Japanese black bears (Ursus thibetanus japonicus), a subspecies of Asiatic black bears, undergo annual cycles in body mass characterized by fattening in autumn and weight loss in winter. During the hibernation period in winter, bears rely on fat stores as their main energy source,1 and pregnant bears give birth and nurse cubs.2,3 Therefore, the accumulation of body fat in autumn is critical to the survival and reproduction of bears.
Fruits and nuts, which contain large amounts of carbohydrates, are the main foods available to bears before hibernation. It is speculated that bears convert these carbohydrates into body fat. However, the metabolic mechanisms involved in the regulation of body mass or body fat have yet to be elucidated.
The distinct seasonal fluctuation in the amount of body fat without adverse health effects in hibernating species makes such species ideal subjects for studying body mass regulation and obesity. Body mass gain is not simply a consequence of increased food intake. Various other factors, such as metabolic enzymes, hormones, and metabolic rate, are related to the circannual rhythm of energy balance and fat metabolism.4 For example, basal insulin concentration reportedly correlates with body mass in fat-storing hibernators.5–7 In marmots, glucose uptake in peripheral tissues is significantly higher in summer than in other seasons, and both hyperinsulinemia and peripheral insulin resistance develop in autumn.7 These findings suggest that glucose metabolism, controlled by insulin, alters in relation to the change of body mass (body fat) in animals that hibernate.
One method to assess pancreatic beta-cell function and glucose clearance in peripheral tissues is the IVGTT. In humans, dogs, and cats, this test is used to assess glucose tolerance or to diagnose diabetes mellitus.8–10 To our knowledge, the IVGTT has never been performed in Japanese black bears, and few if any studies on pancreatic function have involved bears. To perform an IVGTT in bears, anesthesia must be maintained for a long period, and anesthetics may have undesirable effects on carbohydrate metabolism. However, there is little information about anesthesia in Japanese black bears, and there are few reports on the effects of anesthetics on carbohydrate and lipid metabolism in this species. Consequently, anesthetic protocols are needed that are safe and effective in bears and whether these protocols interfere with IVGTT results needs to be determined.
In bears, a 1:1 mixture of TZ is commonly used for anesthesia11–14 because IM administration of TZ induces reliable anesthesia with predictable signs of recovery and has a wide margin of safety.15,16 The disadvantages of using TZ are the lack of specific antagonists or reversal agents and the finding that additional doses prolong recovery time.16 Despite the common use of TZ in bears, the effects of TZ on carbohydrate metabolism and insulin secretion in bears are unknown.
Medetomidine HCl is a specific α2-adrenergic receptor agonist and is widely used in veterinary medicine as a sedative, analgesic, and muscle relaxant.17 The combination of TZ with medetomidine has been used for the immobilization of bears15,18,19 because medetomidine administration allows the dose of TZ required to achieve immobilization to be decreased and because the effects of medetomidine are readily and rapidly reversed with subsequent atipamezole administration. In many species, medetomidine administration yields undesirable effects such as hyperglycemia, hypoinsulinemia, and bradycardia.17,20,21 However, no reports exist of the effect of medetomidine on carbohydrate metabolism in bears. Acepromazine maleate and butorphanol tartrate are other sedatives that might be combined with TZ for anesthesia in bears, particularly because the effects of these agents on glucose metabolism are minimal in dogs.22,23 The purpose of the study reported here was to establish a safe and effective anesthetic protocol with minimal effects on blood biochemical values and IVGTT results in Japanese black bears.
Materials and Methods
Animals—Sixteen adult (5- to 17-year-old) nonpregnant female Japanese black bears kept in Ani Mataginosato Bear Park, Akita Prefecture, Japan, were used in the study. The bears hibernated in indoor rooms without feed (but with water) from December to mid-April. The study was conducted between May and June 2010. During the active season from May to November, all bears were provided with approximately 2 kg of dried corn/d with some fruits, vegetables, and commercial bear food pellets as supplements. Water was freely available at all times. The study was performed in accordance with the guidelines of the Animal Care and Use Committee of Hokkaido University
Study design—The experiment was performed indoors during the morning, and bears were not fed after 4 pm the previous day. Bears were randomly assigned to 4 premedication groups (4 bears/group): TZa (9 mg/kg) alone with no premedication, acepromazineb (0.1 mg/kg) followed by TZ (6 mg/kg), butorphanolc (0.3 mg/kg) followed by TZ (6 mg/kg), or medetomidined (40 μg/kg) followed by TZ (3 mg/kg).
The TZ solution was prepared by reconstituting powdered TZ with 5 mL of diluent to achieve a concentration of 300 mg/mL. The doses of acepromazine and butorphanol used were estimated from published information on dogs and cats.24 The doses for TZ alone and TZM were established through evaluation of previous studies11,25 on TZ and TZM use in Japanese black bears. The dose of TZ in combination with acepromazine or butorphanol was established on the basis of results of preliminary experiments that showed > 5 mg of TZ/kg would be necessary to immobilize the bears (unpublished data).
The dose of premedication was determined on the basis of estimated body weight and was subsequently administered IM via blow dart to bears assigned to treatments involving a premedication. The TZ was administered in a similar manner to all bears 30 minutes after premedication. When the effect of the TZ was insufficient, additional doses of TZ (approximately a third of the original dose) were given IM at approximately 45 minutes after the first dose of TZ was administered.
Once immobilization (anesthetic induction) was achieved, the bears were weighed and handled. Final doses of TZ were calculated on the basis of actual body weights. Catheterse were inserted into both external jugular veins: one for anesthetic (TZ) infusion and glucose injection and the other for blood sample collection. The bears were kept immobilized throughout the experiment by continuous IV infusion of TZ dissolved in 500 mL of acetated Ringer's solutionf at a rate of 1 to 3 mg/kg/h.
Recording and monitoring—Induction time was defined as the interval from TZ administration to the point at which bears moved to sternal or lateral recumbency and no response could be elicited to auditory stimulation from clapping or shouting. Monitoring of rectal temperature, pulse rate, and respiratory rate began 2 hours after the initial dose of TZ was administered and was repeated every 15 minutes until conclusion of the IVGTT. Mean ambient temperatures during the experiments ranged between 9.8° and 23.1°C. Bears were kept warm with an electric compact heater to maintain a rectal temperature > 33°C. Pulse rate was monitored with a stethoscope, and respiratory rate was determined from the number of thoracic excursions observed during a 1-minute period.
IVGTT and blood sample collection—Three baseline blood samples were collected from the catheter and placed into evacuated tubes at 60, 90, and 120 minutes after the administration of TZ, corresponding to 70, 40, and 10 minutes prior to glucose injection. A sterile solution containing 50% glucose (0.5 g/kg) was injected for 1 to 3 minutes at 130 minutes after TZ administration. The endpoint of the injection period was deemed time 0 for the IVGTT.
Blood samples were collected via the catheter at 0, 10, 30, 60, 90, 120, 150, and 180 minutes after glucose injection and placed into evacuated tubes. Immediately after each sample was collected, approximately 1 mL of heparinized saline (0.9% NaCl) solution was used to flush the catheter.
After the IVGTT was finished, the continuous IV infusion of TZ was discontinued. Naloxone hydrochlorideg (0.04 mg/kg, IV; butorphanol antagonist) or atipamezole HClh (200 μg/kg, IM; medetomidine antagonist) was administered to bears in the TZB and TZM groups, respectively, to facilitate anesthetic recovery. Bears in the other groups received no additional treatment.
Blood sample processing—Evacuated tubes containing a mixture of sodium fluoride, heparin sodium, and EDTA-2Na as anticoagulant were used for plasma glucose determination. Tubes containing EDTA-2K were used to determine plasma triglycerides and total cholesterol concentrations, and heparin sodium was used as the anticoagulant to determine plasma ALT and AST activities. Blood samples were also placed in evacuated tubes without anticoagulant and allowed to clot. All samples were chilled on ice, then underwent centrifugation at 1,880 × g for 10 minutes within 10 (for plasma) or 30 (for serum) minutes after sample collection. Separated serum (for NEFA and insulin) and plasma (for glucose, triglycerides, total cholesterol, ALT, and AST assays) were stored at −30°C for approximately 2 weeks until assayed.
Blood biochemical analyses—Analytes other than glucose and insulin were analyzed in the serum or plasma harvested from blood samples obtained 60 minutes after the administration of TZ, with the exception of the sample for 1 bear in the TZB group. For that bear, the sample obtained 90 minutes after the TZ administration was used because the 60-minute sample could not be obtained. Baseline plasma concentrations of glucose and serum concentrations of insulin were analyzed in the samples obtained 60, 90, and 120 minutes after the administration of TZ. Plasma glucose, triglycerides, and total cholesterol concentrations and plasma ALT and AST activities were measured with an automatic blood analyzer.i Serum NEFA concentrations were measured with a commercial kit.j Serum insulin concentration was measured with an ELISA developed for dog insulin concentration measurement,k with purified dog insulinl used as the standard. The intra-assay and interassay coefficients of variation for the ELISA were 3.6% and 8.0%, respectively.
IVGTT analysis—Values of t1/2 and the K were determined from the results of each IVGTT. To calculate the t1/2, linear regression analysis of a semilogarithmic plot of plasma glucose concentration versus time between 10 and 30 minutes after glucose injection was used. The K value was calculated by use of the following formula: K = (0.693/t1/2) × 100.9
Areas under the glucose and insulin curves (AUCglucose and AUCinsulin) were calculated as incremental area above zero with statistical software.m The AUC-glucose and AUCinsulin were determined from 10 minutes before (—10 minutes) to 10 minutes after glucose injection and from −10 minutes to 180 minutes after injection (end of the IVGTT).
Statistical analysis—Unless otherwise stated, statistical analyses were performed with statistical software.m To analyze the effects of 4 anesthetic combinations on the selected blood variables (triglycerides, total cholesterol, and NEFA concentrations and AST and ALT activities), the differences in these variables among groups were evaluated via the Kruskal-Wallis test. Differences in body weight, total TZ dose, and induction time among groups were also evaluated with the Kruskal-Wallis test. When a statistical test revealed a significant difference, the Dunn multiple comparison test was performed. Differences in rectal temperatures, pulse rates, and respiratory rates among groups were evaluated similarly. Changes in those 3 variables over time (the changes from the values at 2 hours after TZ injection [10 minutes before glucose injection]) in each group were evaluated via the Steel multiple comparison test with the aid of statistical software.n
To analyze the effects of 4 anesthetic combinations on IVGTT results, differences in baseline glucose and insulin concentrations and in values calculated from the results of each IVGTT (peak plasma glucose and serum insulin concentrations, t1/2, K, AUCglucose, and AUCinsulin) among groups were evaluated with the Kruskal-Wallis test followed by the Dunn multiple comparison test. Again, because there were no significant differences among the plasma glucose and serum insulin concentrations measured at the 3 points before glucose injection, the means of these 3 baseline values were used to represent the pretest values. To identify any significant changes from baseline values in the serial measurements of glucose and insulin concentrations performed during IVGTTs, the Steel multiple comparison test was used. Values of P < 0.05 were considered significant. All data are reported as mean ± SD.
Results
Animals—Body weights of the bears, doses of TZ required for anesthetic induction, doses of premedication, and induction times in each group were summarized (Table 1). No significant differences were detected in body weight among groups. No bears, except for 2 bears in the TZM group that became laterally or sternally recumbent, had signs of sedation for 30 minutes after premedication administration (acepromazine, butorphanol, or medetomidine). Administration of TZ caused the bears to become recumbent within a mean ± SD time of 13.1 ± 4.8 minutes (range, 6 to 26 minutes), with no significant differences among groups.
Mean ± SD body weight, dose of TZ and premedication for anesthetic induction, and induction time in Japanese black bears (4/group) immobilized with TZ (9 mg/kg) alone (no premedication), acepromazine maleate (0.1 mg/kg) followed by TZ (6 mg/kg), butorphanol tartrate (0.3 mg/kg) followed by TZ (6 mg/kg), or medetomidine HCl (40 μg/kg) followed by TZ (3 mg/kg).
| Group | Additional dose required | Body weight (kg) | TZ dose (mg/kg) | Total dose of premedication (mg/kg) | Induction time (min) | ||
|---|---|---|---|---|---|---|---|
| Initial | Additional | Total | |||||
| TZ alone | 71.3 ± 21.3 | 9.1 ± 0.9 | 2.1 ± 2.6 | 11.2 ± 2.0 | — | 45.5 ± 33.8 | |
| Yes (n = 2) | 85.0 ± 17.0 | 8.7 ± 1.1 | 4.2 ± 1.6 | 12.8 ± 0.4 | — | 74.5 ± 7.8 | |
| No (n = 2) | 57.5 ± 17.7 | 9.5 ± 0.7 | — | 9.5 ± 0.7 | — | 16.5 ± 0.7 | |
| TZA | 66.0 ± 14.6 | 6.6 ± 0.9 | 0.5 ± 1.0 | 7.1 ± 1.1 | 0.110 ± 0.015 | 37.5 ± 12.2 | |
| Yes (n = 1) | 73.0 | 6.2 | 2.1 | 8.2 | 0.103 | 52.0 | |
| No (n = 3) | 63.7 ± 16.9 | 6.8 ± 1.0 | — | 6.8 ± 1.0 | 0.113 ± 0.018 | 32.7 ± 9.1 | |
| TZB | 72.0 ± 17.4 | 6.5 ± 0.5 | 2.7 ± 2.0 | 9.2 ± 1.9 | 0.325 ± 0.024 | 61.3 ± 19.6 | |
| Yes (n = 3) | 78.3 ± 14.6 | 6.4 ± 0.5 | 3.6 ± 1.1 | 10.0 ± 1.2 | 0.320 ± 0.027 | 70.7 ± 6.7 | |
| No (n = 1) | 53.0 | 6.8 | — | 6.8 | 0.340 | 33.0 | |
| TZM | 68.8 ± 6.6 | 3.3 ± 0.3 | 0.3 ± 0.6 | 3.6 ± 0.9* | 0.047 ± 0.013 | 30.5 ± 23.0 | |
| Yes (n = 1) | 64.0 | 3.8 | 1.2 | 5.0 | 0.050 | 64.0 | |
| No (n = 3) | 70.3 ± 7.0 | 3.1 ± 0.2 | — | 3.1 ± 0.2 | 0.041 ± 0.004 | 19.3 ± 6.7 | |
Value differs significantly (P < 0.05) from the value for TZ alone.
— = Not applicable.
Acepromazine, butorphanol, and medetomidine were administered IM as premedications 30 minutes before TZ was administered IM. One hour after TZ administration, anesthesia was maintained with a continuous IV infusion of TZ (1 to 3 mg/kg/h). Induction time was defined as the interval from TZ administration to the point at which bears moved to sternal or lateral recumbency and no response could be elicited to auditory stimulation from clapping or shouting.
Within the TZ, TZA, and TZB groups, 2, 1, and 3 bears, respectively, needed additional doses of TZ to achieve immobilization; consequently, total doses of TZ for these groups were not significantly different. In contrast, sufficient anesthetic depth was achieved with a significantly lower dose of TZ for bears in the TZM group (3.6 ± 0.9 mg/kg) than for bears that received TZ alone (11.2 ± 2.0 mg/kg). Additional doses of TZ prolonged the induction time, but the mean induction time of each group was not significantly different.
Physiologic effects—Rectal temperatures before glucose injection (2 hours after TZ administration) were not significantly different among groups (Table 2), and no significant (P = 0.10) correlation was found between rectal and ambient temperatures at this point. For all groups, there were no significant changes in rectal temperatures over time, although temperatures decreased gradually in the TZA, TZB, and TZM groups (Figure 1). Mean rectal temperatures at the end of the experiment in each group were 35.8° ± 1.0°C for TZ alone, 35.8° ± 1.3°C for TZA, 35.3° ± 0.4°C for TZB, and 34.6° ± 1.0°C for TZM.
Mean ± SD baseline plasma triglycerides concentration, total cholesterol concentration, and AST activity and ALT activity and serum NEFA concentration in the bears in Table 1.
| Variable | TZ alone | TZA | TZB | TZM |
|---|---|---|---|---|
| Triglycerides (mmol/L) | 3.5 ± 0.7 | 3.3 ± 0.7 | 3.5 ± 0.6 | 3.1 ± 0.3 |
| Total cholesterol (mmol/L) | 8.0 ± 1.8 | 7.7 ± 1.6 | 7.6 ± 0.8 | 8.0 ± 0.2 |
| NEFA (mmol/L) | 0.23 ± 0.21 | 0.77 ± 0.79 | 0.28 ± 0.22 | 0.29 ± 0.15 |
| AST (U/L) | 95.0 ± 9.1 | 105.3 ± 41.5 | 90.3 ± 16.7 | 100.3 ± 12.3 |
| ALT (U/L) | 23.3 ± 6.0 | 21.8 ± 7.1 | 21.8 ± 7.6 | 23.0 ± 2.7 |
Changes in mean ± SD rectal temperature (A), pulse rate (B), and respiratory rate (C) over time in bears (4/group) immobilized with TZ (9 mg/kg) alone (no premedication; black circles), acepromazine maleate (0.1 mg/kg) followed by TZ (6 mg/kg; white circles), butorphanol tartrate (0.3 mg/kg) followed by TZ (6 mg/kg; triangles), or medetomidine HCl (40 μg/kg) followed by TZ (3 mg/kg; squares). Acepromazine, butorphanol, and medetomidine were administered IM 30 minutes before TZ was administered IM in all groups. One hour after TZ administration, anesthesia was maintained with a continuous IV infusion of TZ (1 to 3 mg/kg/h). a,bValues with different letters are significantly (P < 0.05) different among groups at 2 hours after TZ administration.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1282
Pulse rates at 2 hours after TZ administration were significantly (P < 0.05) higher in the TZA (129 ± 23 beats/min) group than in the TZB (55 ± 29 beats/min) and TZM (47 ± 7 beats/min) groups. After the 2-hour measurement point, there were no significant changes in pulse rates over time in all groups. Respiratory rates at 2 hours after TZ administration were not significantly different among groups. After the 2-hour measurement point, some variability in respiratory rates was observed among groups, but there were no significant changes over time in any group. The TZB group had a lower respiratory rate than did the other groups, and the mean respiratory rate throughout the experiment in the TZB group was 10 ± 3 breaths/min.
Induction and maintenance of anesthesia during the experiment were safely achieved in all bears. No bears died or developed noticeable physical problems. However, vomiting was observed during induction of anesthesia in 1 bear from the TZA group. Additionally, 2 and 3 bears that received TZ alone and TZA, respectively, had increased salivation during the experiment.
Hematologic effects—Baseline concentrations of plasma lipids and activities of hepatic enzymes did not differ significantly among the groups (Table 2). Baseline concentrations of plasma glucose concentration were significantly higher in the TZM group (6.9 ± 1.2 mmol/L) than in the TZ-alone (4.5 ± 0.4 mmol/L), TZA (4.7 ± 0.7 mmol/L), and TZB (4.2 ± 0.1 mmol/L) groups (Table 3). Plasma glucose concentrations peaked immediately after glucose injection (0 minutes) in all groups and then decreased gradually over time, but there were no significant differences in peak values among the groups (Figure 2). For bears in the TZ-alone, TZA, and TZB groups, plasma glucose concentrations decreased at a similar rate and returned to baseline concentrations within 60 minutes after glucose injection. In contrast, plasma glucose concentrations remained high until 90 minutes after glucose injection for bears in the TZM group.
Mean ± SD plasma glucose and serum insulin concentrations before (baseline; −10 minutes) and after IV glucose injection in the bears in Table 1.
| Variable | TZ alone | TZA | TZB | TZM |
|---|---|---|---|---|
| Glucose | ||||
| Baseline (mmol/L) | 4.5 ± 0.4a | 4.7 ± 0.7a | 4.2 ± 0.1a | 6.9 ± 1.2b |
| Peak (mmol/L) | 26.4 ± 3.2 | 25.4 ± 3.4 | 29.3 ± 3.7 | 28.5 ± 2.5 |
| t1/2 (min) | 16.0 ± 8.0a | 24.0 ± 10.4a,b | 22.5 ± 13.5a,b | 51.5 ± 8.1b |
| K (percentage/min) | 5.2 ± 2.3a | 3.3 ± 1.5a,b | 3.9 ± 1.8a,b | 1.4 ± 0.2b |
| AUCglucose (mmol/L/min) | ||||
| -10 to 10 min | 416 ± 47a | 415 ± 37a,b | 444 ± 39a,b | 513 ± 15b |
| -10 to 180 min | 1,306 ± 196a | 1,508 ± 205a,b | 1,420 ± 191a,b | 2,583 ± 261b |
| Insulin | ||||
| Baseline (pmol/L) | 108 ± 88a | 79 ± 32a | 136 ± 87a | 23 ± 17b |
| Peak (pmol/L) | 700 ± 125 | 903 ± 519 | 691 ± 108 | 232 ± 160 |
| AUCinsulin (pmol/L/min) | ||||
| -10 to 10 min | 11,659 ± 3,169a,b | 9,890 ± 4,265a,b | 12,484 ± 2,985a | 563 ± 329b |
| -10 to 180 min | 39,435 ± 10,620 | 59,127 ± 19,755 | 54,888 ± 17,413 | 27,102 ± 14,144 |
Values with different superscript letters differ significantly (P<0.05) among groups.
Mean ± SD plasma glucose (black circles) and serum insulin (white circles) concentrations before and after IV glucose injection (0.5 g/kg) in TZ-anesthetized bears (4/group) immobilized with TZ (9 mg/kg) alone (no premedication; A), acepromazine maleate (0.1 mg/kg) followed by TZ (6 mg/kg; B), butorphanol tartrate (0.3 mg/kg) followed by TZ (6 mg/kg; C), or medetomidine HCl (40 μg/kg) followed by TZ (3 mg/kg; D). The 0-minute time point represents the point of glucose injection during an IVGTT. a,bValues with different letters are significantly (P < 0.05) different from the baseline value (mean of the 3 values measured before glucose injection but after continuous IV infusion of TZ began).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1282
The t1/2 and K values in the TZM group were significantly longer and slower, respectively, compared with the values in the TZ-alone group (P < 0.05). The AUCglucose from 10 minutes before (—10 minutes) to 10 minutes after glucose administration and from −10 to 180 minutes was significantly (P < 0.05) higher in the TZM group than in the TZ-alone group (Table 3).
Baseline serum insulin concentrations were significantly lower in the TZM group (23 ± 17 pmol/L) than in the TZ-alone (108 ± 88 pmol/L), TZA (79 ± 32 pmol/L), and TZB (136 ± 87 pmol/L) groups. Insulin concentrations after glucose injection varied according to the type of premedication. For bears that received TZ alone or TZB, the peak insulin concentration was observed within 10 minutes after glucose injection (Figure 2). The insulin response after glucose injection was slightly delayed for bears in the TZA group, and the peak concentration was observed 30 minutes after glucose injection. For these 3 groups, serum insulin concentrations returned to baseline values by 60 minutes after glucose injection. In contrast, the insulin response for the TZM group was delayed, with the peak concentration at 90 minutes after glucose injection. However, the peak insulin concentration was not significantly different from the peaks in the other 3 groups.
The AUCinsulin from −10 minutes to 10 minutes in the TZM group was approximately a twentieth of the values of the other 3 groups, with a significant difference evident between the TZB and TZM groups. However, there were no significant differences among the groups in AUCinsulin from −10 minutes to 180 minutes.
Discussion
The safety and efficacy of 4 combinations of anesthetics in Japanese black bears were evaluated in the present study. Results indicated that the 4 administered combinations of TZ with and without premedications yielded sufficient anesthesia without noticeable important physical problems. Approximately 7 and 9 mg of TZ/kg was necessary to induce anesthesia in bears premedicated with 0.1 mg of acepromazine/kg and 0.3 mg of butorphanol/kg, respectively.
The mean anesthetic induction time of 4 bears was considerably longer than that in previous reports of bears immobilized by TZ14 or TZM,19 in which the definition of induction time was similar to that used in the present study. Many factors, such as doses of drugs received, physical condition of the bear, and sensitivity to the anesthetic used, may influence the induction time. A possible explanation for the prolonged induction time in the present study is that the drugs administered for initial immobilization may have been injected into fat tissue rather than in muscle. The target for the blow dart containing the drugs was the hindquarters in most of the bears that required additional doses. Because all bears were in good nutritional status, they may have had a thick fat layer in the hindquarters, slowing drug absorption into the body.
In general, thermoregulation is impaired when animals are anesthetized, and when exposed to a cold environment, most animals without an external heat source develop hypothermia.26 Hypothermia is an undesirable effect of phenothiazine and α2-adrenergic receptor agonist administration.27,28 Therefore, care will need to be taken to mitigate the hypothermia induced by the drug combinations used in the present study when administered in a cold environment.
The reference interval for resting pulse rates of bears is 60 to 90 beats/min,29 and pulse rates observed in the TZB group were within this range throughout the experiment. Pulse rates in the bears that received TZ alone were in agreement with those in a previous study11 on immobilization of Japanese black bears with TZ. Bears that received TZ alone or TZA had pulse rates that were slightly higher than the upper reference limit. In dogs, administration of TZ commonly yields an increase in pulse rate30 and appears to do the same in bears. The lower pulse rate in the TZM group was similar to that noted in previous studies16,18 involving bears and was considered clinically unimportant.
The reference interval for respiratory rate in bears is 15 and 30 breaths/min,29 and respiratory rates observed in bears that received TZ alone, TZA, and TZM in the present study were mostly within this range throughout the experiment. The mild respiratory depression observed in the TZB group was considered to have little clinical importance. Although significant differences were not detected in respiratory rates within each group during the experiment, respiratory rates in bears that received TZ alone or TZA groups increased over time.
Acepromazine administration generally has a mild suppressive effect on respiratory rate,24 and TZ administration has been shown to have a minimal adverse effect on the respiratory system in bears.15 However, when high doses of TZ are administered, significant respiratory depression develops.30 In the present study, a high dose of TZ was administered for anesthetic induction, and after initial immobilization, anesthesia was maintained by continuous IV infusion of TZ at a lower dose. Therefore, the lower respiratory rates evident in bears that received TZ alone or TZA early in the course of anesthesia may have been attributable to an initial high dose of TZ. The gradual increase in respiratory rate during anesthesia may have been due to a progressive reduction in blood TZ concentration. In contrast, the constantly low respiratory rates in the TZB group may have been attributable to the respiratory depressant effect of butorphanol.
Little information is available regarding blood biochemical values in Japanese black bears, and reference intervals have yet to be established. However, plasma total cholesterol concentration and AST and ALT activities in the present study were within the physiologic reference range for American black bears (Ursus americanus).31 Therefore, we suggest that effects of the drugs used on these variables were negligible. Previous studies32–37 have shown that blood triglycerides concentrations vary somewhat during the active season for bears, ranging from approximately 1 to 4 mmol/L, and the plasma triglycerides concentrations observed in the present study were within this range. Compared with reported blood NEFA concentrations in captive32 and wild38 American black bears anesthetized with a combination of ketamine and xylazine HCl, the values in the present study were low. Some reports exist of the suppression of plasma NEFA concentration by medetomidine and xylazine administration in dogs,20 cats,21 and cattle39 and by acepromazine administration in dogs.22 However, noticeable suppression of NEFA concentrations by these drugs was not evident in the study bears.
Baseline glucose values in bears that received TZ alone, TZA, and TZB were within the physiologic reference interval for American black bears,31 and the baseline insulin concentrations were comparable with the summer concentrations in American black bears.6 In contrast, administration of α2-adrenergic receptor agonists, such as xylazine and medetomidine, reportedly causes hyperglycemia in cats and rats by suppressing insulin secretion by the pancreas,40,41 and results of the present study suggested that medetomidine had the same effect in Japanese black bears.
To the authors' knowledge, the IVGTT has never before been performed in Japanese black bears, yet anesthesia is necessary to perform any medical examination on bears. For these reasons, the usual response to the IVGTT in nonanesthetized bears is unknown. In nonanesthetized dogs with a usual response to the IVGTT, plasma glucose concentration returns to the baseline concentration within 60 minutes after glucose injection and the insulin response is observed within 5 minutes.9 The serum insulin concentration also returns to the baseline concentration by 60 minutes after glucose injection.9 Although some variability in insulin response to glucose injection was observed in the present study, results of the IVGTTs in bears that received TZ alone, TZA, or TZB were comparable with the usual response in nonanesthetized dogs. Moreover, the t1/2 and K values of the bears in these 3 groups were similar to the values of healthy dogs (mean ± SD t1/2, 26 ± 9 minutes; K, 2.76 ± 0.91%/min).9 Therefore, we suggest that the effect of TZ alone, TZA, or TZB on the IVGTT performed in Japanese black bears is minimal.
The longer t1/2, slower K, and higher AUCglucose in the TZM group suggested a reduction in glucose tolerance, compared with tolerance in the other groups. The insulin response to glucose injection in the TZM group was noticeably suppressed, particularly during the early phase of the IVGTT, as indicated by the noticeable decrease of AUCinsulin from 10 minutes before to 10 minutes after glucose injection. In dogs, the suppression of insulin secretion by medetomidine is sustained for approximately 2 to 3 hours.20,42 In the present study, serum insulin concentration increased gradually from 30 minutes after glucose injection (approx 3 hours after medetomidine administration). The suppression of insulin secretion by medetomidine may be sustained for approximately 3 hours in bears as occurs in dogs.
In the present study, induction and maintenance of anesthesia with TZ, with and without premedications, were safe and effective in Japanese black bears, with little adverse effects on cardiopulmonary function. However, the hypothermia that results from anesthesia needs to be addressed when bears are anesthetized in cold environments. Administration of TZ alone, TZA, or TZB appeared to have little effects on baseline values of serum or plasma biochemical variables and the results of IVGTTs. We therefore conclude that these 3 combinations can be used for chemical immobilization to allow performance of IVGTTs in Japanese black bears. In contrast, medetomidine, which appeared to have hyperglycemic and hypoinsulinemic effects, is inappropriate as a premedication in bears when evaluating glucose metabolism controlled by insulin.
ABBREVIATIONS
| ALT | Alanine aminotransferase |
| AST | Aspartate aminotransferase |
| AUCglucose | Area under the glucose curve |
| AUCinsulin | Area under the insulin curve |
| IVGTT | IV glucose tolerance test |
| K | Fractional clearance rate |
| NEFA | Nonesterified fatty acid |
| t1/2 | Half-life for glucose disappearance from serum |
| TZ | Tiletamine-zolazepam |
| TZA | Tiletamine-zolazepam and acepromazine maleate |
| TZB | Tiletamine-zolazepam and butorphanol tartrate |
| TZM | Tiletamine-zolazepam and medetomidine |
Zoletil 100, Virbac Laboratory, Carrors, France.
A.C.P. 10 Injection 100 mL, Delvet Pty Ltd, Asquith, NSW, Australia.
Vetorphale, Meiji Seika Kaisha Ltd, Tokyo, Japan.
Domitor, Nippon Zenyaku Kogyo Co Ltd, Fukushima, Japan.
SurFlash I.V. Catheters, TERUMO Co, Tokyo, Japan.
SOLACET F, Terumo Corp, Tokyo, Japan.
Naloxone hydrochloride IV injection 0.2 mg, Daiichi Sankyo Co Ltd, Tokyo, Japan.
Antisedan, Nippon Zenyaku Kogyo Co Ltd, Fukushima, Japan.
DRI-CHEM 7000, FUJIFILM Medical Co Ltd, Tokyo, Japan.
NEFA C, Wako Pure Chemical Industries Ltd, Osaka, Japan.
Insulin ELISA Kit/Dog, Morinaga Institute of Biological Science Inc, Kanagawa, Japan.
Dog Insulin Standard, 2.56 ng, Morinaga Institute of Biological Science Inc, Kanagawa, Japan.
Prism, version 5, GraphPad Software Inc, San Diego, Calif.
JMP, version 9, SAS Institute Inc, Japan, Tokyo, Japan.
References
1 Nelson RA, Folk GE Jr, Pfeiffer EW, et al. Behavior, biochemistry, and hibernation in black, grizzly, and polar bears. Ursus 1983; 5: 284–290.
2 Folk GE Jr. Physiological observations of subarctic bears under winter den conditions. In: Fisher KC, Dawe AR, Lyman CP, et al, eds. Mammalian hibernation. New York: American Elsevier Publishing Co Inc, 1967;75–85.
3 Young RA. Fat, energy, and mammalian survival. Am Zool 1976; 16: 699–710.
4 Dark J. Annual lipid cycles in hibernators: integration of physiology and behavior. Annu Rev Nutr 2005; 25: 469–497.
5 Boswell T, Woods SC, Kenagy GJ. Seasonal changes in body mass, insulin, and glucocorticoids of free living golden mantled ground squirrels. Gen Comp Endocrinol 1994; 96: 339–346.
6 Palumbo PJ, Wellik DL, Bagley NA, et al. Insulin and glucagon responses in the hibernating black bear. Ursus 1983; 5: 291–296.
7 Tokuyama K, Galantino HL, Green R, et al. Seasonal glucose uptake in marmots (Marmota flaviventris): the role of pancreatic hormones. Comp Biochem Physiol A Comp Physiol 1991; 100: 925–930.
8 Brunzell JD, Robertson RP, Lerner RL, et al. Relationships between fasting plasma glucose levels and insulin secretion during intravenous glucose tolerance tests. J Clin Endocrinol Metab 1976; 42: 222–229.
9 Kaneko JJ, Mattheeuws D, Rottiers RP, et al. Glucose tolerance and insulin response in diabetes mellitus of dogs. J Small Anim Pract 1977; 19: 85–94.
10 Link KRJ, Rand JS. Reference values for glucose tolerance and glucose tolerance status in cats. J Am Vet Med Assoc 1998; 213: 492–496.
11 Asano M, Tsubota T, Komatsu T, et al. Immobilization of Japanese black bears (Ursus thibetanus japonicus) with tiletamine hydrochloride and zolazepam hydrochloride. J Vet Med Sci 2007; 69: 433–435.
12 Taylor WP Jr, Reynolds HV, Ballard WB. Immobilization of grizzly bears with tiletamine hydrochloride and zolazepam hydrochloride. J Wildl Manage 1989; 53: 978–981.
13 Haigh JC, Stirling I, Broughton E. Immobilization of polar bears (Ursus maritimus phipps) with a mixture of tiletamine hydrochloride and zolazepam hydrochloride. J Wildl Dis 1985;21: 43–47.
14 Bush M, Custer RS, Smith EE. Use of dissociative anesthetics for the immobilization of captive bears: blood gas, hematology and biochemistry values. J Wildl Dis 1980; 16: 481–489.
15 Caulkett NA, Cattet MR, Caulkett JM, et al. Comparative physiologic effects of Telazol, medetomidine-ketamine, and medetomidine-Telazol in captive polar bears (Ursus maritimus). J Zoo Wildl Med 1999; 30: 504–509.
16 Caulkett NA. Bears. In: West G, Heard D, Caulkett NA, eds. Zoo animal and wildlife immobilization and anesthesia. Ames, Iowa: Blackwell Publishing Professional, 2007;409–415.
17 Sinclair MD. A review of the physiological effects of α2-agonists related to the clinical use of medetomidine in small animal practice. Can Vet J 2003; 44: 885–897.
18 Caulkett NA, Cattet MRL. Physiological effects of medetomidine-zolazepam-tiletamine immobilization in black bears. J Wildl Dis 1997; 33: 618–622.
19 Cattet MRL, Caulkett NA, Polischuk SC, et al. Reversible immobilization of free-ranging polar bears with medetomidine-zolazepam-tiletamine and atipamezol. J Wildl Dis 1997; 33: 611–617.
20 Ambrisko TD, Hikasa Y. Neurohormonal and metabolic effects of medetomidine compared with xylazine in Beagle dogs. Can J Vet Res 2002; 66: 42–49.
21 Kanda T, Hikasa Y. Neurohormonal and metabolic effects of medetomidine compared with xylazine in healthy cats. Can J Vet Res 2008; 72: 278–286.
22 Ionut V, Kirkman EL, Bergman RN. Investigation of the effect of acepromazine on intravenous glucose tolerance tests in dogs. Am J Vet Res 2004; 65: 1124–1127.
23 Ambrisko TD, Hikasa Y, Sato K. Influence of medetomidine on stress-related neurohormonal and metabolic effects caused by butorphanol, fentanyl, and ketamine administration in dogs. Am J Vet Res 2005; 66: 406–412.
24 Paddleford RR. Preanesthetic agents. In: Paddleford RR, ed. Manual of small animal anesthesia. 2nd ed. Philadelphia: WB Saunders Co, 1999;16–36.
25 Nakamura S, Okano T, Yoshida Y, et al. Use of bioelectrical impedance analysis to measure the fat mass of the Japanese black bear (Ursus thibetanus japonicus). Jpn J Zoo Wildl Med 2007; 13: 1–6.
26 Ko JCH, West G. Thermoregulation. In: West G, Heard D, Caulkett NA, eds. Zoo animal and wildlife immobilization and anesthesia. Ames, Iowa: Blackwell Publishing Professional, 2007;111–113.
27 Plumb DC. Acepromazine maleate. In: Veterinary drug handbook. 6th ed. Ames, Iowa: Blackwell Publishing Professional, 2008;3–5.
28 Plumb DC. Medetomidine HCl. In: Veterinary drug handbook. 6th ed. Ames, Iowa: Blackwell Publishing Professional, 2008;562–564.
29 Wallach J. Ursidae. In: Fowler ME, ed. Zoo and wild animal medicine. Philadelphia: WB Saunders Co, 1978;628–637.
30 Plumb DC. Tiletamine HCl/zolazepam HCl. In: Veterinary drug handbook. 6th ed. Ames, Iowa: Blackwell Publishing Professional, 2008;882–884.
31 Ramsay EC. Ursidae and Hyaenidae. In: Fowler ME, Miller RE, eds. Zoo and wild animal medicine. 5th ed. Philadelphia: WB Saunders Co, 2003;523–538.
32 Frank N, Elliott SB, Allin SB, et al. Blood lipid concentrations and lipoprotein patterns in captive and wild American black bears (Ursus americanus). Am J Vet Res 2006; 67: 335–341.
33 Schroeder MT. Blood chemistry, hematology, and condition evaluation of black bears in northcoastal California. Ursus 1987;7: 333–349.
34 Hellgren EC, Rogers LL, Seal US. Serum chemistry and hematology of black bears: physiological indices of habitat quality or seasonal patterns? J Mammal 1993; 74: 304–315.
35 Lohuis TD, Beck TDI, Harlow HJ. Hibernating black bears have blood chemistry and plasma amino acid profiles that are indicative of long-term adaptive fasting. Can J Zool 2005; 83: 1257–1263.
36 Huber D, Kusak J, Žvorc Z, et al. Effects of sex, age, capturing method, and season on serum chemistry values of brown bears in Croatia. J Wildl Dis 1997; 33: 790–794.
37 Tryland M, Brun E, Derocher AE, et al. Plasma biochemical values from apparently healthy free-ranging polar bears from Svalbard. J Wildl Dis 2002; 38: 566–575.
38 LeBlanc PJ, Obbard M, Battersby BJ, et al. Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus. J Comp Physiol B 2001; 171: 327–334.
39 Scholtysik G, Regli F, Bruckmaier RM, et al. The α2-adrenoceptor agonists xylazine and guanfacine exert different central nervous systems, but comparable peripheral effects in calves. J Vet Pharmacol Ther 1998; 21: 477–484.
40 Feldberg W, Symonds HW. Hyperglycaemic effect of xylazine. J Vet Pharmacol Ther 1980; 3: 197–202.
41 Angel I, Langer SZ. Adrenergic-induced hyperglycemia in anaesthetized rats: involvement of peripheral α2-adrenoceptors. Eur J Pharmacol 1988; 154: 191–196.
42 Burton SA, Lemke KA, Ihle SL, et al. Effects of medetomidine on serum insulin and plasma glucose concentrations in clinically normal dogs. Am J Vet Res 1997; 58: 1440–1442.
