Platelet proteome dynamics in hibernating 13-lined ground squirrels.

Hibernating mammals undergo a dramatic drop in temperature and blood flow during torpor, yet avoid stasis blood clotting through mechanisms that remain unspecified. The effects of hibernation on hemostasis are especially complex, as cold temperatures generally activate platelets, resulting in platelet clearance and cold storage lesions in the context of blood transfusion. With a hibernating body temperature of 4°C-8°C, 13-lined ground squirrels (Ictidomys tridecemlineatus) provide a model to study hemostasis as well as platelet cold storage lesion resistance during hibernation. Here, we quantified and systematically compared proteomes of platelets collected from ground squirrels at summer (active), fall (entrance), and winter (topor) to elucidate how molecular-level changes in platelets may support hemostatic adaptations in torpor. Platelets were isolated from a total of 11 squirrels in June, October, and January. Platelet lysates from each animal were digested with trypsin prior to 11-plex tandem mass tag (TMT) labeling, followed by LC-MS/MS analysis for relative protein quantification. We measured >700 proteins with significant variations in abundance in platelets over the course of entrance, torpor, and activity-including systems of proteins regulating translation, secretion, metabolism, complement, and coagulation cascades. We also noted species-specific differences in levels of hemostatic, secretory, and inflammatory regulators in ground squirrel platelets relative to human platelets. Altogether, we provide the first ever proteomic characterization of platelets from hibernating animals, where systematic changes in metabolic, hemostatic, and other proteins may account for physiological adaptations in torpor and also inform translational effort to improve cold storage of human platelets for transfusion.

Physiological Stress Integrates Resistance to Rattlesnake Venom and the Onset of Risky Foraging in California Ground Squirrels.

Using venom for predation often leads to the evolution of resistance in prey. Understanding individual variation in venom resistance is key to unlocking basic mechanisms by which antagonistic coevolution can sustain variation in traits under selection. For prey, the opposing challenges of predator avoidance and resource acquisition often lead to correlated levels of risk and reward, which in turn can favor suites of integrated morphological, physiological and behavioral traits. We investigate the relationship between risk-sensitive behaviors, physiological resistance to rattlesnake venom, and stress in a population of California ground squirrels. For the same individuals, we quantified foraging decisions in the presence of snake predators, fecal corticosterone metabolites (a measure of "stress"), and blood serum inhibition of venom enzymatic activity (a measure of venom resistance). Individual responses to snakes were repeatable for three measures of risk-sensitive behavior, indicating that some individuals were consistently risk-averse whereas others were risk tolerant. Venom resistance was lower in squirrels with higher glucocorticoid levels and poorer body condition. Whereas resistance failed to predict proximity to and interactions with snake predators, individuals with higher glucocorticoid levels and in lower body condition waited the longest to feed when near a snake. We compared alternative structural equation models to evaluate alternative hypotheses for the relationships among stress, venom resistance, and behavior. We found support for stress as a shared physiological correlate that independently lowers venom resistance and leads to squirrels that wait longer to feed in the presence of a snake, whereas we did not find evidence that resistance directly facilitates latency to forage. Our findings suggest that stress may help less-resistant squirrels avoid a deadly snakebite, but also reduces feeding opportunities. The combined lethal and non-lethal effects of stressors in predator-prey interactions simultaneously impact multiple key traits in this system, making environmental stress a potential contributor to geographic variation in trait expression of toxic predators and resistant prey.

Pharmacokinetic Profiles of Gabapentin after Oral and Subcutaneous Administration in Black-tailed Prairie Dogs (Cynomys ludovicianus).

In veterinary and human medicine, gabapentin (a chemical analog of γ-aminobutyric acid) is commonly prescribed to treat postoperative and chronic neuropathic pain. This study explored the pharmacokinetics of oral and subcutaneous administration of gabapentin at high (80 mg/kg) and low (30 mg/kg) doses as a potential analgesic in black-tailed prairie dogs (Cynomys ludovicianus; n = 24). The doses (30 and 80 mg/kg) and half maximal effective concentration (1.4 to 16.7 ng/mL) for this study were extrapolated from pharmacokinetic efficacy studies in rats, rabbits, and cats. Gabapentin in plasma was measured by using an immunoassay, and data were evaluated using noncompartmental analysis. The peak plasma concentrations (mean ±1 SD) were 42.6 ±14.8 and 115.5 ±15.2 ng/mL, respectively, after 30 and 80 mg/kg SC and 14.5 ±3.5 and 20.7 ±6.1 ng/mL after the low and high oral dosages, respectively. All peak plasma concentrations of gabapentin occurred within 5 h of administration. Disappearance half-lives for the low and high oral doses were 7.4 ± 6.0 h and 5.0 ± 0.8 h, respectively. The results of this study demonstrate that oral administration of gabapentin at low (30 mg/kg) doses likely would achieve and maintain plasma concentrations at half maximum effective concentration for 12 h, making it a viable option for an every 12-h treatment.

AMPK activation, eEF2 inactivation, and reduced protein synthesis in the cerebral cortex of hibernating chipmunks.

During hibernation, mammalian cells are exposed to severe environmental stressors such as low temperature, lowered O2 supply, and glucose deficiency. The cellular metabolic rate is markedly reduced for adapting to these conditions. AMP-activated protein kinase (AMPK) senses the cellular energy status and regulates metabolism. Therefore, we examined AMPK signaling in several brain regions and peripheral tissues in hibernating chipmunk. Eukaryotic elongation factor 2 (eEF2) is a downstream target of AMPK. Phosphorylation of eEF2, indicating its inactivation, is enhanced in the cerebral cortex of hibernating chipmunks. The study indicated that the sequential regulation of AMPK-mammalian target of rapamycin complex 1-eEF2 signaling was altered and protein synthesis ability was reduced in the cerebral cortex of hibernating chipmunks.

Experimental Increases in Glucocorticoids Alter Function of the HPA Axis in Wild Red Squirrels without Negatively Impacting Survival and Reproduction.

Hormones such as glucocorticoids (colloquially referred to as "stress hormones") have important effects on animal behavior and life-history traits, yet most of this understanding has come through correlative studies. While experimental studies offer the ability to assign causality, there are important methodological concerns that are often not considered when manipulating hormones, including glucocorticoids, in wild animals. In this study, we examined how experimental elevations of cortisol concentrations in wild North American red squirrels (Tamiasciurus hudsonicus) affected their hypothalamic-pituitary-adrenal (HPA) axis reactivity and life-history traits, including body mass, litter survival, and adult survival. The effects of exogenous cortisol on plasma cortisol concentrations depended on the time between treatment consumption and blood sampling. In the first 9 h after consumption of exogenous cortisol, individuals had significantly higher true baseline plasma cortisol concentrations, but adrenal gland function was impaired as indicated by their dampened response to capture and handling and to injections of adrenocorticotropic hormone compared to controls. Approximately 24 h after consumption of exogenous cortisol, individuals had much lower plasma cortisol concentrations than controls, but adrenal function was restored. Corticosteroid-binding globulin (CBG) concentrations were also significantly reduced in squirrels treated with cortisol. Despite these profound shifts in the functionality of the HPA axis, squirrel body mass, offspring survival, and adult survival were unaffected by experimental increases in cortisol concentrations. Our results highlight that even short-term experimental increases in glucocorticoids can affect adrenal gland functioning and CBG concentrations but without other side effects.

Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel.

Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.

Cardiovascular resistance to thrombosis in 13-lined ground squirrels.

13-lined ground squirrels (Ictidomys tridecemlineatus) enter hibernation as a survival strategy during extreme environmental conditions. Typical ground squirrel hibernation is characterized by prolonged periods of torpor with significantly reduced heart rate, blood pressure, and blood flow, interrupted every few weeks by brief interbout arousals (IBA) during which blood flow fluctuates dramatically. These physiological conditions should increase the risk of stasis-induced blood clots and myocardial ischemia. However, ground squirrels have adapted to survive repeated bouts of torpor and IBA without forming lethal blood clots or sustaining lethal ischemic myocardial damage. The purpose of this study was to determine if ground squirrels are resistant to thrombosis and myocardial ischemia during hibernation. Blood markers of coagulation, fibrinolysis, thrombosis, and ischemia, as well as histological markers of myocardial ischemia were measured throughout the annual hibernation cycle. Hibernating ground squirrels were also treated with isoprenaline to induce myocardial ischemia. Thrombin-antithrombin complex levels were significantly reduced (p < 0.05) during hibernation, while D-dimer level remained unchanged throughout the annual cycle, both consistent with an antithrombotic state. During torpor, the ground squirrels were in a hyperfibrinolytic state with an elevated ratio of tissue plasminogen activator complexed with plasminogen activator inhibitor to total plasminogen activator inhibitor (p < 0.05). Histological markers of myocardial ischemia were reversibly elevated during hibernation with no increase in markers of myocardial cell death in the blood. These data suggest that ground squirrels do not form major blood clots during hibernation through suppression of coagulation and a hyperfibrinolytic state. These animals also demonstrate myocardial resistance to ischemia.

HEMATOLOGIC AND BIOCHEMICAL VALUES OF THE JUVENILE EASTERN GRAY SQUIRREL (SCIURUS CAROLINENSIS).

Venous blood samples were collected from 64 apparently healthy juvenile Eastern gray squirrels (Sciurus carolinensis) after sedation with midazolam at the Wildlife Center of Texas located in Houston, Texas, during 2012. Blood gas (pH, PCO2, PO2, base excess, bicarbonate, oxygen saturation), electrolyte (sodium, potassium), biochemical (total CO2, ionized calcium, glucose), and hematologic parameters (hematocrit, hemoglobin, complete blood count) were determined using the i-STAT point-of-care analyzer. Sex did not affect any analyte. All squirrels recovered uneventfully and were successfully rehabilitated and released. Most values were as expected based on comparison to other young rodent species. These analyte data for healthy juvenile Eastern gray squirrels may be useful in assessment of Eastern gray squirrel population health and management and treatment of individual squirrels presented in need of medical care.

Trade-offs between immunity and testosterone in male African ground squirrels.

The immunocompetence handicap hypothesis (ICHH) proposes that testosterone has both beneficial effects on male reproductive potential and negative effects by suppressing the immune system. However, support for the ICHH has been variable and an alternative hypothesis suggests that testosterone may be acting indirectly via cortisol to suppress immunity (the stress-linked ICHH). A third hypothesis is that increased energetic investment in immunity results in the suppression of testosterone. We tested these hypotheses in male Cape ground squirrels (Xerus inauris) through two separate manipulations: first, by triggering a strong immune response using a lipopolysaccharide (LPS) injection and, secondly, by increasing circulating testosterone using silastic testosterone implants. Responding to an immune challenge significantly reduced testosterone, supporting the immune suppression hypothesis, while increasing circulating testosterone had no effect on immunocompetence, body mass, ectoparasite abundances or cortisol levels, failing to support either the ICHH or stress-linked ICHH. Our results add to the increasing body of literature that challenges the ICHH, and we conclude that the trade-off between testosterone and immunity is mediated through immune activation and not through testosterone in male Cape ground squirrels. Being able to test the ICHH, stress-linked ICHH and immune suppression hypotheses in a free-ranging mammal gives us a unique opportunity to examine the mechanisms mediating this trade-off.

Seasonal changes in acute stressor-mediated plasma glucocorticoid regulation in New World flying squirrels.

Southern flying squirrels have higher circulating cortisol levels than most vertebrates. However, regulation of tissue exposure to cortisol by the hormone's carrier protein, corticosteroid-binding globulin (CBG), appears to be altered due to lower-than-expected CBG expression levels, and a reduced affinity for cortisol. To assess the capacity of flying squirrels to regulate acute stress-mediated cortisol levels, we used the dexamethasone (DEX) suppression test followed by the adrenocorticotropic hormone (ACTH) stimulation test in both the breeding and non-breeding seasons, and quantified resultant changes in plasma cortisol and relative CBG levels. Regulation of cortisol via negative feedback, and the acute stress response appeared to function as they do in other vertebrates during the breeding season, but response to DEX in the non-breeding season showed that the sensitivity of the negative feedback mechanism changed across seasons. The relatively high concentrations of DEX required to induce negative feedback suggests that southern flying squirrels have a reduced sensitivity to cortisol compared with other vertebrates, and that high circulating cortisol levels may be required to compensate for low target tissue responsiveness in this species. Cortisol, but not CBG levels, were higher during the non-breeding than breeding season, and females had higher cortisol and CBG levels than males. Our data suggest that flying squirrel cortisol levels are regulated by negative feedback at a higher set point than in related species. Seasonal changes in cortisol levels, target tissue sensitivity to DEX, and in the capacity to respond to stressors appear to be part of the underlying physiology of southern flying squirrels, and may be required to maximize fitness in the face of tradeoffs between survival and reproduction.

Influence of Isoflurane Anesthesia on Plasma Thyroxine Concentrations in Black-tailed Prairie Dogs (Cynomys ludovicianus).

Anesthesia can affect measured thyroxine (total T4) concentrations in humans and animals, but its effect in black-tailed prairie dogs (Cynomys ludovicianus) has not yet been studied. We used isoflurane to anesthetize 12 prairie dogs for 60 min. Blood samples were obtained from each animal immediately after anesthesia induction and at 30 and 60 min and used for analysis of plasma T4 concentration. The plasma T4 concentration (mean ± 1 SD) was significantly decreased from baseline (3.49 ± 0.52 µg/dL) at both 30 min (3.24 ± 0.52 µg/dL) and 60 min (3.27 ± 0.65 µg/dL) after induction. Compared with baseline, some of the T4 trends were inconsistent between animals, and individual variability in response was responsible for 86% of the overall variability. Regardless of the observed change under isoflurane anesthesia, all measurements in all prairie dogs and at all time points (2.4 to 4.4 µg/dL) were within the reported normal plasma T4 reference range for this species. In conclusion, isoflurane anesthesia appears to cause a significant but inconsistent reduction in plasma T4 concentrations in black-tailed prairie dogs, but because values remain within normal basal levels, the clinical importance of this effect is likely minimal.

Pharmacokinetics of Single-dose Subcutaneous Meloxicam Injections in Black-tailed Prairie Dogs (Cynomys ludovicianus).

This study evaluated the pharmacokinetic profile of a single dose of meloxicam (1.0 mg/kg) administered subcutaneously (n = 6) or intravenously (n = 2) to black-tailed prairie dogs (Cynomys ludovicianus). Blood was collected immediately before (time 0) and at 0.5, 1, 2, 4, 8, 12 and 24 h after drug administration. Plasma meloxicam concentrations were quantified with HPLC-mass spectrometry, and noncompartmental pharmacokinetic analysis was performed. The peak plasma concentrations, time to peak plasma concentration, and terminal half-life of meloxicam after subcutaneous administration (median [minimum-maximum]) were 4.30 (3.00-4.89) µg/mL, 2.00 (0.62-4.00) h, and 11.88 (7.35-18.64) h, respectively. Plasma concentrations of meloxicam for prairie dogs in the present study showed high absorption and slow elimination after drug administration. The results of this study suggest that a 1.0-mg/kg SC dose of meloxicam administered every 24 h might be excessive for prairie dogs, although the ideal therapeutic dose in terms of safety and efficacy is unknown in this species.

Seasonal expression of luteinizing hormone receptor and follicle stimulating hormone receptor in testes of the wild ground squirrels (Citellus dauricus Brandt).

The objective of this study was to evaluate whether luteinizing hormone (LH), follicle stimulating hormone (FSH) and their receptors luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) play roles in the seasonal spermatogenesis of the wild ground squirrels. To that end, we characterized the testicular immunolocalization of LHR and FSHR, their expression on both mRNA and protein levels, as well as serum concentrations of LH and FSH in male wild ground squirrels throughout the annual reproductive cycle. Histologically, all types of spermatogenic cells including mature spermatozoa were identified in the breeding season (April), while spermatogonia and primary spermatocytes were observed in the non-breeding season (June), and spermatogonia, primary spermatocytes and secondary spermatocytes were found in pre-hibernation (September). LHR was present in Leydig cells during the whole periods with more intense staining in the breeding season; Stronger immunostaining of FSHR was observed in Sertoli cells during the breeding season compared to the non-breeding season and pre-hibernation. Consistently, the mRNA and protein levels of LHR and FSHR were higher in testes of the breeding season, and then decreased to a relatively lower level in the non-breeding season and pre-hibernation. Meanwhile, serum LH and FSH concentrations were significantly higher in the breeding season than those in the non-breeding season and pre-hibernation. These results suggested that gonadotropins and its receptors, LHR and FSHR may be involved in the regulation of seasonal changes in testicular functions of the wild ground squirrels.

When the ball is in the female's court: How the scramble-competition mating system of the North American red squirrel has shaped male physiology and testosterone dynamics.

Male reproductive success in most mammals is determined by their success in direct inter-male competition through aggression and conflict, resulting in female-defense mating systems being predominant. This is linked to male testosterone levels and its dynamics. However, in certain environments, a scramble-competition mating system has evolved, where female reproductive behavior takes precedence and male testosterone dynamics are unlikely to be related to inter-male competition. We studied the North American red squirrel (Tamiasciurus hudsonicus), a species with a well-established scramble-competition system. Using an ACTH hormonal challenge protocol as a proxy for competitive interactions, we compared the testosterone dynamics in breeding males in late winter with that in nonbreeding males in late spring in the Yukon. To gain an integrated picture of their physiological state, we also assessed changes in their stress response, body mass, energy mobilization, and indices of immune function. Testosterone levels at the base bleed were high in breeding males (2.72ng/mL) and virtually absent in non-breeding males (0.04ng/mL). Breeding males were in better condition (heavier body mass, higher hematocrit, and higher erythrocytes), had higher indices of immune function (neutrophil:lymphocyte ratio), but a similar ability to mobilize energy (glucose) compared with non-breeding males. Though total cortisol was higher in non-breeding males, free cortisol was twice as high in breeding males as their corticosteroid binding globulin levels were half as high. In response to the ACTH challenge, testosterone levels in breeding males declined 49% over the first hour and increased 36% over the next hour; in non-breeding males levels showed no change. Free cortisol increased only modestly (26% in breeding males; 23% in non-breeding males). Glucose levels changed similarly in breeding and nonbreeding males, declining for the first 30min and then increasing for the next 60min. Thus, testosterone and components of the stress axis function in a profoundly different manner in male red squirrels than in males of mammals with female-defense mating systems. There are four probable interrelated reasons for these adaptations in male red squirrels: the marginal benefits of each mating, the constraints of mate searching away from their own resource-based territories, energy mobilization in a harsh environment, and a long life span.

Seasonal changes of androgen receptor, estrogen receptors and aromatase expression in the hippocampus of the wild male ground squirrels (Citellus dauricus Brandt).

The wild ground squirrel is a typical seasonal breeder whose annual life cycle can be roughly divided into the breeding season, the post-breeding season and hibernation. Our previous study has reported the seasonal changes in the expressions of androgen receptor (AR), estrogen receptors α and ß (ERα and ERß), and aromatase cytochrome P450 (P450arom) in the hypothalamus of male wild ground squirrels. To further seek evidence of seasonal expression of steroid hormone receptors and steroid hormone synthases in other brain regions, we investigated the protein and mRNA expressions of AR, ERα, ERß and P450arom in the hippocampus of the male wild ground squirrels during these different reproductive periods. Histological observation showed that the number of pyramidal cells in Cornu Ammonis 1 (CA1) increased in the breeding season. Both protein and mRNA of AR, ERα, ERß and P450arom were present in CA1 and CA3 of all seasons. There was significant increment in the immune-signal intensity and mRNA level of AR and ERα during the pre-hibernation, whereas those of ERß and P450arom were higher during the post-breeding season. In addition, the profile of plasma testosterone concentration showed the nadir in the post-breeding season, a marked elevation in the pre-hibernation, and the summit in the breeding season. These findings suggested that the hippocampus may be a direct target of androgen and estrogen; androgen may play important regulatory roles through its receptor and/or the aromatized estrogen in the hippocampus of the wild male ground squirrels.

A dramatic blood plasticity in hibernating and 14-day hindlimb unloading Daurian ground squirrels (Spermophilus dauricus).

We compared the effects of hibernation inactivity and 14-day hindlimb unloading in non-hibernating period on biochemical, rheological, and hematological parameters of blood in Daurian ground squirrels (Spermophilus dauricus). Twenty-four squirrels were randomly divided into four groups: control (CON), hibernation (HIB), post-hibernation (POST), and 14-day hindlimb unloading (HU). The results showed that serum enzymes (L-lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) activities decreased in HIB, POST, and HU squirrels compared with CON. Total protein (including albumin and globulin) maintained in HIB but decreased in HU compared with CON. Total cholesterol and high-density lipoprotein-cholesterol increased in HIB but maintained in HU and POST compared with CON. Meanwhile, serum creatinine decreased and urea increased in HU compared with CON. All blood ions concentrations were unchanged in HIB, POST, and HU squirrels compared with CON except calcium which increased in HIB compared with CON, and phosphorus which increased in HIB and POST compared with CON. Most of detected serum biochemical analytes in POST recovered to the CON level. Blood viscosity, which was unchanged in all shear rates in HU, increased in HIB and recovered in POST in lower shear rates compared with CON. Erythrocyte and corpuscular volume decreased in HIB and HU but maintained in POST compared with CON. All the routine hematological parameters recovered in POST as compared with CON except platelet, which decreased in HIB and POST but maintained in HU compared with CON. In conclusion, our results suggested a remarkable ability to maintain blood homeostasis in hibernating squirrels.

PLASMA THYROXINE (T4) CONCENTRATION IN ZOO-KEPT BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

This study was conducted to determine plasma thyroxine (T4) concentrations in zoo-kept black-tailed prairie dogs ( Cynomys ludovicianus ). Thirty-one healthy prairie dogs of both sexes were studied as part of their annual clinical health evaluation, performed under general isoflurane anesthesia. Each animal underwent a complete physical examination, complete blood count, plasma biochemistry, and venous blood gas analysis. Heparinized venous blood samples were collected individually and processed for plasma T4 analysis using a veterinary biochemistry analyzer. The median plasma T4 concentration for the prairie dogs in this study was 4.1 µg/dl (minimum = 0.6 µg/dl; maximum = 8.0 µg/dl). The mean ± standard deviation plasma T4 concentration was 4.49 ± 2.39 µg/dl. No significant differences were found for varying ages, sexes, weights, or housing systems. The data presented in this report can promote better physiologic understanding and improve clinical management of this rodent species.

Pharmacokinetic Profiles of Meloxicam and Sustained-release Buprenorphine in Prairie Dogs (Cynomys ludovicianus).

In this study, we evaluated the pharmacokinetic profiles of meloxicam and sustained-release (SR) buprenorphine in prairie dogs. The 4 treatment groups were: low-dose meloxicam (0.2 mg/kg SC), high-dose meloxicam (4 mg/kg SC), low-dose buprenorphine SR (0.9 mg/kg SC), and high-dose buprenorphine SR (1.2 mg/kg SC). The highest plasma concentrations occurred within 4 h of administration for both meloxicam treatment groups. The therapeutic range of meloxicam in prairie dogs is currently unknown. However, as compared with the therapeutic range documented in other species (0.39 - 0.91 µg/mL), the mean plasma concentration of meloxicam fell below the minimal therapeutic range prior to 24 h in the low-dose group but remained above therapeutic levels for more than 72 h in the high-dose group. These findings suggest that the current meloxicam dosing guidelines may be subtherapeutic for prairie dogs. The highest mean plasma concentration for buprenorphine SR occurred at the 24-h time point (0.0098 µg/mL) in the low-dose group and at the 8-h time point (0.015 µg/mL) for the high-dose group. Both dosages of buprenorphine SR maintained likely plasma therapeutic levels (0.001 µg/mL, based on previous rodent studies) beyond 72 h. Given the small scale of the study and sample size, statistical analysis was not performed. The only adverse reactions in this study were mild erythematous reactions at injection sites for buprenorphine SR.

Techniques for Nonterminal Blood Sampling in Black-Tailed Prairie Dogs (Cynomys ludovicianus).

Black-tailed prairie dogs (Cynomys ludovicianus) are used as an animal model for research on gallbladder stones and several infectious diseases. A comprehensive, instructive resource regarding the appropriate techniques for venipuncture and collection of nonterminal blood samples in this species has not yet been published. Blood samples (1 mL or larger) were readily obtained from the jugular vein, femoral vein, or cranial vena cava, whereas peripheral sites, such as the cephalic vein, saphenous vein, and tarsal vein, mainly were useful for obtaining smaller volumes. The detailed and illustrated information presented here can aid clinicians and researchers in performing venipuncture, anesthesia, and handling of this species.

No safety in the trees: Local and species-level adaptation of an arboreal squirrel to the venom of sympatric rattlesnakes.

Within some species, squirrels respond to variable selection from venomous snake predators by showing population-level variation in resistance, while between species, some rattlesnakes possess venom that is more effective at overcoming venom resistance in different species of squirrels. A functional evaluation of resistance variation to venom within and between species of squirrels and snakes can link resistance variation to its evolutionary causes across these different evolutionary scales. To do this, we compared the effectiveness of squirrel sera in inhibiting rattlesnake (Crotalus spp.) venom metalloproteinase activity between populations and between species to test for a response to local variation in selection from a single rattlesnake predator and for specialization of two resistant squirrel species to each of their distinct sympatric snake predators. We found that Timber Rattlesnake (Crotalus horridus) venom inhibition by Eastern gray squirrels (Sciurus carolinensis) is higher at a site where the rattlesnakes are present, which suggests selection may maintain venom resistance in populations separated by short distances. Next, we performed a reciprocal cross of venoms and sera from two rattlesnake and two squirrel species. This showed that squirrel resistance is lower when tested against venom from allopatric compared to sympatric rattlesnake species, demonstrating that squirrel inhibitors are specialized to sympatric venom and suggesting a tradeoff in terms of specialization to the venom of a specific species of rattlesnake predator. This pattern can be explained if inhibitors must recognize venom proteins and resistance evolution tracks venom evolution.