研究者データベース

坪田 敏男(ツボタ トシオ)
獣医学研究院 獣医学部門 環境獣医科学分野
教授

基本情報

所属

  • 獣医学研究院 獣医学部門 環境獣医科学分野

職名

  • 教授

学位

  • 獣医学博士(北海道大学)

連絡先

  • tsubotavetmed.hokudai.ac.jp

ホームページURL

J-Global ID

研究キーワード

  • 野生動物   ツキノワグマ   ステロイド産生能   ステロイド合成酵素   生殖異常   環境ホルモン   性ステロイドホルモン   卵子体外培養   テストステロン   ダイオキシン   モニター生物   体外受精   PCB   クマ   黄体   胎盤   イヌ   エストロジェン   DDT   インスリン   静注糖負荷試験   冬眠   体脂肪   脂肪肝   ネコ   肥満   ステロイド合成   猛禽類   トビ   生殖周期   野生動物医学   Zoo and Wildlife Medicine   

研究分野

  • ライフサイエンス / 獣医学 / 野生動物学
  • 自然科学一般 / 地球生命科学 / 野生動物
  • 環境・農学 / 生物資源保全学 / クマ類

職歴

  • 2007年04月 - 現在 北海道大学 (連合)獣医学研究i院 教授
  • 2003年04月 - 2007年03月 岐阜大学応用生物科学部 教授
  • 2001年04月 - 2003年03月 岐阜大学農学部 教授
  • 1995年03月 - 2001年03月 岐阜大学農学部助教授
  • 1988年04月 - 1995年02月 岐阜大学農学部獣医学科助手
  • 1991年11月 - 1993年10月 イリノイ大学アーバナ・シャンペーン校(米国)客員研究員

学歴

  •         - 1988年   北海道大学   獣医学研究科   予防治療学専攻
  •         - 1988年   北海道大学
  •         - 1983年   北海道大学   獣医学部   獣医
  •         - 1983年   北海道大学

所属学協会

  • ヒトと動物の関係学会   日本野生動物医学会   アメリカ野生動物獣医学会(American Association of Wildlife Ueterinarians)   アメリカ動物園獣医学会(American Association of Zoo Ueterinarians)   繁殖学会(The Society for the Study of Reproduction)   国際クマ学会(International Bear Association)   日本比較内分泌学会   日本哺乳類学会   日本繁殖生物学会   日本獣医学会   

研究活動情報

論文

  • Genetic population structure of invasive raccoons (Procyon lotor) in Hokkaido, Japan: Unique phenomenon caused by pet escape or abandonment.
    Okuyama MW, Shimozuru M, Nakai M, Yamaguchi E, Fujii K, Shimada K, Ikeda T, Tsubota T
    Scientific Reports in press  2020年 [査読有り][通常論文]
  • Male reproductive input, breeding tenure, and turnover in high-density brown bear populations on the Shiretoko Peninsula, Hokkaido, Japan.
    Michito Shimozuru, Yuri Shirane, Mina Jumbo, Masami Yamanaka, Masanao Nakanishi, Tsuyoshi Isinazaka, Shinsuke Kasai, Takane Nose, Yasushi Fujimoto, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota
    Canadian Journal of Zoology 98 175 - 185 2020年 [査読有り][通常論文]
  • Aya Osaki, Mariko Sashika, Go Abe, Kohei Shinjo, Ayako Fujimoto, Mariko Nakai, Michito Shimozuru, Toshio Tsubota
    BMC ecology 19 1 35 - 35 2019年09月11日 [査読有り][通常論文]
     
    BACKGROUND: In Japan, invasive raccoons cause severe ecological and social problems by transmitting pathogens to humans, livestock, and native species, causing substantial crop damage, and competing with native species. Possible competition between invasive raccoons and native raccoon dogs is of concern in Japan because Japanese raccoon dogs have a limited distribution and are native only to Japan and the two species have similar characteristics. We assessed potential competition between raccoons and raccoon dogs by comparing feeding habits and habitat use. RESULTS: Both species were captured in Hokkaido, Japan from 2004 to 2017. More raccoons were captured close to agricultural land at the forest periphery (70.1%, 358/511); conversely, more raccoon dogs were captured in the forest core (74.9%, 253/338). Feeding habits were then examined by fecal analysis and stable isotope analyses. Fecal analysis revealed both species to be opportunistic omnivores that consumed easily found food items. However, raccoon feces contained more crops, whereas raccoon dog feces contained more insects, reflecting the different locations in which the species were trapped. Moreover, stable isotope ratios were significantly higher in raccoons than raccoon dogs (Corn has the highest carbon stable isotope (δ13C) value, and amphibians and reptiles are high in nitrogen stable isotope (δ15N); forest resources such as insects and wild fruits are low in δ13C and δ15N). CONCLUSIONS: We conclude that both species ate similar food types, but their food preferences appeared to differ. Raccoon and raccoon dog habitat use also differed, possibly because the two species inhabited areas where they could easily obtain their preferred foods. Therefore, the current feeding habits and habitat use of raccoons do not appear to overlap sufficiently with those of raccoon dogs to impact the latter. The results of this study, particularly the stable isotope data, may provide a useful precedent for future studies of competition in medium-sized mammals, particularly canids.
  • Nurliani A, Sasaki M, Budipitojo T, Tsubota T, Suzuki M, Kitamura N
    The Journal of veterinary medical science 81 9 1285 - 1290 2019年09月 [査読有り][通常論文]
  • Shirane, Yuri, Shimozuru, Michito, Yamanaka, Masami, Tsuruga, Hifumi, Nakanishi, Masanao, Ishinazaka, Tsuyoshi, Nose, Takane, Kasai, Shinsuke, Shirayanagi, Masataka, Masuda, Yasushi, Fujimoto, Yasushi, Mano, Tsutomu, Sashika, Mariko, Tsubota, Toshio
    JOURNAL OF MAMMALOGY 100 4 1317 - 1326 2019年07月 [査読有り][通常論文]
     
    Natal dispersal likely plays an important role in avoiding inbreeding among large carnivores. We tested the hypothesis that male-biased dispersal reduces close inbreeding by limiting the spatial overlap of opposite-sex pairs of close relatives in brown bears (Ursus arctos) in the Shiretoko Peninsula, Hokkaido, Japan. We genotyped 837 individuals collected in 1998-2017 at 21 microsatellite loci and performed parentage analysis. To calculate natal dispersal distance, we considered the site where the mother was identified as the birthplace of her offspring, and the site where the offspring were identified as their dispersed place. As predicted, we found that dispersal distances were significantly greater for males (12.4 km +/- 1.0) than for females (7.7 km +/- 0.9), and those for males increased from 3 years old, indicating that males begin to disperse around the time sexual maturation begins. Relatedness decreased with distance among pairs of females, and the mean relatedness was significantly higher between pairs of females than between pairs of males or between female-male pairs within 3 km. Closely related female-male pairs rarely (5-6%) resided in close proximity (< 3 km), compared with pairs of closely related females. Our study revealed that the potential for close inbreeding was low in Hokkaido brown bears because males are effective dispersers.
  • Shimozuru M, Shirane Y, Tsuruga H, Yamanaka M, Nakanishi M, Ishinazaka T, Kasai S, Nose T, Masuda Y, Fujimoto Y, Mano T, Tsubota T
    The Journal of heredity 110 3 321 - 331 2019年05月 [査読有り][通常論文]
  • Paudel S, Nakajima C, Mikota SK, Gairhe KP, Maharjan B, Subedi S, Poudel A, Sashika M, Shimozuru M, Suzuki Y, Tsubota T
    Emerging infectious diseases 25 5 1031 - 1032 2019年05月 [査読有り][通常論文]
  • Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota
    PloS one 14 4 e0215489  2019年 [査読有り][通常論文]
     
    Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to "muscle atrophy resistance" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization.
  • Kadariya, R, Shimozuru, M, Maldonado, J.E, Moustafa, M.A.M, Sashika, M, Tsubota, T
    PloS one in press 12 e0207662  2018年12月 [査読有り][通常論文]
  • Shirane Yuri, Shimozuru Michito, Yamanaka Masami, Tsuruga Hifumi, Hirano Saiko, Nagano Natsuo, Moriwaki Jun, Nakanishi Masanao, Ishinazaka Tsuyoshi, Nose Takane, Kasai Shinsuke, Shirayanagi Masataka, Masuda Yasushi, Fujimoto Yasushi, Osada Masahiro, Akaishi Masao, Mano Tsutomu, Masuda Ryuichi, Sashika Mariko, Tsubota Toshio
    EUROPEAN JOURNAL OF WILDLIFE RESEARCH 64 6 65  2018年12月 [査読有り][通常論文]
  • Jun Moriwaki, Ryosuke Omori, Michito Shimozuru, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota
    Japanese Journal of Veterinary Research 66 2 71 - 81 2018年05月01日 [査読有り][通常論文]
     
    Chest girth and body mass of 3,576 brown bears (Ursus arctos yesoensis) harvested for conflict management in Hokkaido, Japan during 1991-2012 were used to establish methods to assess body condition and to compare the body condition of bears by sex, month, year, and reproductive status. The body mass was estimated based on the chest girth in cases with no measurements of the bear body mass. Using the measured and estimated body mass, a growth curve by age was demonstrated to ascertain the mean asymptotical body mass (245 kg for males, 114 kg for females) and ages at 95% asymptotic body mass (14.2 years for males, 7.1 years for females). The body condition value of bears was evaluated as body mass difference (kg) between the individual body mass and the standard body mass as estimated from the growth curve. Body condition value changed seasonally with a low in summer and the highest in the autumnal hyperphagic period. Female body condition value was higher than the males during September. Fluctuation in annual body condition value was found for females however, there was no difference between solitary adult females and females with offspring (cubs, yearlings, or offspring of unknown age). No significant fluctuation was found for males. Our body condition evaluation method using chest girth and body mass of brown bears is useful to elucidate different trends across sex, year, and season.
  • Tapiwa Lundu, Kentaro Yoshii, Shintaro Kobayashi, Shigeru Morikawa, Toshio Tsubota, Naoaki Misawa, Daisuke Hayasaka, Hiroaki Kariwa
    Japanese Journal of Veterinary Research 66 1 21 - 28 2018年 [査読有り][通常論文]
     
    Severe fever with thrombocytopenia syndrome (SFTS) is a newly recognized zoonosis that occurs in China, Japan, and South Korea and is caused by the SFTS virus (SFTSV), which is in the genus Phlebovirus, family Phenuiviridae. Since its discovery in Japan in 2013, SFTS has been reported in the western parts of the country. To elucidate the distribution of SFTSV, we conducted a serological survey of deer and rodents. Serum was screened using enzyme-linked immunosorbent assay (ELISA) and suspected cases were further tested with an indirect immunofluorescence antibody (IFA) assay. Serum samples from 315 deer from Hokkaido (non-endemic area), 41 deer from Miyazaki (endemic area), and 910 rodents from six locations in Japan were tested. Of the 41 deer from Miyazaki, 2 (4.9%) had high ELISA optical density (OD) values (0.1 < OD < 0.3) and a positive IFA result. All of the deer samples from Hokkaido were negative by ELISA (OD < 0.1). No SFTSV-positive rodents were found. Our results indicate that deer in Miyazaki were exposed to SFTSV, unlike deer from Hokkaido (P < 0.05).
  • Mohamed Abdallah Mohamed Moustafa, Michito Shimozuru, Wessam Mohamed, Kyle Rueben Taylor, Ryo Nakao, Mariko Sashika, Toshio Tsubota
    PARASITOLOGY RESEARCH 116 8 2321 - 2325 2017年08月 [査読有り][通常論文]
     
    Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.
  • Michito Shimozuru, Masami Yamanaka, Masanao Nakanishi, Jun Moriwaki, Fumihiko Mori, Masakatsu Tsujino, Yuri Shirane, Tsuyoshi Ishinazaka, Shinsuke Kasai, Takane Nose, Yasushi Masuda, Toshio Tsubota
    PLOS ONE 12 4 2017年04月 [査読有り][通常論文]
     
    servation and management of wildlife. In this study, we investigated the demographic parameters, including age of primiparity, litter size, inter-birth interval, reproductive rate, and cub survival rate, of Hokkaido brown bears (Ursus arctos yesoensis) in the Rusha area on the Shiretoko Peninsula, Hokkaido, Japan, based on a long-term, individual-based monitoring survey. A total of 15 philopatric females were observed nearly every year from 2006 to 2016, and these observations were used to estimate reproductive parameters. The mean age of primiparity was 5.3 +/- 0.2 (SE) years (n = 7, 95% CI = 5.0-5.6). We observed 81 cubs in 46 litters from 15 bears. Litter size ranged from one to three cubs, and averaged 1.76 +/- 0.08 (SE) cubs/litter (95% CI = 1.61-1.91). Inter-birth intervals ranged from 1 to 4 years, and the mean value was estimated as 2.43 (95% CI = 2.16-2.76) and 2.53 (95% CI = 2.26-2.85) years in all litters and in litters that survived at least their first year, respectively. The reproductive rate was estimated from 0.70 to 0.76 young born/year/reproductive adult female, depending on the method of calculation. The cub survival rate between 0.5 and 1.5 years ranged from 60 to 73%. Most cub disappearances occurred in July and August, suggesting that cub mortality is mainly due to poor nutrition in the summer. All reproductive parameters observed in the Rusha area on the Shiretoko Peninsula fell within the range reported in Europe and North America, and were among the lowest or shortest age of primiparity, litter size, and inter-birth intervals, and ranked at a high level for reproductive rate.
  • Elzahara Elbaz, Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Wessam Ahmed Mohamed Mohamed, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Emad Elsayed Ahmed Younis, Sabry Ahmed El-Khodery, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 8 5 802 - 807 2017年 [査読有り][通常論文]
     
    Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido.
  • Sarad Paudel, Janine L. Brown, Sharada Thapaliya, Ishwari P. Dhakal, Susan K. Mikota, Kamal P. Gairhe, Michito Shimozuru, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 11 1713 - 1716 2016年11月 [査読有り][通常論文]
     
    We compared cortisol and thyroid hormone (T3 and T4) concentrations between tuberculosis (TB)-suspected (n=10) and healthy (n=10) elephants of Nepal. Whole blood was collected from captive elephants throughout Nepal, and TB testing was performed using the ElephantTB STAT-PAK (R) and DPP VetTB (R) serological assays that detect antibodies against Mycobacterium tuberculosis and M bovis in elephant serum. Cortisol, T3 and T4 were quantified by competitive enzyme immunoassays, and the results showed no significant differences in hormone concentrations between TB-suspect and healthy elephants. These preliminary data suggest neither adrenal nor thyroid function is altered by TB disease status. However, more elephants, including those positively diagnosed for TB by trunk wash cultures, need to be evaluated over time to confirm results.
  • Sarad Paudel, Marvin A. Villanueva, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Suraj Subedi, Nabin Rayamajhi, Mariko Sashika, Michito Shimozuru, Takashi Matsuba, Yasuhiko Suzuki, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 7 1117 - 1121 2016年07月 [査読有り][通常論文]
     
    We developed an interferon-gamma release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-gamma (rEIFN-gamma) as well as native interferon-gamma from the Asian elephants was performed using anti-elephant IFN-gamma rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-gamma rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-gamma in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.
  • Michito Shimozuru, Akiko Nagashima, Jun Tanaka, Toshio Tsubota
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 196 38 - 47 2016年06月 [査読有り][通常論文]
     
    Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period. (C) 2016 Elsevier Inc. All rights reserved.
  • Daisuke Fukui, Makiko Nakamura, Tsuyoshi Yamaguchi, Makiko Takenaka, Mami Murakami, Tokuma Yanai, Hideto Fukushi, Kazumi Yanagida, Gen Bando, Keita Matsuno, Masashi Nagano, Toshio Tsubota
    JOURNAL OF WILDLIFE DISEASES 52 2 230 - 241 2016年04月 [査読有り][通常論文]
     
    In 2006-10, an epizootic of emerging avian pox occurred in Carrion Crows (Corvus corone) and Large-billed Crows (Corvus macrorhynchos), leading to mortality of juvenile crows in Hokkaido, the northernmost island of Japan. We diagnosed 27 crows with proliferative skin lesions (19 carcasses and eight biopsied cases [one in zoo captivity]) as avian pox clinically, histopathologically by detection of Avipoxvirus-specific 4b core protein (P4b) gene, and epidemiologically. The fatal cases demonstrated intensively severe infection and aggressive lesions with secondary bacterial infection. Since the first identification of avian pox in Sapporo, Japan, in 2006, the frequency of mortality events has increased, peaking in 2007-08. Mortalities have subsequently occurred in other areas, suggesting disease expansion. In Sapporo, prevalence of avian pox evaluated by field censuses during 2007-12 was 17.6% (6.6-27.2%), peaked during 2007-08 and 2008-09, and then decreased. All diseased crows were juveniles, except for one adult. The number of crows assembling in the winter roosts had been stable for >10 yr; however, it declined in 2007-08, decreased by about 50% in 2008-09, and recovered to the previous level in 2009-10, correlated with the avian pox outbreak. Thus, avian pox probably contributed to the unusual crow population decline. All P4b sequences detected in six specimens in Sapporo were identical and different from any previously reported sequences. The sequence detected in the zoo-kept crow was distinct from any reported clades, and interspecies transmission was suspected. This report demonstrates an emerging novel avian pox in the Japanese avifauna and in global populations of Carrion Crows and Large-billed Crows. Longitudinal monitoring is needed to evaluate its impact on the crow population.
  • Jeewan Thapa, Sarad Paudel, Amir Sadaula, Yogendra Shah, Bhagwan Maharjan, Gretchen E. Kaufman, Deborah McCauley, Kamal P. Gairhe, Toshio Tsubota, Yasuhiko Suzuki, Chie Nakajima
    EMERGING INFECTIOUS DISEASES 22 3 570 - 572 2016年03月 [査読有り][通常論文]
  • Jun Moriwaki, Michito Shimozuru, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota
    URSUS 27 2 99 - 109 2016年 [査読有り][通常論文]
     
    The reproductive tracts of 130 adult female brown bears (Ursus arctos) in Hokkaido, Japan, euthanized during 2010-2013 for conflict management, were examined for the presence and number of corpora lutea (CL) in the ovary and placental scars (PS) in the uterine horns. We scored PS on a 0-5 scale based on morphological features including their rim and color. They were characterized as representing either current-year parturition, as determined by accompanying cubs, or previous year parturition, as determined by accompanying yearlings. In total, 128 females were divided into 3 reproductive categories: 44 females with current-year parturition, 39 with previous-year parturition, and 45 solitary females that had not given birth for >2 years. The estimated ovulation rate was 2.21 +/- 0.55 (mean +/- SD), the implantation rate was 1.91 +/- 0.74, and the litter size was 1.54 +/- 0.51. Implantation rates were lower than ovulation rates and changed annually during 2010-2013. Systematic observation of PS and CL allowed us to evaluate the reproductive history and parameters in Hokkaido brown bears and ascertain their annual variation.
  • Mohamed Abdallah Mohamed Moustafa, Kyle Taylor, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Roberto Rosa, May June Thu, Annapaola Rizzoli, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 7 5 922 - 928 2016年 [査読有り][通常論文]
     
    Many of the emerging infectious diseases originate in wildlife and many of them are caused by vector borne pathogens. In Japan, zoonotic tick-borne pathogens (TBPs) are frequently detected in both ticks and wildlife. Here, we studied the infection rates of potentially zoonotic species, including Anaplasma, Ehrlichia, Neoehrlichia and Babesia spp., in Hokkaido's most abundant small mammals as they relate to variable extrinsic factors that might affect the infection rates of these pathogens. A total of 412 small mammals including 64 Apodemus argenteus, 219 Apodemus speciosus, 78 Myodes rufocanus, 41 Myodes rutilus, 6 Myodes rex and 4 Sorex unguiculatus were collected from Furano and Shari sites in Hokkaido, Japan, in 2010 and 2011 and were examined by multiplex PCR for TBPs. A reverse line blot hybridization (RLB) was then developed for the specific detection of 13 potentially zoonotic TBPs. A total of 4 TBPs were detected: Anaplasma sp. AP-sd, Ehrlichia muris, Candidatus Neoehrlichia mikurensis and Babesia microti. The infection rates were 4.4% (18/412), 1.2% (5/412), 13.1% (54/412) and 17.2% (71/412), respectively. The infection rates of each of the detected TBPs were significantly correlated with host small mammal species. A total of 22 (two triple and 20 double) co-infection cases were detected (5.3%). The most frequent co-infection cases occurred between Candidatus N. mikurensis and B. microti 68.2% (15/22). Further studies are required to examine human exposure to these zoonotic TBPs in Hokkaido. (C) 2016 Elsevier GmbH. All rights reserved.
  • Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Kyle Taylor, Ryo Nakao, Mariko Sashika, Michito Shimozuru, Toshio Tsubota
    INFECTION GENETICS AND EVOLUTION 36 268 - 274 2015年12月 [査読有り][通常論文]
     
    A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan. (C) 2015 Elsevier B.V. All rights reserved.
  • Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota
    FASEB JOURNAL 29 2015年04月 [査読有り][通常論文]
  • Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Shin-ichi Saga, Hideto Okuyama, Daisuke Fukui, Gen Bando, Masashi Nagano, Seiji Katagiri, Yoshiyuki Takahashi, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 61 1 61 - 66 2015年02月 [査読有り][通常論文]
     
    Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17 beta and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17 beta, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17 beta after copulation.
  • Aksorn Saengtienchai, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayu Kakehi, Nesta Bortey-Sam, Wageh Sobhy Darwish, Toshio Tsubota, Masanori Terasaki, Amnart Poapolathep, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 33 9 2062 - 2069 2014年09月 [査読有り][通常論文]
     
    Interspecific differences in xenobiotic metabolism are a key to determining relative sensitivities of animals to xenobiotics. However, information on domesticated livestock, companion animals, and captive and free-ranging wildlife is incomplete. The present study evaluated interspecific differences in phase II conjugation using pyrene as a nondestructive biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Polycyclic aromatic hydrocarbons and their metabolites have carcinogenic and endocrine-disrupting effects in humans and wildlife and can have serious consequences. The authors collected urine from 16 mammalian species and analyzed pyrene metabolites. Interspecific differences in urinary pyrene metabolites, especially in the concentration and composition of phase II conjugated metabolites, were apparent. Glucuronide conjugates are dominant metabolites in the urine of many species, including deer, cattle, pigs, horses, and humans. However, they could not be detected in ferret urine even though the gene for ferret Uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) 1A6 is not a pseudogene. Sulfate conjugates were detected mainly in the urine of cats, ferrets, and rabbits. Interestingly, sulfate conjugates were detected in pig urine. Although pigs are known to have limited aryl sulfotransferase activity, the present study demonstrated that pig liver was active in 1-hydroxypyrene sulfation. The findings have some application for biomonitoring environmental pollution. (c) 2014 SETAC
  • Minami W. Okuyama, Michito Shimozuru, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 5 773 - 775 2014年05月 [査読有り][通常論文]
     
    A genetic method for sex determination in raccoons was developed based on nucleotide differences of the zinc finger protein genes ZFX and ZFY. Four novel internal primers specific for ZFX or ZFY were designed. PCR amplification using two primer sets followed by agarose gel electrophoresis enabled sex determination. 141-bp and 447-bp bands were in both sex, and 346-bp band was specific only in male with primer set I. 345-bp and 447-bp bands were in both sex, and 141-bp band was specific only in male with primer set II, which could distinguish raccoon's electrophoresis pattern from three native carnivores in Hokkaido. This method will be useful for conservation genetics studies or biological analyses of raccoons.
  • Sarad Paudel, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Michito Shimozuru, Yasuhiko Suzuki, Toshio Tsubota
    TUBERCULOSIS 94 3 287 - 292 2014年05月 [査読有り][通常論文]
     
    Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. (C) 2014 Elsevier Ltd. All rights reserved.
  • Minami W. Okuyama, Michito Shimozuru, Yojiro Yanagawa, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 2 155 - 161 2014年04月 [査読有り][通常論文]
     
    The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3 beta HSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3 beta HSD expression, suggesting that 3 beta HSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.
  • 2-1.全国のクマ分布の概要,過去との比較
    佐藤喜和, 中下留美子, 坪田敏男, 中島亜美
    日本クマネットワーク(編)「ツキノワグマおよびヒグマの分布域拡縮の現況把握と軋轢防止および危機個体群回復のための支援事業」報告書 4 - 13 2014年03月 [査読無し][通常論文]
  • Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 1 62 - 67 2014年02月 [査読有り][通常論文]
     
    In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.
  • Kyunglee Lee, Ai Takano, Kyle Taylor, Mariko Sashika, Michito Shimozuru, Satoru Konnai, Hiroki Kawabata, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 5 6 841 - 847 2014年 [査読有り][通常論文]
     
    A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks. (C) 2014 Elsevier GmbH. All rights reserved.
  • Minami W. Okuyama, Michito Shimozuru, Go Abe, Mariko Nakai, Mariko Sashika, Ken-Ichiro Shimada, Nobuhiro Takahashi, Daisuke Fukui, Ryohei Nakamura, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 4 361 - 367 2013年08月 [査読有り][通常論文]
     
    The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.
  • Mohamed Abdallah Mohamed Moustafa, Mohamed Nour-Eldin Ismail, Adel El Sayed Ahmed Mohamed, Alsagher Omran Ali, Toshio Tsubota
    Journal of Wildlife Diseases 49 3 750 - 754 2013年07月01日 [査読有り][通常論文]
     
    We report blood parameters for 18 adult and 12 juvenile, free-ranging, female Nile monitors (Varanus niloticus) in Egypt. We report differences between adult and young monitors and compare parameters to those of other reptile species. © Wildlife Disease Association 2013.
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 785 - 790 2013年06月 [査読有り][通常論文]
     
    The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
  • Michito Shimozuru, Ruriko Iibuchi, Takuro Yoshimoto, Akiko Nagashima, Jun Tanaka, Toshio Tsubota
    JOURNAL OF MAMMALOGY 94 3 618 - 627 2013年06月 [査読有り][通常論文]
     
    Bears from the family Ursidae are the only terrestrial mammals that go through gestation, parturition, and lactation during hibernation. This is the 1st study to examine the influence of reproductive status (i.e., nonpregnant, pseudopregnant, or pregnant) on body temperature and blood biochemical profiles in hibernating black bears. Pregnant bears appeared to have higher and more stable body temperatures (37-38 degrees C) than nonpregnant ones (34-36 degrees C) during pregnancy, which was followed by a rapid drop to levels comparable to those of nonpregnant individuals after parturition. In midpregnancy (i.e., January), pregnant bears had higher blood glucose and lower triglyceride concentrations than did nonpregnant ones, whereas blood concentrations of free fatty acids, glycerol, and ketone bodies did not differ significantly. Plasma urea, creatinine, and urea/creatinine levels were significantly lower in pregnant bears than in nonpregnant ones. Pseudopregnant bears showed similar changes in body temperature and blood profiles to pregnant ones, but blood glucose levels in January were significantly lower than those in pregnant bears. These results suggest that pregnant bears maintain homeothermic conditions and supply nutrients to the fetus by stimulating thermogenesis, gluconeogenesis, and urea recycling during hibernation. In addition, these physiological changes appear to be mainly caused by corpus luteum-derived factors (e.g., progesterone) but also were facilitated by placental factors.
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 785 - 790 2013年06月 [査読有り][通常論文]
     
    The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
  • Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 2 159 - 167 2013年04月 [査読有り][通常論文]
     
    There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.
  • Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 2 215 - 218 2013年02月 [査読有り][通常論文]
     
    In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038).
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    VECTOR-BORNE AND ZOONOTIC DISEASES 13 2 92 - 97 2013年02月 [査読有り][通常論文]
     
    To clarify how Borrelia miyamotoi is maintained in the environment in Hokkaido, we examined Ixodes persulcatus for its prevalence among wild rodents and its tick vector by detecting a portion of the borrelial flaB gene in rodent urinary bladder and blood samples, and from whole ticks. We compared B. miyamotoi infection rates to Borrelia garinii and Borrelia afzelii, which are human Lyme disease pathogens also carried by wild rodents, and which are transmitted by the same vector tick. Whereas B. garinii and B. afzelii showed age dependence of infection rates among wild rodents (18.4% and 9.9% among adults and 6.0% and 3.4% among sub-adults, respectively) when looking at urinary bladder samples, B. miyamotoi infection rates were not age dependent for either blood (4.2% among adults, and 7.9% among sub-adults) or urinary bladder samples (1.0% among adults, and 1.7% among sub-adults). Moreover, while B. garinii and B. afzelii infection rates showed increases across months (June, July [p < 0.05] and August [p < 0.01] had higher rates than in May for adult rodents with B. garinii, and July and August had higher rates than in May [p < 0.01] for adult rodents with B. afzelii), B. miyamotoi infection rates did not show significant month dependence. These differences in month and age dependence led us to suspect that B. miyamotoi may not develop persistent infections in wild rodents, as B. garinii and B. afzelii are thought to. Furthermore, we examined the extent of rodent exposure to I. persulcatus nymphs and larvae throughout most of the tick's active season (May through September), and determined that B. miyamotoi infection rates in sub-adult rodents were correlated with larval burden (p < 0.01), suggesting that larvae may be very important in transmission of B. miyamotoi to wild rodents.
  • Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 2 215 - 218 2013年02月 [査読有り][通常論文]
     
    In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038).
  • Takanori Kooriyama, Michiko Okamoto, Tomoyuki Yoshida, Toshisada Nishida, Toshio Tsubota, Akatsuki Saito, Masaki Tomonaga, Tetsuro Matsuzawa, Hirofumi Akari, Hidekazu Nishimura, Takako Miyabe-Nishiwaki
    PRIMATES 54 1 89 - 98 2013年01月 [査読有り][通常論文]
     
    Emerging infectious diseases (EIDs) in wildlife are major threats both to human health and to biodiversity conservation. An estimated 71.8 % of zoonotic EID events are caused by pathogens in wildlife and the incidence of such diseases is increasing significantly in humans. In addition, human diseases are starting to infect wildlife, especially non-human primates. The chimpanzee is an endangered species that is threatened by human activity such as deforestation, poaching, and human disease transmission. Recently, several respiratory disease outbreaks that are suspected of having been transmitted by humans have been reported in wild chimpanzees. Therefore, we need to study zoonotic pathogens that can threaten captive chimpanzees in primate research institutes. Serological surveillance is one of several methods used to reveal infection history. We examined serum from 14 captive chimpanzees in Japanese primate research institutes for antibodies against 62 human pathogens and 1 chimpanzee-borne infectious disease. Antibodies tested positive against 29 pathogens at high or low prevalence in the chimpanzees. These results suggest that the proportions of human-borne infections may reflect the chimpanzee's history, management system in the institute, or regional epidemics. Furthermore, captive chimpanzees are highly susceptible to human pathogens, and their induced antibodies reveal not only their history of infection, but also the possibility of protection against human pathogens.
  • Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura
    Journal of Reproduction and Development 59 2 159 - 167 2013年 [査読有り][通常論文]
     
    There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale. © 2013 by the Society for Reproduction and Development.
  • TERAGUSHI Tomoko, AMAKI Ryota, YOKOHATA Yasushi, ITANI Miki, TSUBOTA Toshio, SUDO Akiko, YATA Shinpei
    日本野生動物医学会誌 18 33 - 39 2013年 [査読有り][通常論文]
  • Takanori Kooriyama, Hideo Hasegawa, Michito Shimozuru, Toshio Tsubota, Toshisada Nishida, Takashi Iwaki
    PRIMATES 53 4 365 - 375 2012年10月 [査読有り][通常論文]
     
    Parasitological surveillance in primates has been performed using coprological observation and identification of specimens from chimpanzees (Pan troglodytes schweinfurthii) in Mahale Mountains National Park, Tanzania (Mahale). In this study, we conducted coprological surveillance to identify the fauna of parasite infection in five primate species in Mahale: red colobus (Procolobus badius tephrosceles), red-tailed monkeys (Cercopithecus ascanius schmidti), vervet monkeys (Cercopithecus aethiops pygerythrus), yellow baboons (Papio cynocephalus), and chimpanzees. Fecal samples were examined microscopically, and parasite identification was based on the morphology of cysts, eggs, larvae, and adult worms. Three nematodes (Oesophagostomum spp., Strongyloides sp., and Trichuris sp.), Entamoeba coli, and Entamoeba spp. were found in all five primate species. The following infections were identified: Bertiella studeri was found in chimpanzees and yellow baboons; Balantidium coli was found in yellow baboons; three nematodes (Streptopharagus, Primasubulura, an undetermined genus of Spirurina) and Dicrocoeliidae gen. sp. were found in red-tailed monkeys, vervet monkeys, and yellow baboons; Chitwoodspirura sp. was newly identified in red colobus and red-tailed monkeys; Probstmayria gombensis and Troglocorys cava were newly identified in chimpanzees, together with Troglodytella abrassarti; and Enterobius sp. was newly identified in red colobus. The parasitological data reported for red colobus, vervet monkeys, and yellow baboons in Mahale are the first reports for these species.
  • Michito Shimozuru, Akari Kamine, Toshio Tsubota
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 163 2 254 - 261 2012年10月 [査読有り][通常論文]
     
    Hibernating bears survive up to 6 months without feeding by utilizing stored body fat as fuel. To investigate how bears maintain energy homeostasis during hibernation, we analyzed changes in mRNA expression of hepatic genes involved in energy metabolism throughout the hibernation period in captive, adult, female Japanese black bears (Ursus thibetanus japonicus). Real-time PCR analysis revealed down-regulation of glycolysis(e.g.. glucokinase). amino acid catabolism- (e.g., alanine aminotransferase) and de novo lipogenesis-related genes (e.g., acetyl-CoA carboxylase 1), and up-regulation of gluconeogensis- (e.g., pyruvate carboxylase), beta-oxidation- (i.e., uncoupling protein 2) and ketogenesis-related genes (i.e., 3-hydroxy-3-methylglutary-CoA synthase 2), during hibernation, compared to the active period (June). In addition, we found that glycolysis-related genes (i.e., glucokinase and pyruvate kinase) were more suppressed in the early phase of hibernation (January) compared to the late phase (March). One week after the commencement of feeding in April, expression levels of most genes returned to levels comparable to those seen in June, but beta-oxidation-related genes were still up-regulated during this period. These results suggest that the modulation of gene expression is not static, but changes throughout the hibernation period. The transcriptional modulation during hibernation represents a unique physiological adaptation to prolonged fasting in bears. (C) 2012 Elsevier Inc. All rights reserved.
  • Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota
    AMERICAN JOURNAL OF VETERINARY RESEARCH 73 8 1282 - 1289 2012年08月 [査読有り][通常論文]
     
    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 HCI and zolazepam HCI (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 HCI (40 mu g/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. (Am J Vet Res 2012;73:1282-1289)
  • Michito Shimozuru, Kamine Akari, Toshio Tsubota
    CANADIAN JOURNAL OF ZOOLOGY 90 8 945 - 954 2012年08月 [査読有り][通常論文]
     
    Body fat accumulation in the prehibernation period is crucial for survival and reproduction during hibernation for bears. Bear body mass increases rapidly during their autumnal hyperphagia phase, which is attributed not only to an increase in food availability, but also to physiological changes in lipid metabolism. To test this hypothesis, we investigated changes in blood biochemical values and mRNA expression levels of hepatic genes involved in lipid metabolism during the active period (June, August, October, and November) in Japanese black bears (Ursus thibetanus japonicus Schlegel, 1857), which were fed a constant ration throughout this period. Blood biochemical analysis revealed that plasma triglyceride concentrations decreased in October and November, implying that peripheral triglyceride uptake was accelerated in autumn. The liver was sampled by needle biopsy. Real-time polymerase chain reaction (PCR) analysis revealed that mRNA expressions of enzymes involved in glycolysis (glucokinase), as well as fatty acid and triglyceride synthesis (ATP-citrate lyase, acetylCoA carboxykinase 1, fatty acid synthase, and diacylglycerol O-acyltransferase 2), increased in November, which suggests that hepatic lipogenesis becomes accelerated during the hyperphagia phase. These results suggest that lipid metabolism is seasonally controlled even without changes in food intake. These physiological changes seen in the prehibernation period would contribute to the rapid mass gain necessary for hibernation.
  • Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota
    AMERICAN JOURNAL OF VETERINARY RESEARCH 73 8 1282 - 1289 2012年08月 [査読有り][通常論文]
     
    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 HCI and zolazepam HCI (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 HCI (40 mu g/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. (Am J Vet Res 2012;73:1282-1289)
  • Minami W. Okuyama, Michito Shimozuru, Nobuhiro Takahashi, Daisuke Fukui, Ryohei Nakamura, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 6 727 - 732 2012年06月 [査読有り][通常論文]
     
    Feral raccoons (Procyon lotor) have been increasing in number since 1979 and are currently subject to pest control in Hokkaido. One of the reasons for the increase in numbers is thought to be the high reproductive potential of raccoons, but little is known about their reproduction. The main aim of this study was to clarify seasonal changes in spermatogenesis and peripheral testosterone concentration of raccoons in Hokkaido. In the present study, external characteristics and histology of the testis and epididymis and the plasma testosterone concentration were investigated in 68 feral, male raccoons culled for pest control and once a month in one live, captive male. The feral males exhibited seasonal changes in spermatogenesis, showing active spermatogenesis in autumn, winter and spring (October June) with noted spermatogenesis and inactive spermatogenesis in summer (July September) with lower mean levels of spermatozoa in the cauda epididymis. Even in the inactive period, spermatozoa were observed in about half of the individuals (14/26); therefore, individuals producing spermatozoa existed every month throughout the year. Testosterone concentrations were significantly high in the winter mating season. In the captive male, the testosterone concentrations were low from June to August, and spermatozoa could not be observed from July to September. These results suggest that raccoons exhibit seasonality of reproduction, but the time and duration of spermatogenetic decline varies widely among individuals. This individual variation in the inactive period is a feature of male raccoon reproduction and is unique among seasonally breeding mammals.
  • Qiang Weng, Toshio Tsubota, Mingdao Dai, Jiaju Weng, Yang Tian, Meiyu Xu, Gen Watanabe, Kazuyoshi Taya
    ANIMAL SCIENCE JOURNAL 83 7 535 - 542 2012年 [査読有り][通常論文]
     
    The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3 beta HSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3 beta HSD, P450c17 and P450arom was measured in the testes during the breeding season. 3 beta HSD was found in Leydig cells during the breeding and non-breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non-breeding season. The localization of P450arom changed seasonally: no immunostaining in the non-breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3 beta HSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process.
  • Takano, Ai, Sugimori, Chieko, Fujita, Hiromi, Kadosaka, Teruki, Taylor, Kyle R., Tsubota, Toshio, Konnai, Satoru, Tajima, Tomoko, Sato, Kozue, Watanabe, Haruo, Ohnishi, Makoto, Kawabata, Hiroki
    TICKS AND TICK-BORNE DISEASES 3 4 259 - 261 2012年 [査読有り][通常論文]
     
    A novel relapsing fever Borrelia sp. was found in Amblyomma geoemydae in Japan. The novel Borrelia sp. was phylogenetically related to the hard (ixodid) tick-borne relapsing fever Borrelia app. Borrelia miyamotoi and B. lonestari. The novel relapsing fever Borrelia sp. was detected in 39 A. geoemydae (39/274: 14.2%), of which 14(14/274: 5.1%) were co-infected with the novel relapsing fever Borrelia sp. and Borrelia sp. tAG, one of the reptile-associated borreliae. Transstadial transmission of the novel relapsing fever Borrelia sp. occurred in the tick midgut and the salivary glands, although Borrelia sp. tAG was only detected in the tick midgut. The difference of the borrelial niche in molted ticks might be associated with borrelial characterization. (c) 2012 Published by Elsevier GmbH.
  • Ai Takano, Minoru Nakao, Toshiyuki Masuzawa, Nobuhiro Takada, Yasuhiro Yano, Fubito Ishiguro, Hiromi Fujita, Takuya Ito, Xiaohang Ma, Yozaburo Oikawa, Fumihiko Kawamori, Kunihiko Kumagai, Toshiyuki Mikami, Nozomu Hanaoka, Shuji Ando, Naoko Honda, Kyle Taylor, Toshio Tsubota, Satoru Konnai, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata
    JOURNAL OF CLINICAL MICROBIOLOGY 49 5 2035 - 2039 2011年05月 [査読有り][通常論文]
     
    Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
  • Keiko Ogawa, Keiji Yamaguchi, Masatsugu Suzuki, Toshio Tsubota, Kenji Ohya, Hideto Fukushi
    JOURNAL OF WILDLIFE DISEASES 47 2 261 - 270 2011年04月 [査読有り][通常論文]
     
    Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to irvestigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the Wild macaque population in Shimokita Peninsula, a rural area of japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PEG E), and microbial sensitivity tests. In total, 9,18 F., colt strains were isolated from 159 fecal samples from wild macaques, and 42 E. cell were isolated from 17 samples from captive macaques. None of the virulence genes cue, six, cif, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates hail no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. cell Was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.
  • Atsushi Yamanaka, Makoto Asano, Masatsugu Suzuki, Toshio Mizoguchi, Toru Oi, Michito Shimozuru, Toshio Tsubota
    ZOOLOGICAL SCIENCE 28 2 105 - 111 2011年02月 [査読有り][通常論文]
     
    We evaluated the stored body fat of Japanese black bears (Ursus thibetanus japonicus) killed as nuisances in Gifu and Fukushima prefectures, Japan, during 2005-2007. We employed femur marrow fat (FMF), modified kidney fat index (mKFI), and abdominal subcutaneous fat (ASF) as indices for quantitative evaluation. We examined the basic characteristics of these indices, such as seasonality, age and sex dependency, and the quantitative relationship among them. mKFI and ASF increased towards the beginning of the denning period (December), while FMF was relatively stable throughout the sampling period (July-December). In cubs, all indices showed significantly lower values than in the older age classes. There seemed to be a catabolizing order between FMF and mKFI, but not between mKFI and ASF. We also evaluated the yearly change in the indices, and discussed its relevance to the incidence of bear intrusion into human residential areas. Bears nuisance-killed in summer (July-September) 2006 had a significantly larger amount of stored body fat than those killed in summer 2007, although the number of nuisance kills was larger in 2006 than in 2007. This suggests that poor nutritional condition is not a direct cause of bear intrusion.
  • 獣医学における保全医学の展開-生物多様性と野生動物感染症-(緒言)
    坪田敏男, 村田浩一
    獣医畜産新報 64 1 7-8  2011年01月01日 [査読無し][通常論文]
  • エゾヒグマ(Ursus arctos yesoensis)におけるHepatozoon ursiの疫学調査
    久保正仁, 千葉依里, 飯渕るり子, 坪田敏男, 佐々木基樹, 酒井洋樹, 柳井徳磨
    Jpn.J.Zoo Wildl.Med. 15 2 111 - 113 2010年12月 [査読有り][通常論文]
  • I. Adachi, S. Kusuda, E. Nagao, Y. Taira, M. Asano, T. Tsubota, O. Doi
    Theriogenology 74 8 1499 - 1503 2010年11月 [査読無し][通常論文]
     
    Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy. © 2010 Elsevier Inc.
  • Ruriko Iibuchi, Michito Shimozuru, Akari Kamine, Junko Nio-Kobayashi, Toshihiko Iwanaga, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 2 236 - 242 2010年04月 [査読有り][通常論文]
     
    The Japanese black bear (Ursus thibetanus japonicus) is a typical seasonal breeder that has a mating season in early summer. Spermatogenesis and testicular steroidogenesis are known to develop and regress annually; however, its molecular mechanism has not yet been investigated. In the present study, we clarified the mRNA sequence of 5 steroidogenic enzymes (P450scc, 3 beta HSD, P450c17, 17 beta HSD3 and P450arom) using RT-PCR and RACE methods and the localization of these gene expressions in the bear testis using an in situ hybridization technique. The amino acid sequence deduced from each mRNA sequence had high homology with the corresponding sequences of other species and possessed a motif typical of the P450 family or short chain alcohol dehydrogenase family. Expression of P450scc, 3 beta HSD and P450c17 mRNA in interstitial tissue indicated that conversion from cholesterol to androstenedione occurs in Leydig cells. On the other hand, the mRNA of 17 beta HSD3, which plays a central role in synthesizing testosterone, was detected not only in the interstitium but also inside the seminiferous tubules, along the basement membrane. P450arom mRNAs were distributed in the seminiferous tubules. These results suggest the possibility of testosterone and estradiol-17 beta synthesis inside the seminiferous tubules in the bear testis. We expect that the results of this study will be useful for further investigation of the molecular mechanism of steroidogenic seasonality in the bear testis.
  • Emiko Nakagawa, Mari Kobayashi, Masatsugu Suzuki, Toshio Tsubota
    ZOOLOGICAL SCIENCE 27 3 263 - 268 2010年03月 [査読有り][通常論文]
     
    The harbor seal (Phoca vitulina) and spotted seal (Phoca largha) are the main seal species around Hokkaido, Japan. While some investigations have been conducted on the ecology and morphology of these two species, there is a lack of genetic information. We studied variation in mitochondrial cytochrome b sequences in the two species. Fifteen haplotypes were observed in 39 harbor seals from Erimo, Akkeshi, and Nosappu, and 23 were observed in 31 spotted seals from Erimo, Akkeshi, Nosappu, Rausu, Yagishiri Island, and Hamamasu. Phylogenetic trees showed two harbor seal lineages: Group I contained primarily haplotypes from Erimo, and Group II contained haplotypes from Akkeshi and Nosappu. Because the Erimo population had fewer haplotypes and less nucleotide diversity than the Akkeshi and Nosappu populations, we considered it to be isolated from the others. In contrast, genetic variance within populations of spotted seals (97.3%) was far higher than that among populations (2.7%), determined by analysis of molecular variance. There were no significant difference among the spotted seal populations, indicating the absence of distinct lineages around Hokkaido. The differences in the genetic population structure between the two species could have been generated by their ecological differences. This study provides basic genetic information on these seal species and will contribute to the conservation and management of fisheries and seals throughout Hokkaido.
  • Daisuke Hayakawa, Motoki Sasaki, Masatsugu Suzuki, Toshio Tsubota, Hiromasa Igota, Koichi Kaji, Nobuo Kitamura
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 1 117 - 123 2010年02月 [査読有り][通常論文]
     
    Testicular steroidogenesis and spermiatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P45007, 3 beta HSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3 beta HSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.
  • Masahito Kubo, Tsukasa Okano, Keiko Ito, Toshio Tsubota, Hiroki Sakai, Tokuma Yanai
    PARASITOLOGY RESEARCH 106 1 213 - 219 2009年12月 [査読有り][通常論文]
     
    A total of 65 free-living carnivores collected on Honshu Island, Japan were examined for muscular Sarcocystis species infections. Among them, 12 Japanese raccoon dogs (Nyctereutes procyonoides viverrinus), one Japanese red fox (Vulpes vulpes japonica), three Japanese martens (Martes melampus melampus), and two Japanese badgers (Meles meles anakuma) were found to have sarcocysts in their muscles. No inflammatory reactions associated with sarcocysts were observed. Ultrastructurally, the sarcocysts detected in the Japanese raccoon dogs, Japanese red fox, and Japanese martens were similar to each other, with the sarcocyst wall being thin and exhibiting minute undulations. On the other hand, the sarcocysts detected in the Japanese badgers had a thick cyst wall with numerous finger-like protrusions which contained microtubules. The species of Sarcocystis in Japanese carnivores remain to be determined. This is the first published report on muscular sarcocystosis in Japanese carnivores.
  • Tsukasa Okano, Tetsuma Murase, Sachiko Nakamura, Takeshi Komatsu, Toshio Tsubota, Makoto Asano
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 194 - 199 2009年04月 [査読有り][通常論文]
     
    The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 +/- 0.4, 4.5 +/- 0.3, 10.4 +/- 0.7 and 69.6 +/- 3.1 mu m (mean +/- SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 +/- 0.36 ml, 7.3 +/- 0.4, 659 +/- 644 x 10(6)/ml, 214 +/- 208 x 10(6) +/- 82.9 +/- 9.6%, 89.3 +/- 9.5% and 97.0 +/- 3.2% (mean +/- SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean +/- SD%; n=23) were simply bent tail (19.9 +/- 22.6), distal droplets (13.5 +/- 11.7), proximal droplets (9.6 +/- 7.8), teratoid spermatozoa (6.7 +/- 10.7), knobbed acrosome (4.9 +/- 8.6), acrosome damage (3.7 +/- 2.8) and bent midpiece (3.7 +/- 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.
  • Sachiko Nakamura, Naohito Nishii, Atsushi Yamanaka, Hitoshi Kitagawa, Makoto Asano, Toshio Tsubota, Masatsugu Suzuki
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 110 - 115 2009年04月 [査読有り][通常論文]
     
    To verify target organ(s) of leptin in the reproductive system of the Japanese black bear, we examined the expression of leptin receptor (Ob-R) protein in ovaries and uteri collected from July to December 2006 by immunohistochemical techniques. Eleven of 22 female Japanese black bears examined had corpora lutea (CLs) in their ovaries and were thought to have undergone or to have terminated delayed implantation in the early pregnancy stage. The CLs were classified into 3 types based on morphological features. The maximum diameters of Type 1 CLs ranged 3 to 7 mm, and the luteal cells contained numerous vacuoles in the cytoplasm, which suggests that this type of CL was funcional. The maximum diameters of Type 2 CLs were approximately 7 mm, and the luteal cells contained fewer vacuoles in the cytoplasm, which suggests that this type of CL was in the early stage of regression. Finally, the maximum diameters of Type 3 CLs were approximately 1 mm, and these CLs contained collagen fibers among their luteal cells, which suggests that this type of CL had regressed. The 3 types of CLs showed different reactions to Ob-R, with positive staining in Type 1, much less positive staining in Type 2 and nearly negative staining in Type 3. In bears having CLs with functional and regressive features (Type I and 2 CLs), Ob-R was also immunolocalized in the developed glandular and ductal endometrial epithelium. In contrast, the Ob-R was absent in the undeveloped endometrial epithelium in bears with regressed (Type 3 CLs) or no CLs. These findings suggested that leptin directly targets CLs and the endometrium so as to develop and maintain them during the delayed implantation period in Japanese black bears.
  • Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Shin-ichi Saga, Hideto Okuyama, Daisuke Fukui, Gen Bandou, Seiji Katagiri, Yoshiyuki Takahashi, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 143 - 148 2009年04月 [査読有り][通常論文]
     
    In sika deer, the normal method of estimating fetal age, based on fetal weight, is not applicable during the early pregnancy period. The objective of the present study was to describe the growth and development of sika deer fetuses and to establish a method for fetal age estimation during early pregnancy using ultrasonography. Five captive female Hokkaido sika deer (Cervus nippon yesoensis) were observed for estrus and mated (day 0) with an intact male. At two- or three-day intervals, fetuses were observed by rectal ultrasonographic scans until 59-61 days of gestation. The straight crown-rump length (SCRL), curved crown-rump length (CCRL), head length (HL), trunk depth (TD) and heart rate (HR) of the fetuses were measured. Linear regression equations were computed for each measurement together with fetal age. Analyses were conducted after transformation to a natural logarithm for SCRL and CCRL. All equations were significant (P<0.001), with SCRL becoming measurable earlier (day 20) than the others and yielding the best correlation (Days = -2.08+14.15 LnX: X=SCRL, Ln=natural logarithm). Therefore, we concluded that a precise estimation of fetal age in early gestation is best performed using SCRL measurements.
  • Toshio Tsubota, Miho Sato, Tsukasa Okano, Sachiko Nakamura, Makoto Asano, Takeshi Komatsu, Haruki Shibata, Masayuki Saito
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 12 1399 - 1403 2008年12月 [査読有り][通常論文]
     
    In the present study, assay of the serum leptin concentration of the Japanese black bear (Ursus thibetanus japonicus) was attempted using a canine-leptin-specific sandwich enzyme-linked immunosorbent assay (ELISA). The dose-response Curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September, gradually increased from September or October, and then remarkably increased in late November. We conclude that this method may be useful for measuring bear serum leptin concentration and that the serum leptin concentration changes annually with a peak in late November.
  • Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Seiji Katagiri, Toshio Tsubota
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 3 139 - 149 2008年11月 [査読有り][通常論文]
     
    Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ER alpha) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestational status was unknown: one female had only one developing corpus luteum: <= Day 4 (metestrus) and two females had two corpora lutea, one of which was at the developing stage equivalent to diestrus or early pregnancy: > Day 7 (diestrus). Staining of ER(x and PR was clear in all cell types during metestrus. During diestrus, the presence of ERa was also clear in deep glandular epithelium, stroma and myometrium., whereas it was suppressed in luminal epithelium and shallow glandular epithelium. Staining of PR was suppressed in luminal epithelium but was detectable in other cell types. Staining of ERa in all cell types and PR in luminal epithelium and glandular epithelium became undetectable by Day 28. PR was presented in stroma and myometrium. throughout pregnancy. The distribution pattern of ERa and PR was different during diestrus from that in a ruminant. This could be attributed to estrogen secretion from the maturing and ovulating follicles in the presence of developed corpus luteum.
  • S. Nakamura, T. Okano, H. Shibata, M. Saito, T. Komatsu, M. Asano, M. Sugiyama, T. Tsubota, M. Suzuki
    CANADIAN JOURNAL OF ZOOLOGY-REVUE CANADIENNE DE ZOOLOGIE 86 9 1042 - 1049 2008年09月 [査読無し][通常論文]
     
    As a first step to study the relationship between fat accumulation and reproductive success in Japanese black bears (Ursus thibetanus japonicus Schlegel, 1857) with the focus on leptin, we determined leptin cDNA sequences in the bears. Next, we studied the possibility of white adipose tissue (WAT) as a leptin secretion source by observing the changes of leptin mRNA expression in WAT by semiquantitative real-time reverse transcript-polymerase chain reaction, the index of WAT fat-cell size, and serum leptin concentration in pregnant bears. Then, based on our results, we discussed roles of leptin in those bears. The amino acid sequences of leptin from the bears were highly identical to that of other carnivores. The expression of leptin mRNA in WAT was detected from September to January, with a tendency to increase in late November and January; the relationship between changes in the index of WAT fat-cell size and those in serum leptin concentration was high (r = 0.55, P < 0.01), with an increase in both in mid-November. These results suggested that leptin was mainly secreted from WAT in bears and that serum leptin concentrations might reflect their nutritional condition. Moreover, leptin might serve as an indicator of their fat mass, which would affect their survival during hibernation and their reproductive success.
  • Tatsuya Kunisue, Nozomi Takayanagi, Tomohiko Isobe, Shin Takahashi, Susumu Nakatsu, Toshio Tsubota, Keisuke Okumoto, Sumio Bushisue, Kazuyuki Shindo, Shinsuke Tanabe
    ENVIRONMENTAL SCIENCE & TECHNOLOGY 42 3 685 - 691 2008年02月 [査読有り][通常論文]
     
    The present study investigated concentrations and patterns of brominated flame retardants, such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs), and persistent organochlorines (OCs) in liver and adipose tissues of raccoon dogs (RD: Nyctereutes procyonoides) collected from two metropolises and a local prefecture in Japan during 2001-2006. Relatively high concentrations of PBDEs were found in RD livers, while HBCD levels were the lowest among the measured organohalogen compounds. Among PBDE congeners, BDE 209 was predominant in RDs from all the regions, indicating that pollution derived from the technical decaBDE product is extensive across Japan. On the other hand, concentrations of tetra- to nona-BDE congeners in RDs from a metropolis were significantly higher than those from the other two regions, implying that there were regional differences in the past usage of the technical tetraBDE and octaBDE products. Such a regional difference was also observed for HBCD levels. Lipid-normalized concentration ratios of liver to adipose tissue L/A ratio) for tri to hepta-BDE congeners were lower than 1.0 in the investigated eight RDs, suggesting lipid-dependent accumulation. However, the L/A ratios of BDE 209 exceeded 1.0 in all the specimens, suggesting hepatic retention of this compound. In addition, lipid-dependent accumulation of alpha-HBCD was observed, but the L/A ratios of gamma-HBCD were greater than 1.0 in some specimens. These results indicate that Japanese RDs have been recently exposed to BDE 209 and gamma-HBCD and accumulated both these compounds preferentially in blood-rich organs, probably due to their binding to proteins and/or rapid biotransformation, as reported in experimental rodents.
  • Ruriko Iibuchi, Junko Nio-Kobayashi, Sachiko Nakamura, Makoto Asano, Toshihiko Iwanaga, Toshio Tsubota
    BIOLOGY OF REPRODUCTION 297 - 297 2008年 [査読有り][通常論文]
  • Masahito Kubo, Keiya Kobayashi, Toshiaki Masegi, Hiroki Sakai, Toshio Tsubota, Makoto Asano, Miki Itani, Tokuma Yanai
    JOURNAL OF WILDLIFE DISEASES 43 3 542 - 544 2007年07月 [査読有り][通常論文]
     
    A free-flying Great Egret (Ardea alba) captured in Gifu, central Japan, in May 2006 had a large mass on the right carpal joint. The tumor was diagnosed as chondrosarcoma by histopathologic examination.
  • Makoto Asano, Toshio Tsubota, Takeshi Komatsu, Atsushi Katayama, Tsukasa Okano, Sachiko Nakamura
    JOURNAL OF VETERINARY MEDICAL SCIENCE 69 4 433 - 435 2007年04月 [査読有り][通常論文]
     
    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 I 1 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.
  • Okano, T, Nakamura, S, Nakashita, R, Komatsu, T, Murase, T, Asano, M, Tsubota, T
    Jpn. J. Zoo Wildl. Med. 12 61 - 70 2007年 [査読無し][通常論文]
  • Tatsuya Kunisue, Nozonu Takayanagi, Toshio Tsubota, Shinsuke Tanabe
    CHEMOSPHERE 66 2 203 - 211 2007年01月 [査読有り][通常論文]
     
    The present study determined the accumulation features of persistent organochlorines (OCs) such as polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB), and chlordane compounds (CHLs) in wild raccoon dogs (RDs; Nyclereutes procyonoides) collected from Kanagawa prefecture in Japan during 2001. In livers of RDs, CHLs were remarkably dominant (20 times higher than PCBs) followed by PCBs > DDTs > HCHs > HCB, whereas the chemicals in muscles were in the order of CHLs > PCBs > HCHs > DDTs > HCB. The accumulation pattern of OCs in RDs was different from those in Japanese humans and avian species reported previously, which generally accumulate higher levels of DDTs and PCBs than CHLs. This result indicates that RDs have been exposed to relatively high levels of CHLs and have high metabolic and elimination capacity for DDTs. In fact, CHL levels in RDs were higher than those in humans and some avian species, while DDT levels in RDs were much lower than other animals. In particular, extremely high accumulation levels of oxychlordane, which is a metabolite from chlordanes and nonachlors, were observed in RD livers. The higher toxic potency of oxychlordane than parent compounds may suggest that RDs are at high risk by this metabolite. On lipid weight basis, PCBs, HCHs and HCB levels were almost similar in livers and Muscles, suggesting that the tissue distribution of these compounds principally followed the lipid-dependent accumulation. However, accumulation levels of oxychlordane and p,p'-DDD in livers were significantly higher than those in muscles, and concentration ratios of liver to muscle (L/M ratios) of these Compounds were greater than 1.0 in all the specimens. This phenomenon was similar to PCDD/DF congener accumulation patterns observed previously in RDs. When relationships between hepatic TEQs and L/M ratios were examined for oxychlordane and p,p'-DDD, L/M ratios for these compounds significantly increased with hepatic TEQ levels, suggesting their hepatic sequestration in TEQs-dependent manner.
  • Tsukasa Okano, Sachiko Nakamura, Rumiko Nakashita, Takeshi Komatsu, Tetsuma Murase, Makoto Asano, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 10 1133 - 1137 2006年10月 [査読有り][通常論文]
     
    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.
  • Tsukasa Okano, Sachiko Nakamura, Takeshi Komatsu, Tetsuma Murase, Kiyoshi Miyazawa, Makoto Asano, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 10 1101 - 1104 2006年10月 [査読有り][通常論文]
     
    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%.
  • T. Okano, T. Murase, C. Yayota, T. Komatsu, K. Miyazawa, M. Asano, T. Tsubota
    Animal Reproduction Science 95 1-2 134 - 143 2006年09月 [査読有り][通常論文]
     
    The Japanese black bear (Ursus thibetanus japonicus) is an endangered species in some areas of Japan, and semen collection and cryopreservation are important means to preserve genetic resources and prevent extinction. The aim of the present study was to improve the methods for electroejaculation and cryopreservation in these bears. We collected the semen from captive mature Japanese black bears during the mating season by the electroejaculation method using maximum stimulus voltages of 10 V (n = 9) and 7 V (n = 15), and compared the characteristics of fresh semen after collection. None of the variables examined (i.e. total sperm count, motility, viability, morphology, and rate of urine contamination) were statistically different between the two electro-stimulus voltages. After the semen obtained was cryopreserved, the effects of three different diluents (egg yolk-TRIS-citrate-glucose, egg yolk-TRIS-citrate-fructose, and egg yolk-TRIS-citrate-fructose-lactose-raffinose n = 10) and two different incubation times (1-1 and 3-0 h of cooling-glycerol equilibration times n = 12) on frozen-thawed spermatozoa. None of the variables examined (i.e. motility, viability and morphology) were statistically different among the three diluents and between the two incubation times. The results indicate that, in the collection and cryopreservation of Japanese black bear semen: (1) a maximum voltage of 7 V may be enough to obtain ejaculates (2) the three diluents examined were useful and (3) glycerol equilibration time may be omitted with prior cooling for 3 h. © 2005 Elsevier B.V. All rights reserved.
  • Qiang Weng, Tetsuma Murase, Makoto Asano, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 9 999 - 1002 2006年09月 [査読有り][通常論文]
     
    We investigated the distribution of 3 types of steroidogenic enzymes, P450scc, 3 beta HSD, and P450c17, in wild raccoon dog ovaries by immunohistochemistry. Six pairs of ovaries were obtained from wild raccoon dogs between 2001 and 2003, with 3 of the 6 pairs of ovaries containing corpora lutea. P450scc, 3 beta HSD, and P450c17 were localized in the granulosa and theca cells of these raccoon dogs. Furthermore, lutein cells were stained positively for P450sce and 3 beta HSD in the pregnant and non-pregnant raccoon dogs. These results suggest that granulosa and theca cells may synthesize progesterone and androgens, which may play an important role in follicular development, and that lutein cells are a major source of progesterone in wild raccoon dogs.
  • 中村幸子, 岡野司, 柴田治樹, 斉藤昌之, 小松武志, 淺野玄, 坪田敏男
    J Reprod Dev 52 Supplement J115  2006年08月 [査読有り][通常論文]
  • Qiang Weng, Mohamed S. Medan, Meiyu Xu, Toshio Tsubota, Gen Watanabe, Kazuyoshi Taya
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 52 4 503 - 510 2006年08月 [査読有り][通常論文]
     
    Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin a and inhibin/activin (beta(A) and beta(B)) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidinbiotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin u., inhibin/activin beta(A) and inhibin/activin beta(B). The inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, beta(A), and beta(B) subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and nonbreeding seasons.
  • Tatsuya Kunisue, Michio X. Watanabe, Hisato Iwata, Toshio Tsubota, Fumio Yamada, Masatoshi Yasuda, Shinsuke Tanabe
    Environmental Pollution 140 3 525 - 535 2006年04月 [査読無し][通常論文]
     
    The present study determined the contamination levels and congener-specific accumulation features of dioxins and related compounds (DRCs) in wild terrestrial mammals such as large Japanese field mice (LJFM), lesser Japanese moles (LJMs), and raccoon dogs (RDs) collected from Kanto region in Japan during 2001. The toxic equivalent quantity (TEQ) levels in the carcasses or adipose tissues were in the order of RDs ≥ LJMs > LJFM. Comparison of DRC congener profiles in the three species and principal component analysis (PCA) demonstrated a higher contribution of OCDD, T4CB77, and P 5CB118 in LJMs. Analysis of liver-adipose distribution of DRC congeners in RDs showed that livers contained significantly higher TEQs than adipose tissues, indicating that liver is a depository organ and critical for determining the toxicokinetics of DRCs. As for most T4, P 5, H6CDD/DFs and for P5CB126, H 6CB169 and mono-ortho PCB congeners, their liver/adipose concentration ratios in RDs revealed a tendency to increase with hepatic TEQ levels, suggesting TEQ-dependent hepatic sequestration. © 2005 Elsevier Ltd. All rights reserved.
  • Qiang Weng, Mohamed S. Medan, Tsukasa Okano, Tetsuma Murase, Toshio Tsubota, Meiyu Xu, Gen Watanabe, Kazuyoshi Taya
    ENDOCRINE 29 2 345 - 350 2006年04月 [査読有り][通常論文]
     
    The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of inhibin a and inhibin/activin (beta(A) and beta(B)) subunits during the breeding season in the wild male Japanese black bear. Histological observations of testes were performed and seminiferous tubule diameters were measured. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin a, inhibin/activin beta(A), and inhibin/activin beta(B) during the breeding season. Serum concentrations of immunoreactive (ir-)inhibin, testosterone, and luteinizing hormone (LH) were measured by radioimmunoassay. Higher values of seminiferous tubule diameters and all types of spermatogenic cells including mature-phase spermatozoa were found during the breeding season. There were seasonal changes in serum concentrations of ir-inhibin, testosterone, and LH. Ir-inhibin was positively correlated with testosterone, and LH. in addition, immunoreactivity of inhibin alpha, beta(A), and beta(B) subunits were also detected in Sertoli and Leydig cells during the breeding season. These results suggest that Japanese black bear testes may secrete bioactive inhibins during the breeding season and that the circulating inhibin may be a useful indicator of the testicular function in wild male Japanese black bears.
  • T Mano, T Tsubota
    JOURNAL OF MAMMALOGY 83 4 1026 - 1034 2002年11月 [査読有り][通常論文]
     
    We investigated reproductive characteristics of brown bears (Ursus arctos yesoensis) in the Oshima Peninsula, Hokkaido, Japan, based on the study of individuals harvested from 1983 to 1987. Analyses were based on age-class and reproductive status data determined for 196 females killed during the study period. The minimum age at 1st parturition was 4 years, but frequency of reproductive success among females <6 years old was notably lower than that among females greater than or equal to6 years of age. Females <6 years old were more apt to produce single offspring and lose them during the 1st year. There appeared to be few changes in frequency of reproducing cubs among females 6-20 years of age. Females typically separated from young when young were 15-27 months old. For mature adult females, mean litter size was 1.8, and interval between births was 2.3-3.0 years. Embryo loss and neonatal mortality were uncommon.
  • Analysis of Causes of Bark Stripping by the Japanese Black Bear (Ursus thibetanus japonicus).
    Yoshida Y, Hayashi S, Horiuchi M, Tsubota T, Murase T, Okano T, Sato M, Yamamoto K
    Mammalian Science 42 35 - 43 2002年 [査読有り][通常論文]
  • Ohdachi, S, T. Aoi, T. Mano, T. Tsubota
    Journal of the Mammalogical Society of Japan 17 1 27 - 47 一般社団法人 日本哺乳類学会 1992年 [査読有り][通常論文]
     
    The growth, sexual dimorphism, and geographical variation in skulls of 596 brown bears (Ursus arctos) hunted in Hokkaido, Japan from 1971 to 1986 were investigated. Most cranial traits continued to increase after the age of sexual maturity, and in particular skulls became progressively wider with age. Skulls of males, even those of cubs and yearlings, were larger than those of females, and the difference became significant after two years of age. Skull dimensions tended to increase from south to north, in Hokkaido, and bears from the north-eastern region possessed higher brain cases and longer mandibles relative to skull length and width, than those in the southern region.
  • T TSUBOTA, H KANAGAWA, T MANO, T AOI
    BEARS - THEIR BIOLOGY AND MANAGEMENT 8 125 - 128 1990年 [査読有り][通常論文]

書籍

作品等

  • ツキノワグマ生息実態調査報告書(共著)、岐阜県根尾村
    2000年
  • 訪問紀:サンディエゴ動物園希少種繁殖センター(CRES)、J. Reprod. Dev. 46/4:(]G0008[)-(]G0009[)
    2000年
  • 欧米の野生動物医学事情-2ヵ月間の研修より-、Zoo and Wildlife News 10:9-12
    2000年
  • 内分泌攪乱化学物質による野生生物影響実態調査マニュアル(共著)
    1999年
  • 環境ホルモンと生殖異常(単著)環境保全(岐阜大学環境保全広報)創刊号:5-10
    1999年
  • 第21回総会記念講演「環境ホルモンと生殖異常」(単著)自治研ぎふ(岐阜県地方自治研究センター広報)64:12-20
    1999年
  • 野生生物のダイオキシン類汚染状況調査マニュアル(共著)
    1998年
  • ジャイアントパンダの繁殖に関するワークショップに参加して,岐阜県獣医師会報37:77-79
    1997年
  • ニホンジカ黄体退縮物の経産指標としての有効性,岐阜県獣医師会報36:20-25
    1996年
  • クマ,畜産の研究50(I):145-150
    1996年
  • 天然林におけるニホンツキノワグマの生息可能密度-ブナの豊凶に伴うツキノワグマの春と秋の食性の年次変動-、第4期プロ・ナトゥーラ・ファンド助成成果報告書17-25
    1995年
  • 岐阜県ツキノワグマ個体群指標調査報告書(岐阜県林政部自然環境保全課編),35pp
    1995年
  • クマの繁殖生理、はばたき(神戸市立王子動物園発行)36:9-10
    1994年
  • ツキノワグマによる白山高山帯の利用について,天然林におけるツキノワグマの生態調査報告書(1989-1994年):19-26
    1994年
  • アメリカでの研究生活を終えて、月輪:75-78
    1994年
  • グリズリーの楽園-カトマイ国立公園-、Field Note40:3-6
    1993年
  • ミネソタ州のクロクマ調査、月輪5:78-81
    1993年
  • アメリカでのクマ研究事情Part1-イリノイでのクマ研究-、月輪4:83-85
    1992年
  • SSRに参加して-とくにアメリカでの野生動物の繁殖生理学的研究、比較内分泌学会ニュース67:7-8
    1992年
  • Denali National Parkでのクマの研究と保護管理、Field Note36:13-16
    1992年
  • エゾヒグマの繁殖生理、遺伝45(7):77-81
    1991年
  • クマ類の繁殖特性、日本科学者会議岐阜支部会報38:3-22
    1991年
  • 日本産クマ類の研究概観、高教研会誌、20:96-99
    1991年
  • 野生クマへの研究アプローチ-野生動物との共存への道-、岐阜県獣医師会報、28:9-14
    1991年
  • 日本産クマ類の研究紹介、岐阜県高等学校教育研究会生物教育研究部会雑誌34:8-15
    1990年
  • 1989年ツキノワグマ夏季白山高山帯調査報告、月輪2:14-20
    1990年
  • 1989年白川村ツキノワグマ生態調査報告(共著)、月輪2:1-11
    1990年

その他活動・業績

  • 奥山みなみ, 下鶴倫人, 中井真理子, 藤井啓, 山口英美, 島田健一郎, 池田透, 坪田敏男 日本獣医学会学術集会講演要旨集 159th 515 2016年08月30日 [査読無し][通常論文]
  • 南アジアはMycobacterium orygisの風土病地域である(South Asia is an endemic region for Mycobacterium orygis)
    Thapa Jeewan, 中島 千絵, Rahim Zeaur, Paudel Sarad, Shah Yogendra, Maharian Bhagwan, Poudel Ajay, Gairhe Kamal Prasad, 坪田 敏男, 鈴木 定彦 日本細菌学雑誌 71 (1) 159 -159 2016年02月 [査読無し][通常論文]
  • 高江洲昇, 伊藤真輝, 菅原里沙, 柳川洋二郎, 永野昌志, 佐鹿万里子, 下鶴倫人, 坪田敏男 日本獣医学会学術集会講演要旨集 157th 547 2014年08月11日 [査読無し][通常論文]
  • 高野愛, 川端寛樹, 大久保(佐藤)梢, 中尾稔, 伊東拓也, TAYLOR Kyle, 李景利, 坪田敏男, 今内覚, 吉村英紘, 豊間根耕地, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 156th 304 2013年08月30日 [査読無し][通常論文]
  • 高野愛, 佐藤梢, 中尾稔, 伊東拓也, TAYLOR Kyle, 今内覚, 坪田敏男, 高田伸弘, 川端寛樹 衛生動物 64 (2) 113 2013年06月15日 [査読無し][通常論文]
  • 苅和 宏明, 尾崎 由佳, 真田 崇弘, 池中 良徳, 石塚 真由美, 坪田 敏男, 好井 健太朗, 吉松 組子, 有川 二郎, 高島 郁夫 獣医畜産新報 66 (4) 262 -264 2013年04月 [査読無し][通常論文]
     
    ハンタウイルスはげっ歯類媒介性の人獣共通感染症の病原体で、腎症候性出血熱(HFRS)またはハンタウイルス肺症候群(HPS)を引き起こす。近年の日本におけるげっ歯類のハンタウイルス感染状況を明らかにするために、1994年から2010年にかけて国内の様々な地域で捕獲された1658匹のげっ歯類の血清について、抗ハンタウイルス抗体の検出を行った。840例のRattus属げっ歯類(ドブネズミとクマネズミ)は全て抗体陰性であった。北海道以外の地域で捕獲された野生げっ歯類113例はいずれも抗体陰性であったのに対し、北海道で捕獲された705例の野性げっ歯類のうち、エゾヤチネズミの7.4%(26/352)とアカネズミの1.2%(2/168)が抗体を保有していた。(著者抄録)
  • 高江洲昇, 柳川洋二郎, 永野昌志, 兼子明久, 今井啓雄, 岡本宗裕, 下鶴倫人, 坪田敏男 日本野生動物医学会大会・講演要旨集 19th 72 2013年 [査読無し][通常論文]
  • 小林すみよ, 柳川洋二郎, 坪田敏男, 永野昌志 日本野生動物医学会大会・講演要旨集 19th 95 2013年 [査読無し][通常論文]
  • 高野愛, 杉森千恵子, 藤田博己, 角坂照貴, TAYLOR Kyle, 坪田敏男, 今内覚, 田島朋子, 佐藤梢, 渡邉治雄, 大西真, 川端寛樹 大原綜合病院年報 52 106 -107 2012年12月15日 [査読無し][通常論文]
  • 高野愛, 杉森千恵子, 藤田博己, 角坂照貴, TAYLOR Kyle, 坪田敏男, 今内覚, 田島朋子, 佐藤梢, 渡邉治雄, 大西真, 川端寛樹 日本獣医学会学術集会講演要旨集 154th 270 2012年08月31日 [査読無し][通常論文]
  • 高見 一利, 渡邊 有希子, 坪田 敏男, 福井 大祐, 大沼 学, 山本 麻衣, 村田 浩一 Japanese journal of zoo and wildlife medicine = 日本野生動物医学会誌 17 (2) 33 -42 2012年06月01日 [査読無し][通常論文]
  • 條野真奈美, 小林由美, 浅沼武敏, 浅沼武敏, 坪田敏男, 桜井泰憲 北海道大学水産科学研究い報 62 (1) 1 -7 2012年03月28日 [査読無し][通常論文]
  • Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (1) 5 -13 2012年02月 [査読無し][通常論文]
     
    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.
  • 新井智, 浜田雅史, 多屋馨子, 岡部信彦, TAYLOR Kyle, 西澤次訓, 坪田敏男, 大館智志, 森川茂 現在、国内で分離・同定できないウイルス性出血熱等の診断等の対応方法に関する研究 平成23年度 総括・分担研究報告書 50 -55 2012年 [査読無し][通常論文]
  • Nakashita Rumiko, Suzuki Yaeko, Akamatsu Fumikazu, Naito Yuichi I, Sato-Hashimoto Miho, Tsubota Toshio Researches in organic geochemistry 27 (0) 73 -79 2011年09月30日 [査読無し][通常論文]
     
    Several recent studies have suggested an innovative method for estimating the food sources, feeding habits, and trophic position of consumers in food webs, based on the compound-specific stable isotope analysis (CSIA) of amino acids. In this study, we used CSIA to study terrestrial mammals in a controlled feeding experiment with captive Asiatic black bears (Ursus thibetanus). The trophic position is estimated to be 1.7-1.9 for the bears, which is consistent with their actual trophic position (approximately 2). We also investigated the feeding habits of six wild Asiatic black bears (the same...
  • 尾崎由佳, 萩谷友洋, 真田崇弘, 瀬戸隆弘, TAYLOR Kyle, 吉川佳佑, IVANOV Leonid, 好井健太朗, 坪田敏男, 池中良徳, 石塚真由美, 吉松組子, 有川二郎, 苅和宏明 日本獣医学会学術集会講演要旨集 152nd 256 2011年08月31日 [査読無し][通常論文]
  • 佐々木基樹, 坪田敏男, 石川創, 茂越敏弘, 大隅清治, 手塚雅文, 宮本明夫, 福井豊, 北村延夫 北海道獣医師会雑誌 55 (8) 410 2011年08月10日 [査読無し][通常論文]
  • 佐鹿万里子, 阿部豪, 奥山みなみ, 坪田敏男 北海道獣医師会雑誌 55 (8) 409 2011年08月10日 [査読無し][通常論文]
  • 尾崎由佳, 萩谷友洋, 真田崇弘, 瀬戸隆弘, TAYLOR Kyle, 吉川佳佑, IVANOV Leonid I, 好井健太朗, 坪田敏男, 池中良徳, 石塚真由美, 吉松組子, 有川二郎, 苅和宏明 北海道獣医師会雑誌 55 (8) 415 2011年08月10日 [査読無し][通常論文]
  • 大井徹, 中下留美子, 岡輝樹, 大西尚樹, 高橋裕史, 正木隆, 阿部真, 宮本麻子, 佐野真琴, 坪田敏男, 山崎晃司 森林総合研究所研究成果選集 2011 22 -23 2011年07月 [査読無し][通常論文]
  • 坪田敏男, 平野沙衣子, 藤本靖, 上野洋一, 長田雅裕, 郡山尚紀, 下鶴倫人 日本生態学会大会講演要旨集 58th 211 2011年03月08日 [査読無し][通常論文]
  • Atsushi Yamanaka, Kiyoshi Yamauchi, Tsunenori Tsujimoto, Toshio Mizoguchi, Toru Oi, Seigo Sawada, Michito Shimozuru, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 59 (1) 31 -39 2011年02月 [査読無し][通常論文]
     
    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 >= 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.
  • 川端 寛樹, 及川 陽三郎, 川森 文彦, 熊谷 邦彦, 三上 稔之, 花岡 希, 安藤 秀二, 本田 尚子, Kyle Taylor, 坪田 敏男, 今内 覚, 高野 愛, 渡邉 治雄, 大西 真, 中尾 稔, 増沢 俊幸, 高田 伸弘, 矢野 泰弘, 石畝 史, 藤田 博己, 伊東 拓也 日本衛生動物学会全国大会要旨抄録集 63 (0) 69 -69 2011年 [査読無し][通常論文]
     
    Multi-locus sequence typing of <I>Borrelia garinii</I> isolates from humans in Japan, and comparison with environmental isolates was performed. All isolates could be divided into two groups, and an association was detected between clinical and rodent isolates, suggesting that Japanese clinical isolates of <I>B.garinii</I> come from rodents via ticks.
  • 川端寛樹, 石ぐろ史, 藤田博己, 高田伸弘, 矢野泰弘, 増澤俊幸, 中尾稔, 伊東拓也, 及川陽三郎, TAYLOR Kyle, 坪田敏男, 今内覚, 川森文彦, 三上稔之, 熊谷邦彦, 高野愛, 安藤秀二, 花岡希, 本田尚子, 渡邉治雄, 大西真 ワンヘルス理念に基づく動物由来感染症制御に関する研究 平成22年度 総括・分担研究報告書 17 -20 2011年 [査読無し][通常論文]
  • 坪田 敏男, 植木 玲一, 坪田 あゆみ Bears Japan 11 (1) 10 -13 2010年07月 [査読無し][通常論文]
  • 加味根 あかり, 下鶴 倫人, 坪田 敏男 獣医畜産新報 63 (5) 367 -370 2010年05月 [査読無し][通常論文]
  • Ruriko Iibuchi, Akari Kamine, Michito Shimozuru, Junko Nio-Kobayashi, Gen Watanabe, Kazuyoshi Taya, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (4) 185 -196 2010年02月 [査読無し][通常論文]
     
    Male Japanese black bears (Ursus thibetanus japonicus) have an explicit reproductive cycle. The objective of this study was to clarify the variation of plasma testosterone, FSH, inhibin, LH levels and testicular gonadotropin receptor mRNA expression of male bears associated with their testicular activity. Notably, this study investigated peripheral FSH concentration and localization of gonadotropin receptor mRNAs for the first time in male bears. Blood and testicular tissue samples were taken from captive, mature, male Japanese black bears during testicular active, regressive and recrudescent phases. Plasma hormone concentrations were measured by immunoassays, and gonadotropin receptor mRNA expression in the testis was investigated by in situ hybridization technique and also by real-time PCR. There were significant variations in plasma testosterone and inhibin concentrations. Changes in FSH concentration preceded these hormones with a similar tendency. Hormones started to increase during denning, and achieved the highest values at the end of the recrudescent phase for FSH and in the active phase for testosterone and inhibin. These changes in hormone concentrations were accompanied by testicular growth. In situ hybridization analysis revealed that FSH and LH receptor mRNA was possibly expressed in Sertoli cells and Leydig cells, respectively, as they are in other mammals. However, neither plasma LH concentration nor testicular gonadotropin receptor mRNA expression level varied significantly among the sampling months. These results suggest that FSH, inhibin and testosterone have roles in testicular activity in male bears. This study provides important endocrine information for comprehending seasonal reproductivity in male Japanese black bears.
  • Daisuke Hayakawa, Motoki Sasaki, Masatsugu Suzuki, Toshio Tsubota, Hiromasa Igota, Koichi Kaji, Nobuo Kitamura JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 (1) 117 -123 2010年02月 [査読無し][通常論文]
     
    Testicular steroidogenesis and spermiatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P45007, 3 beta HSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3 beta HSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.
  • Emiko Nakagawa, Mari Kobayashi, Masatsugu Suzuki, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (3) 147 -162 2009年11月 [査読無し][通常論文]
     
    We examined morphological growth variations in skull features between the Kuril harbor seal and the spotted seal in Hokkaido, Japan. Skulls from 80 Kuril harbor seals and 41 spotted seals were collected, and we measured 29 metric and 6 non-metric cranial characteristics. Three growth classes were defined according to the postnatal developmental stage: pups (0 year), subadults (1-4 years old) and adults (more than 5 years old). We detected sexual dimorphism in Kuril harbor seal pups, subadults, and adults. Although interspecies differences were detected in each growth class, Kuril harbor seals were larger and more massive than spotted seals; this feature was already detectable in pups. We did not detect certain cranial characteristics with which to identify the two species, but it was possible to identify any unknown specimens to their species, sex, and growth class using the cranial data generated in this study. Using 6 non-metric cranial characteristics, we identified significant interspecies differences with regard to the shape of the temporozygomatic suture and the extent of the nasal-incisive suture; the shape of the temporozygomatic suture and the shape of the nares were indicators of growth class in Kuril harbor seals. Although non-metric cranial characteristics have a lower discriminating power than metric characteristics, they are easy to use in the field even by inexperienced researchers.
  • 山中 淳史, 坪田 敏男 森林科学 : 日本林学会会報 0 (57) 9 -12 2009年10月01日 [査読無し][通常論文]
  • Ruriko Iibuchi, Noriko Nakano, Tadashi Nakamura, Tadasu Urashima, Michito Shimozuru, Tetsuma Murase, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (1) 13 -22 2009年05月 [査読無し][通常論文]
     
    Japanese black bears, Ursus thibetanus japonicus, have been classified as a vulnerable species so that data on reproduction are needed to maintain and/or extend their population. They are known to have a peculiar style of reproduction, giving birth to their neonates and raising them during denning, a period of complete fasting. In this study, we investigated the metabolic rate and milk composition of mother bears raising neonates, and the changes in body weight of the neonates under captive conditions. Seven female bears kept in dens were weighed once a month, and the amount of energy they used was calculated. From birth, cubs were also weighed and their growth rate was determined. In addition, the milk composition was analyzed to investigate its characteristics. As a result, it was found that mother bears used 34% more energy than did solitary females. There was no significant difference in the energy used for nursing whether they had single or twin cubs. On the other hand, the body weight gain of single cubs was significantly higher than that of twin cubs, suggesting that the growth of the cubs was highly affected by the suppression of mother's energy consumption during the fasting period. The milk had high fat and low sugar concentrations. This indicates that mother bears used the fat accumulated prior to denning for their main energy source when raising cubs. Considering all results together, Japanese black bears showed remarkable efficiency in the use of energy for reproduction during the fasting period.
  • 飼育下チンパンジーにおける上部気道感染細菌の検索
    郡山尚紀, 宮部貴子, 西田利貞, 坪田敏男 第15回野生動物医学会 (2009/9 富山) 2009年 [査読無し][通常論文]
  • 坪田 敏男, 佐藤 美穂, 岡野 司, 中村 幸子, 淺野 玄, 小松 武志, 柴田 治樹, 斉藤 昌之 The journal of veterinary medical science 70 (12) 1399 -1403 2008年12月 [査読無し][通常論文]
     
    In the present study, assay of the serum leptin concentration of the Japanese black bear (Ursus thibetanus japonicus) was attempted using a canine-leptin-specific sandwich enzyme-linked immunosorbent assay (ELISA). The dose-response Curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September, gradually increased from September or October, and then remarkably increased in late November. We conclude that this method may be useful for measuring bear serum leptin concentration and that the serum leptin concentration changes annually with a peak in late November.
  • Yanagawa Yojiro, Matsuura Yukiko, Suzuki Masatsugu, Katagiri Seiji, Tsubota Toshio Japanese Journal of Veterinary Research 56 (3) 139 -149 2008年11月 [査読無し][通常論文]
     
    Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestatio...
  • Masahito Kubo, Shigehiko Uni, Takeshi Agatsuma, Mitsuru Nagataki, Roger J. Panciera, Toshio Tsubota, Sachiko Nakamura, Hiroki Sakai, Toshiaki Masegi, Tokuma Yanai PARASITOLOGY INTERNATIONAL 57 (3) 287 -294 2008年09月 [査読無し][通常論文]
     
    Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. spa, in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7 +/- 4.6 x 42.7 +/- 4.5 mu m in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0 +/- 0.7 x 1.8 +/- 0.3 mu m in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9 +/- 0.3 x 3.3 +/- 0.2 mu m. The analyses of the 18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis fava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 mu m and 331.8 x 231.7 mu m, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2 +/- 2.5 x 27.0 +/- 2.9 mu m in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2 +/- 1.4 x 3.5 +/- 0.5 mu m in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  • 中村 幸子, 岡野 司, 吉田 洋, 松本 歩, 村瀬 豊, 加藤 春喜, 小松 武志, 淺野 玄, 鈴木 正嗣, 杉山 誠, 坪田 敏男 Japanese journal of zoo and wildlife medicine 13 (1) 15 -20 2008年03月 [査読無し][通常論文]
     
    Bioelectrical impedance analysis(BIA)によるニホンツキノワグマ(Ursus thibetanus japonicus)(以下,クマ)の体脂肪量FM測定法確立を試みた。クマを横臥位にし,前肢および後肢間の電気抵抗値を測定した。その値をアメリカクロクマに対する換算式に当てはめ,クマのFMを求めた。2005年9月から翌年の1月までの間,飼育下クマを用いて体重BMおよびFMを測定したところ,BMとFMの変動は高い相関(r=0.89)を示した。よって,秋のBM増加はFM増加を反映していること,ならびにBIAがクマのFM測定に応用可能であることが示された。飼育クマの体脂肪率FRは,9月初旬で最も低く(29.3±3.3%),12月に最も高い値(41.6±3.0%)を示した。彼らの冬眠開始期までの脂肪蓄積量(36.6kg)は約252,000kcalに相当し,冬眠中に1,900kcal/日消費していることが示唆された。一方,2006年6月から11月までの岐阜県および山梨県における野生個体13頭の体脂肪率は,6.9〜31.7%であった。野生個体のFRは飼育個体に比較して低かった。BIAを用いて,ニホンツキノワグマの栄養状態が評価でき,この方法は今後彼らの環境評価指標のツールとしても有用であると思われる。
  • 杉山誠, 安部昌子, 川口まり子, 長谷部文子, 伊藤直人, 宮澤清志, 村瀬哲磨, 坪田敏男, 石黒直隆, 淺野玄, 伊藤雅, 山下照夫, 榮賢司 食肉における家畜・家禽のウイルス疾病に関する研究 平成18年度 総括・分担研究報告書 49 -58 2007年 [査読無し][通常論文]
  • 坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (23) 1 -2 2006年12月01日 [査読無し][通常論文]
  • 国内の野生動物における日本脳炎ウイルスに対する血清疫学調査−イノシシが日本脳炎ウイルスの増殖動物である可能性−.
    杉山 誠, 高木愛香, 源 宣之, 伊藤直人, 淺野 玄, 坪田敏男, 石黒直隆, 伊藤 雅, 山下照夫, 榮 賢司 日本獣医畜産新報 JVM 59 284 -285 2006年 [査読無し][通常論文]
  • Q Weng, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya EXPERIMENTAL ANIMALS 54 (5) 451 -454 2005年10月 [査読無し][通常論文]
     
    The localizations of steroidogenic enzymes (P450scc, 3 beta HSD, P450c17 and P450arom) in testes of Shiba goats were investigated by immunohistochemistry. P450scc, 3 beta HSD, P450c17 and P450arom were detected in all Leydig cells of adults. P450scc and P450c17 were observed in most Leydig cells in the fetus (90 days) and neonate (15 days). 3 beta HSD and P450arom were found in some Leydig cells of the fetus with weak immunostaining but the numbers of immunopositive Leydig cells and intense immunostaining were increased in Leydig cells of the neonate. These results suggest that Shiba goat testes have the ability to synthesize progestin, androgen and estrogen in the fetus, neonate and adult, and synthesis of these steroid hormones showed an age-related rise.
  • Q Weng, MS Medan, G Watanabe, T Tsubota, Y Tanioka, K Taya JOURNAL OF REPRODUCTION AND DEVELOPMENT 51 (3) 299 -304 2005年06月 [査読無し][通常論文]
     
    The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Gottingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3 beta HSD, P450c17 and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.
  • Q Weng, T Murase, M Asano, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (6) 603 -605 2005年06月 [査読無し][通常論文]
     
    A wild raccoon dog (Nyctereutes procynoides) which died due to a traffic accident on 18 October 2001, and was determined to be 4.5 years old, was examined. Visual appearance of the external genitalia in this animal showed to be female with a large penis-like clitoris protruding from the vulvar juncture. Visual examination of the internal genitalia revealed that the animal possessed both testes and uterus. Histological appearance of the removed gonads showed only Sertoli cells but no spermatogenesis. Using polymerase chain reaction with skin biopsy directed against the sex-determining region on the Y chromosome (SRY) gene, the genomic SRY gene was expressed as a single band and sequenced. Based on these findings, this raccoon dog was diagnosed as male pseudohermaphrodite.
  • WENG Qiang, MEDAN Mohamed S, WATANABE Gen, TSUBOTA Toshio, TANIOKA Yoshikuni, TAYA Kazuyoshi The Journal of reproduction and development 51 (3) 299 -304 2005年06月 [査読無し][通常論文]
     
    9月(n=2)、2月(n=2)および6月(n=2)に採取した6頭の雄ゲッチンゲンミニブタの精巣を用いて、ステロイドホルモン合成酵素の免疫局在を調べた。免疫組織化学染色には、choleserol side-chain cleavage cyochrome P450(P450scc)、3 β-hydroxy seroid dehydrogenase (3βHSD)、17α-hydroxylase cyochrome P450(P450cl7)およびaro-maase cyochrome P450(P450arom)に対するポリクローン抗体を用いた。9月、2月および6月のいずれの時期に採取した精巣でも、精細管内に精祖細胞から伸長精子細胞が確認され、採取時期による差は認められなかった。精巣組織中のライディッヒ細胞に、P450scc、P450cl7、3βHSDおよびP450aromの局在が認められた。以上の結果から、雄ゲッチンゲンミニブタにおいては、年間を通して精子形成が行われており、ライディッヒ細胞がプロジェステロン、アンドロジェンおよびエストロジェンの分泌細胞であろうと推察された。
  • Q Weng, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya JOURNAL OF REPRODUCTION AND DEVELOPMENT 51 (2) 247 -252 2005年04月 [査読無し][通常論文]
     
    In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome 17 alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3 beta HSD, P450c17 and P450arom. The distribution of P450scc, 3 beta HSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.
  • M Sasaki, H Endo, O Wiig, AE Derocher, T Tsubota, H Taru, M Yamamoto, K Arishima, Y Hayashi, N Kitamura, J Yamada ANNALS OF ANATOMY-ANATOMISCHER ANZEIGER 187 (2) 153 -160 2005年04月 [査読無し][通常論文]
     
    The hindlimbs of the Malayan sun bear (Helarctos malayanus), the polar bear (Ursus maritimus), the brown bear (Ursus arctos) and the giant panda (Ailuropodo melanoleuca) have been anatomically and osteometrically studied. The Musculus tibialis cranialis of the Malayan sun bear and the giant panda possessed a well-developed rich fleshy portion until the distal end of the tibia. In the polar bear and the brown bear, however, the fleshy portion of the M. tibialis cranialis was not developed until the distal, end of the tibia. The tendon of the M. tibialis cranialis inserting on the proximal end of the Ossa metatarsalia was shorter in the Malayan sun bear and the giant panda than in the polar bear and the brown bear. In the Malayan sun bear and the giant panda, moreover, the M. popliteus was attached more distally to the tibia than in the polar bear and the brown bear. The stable dorsiflexion and supination of the foot and the efficient pronation of the crus are important for skillful tree climbing. The present study suggests that the Malayan sun bear and the giant panda have hindlimbs especially adapted to tree climbing by the well-developed fleshy portion of the A tibialis cranialis reaching the distal end of the tibia, its short tendon, and the A popliteus inserting near the distal end of the tibia. © 2005 Elsevier GmbH. All rights reserved.
  • Durrant Barbara, 赤木 智香子, 坪田 敏男 Journal of Reproduction and Development 51 (1) 巻頭10 -12 2005年02月 [査読無し][通常論文]
  • Rapid, easy and reliable sexing method for falconiformes by PCR
    Jpn. J. Zoo Wildl. Med. 10: 79-84 2005年 [査読無し][通常論文]
  • 長 雄一, 赤松 里香, 高田 雅之, 大橋 和彦, 岡崎 克則, 金子 正美, 遠藤 大二, 浅川 満彦, 坪田 敏男, 淺野 玄 日本生態学会大会講演要旨集 52 (0) 432 -432 2005年 [査読無し][通常論文]
     
    生態学会2004年大会において「野生鳥類の大量死リスク評価につながる病原体データベースの基本コンセプトについて」を発表し、病原体情報の収集分析のために、広域サンプリング_-_>病原体タイプの同定_-_>情報管理・蓄積_-_>情報解析_-_>情報公開及び活用といった基本的なコンセプト(設計図)を提示した。
    本発表においては、これらのコンセプトに基づいたシステムの実際的な運用例等を示すとともに、2003年度に実施したサンプリングにより把握できた野生鳥類における病原体保有動態について中間的な報告を行う。また、実際に罹病あるいは死亡した個体に関する病原体(死因)情報のフォーマットや、「情報の共通化・共有化」等の具体的な運用面の問題についても述べる。さらに野生鳥類の移動_-_分散様式・病原体の伝播_-_発病様式・湖沼等の生息環境の空間構造・人間活動との相互作用を視野においた野生鳥類大量死発生予測モデリングについて手法検討を行う。
  • T Urashima, T Nakamura, K Teramoto, Arai, I, T Saito, T Komatsu, T Tsubota COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 139 (4) 587 -595 2004年12月 [査読無し][通常論文]
     
    Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by H-1-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following: Neu5Ac(alpha2-3)Gal(beta1-4)Glc Neu5 Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1 -3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Neu5Ac(alpha2-6)Gal(beta1-4)GIcNAc(beta1-6)]Gal(beta1-4)Glc (C) 2004 Elsevier Inc. All rights reserved.
  • T Murase, N Imaeda, N Kondoh, T Tsubota JOURNAL OF REPRODUCTION AND DEVELOPMENT 50 (6) 667 -674 2004年12月 [査読無し][通常論文]
     
    Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17C for up to 3 days, were washed and preincubated for 10 min with C-2-ceramide, an analogue of endogenous ceramide, C-2-dihydroceramide (C-2-DH-ceramide), a negative control to C-2-ceramide, or with (1S,2R)-D-erythro-2(N-myristoylamino)- 1-phenyl-1-propanol (D-crythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 muM A23187 (Ca2+/A23187) at 37C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+ /A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C-2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+ /A23187 in a dose-dependent manner, whereas C-2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.
  • D Hayakawa, M Sasaki, C Akabane, N Kitamura, T Tsubota, M Suzuki, J Yamada JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1463 -1466 2004年11月 [査読無し][通常論文]
     
    The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.
  • T Okano, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1371 -1376 2004年11月 [査読無し][通常論文]
     
    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.
  • T Okano, T Murase, M Asano, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1359 -1364 2004年11月 [査読無し][通常論文]
     
    This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4degreesC (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4degreesC (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 X 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.
  • 翁 強, 村瀬 哲磨, 淺野 玄, 坪田 敏男 Japanese journal of zoo and wildlife medicine 9 (2) 65 -70 2004年09月 [査読無し][通常論文]
     
    2001年11月〜2003年4月の間に,神奈川県において野生雌タヌキ5頭(11月,2月,3月および4月)の死体を収集した。死体より卵巣を摘出した後,一方を液体窒素で急速凍結するとともに,残りをホルマリン固定し,パラフィン切片を作製した。免疫組織化学を実施し,P450aromの局在を確定した。また,凍結保存した卵巣からmRNAを抽出した後,RT-PCR法にてcDNA断片を増幅した。増幅したDNAのうちP450aromをコードすると予想されるサイズのバンドを確認し,増幅DNA断片のクローニングを行った。プラスミドにインサートされたことを確認した後,シークエンスを行った。免疫組織化学の結果,P450aromは内卵胞膜細胞および卵胞上皮細胞(顆粒層細胞)に認められた。また,3月における黄体細胞および4月における妊娠黄体を構成するほとんどの細胞の細胞質に陽性反応が認められた。PCRによりP450arom cDNAバンドの大きさは289bpであった。また,シークエンスにより得られたP450aromの塩基配列はヒト,ウシおよびラットのそれと76.3%,78.2%および72.6%の相同性を示した。以上の結果より,野生タヌキにおいて卵胞上皮細胞(顆粒層細胞),内卵胞膜細胞および黄体細胞がP450aromを発現し,エストロジェン合成能を有することが示唆された。
  • WR Boone, BB Keck, JC Catlin, KJ Casey, ET Boone, PS Dye, RJ Schuett, T Tsubota, JC Bahr THERIOGENOLOGY 61 (6) 1163 -1169 2004年04月 [査読無し][通常論文]
     
    The objective of this study was to determine if black bears are induced ovulators. We conducted a single experiment with two replicates; each replicate was divided into two arms: females exposed to male bears and females without male exposure. We used laparoscopy to examine ovaries for corpora lutea and measured serum progesterone, concentrations. Six of the seven isolated females failed to ovulate, while seven of the eight females exposed to males produced one to four corpora lutea. Furthermore, isolated females had significantly lower progesterone concentrations than females exposed to males. Thus, our data suggest that the American black bear is an induced ovulator. These results may aid biologists in their efforts to reproduce ursids in controlled environments. (C) 2003 Elsevier Inc. All rights reserved.
  • 伊藤 英之, 須藤-山地 明子, 阿部 素子, 村瀬 哲麿, 坪田 敏男 Japanese journal of zoo and wildlife medicine 9 (1) 39 -43 2004年03月 [査読無し][通常論文]
     
    近年,イヌワシ(Aquila chrysaetos)やクマタカ(Spizaetus nipalensis)のような猛禽類は生息地の破壊等により減少しており,生息地や遺伝的多楡院の保全が重要となっている。しかしながら,猛禽類に開する遺伝的な情報はまだ少ない。本研究では,ハヤブサ(Falco peregrinus)とシロハヤブサ(Falco rusticalus)において開発された8つのマイクロサテライトマーカーを6種の猛禽類,トビ(Milvus migrans),オオタカ(Accipiter gentiles),チョウゲンボウ(Falco tinnunculus),イヌワシ,チュウヒ(Circus spilonotus),クマタカヘの応用を試みた。今回用いたマイクロサテライトマーカーは,1つのマイクロサテライトマーカーを除き,少なくとも1種以上の種において遺伝的多型を検出し,猛禽類における有用性を示した。我々は猛禽類間におけるマイクロサテライトマーカーの種間増幅の有用性を示した。この結果は今後猛禽類の保全や研究に有用であると考えられる。
  • Y Matsuura, M Suzuki, D Hayakawa, M Asano, M Sasaki, N Kitamura, J Yamada, T Tsubota, N Ohtaishi JAPANESE JOURNAL OF VETERINARY RESEARCH 51 (3-4) 167 -172 2004年02月 [査読無し][通常論文]
     
    We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 P HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.
  • 坪田 敏男, 淺野 玄 獣医畜産新報 57 (2) 142 -144 2004年02月 [査読無し][通常論文]
  • Y Matsuura, M Suzuki, D Hayakawa, M Asano, M Sasaki, N Kitamura, J Yamada, T Tsubota, N Ohtaishi JAPANESE JOURNAL OF VETERINARY RESEARCH 51 (3-4) 167 -172 2004年02月 [査読無し][通常論文]
     
    We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 P HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.
  • Jpn. J. Zoo Wildl. Med. 9: 39-43 2004年 [査読無し][通常論文]
  • AS Mweene, H Fukushi, GS Pandey, M Syakalima, M Simuunza, M Malamo, A Nambota, KL Samui, T Tsubota, Y Nakazato, M Onuma, J Yasuda REVUE SCIENTIFIQUE ET TECHNIQUE DE L OFFICE INTERNATIONAL DES EPIZOOTIES 22 (3) 873 -877 2003年12月 [査読無し][通常論文]
     
    A study was conducted to determine the prevalence of bovine herpesvirus-1 (BHV-1), which causes infectious bovine rhinotracheitis, in cattle destined for market in Southern Province, Zambia. A total of 116 nasal secretion samples were tested using the direct fluorescent antibody test, while blood samples from the same cattle were examined by a commercial enzyme-linked immunosorbent assay kit. The prevalence of the BHV-1 antigens in cattle was 23.28% (27/116), while the mean prevalence of the BHV-1 antibodies was 48.28% (56/116). This study showed that cattle in transit to markets could easily spread the virus, which was reactivated by the stress of trekking for long distances under unfavourable conditions, to the other cattle with which they came into contact. Thus, these transit cattle posed a serious threat to other bovines. Systems of cattle trading where cattle must be transported a long way to market should be reviewed by the authorities to minimise the conditions that may exacerbate the spread of infection.
  • W Qiang, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1087 -1092 2003年10月 [査読無し][通常論文]
     
    Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3,3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.
  • T Okano, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1093 -1099 2003年10月 [査読無し][通常論文]
     
    Twenty-one wild mate 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/ml, 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.
  • W Qiang, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1087 -1092 2003年10月 [査読無し][通常論文]
     
    Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3,3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.
  • 長谷川 淳, 松田 宗明, 河野 公栄, 須藤 明子, 坪田 敏男, 平岡 考, 脇本 忠明 環境化学 : journal of environmental chemistry 13 (3) 765 -779 2003年09月24日 [査読無し][通常論文]
  • H. Fujise, T. Nakayama, N. Iwase, T. Tsubota, T. Komatsu Comparative Clinical Pathology 12 (1) 33 -39 2003年05月 [査読無し][通常論文]
     
    Cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBC) of the Japanese black bear and compared with that of the normal dog (LK dog), which possesses RBC with high sodium (Na) and low potassium (K). Unlike the RBC of the American black bear, RBC from the Japanese black bear failed to exhibit K congener rubidium (Rb) influxes through Na-K pump and K-chloride (Cl) cotransport. In the RBC of the Japanese black bear, treatment with N-ethyl maleimide (NEM), calcium (Ca) ionophore A23187 and EGTA, and hyposmotic medium failed to elicit K-Cl cotransport, whereas these perturbations elicited K-Cl cotransport in the LK dog RBC. The Ca uptake was measured in RBC of both the Japanese black bear and the LK dog, and the fluxes were enhanced in hyposmotic medium and inhibited with quinidine, an inhibitor of Na/Ca exchange transporter. Extracellular Ca-dependent Na efflux was detected in RBC of both the Japanese black bear and the LK dog, and it was also inhibited with quinidine. The Ca uptake and Na efflux were probably mediated by Na/Ca exchange transporter. RBC of both the Japanese black bear and the LK dog were restored to their original volume after being swollen in hyposmotic medium with, but not without, Ca. Thus, Na/Ca exchange transporter causes regulatory volume decrease in the RBC of both the Japanese black bear and the LK dog.
  • 村田 浩一, 坪田 敏男 獣医畜産新報 56 (4) 281 -283 2003年04月 [査読無し][通常論文]
  • H Ito, A Sudo-Yamaji, M Abe, T Murase, T Tsubota ZOOLOGICAL SCIENCE 20 (3) 339 -344 2003年03月 [査読無し][通常論文]
     
    A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z From sequence analysis, a T-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the T-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.
  • H Ito, A Sudo-Yamaji, M Abe, T Murase, T Tsubota ZOOLOGICAL SCIENCE 20 (3) 339 -344 2003年03月 [査読無し][通常論文]
     
    A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z From sequence analysis, a T-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the T-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.
  • 長谷川淳, 松田宗明, 河野公栄, 須藤明子, 坪田敏男, 平岡考, 脇本忠明 環境科学 13 (3) 765 -779 2003年 [査読無し][通常論文]
  • Fujise, H., Nakayama, T., Iwase, N., Tsubota, T. & Komatsu, T.: Comparison of cation transport and regulatory volume decrease between the red blood cells from the Japanese black bear and the dog(jointly worked)
    Comp. Clinic. Pathol (12) 33 -39 2003年 [査読無し][通常論文]
  • Hideyuki Ito, Akiko Sudo-Yamaji, Motoko Abe, Tetsuma Murase, Toshio Tsubota Zool. Sci 20 (3) 339 -344 2003年 [査読無し][通常論文]
  • 山本 かおり, 河村 篤紀, 坪田 敏男, 釣賀 一二三, 小松 武志, 村瀬 哲磨, 喜多 功, 工藤 忠明 Japanese journal of zoo and wildlife medicine 7 (2) 103 -108 2002年09月 [査読無し][通常論文]
     
    本研究では,飼育条件下のニホンツキノワグマにおいてDNAフィンガープリント法による父子判定の有用性を検討した。制限酵素Hinf Iおよび(GATA)_4プローブを用いたDNAフィンガープリントはニホンツキノワグマの個体識別および父子判定に有用であることが示された。1995年から1997年の間に11頭の母グマから生まれた13頭の子グマと22頭の父親候補の雄グマについて父子判定を行った結果,7頭の雄グマが父親と判定された。特に2頭の雄グマが8頭の子グマの父親と判定された。本研究では,飼育条件下において雌グマが多くの雄グマとの交尾の機会をもっても,ある特定の雄の繁殖成功が高くなることが示された。
  • K Yamamoto, T Tsubota, T Komatsu, A Katayama, T Murase, Kita, I, T Kudo J. Vet. Med.Sci. 64 (6) 505 -508 2002年06月 [査読無し][通常論文]
     
    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.
  • K Yamamoto, T Tsubota, T Komatsu, A Katayama, T Murase, Kita, I, T Kudo JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (6) 505 -508 2002年06月 [査読無し][通常論文]
     
    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.
  • 坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (14) 7 -8 2002年06月01日 [査読無し][通常論文]
  • 坪田 敏男, 瀧紫 珠子, 須藤 明子, 村瀬 哲磨, 野田 亜矢子, 柵木 利昭, 源 宣之 Japanese journal of zoo and wildlife medicine 7 (1) 69 -74 2002年03月 [査読無し][通常論文]
     
    人間の営みによって作り出された化学物質が,長期間分解されることなく環境に蓄積し,内分泌攪乱化学作用によって人や野生動物の生殖に異常をもたらすことが解明されつつある。内分泌攪乱化学物質には,DDTなどの農薬,PCB類などの工業化学物質,ダイオキシンなどの非意図的生成物,合成女性ホルモンとして使われたDESなどの医薬品などが含まれる。これまでに,アメリカ合衆国のアポプカ湖でのワニの個体数減少,ミンクやカワウソの繁殖率の低下,猛禽類の卵殻の薄化や孵化率の低下,イルカやアザラシの大量死,イボニシでのインポセックス,コイの雌雄同体化,ホッキョクグマの生殖能力の低下や間性といったさまざまな生殖異常が内分泌攪乱作用によって引き起こされている。日本においては平成10年度より内分泌攪乱化学物質およびダイオキシン類による野生生物への影響実態調査が開始され,さまざまな野生動物,とくに海獣類や猛禽類における内分泌攪乱化学物質の蓄積が認められた。今後さらに影響実態を究明し,内分泌攪乱化学物質問題を解決していく必要がある。
  • 野生動物における内分泌攪乱化学物質の蓄積濃度と生殖への影響(共著) 2002
    Jpn. J. Zoo Wildl. Med. 7, 69‐74 2002年 [査読無し][通常論文]
  • ニホンツキノマグワ(Ursus thibetanusjaponicus)によるクマハギの発生原因の検討(共著)
    哺乳類科学 42, 35‐43 2002年 [査読無し][通常論文]
  • Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats (共著)
    Bull. Environ. Contam. Toxicol. 67, 438‐445 2001年 [査読無し][通常論文]
     
    2001
  • Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita J. Reprod. Dev. 47 (4) 237 -243 2001年 [査読無し][通常論文]
     
    2001
  • Changes in sex steroids, gonadotropins, prolactin and inhibin in pregnant and nonpregnant black bears (Ursus thibetanus japonicus) (共著)
    Biol. Reprod 65. 1006‐1013 2001年 [査読無し][通常論文]
  • Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 47 (2) 109 -112 2001年 [査読無し][通常論文]
     
    This study examined differences in the penetration of various batches of mixed cervical mucus by bull spermatozoa. Mucus was collected from different numbers (2, 2, 3, 4 or 7) of cows or heifers in estrus and a part of the sample was individually, and the rest was mixed, and stored frozen. The distance traveled by the most advanced sperm cell in frozen-thawed mucus during incubation at 38 C for 10 min was measured (sperm penetration). Variation of sperm penetration in the individual mucus samples was reduced by mixing but the difference among the batches of mixed mucus was significant (p< 0.0002). Nevertheless, the number of animals from which mucus was collected and mixed did not seem to influence the sperm penetration of the different batches. Unexpectedly it was observed that some individual and mixed mucus samples had reduced viscosity after freezing and thawing and sperm penetration in the mixed mucus samples showed a significant, negative correlation with the proportion of mucus of reduced viscosity contained in the batch (r=-0.969, p< 0.01). These results suggest that the difference in the sperm penetration of mixed mucus may probably be rather independent of the number of mucus donors.
  • Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats (共著)
    Bull. Environ. Contam. Toxicol. 67, 438‐445 2001年 [査読無し][通常論文]
  • Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 47 (4) 237 -243 2001年 [査読無し][通常論文]
     
    To develop new tests to predict bull subfertility, a mucus penetration test, in which penetration of cervical mucus by spermatozoa is quantitated, was applied using frozen-thawed semen and a test to examine the ability of spermatozoa to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187 (A23187 stimulation test) was attempted. Frozen-thawed spermatozoa from 4 Japanese black bulls (2 fertile and the other 2 subfertile) were analyzed by standard semen analysis such as sperm concentration, motility, viability and morphology, the mucus penetration test and the A23187 stimulation test, and the relationship of the test results to fertility status was investigated retrospectively. The results of the standard semen analysis and the mucus penetration test did not reveal any remarkable differences among the bulls, showing no relationship to fertility status. However, when spermatozoa were stimulated with 3 mM Ca2+ and 1 μM A23187, the bulls all showed a time-dependent induction of the acrosome reaction but the subfertile bulls showed a slower and lower induction than the fertile bulls. These results suggest that A23187 stimulation test may be useful when abnormal sperm characteristics cannot be found by standard semen analysis or the mucus penetration test.
  • Changes in sex steroids, gonadotropins, prolactin and inhibin in pregnant and nonpregnant black bears (Ursus thibetanus japonicus) (共著)
    Biol. Reprod 65, 1006‐1013 2001年 [査読無し][通常論文]
  • Michelo SYAKALIMA, Kennedy CHOONGO, Yoshikazu NAKAZATO, Misao ONUMA, Chihiro SUGIMOTO, Toshio TSUBOTA, Hideto FUKUSHI, Mitsutoshi YOSHIDA, Tadashi ITAGAKI, Jun YASUDA J. Vet. Med. Sci. 63 (3) 315 -318 2001年 [査読無し][通常論文]
  • Immunolocalization of steroidogenic enzymes in the corpus luteum and the placenta of the Japanese black bear ┣DBUrsus thibetanus japonicus(/)-┫DB, during pregnancy.(共著)
    Reproduction 121 587 -594 2001年 [査読無し][通常論文]
  • 坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (10) 9 -12 2000年06月01日 [査読無し][通常論文]
  • M Sato, T Tsubota, K Yamamoto, T Komatsu, Y Hashimoto, A Katayama, T Hazumi, Kita, I, T Kudo JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (4) 415 -420 2000年04月 [査読無し][通常論文]
     
    Progesterone(P-4) and estradiol-17 beta (E-2) 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 P-4 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 P-4 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 P-4 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 E-2 concentrations varied from April to October but showed low levels in November and December, and became high in January. Serum E-2 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 E-2 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 P-4 concentrations in eight bears led to expectations of pregnancy, whereas serum E-2 concentrations showed no distinct evidence related to pregnancy. These results, particularly in captive pregnant bears, indicate that a marked increase of P-4 in December might be accompanied by reactivation of the corpus luteum preceding implantation. Furthermore, changes in E-2 concentrations suggested the possibility that a decline in December and an increase in January are associated with implantation and parturition, respectively.
  • 糞分析からみたニホンツキノワグマ(┣DBUrsus(/)-┫DB ┣DBthibetanus(/)-┫DB ┣DBjaponicus(/)-┫DB)の食性-岐阜県本巣郡根尾村における事例-(共著)
    中森研 48 149 -152 2000年 [査読無し][通常論文]
  • T Urashima, W Sumiyoshi, T Nakamura, Arai, I, T Saito, T Komatsu, T Tsubota BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1472 (1-2) 290 -306 1999年10月 [査読無し][通常論文]
     
    Two trisaccharides, two tetrasaccharides, one penta-, one hexa-, two hepta-, one deca- and two undeca-saccharides were isolated from several Japanese black bear milk samples by chloroform/methanol extraction, gel filtration and preparative thin-layer chromatography. The oligosaccharides were characterized by H-1-NMR as follows: Gal(alpha 1-3)Gal(beta 1-4)Glc (alpha 3'-galactosyllactose), Fuc(alpha 1-2)Gal(beta 1-4)Glc (2'-fucosyllactose), Gal(alpha 1-3)(Fuc(alpha 1-2)]Gal(beta 1-4)Glc (B-tetrasaccharide), Gal(alpha 1-3)Gal(beta 1-4)(Fuc(alpha 1-3))Glc, Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (B-pentasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (monofucosylhexasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Gl (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3){Gal(alpha 1-3)Gal(beta 1-4)[Fuc(a 1-3)]GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (difucosyldecasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GIcNAc(beta 1-3){Gal-(alpha 1-3) Gal(beta 1-4)[Fuc(alpha 1-3)GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (trifucosylundecasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3){Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (trifucosylundecasaccharide). Lactose was present only in trace amounts. B-pentasaccharide was a dominant saccharide in early lactation milk, while alpha 3'-galactosyllactose was dominant in milk, later. The milk oligosaccharides of the Japanese black bear were compared with those of the Ezo brown bear. (C) 1999 Elsevier Science B.V. All rights reserved.
  • Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 45 (6) 425 -428 1999年 [査読無し][通常論文]
     
    This study was carried out to simplify the previous method for the mucus penetration test in Japanese beef cattle. Frozen-thawed semen was brought into contact with frozen-thawed estrous cervical mucus loaded in a rectangular capillary tube under the following conditions for placement of the tube and temperature of incubation: 1) horizontal/38 C (the previous method, Horizontal 38 C), 2) upright/38 C (Upright 38 C), or 3) upright/room temperature (Upright RT). The previous method included fixing the capillary tube with a mixture of vaseline and paraffin and a cover of semen with paraffin oil and a cover slip on a glass slide. This procedure was simplified by use of a small vial, in which semen was placed and covered with paraffin oil in Upright 38 C and Upright RT. Mucus penetration, expressed as the distance travelled by the sperm cell that penetrated the mucus the farthest in 10 min, was similar to Horizontal 38 C and Upright 38 C but was significantly lower with Upright RT. Intra-assay and inter-assay coefficients of variation in Upright 38 C were below 5%. Thus this study simplified the technique of the mucus penetration test using Upright 38 C.
  • T Tsubota, DL Garshelis, RA Nelson, JM Bahr JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (1) 81 -83 1999年01月 [査読無し][通常論文]
     
    Serum sex steroid and prolactin profiles were examined in the male American black bear, Ursus americanus during denning. Sera collected in December and the following March from 8 denning male black bears in Minnesota, U.S.A. were assayed for testosterone, estradiol-17 beta and prolactin. Eight bears were confirmed to be the denning mode based on a serum urea to creatinine ratio less than 10. Serum testosterone concentrations tended to increase from December to the subsequent March whereas serum estradiol-17 beta concentrations tended to decrease during this period. There were few changes in serum prolactin concentrations between December and March. These findings suggest that spermatogenesis and testicular steroidogenesis initiated during denning may be influenced by changes in serum sex steroid concentrations in the American black bear.
  • T Tsubota, L Howell-Skalla, WR Boone, DL Garshelis, JM Bahr ANIMAL REPRODUCTION SCIENCE 53 (1-4) 107 -118 1998年10月 [査読無し][通常論文]
     
    Identifying steroid and pituitary hormone profiles in the female black bear (Ursus americanus) throughout pregnancy may provide a greater understanding of the reproductive cycle and indicate which hormones are required for implantation. Our objective was to assess endocrine activity in black bears oestrus onset, at oestrus, during pregnancy and after parturition. Serum samples were obtained from 12 captive, 16 uncollared and five radiocollared free-ranging female black bears from March through the end of December and assayed for serum progesterone, oestradiol, luteinizing hormone (LH) and prolactin (PRL). In captive bears, progesterone concentrations were low at days 0-10 after oestrus and increased significantly days 25-35 and 45-52 after oestrus, Oestradiol concentrations were high at oestrus (day 0) and days 4-10 after oestrus and then decreased days 25-35 and 45-52 after oestrus. LH concentrations were not significantly different throughout the sampling period. Changes in PRL concentrations pattern were similar to those of oestradiol, with elevated levels at oestrus and days 4-10 after oestrus, followed by a significant decrease 45-52 days after oestrus. In non-collared free-ranging bears, progesterone concentrations increased gradually after mating with a further significant increase in November-December. Oestradiol concentrations were highest in March (before mating) and in June (during mating) followed by a significant decrease in July (early delay period) and November-December (peri-implantation period). LH concentrations were low until November-December and then increased significantly. PRL concentrations were low in March (before mating), increased significantly during the mating season in June, decreased slightly in July, and were low in November-December (peri-implantation period). In radiocollared free-ranging bears, serum progesterone concentrations were elevated in pregnant bears in December and extremely low in lactating and non-lactating bears in March. Oestradiol levels were slightly higher in pregnant bears in December than in non-lactating or lactating bears in March. PRL concentrations were considerably higher in lactating bears in March than in pregnant bears in December. Our results suggest that: (1) serum progesterone concentrations are low, but detectable during the early delay implantation period and greatly elevated during the peri-implantation period; (2) serum oestradiol concentrations are elevated at oestrus and decline during the delay period; (3) LB may be involved in luteal activation; and (4) the decline of serum PRL concentrations during short days may be necessary for implantation to occur. (C) 1998 Elsevier Science B.V. All rights reserved.
  • T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki ANATOMIA HISTOLOGIA EMBRYOLOGIA-JOURNAL OF VETERINARY MEDICINE SERIES C-ZENTRALBLATT FUR VETERINARMEDIZIN REIHE C 27 (3) 209 -213 1998年06月 [査読無し][通常論文]
     
    The seasonal changes of the cytoskeletal protein expressions were immunohistochemically investigated in the testes of Japanese black bear, Ursus thibetanus japonicus. A strong immunoreaction for alpha-smooth muscle actin is restricted to the vascular smooth muscle cells and the peritubular cells which surround the seminiferous tubules by several layers throughout the year. Weak immunoreactions for B4 antigen and desmin were observed in the vascular smooth muscle cells and in a part of peritubular cells throughout the year. A strong immunoreaction for vimentin was also detected in the fibroblasts and Leydig cells, in addition to the vascular smooth muscle and epithelial cells and the peritubular cells throughout the year. A strong alpha-tubulin immunoreaction was detected in the elongating spermatids during the acrosome phase of spermiogenesis in May and June. The cytoplasm of several Sertoli cells was faintly immunoreacted for vimentin in the basal and lateral region, while an intense alpha-tubulin reaction was seen in the entire cytoplasm in May, April and June. In November, January and March, the immunoreactions for vimentin and alpha-tubulin strongly accumulate in a perinuclear region of Sertoli cells when developmental spermatids are not seen in the seminiferous tubules. These accumulations in the immunoreactions for vimentin and alpha-tubulin seem to be caused by the reduction in size of Sertoli cells cytoplasm with season. However, the seasonal changes of distributions in the cytoskeletal proteins are obscure in the bear testes. These results suggest that the contents of cytoskeletal proteins may not change in relation to the morphological differences with season in the testes of the seasonal breeders.
  • Y Atoji, Y Yamamoto, T Komatsu, Y Suzuki, T Tsubota ANATOMICAL RECORD 250 (3) 251 -267 1998年03月 [査読無し][通常論文]
     
    Background: The circumanal glands of the dog are thought to be a glandular tissue, but there is some controversy as to whether they should be classified as exocrine or endocrine. In this study, we examined the nature of the circumanal glands to determine whether they should be described as exocrine, endocrine, or something else altogether. In addition, we investigated the cell degeneration in lobules of the circumanal glands in relation to the apocrine glands. Methods: Light microscopic observations were made of paraffin sections stained with hematoxylin and eosin, and after immunohistochemical staining with antibodies against a-smooth muscle actin, keratin, filaggrin, and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD). Samples were also examined by electron microscopy after fixation by aldehyde perfusion. Results: The lobules of circumanal glands could be divided into two types on the basis of the presence or absence of cysts. Four layers (I-IV) were detected in the lobules with cysts. The outermost layer (layer I or the basal layer) consisted of flattened cells that contained bundles of tonofilaments and were stained immunohistochemically with the antibody against keratin. Layer II (the polyhedral or "spinous" layer) consisted of polyhedral cells that contained bundles of tonofilaments. These cells were connected to adjacent cells by desmosomes, interdigitations, and gap junctions, and they were immunopositive for keratin. A small number of polyhedral cells were immunopositive for 3 beta-HSD. Layer III (the granular layer) was composed of flattened cells that contained hematoxylin-stainable granules and were moderately immunopositive for filaggrin. The innermost layer (layer IV or the horny layer) consisted of keratin. Lobules without cysts consisted only of layer I (the basal layer) and layer II (the polyhedral layer). Lobules of the circumanal glands were not directly connected to apocrine glands. Polyhedral cells degenerated and were phagocytosed by basal cells at a periphery of lobules. Then, basal cells phagocytosing degenerated polyhedral cells escaped from lobules, moved into the walls of apocrine glands, and, finally, dropped into the lumen of apocrine glands. Conclusions: Lobules of the circumanal glands have many characteristics of epidermis (a basal layer, a polyhedral or "spinous layer," a granular layer, and a horny layer) and they should not be classified as glandular tissue. The cysts in lobules can be interpreted as "closed hair canals." We suggest that steroid metabolism might occur in the polyhedral cells of the lobules. (C) 1998 Wiley-Liss, Inc.
  • 坪田 敏男, 溝口 紀泰, 喜多 功 Japanese journal of zoo and wildlife medicine 3 (1) 17 -24 1998年03月 [査読無し][通常論文]
     
    ニホンツキノワグマUrsus thibetanus japonicusは, 本州, 四国および九州に生息する大型哺乳動物の一種である。しかしながら, 最近では, 九州はほぼ絶滅状態となり, さらに四国山地, 西中国地域, 東中国山地および紀伊半島が絶滅のおそれのある地域となっている。1990年から1994年にかけて岐阜県白川村において直接観察, 痕跡調査(糞分析)およびラジオトラッキングといった生態調査が行われた。その結果, ツキノワグマの春と秋の食物種がブナ林という生息環境と密接に関係していることが示された。すなわち, ツキノワグマは, ブナ豊作年にはブナの花芽や種子を食べ, 一方ブナ不作年には他の食物を利用していた。また, 1992年から1993年のツキノワグマの行動圏が求められ, その平均値は雄で6.4km^2, 雌で3.4km^2であった。主に飼育下でのツキノワグマの繁殖生理学的研究により, 雄では季節繁殖性が顕著に認められること, また雌では着床遅延や冬眠中の出産といったクマ類特有の繁殖生理機構を有していることが解明された。これらの結果より, 将来にわたってツキノワグマを保護していくためには, 繁殖の成功につながる十分な食物環境を確保することが肝要であると結論づけることができる。
  • ニホンツキノワグマUrsus thibetanus japonicusの生態と生理に関する野生動物医学的研究(共著)
    3 17 -24 1998年 [査読無し][通常論文]
  • 哺乳類の生物学
    寸胴 25 1 -2 1998年 [査読無し][通常論文]
  • ニホンツキノワグマの繁殖メカニズム
    第2回東中国クマ集会報告書 17 -29 1998年 [査読無し][通常論文]
  • ツキノワグマの生態と保護管理
    はくさん 25 (3) 7 1998年 [査読無し][通常論文]
  • クマの繁殖
    畜産の研究 52 (1) 205 -210 1998年 [査読無し][通常論文]
  • 飼育条件下におけるニホンツキノワグマ(┣DBUrsus(/)-┫DB ┣DBthibetanus(/)-┫DB ┣DBjaponicus(/)-┫DB)の性行動の観察(共著)
    44 j13-j18 1998年 [査読無し][通常論文]
  • ニホンツキノワグマ┣DBUrsus(/)-┫DB ┣DBthibetanus(/)-┫DB ┣DBjaponicus(/)-┫DBの生態と生理に関する野生動物医学的研究(共著)
    3 17 -24 1998年 [査読無し][通常論文]
  • WR Boone, JC Catlin, KJ Casey, ET Boone, PS Dye, RJ Schuett, JO Rosenberg, T Tsubota, JM Bahr URSUS, VOL 10 - 1998 10 503 -505 1998年 [査読無し][通常論文]
     
    We visually verified corpora lutea and measured serum progesterone concentrations during the estrous cycle in 8 semi-captive black bears (Ursus americanus) in South Dakota. Our data suggested that black bears ovulate after they mate, indicating that black bears are induced ovulators. These preliminary findings may aid scientists in their efforts to save endangered bear species through controlled breeding programs.
  • 坪田 敏男 畜産の研究 52 (1) 205 -210 1998年01月 [査読無し][通常論文]
  • T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki ARCHIVES OF HISTOLOGY AND CYTOLOGY 60 (3) 225 -234 1997年08月 [査読無し][通常論文]
     
    The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year, Lipid inclusions of both electron-dense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity.
  • T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki ARCHIVES OF HISTOLOGY AND CYTOLOGY 60 (3) 225 -234 1997年08月 [査読無し][通常論文]
     
    The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year, Lipid inclusions of both electron-dense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity.
  • T Komatsu, T Tsubota, Y Yamamoto, Y Atoji, Y Suzuki JOURNAL OF VETERINARY MEDICAL SCIENCE 59 (7) 521 -529 1997年07月 [査読無し][通常論文]
     
    Seasonal changes in sites of immunostaining of steroidogenic enzymes were examined in testes of the Japanese black bear, Ursus thibetanus japonicus. In addition, serum concentrations of testosterone and estradiol-17 beta were investigated by radioimmunoassay, and the seasonal changes were compared with the results of immunostaining. On the basis of morphological observations of spermatogenic activity, the reproductive cycle was divided into five periods: an active period in May and June; a degenerative period in November; a resting period in January; an early-resumptive period in March; and a late-resumptive period in April. Serum concentrations of testosterone differed with season accompanied by differences in spermatogenic activity, with baseline levels in November and January, increasing levels in March and April, and high levels in May, and April and June of the next year. Immunoreactivities specific for cholesterol side-chain cleaving cytochrome P450, 17 alpha-hydroxylase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were observed in Leydig cells throughout the year. Only the percentages of Leydig cells immunopositive for 3 beta HSD exhibited seasonal differences that correlated with serum concentrations of testosterone. Aromatase cytochrome P450 (P450arom) was immunolocalized in Leydig and Sertoli cells throughout the year, in spermatids in May, and April and June of the next year and in myoid cells in January and March. The percentages of Leydig cells immunopositive for this enzyme increased in May, and January, March and June of the next year. On the other hand, no pattern of seasonal change in serum estradiol-17 beta concentration was observed. These results suggest that 3 beta HSD is a key enzyme in the regulation of the testosterone production in Leydig cells. Furthermore, estrogen derived from Leydig and myoid cells seems to play a role in the regulation of Leydig cells by negative feedback as a paracrine and/or autocrine mediator.
  • 坪田 敏男 獣医畜産新報 50 (5) 413 -416 1997年05月 [査読無し][通常論文]
  • 厳寒の地に生きるホッキョクグマ
    ニュートン 17 (8) 30 -37 1997年 [査読無し][通常論文]
  • 雄の繁殖学
    獣医畜産新報 50 (5) 413 -416 1997年 [査読無し][通常論文]
  • ブナの豊凶がツキノワグマの食性に与える影響-ブナとミズナラの種子落下量の年次変動に関連して-(共著)
    哺乳類科学 36 33 -44 1997年 [査読無し][通常論文]
  • T Tsubota, L HowellSkalla, H Nitta, Y Osawa, JI Mason, PG Meiers, RA Nelson, JM Bahr JOURNAL OF REPRODUCTION AND FERTILITY 109 (1) 21 -27 1997年01月 [査読無し][通常論文]
     
    American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March; 15 lune, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggest that aromatase and oestrogen may play a role in re-initiating spermatogenesis.
  • T Tsubota, S Taki, K Nakayama, JL Mason, S Kominami, N Harada, Kita, I BIOLOGY OF REPRODUCTION 56 30 -30 1997年 [査読無し][通常論文]
  • H Araki, T Tsubota, N Maeda, N Harada, S Kominami, JJ Mason, Kita, I JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (8) 787 -790 1996年08月 [査読無し][通常論文]
     
    Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.
  • 喜多 功, 鈴木 法子, 丹羽 範郎, 坪田 敏男 Japanese journal of zoo and wildlife medicine 1 (2) 113 -117 1996年08月 [査読無し][通常論文]
     
    成熟雌ニホンカモシカCapricornis crispus30頭の子宮壁動脈を組織学的に検索した結果, 経産カモシカの子宮壁動脈に妊娠性硬変が見出された。すなわち, 未経産成雌の子宮壁動脈では, 内弾性板は薄く, その屈曲は単純であったが, 1〜3産例の子宮壁動脈では, 内弾性板は厚く明瞭で, 複雑な屈曲を示していた。5産以上の例では, 子宮壁動脈に2層以上の弾性板様構造物が出現し, 中膜における弾性線維の増加と平滑筋細胞の核の変性が観察された。8産以上になると子宮壁動脈の中膜に弾性線維の変性(エラストージス)および平滑筋細胞の核の変性・消失が起こった。以上のことから, 子宮壁の組織学的検査によって, 産歴不明のカモシカ個体における経産の判定および経産回数の推定が可能と思われた。
  • 荒城 寛, 坪田 敏男, 前田 菜穂子, 原田 信広, 小南 思郎, Mason J.I, 喜多 功 The journal of veterinary medical science 58 (8) 787 -790 1996年08月 [査読無し][通常論文]
     
    交尾期にあるエゾヒグマの卵巣を用いて4種のステロイド合成酵素の局在を免疫組織化学的に調べた. この卵巣は, 大型卵胞を有していたことより, おそらく卵胞期のものと考えられた. 大型卵胞においては, P450 sccおよび3βHSDは内卵胞膜細胞と顆粒層細胞で認められた. P450 c17は内卵胞膜細胞で認められたが, 顆粒層細胞では認められなかった. また, P450 aromは顆粒層細胞で認められた. しかし, 中型卵胞においては, P450 sccおよび3βHSDは内卵胞膜細胞にのみ認められ, P450 aromは内卵胞膜細胞と顆粒層細胞のいずれにも認められなかった. 間質細胞では, P450 aromを除く全ての酵素が認められた. 以上の結果は, エゾヒグマの卵胞におけるエストロジェンの産生機序は卵胞膜細胞と顆粒層細胞の両者の関連によるものであり, いわゆる2細胞説に従うこと, 大型卵胞の顆粒層細胞はプレグネノロンおよびプロジェステロンの産生能力を有すること, さらに間質がステロイド合成能力を有することを示唆している.
  • T Komatsu, Y Yamamoto, T Tsubota, Y Atoji, Y Suzuki JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (4) 329 -335 1996年04月 [査読無し][通常論文]
     
    Spermatogenic cycle in the testis of the Japanese black bear (Selenarctos thibetanus japonicus) was studied by light and transmission electron microscopy. By light microscopy, spermatids were allocated into eleven steps based on morphological changes in the nucleus and the acrosome of spermatids. Cellular associations of the seminiferous epithelium were allocated into eight stages based on the changes in the nucleus and acrosome of spermatids, appearance of meiotic figures and time of spermiation. Cross-sections of the seminiferous tubule seldom contained more than one type of stage. Spermatids at steps 1-2 had the well-developed Golgi complex. The crescent-shaped Golgi complex was accompanied by the acrosome extending over the nucleus at steps 3-5. At step 6, spermatids faced the base, and the outer membrane of the acrosome converged upon the plasma membrane of spermatids. The acrosome projected into the cytoplasm of Sertoli cells at step 9. At step 11, most of the cytoplasm was phagocytosed by Sertoli cells, and spermatids were released in the lumen to become spermatozoa.
  • 小松 武志, 山本 欣郎, 坪田 敏男, 阿閉 泰郎, 鈴木 義孝 The journal of veterinary medical science 58 (4) 329 -335 1996年04月 [査読無し][通常論文]
     
    野生個体1頭と飼育個体3頭から採取した精巣組織材料を用いて, ニホンツキノワグマ(Selenarctos thibetanus japonicus)における精子形成サイクルについて, 光学顕微鏡および電子顕微鏡による観察を行った. 光顕による観察から, 精子細胞の核およびアクロソームの形態学的変化に基づいて, 精子細胞を11ステップに分類した. さらに, この精子細胞の形態学的変化, 減数分裂像および精子細胞の管腔への遊離時期を指標にして, 精上皮サイクルを8ステージに区分した. 一つの精細管横断面は, 大抵単一のステージによって占められていた. ステップ1-2の精子細胞は, よく発達したゴルジ装置を持ち, ステップ3-5では核膜表面を被うアクロソームに沿って, 三日月状に観察された. ステップ6の精子細胞は先端を基底膜方向に向け, 精子細胞の細胞膜とアクロソーム外膜とが接着した. ステップ9においては, アクロソームがセルトリ細胞の細胞質ヘ突出している像が観察され, ステップ11になると, 精子細胞の細胞質のほとんどがセルトリ細胞に取り込まれ, 精子細胞そのものは精子として管腔へ遊離した.
  • 片山 敦司, 坪田 敏男, 山田 文雄, 喜多 功, 千葉 敏郎 Japanese journal of zoo and wildlife medicine 1 (1) 26 -32 1996年02月 [査読無し][通常論文]
     
    1991年3月から1993年8月までの間に, 岐阜県および京都府で捕殺された雌ニホンツキノワグマ(Selenarctos thibetanus japonicus)19頭の生殖器の肉眼的および組織学的観察により, 性成熟年齢, 排卵数, 着床数, 一腹産子数および繁殖歴などを推定した。卵巣の重量および大きさは加齢に伴って増加の傾向を示した。その傾向は未成熟個体で顕著であり, 性成熟個体で緩やかであった。黄体および黄体退縮物の存在を性成熟の基準とした場合, 4歳以上の全ての個体は性成熟に達していると判定された。しかし, 4歳未満でも性成熟に達する例も存在することが示唆され, 性成熟に達する年齢には個体差があることがうかがわれた。黄体, 黄体退縮物および胎盤痕の観察と連れ子の数から平均排卵数は1.89, 平均着床数は2.00, および平均連れ子頭数は, 1.86と算定された。さらに, 黄体退縮物の組織学的観察により, 捕殺時点における過去の総排卵数の推定を試みた。その結果, 黄体および黄体退縮物の数と交尾期経過回数には正の相関が認められた。しかし, 黄体およびその退縮物の数にはばらつきがあり, 交尾期経過回数との間に大きな差が認められる例もあった。
  • ニホンカモシカ┣DBCapricornis(/)-┫DB ┣DBcrispus(/)-┫DB 子宮壁動脈の妊娠性硬変-経産歴との関連において(共著)
    1 113 -117 1996年 [査読無し][通常論文]
  • ニホンツキノワグマ(Selenarctos thibetanus japonicus)の繁殖指標としての卵巣と子宮の形態学的研究(共著)
    1 26 -32 1996年 [査読無し][通常論文]
  • T Tsubota, H Araki, JI Matson, S Kominami, N Harada, Kita, I BIOLOGY OF REPRODUCTION 54 262 -262 1996年 [査読無し][通常論文]
  • Kita, I, M Eguchi, S Takatsuki, T Koizumi, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 57 (6) 1029 -1033 1995年12月 [査読無し][通常論文]
     
    Corpora lutea of the sika deer (Cervus nippon, Cervidae) were histologically examined in order to elucidate their retrograding processes after parturition. They showed a remarkable regression within one month as luteal cells decreased in number and, by nine months after parturition, turned into masses of blood vessels containing many arterioles with a diameter of 30 mu m or less. In retrograde corpora lutea (RCL), older than 1.5 years after parturition, arterioles of these dimensions decreased markedly, while those measuring 50 mu m or more became dominant. These findings indicate that an examination of RCL can distinguish females less than nine months after parturition from those more than 1.5 years after parturition. Based on the findings of RCL from the ovaries of a 10.5-year-old female, it is suggested that RCL remain in the ovaries for at least 8.5 years after parturition. It was, however, difficult to distinguish RCL of pregnancy from retrograde accessory corpora lutea (RACL). In order to arrive at a complete count of RCL, the ovaries should be examined at thicknesses of 0.5 mm. To check the presence/absence of RCL of pregnancy within nine months or less after parturition, sectioning at thicknesses of 2 mm is sufficient.
  • 喜多 功, 江口 雅子, 高槻 成紀, 小泉 透, 坪田 敏男 The journal of veterinary medical science 57 (6) 1029 -1033 1995年12月 [査読無し][通常論文]
     
    分娩後におけるニホンジカ黄体の退縮過程について組織学的研究を行った. 黄体は分娩後1か月以内に黄体細胞の減少と共に急激にその大きさを減じ, 分娩後9か月迄に直径30μm以下の小動脈を多く含む血管の集積に変った. 分娩後1.5年以上の退縮物では直径30μm以下の小動脈が著しく減少して, 直径50μm以上の小動脈が多数を占めた. 従って, 黄体退縮物の検査によって分娩後9か月未満の個体と1.5年以上の個体とを区別することが可能である. 10.5歳の個体における退縮物の所見に基づき, 退縮物は分娩後少なくとも8.5年は卵巣中に残存すると推定された. 妊娠黄体退縮物と副黄体退縮物とを組織学的に区別することは困難であった. すべての黄体退縮物を洩れなく数えるためには, 卵巣を0.5mm間隔で観察する必要があるが, 分娩後9か月以内の黄体退縮物の有無を調べるだけならば, 卵巣を2mm間隔で観察すればよい.
  • T TSUBOTA, RA NELSON, JD THULIN, L HOWELL, JM BAHR JOURNAL OF REPRODUCTION AND FERTILITY 104 (2) 187 -191 1995年07月 [査読無し][通常論文]
     
    Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig I-125-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4-14.8 ng ml(-1); t = 15-30 min, 18.4-28.7 ng ml(-1)). The sensitivity of the assay was 0.08 ng per tube. Intra- and interassay coefficients of variation were 5.5% (n = 6) and 5.7% (n = 6), respectively. Serum concentrations of prolactin changed seasonally, with the lowest concentrations in December (mean +/- SD = 1.1 +/- 0.1 ng ml(-1)); this was followed by a gradual increase between January (2.6 +/- 0.6 ng ml(-1)) and April (6.4 +/- 1.2 ng ml(-1)) and the highest concentrations in May (17.6 +/- 4.7 ng ml(-1)), preceding peak testosterone concentrations in June. The observation that prolactin secretion increased with increasing daylength suggests that photoperiod may be an external regulator. The presence of high concentrations of prolactin before peak testosterone concentrations suggests that prolactin may play a role in regulating seasonal changes in the testes.
  • T TSUBOTA, H NITTA, Y OSAWA, JI MASON, KITA, I, T TIBA, JM BAHR JOURNAL OF REPRODUCTION AND FERTILITY 101 (3) 557 -561 1994年08月 [査読無し][通常論文]
     
    The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.
  • Toshio TSUBOTA, Naoko MAEDA, Hiroshi KANAGAWA J. Mamm. Soc. Japan 19 (2) 75 -82 1994年 [査読無し][通常論文]
  • T TSUBOTA, WR BOONE, DL GARSHELIS, RA NELSON, JM BAHR BIOLOGY OF REPRODUCTION 50 82 -82 1994年 [査読無し][通常論文]
  • T TSUBOTA, H NITTA, Y OSAWA, JI MASON, KITA, I, T TIBA, JM BAHR GENERAL AND COMPARATIVE ENDOCRINOLOGY 92 (3) 439 -444 1993年12月 [査読無し][通常論文]
  • T TIBA, Y SHIMIZU, KITA, I, T TSUBOTA ANATOMIA HISTOLOGIA EMBRYOLOGIA-JOURNAL OF VETERINARY MEDICINE SERIES C-ZENTRALBLATT FUR VETERINARMEDIZIN REIHE C 22 (3) 254 -263 1993年09月 [査読無し][通常論文]
     
    It was investigated in the domestic fowl, whether a mechanism for a synchronous stan of the proliferation of the differentiating spermatogonia in a transversely cut seminiferous tubule (called the ''co-ordination' between germ cell association) operates effectively in this species. A trapezoid-shaped cellular association demarcated by two perpendiculars from the tubular lumen to the basement membrane was the unit of observation (provisionally called a ''column'' Each column is composed of a bundle of elongate spermatids and the neighbouring, various types of germ cells). The extent of closeness of the co-ordination was examined in a total of 2,650 columns, based on the continuity of the spermiogenic sptep-number of the youngest generation of spermatids within adjacent columns. In no case did all columns in a tubular cross section show the same-step spermatids. In most cases (50.2 % of 2,650 columns), the same number was kept in only 2 to 6 adjacent columns. The arrangement of different but consecutive numbers (e. g., 1-2-3 or 1-2-1) was observed in a total of 882 columns. The co-ordination mechanism, therefore, is considered to work, albeit incomplete, among a limited number of adjacent columns in this species.
  • T TIBA, K YOSHIDA, M MIYAKE, K TSUCHIYA, KITA, I, T TSUBOTA ANATOMIA HISTOLOGIA EMBRYOLOGIA-JOURNAL OF VETERINARY MEDICINE SERIES C-ZENTRALBLATT FUR VETERINARMEDIZIN REIHE C 22 (3) 241 -253 1993年09月 [査読無し][通常論文]
     
    A cellular association demarcated by two perpendiculars which were drawn between adjacent bundles of elongate spermatids from the tubular lumen to the basement membrane, was made the unit of histometrical observation in this study (provisionally called a ''column''). Cell counting revealed that the average numbers per column of various types of germ cells do not show any significant differences among 5 fowls and between paired testes. The frequency of spermiogenic steps (numbered 1-8) was investigated in each column. A definite and common pattern was found in the frequency distribution in the 5 fowls observed. A relationship between spermiation and younger spermatid steps was also investigated in each column. The spermiation was found at different steps, but most frequently at step 2 (30.6 %). Based on these observations and referring to other author's information, an average time interval between two successive spermiations was calculated roughly at 3.3 +/- 1.2 days. Theoretically, this value is equal to an average length of one epithelial cycle. Such a variable cycle may have caused irregular cellular associations in this species.
  • T TSUBOTA, H KANAGAWA, K YAMAMOTO, T MANO, M YAMANAKA, KITA, I, T TIBA JOURNAL OF VETERINARY MEDICAL SCIENCE 54 (1) 1 -5 1992年02月 [査読無し][通常論文]
     
    Serum progesterone (P) concentrations using P-EIA kit (Ovucheck, Cambridge Life Science Co., Ltd.) were examined in 8 captive and 7 free-ranging female Hokkaido brown bears (Ursus arctos yesoensis). The intra- and inter-assay coefficients of variation were 8.9%, 12.6% and 16.6%, 22.7%, respectively, based on 2 serum samples. There was a significant correlation between EIA and radioimmunoassay results based on 64 serum samples (r = 0.725; p < 0.01). Serum P concentrations were examined in 5 pregnant, 2 solitary non-pregnant bears and a lactating non-pregnant bear in captivity. Annual changes of P levels in pregnant bears were observed as a small elevation during the mating season (May-June), a re-elevation in September-October and a sharp elevation in November-December. The sharp elevation was suspected to reflect changes when implantation occurred. Annual changes of P levels in solitary non-pregnant bears were similar to those in pregnant bears. An annual change of P levels in a lactating non-pregnant bear maintained levels under 5 ng/ml. Two of 7 free-ranging bears exhibited P levels over 1 ng/ml and the birth of cubs was confirmed in the following year in 1 of the 2 bears. P concentrations of other free-ranging bears exhibited less than 1 ng/ml, and these bears were considered to be non-pregnant. It was concluded that P-EIA kit was available for measuring P concentrations in Hokkaido brown bears.
  • 坪田 敏男, 金川 弘司, 山本 聖子, 間野 勉, 山中 正実, 喜多 功, 千葉 敏郎 日本獣医学雑誌 54 (1) 1 -5 1992年02月 [査読無し][通常論文]
     
    飼育下8頭および野生7頭の雌エゾヒグマについて, プロジェステロン(P)測定用エンザイムイムノアッセイ(EIA)キット(「オブチェック」ケンブリッジ・ライフ・サイエンス社)を用いて血清中P値を測定し, その有効性を検討した. 本キットによる2検体の測定内および測定間変動係数は, それぞれ8.9%, 12.6%および16.6%, 22.7%と比較的良好な成績であった. ラジオイムノアッセイ法との相関関係については, 64サンプルで相関係数r=0.725と高い相関が認められた(p<0.01). 飼育エゾヒグマでは, 妊娠個体5頭, 非妊娠単独個体2頭および非妊娠子連れ個体1頭についてP値が調べられた. 妊娠個体のP値は, 交尾期(5〜6月)後の小さな上昇, 9〜10月にかけての2回目の上昇, さらに11〜12月にかけての大きな上昇として観察された. この最後の大きなP値上昇は, 着床に伴う変化と推測される. 非妊娠単独個体のP値変化は, 妊娠個体のP値変化と類似した. 非妊娠子連れ個体のP値は, 6〜12月まで5 ng/ml以下の値を持続した. 野生エゾヒグマ7頭中2頭は, 1 ng/ml以上の値を示し, そのうちの1頭では出産が確認された. 他の5頭はいずれも1 ng/ml以下の低値であり, 非妊娠個体と考えられた. エゾヒグマでのP-EIAキットによるP値の測定は有効であると...
  • 坪田 敏男 生物科学 43 (4) p190 -194 1991年11月 [査読無し][通常論文]
  • 坪田 敏男 遺伝 45 (7) p77 -81 1991年07月 [査読無し][通常論文]
  • T TSUBOTA, Y TAKAHASHI, H KANAGAWA, K GOHDA JOURNAL OF VETERINARY MEDICAL SCIENCE 53 (1) 141 -142 1991年02月 [査読無し][通常論文]
  • クマ類の繁殖特性
    生物科学 43 (4) 1 -5 1991年 [査読無し][通常論文]
  • Reproductive characteristics of bears
    43 (4) 1 -5 1991年 [査読無し][通常論文]
  • 坪田 敏男 家畜繁殖学雑誌 36 (5) p1 -10 1990年12月 [査読無し][通常論文]
  • 家畜繁殖誌 36,1-10 1990年 [査読無し][通常論文]
  • Studies on reproductive physiology of Hokkaido brown bear, Ursus arctos yesoensis
    Jpn. J. Anim. Reprod 36,1-10 1990年 [査読無し][通常論文]
  • Tsubota Toshio, Kanagawa Hiroshi Journal of the Mammalogical Society of Japan 14 (1) 11 -17 1989年07月 [査読無し][通常論文]
  • 坪田 敏男, 金川 弘司 家畜繁殖学雑誌 32 (4) 184 -187 1986年12月 [査読無し][通常論文]
  • 坪田 敏男 家畜繁殖学雑誌 31 (4) 203 -210 1985年12月 [査読無し][通常論文]
  • 坪田 敏男 動物と自然 15 (12) p20 -24 1985年11月 [査読無し][通常論文]
  • TSUBOTA Toshio Japanese journal of veterinary research 33 (1) 1985年04月30日 [査読無し][通常論文]

受賞

  • 2003年 日本獣医学会賞
  • 1990年 家畜繁殖学会賞(島村賞)

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2009年 -2011年 
    代表者 : 坪田 敏男, 下鶴 倫人
     
    本研究では、なぜクマは体脂肪率30~40%の肥満状態でも脂肪肝や高脂肪血症を発症しないのか、その特徴的な体脂肪蓄積メカニズムを明らかにすることを目的にして次のような実験を行った。実験は、秋田県北秋田市マタギの里阿仁クマ牧場において、5~11月にかけて月1回行った。1回の実験に4頭のツキノワグマを用いた。塩酸チレタミンと塩酸ゾラゼパムの混合薬(Zoletil 100、Virbac、フランス)9mg/kgにより不動化を行い、麻酔状態下で以下のサンプリングおよび実験を行った。なお、使用したツキノワグマは前日の夕方5:00以降は絶食状態とした。1)基底レベルの血中グルコース、インスリン、中性脂肪、コレステロールおよび遊離脂肪酸濃度を測定した。血中グルコースおよびインスリン濃度には月別の変化は見られなかったが、血中中性脂肪、コレステロールおよび遊離脂肪酸濃度は冬眠前に減少する傾向にあった。2)グルコース投与後0~180分までおよそ30分間隔で血中グルコースおよびインスリン濃度を測定した。その結果、グルコース投与後血中グルコース濃度は一過性に上昇した後、徐々に減少し180分後にはおよそ基底値まで減少した。減少のスピードに月別変化はみられなかった。一方、血中インスリン濃度はグルコース投与直後から上昇し、およそ30分でピークに達した。この時のインスリン上昇度は11月で最も著しい傾向を示した(...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2005年 -2007年 
    代表者 : 源 宣之, 福士 秀人, 福士 秀人, 杉本 千尋, 柵木 利昭, 坪田 敏男, 安田 準, 伊藤 直人, 淺野 玄
     
    近年,SARSやトリインフルエンザなどの新たな感染症が次々と発生し,社会をパニックに陥れている.これらの病原体の自然界における本来の住処は不明なことが多いが,各種野生動物の関与が示唆されている.したがって,環境破壊によるそれらの動物の生息分布域の変化が新たな感染症の出現の主な原因と考えられている.そこで,本課題では,各種動物が多数棲息し,毎年新たな感染症が突然出現しているアフリカの野生動物における種々の人獣共通病原体の保有状況を調べ,それと環境変化との因果関係を推定ることにより,新たな感染症の出現動向を予知すると共にその予防に役立てることを目的とした.また,これまでに東南アジアで行ってきた狂犬病に関する海外調査をアフリカ地域に拡大し,狂犬病ウイルスの世界規模の感染動態を検討した.1年次である2005年はザンビア共和国およびエジプトを対象国とし,検体収集ならびに現地協力機関との連携確認ならびに今後の協力体制を構築した.2年次である2006年はザンビア共和国,マラウイ共和国およびエジプトを対象国とし,前年に引き続き検体収集を行った.また,現地協力者の研究支援を行った.最終年度となった2007年はザンビア共和国およびエジプトに加え,タンザニア共和国を訪問し,検体収集ならびに現地協力者の研究支援および今後の研究連携の確認を行った.一連の研究により,アフリカにおける狂犬病ウイルスの生態...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2003年 -2006年 
    代表者 : 坪田 敏男, 村瀬 哲磨
     
    本研究では、希少野生動物から非侵襲的に得られる糞の中に含まれる性ステロイドホルモンの分子生物学的微量測定により各種希少野生動物の生殖周期をモニタリングすること、および希少野生動物の個体数回復を目指して飼育条件下において人工繁殖の臨床応用を実践することを目的とした。本研究期間の4年間に、希少野生動物であるツシマヤマネコとツキノワグマにおいて以下のような実験を行った。1.ツシマヤマネコおよびツキノワグマにおける糞中性ステロイドホルモン濃度の測定1)ツシマヤマネコのモデル動物としてイエネコにおいてTR-FIAキットを用いて糞中エストラジオールおよびプロジェステロン濃度測定法を確立した。2)ツシマヤマネコにおいてTR-FIA法での糞中性ステロイドホルモン濃度測定により生殖周期のモニタリングを行った。3)ツキノワグマの糞中性ステロイドホルモン濃度微量測定については、ER Cell linesを探索し、予備的に測定を行うに留まった。2.ツキノワグマにおける凍結精液を用いた人工授精に関する研究1)飼育下雄グマより麻酔下で電気刺激射精法により精液を採取した。2)精子の運動性、精子数および奇形率などの検査を行なった後、精液を凍結保存した。3)凍結時の希釈液および平衡時間の検討を行い、より精液性状の高い凍結保存法を確立した。4)雌グマの排卵様式を知るために、交尾期の血中プロジェステロン濃度測定を...
  • 野生動物の救護に関する研究
    研究期間 : 2003年
  • Wildlife rescue
    研究期間 : 2003年
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2001年 -2002年 
    代表者 : 福井 豊, 佐々木 基樹, 手塚 雅文, 浦島 匡, 坪田 敏男, 中村 正
     
    事業報告:福井,坪田,浦島,手塚,佐々木はオスロ大学動物学博物館,ノルウェー極地研,スバールバル大学およびトロムソ大学を訪問し、アザラシの生息状況、体内汚染、クジラ,クマの繁殖生理、哺乳類の乳腺進化についての研究交流を行った。佐々木はオスロ大学においてホッキョクギツネ,ホッキョクグマの形態観察を行い、手塚はノルウェー近海域におけるホッキョクグマ,アザラシの体内におけるPCB汚染の実態調査を行った。スミソニアン動物学研究所のDr.O.oftedalを招聘し、"The origin and early evolution of lactation"と題する講演会を開催した。また、立教大学阿部教授による「環境教育の最前線」と題する講演会を開催した。「野生動物の生命と環境」に関するワークショッブを開催し、福井,坪田,浦島,手塚,佐々木,中村,阿部および日本鯨類研究所の石川が各々の研究成果を報告した。成果報告:福井は北大西洋のヒゲクジラ類の繁殖生理の解明を目的として、索餌期におけるミンククジラおよびニタリクジラの血清中性ホルモン濃度と精細管内組織像の関連性を検討した。坪田は野生動物の人工繁殖への応用を目的として、ビーグル犬ならびに飼育ツキノワグマにおける精液採集、凍結保存、糞中性ステロイドホルモンの測定を行った。佐々木はホッキョクギツネの咀嚼筋の形態および構成を肉眼解剖学的に検索し、さ...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    研究期間 : 2001年 -2001年 
    代表者 : 坪田 敏男, 村瀬 哲磨
     
    これまでに内分泌撹乱化学物質(EDs)が猛禽類に何らかの影響を与えているとする報告が数多くみられる。PCB類はハクトウワシにおいて生殖低下と関連付けられ、DDEはハヤブサやチョウゲンボウにおいて卵殻薄化との関連が示唆されている。わが国でも猛禽類の個体数減少及び繁殖状況の悪化が報じられている。その主たる要因は生息環境の悪化にあると思われるが、EDsも何らかの影響を与えていると推測されている。そこで本研究では、猛禽類において影響が大きいと考えられるダイオキシン類、PCB類およびDDT類の蓄積濃度とその影響を調べた。また、EDsの繁殖学的影響を明らかにするにあたり、猛禽類の繁殖生理解明の端緒としてトビの精巣の形態学的観察を行った。その結果、ダイオキシン類とP, P'-DDEに関して、何らかの影響が及んでいる可能性を否定できない蓄積が多くの猛禽類に認められた。繁殖期成鳥の検体が少なく、配偶子形成に関して十分な情報を得られなかったが、性腺に組織学的異常は認められず、これらの物質が発生学的影響を与えている可能性は低いと思われた。ダイオキシン類の蓄積傾向は種や生息地による食性の違いを反映していることが示唆され、今後食性を把握することにより猛禽類が地域の汚染状況のモニター生物として有用になり得ることが考えられた。しかし、影響や蓄積傾向を明らかにするには、食性や薬物代謝能、対象物質に対する感受...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 1999年 -2001年 
    代表者 : 安田 準, 杉本 千尋, 小沼 操, 坪田 敏男, 稲垣 匡, 福士 秀人, 吉田 光敏
     
    ウシのヘルペスウイルス1(BHV-1)は23.2%で抗原陽性、48.2%で抗体陽性であったことから、ザンビアでのBHV-1感染は既に広く拡散しているごとが示唆された。ザンビアにおける狂犬病発生率は3%〜28.8%で、イヌが75%ウシが13%を占めた。狂犬病ウィルス遺伝子解析からイヌとヌーの塩基配列は93〜99%の相同性があり、ヌーはイヌから感染を受けたと考えられた。ウシとレチュエに寄生する肝蛭は外部形態から野生動物由来のF.giganticaと同定した。遺伝子塩基配列はレチュエはC型のみ、ウシはA、B、C型であり、ウシのC型はレチュエからウシに伝播したと考えられた。レチュエでウシと同様なMycobacterium bovisによる泌尿生殖器結核感染症が確認され、尿が感染源となることが示唆された。DNA解析ではレチュエ株とウシ株は相同性があり、他の野生動物への感染の可能性を指摘した。レチュエは血液塗抹、PCRともタイレリア原虫は検出できなかった。ウシは血液塗抹で確認できなかったが、PCRでは7%でrRNA遺伝子が検出され、4種類のタイレリア種が同定された。アフリカ大陸に普遍的に存在し、バッファローでの感染も知られており、ウシとバッファローとの感染環が成立していると考えられた。シマウマで分離されたRhodococcus equiは病原性マーカータンパク質やプラスミドは全例保有せず...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1999年 -2000年 
    代表者 : 坪田 敏男, 村瀬 哲磨, 喜多 功
     
    1.野生動物における生殖異常の検出野外で死亡したタヌキ、クマ、ハクビシン、ネズミ、猛禽類の死体を収集し臓器材料を得た。また、飼育環境下にあったオットセイ、ラッコ、アシカ、イルカについても死体より臓器材料を採取した。これらの動物の肝臓および脂肪に含まれる内分泌かく乱化学物質(PCB、HCH、DDTおよびその異性体、有機スズ、各種農薬)の蓄積濃度を測定した(環境庁調査および基盤研究(A)による)。生殖器の組織標本を作製し病理学的検査を行った。これまでにタヌキ1例に精巣腫瘍が認められている。2.生殖異常をひき起こすメカニズムの解明性腺内でのステロイド合成を調節するステロイド合成酵素の発現量を調べる目的で、代表的なステロイド合成酵素である3βHSD、P450c17およびP450aromのmRNA断片の塩基配列をイヌおよびネコで決定した。今後これらをプローブとして使ってNorthern blottingやin situ hybridizationを行い、各ステロイド合成酵素の発現量および発現部位を調べる。その上で内分泌かく乱化学物質がこのステロイド合成にどのような影響を与えているか検討する。卵子の発育能および胎子の性分化への影響については引き続き実験中である。
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 1998年 -2000年 
    代表者 : 柵木 利昭, 坪田 敏男, 工藤 忠明, 源 宣之, 福士 秀人, 鈴木 義孝
     
    本研究によって得られた主な野生動物の病態と生態に関する調査結果は以下の通りである。1)再興感染症であるニホンカモシカから羊へのパラポックス感染症の伝搬の可能性を明らかにする目的で、カモシカ由来のパラポックスウイルスを用いて羊への接種実験を試みた。その結果、ニホンカモシカのパラポックス感染症が羊へ伝搬する可能性が示された。2)トムソンガゼルの集団斃死例から分離されたガゼルヘルペスウィルス1型は、新興感染症のウマヘルペスウイルスであることが判り、EHV-9として提唱した。EHV-9はこれまで検索したウマ、ヤギ、ウシ、ブタ、イヌ、ハムスター、マウス、モルモットのすべてに脳炎を引き起こすことが明らかとなった。この脳炎はウマ、ブタ及びウシを除き総て致死的であった。EHV-9は強い神経向性を有するウイルスであり広範囲の動物に強い毒力を持っていることが明らかになった。3)中部地区を中心とした各種野生動物における有機塩素系薬剤、PCBs,DDTs,HCHなどの残留濃度を測定したところ、陸生動物に比べて鳥類中でも猛禽類が各薬剤を高濃度保有していることが明らかになった。また、その生体への影響についても実験的に調査した。4)ツキノワグマおよびキタオットセイの繁殖に関わる生態学的および生理学的知見が得られた。また、希少野生動物の人工繁殖に向けてツキノワグマをモデルとした精液の凍結保存に関する技術開発...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1996年 -1996年 
    代表者 : 坪田 敏男
     
    本研究の目的は、各種雄動物における精巣でのステロイド産生能を検討することであった。そこで、イヌとツキノワグマの精巣を研究材料として、免疫組織化学および内分泌学的にステロイド産生能を検討した。方法は、イヌおよびツキノワグマの精巣を外科手術的に採取し、10%ホルマリン液で固定した。ツキノワグマについては各季節に採材を行なった。固定された材料は、常法にしたがってパラフィン切片とした。免疫染色は、キットを用いてABC法で行なった。使用した抗体は、4種類のステロイド合成酵素、P450scc、3βHSD、P450c17およびP450aromに対する抗血清であった。さらに、ツキノワグマについては血液を採取して、血液中のステロイド濃度をラジオイムノアッセイによって測定した。その結果、イヌでは、ライディッヒ細胞がアンドロジェン産生能を有し、また、精母細胞および精子細胞がエストロジェン産生能を有していた。ツキノワグマでは、交尾期にはやはりライディッヒ細胞がアンドロジェン産生能を有していたが、他の季節(非交尾期)でもP450sccおよびP450c17の染色性は変化しなかった。一方3βHSDについては、交尾期に比べて非交尾期に陽性細胞が顕著に減少した。また、交尾期には精子細胞がエストロジェン産生能を有していた。血中テストロン濃度は交尾期に高値を示し、非交尾期に底値を示した。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1995年 -1995年 
    代表者 : 坪田 敏男
     
    本研究の目的は、各種雌動物における卵巣および胎盤でのステロイド産生能を検討することであった。そこで、ネコとツキノワグマの卵巣および胎盤を研究材料として免疫組織化学および内分泌学的にステロイド産生能の検討を行った。方法は、妊娠中のネコおよび妊娠していたと思われるツキノワグマの卵巣および胎盤を採集し、10%ホリマリン液で固定した。ネコについては生体より外科手術的に採取した。固定された材料は常法にしたがってパラフィン切片とした。免疫染色は、キットを用いてABC法で行った。使用した抗体は、4種類のステロイド合成酵素、P450scc、3βHSD、P450c17およびP450aromに対する抗血清であった。さらに、ツキノワグマについては妊娠雌より血液を採集して、血清中のステロイド濃度をラジオイムノアッセイによって測定した。その結果ネコでは、1)妊娠期のプロジェステロン産生母地は黄体から胎盤に移るが、妊娠後半期にも黄体はプレグネノロンの産生能を有する 2)胎盤の栄養膜合胞体層はエストロジェン(妊娠期間全体)およびプロジェステロン(妊娠後半期)の産生能を有し、加えて妊娠末期には脱落膜細胞がエストロジェンおよびプレグネノロンの産生能を有することが推察された。ツキノワグマでは、1)着床遅延初期には黄体が主要なプロジェステロン産生母地であるが、少量ながらエストロジェンも産生されている 2)黄体での...
  • 哺乳動物の季節繁殖性に関する研究
  • 内分泌かく乱化学物資による野生動物への影響
  • 野生動物の繁殖生理に関する研究
  • Seasonality on reproducction of mammals
  • Affects of endocrine disruptours on wildlife
  • Reproductive physiology of bears

教育活動情報

主要な担当授業

  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
  • 獣医科学特別研究
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 動物福祉学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 動物福祉、伴侶動物、産業動物、実験動物、野生動物、展示動物
  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 野生動物学演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 保全生態学、野生動物医学、保全医学、生態系、保護管理
  • 獣医科学特論演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 動物行動学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 獣医科学基礎科目A 野生動物/保全医学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 環境と人間
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 脊椎動物、進化、生態、生理、生物多様性
  • 獣医科学特別研究
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 野生動物学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 野生動物、野生動物医学、保全医学、保全生態学、環境、生態系、生物多様性、保護管理
  • 獣医科学特論演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 動物園学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 動物園、水族館、展示動物、生息域外保全、環境教育、環境エンリッチメント、動物福祉
  • 獣医科学基礎科目 環境獣医科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 国際交流Ⅱ
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 国際本部
    キーワード : wildlife, veterinary medicine, conservation medicine, Hokkaido
  • 先端獣医科学科目 野生動物/保全医学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 保全生態・野生動物医学演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 研究・臨床セミナー
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • アドバンスト演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部

大学運営

学内役職歴

  • 2017年4月1日 - 2019年3月31日 教育研究評議会評議員

委員歴

  • 1998年 - 2000年   ヒトと動物の関係学会   評議員   ヒトと動物の関係学会
  • 1998年 - 2000年   日本野生動物医学会   事務局長   日本野生動物医学会
  • 日本哺乳類学会   編集委員   日本哺乳類学会
  • 日本繁殖生物学会   編集委員   日本繁殖生物学会
  • 日本獣医学会   評議員   日本獣医学会


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