Changes in blood glucose and insulin responses to intravenous glucose tolerance tests and blood biochemical values in adult female Japanese black bears (Ursus thibetanus japonicus)

The metabolic mechanisms to circannual changes in body mass of bears have yet to be elucidated. We hypothesized that the Japanese black bear (Ursus thibetanus japonicus) has a metabolic mechanism that efficiently converts carbohydrates into body fat by altering insulin sensitivity during the hyperphagic stage before hibernation. To test this hypothesis, we investigated the changes in blood biochemical values and glucose and insulin responses to intravenous glucose tolerance tests (IVGTT) during the active season (August, early and late November). Four, adult, female bears (5-17 years old) were anesthetized with 6 mg/kg TZ (tiletamine HCl and zolazepam HCl) in combination with 0.1 mg/kg acepromazine maleate. The bears were injected intravenously with glucose (0.5 g/kg of body mass), and blood samples were obtained before, at, and intermittently after glucose injection. The basal triglycerides concentration decreased significantly with increase in body mass from August to November. Basal levels of plasma glucose and serum insulin concentrations were not significantly different among groups. The results of IVGTT demonstrated the increased peripheral insulin sensitivity and glucose tolerance in early November. In contrast, peripheral insulin resistance was indicated by the exaggerated insulin response in late November. Our findings suggest that bears shift their glucose and lipid metabolism from the stage of normal activity to the hyperphagic stage in which they show lipogenic-predominant metabolism and accelerate glucose uptake by increasing the peripheral insulin sensitivity.     

Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.     

Incidence of ovulation without coital stimuli in captive Japanese black bears (Ursus thibetanus japonicus) based on serum progesterone profiles

The purpose of this study was to ascertain whether or not Japanese black bears (Ursus thibetanus japonicus) are induced ovulators. The progesterone levels of female bears kept with a male and allowed to mate (n=2) and female bears allowed contact with a male through bars but not allowed to mate (n=6) during the mating season increased significantly in late October. Based on this result, the female bears were considered to have ovulated. Females isolated from males (n=3) were ovarioectomized after the mating season, and no corpora lutea were observed, indicating they had not ovulated. These findings suggest that Japanese black bears may be induced ovulators that ovulate with stimuli from males and without coitus at a high rate.     

Characteristics of a hepatozoonosis in lungs of Japanese black bears (Ursus thibetanus japonicus)

In a survey of pathologic agents in the wild animals of central Japan, we found a Hepatozoon sp. in the lungs of Japanese black bears in Fukui, Shiga, and Gifu Prefectures, Japan. Histopathologic examination of organs and tissues from the 18 bears inspected showed hepatozoonosis in all. Immature and mature meronts were found in all lobules and between alveoli, with a few found between pleura and in connective tissue. In the lungs, inflammatory cells were not found around meronts, merozoites, or tubercles made of macro-phages including zoites, but inflammatory cells were found around degenerating cells, zoites, and tubercles. A Hepatozoon sp. has not been reported as being detected in bears of any species before.     

Immobilization of Japanese black bears (Ursus thibetanus japonicus) with tiletamine hydrochloride and zolazepam hydrochloride

The effect of anesthetizing with a 1:1 combination of tiletamine hydrochloride and zolazepam hydrochloride (TZ) was evaluated in 75 Japanese black bears. TZ was administered to 43 captive and 11 wild, 8 captives and 13 hibernating captive bears at the doses of approximately 9.0 mg/kg (usual dosage), 18.0 mg/kg (high dosage) and 5.0 mg/kg (low dosage), respectively. Sufficient anesthetic effects were achieved in all bears, and rectal temperatures, heart rates and respiratory rates did not change significantly during an hour handling. Complete blood cell examinations showed no abnormal data. A combination of TZ would be an efficient and safe drug for chemical immobilization of Japanese black bears.     

Characteristics of frozen-thawed spermatozoa cryopreserved with different concentrations of glycerol in captive Japanese black bears (Ursus thibetanus japonicus)

Seven mature Japanese black bears were used as semen donors, and a total of 7 semen samples collected from the animals by the electroejaculation method were cryopreserved in liquid nitrogen. Egg yolk-TRIS-citrate-glucose extender was used, and the effects of different final concentrations of glycerol, at 4-12% (v/v), on frozen-thawed spermatozoa were examined. No significant difference was observed in percent motility or percent abnormal morphology of frozen-thawed spermatozoa among the different glycerol concentrations. Percent viability and percent intact acrosomes of spermatozoa cryopreserved with 4 and 6% glycerol were significantly higher than those with 10 and 12% glycerol. These results suggest that a suitable glycerol concentration for freezing Japanese black bear semen within the range tested would be 4-6%.     

Morphological and molecular phylogenetical identification of Tricodectes pinguis from Japanese black bears (Ursus thibetanus japonicus) in Aomori Prefecture,Japan

Trichodectes pinguis, referred to commonly as the bear-biting louse, has been reported in several bear species. However, graphical (blurred or coarse) and genetic information on the louse is limited. In this study, we identified T. pinguis collected from Japanese black bears in the Aomori Prefecture, Japan. We confirmed 12S rDNA sequences derived from the collected T. pinguis and performed molecular phylogenetic analysis based on 12S rDNA. The analysis revealed the parasitic louse to be T. pinguis. Interestingly, the body size of T. pinguis found in this study was smaller than the previous recorded body size of them in Japan and Turkey. To better understand the biting louse infesting bears, morphometric and genetic information from other bear hosts needs to be accumulated.     

Serum progesterone and estradiol-17beta concentrations in captive and free-ranging adult female japanese black bears (Ursus thibetanus japonicus)

Progesterone (P4) and estradiol-17beta (E2) concentrations were measured in serum samples obtained from 23 captive and 23 free-ranging adult female Japanese black bears. We then determined the relationship between changes in these sex steroid hormones and pregnancy. In all captive bears, which included animals of both known and unknown reproductive status, serum P4 concentrations were low from April to July, then tended to become higher after August. The levels then became much higher still in November and December, but returned to low levels in March. Serum P4 concentrations in eight captive pregnant bears, which had parturitions the following spring, increased gradually from August (0.5-2.4 ng/ml) to October (0.9-3.6 ng/ml), and achieved significantly higher maximum levels in December (7.2-18.0 ng/ml). Thereafter, serum P4 concentrations tended to decrease (3.5-6.4 ng/ml in January and 0.3-0.7 ng/ml in March). In all captive bears, serum E2 concentrations varied from April to October but showed low levels in November and December, and became high in January. Serum E2 concentrations in the eight pregnant bears were high in May (95.6-191.4 pg/ml) and varied from August to October (35.6-143.3 pg/ml). Subsequently, serum E2 concentrations in December dropped to significantly lower minimum levels (5.3-11.9 pg/ml) and increased again in January (67.6-153.1 pg/ml). Among the free-ranging bears, the data on serum P4 concentrations in eight bears led to expectations of pregnancy, whereas serum E2 concentrations showed no distinct evidence related to pregnancy. These results, particularly in captive pregnant bears, indicate that a marked increase of P4 in December might be accompanied by reactivation of the corpus luteum preceding implantation. Furthermore, changes in E2 concentrations suggested the possibility that a decline in December and an increase in January are associated with implantation and parturition, respectively.     

Spermatogenesis, serum testosterone levels and immunolocalization of steroidogenic enzymes in the wild male Japanese black bear (Ursus thibetanus japonicus)

Twenty-one wild male Japanese black bears (Ursus thibetanus japonicus) were captured in the summer-autumn of 1998-2000 in the vicinity of Neo Village, Gifu Prefecture. Testes were measured, and testicular samples were biopsied and observed histologically. Four steroidogenic enzymes, i.e., cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17-alpha hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) were immunolocalized. Serum testosterone concentrations were measured by radioimmunoassay. Testis size changed little from 1-3 years of age, increased rapidly at 4 years, and attained its peak at 5 years. Serum testosterone concentrations ranged from 0.05 to 1.78 ng/m l, and the mean +/- standard deviation was 0.43 +/- 0.48 ng/ml. Age of sexual maturation in wild male Japanese black bears was estimated to be 3-4 years. Seasonal changes in spermatogenesis were obvious; active in June, July and August, degenerated by September. Leydig cells, Sertoli cells and germ cells have the capability of synthesizing androgen, and Leydig cells, Sertoli cells, spermatids and spermatogonia have the capability of synthesizing estrogen in Japanese black bears.     

Estimating the success rate of ovulation and early litter loss rate in the Japanese black bear (Ursus thibetanus japonicus) by examining the ovaries and uteri

In order to develop a method for estimating the success/failure rates of reproductive processes, especially those of ovulation and neonate nurturing, in the Japanese black bear (Ursus thibetanus japonicus), we examined offspring status, corpora lutea (CLs), placental scars (PSs) and corpora albicantia (CAs) in 159 females (0-23 years old) killed as nuisances on Honshu Island of Japan during 2001-2009. PSs were found to remain in the uterus at least until November of the year of parturition. CA detectability began to decline after September of the year of parturition. Monthly and age-specific proportions of CL-present females revealed that the post-mating season starts in August, and that the age of first ovulation is 4 years. These results indicate that the success rate of ovulation (SRO: the probability that solitary/non-lactating mature females actually succeed in ovulation) can be estimated by calculating the proportion of CL-present females among > or = 4-year-old females without PSs captured from August to November; the early litter loss rate (ELLR: the probability that parenting females lose all of their cubs [0-year-old offspring] before mating season) can be estimated by calculating the proportion of CL-present females among those with PSs and CAs captured in August or later. The estimated values of SRO and ELLR were 0.93 (62/67) and 0.27 (6/22), respectively.     

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.     

Detection and prevalence of four different hemotropic Mycoplasma spp. in Eastern North Carolina American black bears (Ursus americanus)

Hemotropic Mycoplasma spp. are globally emerging, obligate parasitic, epierythrocytic bacteria that infect many vertebrates, including humans. Hemoplasma infection can cause acute life-threatening symptoms or lead to a chronic sub-clinical carrier state. Hemotropic Mycoplasma spp. transmission, prevalence, and host specificity are uncertain. The purpose of this study was to determine the molecular prevalence of Mycoplasma species in blood from 68 free-ranging black bears from the eastern coast of North Carolina. DNA amplification of Mycoplasma 16S rRNA gene identified four distinct species infecting 34/68 (50%) of the black bear blood samples, including Candidatus M. haematoparvum. The high prevalence of hemotropic Mycoplasma infection in this wildlife species highlights the importance of understanding intra and inter species transmission. Black bears may play a role in the transmission of hemotropic Mycoplasma spp. between animals, arthropod vectors, and humans. Further studies are needed to elucidate black bears as a potential reservoir for hemotropic Mycoplasma infections.     

Haematological and serum biochemical values of southern chamois (Rupicapra pyrenaica)

Blood samples were taken from 75 free-ranging southern chamois (Rupicapra pyrenaica) captured in drive-nets in Catalonia, north-eastern Spain, and 20 haematological and 24 serum biochemical variables were analysed. The values were similar to those of other species of the Caprinae subfamily, except for cortisol, the concentration of which was higher. The red blood cell count (RBC), platelets and leucocytes, and the concentrations of cortisol, lactate, muscular enzymes and gamma-globulins were higher in summer than in spring, whereas the mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, and the concentrations of cholesterol, total bilirubin and creatinine were lower. Adult males had higher RBCs and haemoglobin values than females in summer, and lower leucocyte, lymphocyte and neutrophil counts than females and yearling males. The concentrations of triglycerides, total bilirubin, lactate, creatinine, urea, chloride and alpha2-globulins were higher in adult males than yearling males. In summer the adult females had higher values for platelets, lymphocytes, cortisol, sodium and muscular enzymes.     

Comparison of biochemical values in serum and plasma, fresh and frozen plasma, and hemolyzed samples from orange-winged Amazon parrots (Amazona amazonica)

BACKGROUND: To the authors' knowledge, on the basis of sample type, storage condition, or hemolysis, differences in serum and plasma biochemical values have not been evaluated in orange-winged Amazon parrots (Amazona amazonica). OBJECTIVES: The purpose of this study was to compare values for biochemical analytes in serum vs plasma, fresh vs frozen plasma, and nonhemolyzed vs hemolyzed samples in orange-winged Amazon parrots. We also compared differences in serum and plasma yield from whole-blood aliquots. METHODS: Fifteen biochemical analytes were evaluated in paired serum and plasma, fresh and frozen plasma, nonhemolyzed and hemolyzed serum and plasma samples from orange-winged Amazon parrots (n = 10) using a wet reagent analyzer. Hemolysis was assessed qualitatively (visually) and quantitatively (hemoglobin [Hgb] measured spectrophotometrically). Serum and plasma yields from 500-microl whole-blood aliquots were determined from centrifuged samples. RESULTS: Analyte values significantly differed among sample groups, but were still within published reference intervals, with the exception of increases in potassium concentration in markedly hemolyzed serum and plasma samples. Clinically important changes in hemolyzed serum and plasma samples included increases in potassium, phosphorus, and albumin concentrations and lactate dehydrogenase activity. The degree of hemolysis assigned qualitatively did not correlate with quantitative Hgb concentration. A significantly greater yield of plasma (288 +/- 13 microL) than serum (241 +/- 44 microL) was obtained. CONCLUSIONS: Significant differences may occur in different sample types, however, only changes in potassium, phosphorus, albumin, and lactate dehydrogenase values in hemolyzed samples were considered clinically relevant. Lack of agreement between qualitative and quantitative Hgb concentration indicates the unreliability of visual estimation. Based on higher sample yield, and lack of clinically relevant differences from serum, plasma is a better sample choice for clinical chemistry analysis in birds.     

Mobility of the forearm skeleton in the Asiatic black (Ursus thibetanus), brown (U. arctos) and polar (U. maritimus) bears

In several primates and carnivores, pronation/supination angles of the forearm skeleton were examined, and it is thought that a larger angle is useful to acquire dexterous behaviors in feeding and/or life style, including climbing. In this study, the pronation/supination angles in Asiatic black, brown and polar bears were nondestructively examined. These specimens were classified as adult or non-adult. Three or four carcasses of each group of Asiatic black and brown bears were used for CT analysis, whereas only one adult polar bear was used. The forearms were positioned within the gantry of a CT scanner in both maximally supinated and pronated states. Extracted cross-sectional CT images of two positions were superimposed by overlapping the outlines of each ulna. The centroids of the radii were detected, and then the centroid of each radius and the midpoint of a line which connects between both ends of the surface of each radius facing the ulna, were connected by lines to measure the angle of rotation as an index of pronation/supination. In adult brown and polar bears, the angles were smaller as compared with the other groups (Asiatic black and non-adult brown bears). Asiatic black and non-adult brown bears can climb trees, whereas adult brown bears and polar bears cannot. This suggests that the pronation/supination angle is related to arboreal activity in Ursidae.     

Comparison of serum and plasma for determination of blood biochemical values in Malaysian flying foxes (Pteropus vampyrus).

The effects of the anticoagulant sodium heparin and time of centrifugation on 20 biochemical analytes in the blood of Malaysian flying foxes (Pteropus vampyrus) were evaluated. Paired plasma and serum samples were centrifuged at 1 hr and 6 hr postcollection. Heparinization and time of centrifugation did not significantly affect albumin, cholesterol, triglycerides, amylase, blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransaminase, alkaline phosphatase, calcium, sodium, and total carbon dioxide levels. Plasma was associated with higher globulin and lower potassium values. Glucose and chloride levels decreased significantly over time, whereas phosphorus levels increased. Serum creatine kinase activity at 6 hr postcollection was significantly higher than the other creatine kinase means. Sodium levels were not significantly affected by sodium heparin as used as an anticoagulant in this study.     

Assessment of the effects of intramuscular administration of alfaxalone with and without medetomidine in Horsfield's tortoises (Agrionemys horsfieldii)

ObjectiveTo characterise four different intramuscular (IM) anaesthetic protocols, two with alfaxalone and two with alfaxalone in combination with medetomidine in terrestrial tortoises.Study designBlinded, randomized, cross‐over experimental study.AnimalsNine healthy adult male Horsfield's tortoises (Agrionemys horsfieldii).MethodsEach tortoise was randomly assigned to one of four different protocols: 1) 10 mg kg^?1 alfaxalone; 2) 10 mg kg^?1 alfaxalone + 0.10 mg kg^?1 medetomidine; 3) 20 mg kg^?1 alfaxalone; and 4) 20 mg kg^?1 alfaxalone + 0.05 mg kg^?1 medetomidine. During the experiment, the following variables were recorded: heart rate; respiratory rate; peripheral nociceptive responses; muscle strength; ability to intubate; palpebral, corneal and tap reflexes; and cloacal temperature.ResultsProtocols 1 and 2 resulted in moderate sedation with no analgesia, and moderate to deep sedation with minimal analgesia, respectively. Protocols 3 and 4 resulted in deep sedation or anaesthesia with variable analgesic effect; these two protocols had the longest total anaesthetic time and allowed intubation in 6/9 and 8/9 tortoises respectively. The total anaesthesia/sedation time produced by alfaxalone was significantly increased (p <0.05) by the addition of medetomidine. There were no significant differences regarding time to plateau phase and duration of plateau phase. Baseline heart rate of 53 ± 6 beats minute^?1 decreased significantly (p <0.05) with all protocols, and was lower (p <0.05) in protocols 3 and 4. Heart rate increased after atipamezole administration, but the increase was transient. In two tortoises, extreme bradycardia with no cardiac activity for 10 minutes was observed with protocols 3 and 4.Conclusion and clinical relevanceAlfaxalone 10 and 20 mg kg^?1 IM can be used for sedation for non‐painful procedures. Alfaxalone in combination with medetomidine can be used for deeper sedation or anaesthesia, but the observed respiratory and cardiovascular depression may limit its use.     

Effects of body weight and season on serum lipid concentrations in sloth bears (Melursus ursinus ursinus)

Serum lipid levels were measured in 66 healthy sloth bears (Melursus ursinus ursinus) living under semicaptive conditions with access to natural food resources in the Bannerghatta Biological Park (Karnataka, India), a portion of their native habitat range in the Indian peninsula. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein cholesterol levels were analyzed. The effects of age, body weight, and season on these lipid parameters were statistically evaluated. There were no correlations between age and any of the serum lipid parameters analyzed. Positive correlations of body weight to both triglyceride and HDL cholesterol levels in these bears were identified. In addition, seasonal trends in physiological serum lipid values, potentially due to variations in the sloth bear diet, were identified. Serum triglyceride levels were higher during postmonsoon season and cholesterol levels were higher during winter compared to other seasons. Serum lipid values obtained from sloth bears in this study were also compared to previously published data on other members of the family Ursidae. This is the first report of serum lipid values as a reference for sloth bears. These values can be used as sensitive predictors of overall health and nutritional status to aid in the captive management and feeding of these bears.     

Effects of dietary copper on elemental balance,plasma minerals and serum biochemical parameters of growing-furring male mink(Mustela vison)

The objectives of this study were to study the effects of different levels of dietary copper on copper and zinc balance, plasma minerals and serum biochemical parameters of mink in the growing-furring periods. One hundred and five standard dark male mink were randomly assigned to seven groups with the following dietary treatments: basal diet with no supplemental Cu(Control); basal diet supplemented with either 6,12, 24, 48, 96, or 192 mg/kg Cu from copper sulfate,respectively. The average daily gain(ADG) linearly(P = 0.0026, P = 0.0006) responded to increasing levels of Cu; maximal growth was seen in the Cu24 group. Feed efficiency tended to improve with the increase of dietary copper level(linear P = 0.0010, quad, P = 0.0011). Fecal copper, urinary copper, retention copper responded in a linear(P 0.05) fashion with increasing level of Cu. The effect of level of Cu was linear(P 0.001) for plasma Cu concentration. The serum glutamic-oxalacetic transaminase(GOT) and glutamic-pyruvic transaminase(GPT) activities were increased linearly(P 0.05) with dose of Cu, but serum total protein(TP) and albumin(ALB) concentrations decreased linearly(P 0.05) as dietary copper levels increased. Effect of level of Cu was linear(P 0.001) for serum ceruloplasmin(CER) concentration or Cu-Zn superoxide dismutase(Cu-Zn SOD) activity. Supplemental dose of Cu linearly decreased serum triglyceride(TG)(P = 0.011) and total cholesterol(TC)(P = 0.007). Our results indicated that the activity of Cu-dependent enzymes was enhanced by increasing dietary Cu concentration and that supplementation of Cu in the diet of mink could alter the plasma lipid profile and copper concentration.