTS: Captive grizzly bears housed in constant darkness during winter dormancy expressed circadian rhythms of activity and Tb. The rhythm period of juvenile bears was significantly shorter than that of adult bears. However, the period of activity rhythms in adult captive bears was virtually identical to that of adult wild denning bears as was the strength of the activity rhythms. Similar to what has been found in other mammals, a single light exposure during the bear's active period delayed subsequent activity onsets whereas these were advanced when light was applied during the bear's inactive period. Lastly, in vitro studies confirmed the expression of molecular circadian rhythms with a period comparable to the bear's own behavioral rhythms. CONCLUSIONS: Based on these findings we conclude that the circadian system is functional in torpid bears and their peripheral tissues even when housed in constant darkness, is responsive to phase-shifting effects of light, and therefore, is a normal facet of torpid bear physiology.

Staying cool in a changing landscape: the influence of maximum daily ambient temperature on grizzly bear habitat selection.

To fulfill their needs, animals are constantly making trade-offs among limiting factors. Although there is growing evidence about the impact of ambient temperature on habitat selection in mammals, the role of environmental conditions and thermoregulation on apex predators is poorly understood. Our objective was to investigate the influence of ambient temperature on habitat selection patterns of grizzly bears in the managed landscape of Alberta, Canada. Grizzly bear habitat selection followed a daily and seasonal pattern that was influenced by ambient temperature, with adult males showing stronger responses than females to warm temperatures. Cutblocks aged 0-20 years provided an abundance of forage but were on average 6 °C warmer than mature conifer stands and 21- to 40-year-old cutblocks. When ambient temperatures increased, the relative change (odds ratio) in the probability of selection for 0- to 20-year-old cutblocks decreased during the hottest part of the day and increased during cooler periods, especially for males. Concurrently, the probability of selection for 21- to 40-year-old cutblocks increased on warmer days. Following plant phenology, the odds of selecting 0- to 20-year-old cutblocks also increased from early to late summer while the odds of selecting 21- to 40-year-old cutblocks decreased. Our results demonstrate that ambient temperatures, and therefore thermal requirements, play a significant role in habitat selection patterns and behaviour of grizzly bears. In a changing climate, large mammals may increasingly need to adjust spatial and temporal selection patterns in response to thermal constraints.

Toward the Restoration of Caribou Habitat: Understanding Factors Associated with Human Motorized Use of Legacy Seismic Lines.

Populations of boreal and southern mountain caribou in Alberta, Canada, are declining, and the ultimate cause of their decline is believed to be anthropogenic disturbance. Linear features are pervasive across the landscape, and of particular importance, seismic lines established in the 1900s (legacy seismic lines) are slow to regenerate. Off-highway vehicles are widely used on these seismic lines and can hamper vegetative re-growth because of ongoing physical damage, compaction, and active clearing. Restoration of seismic lines within caribou range is therefore a priority for the recovery of threatened populations in Alberta, but a triage-type approach is necessary to prioritize restoration and ensure conservation resources are wisely spent. To target restoration efforts, our objective was to determine factors that best explained levels of off-highway vehicles use on seismic lines intersecting roads. We investigated the relative importance of local topography, vegetation attributes of seismic lines, and broad-scale human factors such as the density of infrastructures and the proximity to recreation campsites and towns to explain the observed levels of off-highway vehicles use. We found that off-highway vehicles use was mainly associated with local topography and vegetation attributes of seismic lines that facilitated ease-of-travel. Broad-scale landscape attributes associated with industrial, recreation access, or hunting activities did not explain levels of off-highway vehicles use. Management actions aimed at promoting natural regeneration and reduce ease-of-travel on legacy seismic lines within caribou ranges can be beneficial to caribou recovery in Alberta, Canada, and we therefore recommend restrictions of off-highway vehicles use on low vegetation, dry seismic lines in caribou ranges.

EFFECT OF ACTIVE COOLING AND α-2 ADRENOCEPTOR ANTAGONISM ON CORE TEMPERATURE IN ANESTHETIZED BROWN BEARS (URSUS ARCTOS).

Hyperthermia is a common complication during anesthesia of bears, and it can be life threatening. The objective of this study was to evaluate the effectiveness of active cooling on core body temperature for treatment of hyperthermia in anesthetized brown bears (Ursus arctos). In addition, body temperature after reversal with atipamezole was also evaluated. Twenty-five adult and subadult brown bears were captured with a combination of zolazepam-tiletamine and xylazine or medetomidine. A core temperature capsule was inserted into the bears' stomach or 15 cm into their rectum or a combination of both. In six bears with gastric temperatures≥40.0°C, an active cooling protocol was performed, and the temperature change over 30 min was analyzed. The cooling protocol consisted of enemas with 2 L of water at approximately 5°C/100 kg of body weight every 10 min, 1 L of intravenous fluids at ambient temperature, water or snow on the paws or the inguinal area, intranasal oxygen supplementation, and removing the bear from direct sunlight or providing shade. Nine bears with body temperature>39.0°C that were not cooled served as control for the treated animals. Their body temperatures were recorded for 30 min, prior to administration of reversal. At the end of the anesthetic procedure, all bears received an intramuscular dose of atipamezole. In 10 bears, deep rectal temperature change over 30 min after administration of atipamezole was evaluated. The active cooling protocol used in hyperthermic bears significantly decreased their body temperatures within 10 min, and it produced a significantly greater decrease in their temperature than that recorded in the control group.

Idiosyncratic responses of grizzly bear habitat to climate change based on projected food resource changes.

Climate change vulnerability assessments for species of conservation concern often use species distribution and ecological niche modeling to project changes in habitat. One of many assumptions of these approaches is that food web dependencies are consistent in time and environmental space. Species at higher trophic levels that rely on the availability of species at lower trophic levels as food may be sensitive to extinction cascades initiated by changes in the habitat of key food resources. Here we assess climate change vulnerability for Ursus arctos (grizzly bears) in the southern Canadian Rocky Mountains using projected changes to 17 of the most commonly consumed plant food items. We used presence-absence information from 7088 field plots to estimate ecological niches and to project changes in future distributions of each species. Model projections indicated idiosyncratic responses among food items. Many food items persisted or even increased, although several species were found to be vulnerable based on declines or geographic shifts in suitable habitat. These included Hedysarum alpinum (alpine sweet vetch), a critical spring and autumn root-digging resource when little else is available. Potential habitat loss was also identified for three fruiting species of lower importance to bears: Empetrum nigrum (crowberry), Vaccinium scoparium (grouseberry), and Fragaria virginiana (strawberry). A general trend towards uphill migration of bear foods may result in higher vulnerability to bear populations at low elevations, which are also those that are most likely to have human-bear conflict problems. Regardless, a wide diet breadth of grizzly bears, as well as wide environmental niches of most food items, make climate change a much lower threat to grizzly bears than other bear species such as polar bears and panda bears. We cannot exclude, however, future alterations in human behavior and land use resulting from climate change that may reduce survival rates.

Evaluation of the accuracy of different methods of monitoring body temperature in anesthetized brown bears (Ursus arctos).

There is some evidence that the handheld rectal thermometer does not accurately measure core temperature in bears. The objective of this study was to compare body temperature measured by the handheld digital thermometer (HDT), deep rectally inserted core temperature capsules (CTCs), and gastrically inserted CTCs in anesthetized brown bears (Ursus arctos). Twenty-two brown bears were immobilized with a combination of zolazepam-tiletamine and xylazine or medetomidine. After immobilization, one CTC was inserted 15 cm deep into the animal's rectum (DRTC) with a standard applicator, and another CTC was inserted into the stomach (GTC) via a gastric tube inserted orally. Temperature was measured every 5-10 min with an HDT. Paired temperature data points were analyzed with the Bland-Altman technique for repeated measurements and regression analysis with a significance level of 0.05. The mean difference ± SD of the difference between HDT and GTC readings was 0.27 ± 0.47 degrees C and the 95% limits of agreement (LoA) were 1.20 and -0.66 degrees C. The determination coefficient (r2) found between these methods was 0.68 (P < 0.0001). The mean difference ± SD of the difference between HDT and DRTC readings was 0.36 ± 0.32 degreesC and the 95% LoA were 1.0 and -0.28 degrees C. The r2 between HDT and DRTC was 0.83 (P < 0.0001). The mean difference ± SD of the difference between the two insertions of the VitalSense capsules was -0.06 ± 0.24 degrees C and the 95% LoA were 0.42 and -0.54 degrees C. The r2 found between GTC and DRTC was 0.91 (P < 0.0001). This study demonstrates that DRTC provided accurate measurement of core temperature and that HDT did not accurately measure core temperature, compared with GTC in anesthetized brown bears.

Environmental, biological and anthropogenic effects on grizzly bear body size: temporal and spatial considerations.

BACKGROUND: Individual body growth is controlled in large part by the spatial and temporal heterogeneity of, and competition for, resources. Grizzly bears (Ursus arctos L.) are an excellent species for studying the effects of resource heterogeneity and maternal effects (i.e. silver spoon) on life history traits such as body size because their habitats are highly variable in space and time. Here, we evaluated influences on body size of grizzly bears in Alberta, Canada by testing six factors that accounted for spatial and temporal heterogeneity in environments during maternal, natal and 'capture' (recent) environments. After accounting for intrinsic biological factors (age, sex), we examined how body size, measured in mass, length and body condition, was influenced by: (a) population density; (b) regional habitat productivity; (c) inter-annual variability in productivity (including silver spoon effects); (d) local habitat quality; (e) human footprint (disturbances); and (f) landscape change. RESULTS: We found sex and age explained the most variance in body mass, condition and length (R(2) from 0.48-0.64). Inter-annual variability in climate the year before and of birth (silver spoon effects) had detectable effects on the three-body size metrics (R(2) from 0.04-0.07); both maternal (year before birth) and natal (year of birth) effects of precipitation and temperature were related with body size. Local heterogeneity in habitat quality also explained variance in body mass and condition (R(2) from 0.01-0.08), while annual rate of landscape change explained additional variance in body length (R(2) of 0.03). Human footprint and population density had no observed effect on body size. CONCLUSIONS: These results illustrated that body size patterns of grizzly bears, while largely affected by basic biological characteristics (age and sex), were also influenced by regional environmental gradients the year before, and of, the individual's birth thus illustrating silver spoon effects. The magnitude of the silver spoon effects was on par with the influence of contemporary regional habitat productivity, which showed that both temporal and spatial influences explain in part body size patterns in grizzly bears. Because smaller bears were found in colder and less-productive environments, we hypothesize that warming global temperatures may positively affect body mass of interior bears.

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus).

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.

Using digital time-lapse cameras to monitor species-specific understorey and overstorey phenology in support of wildlife habitat assessment.

Critical to habitat management is the understanding of not only the location of animal food resources, but also the timing of their availability. Grizzly bear (Ursus arctos) diets, for example, shift seasonally as different vegetation species enter key phenological phases. In this paper, we describe the use of a network of seven ground-based digital camera systems to monitor understorey and overstorey vegetation within species-specific regions of interest. Established across an elevation gradient in western Alberta, Canada, the cameras collected true-colour (RGB) images daily from 13 April 2009 to 27 October 2009. Fourth-order polynomials were fit to an RGB-derived index, which was then compared to field-based observations of phenological phases. Using linear regression to statistically relate the camera and field data, results indicated that 61% (r (2) = 0.61, df = 1, F = 14.3, p = 0.0043) of the variance observed in the field phenological phase data is captured by the cameras for the start of the growing season and 72% (r (2) = 0.72, df = 1, F = 23.09, p = 0.0009) of the variance in length of growing season. Based on the linear regression models, the mean absolute differences in residuals between predicted and observed start of growing season and length of growing season were 4 and 6 days, respectively. This work extends upon previous research by demonstrating that specific understorey and overstorey species can be targeted for phenological monitoring in a forested environment, using readily available digital camera technology and RGB-based vegetation indices.

Landscape condition influences energetics, reproduction, and stress biomarkers in grizzly bears.

Environmental change has been shown to influence mammalian distribution, habitat use, and behavior; however, few studies have investigated the impact on physiological function. This study aimed to determine the influence of landscape condition on the expression of target proteins related to energetics, reproduction, and stress in grizzly bears. We hypothesized that changes in landscape condition explains protein expression. Skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada from 2013-2019 (n = 86 individuals). We used an information theoretic approach to develop 11 a priori candidate generalized linear mixed models to explain protein expression. We compared models using Akaike Information Criteria (AICc) weights and averaged models with ΔAICc < 2 for each protein. Food resources, represented by increased distance to coal mines and decreased crown closure, positively influenced energetic proteins (adiponectin and alpha-1-acid glycoprotein). Proteins related to reproduction (ceruloplasmin and serpin B5) were positively associated with increased wetland and upland food resources in addition to movement, but negatively associated with increased distance to roads. One stress related protein, complement C3, was positively influenced by increased percent conifer. Given the need to detect emerging threats to wildlife, we suggest the assessment of physiological function will lead to improved monitoring of species in rapidly changing landscapes.

Protein biomarkers in serum as a conservation tool to assess reproduction: a case study on brown bears (Ursus arctos).

Monitoring the reproductive characteristics of a species can complement existing conservation strategies by understanding the mechanisms underlying demography. However, methodology to determine important aspects of female reproductive biology is often absent in monitoring programs for large mammals. Protein biomarkers may be a useful tool to detect physiological changes that are indicative of reproductive state. This study aimed to identify protein biomarkers of reproductive status in serum collected from free-ranging female brown bears (Ursus arctos) in Alberta, Canada, from 2001 to 2018. We hypothesized that the expression of proteins related to reproduction in addition to energetics and stress can be used to answer specific management-focused questions: (i) identify when a female is pregnant, (ii) detect if a female is lactating, (iii) determine age of sexual maturity (i.e. primiparity) and (iv) assess female fertility (i.e. reproduction rate). Furthermore, we investigated if silver spoon effects (favourable early life conditions provide fitness benefits through adulthood) could be determined using protein expression. A target panel of 19 proteins with established relationships to physiological function was measured by peptide-based analysis using liquid chromatography and multiple reaction monitoring mass spectrometry and their differential expression was evaluated using a Wilcoxon signed-rank test. We found biomarkers of pregnancy (apolipoprotein B-100 and afamin), lactation (apolipoprotein B-100 and alpha-2-macroglobulin) and sexual maturity (corticosteroid-binding globulin), but there were no statistically significant relationships with protein expression and fertility. The expression of proteins related to reproduction (afamin) and energetics (vitamin-D binding protein) was associated with the nutritional quality of the individual's present habitat rather than their early life habitat. This study highlights potential biomarkers of reproductive status and provides additional methods for monitoring physiological function in wildlife to inform conservation.

Correcting for enzyme immunoassay changes in long term monitoring studies.

Enzyme immunoassays (EIAs) are a common tool for measuring steroid hormones in wildlife due to their low cost, commercial availability, and rapid results. Testing technologies improve continuously, sometimes requiring changes in protocols or crucial assay components. Antibody replacement between EIA kits can cause differences in EIA sensitivity, which can hinder monitoring hormone concentration over time. The antibody in a common cortisol EIA kit used for long-term monitoring of stress in wildlife was replaced in 2014, causing differences in cross reactivity and standard curve concentrations. Therefore, the objective of this study was to develop a method to standardize results following changes in EIA sensitivity. We validated this method using cortisol concentrations measured in the hair of brown bears (Ursus arctos).•We used a simple linear regression to model the relationship between cortisol concentrations using kit 1 and kit 2.•We found a linear relationship between the two kits (R2 = 0.85) and used the regression equation (kit2 = (0.98 × kit1) + 1.65) to predict cortisol concentrations in re-measured samples.•Mean predicted percent error was 16% and 72% of samples had a predicted percent error <20%, suggesting that this method is well-suited for correcting changes in EIA sensitivity.

Cortisol levels in blood and hair of unanesthetized grizzly bears (Ursus arctos) following intravenous cosyntropin injection.

Hair cortisol concentration (HCC) is being used increasingly to evaluate long-term stress in many mammalian species. Most of the cortisol is assumed to passively diffuse from circulating blood into hair follicles and gradually accumulate in growing hair. However, our research with free-ranging grizzly bears (Ursus arctos) suggests HCC increases significantly within several hours following capture, a time too brief to be explained by this mechanism alone. In this study with captive grizzly bears, we sought to determine if a brief spike in blood cortisol concentration, thus mimicking a single stressful event, would cause an increase in HCC over a 7-day period. To do this, we administered a single intravenous dose (5 µg/kg) of cosyntropin to three captive unanaesthetised adult female grizzly bears on two occasions, during April when hair growth was arrested and during August when hair was growing. In both trials, the cosyntropin caused a two-fold or greater increase in serum cortisol levels within 1 hr but did not appear to influence HCC at 1, 48, and 168 hr following cosyntropin administration. We conclude the cosyntropin-induced cortisol spike was likely insignificant when compared to the adrenocortical response that occurs in free-ranging bears when captured. We suggest further study with a larger sample of captive bears to evaluate the combined effects of anaesthesia and multiple doses of cosyntropin administered over several hours would better simulate the adrenocortical response of free-ranging grizzly bears during capture.

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

Development and validation of protein biomarkers of health in grizzly bears.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears (Ursus arctos) using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.