Alina L. Evans

Biomedical Protocols for Free-ranging Brown Bears, Gray Wolves, Wolverines and Lynx Editors

PREFACE Compilation of this document was initiated by the Norwegian Directorate for Nature Management in order to establish recommended protocols for capture, chemical immobilization, anesthesia and radiotagging of free-ranging brown bears (Ursus arctos), gray wolves (Canis lupus), wolverines (Gulo gulo) and Eurasian lynx (Lynx lynx). In addition, procedures to ensure proper sampling of biological materials for management, research and banking purposes have been included. The current protocols are based on nearly 3,000 captures of free-ranging brown bears, wolves, wolverines and lynx carried out from 1984 through 2012 in Scandinavia. Some of the results have been published as peer reviewed papers, conference presentations, theses, and reports. However, a large amount of data are still on file and will be published in the future. In addition, comprehensive reviews of the global literature on brown bears, wolves, wolverines and lynx have been carried out in order to include pertinent information from other sources. These protocols have been approved by all ongoing research projects on brown bears, wolves, wolverines and lynx in Scandinavia. We thank the contributors for their cooperative efforts. We also thank the Norwegian Directorate for Nature Management for their support.

Capture, anesthesia, and disturbance of free-ranging brown bears (Ursus arctos) during hibernation.

We conducted thirteen immobilizations of previously collared hibernating two- to four-year-old brown bears (Ursus arctos) weighing 21–66 kg in central Sweden in winter 2010 and 2011 for comparative physiology research. Here we report, for the first time, an effective protocol for the capture and anesthesia of free-ranging brown bears during hibernation and an assessment of the disturbance the captures caused. Bears were darted in anthill, soil, or uprooted tree dens on eleven occasions, but two bears in rock dens fled and were darted outside the den. We used medetomidine at 0.02–0.06 mg/kg and zolazepam-tiletamine at 0.9–2.8 mg/kg for anesthesia. In addition, ketamine at 1.5 mg/kg was hand-injected intramuscularly in four bears and in six it was included in the dart at 1.1–3.0 mg/kg. Once anesthetized, bears were removed from the dens. In nine bears, arterial blood samples were analyzed immediately with a portable blood gas analyzer. We corrected hypoxemia in seven bears (PaO2 57–74 mmHg) with supplemental oxygen. We placed the bears back into the dens and antagonized the effect of medetomidine with atipamezole. Capturing bears in the den significantly increased the risk of den abandonment. One of twelve collared bears that were captured remained at the original den until spring, and eleven, left their dens (mean ± standard deviation) 3.2±3.6 (range 0.5–10.5) days after capture. They used 1.9±0.9 intermediate resting sites, during 6.2±7.8 days before entering a new permanent den. The eleven new permanent dens were located 730±589 m from the original dens. We documented that it was feasible and safe to capture hibernating brown bears, although they behaved differently than black bears. When doing so, researchers should use 25% of the doses used for helicopter darting during the active period and should consider increased energetic costs associated with den abandonment.

Factors Affecting Date of Implantation, Parturition, and Den Entry Estimated from Activity and Body Temperature in Free-Ranging Brown Bears

Knowledge of factors influencing the timing of reproduction is important for animal conservation and management. Brown bears (Ursus arctos) are able to vary the birth date of their cubs in response to their fat stores, but little information is available about the timing of implantation and parturition in free-ranging brown bears. Body temperature and activity of pregnant brown bears is higher during the gestation period than during the rest of hibernation and drops at parturition. We compared mean daily body temperature and activity levels of pregnant and nonpregnant females during preimplantation, gestation, and lactation. Additionally we tested whether age, litter size, primiparity, environmental conditions, and the start of hibernation influence the timing of parturition. The mean date of implantation was 1 December (SD = 12), the mean date of parturition was 26 January (SD = 12), and the mean duration of the gestation period was 56 days (SD = 2). The body temperature of pregnant females was higher during the gestation and lactation periods than that of nonpregnant bears. The body temperature of pregnant females decreased during the gestation period. Activity recordings were also used to determine the date of parturition. The parturition dates calculated with activity and body temperature data did not differ significantly and were the same in 50% of the females. Older females started hibernation earlier. The start of hibernation was earlier during years with favorable environmental conditions. Dates of parturition were later during years with good environmental conditions which was unexpected. We suggest that free-ranging pregnant brown bears in areas with high levels of human activities at the beginning of the denning period, as in our study area, might prioritize investing energy in early denning than in early parturition during years with favorable environmental conditions, as a strategy to prevent disturbances caused by human.

Brown Bears (Ursus arctos) Seem Resistant to Atherosclerosis ­Despite Highly Elevated Plasma Lipids during Hibernation and Active State

Hibernation is an extreme physiological challenge for the brown bear (Ursus arctos) in which metabolism is based mainly on lipids. The study objective was to compare plasma lipids in hibernating and active free‐ranging brown bears and relate them to arterial histopathology. Blood was drawn from seven immobilized free‐ranging brown bears (three females, 2–3 years old) during hibernation in February and from the same bears while active in June and analyzed by enzymatic and automated hematology methods within 48 hours of sampling. Left anterior descending coronary arteries and aortic arches from 12 bears (six females, 1.5–12 years old) killed in hunting were examined by histopathology. Total plasma cholesterol decreased from hibernation to the active period (11.08 ± 1.04 mmol/L vs. 7.89 ± 1.96 mmol/L, P= 0.0028) as did triglyceride (3.16 ± 0.62 mmol/L vs. 1.44 ± 0.27 mmol/L, P= 0.00012) and LDL cholesterol (4.30 ± 0.71 mmol/L vs. 2.02 ± 1.03 mmol/L, P= 0.0075), whereas HDL cholesterol was unchanged. No atherosclerosis, fatty streaks, foam cell infiltration, or inflammation were seen in any arterial samples. Brown bears tolerate elevated cholesterol levels, obesity, physical inactivity, and circulatory slow flow during hibernation without signs of atherosclerosis. This species might serve as a reverse translational model for atherosclerosis resistance. Clin Trans Sci 2012; Volume 5: 269–272

HEMATOLOGY AND SERUM CHEMISTRY REFERENCE RANGES OF FREE-RANGING MOOSE (ALCES ALCES) IN NORWAY

Baseline reference ranges of serum chemistry and hematology data can be important indicators for the status of both individuals or populations of wild animals that are affected by emerging pathogens, toxicants, or other causes of disease. Frequently, reference ranges for these values are not available for wildlife species or subspecies. We present hematologic and serum chemistry reference ranges for moose (Alces alces) adults, yearlings, and calves in Norway sampled from 1992–2000. Additionally, we demonstrated that both induction time and chase time were correlated with initial rectal temperature, although they were not significantly correlated with cortisol, aspartate aminotransferase, glucose, or creatine kinase. Overall, the reference ranges given here are similar to those given for American moose, with a few differences that can be attributed to environment, testing methodology, or subspecies or species status. This is the first report, to our knowledge, of reference ranges for moose in Norway.

Body temperature during hibernation is highly correlated with a decrease in circulating innate immune cells in the brown bear (Ursus arctos) : a common feature among hibernators?

Background: Hibernation involves periods of severely depressed metabolism (torpor) and decreases in body temperature (Tb). Small arctic mammals (<5kg), in which Tb generally drop drastically, display leukopenia during hibernation. This raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low Tb. The present study examined immune cell counts in brown bears (Ursus arctos), where torpor is only associated with shallow decreases in Tb. The results were compared across hibernator species for which immune and Tb data were available. Methods and Results: The white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time. Tb dropped from 39.6±0.8 to 33.5±1.1°C during hibernation. Blood neutrophils and monocytes were lower during hibernation than during the active period (47%, p= 0.001; 43%, p=0.039, respectively), whereas no change in lymphocyte counts was detected (p=0.599). Further, combining our data and those from 10 studies on 9 hibernating species suggested that the decline in Tb explained the decrease in innate immune cells (R2=0.83, p<0.0001). Conclusions: Bears have fewer innate immune cells in circulation during hibernation, which may represent a suppressed innate immune system. Across species comparison suggests that, both in small and large hibernators, Tb is the main driver of immune function regulation during winter dormancy. The lack of a difference in lymphocyte counts in this context requires further investigations.

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.

Haematological and biochemical reference intervals for free-ranging brown bears (Ursus arctos) in Sweden

BackgroundEstablishment of haematological and biochemical reference intervals is important to assess health of animals on individual and population level. Reference intervals for 13 haematological and 34 biochemical variables were established based on 88 apparently healthy free-ranging brown bears (39 males and 49 females) in Sweden. The animals were chemically immobilised by darting from a helicopter with a combination of medetomidine, tiletamine and zolazepam in April and May 2006–2012 in the county of Dalarna, Sweden. Venous blood samples were collected during anaesthesia for radio collaring and marking for ecological studies. For each of the variables, the reference interval was described based on the 95% confidence interval, and differences due to host characteristics sex and age were included if detected. To our knowledge, this is the first report of reference intervals for free-ranging brown bears in Sweden.ResultsThe following variables were not affected by host characteristics: red blood cell, white blood cell, monocyte and platelet count, alanine transaminase, amylase, bilirubin, free fatty acids, glucose, calcium, chloride, potassium, and cortisol. Age differences were seen for the majority of the haematological variables, whereas sex influenced only mean corpuscular haemoglobin concentration, aspartate aminotransferase, lipase, lactate dehydrogenase, β-globulin, bile acids, triglycerides and sodium.ConclusionsThe biochemical and haematological reference intervals provided and the differences due to host factors age and gender can be useful for evaluation of health status in free-ranging European brown bears.

Physiological evaluation of free-ranging moose (Alces alces) immobilized with etorphine-xylazine-acepromazine in Northern Sweden.

BackgroundEvaluation of physiology during capture and anesthesia of free-ranging wildlife is useful for determining the effect that capture methods have on both ecological research results and animal welfare. This study evaluates capture and anesthesia of moose (Alces alces) with etorphine-xylazine-acepromazine in Northern Sweden.MethodsFifteen adult moose aged 3–15 years were darted from a helicopter with a combination of 3.37 mg etorphine, 75 mg xylazine, and 15 mg acepromazine. Paired arterial blood samples were collected 15 minutes apart with the first sample at 15–23 minutes after darting and were analyzed immediately with an i-STAT®1 Portable Clinical Analyzer.ResultsAll animals developed hypoxemia (PaO2 <10 kPa) with nine animals having marked hypoxemia (PaO2 5.5-8 kPa). All moose were acidemic (ph<7.35) with nine moose having marked acidemia (pH<7.20). For PaCO2, 14 moose had mild hypercapnia (PaCO2 6-8 kPa) and two had marked hypercapnia (PaCO2>8 kPa). Pulse, respiratory rate, pH and HCO3 increased significantly over time from darting whereas lactate decreased.ConclusionsThe hypoxemia found in this study is a strong indication for investigating alternative drug doses or combinations or treatment with supplemental oxygen.

Effective Immobilizing Doses of Medetomidine-Ketamine in Free-ranging, Wild Norwegian Reindeer (Rangifer tarandus tarandus)

Combinations of medetomidine and ketamine were evaluated in free-ranging, wild Norwegian reindeer (Rangifer tarandus tarandus) as part of a reintroduction program in southwestern Norway in November 1995 and November 1996. The drugs were administered by dart from a helicopter. The mean (SD) effective immobilizing doses for 29 adults (8 males, 21 females) were 0.21 (0.04) mg medetomidine/kg and 1.0 (0.2) mg ketamine/ kg based on estimated body mass. There was no significant difference in mean induction times between males and females. However, animals with optimal hits (shoulder or thigh muscles; n=16) had a significantly shorter (P<0.05) mean induction time than did animals with suboptimal hits (abdomen or flank; n=13), 5.6 (2.2) min and 11.1 (4.7) min, respectively. Inductions were calm, and immobilized animals were maintained in sternal recumbency. Clinical side effects included hypoxemia and hyperthermia in most animals. For reversal, all animals received 5 mg atipamezole per mg medetomidine, half intravenously and half intramuscularly, and the mean (SD) time to standing was 3.7 (3.6) min.