亚洲黑熊源阴沟肠杆菌的分离鉴定及耐药性分析

为明确贵阳市某公园动物园1只亚洲黑熊死亡的原因,本试验采集死亡黑熊内脏器官,进行细菌分离培养、菌体形态观察、生理生化鉴定、16S rRNA基因序列分析和小鼠感染试验。结果显示,从死亡黑熊肺脏样本分离出一种圆形、凸起、表面光滑、边缘整齐、中等大小的乳白色菌落,经染色镜检为呈单在或双排列的革兰氏阴性小杆菌。该分离菌可利用葡萄糖、蔗糖、甘露醇、山梨醇和枸橼酸盐,不发酵乳糖,不产生硫化氢,精氨酸脱羧酶阳性,赖氨酸脱羧酶阴性,符合阴沟肠杆菌生化特征。经细菌16S rRNA通用引物扩增,可得到大小为1 419 bp的片段,与NCBI上相应序列进行BLAST比对,结果显示与阴沟肠杆菌序列相似度达99.9%,确认该分离菌为阴沟肠杆菌。用该分离菌腹腔接种小鼠,可致死小鼠,半数致死量（LD₅₀）为7.94×10⁸ CFU/mL。药敏试验发现,该分离菌对丁胺卡那霉素、氧氟沙星、诺氟沙星和环丙沙星等常用抗菌药均保持较高的敏感性。本试验结果表明,阴沟肠杆菌是导致该亚洲黑熊死亡的病原。     

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.     

Sex identification of Japanese black bear,Ursus thibetanus japonicus,by PCR based on amelogenin gene

A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.     

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.     

亚洲黑熊主要组织相容性复合体DQA及DRB基因外显子2多态性分析

试验旨在分析亚洲黑熊主要组织相容性复合体(major histocompatibility complex,MHC) DQA和DRB基因外显子2多态性。采用PCR扩增和克隆测序等方法对来自云南地区的40只亚洲黑熊DNA样本进行研究。结果发现,在40只亚洲黑熊样本中检测出了9个DQA外显子2等位基因和13个DRB外显子2等位基因,和大熊猫相比,亚洲黑熊在DQA和DRB基因外显子2位点上有较高的多态性。通过对推导出的氨基酸序列抗原结合位点的同义替换和非同义替换的比较,证明了DQA和DRB位点的平衡选择作用。     

Serial Transrectal Ultrasonography for Monitoring the Reproductive Activity of the Asiatic Black Bear (Ursus thibetanus ussuricus)

This study evaluated the structural changes in the reproductive tract of Asiatic black bears using serial transrectal ultrasonography. In addition, the ultrasonographic observations were compared with the results of vaginal cytology and hormonal analyses. The collection of blood for hormonal analysis, vaginal cytology and transrectal ultrasonography was performed in two bears (Bears 1 and 2) from June 2011 to August 2013 without mating and in a third bear (Bear 3) from April to December 2012, allowing natural mating. Serial ultrasonographic observations showed cyclic changes in ovarian structures (e.g. emergence of small follicles, growth and ovulation of dominant follicles and corpus luteum (CL) formation) during the reproductive cycles of the three bears. The diameter of the uterine horns remained similar throughout the reproductive cycle in Bears 1 and 2, and it remained similar from April until October, but an enlargement containing foetuses was observed in Bear 3 in December. The ultrasonographic observations were consistent with the data obtained through vaginal cytology and progesterone analysis during the reproductive cycle. An average of 4.0 (±0.4) dominant follicles was observed during the oestrous stage (May‐August), during which the superficial cells accounted for >90% of the total vaginal cells. In addition, the detection of an average of 2.6 (±0.2) CL was associated with increased plasma progesterone concentrations (3.0 ± 0.4 ng/ml) between June and December (near hibernation). In conclusion, serial transrectal ultrasonography demonstrated yearly oestrous (ovulation) cycles via follicular dynamics and CL formation on ovaries, accordingly with vaginal cytology and hormonal level in the Asiatic black bear.     

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.     

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.     

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%.     

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.     

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.     

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.     

周岁黑熊脾脏的组织学和组织化学研究

采用石蜡和冰冻切片方法，进行３Ｍ·Ｅ和Ｗｉｌｄｅｒ染色，并以酸性α－醋酸萘醋酶（ＡＮＡＥ）法显示Ｔ淋巴细胞的分布。结果表明：周岁黑熊（Ｓｅｌｅｎａｒｃｔｏｓｔｈｉｂｅｔａｎｕｓ）脾脏小梁较多，脾窦发育不良．动脉周围淋巴鞘发达；鞘动脉位于脾索内，靠近边缘区；椭球厚为３０－４０μｍ。ＡＮＡＥ阳性反应的淋巴细胞主要分布于白髓的中央动脉周围淋巴鞘内，其次是脾小结外周，边缘区和椭球内。     

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.     

辽宁省黑熊资源现状及保护

1996～2000年采取样带法实地调查和分布区社会调查相结合方法,对辽宁省黑熊资源进行了系统调查.查清了黑熊在辽宁省分布于3市6县(市、区),具体分布地点有20个乡镇,分布面积3594.21 km2,种群数量总计347只,平均种群密度0.10只/hm2.在调查的基础上,分析了影响黑熊资源动态变化的原因,提出了加强保护管理的措施.     

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.     

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.     

圈养黑熊冬季昼间行为节律和时间分配

2014年12月—2015年2月,采用目标取样法和即时记录法对福建归真堂黑熊生态养殖基地的24头圈养黑熊进行了行为观察和记录。通过预观察到的行为构建的行为谱包括休息、食粪、饮水、排粪、排尿、走动、咂舌、攀爬、舔舐、搔痒、啃趾甲、张望、嗅闻、玩耍、示警、打斗、刻板动作、探究、育幼,对相关行为进行归类,包括休息、饮水、排遗、运动、警戒、体表护理、社群、育幼、刻板、其他计10类行为。为尽量避免人为干扰对试验的影响,避开饲养员饲喂、取胆时间段,选取9:00—15:55对目标黑熊进行正式观察。观察结束后经统计分析,圈养黑熊冬季昼间用于休息的时间最多,为50.43%,其次是刻板行为25.35%、运动行为8.63%、警戒行为8.42%。表现较为明显的行为节律,11:00—11:55时是黑熊的主要昼间休息时间。产仔雌性黑熊冬季昼间行为的时间分配和节律和其它黑熊保持一致,同样用于休息的时间最多,且最高峰在11:00—11:55时。     

Effects of intramuscular administration of tiletamine-zolazepam with and without sedative pretreatment on plasma and serum biochemical values and glucose tolerance test results in Japanese black bears (Ursus thibetanus japonicus)

Objective-To establish a safe anesthetic protocol with little effect on blood biochemical values and IV glucose tolerance test (IVGTT) results in Japanese black bears (Ursus thibetanus japonicus). Animals-16 captive female Japanese black bears (5 to 17 years of age). Procedures-Bears were randomly assigned to 4 treatment groups (4 bears/group) in which various treatment combinations were administered via blow dart: tiletamine HCl and zolazepam HCl (9 mg/kg) alone (TZ), TZ (6 mg/kg) and acepromazine maleate (0.1 mg/kg), TZ (6 mg/kg) and butorphanol tartrate (0.3 mg/kg), or TZ (3 mg/kg) and medetomidine HCl (40 μg/kg). Glucose injection for the IVGTT was started 130 minutes after TZ administration. Blood samples were obtained before, at, and intermittently after glucose injection for measurement of biochemical variables as well as plasma glucose and serum insulin concentrations during the IVGTT. Rectal temperature, pulse rate, and respiratory rate were assessed every 15 minutes during the experiment. Results-Induction and maintenance of anesthesia were safely achieved with little adverse effect on cardiopulmonary function when each of the 4 anesthetic regimens was used, although mild hypothermia was induced. No difference was evident between treatment groups in blood biochemical values. Blood glucose and insulin concentration profiles during the IVGTT were similar among the bears given TZ, with or without acepromazine or butorphanol, but hyperglycemia and hypoinsulinemia developed in bears given TZ with medetomidine. Conclusions and Clinical Relevance-All 4 anesthetic regimens yielded chemical restraint without affecting clinical and biochemical values in bears, but medetomidine appeared to affect IVGTT results. For this reason, medetomidine should not be used when anesthetizing bears for IVGTTs.     

中国黑熊及其养殖业的发展现状

如何认识、正确处理野生动物的保护与利用之间的关系是极为重要的。与中国传统医药发展息息相关的黑熊养殖业,在中国已经存在20多年了。目前已完成了自野外捕获向自我繁殖以维持种群发展的关键转折,实现了养殖技术的升级、取胆技术的革新、养殖规模呈现集约化发展趋势。本文概述了中国野生黑熊分布和种群数量,为保护黑熊所采取的各种措施和成效,以及黑熊养殖业的历史和发展现状。介绍了国内与黑熊保护和驯养有关的法律和政策,在此基础上,详细分析了野生黑熊保护与黑熊养殖业的关系,揭示二者间即保护与可持续利用的内在关联,并对黑熊养殖业涉及的问题进行了解析。以化解黑熊保护与利用之间的矛盾,引导黑熊养殖业迈向健康发展之路。