Researcher Database

Toshio Tsubota
Faculty of Veterinary Medicine Veterinary Medicine Environmental Veterinary Sciences
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Veterinary Medicine Veterinary Medicine Environmental Veterinary Sciences

Job Title

  • Professor

Degree

  • (BLANK)(Hokkaido University)

Contact information

  • tsubotavetmed.hokudai.ac.jp

J-Global ID

Research Interests

  • 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   

Research Areas

  • Life sciences / Veterinary medicine / Wildlife biology and medicine
  • Natural sciences / Biogeoscience / Wildlife
  • Environmental science/Agricultural science / Biological resource conservation / Bears

Academic & Professional Experience

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

Education

  •        - 1988  Hokkaido University
  •        - 1988  Hokkaido University  Graduate School, Division of Veterinary Medicine
  •        - 1983  Hokkaido University  School of Veterinary Medicine
  •        - 1983  Hokkaido University  Faculty of Veterinary Medicine

Association Memberships

  • 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   

Research Activities

Published Papers

  • 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 [Refereed][Not invited]
  • 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 [Refereed][Not invited]
  • Aya Osaki, Mariko Sashika, Go Abe, Kohei Shinjo, Ayako Fujimoto, Mariko Nakai, Michito Shimozuru, Toshio Tsubota
    BMC ecology 19 (1) 35 - 35 2019/09/11 [Refereed][Not invited]
     
    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 0916-7250 2019/09 [Refereed][Not invited]
  • 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 0022-2372 2019/07 [Refereed][Not invited]
     
    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 0022-1503 2019/05 [Refereed][Not invited]
  • 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 1080-6040 2019/05 [Refereed][Not invited]
  • Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota
    PloS one 14 (4) e0215489  2019 [Refereed][Not invited]
     
    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 JE, Moustafa MAM, Sashika M, Tsubota T
    PloS one in press (12) e0207662  2018/12 [Refereed][Not invited]
  • 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  1612-4642 2018/12 [Refereed][Not invited]
  • Moriwaki Jun, Omori Ryosuke, Shimozuru Michito, Tsuruga Hifumi, Mano Tsutomu, Tsubota Toshio
    JAPANESE JOURNAL OF VETERINARY RESEARCH 66 (2) 71 - 81 0047-1917 2018/05 [Refereed][Not invited]
  • Lundu Tapiwa, Yoshii Kentaro, Kobayashi Shintaro, Morikawa Shigeru, Tsubota Toshio, Misawa Naoaki, Hayasaka Daisuke, Kariwa Hiroaki
    JAPANESE JOURNAL OF VETERINARY RESEARCH 66 (1) 21 - 28 0047-1917 2018/02 [Refereed][Not invited]
  • Mohamed Abdallah Mohamed Moustafa, Michito Shimozuru, Wessam Mohamed, Kyle Rueben Taylor, Ryo Nakao, Mariko Sashika, Toshio Tsubota
    PARASITOLOGY RESEARCH 116 (8) 2321 - 2325 0932-0113 2017/08 [Refereed][Not invited]
     
    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) 1932-6203 2017/04 [Refereed][Not invited]
     
    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 1877-959X 2017 [Refereed][Not invited]
     
    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 0916-7250 2016/11 [Refereed][Not invited]
     
    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 0916-7250 2016/07 [Refereed][Not invited]
     
    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 1096-4959 2016/06 [Refereed][Not invited]
     
    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 0090-3558 2016/04 [Refereed][Not invited]
     
    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 1080-6040 2016/03 [Refereed][Not invited]
  • Jun Moriwaki, Michito Shimozuru, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota
    URSUS 27 (2) 99 - 109 1537-6176 2016 [Refereed][Not invited]
     
    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 1877-959X 2016 [Refereed][Not invited]
     
    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 1567-1348 2015/12 [Refereed][Not invited]
     
    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.
  • Altered signaling pathway governing protein metabolism in skeletal muscle of the Japanese black bear during hibernation
    Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota
    FASEB JOURNAL 29 0892-6638 2015/04 [Refereed][Not invited]
  • 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 0916-8818 2015/02 [Refereed][Not invited]
     
    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 0730-7268 2014/09 [Refereed][Not invited]
     
    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 0916-7250 2014/05 [Refereed][Not invited]
     
    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 1472-9792 2014/05 [Refereed][Not invited]
     
    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 0916-8818 2014/04 [Refereed][Not invited]
     
    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 2014/03 [Not refereed][Not invited]
  • 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 0916-8818 2014/02 [Refereed][Not invited]
     
    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 1877-959X 2014 [Refereed][Not invited]
     
    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 0916-8818 2013/08 [Refereed][Not invited]
     
    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.
  • Moustafa Mohamed, Abdallah Mohamed, Ismail Mohamed Nour-Eldin, Mohamed Adel El Sayed Ahmed, Ali Alsagher Omran, Tsubota Toshio
    JOURNAL OF WILDLIFE DISEASES 49 (3) 750 - 754 0090-3558 2013/07 [Refereed][Not invited]
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (6) 785 - 790 0916-7250 2013/06 [Refereed][Not invited]
     
    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 0022-2372 2013/06 [Refereed][Not invited]
     
    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 0916-7250 2013/06 [Refereed][Not invited]
     
    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 0916-8818 2013/04 [Refereed][Not invited]
     
    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 0916-7250 2013/02 [Refereed][Not invited]
     
    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 1530-3667 2013/02 [Refereed][Not invited]
     
    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 0916-7250 2013/02 [Refereed][Not invited]
     
    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 0032-8332 2013/01 [Refereed][Not invited]
     
    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.
  • TERAGUSHI Tomoko, AMAKI Ryota, YOKOHATA Yasushi, ITANI Miki, TSUBOTA Toshio, SUDO Akiko, YATA Shinpei
    Japanese Journal of Zoo and Wildlife Medicine 18 33 - 39 2013 [Refereed][Not invited]
  • Sasaki M, Amano Y, Hayakawa D, Tsubota T, Ishikawa H, Mogoe T, Ohsumi S, Tetsuka M, Miyamoto A, Fukui Y, Budipitojo T, Kitamura N
    The Journal of reproduction and development 0916-8818 2012/12 [Refereed][Not invited]
  • Takanori Kooriyama, Hideo Hasegawa, Michito Shimozuru, Toshio Tsubota, Toshisada Nishida, Takashi Iwaki
    PRIMATES 53 (4) 365 - 375 0032-8332 2012/10 [Refereed][Not invited]
     
    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 1096-4959 2012/10 [Refereed][Not invited]
     
    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 0002-9645 2012/08 [Refereed][Not invited]
     
    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 0008-4301 2012/08 [Refereed][Not invited]
     
    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 0002-9645 2012/08 [Refereed][Not invited]
     
    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 0916-7250 2012/06 [Refereed][Not invited]
     
    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 1344-3941 2012 [Refereed][Not invited]
     
    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 1877-959X 2012 [Refereed][Not invited]
     
    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 0095-1137 2011/05 [Refereed][Not invited]
     
    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 0090-3558 2011/04 [Refereed][Not invited]
     
    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 0289-0003 2011/02 [Refereed][Not invited]
     
    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  0447-0192 2011/01/01 [Not refereed][Not invited]
  • Hepatozoon sp. Infection in Hokkaido brown bears (Ursus arctos yesoensis)
    Kubo, M, Chiba, E, Iibuchi, R, Tsubota, T, Sasaki M, Sakai, H, Yanai, T
    Jpn.J.Zoo Wildl.Med. 15 (2) 111 - 113 2010/12 [Refereed][Not invited]
  • Ruriko Iibuchi, Michito Shimozuru, Akari Kamine, Junko Nio-Kobayashi, Toshihiko Iwanaga, Toshio Tsubota
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 (2) 236 - 242 0916-8818 2010/04 [Refereed][Not invited]
     
    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 0289-0003 2010/03 [Refereed][Not invited]
     
    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 0916-8818 2010/02 [Refereed][Not invited]
     
    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.
  • I. Adachi, S. Kusuda, E. Nagao, Y. Taira, M. Asano, T. Tsubota, O. Doi
    Theriogenology 74 1499 - 1503 0093-691X 2010 [Not refereed][Not invited]
     
    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
  • Masahito Kubo, Tsukasa Okano, Keiko Ito, Toshio Tsubota, Hiroki Sakai, Tokuma Yanai
    PARASITOLOGY RESEARCH 106 (1) 213 - 219 0932-0113 2009/12 [Refereed][Not invited]
     
    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 0916-8818 2009/04 [Refereed][Not invited]
     
    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 0916-8818 2009/04 [Refereed][Not invited]
     
    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 0916-8818 2009/04 [Refereed][Not invited]
     
    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 0916-7250 2008/12 [Refereed][Not invited]
     
    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 0047-1917 2008/11 [Refereed][Not invited]
     
    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 0008-4301 2008/09 [Not refereed][Not invited]
     
    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 0013-936X 2008/02 [Refereed][Not invited]
     
    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.
  • Expressions of steroidogenic enzymes mRNA in the testes of Japanese black bears (Ursus thibetanus japonicus) as revealed by in situ hybridization
    Ruriko Iibuchi, Junko Nio-Kobayashi, Sachiko Nakamura, Makoto Asano, Toshihiko Iwanaga, Toshio Tsubota
    BIOLOGY OF REPRODUCTION 297 - 297 0006-3363 2008 [Refereed][Not invited]
  • Masahito Kubo, Keiya Kobayashi, Toshiaki Masegi, Hiroki Sakai, Toshio Tsubota, Makoto Asano, Miki Itani, Tokuma Yanai
    JOURNAL OF WILDLIFE DISEASES 43 (3) 542 - 544 0090-3558 2007/07 [Refereed][Not invited]
     
    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 0916-7250 2007/04 [Refereed][Not invited]
     
    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 [Not refereed][Not invited]
  • Tatsuya Kunisue, Nozonu Takayanagi, Toshio Tsubota, Shinsuke Tanabe
    CHEMOSPHERE 66 (2) 203 - 211 0045-6535 2007/01 [Refereed][Not invited]
     
    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 0916-7250 2006/10 [Refereed][Not invited]
     
    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 0916-7250 2006/10 [Refereed][Not invited]
     
    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%.
  • Qiang Weng, Tetsuma Murase, Makoto Asano, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (9) 999 - 1002 0916-7250 2006/09 [Refereed][Not invited]
     
    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.
  • NAKAMURA SACHIKO, OKANO TSUKASA, SHIBATA HARUKI, SAITO MASAYUKI, KOMATSU TAKESHI, ASANO GEN, TSUBOTA TOSHIO
    J Reprod Dev 52 (Supplement) J115  0916-8818 2006/08 [Refereed][Not invited]
  • Qiang Weng, Mohamed S. Medan, Meiyu Xu, Toshio Tsubota, Gen Watanabe, Kazuyoshi Taya
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 52 (4) 503 - 510 0916-8818 2006/08 [Refereed][Not invited]
     
    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 140 525 - 535 0269-7491 2006/04/01 [Not refereed][Not invited]
     
    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 P5CB118 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, P5, H6CDD/DFs and for P5CB126, H6CB169 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 1355-008X 2006/04 [Refereed][Not invited]
     
    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.
  • 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 [Refereed][Not invited]
  • OHDACHI Satoshi, AOI Toshiki, MANO Tsutomu, TSUBOTA Toshio
    Journal of the Mammalogical Society of Japan 一般社団法人 日本哺乳類学会 17 (1) 27 - 47 0914-1855 1992 [Refereed][Not invited]
     
    The growth, sexual dimorphism, and geographical variation in skulls of 596 brown bears (<i>Ursus arctos</i>) 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.<br>
  • CORPORA-ALBICANTIA AND PLACENTAL SCARS IN THE HOKKAIDO BROWN BEAR
    T TSUBOTA, H KANAGAWA, T MANO, T AOI
    BEARS - THEIR BIOLOGY AND MANAGEMENT 8 125 - 128 1990 [Refereed][Not invited]
  • Okano, T, Murase, T, Yayota, C, Komatsu, T, Miyazawa, K, Asano, M, Tsubota, T
    Anim.Reprod. Sci. 95 134 - 143 0378-4320 [Refereed][Not invited]
     
    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 s

Books etc

Works

  • ツキノワグマ生息実態調査報告書(共著)、岐阜県根尾村
    2000
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • クマ,畜産の研究50(I):145-150
    1996
  • 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
  • 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
  • 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
  • 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
  • アメリカでの研究生活を終えて、月輪:75-78
    1994
  • グリズリーの楽園-カトマイ国立公園-、Field Note40:3-6
    1993
  • ミネソタ州のクロクマ調査、月輪5:78-81
    1993
  • 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
  • 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
  • Denali National Parkでのクマの研究と保護管理、Field Note36:13-16
    1992
  • エゾヒグマの繁殖生理、遺伝45(7):77-81
    1991
  • 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
  • 日本産クマ類の研究概観、高教研会誌、20:96-99
    1991
  • 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
  • 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
  • 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
  • 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

MISC

  • 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  2016/08/30  [Not refereed][Not invited]
  • 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  2016/02  [Not refereed][Not invited]
  • 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  2014/08/11  [Not refereed][Not invited]
  • 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  2013/08/30  [Not refereed][Not invited]
  • 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  2013/06/15  [Not refereed][Not invited]
  • 【人と動物の共通感染症最前線10】 日本のげっ歯類におけるハンタウイルス感染の血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの分離
    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  2013/04  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 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  2012/12/15  [Not refereed][Not invited]
  • 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  2012/08/31  [Not refereed][Not invited]
  • TAKAMI Kazutoshi, WATANABE Yukiko, TSUBOTA Toshio, FUKUI Daisuke, ONUMA Manabu, YAMAMOTO Mai, MURATA Koichi  Jpn J Zoo Wildl Med  17-  (2)  33  -42  2012/06/01  [Not refereed][Not invited]
  • JONO MANAMI, KOBAYASHI YUMI, ASANUMA TAKETOSHI, ASANUMA TAKETOSHI, TSUBOTA TOSHIO, SAKURAI YASUNORI  北海道大学水産科学研究い報  62-  (1)  1  -7  2012/03/28  [Not refereed][Not invited]
  • Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota  JAPANESE JOURNAL OF VETERINARY RESEARCH  60-  (1)  5  -13  2012/02  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • Nakashita Rumiko, Suzuki Yaeko, Akamatsu Fumikazu, Naito Yuichi I, Sato-Hashimoto Miho, Tsubota Toshio  Researches in organic geochemistry  27-  (0)  73  -79  2011/09/30  [Not refereed][Not invited]
     
    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  2011/08/31  [Not refereed][Not invited]
  • 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  2011/08/10  [Not refereed][Not invited]
  • 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  2011/08/10  [Not refereed][Not invited]
  • 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  2011/08/10  [Not refereed][Not invited]
  • 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  2011/07  [Not refereed][Not invited]
  • 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  2011/03/08  [Not refereed][Not invited]
  • 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  2011/02  [Not refereed][Not invited]
     
    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.
  • Kawabata Hiroki, Oikawa Yozaburo, Kawamori Fumihiko, Kumagai Kunihiko, Mikami Toshiyuki, Hanaoka Nozomu, Ando Shuji, Honda Naoko, Taylor Kyle, Tsubota Toshio, Konnai Satoru, Takano Ai, Watanabe Haruo, Ohnishi Makoto, Nakao Minoru, Masuzawa Toshiyuki, Takada Nobuhiro, Yano Yasuhiro, Ishiguro Fubito, Fujita Hiromi, Itoh Takuya  JSMEZ Annual Meetings Abstracts  63-  (0)  69  -69  2011  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • 坪田 敏男, 植木 玲一, 坪田 あゆみ  Bears Japan  11-  (1)  10  -13  2010/07  [Not refereed][Not invited]
  • 加味根 あかり, 下鶴 倫人, 坪田 敏男  獣医畜産新報  63-  (5)  367  -370  2010/05  [Not refereed][Not invited]
  • Ruriko Iibuchi, Akari Kamine, Michito Shimozuru, Junko Nio-Kobayashi, Gen Watanabe, Kazuyoshi Taya, Toshio Tsubota  JAPANESE JOURNAL OF VETERINARY RESEARCH  57-  (4)  185  -196  2010/02  [Not refereed][Not invited]
     
    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  2010/02  [Not refereed][Not invited]
     
    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  2009/11  [Not refereed][Not invited]
     
    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  2009/10/01  [Not refereed][Not invited]
  • Ruriko Iibuchi, Noriko Nakano, Tadashi Nakamura, Tadasu Urashima, Michito Shimozuru, Tetsuma Murase, Toshio Tsubota  JAPANESE JOURNAL OF VETERINARY RESEARCH  57-  (1)  13  -22  2009/05  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • 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  2008/12  [Not refereed][Not invited]
     
    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  2008/11  [Not refereed][Not invited]
     
    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.
  • 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  2008/09  [Not refereed][Not invited]
     
    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.
  • NAKAMURA Sachiko, OKANO Tsukasa, YOSHIDA Yutaka, MATSUMOTO Ayumi, MURASE Yutaka, KATO Haruki, KOMATSU Takeshi, ASANO Makoto, SUZUKI Masatsugu, SUGIYAMA Makoto, TSUBOTA Toshio  Japanese Journal of Zoo and Wildlife Medicine  13-  (1)  15  -20  2008/03  [Not refereed][Not invited]
     
    Body fat mass(FM) of the Japanese black bear (Ursus thibetanus japonicus) was examined by bioelectrical impedance analysis(BIA) using Model Quantum X (RJL systems). Whole-body resistance was measured between forelimbs and hind limbs in the recumbent position. The FM was calculated by the equations for the American black bear. In 5 captive Japanese black bears, from September 2005 to January 2006, changes in FM and body mass(BM) were highly correlated (r=0.89). This study demonstrates that BIA is useful in the measurement of FM in the Japanese black bear, and that an increase in BM in autumn...
  • 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  [Not refereed][Not invited]
  • 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  2006/12/01  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Q Weng, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya  EXPERIMENTAL ANIMALS  54-  (5)  451  -454  2005/10  [Not refereed][Not invited]
     
    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  2005/06  [Not refereed][Not invited]
     
    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  2005/06  [Not refereed][Not invited]
     
    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.
  • Q Weng, MS Medan, G Watanabe, T Tsubota, Y Tanioka, K Taya  JOURNAL OF REPRODUCTION AND DEVELOPMENT  51-  (3)  299  -304  2005/06  [Not refereed][Not invited]
     
    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, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya  JOURNAL OF REPRODUCTION AND DEVELOPMENT  51-  (2)  247  -252  2005/04  [Not refereed][Not invited]
     
    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  2005/04  [Not refereed][Not invited]
     
    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  2005/02  [Not refereed][Not invited]
  • Rapid, easy and reliable sexing method for falconiformes by PCR
    Jpn. J. Zoo Wildl. Med.  10: 79-84-  2005  [Not refereed][Not invited]
  • Osa Yuichi, Akamatsu Rika, Takada Masayuki, Ohashi Kazuhiko, Okazaki Katsunori, Kaneko Masami, Endoh Daiji, Asakawa Mitsuhiko, Tsubota Toshio, Asano Makoto  Abstracts of the Annual Meeting of the Ecological Society of Japan  52-  (0)  432  -432  2005  [Not refereed][Not invited]
     
    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  2004/12  [Not refereed][Not invited]
     
    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  2004/12  [Not refereed][Not invited]
     
    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  2004/11  [Not refereed][Not invited]
     
    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  2004/11  [Not refereed][Not invited]
     
    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  2004/11  [Not refereed][Not invited]
     
    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.
  • WENG Qiang, MURASE Tetsuma, ASANO Makoto, TSUBOTA Toshio  Japanese Journal of Zoo and Wildlife Medicine  9-  (2)  65  -70  2004/09  [Not refereed][Not invited]
     
    Five pairs of ovaries were obtained from wild adult raccoon dogs (Nyctereutes procynoides) in November 2001, March and April 2002, and February and March 2003. Two of the 5 ovaries (March and April) contained corpora lutea. The purpose of the present study was to investigate immunolocalization of P450arom and its mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) in wild raccoon dog ovaries. Immunoreactive aromatase was detected in granulosa cells, internal theca cells and lutein cells. The P450arom mRNA was expressed in the ovaries of all wild raccoon dogs. We also...
  • WR Boone, BB Keck, JC Catlin, KJ Casey, ET Boone, PS Dye, RJ Schuett, T Tsubota, JC Bahr  THERIOGENOLOGY  61-  (6)  1163  -1169  2004/04  [Not refereed][Not invited]
     
    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.
  • ITO Hideyuki, SUDO YAMAJI Akiko, ABE Motoko, MURASE Tetsuma, TSUBOTA Toshio  Japanese Journal of Zoo and Wildlife Medicine  9-  (1)  39  -43  2004/03  [Not refereed][Not invited]
     
    Some species of raptors, such as the golden eagle (Aquila chrysaetos) and the mountain hawk-eagle (Spizaetus nipalensis), were recently reduced in population by habitat destruction. Conservation of habitats and genetic diversity for raptors is crucial. However, there is little genetic information on raptors. In this study, 8 microsatellite markers isolated from the peregrine falcon (Falco peregrinus) and gyrfalcon (Falco msticolus) were applied to 6 other raptors. The number of loci detected polymorphisms in the black kite, goshawk, kestrel, marsh harrier, golden eagle and mountain hawk-eag...
  • Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season
    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  2004/02  [Not refereed][Not invited]
     
    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  2004/02  [Not refereed][Not invited]
  • 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  2004/02  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • The prevalence of bovine herpevirus-1 in traditional cattle in Southern Province, Zambia
    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  2003/12  [Not refereed][Not invited]
     
    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  2003/10  [Not refereed][Not invited]
     
    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  2003/10  [Not refereed][Not invited]
     
    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  2003/10  [Not refereed][Not invited]
     
    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.
  • HASEGAWA Jun, MATSUDA Muneaki, KAWANO Masahide, SUDO Akiko, TSUBOTA Toshio, HIRAOKA Takashi, WAKIMOTO Tadaaki  Journal of environmental chemistry  13-  (3)  765  -779  2003/09/24  [Not refereed][Not invited]
  • 村田 浩一, 坪田 敏男  獣医畜産新報  56-  (4)  281  -283  2003/04  [Not refereed][Not invited]
  • H Ito, A Sudo-Yamaji, M Abe, T Murase, T Tsubota  ZOOLOGICAL SCIENCE  20-  (3)  339  -344  2003/03  [Not refereed][Not invited]
     
    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  2003/03  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Hideyuki Ito, Akiko Sudo-Yamaji, Motoko Abe, Tetsuma Murase, Toshio Tsubota  Zool. Sci  20-  (3)  339  -344  2003  [Not refereed][Not invited]
  • YAMAMOTO Kaori, KAWAMURA Atsuki, TSUBOTA Toshio, TSURUGA Hifumi, KOMATSU Takeshi, MURASE Tetsuma, KITA Isao, KUDO Tadaaki  Japanese Journal of Zoo and Wildlife Medicine  7-  (2)  103  -108  2002/09  [Not refereed][Not invited]
     
    We examined the availability of DNA fingerprinting in paternity determination of the captive Japanese black bear, Ursus thibetanus japonicus. The DNA fingerprint using the restriction enzyme Hinf I and (GATA)_4 probe was available for individual identification and paternity determination. Eleven females and their 13 cubs born from 1995 to 1997 along with 22 males were tested in the paternity determination. Seven male bears were determined as putative fathers of the 13 cubs. Two males in particular were the putative fathers of 8 cubs. Our study suggests that, under captive conditions, some m...
  • K Yamamoto, T Tsubota, T Komatsu, A Katayama, T Murase, Kita, I, T Kudo  JOURNAL OF VETERINARY MEDICAL SCIENCE  64-  (6)  505  -508  2002/06  [Not refereed][Not invited]
     
    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  2002/06  [Not refereed][Not invited]
     
    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  2002/06/01  [Not refereed][Not invited]
  • TSUBOTA Toshio, TAKI Shizuko, SUDO Akiko, MURASE Tetsuma, NODA Ayako, MASEGI Toshiaki, MINAMOTO Nobuyuki  Japanese Journal of Zoo and Wildlife Medicine  7-  (1)  69  -74  2002/03  [Not refereed][Not invited]
     
    It is being clarified that endocrine disruptors which were produced by human being accumulated without decomposition in the environment for a long time and affected reproduction of human and wild animals. DDT as a organochlorine pesticide, PCBs as industrial products, dioxins as non-intensive products and DES as a synthetic estrogen are categorized into the endocrine disruptor. Population decline of alligators in Lake Apopka in U.S.A., decrease of reproductive rate in mink and river otters, thinning of eggshell or decrease of hatching rate in raptors, large-number death in dolphin and seals...
  • 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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats (共著)
    Bull. Environ. Contam. Toxicol.  67, 438‐445-  2001  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats (共著)
    Bull. Environ. Contam. Toxicol.  67, 438‐445-  2001  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 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  2000/06/01  [Not refereed][Not invited]
  • 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  2000/04  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • 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  1999/10  [Not refereed][Not invited]
     
    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.
  • T Tsubota, DL Garshelis, RA Nelson, JM Bahr  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (1)  81  -83  1999/01  [Not refereed][Not invited]
     
    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  1998/10  [Not refereed][Not invited]
     
    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.
  • Immunohistochemical demonstration of cytoskeletal proteins in the testis of the Japanese black bear, Ursus thibetanus japonicus
    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  1998/06  [Not refereed][Not invited]
     
    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  1998/03  [Not refereed][Not invited]
     
    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.
  • TSUBOTA Toshio, MIZOGUCHI Noriyasu, KITA Isao  Japanese Journal of Zoo and Wildlife Medicine  3-  (1)  17  -24  1998/03  [Not refereed][Not invited]
     
    The Japanese black bear Ursus thibetanus japonicus, a large terrestrial mammal inhabits Honshu, Shikoku and Kyushu islands of Japan. However, Kyushu populations face extinction while populations on Shikoku island, in regions of Chugoku, and on the Kii peninsula are endangered. Ecological surveys such as direct observation, fecal analysis after field-sign detection and radio-tracking were carried out on Japanese black bears in Shirakawa Village, Gifu Prefecture in 1990-1994. Food habits of Japanese black bears in the spring and autumn related to the beech forest habitat of Shirakawa Village....
  • 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  [Not refereed][Not invited]
  • 哺乳類の生物学
    寸胴  25-  1  -2  1998  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • ツキノワグマの生態と保護管理
    はくさん  25-  (3)  7  1998  [Not refereed][Not invited]
  • クマの繁殖
    畜産の研究  52-  (1)  205  -210  1998  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Bears as induced ovulators - A preliminary study
    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  [Not refereed][Not invited]
     
    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  1998/01  [Not refereed][Not invited]
  • T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki  ARCHIVES OF HISTOLOGY AND CYTOLOGY  60-  (3)  225  -234  1997/08  [Not refereed][Not invited]
     
    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  1997/08  [Not refereed][Not invited]
     
    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  1997/07  [Not refereed][Not invited]
     
    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  1997/05  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 雄の繁殖学
    獣医畜産新報  50-  (5)  413  -416  1997  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus
    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  1997/01  [Not refereed][Not invited]
     
    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.
  • H Araki, T Tsubota, N Maeda, N Harada, S Kominami, JJ Mason, Kita, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (8)  787  -790  1996/08  [Not refereed][Not invited]
     
    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.
  • KITA Isao, SUZUKI Noriko, NIWA Norio, TSUBOTA Toshio  Japanese Journal of Zoo and Wildlife Medicine  1-  (2)  113  -117  1996/08  [Not refereed][Not invited]
     
    Histological studies on the artery of the uterine wall were performed in 30 adult female Japanese serows, Capricornis crispus. Gravid sclerosis was found in the myo- and endometrial arteries of parous serows. In nulliparous adult females, the internal elastic lamina is thin and poorly indented, whereas in the females that kidded one to three times, it is thick and full of indentation. In females that kidded more than four times, double- or multilayered elastic laminae, hyperplastic elastic fibers and degenerated nuclei of smooth muscle cells in the media were observed. In females that kidde...
  • H Araki, T Tsubota, N Maeda, N Harada, S Kominami, JJ Mason, Kita, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (8)  787  -790  1996/08  [Not refereed][Not invited]
     
    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.
  • T Komatsu, Y Yamamoto, T Tsubota, Y Atoji, Y Suzuki  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (4)  329  -335  1996/04  [Not refereed][Not invited]
     
    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.
  • T Komatsu, Y Yamamoto, T Tsubota, Y Atoji, Y Suzuki  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (4)  329  -335  1996/04  [Not refereed][Not invited]
     
    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.
  • KATAYAMA Atsushi, TSUBOTA Toshio, YAMADA Fumio, KITA Isao, TIBA Tosiro  Japanese Journal of Zoo and Wildlife Medicine  1-  (1)  26  -32  1996/02  [Not refereed][Not invited]
     
    Female genital organs from 19 Japanese black bears(Selenarctos thibetanus japonicus)were collected at Gifu and Kyoto prefecture during the period from March 1991 to August 1993. Both weight and volume of the ovaries increased according to adding of the age. The existence of corpora lutea and its involuted matters showed Japanese black bear sexually matured at the age of four, though there were differences among individuals. From observation of the genital organs, the mean value of ovulation was indicated 1.89 and that of implantation 2.00. The mean value of cubs having observed by hunters w...
  • 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  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Kita, I, M Eguchi, S Takatsuki, T Koizumi, T Tsubota  JOURNAL OF VETERINARY MEDICAL SCIENCE  57-  (6)  1029  -1033  1995/12  [Not refereed][Not invited]
     
    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.
  • Kita, I, M Eguchi, S Takatsuki, T Koizumi, T Tsubota  JOURNAL OF VETERINARY MEDICAL SCIENCE  57-  (6)  1029  -1033  1995/12  [Not refereed][Not invited]
     
    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.
  • ANNUAL CHANGES IN SERUM CONCENTRATIONS OF PROLACTIN IN CAPTIVE MALE BLACK BEARS (URSUS-AMERICANUS)
    T TSUBOTA, RA NELSON, JD THULIN, L HOWELL, JM BAHR  JOURNAL OF REPRODUCTION AND FERTILITY  104-  (2)  187  -191  1995/07  [Not refereed][Not invited]
     
    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.
  • IMMUNOLOCALIZATION OF STEROIDOGENIC ENZYMES P450SCC, 3-BETA-HSD, P450C17 AND P450AROM IN THE CORPUS-LUTEUM OF THE HOKKAIDO BROWN BEAR (URSUS-ARCTOS YESOENSIS) IN RELATION TO DELAYED IMPLANTATION
    T TSUBOTA, H NITTA, Y OSAWA, JI MASON, KITA, I, T TIBA, JM BAHR  JOURNAL OF REPRODUCTION AND FERTILITY  101-  (3)  557  -561  1994/08  [Not refereed][Not invited]
     
    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  [Not refereed][Not invited]
  • IMMUNOLOCALIZATION OF STEROIDOGENIC ENZYMES, P450SCC, 3-BETA-HSD, P450C17, AND P450AROM IN THE HOKKAIDO BROWN BEAR (URSUS-ARCTOS YESOENSIS) TESTIS
    T TSUBOTA, H NITTA, Y OSAWA, JI MASON, KITA, I, T TIBA, JM BAHR  GENERAL AND COMPARATIVE ENDOCRINOLOGY  92-  (3)  439  -444  1993/12  [Not refereed][Not invited]
  • REGULARITIES AND IRREGULARITIES IN THE STRUCTURE OF THE SEMINIFEROUS EPITHELIUM IN THE DOMESTIC-FOWL (GALLUS-DOMESTICUS) .2. COORDINATION BETWEEN GERM-CELL ASSOCIATIONS
    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  1993/09  [Not refereed][Not invited]
     
    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.
  • REGULARITIES AND IRREGULARITIES IN THE STRUCTURE OF THE SEMINIFEROUS EPITHELIUM IN THE DOMESTIC-FOWL (GALLUS-DOMESTICUS) .1. SUGGESTION OF THE PRESENCE OF THE SEMINIFEROUS EPITHELIAL CYCLE
    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  1993/09  [Not refereed][Not invited]
     
    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  1992/02  [Not refereed][Not invited]
     
    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.
  • T TSUBOTA, H KANAGAWA, K YAMAMOTO, T MANO, M YAMANAKA, KITA, I, T TIBA  JOURNAL OF VETERINARY MEDICAL SCIENCE  54-  (1)  1  -5  1992/02  [Not refereed][Not invited]
     
    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  生物科学  43-  (4)  p190  -194  1991/11  [Not refereed][Not invited]
  • 坪田 敏男  遺伝  45-  (7)  p77  -81  1991/07  [Not refereed][Not invited]
  • クマ類の繁殖特性
    生物科学  43-  (4)  1  -5  1991  [Not refereed][Not invited]
  • Reproductive characteristics of bears
    43-  (4)  1  -5  1991  [Not refereed][Not invited]
  • 坪田 敏男  家畜繁殖学雑誌  36-  (5)  p1  -10  1990/12  [Not refereed][Not invited]
  • 家畜繁殖誌  36,1-10-  1990  [Not refereed][Not invited]
  • Studies on reproductive physiology of Hokkaido brown bear, Ursus arctos yesoensis
    Jpn. J. Anim. Reprod  36,1-10-  1990  [Not refereed][Not invited]
  • TSUBOTA T  Journal of the Mammalogical Society of Japan  14-  (1)  11  -17  1989/07  [Not refereed][Not invited]
  • 坪田 敏男, 金川 弘司  家畜繁殖学雑誌  32-  (4)  184  -187  1986/12  [Not refereed][Not invited]
  • 坪田 敏男  家畜繁殖学雑誌  31-  (4)  203  -210  1985/12  [Not refereed][Not invited]
  • 坪田 敏男  動物と自然  15-  (12)  p20  -24  1985/11  [Not refereed][Not invited]
  • TSUBOTA Toshio  Japanese journal of veterinary research  33-  (1)  1985/04/30  [Not refereed][Not invited]

Awards & Honors

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

Research Grants & Projects

  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2009 -2011 
    Author : 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(...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2005 -2007 
    Author : 源 宣之, Hideto FUKUSHI, 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
     
    Emerging infectious diseases including SARS and avian influenza virus infection triggered the social problems in these years. Sources of these pathogens have been usually unknown, although the involvement of wild animals has been indicated. Furthermore, environmental destruction might be one of the cause of emerging infectious diseases.In this project, we planned to investigate the current situation of zoonoses and other infectious diseases in Africa where many emerging diseases have appeared almost every year in order to evaluate the environment changes and occurrences of the diseases. Add...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2003 -2006 
    Author : Toshio TSUBOTA, 村瀬 哲磨
     
    The purposes of this study were to monitor reproductive cycles by measuring sex steroid hormone concentrations non- invasively using molecular biology technique and to apply artificial breeding methods clinically under captive conditions in order to recover their populations in endangered wildlife. The following studies were conducted for 4 years in Tsushima wild cats and Japanese black bears.1. Measurement of fecal sex steroid hormone concentrations in Tsushima wild cats and Japanese black bears1) The method of measurement for fecal estradiol and progesterone concentrations using TR-FIA ki...
  • 野生動物の救護に関する研究
    Date (from‐to) : 2003
  • Wildlife rescue
    Date (from‐to) : 2003
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2001 -2002 
    Author : Yutaka FUKUI, 佐々木 基樹, 手塚 雅文, 浦島 匡, 坪田 敏男, 中村 正
     
    Project reports: Fukui, Tsubota, Urashima, Tetsuka and Sasaki visited Zoological Museum of Oslo University, Norwegian Polar Institute, Svalbard University and Tromso University, and discussed with the scientists there on the pollutants in wild seals and bears, reproduction strategies of whales and bears as well as on lactation strategies of many mammalian species. Sasaki anatomized polar foxes and polar bears in Oslo University, and Tetsuka searched PCB pollution in polar bears and Arctic seals. Dr. O. Oftedal of Smithonian Institute had a lecture on "the origin and early evolution of lacta...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    Date (from‐to) : 2001 -2001 
    Author : 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...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 1999 -2001 
    Author : Jun YASUDA, 杉本 千尋, 小沼 操, 坪田 敏男, 稲垣 匡, 福士 秀人, 吉田 光敏
     
    The prevalence of the BHV 1 antigens in cattle was 23.28 % while the mean prevalence of the BHV 1 antibodies was 48.28 %. The prevalence of the Rabies in Zambia was 3-28.8 %, especially in dogs and cattle was 75 % and 13 %, respectively. As the nucleotide sequences of dogs showed 93-99 % homology to wild beasts in Zambia, it was concluded that wild beasts were infected Rabies from dogs. The liver flukes obtained from cattle and Kafue lechwe were identified as Fasciola gigantica with normal spermatogenesis ability. The flukes from cattle showed three different genotypes (A, B and C), but tho...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 1999 -2000 
    Author : Toshio TSUBOTA, 村瀬 哲磨, 喜多 功
     
    1) Detection of Reproductive Disorder Caused by Endocrine Disruptors in Wild AnimalsOrgan samples were obtained from dead bodies of wild raccoon dogs, bears, mice and raptors. Dead bodies from captive fur seals, sea lions and dolphins were also used for the samples. Accumulated concentrations of endocrine disruptors (PCB, HCH, DDT and its congeners, butyltins and agricultural chemicals) were measured using the liver and adipose tissue of the samples. Histological specimens of the reproductive tracts were employed for the pathological examination. A case of testicular cancer from a raccoon d...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 1998 -2000 
    Author : Toshiaki MASEGI, 坪田 敏男, 工藤 忠明, 源 宣之, 福士 秀人, 鈴木 義孝
     
    1. To clarify the infectivity of parapox virus re-emerging in Japanese serow in mountains was inoculated to sheep. As a result, parapox virus was proved to spread from serow to sheep easily. This study warns the role of serow as a reservoir in parapox virus infection.2. GHV-1 is a new type emerging equine herpesvirus isolated from a mass outbreak of encephalitis in gazelles. In this study, GHV-1 was designated as EHV-9 based on virological and pathological studies. EHV-9 induced experimentally encephalitis in wide variety of animals including horses, goats, cattle, pigs, dogs, hamsters, mic...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1996 -1996 
    Author : 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))
    Date (from‐to) : 1995 -1995 
    Author : 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

Educational Activities

Teaching Experience

  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Animal Welfare
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Seminar in Wildlife Biology
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Ethology
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Environment and People
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • Wildlife Biology
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • Zoo Science
    開講年度 : 2018
    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
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • Arts and Science Courses in English 2
    開講年度 : 2018
    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
    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
    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
    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
    課程区分 : 学士課程
    開講学部 : 獣医学部

Campus Position History

  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員

Position History

  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員


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