Blood lipid concentrations and lipoprotein patterns in captive and wild American black bears (Ursus americanus)

Nicholas Frank Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

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Sarah B. Elliott Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

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Shawn B. Allin Department of Chemistry, Physics, and Engineering, Division of Science, Spring Hill College, Mobile, AL 36608

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Edward C. Ramsay Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

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Abstract

Objective—To compare blood lipid concentrations and lipoprotein patterns for captive and wild American black bears (Ursus americanus).

Animals—7 captive and 9 wild adult (≥ 4 years old) black bears.

Procedure—Blood was collected from 2 groups of captive black bears (groups A and B) and 1 group of wild black bears (group C). Blood triglyceride (TG) and cholesterol concentrations were compared among groups. Plasma lipoproteins were isolated by use of a self-generating gradient of iodixanol, and lipoprotein patterns were compared between groups A and B.

Results—Captive bears (mean ± SD, 187.8 ± 44.4 kg) weighed significantly more than wild bears (mean, 104.8 ± 41.4 kg), but mean body weight did not differ between groups A and B. Mean blood TG concentrations for groups B (216.8 ± 16.0 mg/dL) and C (190.7 ± 34.0 mg/dL) were significantly higher than that of group A (103.9 ± 25.3 mg/dL). Mean blood cholesterol concentration was also significantly higher for group B (227.8 ± 8.2 mg/dL) than for groups A (171.7 ± 35.5 mg/dL) or C (190.8 ± 26.8 mg/dL). Mean very-low-density lipoprotein TG and low-density lipoprotein cholesterol concentrations were 2- and 3-fold higher, respectively, for group B, compared with concentrations for group A.

Conclusions and Clinical Relevance—Blood lipid concentrations vary significantly among populations of black bears. Plasma lipoprotein patterns of captive bears differed significantly between colonies and may have reflected differences in diet or management practices.

Abstract

Objective—To compare blood lipid concentrations and lipoprotein patterns for captive and wild American black bears (Ursus americanus).

Animals—7 captive and 9 wild adult (≥ 4 years old) black bears.

Procedure—Blood was collected from 2 groups of captive black bears (groups A and B) and 1 group of wild black bears (group C). Blood triglyceride (TG) and cholesterol concentrations were compared among groups. Plasma lipoproteins were isolated by use of a self-generating gradient of iodixanol, and lipoprotein patterns were compared between groups A and B.

Results—Captive bears (mean ± SD, 187.8 ± 44.4 kg) weighed significantly more than wild bears (mean, 104.8 ± 41.4 kg), but mean body weight did not differ between groups A and B. Mean blood TG concentrations for groups B (216.8 ± 16.0 mg/dL) and C (190.7 ± 34.0 mg/dL) were significantly higher than that of group A (103.9 ± 25.3 mg/dL). Mean blood cholesterol concentration was also significantly higher for group B (227.8 ± 8.2 mg/dL) than for groups A (171.7 ± 35.5 mg/dL) or C (190.8 ± 26.8 mg/dL). Mean very-low-density lipoprotein TG and low-density lipoprotein cholesterol concentrations were 2- and 3-fold higher, respectively, for group B, compared with concentrations for group A.

Conclusions and Clinical Relevance—Blood lipid concentrations vary significantly among populations of black bears. Plasma lipoprotein patterns of captive bears differed significantly between colonies and may have reflected differences in diet or management practices.

American black bears (Ursus americanus) are commonly found in zoologic parks, yet blood lipid concentrations have only been examined in wild or newly captured bears.1–3 Those studies focused on physiologic changes that accompany winter denning. Black bears have a remarkable increase in body fat prior to denning, followed by slow consumption of fat during hibernation when they go without food for 3 months or longer.4 Some black bears kept in zoologic parks are encouraged to den during the winter, whereas others remain active throughout the year.

Evaluation of blood lipoproteins in bears has been limited to the measurement of serum cholesterol (LDL and HDL) concentrations in captive brown bears (Ursus arctos), polar bears (Ursus maritimus), spectacled bears (Tremarctos ornatus), and sun bears (Ursus malayanus).5 To our knowledge, lipoproteins have not been isolated from the plasma of wild or captive American black bears.

Lipoproteins have been isolated from plasma of humans6,7 and cattle8 by use of rapid ultracentrifugation with a self-generating gradient of iodixanol. A gradient forms within this nonionic density medium, and lipoproteins are separated in accordance with their densities.6 Fractions are assigned to lipoprotein classes on the basis of plotted TG and cholesterol concentrations. Other advantages of this technique include less time required for centrifugation and ease of evaluating apolipoproteins by use of SDS-PAGE because dialysis is not required before samples are applied to gels.6

The study reported here was conducted to evaluate blood lipid concentrations in captive and wild black bears. We also characterized the lipoprotein composition of ursid plasma and compared the concentrations and composition of plasma lipoproteins from captive black bears kept at 2 zoologic parks.

Materials and Methods

Animals—Sixteen adult (≥ 4 years old) black bears (7 captive and 9 wild) comprised 3 groups for the study. Groups A and B consisted of captive bears, whereas group C consisted of wild bears. Group A consisted of 4 captive adult American black bears (2 females [both of which were 17 years old] and 2 males [12 and 17 years old, respectively]). Three of the bears of group A were related in that the 2 females were siblings and had the same sire as the 17-year-old male. Group B contained 3 captive male American black bears (2 bears were 4 years old and 1 bear was 13 years old). None of the bears of group B were related. Group C consisted of 8 male and 1 female adult (≥ 4 years old) wild American black bears. Ages for captive bears were obtained from records of the zoologic parks; ages for wild bears were estimated on the basis of teeth analysis or physical characteristics.

All captive bears had been at the same zoologic park for at least 2 years. Captive bears were managed in accordance with the feeding and housing practices of the particular facility; no changes were made for the study. Diets fed to bears at both zoologic parks consisted primarily of commercial dry food formulated for dogs.a It was estimated from records that each bear in group A was fed approximately 7,000 kcal/d in the form of the commercial dry food. According to the manufacturer, the commercial dry food contained a minimum of 21.0% crude protein, minimum of 10.0% crude fat (80% from beef or pork fat and 20% from corn oil), maximum of 5.5% crude fiber, and maximum of 10.0% moisture. Each bear of group B received approximately 6,000 kcal/d of another commercial dry food formulated for dogsb that contained a minimum of 26.0% crude protein, minimum of 18.0% fat (71% from poultry fat, 14% from other meat products, and 5% from sunflower oil), maximum of 3.5% fiber, and maximum of 12.0% moisture. Bears in both captive groups also received varying quantities of fruits and vegetables, and group B bears were periodically fed fish as a treat. Analysis of records indicated that each bear of group B received 4 feedings of mackerel or herring (2 or 3 whole fish/feeding) 4 times during the 30-day period preceding collection of blood samples. Exact amounts of the commercial dry foods, fish, or fruits and vegetables consumed by the bears were not measured.

Management practices differed between zoologic parks in that group A bears were allowed to den during the winter preceding collection of blood samples. Bears started denning between December 10 and 12, 2002, and did not resume typical activity until early in March 2003. In contrast, group B bears remained active throughout the winter preceding collection of blood samples.

Collection of samples—Blood samples analyzed in the study were obtained as part of routine health checks performed on captive bears at the request of the zoologic park or as part of a preventative medicine program organized by the Virginia Department of Game and Inland Fisheries. Blood samples were collected while the bears were anesthetized for routine physical examinations during June 2003 (captive bears) or during routine management procedures such as relocation during the period between April 23 and August 27, 2003 (wild bears). All bears were anesthetized by a combination of ketamine and xylazine hydrochloride administered via dart. Food was withheld from captive bears overnight prior to anesthesia. Captive bears were weighed by use of a portable platform scale. Each wild bear was weighed by use of a hanging scale while anesthetized.

Blood samples were collected from captive bears via jugular or femoral venipuncture. Five 10-mL vacutainer tubesc containing EDTA were filled with blood, chilled on ice for 10 minutes, and then stored with ice packs or refrigerated. Blood samples were subjected to low-speed centrifugation (1,000 × g for 10 minutes), and plasma was harvested and stored at −70°C until further analysis. Plasma was harvested within 2 hours after collection of blood samples.

Blood samples were collected from wild bears via femoral venipuncture. Blood was collected into tubes without anticoagulant, and samples were transported on ice to the laboratory within 6 hours after collection. Blood samples were centrifuged; serum was then harvested, initially stored at −20°C, and transported on ice to the University of Tennessee. Serum samples were then stored at −70°C until analyzed.

Lipid concentrations—Commercially available kits were used to quantify concentrations of TG,d cholesterol,e PL,f and NEFAg in plasma, serum, or plasma fractions. Absorbance was measured on a microtiter plate reader.h

Isolation of lipoproteins—Plasma samples were stored at −70°C for approximately 6 months before analysis. Samples were thawed overnight at 4°C and then separated into fractions on the basis of density by use of a 2-layer iodixanol method, as described elsewhere.8 Samples were processed in duplicate. Briefly, 6% and 20% iodixanol solutions were prepared by diluting 60% (wt:vol) iodixanol solutioni with PBS solution. Four milliliters of plasma was then mixed with 1 mL of 60% iodixanol solution to create a 4:1 mixture. One milliliter of 20% iodixanol solution was added to an 11.2-mL ultracentrifuge tube.j This was gently overlaid with 4.5 mL of the 4:1 diluted plasma-iodixanol mixture, then 4.5 mL of 6% iodixanol solution, and finally PBS solution until the tube was full. Each tube was capped, placed in a rotor,k and centrifuged at 340,000 × g for 3 hours at 16°C. After centrifugation, 44 fractions (250 μL/fraction) were collected by use of an automated gradient fractionatorl proceeding from the top of the tube (fraction 1) to the bottom of the tube (fraction 44).

Concentrations of TG and cholesterol were subsequently measured in each fraction, and ranges for each fraction were determined for the 3 major classes of lipoprotein by inspecting TG and cholesterol concentrations in each fraction, bands on agarose gels, and apolipoprotein composition. Very-low-density lipoprotein was found in fractions 1 to 6 inclusive, whereas LDL and HDL were found in fractions 7 to 22 and 23 to 42, respectively. Two additional fractions (43 and 44) were isolated when the gel that collected at the bottom of tubes was withdrawn by the fraction collector. Lipid concentrations within lipoproteins (eg, LDL) were calculated by multiplying the mean concentration for the specific fraction by the total volume of that fraction (eg, 1.5 mL for VLDL) and dividing the product by 3.6 mL, which was the volume of plasma that had been added to each tube. Area under the curve values for VLDL (fractions 1 to 6), LDL (fractions 7 to 22), and HDL (fractions 23 to 42) peaks in TG and cholesterol concentrations were calculated by use of the trapezoidal method and a computer software program.m

Distribution of TG and cholesterol among the 3 classes of lipoprotein were expressed as percentages of the total AUC for TG or cholesterol concentrations.

Lipoprotein analysis by use of agarose gel electrophoresis—Forty-two of 44 fractions isolated from the plasma of a single 4-year-old male bear of group B that weighed 138 kg were electrophoresed on agarose gels by use of a gel lipoprotein electrophoresis system.n A standard solutiono containing human VLDL, LDL, and HDL was also loaded onto each gel. Two microliters of fraction or standard was added to each lane and was allowed to absorb into the agarose for 7 minutes. Gels were then placed in a barbital−sodium barbital buffer and electrophoresed at 80 V for 35 minutes at 20°C. After electrophoresis, gels were dried, stained, and then destained with reagents provided in the gel lipoprotein electrophoresis kit.n Bands of plasma samples were visually compared with results for the standards.

Apolipoprotein analysis by use of SDS-PAGE— Apolipoproteins were separated by use of SDS-PAGE in accordance with a method described elsewhere.9 Fractions were loaded onto 4% to 20% Tris-HCL gradient gels,o and electrophoresis was performed at 30 V for 4 hours at 4°C. Undiluted sample (20 μL/lane) was loaded onto gels for fractions 1 to 27. Fractions 28 to 44 were diluted 1:50 with PBS solution, and then diluted sample (20 μL/lane) was loaded onto gels. Molecular weight standardsq were included on each gel. Bands were visually inspected and compared among fractions.

Statistical analysis—Data were normally distributed, so groups were compared by use of 1-way ANOVA and Student 2-tailed t tests. Statistical analysis was conducted by use of computer software.m Correlations between body weight and lipid concentrations were evaluated by examining Pearson correlation coefficients. Mean ± SD values were reported. Significance was defined at values of P < 0.05.

Results

Mean body weights for groups A and B did not differ from each other but were significantly higher than the mean body weight for group C (Table 1). Groups B and C had significantly higher mean blood TG concentrations than group A, with the highest mean concentration detected for group B. Blood cholesterol concentrations were significantly higher for group B, compared with concentrations for groups A and C. Mean plasma TG and cholesterol concentrations for group B were 109% and 33% higher than, respectively, the mean values for those variables for group A bears. Plasma NEFA concentrations did not differ significantly among groups.

Table 1—

Mean ± SD body weights and plasma lipid concentrations for adult American black bears (Ursus americanus) in 2 zoologic parks (groups A and B) and a group of wild adult black bears (group C).

VariableA (n = 4)B (3)C (9)
Body weight (kg)196.9 ± 50.7a175.7 ± 41.0a104.8 ± 41.4b
TG (mg/dL)103.9 ± 25.3a216.8 ± 16.0b190.7 ± 34.0b
Cholesterol (mg/dL)171.7 ± 35.5a227.8 ± 8.2b190.8 ± 26.8a

Within a row, values with different superscript letters differ significantly (P < 0.05).

Plasma fractions were assigned to lipoprotein classes on the basis of TG and cholesterol concentrations (Figures 1 and 2). Lipoprotein classes were confirmed for 1 bear by examining bands on agarose gels relative to human VLDL, LDL, and HDL standards and apolipoproteins separated by use of SDS-PAGE (Figures 3 and Figures 4). Examination of plasma lipoprotein patterns revealed that group B bears were hyperlipid-emic, relative to bears of group A, and had significantly higher VLDL and LDL total lipid concentrations, compared with concentrations for bears of group A (Table 2). Significantly higher concentrations of TG, cholesterol, and PL were detected within VLDL and LDL isolated from group B bears, compared with concentrations for group A bears. In addition, HDL-TG concentrations were significantly higher in group B bears, compared with concentrations in group A bears. None of the blood lipid or lipoprotein variables examined were significantly correlated with body weight.

Table 2—

Mean ± SD and range values for plasma lipid concentrations within plasma lipoproteins isolated from captive adult black bears in 2 zoologic parks.

VariableGroup A (n = 4)Group B (3)
 Mean ± SDRangeMean ± SDRange
NEFA (μmol/L)463.4 81.2352.3533.0608.9 310.3328.5942.3
VLDL
   TG (mg/dL)48.8 ± 20.3a29.469.998.2 ± 20.0b85.4121.2
   Cholesterol (mg/dL)15.0 ± 7.8a8.423.639.3 ± 3.2b35.741.8
   PL (mg/dL)25.7 ± 9.9a17.036.551.7 ± 2.5b50.054.6
   Total lipid (mg/dL)89.5 ± 38.0a54.8130.0189.2 ± 24.7b208.6221.7
LDL
   TG (mg/dL)35.7 ± 11.0a24.547.289.9 ± 22.2b64.4105.0
   Cholesterol (mg/dL)19.3 ± 7.0a10.526.361.7 ± 13.4b46.369.7
   PL (mg/dL)28.8 ± 9.0a19.737.078.8 ± 10.5b67.888.6
   Total lipid (mg/dL)83.7 ± 26.1a58.5107.6230.4 ± 28.5b213.6263.3
HDL
   TG (mg/dL)12.2 ± 2.2a9.414.616.4 ± 1.2b15.317.6
   Cholesterol (mg/dL)105.7 ± 15.688.6126.4118.0 ± 13.3109.5133.3
   PL (mg/dL)154.1 ± 41.1112.0208.5213.4 ± 7.2208.6221.7
   Total lipid (mg/dL)271.9 ± 50.8212.6326.8347.8 ± 11.2337.4359.6

See Table 1 for key.

Figure 1—
Figure 1—

Mean ± SD TG concentrations in plasma samples obtained from 2 groups (group A, 4 bears; open diamonds; group B, 3 bears; closed diamonds) of captive adult American black bears (Ursus americanus) kept in 2 zoologic parks. Fractions were isolated by use of a self-generating density gradient of iodixanol and correspond to fractions proceeding from the top of the tube (fraction No. 1) to the bottom of the tube (fraction No. 44).

Citation: American Journal of Veterinary Research 67, 2; 10.2460/ajvr.67.2.335

Figure 2—
Figure 2—

Mean ± SD cholesterol concentrations in plasma samples obtained from 2 groups (group A, diamonds; group B, squares) of captive adult American black bears kept in 2 zoologic parks. Fractions were isolated by use of a self-generating density gradient of iodixanol and correspond to fractions proceeding from the top of the tube (fraction No. 1) to the bottom of the tube (fraction No. 44).

Citation: American Journal of Veterinary Research 67, 2; 10.2460/ajvr.67.2.335

Figure 3—
Figure 3—

Gels depicting SDS-PAGE separation of lipoproteins for plasma fractions 1 to 7 (A), 8 to 14 (B), 15 to 21 (C), 22 to 28 (D), 29 to 35 (E), and 36 to 42 (F) obtained from a 4-year-old American black bear of group B. The SDS-PAGE electrophoresis was conducted on 4% to 20% gradient gels with a broad range standard (ie, STD) solution applied to the first lane of each gel.q Fractions were isolated from plasma by use of a self-generating density gradient of iodixanol and correspond to fractions proceeding from the top of the tube (fraction No. 1) to near the bottom of the tube (fraction No. 42). The highest number of the fraction included in each class of lipoprotein is indicated (asterisks). − = Negative charge. 0 = No charge. + = Positive charge.

Citation: American Journal of Veterinary Research 67, 2; 10.2460/ajvr.67.2.335

Figure 4—
Figure 4—

Agarose gels depicting SDS-PAGE separation of lipoproteins for plasma fractions 1 to 9 (A), 10 to 18 (B), 19 to 27 (C), 28 to 36 (D), and 37 to 44 (E) obtained from a 4-year-old American black bear of group B. The SDS-PAGE electrophoresis was conducted on 4% to 20% gradient gels by use of a kit that included a standard (ie, STD) composed of human lipoproteins, which was included in the first lane of each gel. Twenty microliters of undiluted sample was applied to each lane of gels A to C (fractions 1 to 27), whereas samples were diluted 1:50 with PBS solution before 20 μL of the diluted sample was loaded onto gels D and E (fractions 28 to 44). Lane 45 was left empty. Values on the left side represent molecular weight in number of kilodaltons. Bands representing albumin are indicated (arrows). ApoB = Apolipoprotein B. ApoAI = Apolipoprotein A-I. ApoAIV = Apolipoprotein A-IV. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 67, 2; 10.2460/ajvr.67.2.335

Examination of agarose gels for 1 bear confirmed that fractions assigned to the 3 major lipoprotein classes (VLDL, fractions 1 to 6; LDL, fractions 7 to 22; and HDL, fractions 23 to 42) contained the lipoprotein of interest (Figure 3). Fractions 1 through 7 smeared on the gels, but lipoproteins migrated to the pre-β and β regions appropriate for VLDL. A more clearly defined band was seen within the β region for fraction 8, indicating evidence of LDL, and this band was evident up to fraction 25, which suggests that there were traces of LDL in 3 fractions classified as HDL. For fraction 18, a faint band appeared in the α region where HDL is found, and this band was detected in all subsequent fractions. Therefore, traces of HDL were evident in 5 fractions (18 to 22) labeled as LDL.

When plasma fractions from the same bear were analyzed by use of SDS-PAGE, a band representing a large−molecular-weight (> 200 kd) protein (which was assumed to be apolipoprotein B)10 was visible in fractions containing VLDL and LDL (Figure 4). This protein was still evident as a clearly defined band in fraction 22, which was the last fraction included in LDL. Bands differed in intensity among gels because samples loaded onto gels for fractions 28 to 44 were diluted 1:50, but a band assumed to be apolipoprotein A-IV was initially detected at 45 to 66 kd on the gel for fractions 19 to 27 and gained intensity as the fraction number increased. In humans, apolipoprotein A-IV has a molecular weight of 46 kd and is most abundant within HDL.10 Protein migrating to the same point as the 66-kd standard was assumed to be albumin because bovine albumin was contained in the standard solution. A band with a molecular weight close to but less than the 31-kd standard was also found in every fraction and was most abundant within HDL. This protein was assumed to be apolipoprotein AI on the basis of a molecular weight of 28.1 kd reported10 for human apolipoprotein A-I. Apolipoproteins A-I and A-IV were also evident in fractions 43 and 44.

Discussion

A self-generating gradient of iodixanol was successfully used to isolate lipoproteins from the plasma of American black bears. Lipoproteins were separated in a single step that required centrifugation for only 3 hours, compared with other sequential ultracentrifugation procedures11 that require up to 4 days to complete.6 The technique reported here has been successfully used to separate plasma lipoproteins in human6,7 and bovine8 plasma, but to our knowledge, this was the first use of this method to evaluate lipoproteins in ursid plasma. This method was selected because density ranges have not been established for the 3 major classes of lipoprotein (VLDL, LDL, and HDL) in bears. Isolated lipoproteins were assigned to classes on the basis of plotted TG and cholesterol concentrations (Figures 1 and 2). Fraction densities were not measured in our study, but it has been reported6–8 that density gradients are consistently formed within iodixanol after ultracentrifugation. Plasma samples from groups A and B were also processed at the same time with the objective of minimizing the effects of operator error on comparisons between groups.

Results may have been influenced by sample-handling procedures used in the study because lipoproteins should ideally have been isolated from fresh plasma or serum to avoid potential effects of freezing. However, storage of samples at −70°C for 6 months was unlikely to have had an effect on the differences detected among groups. When lipoproteins are isolated from human plasma by use of density-gradient ultracentrifugation after 6 months of storage at −20°C, cholesterol concentrations change only by < 4.1% in LDL and HDL, whereas TG concentrations change by 13% in VLDL and LDL.12 When human plasma is stored at −80°C for 12 months, TG and cholesterol concentrations increase only by 6.8% and 2.2%, respectively.13 Analysis of serum versus EDTA plasma was also considered a potential confounding factor in this study because plasma was collected from captive bears of groups A and B but serum was collected from wild bears of group C. However, the impact of this discrepancy was considered minimal because reported14 mean EDTA plasma-to-serum ratios range from 0.950 to 1.013 and 0.976 to 1.003 for TG and cholesterol concentrations, respectively, on the basis of the analyzer used. Even the largest mean difference detected for TG concentrations (5%)14 would account for only a small part of the 45% difference detected between concentrations for groups A and C.

When blood lipoprotein concentrations were measured in captive bears in another study,5 only serum LDL-C and HDL-C concentrations were examined, and American black bears were not included in the sample population. Serum LDL-C concentrations in that study5 were calculated from HDL-C and TG concentrations, and serum HDL-C concentrations were measured by use of an assay used to measure HDL-C in humans. Mean serum LDL-C concentrations reported in that study5 were 34.6, 261.5, 246.2, and 150.0 mg/dL in captive brown bears (U arctos), polar bears (U maritimus), spectacled bears (T ornatus), and sun bears (U malayanus), respectively, whereas serum HDL-C concentrations in the same bears were 80.8, 223.1, 96.2, and 119.2 mg/dL, respectively. In the study reported here, American black bears in groups A and B had mean plasma LDL-C concentrations of 19.3 and 61.7 mg/dL, respectively, and mean plasma HDL-C concentrations of 105.7 and 118.0 mg/dL, respectively. These mean values were closest to those of captive brown bears, but differences in methods between that other study5 and the study reported here may hinder comparisons.

Blood lipid concentrations have been measured in wild American black bears, and particular attention has been paid to alterations associated with denning.1–3 In 1 study,2 samples obtained from 14 adult female black bears with cubs during denning (between February and April) had mean serum TG, cholesterol, and total lipid concentrations of 350.4, 382.2, and 1,274.2 mg/dL, respectively. In a study15 of wild American black bears located in Ontario, mean plasma TG concentration was 165.4 mg/dL for 24 active males, whereas mean values of 226.9 and 161.5 mg/dL, respectively, were detected for 7 actively lactating and 7 nonlactating female bears. Unfortunately, ages of the bears and month of sample collection were not reported. In that same study,15 mean cholesterol concentrations of 319.2 and 292.3 mg/dL were detected during denning for lactating and nonlactating female bears, respectively. Plasma mean TG and cholesterol concentrations (190.7 mg/dL and 190.8 mg/dL, respectively) detected in the wild bears of the study reported here compare favorably with those of the active males and nonlactating female black bears in Ontario.

Results of the study reported here do not explain differences in blood lipid patterns detected among groups of bears, but effects of body fat mass, diet, and denning practices warrant further study. Body fat mass must be examined because blood lipid values were not correlated with body weight in this study, despite the finding that captive black bears were heavier (88% and 68% for groups A and B, respectively) than wild bears. Body fat mass may be a more important determinant of blood lipid concentrations and composition than body weight in bears, similar to the situation in humans.16 Bioelectric impedance analysis has been used to measure body fat mass in American black bears.17 In the study reported here, group B bears consumed more fat than did the bears of group A, and this difference in diet may have had an effect on the results. Bears of group A were fed a diet consisting primarily of dry food formulated for dogs that contained a minimum of 10% fat, whereas bears of group B received a dry food formulated for dogs that contained a minimum of 18% fat in addition to receiving fish on an irregular basis.

Denning practices may have also influenced blood lipoprotein patterns because group A bears were encouraged to den each year, whereas bears of group B remained active throughout the year. Measurement of changes in body weight before and after denning has revealed that captive black bears lose 15% to 27% of predenning body weight, and wild black bears lose 16% to 37% of predenning body weight.4 Approximately 50% of weight loss during denning is attributable to a reduction in body fat mass, which may affect blood lipid concentrations.17

Bears included in the study reported here were assumed to be healthy, but results of CBC counts and serum biochemical analyses were not examined and bears were not tested for underlying endocrine or metabolic diseases. Hypothyroidism has been reported18 in an American black bear, and this disorder affects lipid metabolism in other animals.19

Because 3 of 4 bears in group A were related, differences between groups may also have been confounded by hereditary factors. A larger number of captive bears must be examined to determine the degree of variability in blood lipid concentrations expected within and among bears kept in identical conditions.

The length of time that feed was withheld from bears before collection of samples may also have confounded results. Food was withheld from captive bears overnight before sample collection, whereas wild bears may have eaten within a few hours preceding collection of samples.

Plasma lipoproteins were isolated from ursid plasma by use of a self-generating gradient of iodixanol, and 3 classes of lipoprotein (VLDL, LDL, and HDL) were identified. Plasma lipid and lipoprotein concentrations appear to differ among populations of captive American black bears, but additional studies are required to establish reference ranges for healthy bears and identify factors that influence these values. Analysis of results of the study reported here suggests that body fat mass, diet, and management practices should be examined in future studies.

LDL

Low-density lipoprotein

HDL

High-density lipoprotein

TG

Triglyceride

PL

Phospholipid

NEFA

Nonesterified fatty acids

VLDL

Very-low-density lipoprotein

AUC

Area under the curve

LDL-C

LDL-cholesterol

HDL-C

HDL-cholesterol

a.

Big Red Nuggets, Southern States Cooperative Inc, Richmond, Va.

b.

Prime Formula, PMI Nutrition, St Louis, Mo.

c.

Vacutainer Systems, Becton-Dickinson, Franklin Lakes, NJ.

d.

Triglyceride E, Wako Chemicals USA Inc, Richmond, Va.

e.

Cholesterol CII, Wako Chemicals USA Inc, Richmond, Va.

f.

Phospholipids B, Wako Chemicals USA Inc, Richmond, Va.

g.

NEFA C, Wako Chemicals USA Inc, Richmond, Va.

h.

ELx800 microplate reader, Bio-Tek, Winooski, Vt.

i.

OptiPrep, Axis-Shield, Oslo, Norway.

j.

Optiseal tube, Beckman Coulter, Fullerton, Calif.

k.

NVT65 rotor, Beckman Coulter, Fullerton, Calif.

l.

Auto Densi-Flow density gradient fractionator, Labconco Corp, Kansas City, Mo.

m.

SAS, version 9.1, SAS Institute Inc, Cary, NC.

n.

Titan gel lipoprotein electrophoresis system, Helena Laboratories, Beaumont, Tex.

o.

Titan gel Lipotrol, Helena Laboratories, Beaumont, Tex.

p.

Ready Gel, Bio-Rad Laboratories, Hercules, Calif.

q.

SDS-PAGE standards broad range, Bio-Rad Laboratories, Hercules, Calif.

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