基本情報

所属

• 獣医学研究院　獣医学部門　環境獣医科学分野

職名

• 教授

学位

• 獣医学博士（北海道大学）

連絡先

• tsubotavetmed.hokudai.ac.jp

ホームページURL

http://wildlife.vetmed.hokudai.ac.jp/

J-Global ID

• 200901050144415901

研究キーワード

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

研究分野

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

担当教育組織

•  学士課程　獣医学部
•  博士課程　獣医学院

職歴

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

学歴

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

所属学協会

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

研究活動情報

論文

• Age estimation based on blood DNA methylation levels in brown bears.

  Shiori Nakamura, Jumpei Yamazaki, Naoya Matsumoto, Miho Inoue-Murayama, Huiyuan Qi, Masami Yamanaka, Masanao Nakanishi, Yojiro Yanagawa, Mariko Sashika, Toshio Tsubota, Hideyuki Ito, Michito Shimozuru

  Molecular ecology resources 2023年03月23日 

  Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.
• The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo.

  Alice C C Lau, Wessam Mohamed Ahmed Mohamed, Ryo Nakao, Manabu Onuma, Yongjin Qiu, Nobuyoshi Nakajima, Michito Shimozuru, Jayasilan Mohd-Azlan, Mohamed Abdallah Mohamed Moustafa, Toshio Tsubota

  Microbial genomics 9 2 2023年02月 

  Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia-positive and Borrelia-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis, Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.
• Regulation of protein and oxidative energy metabolism are down-regulated in the skeletal muscles of Asiatic black bears during hibernation

  Mitsunori Miyazaki, Michito Shimozuru, Yu Kitaoka, Kenya Takahashi, Toshio Tsubota

  Scientific Reports 12 1 2022年11月16日 

  Abstract Hibernating animals exhibit an unexplained physiological characteristic of skeletal muscles being atrophy resistance, in which case muscle mass and strength remain almost unchanged both before and after hibernation. In this study, we examined the alterations in the regulatory systems of protein and energy metabolism in the skeletal muscles of Asiatic black bears during hibernation. Skeletal muscle samples (vastus lateralis muscle) were collected from identical individuals (n = 8) during the active (July) and hibernating (February) periods, while histochemical and biochemical analyses were performed. We observed no significant alterations in body weight, muscle fiber size, and fiber type composition during the active and hibernating periods, indicating that the skeletal muscles of bears are very well preserved during hibernation. In hibernating bear skeletal muscles, both regulatory pathways of muscle protein synthesis (Akt/mechanistic target of rapamycin and mitogen-activated protein kinase systems) and proteolysis (ubiquitin–proteasome and autophagy systems) were down-regulated. Gene expression levels of factors regulating oxidative metabolism were also decreased in hibernating bear skeletal muscles. This is likely an adaptive strategy to minimize the energy wasting of amino acids and lipids during hibernation, which is accompanied by a prolonged period of disuse and starvation.
• Detection of a Babesia sp. genotype closely related to marsupial-associated Babesia spp. in male Haemaphysalis shimoga from Sarawak, Malaysian Borneo.

  Alice Cc Lau, Yongjin Qiu, Mohamed Abdallah Mohamed Moustafa, Ryo Nakao, Michito Shimozuru, Manabu Onuma, Nobuyoshi Nakajima, Mariko Sashika, Jayasilan Mohd-Azlan, Toshio Tsubota

  The Journal of veterinary medical science 84 11 1469 - 1473 2022年09月16日 

  In this study, Babesia screening was conducted in 55 rodents and 160 tick samples collected from primary forests and an oil palm plantation in Sarawak, Malaysian Borneo. PCR targeting the 18S ribosomal DNA revealed the presence of Babesia spp. DNA detected in two questing male Haemaphysalis shimoga ticks collected from the oil palm plantation. Sequence analysis revealed that both sequences were identical and had 98.6% identity to a Babesia macropus sequence obtained from Eastern grey kangaroos (Macropus giganteus) in Australia. Phylogenetic tree revealed clustering with marsupial-associated Babesia spp. in the Babesia sensu stricto clade. Whether or not H. shimoga is the competent vector and the importance of the Babesia sp. detected in this study warrants more investigation.
• Estimation of breeding population size using DNA‐based pedigree reconstruction in brown bears

  Michito Shimozuru, Mina Jimbo, Keisuke Adachi, Kei Kawamura, Yuri Shirane, Yoshihiro Umemura, Tsuyoshi Ishinazaka, Masanao Nakanishi, Mayu Kiyonari, Masami Yamanaka, Yukihiro Amagai, Ayaho Ijuin, Tomoki Sakiyama, Shinsuke Kasai, Takane Nose, Masataka Shirayanagi, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota, Keita Fukasawa, Hiroyuki Uno

  Ecology and Evolution 12 9 2022年09月
• Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

  Kohji Uraguchi, Takao Irie, Hirokazu Kouguchi, Azusa Inamori, Mariko Sashika, Michito Shimozuru, Toshio Tsubota, Kinpei Yagi

  Emerging infectious diseases 28 8 1677 - 1680 2022年08月 

  We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.
• Diet selection and asocial learning: Natal habitat influence on lifelong foraging strategies in solitary large mammals

  Mina Jimbo, Tsuyoshi Ishinazaka, Yuri Shirane, Yoshihiro Umemura, Masami Yamanaka, Hiroyuki Uno, Mariko Sashika, Toshio Tsubota, Michito Shimozuru

  Ecosphere 13 7 2022年07月
• Semen collection by urethral catheterization and electro-ejaculation with different voltages, and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata).

  Noboru Takaesu, Chihiro Kanno, Kosuke Sugimoto, Masashi Nagano, Akihisa Kaneko, Yoriko Indo, Hiroo Imai, Hirohisa Hirai, Munehiro Okamoto, Mariko Sashika, Michito Shimozuru, Seiji Katagiri, Toshio Tsubota, Yojiro Yanagawa

  The Journal of veterinary medical science 84 3 429 - 438 2022年03月15日 

  In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.
• Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park, Nepal

  Rajan Prasad Paudel, Rabin Kadariya, Babu Ram Lamichhane, Naresh Subedi, Mariko Sashika, Michito Shimozuru, Toshio Tsubota

  ECOLOGY AND EVOLUTION 12 3 2022年03月 

  Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 x 4 km grids (n = 45). We collected detection/non-detection data along a 4-km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single-species single-season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model-averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.
• Ovulatory follicle size investigated by ultrasonography and single artificial insemination with ovulation induction in the Japanese macaque (Macaca fuscata)

  Noboru Takaesu, Masako Minami, Masashi Nagano, Akihisa Kaneko, Yoriko Indo, Hiroo Imai, Hirohisa Hirai, Mariko Sashika, Michito Shimozuru, Munehiro Okamoto, Seiji Katagiri, Toshio Tsubota, Yojiro Yanagawa

  Japanese Journal of Veterinary Research 70 1 5 - 17 2022年02月 [査読有り][通常論文]
  
• Diel and monthly activity pattern of brown bears and sika deer in the Shiretoko Peninsula, Hokkaido, Japan

  Kei KAWAMURA, Mina JIMBO, Keisuke ADACHI, Yuri SHIRANE, Masanao NAKANISHI, Yoshihiro UMEMURA, Tsuyoshi ISHINAZAKA, Hiroyuki UNO, Mariko SASHIKA, Toshio TSUBOTA, Michito SHIMOZURU

  Journal of Veterinary Medical Science 84 8 1146 - 1156 2022年
• Supplementing cultured human myotubes with hibernating bear serum results in increased protein content by modulating Akt/FOXO3a signaling.

  Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota

  PloS one 17 1 e0263085  2022年 

  Hibernating bears remain in their dens for 5-7 months during winter and survive without eating or drinking while staying inactive. However, they maintain their physical functions with minimal skeletal muscle atrophy and metabolic dysfunction. In bears, resistance to skeletal muscle atrophy during hibernation is likely mediated by seasonally altered systemic factors that are independent of neuromuscular activity. To determine whether there are components in bear serum that regulate protein and energy metabolism, differentiated human skeletal muscle cells were treated with bear serum (5% in DMEM/Ham's F-12, 24 h) collected during active summer (July) and hibernating winter (February) periods. The serum samples were collected from the same individual bears (Ursus thibetanus japonicus, n = 7 in each season). Total protein content in cultured skeletal muscle cells was significantly increased following a 24 h treatment with hibernating bear serum. Although the protein synthesis rate was not altered, the expression of MuRF1 protein, a muscle-specific E3 ubiquitin ligase was significantly decreased along with a concomitant activation of Akt/FOXO3a signaling. Increased levels of insulin-like growth factor-1 (IGF-1) were also observed in hibernating bear serum. These observations suggest that protein metabolism in cultured human myotubes may be altered when incubated with hibernating bear serum, with a significant increase in serum IGF-1 and diminished MuRF1 expression, a potential target of Akt/FOXO3a signaling. A protein sparing phenotype in cultured muscle cells by treatment with hibernating bear serum holds potential for the development of methods to prevent human muscle atrophy and related disorders.
• Genome Sequences of Two Mycobacterium tuberculosis Isolates from Asian Elephants in Nepal.

  Sarad Paudel, Evan P Brenner, Syeda A Hadi, Yasuhiko Suzuki, Chie Nakajima, Toshio Tsubota, Kamal Prasad Gairhe, Bhagwan Maharjan, Srinand Sreevatsan

  Microbiology resource announcements 10 36 e0061421  2021年09月09日 

  This report describes the genome sequences of two Mycobacterium tuberculosis isolates, S1 and S3, recovered from Asian elephants in Nepal. These genome sequences will enhance our understanding of the genomic epidemiology of Mycobacterium tuberculosis in Asian elephants.
• Dining from the coast to the summit: Salmon and pine nuts determine the summer body condition of female brown bears on the Shiretoko Peninsula

  Yuri Shirane, Mina Jimbo, Masami Yamanaka, Masanao Nakanishi, Fumihiko Mori, Tsuyoshi Ishinazaka, Mariko Sashika, Toshio Tsubota, Michito Shimozuru

  ECOLOGY AND EVOLUTION 11 10 5204 - 5219 2021年05月 

  Body condition in mammals fluctuates depending on energy intake and expenditure. For brown bears (Ursus arctos), high-protein foods facilitate efficient mass gain, while lipids and carbohydrates play important roles in adjusting dietary protein content to optimal levels to maximize energy intake. On the Shiretoko Peninsula, Hokkaido, Japan, brown bears have seasonal access to high-lipid pine nuts and high-protein salmon. To assess seasonal and annual fluctuation in the body condition of adult female brown bears in relation to diet and reproductive status, we conducted a longitudinal study in a special wildlife protection area on the Shiretoko Peninsula during 2012-2018. First, analyses of 2,079 bear scats revealed that pine nuts accounted for 39.8% of energy intake in August and salmon accounted for 46.1% in September and that their consumption by bears varied annually. Second, we calculated the ratio of torso height to torso length as an index of body condition from 1,226 photographs of 12 adult females. Results indicated that body condition continued to decline until late August and started to increase in September when salmon consumption increased. In addition, body condition began to recover earlier in years when consumption of both pine nuts and salmon was high. Furthermore, females with offspring had poorer body condition than solitary females, in particular in late August in years with low salmon consumption. Our findings suggest that coastal and subalpine foods, which are unique to the Shiretoko Peninsula, determine the summer body condition of female brown bears, as well as their survival and reproductive success.
• SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF beta-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS)

  Mohamed Abdallah Mohamed Moustafa, Jason L. Anders, Wessam Mohamed Ahmed Mohamed, Kyunglee Lee, Elzahara Elbaz, Mariko Sashika, Ryo Nakao, Michito Shimozuru, Toshio Tsubota

  JOURNAL OF ZOO AND WILDLIFE MEDICINE 52 1 200 - 205 2021年03月 

  Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and beta-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The beta-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the beta-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.
• Changes in liver microRNA expression and their possible regulatory role in energy metabolism-related genes in hibernating black bears

  Kazuhei Nishida, Michito Shimozuru, Yuko Okamatsu-Ogura, Mitsunori Miyazaki, Tsukasa Soma, Mariko Sashika, Toshio Tsubota

  JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMS AND ENVIRONMENTAL PHYSIOLOGY 191 2 397 - 409 2021年03月 

  Hibernating bears survive up to 6 months without feeding while yet maintaining metabolic homeostasis. We previously reported expression changes in energy metabolism-related genes in the liver of hibernating Japanese black bears. The present study examined the role of microRNAs in the regulation of hepatic gene expression during hibernation. The quantitative analyses revealed significant increases in the expression of 4 microRNAs (miR-221-3p, miR-222-3p, miR-455-3p, and miR-195a-5p) and decreases of 2 microRNAs (miR-122-5p and miR-7a-1-5p) during hibernation. RNA sequencing and in silico target prediction regarding 3 upregulated microRNAs (miR-221-3p, miR-222-3p and miR-455-3p) found 13 target mRNAs with significantly decreased expression during hibernation. The transfection of microRNA mimics into cells showed that miR-222 and miR-455 reduced solute carrier family 16 member 4 (SLC16A4) and fatty acid synthase (FASN) mRNA expression, respectively. Our results suggest that the increased levels of hepatic miRNA during hibernation (miR-222-3p and miR-455-3p) negatively regulate the expression of targeted genes predicted to be involved in the transport of energy source and de novo fatty acid synthesis, is consistent with a regulatory role of these miRNAs in energy metabolism in hibernating black bears.
• Amblyomma testudinarium infestation on a brown bear (Ursus arctos yesoensis) captured in Hokkaido, a northern island of Japan

  Ryo Nakao, Kohei Shinjo, Tomoki Sakiyama, Shohei Ogata, Kodai Kusakisako, Gohta Kinoshita, Doaa Naguib, Elisha Chatanga, Wessam Mohamed Ahmed Mohamed, Mohamed Abdallah Mohamed Moustafa, Keita Matsuno, Takuya Ito, Nariaki Nonaka, Mariko Sashika, Toshio Tsubota, Michito Shimozuru

  PARASITOLOGY INTERNATIONAL 80 2021年02月 

  The tick Amblyomma testudinarium Koch, 1844 (Acari: Ixodidae) is known as a vector of several pathogens such as Rickettsia tamurae and severe fever with thrombocytopenia syndrome (SFTS) virus. This tick species is present in many Asian countries, including Japan, where its distribution is limited to the warm areas of Kanto region and the southwestern region. The present study reports the recovery of a partially engorged A. testudinarium from a wild brown bear captured in Shari town, Hokkaido. In addition to morphological identification, the specimen was genetically characterized by the complete mitochondrial genome sequencing. The results showed that the length of the obtained mitogenome is 14,835 bp that encodes 13 protein-coding, two ribosomal RNA (rRNA) (12S and 16S), and 22 transfer RNA genes with two non-coding control regions. The phylogenetic analysis indicated that our sample clustered with A. testudinarium from Nara, Japan, but separated from A. testudinarium from China. Although the introduction of the tick through livestock transportation cannot be ruled out, the detection of A. testudinarium in Hokkaido prefecture, which is separated from the main island where A. testudinarium is present in the south, may suggest the introduction by migratory birds. This study provides important insights on the distribution and host range of A. testudinarium. This will be useful for the future taxonomic analysis of ticks based on the complete mitogenome sequencing. To our knowledge, this is the northernmost detection point of the tropical tick A. testudinarium.
• Utilizing attached hard ticks as pointers to the risk of infection byBabesiaandTheileriaspecies in sika deer (Cervus nippon yesoensis), in Japan

  Elzahara Elbaz, Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Alice Lau Ching Ching, Michito Shimozuru, Mariko Sashika, Ryo Nakao, Sabry Ahmed El-khodery, Toshio Tsubota

  EXPERIMENTAL AND APPLIED ACAROLOGY 82 3 411 - 429 2020年11月 

  Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the generaIxodesandHaemaphysalis. In total 163 samples (31.4%) were positive forBabesiaandTheileriaspp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes ofTheileriasp.Thrivae (27.0% of ticks; 140/519),Theileria capreoli(10.6%; 55/519),Babesia divergens-like (1.7%; 9/519),Babesiasp. (Bab-SD) (0.6%; 3/519),Babesia microtiU.S. (0.4%; 2/519), andB. microtiHobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.
• Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus)

  Mohamed Abdallah Mohamed Moustafa, Ayaka Sasaki, Michito Shimozuru, Ryo Nakao, Mariko Sashika, Koji Yamazaki, Shinsuke Koike, Junpei Tanaka, Hiroo Tamatani, Masami Yamanaka, Tsuyoshi Ishinazaka, Toshio Tsubota

  PARASITOLOGY RESEARCH 119 11 3739 - 3753 2020年11月 

  Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi,Babesiasp. UR2-like group,Cytauxzoonsp. UR1,Babesiasp. UR1, andBabesia microti) were detected from bear blood DNA samples.B. microtiwas detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens,H. ursi,Babesiasp. UR2-like pathogens, andCytauxzoonsp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence ofH. ursiwere significantly higher in Japanese black bear (0% vs 96.4%) while that ofBabesiasp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence ofBabesiasp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.
• Predation impacts of invasive raccoons on rare native species

  Sakura Oe, Mariko Sashika, Ayako Fujimoto, Michito Shimozuru, Toshio Tsubota

  SCIENTIFIC REPORTS 10 1 2020年11月 

  In Japan, there are concerns that invasive alien raccoons prey on rare native species during their spawning season from late winter to early summer. We investigated raccoon predation impact by examining the predation presence using DNA metabarcoding and extent of predation on rare native species using stable isotope analysis. We captured raccoons in Hokkaido, Japan from April to August in 2018 and 2019. We analysed raccoon faeces and gastric contents by DNA metabarcoding to detect the rare native Hokkaido salamander and Japanese crayfish. Hokkaido salamanders were detected from gastric contents, but Japanese crayfish were not detected in any samples. Stable isotope analysis of raccoon muscle samples and the Bayesian mixing model were used to estimate each food resource's contribution to the raccoon diet. Animal food resources accounted for 70% of total consumed food. The foraging ratios of amphibians and crustaceans were about 9% and 5%, respectively. Raccoons have been found to use amphibians at a higher rate than previously reported, including a rare endangered species, the Hokkaido salamander. Hokkaido salamander and Japanese crayfish spawn in the spring, and increased predation pressure by raccoons may directly impact populations of these rare native species.
• Hair growth in brown bears and its application to ecological studies on wild bears

  Mina Jimbo, Naoya Matsumoto, Hideyuki Sakamoto, Yojiro Yanagawa, Yoshiko Torii, Masami Yamanaka, Tsuyoshi Ishinazaka, Yuri Shirane, Mariko Sashika, Toshio Tsubota, Michito Shimozuru

  MAMMAL STUDY 45 4 337 - 345 2020年10月 

  Hair biomarkers have been widely used to study wildlife physiology and ecology. To apply this technique to practical studies, detailed information on hair growth is needed for each study species. This study measured hair growth patterns, including growth rate, periods of growth and shedding, and hair bulb structural changes in the brown bear. Hair growth was examined monthly in captive bears, and gross observations of fur were performed in wild bears. Guard hairs grew from late April to early October at an approximate rate of 0.5 mm/day for captive bears, and from late May to late September at an approximate rate of 0.7 mm/day for wild bears. Underfur began growing in August and continued into winter. The growth periods for intermediate hairs began at different times and lasted 3-5 months, depending on individuals. Most shedding was complete by August, and all shedding was complete by the end of September. Hair bulbs were classified into three types: white spheres (WS-type), black hooks (BH-type), and white hooks (WH-type). This classification can be used to discriminate between previous year's hair (WS-type) and current year's hair (BH-type or WH-type). The current findings will enhance the understanding of brown bear ecology in the wild.
• Detection of Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia in Feeding Ixodes Ticks and Rodents in Sarawak, Malaysia: New Geographical Records of Borrelia yangtzensis and Borrelia miyamotoi

  Alice C. C. Lau, Yongjin Qiu, Mohamed Abdallah Mohamed Moustafa, Ryo Nakao, Michito Shimozuru, Manabu Onuma, Jayasilan Mohd-Azlan, Toshio Tsubota

  PATHOGENS 9 10 2020年10月 

  Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp.
• Maternal human habituation enhances sons' risk of human-caused mortality in a large carnivore, brown bears

  Michito Shimozuru, Yuri Shirane, Masami Yamanaka, Masanao Nakanishi, Tsuyoshi Ishinazaka, Shinsuke Kasai, Takane Nose, Masataka Shirayanagi, Mina Jimbo, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota

  SCIENTIFIC REPORTS 10 1 2020年10月 

  Human habituation of large carnivores is becoming a serious problem that generates human-wildlife conflict, which often results in the removal of animals as nuisances. Although never tested, human habituation potentially reduces the fitness of adult females by reducing their offspring's survival as well as their own, due to an increased likelihood of human-caused mortality. Here, we tested this hypothesis in brown bears inhabiting Shiretoko National Park, Japan. We estimated the frequency of human-caused mortality of independent young (aged 1-4 years) born to mothers living in areas with different maternal levels of human habituation and different proximities to areas of human activity. The overall mortality rate was higher in males than in females, and in females living near a town than those in a remote area of park. Surprisingly, more than 70% of males born to highly habituated mothers living around a remote wildlife protection area were killed by humans; this proportion is greater than that for males born to less-habituated mothers living in almost the same area. The current study clarified that interactions among maternal human habituation, birthplace (proximity to town), age, and sex determine the likelihood of human-caused mortality of brown bears at an early stage of life.
• Development of a noninvasive photograph-based method for the evaluation of body condition in free-ranging brown bears

  Yuri Shirane, Fumihiko Mori, Masami Yamanaka, Masanao Nakanishi, Tsuyoshi Ishinazaka, Tsutomu Mano, Mina Jimbo, Mariko Sashika, Toshio Tsubota, Michito Shimozuru

  PEERJ 8 2020年09月 

  Body condition is an important determinant of health, and its evaluation has practical applications for the conservation and management of mammals. We developed a noninvasive method that uses photographs to assess the body condition of free-ranging brown bears (Ursus arctos) in the Shiretoko Peninsula, Hokkaido, Japan. First, we weighed and measured 476 bears captured during 1998-2017 and calculated their body condition index (BCI) based on residuals from the regression of body mass against body length. BCI showed seasonal changes and was lower in spring and summer than in autumn. The torso height:body length ratio was strongly correlated with BCI, which suggests that it can be used as an indicator of body condition. Second, we examined the precision of photograph-based measurements using an identifiable bear in the Rusha area, a special wildlife protection area on the peninsula. A total of 220 lateral photographs of this bear were taken September 24-26, 2017, and classified according to bear posture. The torso height:body/torso length ratio was calculated with four measurement methods and compared among bear postures in the photographs. The results showed torso height:horizontal torso length (TH:HTL) to be the indicator that could be applied to photographs of the most diverse postures, and its coefficient of variation for measurements was <5%. In addition, when analyzing photographs of this bear taken from June to October during 2016-2018, TH:HTL was significantly higher in autumn than in spring/summer, which indicates that this ratio reflects seasonal changes in body condition in wild bears. Third, we calculated BCI from actual measurements of seven females captured in the Rusha area and TH:HTL from photographs of the same individuals. We found a significant positive relationship between TH:HTL and BCI, which suggests that the body condition of brown bears can be estimated with high accuracy based on photographs. Our simple and accurate method is useful for monitoring bear body condition repeatedly over the years and contributes to further investigation of the relationships among body condition, food habits, and reproductive success.
• Hibernating bear serum hinders osteoclastogenesis in-vitro

  Alireza Nasoori, Yuko Okamatsu-Ogura, Michito Shimozuru, Mariko Sashika, Toshio Tsubota

  PLOS ONE 15 8 2020年08月 

  Bears do not suffer from osteoporosis during hibernation, which is associated with long-term inactivity, lack of food intake, and cold exposure. However, the mechanisms involved in bone loss prevention have scarcely been elucidated in bears. We investigated the effect of serum from hibernating Japanese black bears (Ursus thibetanus japonicus) on differentiation of peripheral blood mononuclear cells (PBMCs) to osteoclasts (OCs). PBMCs collected from 3 bears were separately cultured with 10% serum of 4 active and 4 hibernating bears (each individual serum type was assessed separately by a bear PBMCs), and differentiation were induced by treatment with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). PBMCs that were cultured with the active bear serum containing medium (ABSM) differentiated to multi-nucleated OCs, and were positive for TRAP stain. However, cells supplemented with hibernating bear serum containing medium (HBSM) failed to form OCs, and showed significantly lower TRAP stain (p< 0.001). On the other hand, HBSM induced proliferation of adipose derived mesenchymal stem cells (ADSCs) similarly to ABSM (p> 0.05), indicating no difference on cell growth. It was revealed that osteoclastogenesis of PBMCs is hindered by HBSM, implying an underlying mechanism for the suppressed bone resorption during hibernation in bears. In addition, this study for the first time showed the formation of bears' OCsin-vitro.
• Morphological and histological studies on the epididymis and deferent duct of the Sunda porcupine (Hystrix javanica)

  Anni Nurliani, Motoki Sasaki, Teguh Budipitojo, Toshio Tsubota, Nobuo Kitamura

  MAMMAL STUDY 45 3 201 - 211 2020年07月 

  The Sunda porcupine (Hystrix javanica) is an endemic species of Indonesia. The population of this species has been decreased by hunting and habitat loss. Understanding of reproductive characteristics is essential for the appropriate management of their population. However, there is little knowledge about reproductive features of this species. The epididymis and deferent duct are important organs for maturation, transport, and storage of sperm. In this study, therefore, the epididymis and deferent duct of Sunda porcupines were examined histologically, histochemically, immunohistochemically, and electron-microscopically to understand their structures and functions. Four adult wild porcupines that inhabit Central Java, Indonesia, were captured in June and September and euthanized. Then, the epididymides and deferent ducts were removed. Principal cells were the main cell type found in the epididymis with stereocilia and apical blebs, but both of these structures were absent in the deferent duct. These observations suggest high secretion and absorption capability by the epididymal epithelium. In the epididymis and deferent duct, PAS-positive principal cells were shown, and the immunoreactivities for 3 P-HSD were found in the epithelium of the deferent ducts. These reults suggest effects on sperm by glycoproteins and steroid metabolites.
• Genetic population structure of invasive raccoons (Procyon lotor) in Hokkaido, Japan: Unique phenomenon caused by pet escape or abandonment.

  Minami W Okuyama, Michito Shimozuru, Mariko Nakai, Emi Yamaguchi, Kei Fujii, Ken-Ichiro Shimada, Tohru Ikeda, Toshio Tsubota

  Scientific reports 10 1 8108 - 8108 2020年05月15日 [査読有り][通常論文]
   

  Phylogeographic studies can resolve relationships between genetic population structure of organisms and geographical distributions. Raccoons have become feral in Japan, and in Hokkaido island, they have been rapidly increasing in number and spreading since the 1970s. We analyzed mitochondrial (mtDNA) and microsatellite DNA to understand the current phylogenetic distribution and invasive founder events. Overall, Hokkaido raccoons maintained high genetic diversity (i.e., the level of heterozygosity was comparable to the original habitat, North America). Based on mtDNA distribution and microsatellite diversity, Hokkaido raccoons were divided into six management units. However, mtDNA haplotype distributions and genetic structures based on microsatellites did not always correspond to each other (e.g., two geographically and genetically separated populations showed similar mtDNA distributions). In addition, a high degree of genetic admixture was observed in every unit, and the degree of genetic differentiation was low even between regions separated by long distances. Compared with other countries in Europe where genetic distribution of introduced raccoons is more clearly structured, the current results represent a unique and complex phenomenon of pet escape/abandonment in Hokkaido: i.e., genetically related colonies were introduced into multiple regions as founder events, resulting in the current state in which raccoons are not clearly genetically differentiated even 40 years after introduction.
• 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 3 175 - 185 2020年 [査読有り][通常論文]
   

  Male reproductive behavior has not been thoroughly investigated in large, non-social mammals. We studied male reproductive input using field observations and microsatellite DNA data for the brown bear (Ursus arctos yesoensis Lydekker, 1897) population in a special wildlife protection area on the Shiretoko Peninsula in Hokkaido, Japan. We identified the father of 82 offspring born between 2006 and 2018 from 54 litters produced by 20 females and revealed that they were sired by a total of 19 males. Overall, paternity was distributed relatively evenly among different males, although litter production tended to be skewed to a few bears in some years. Male breeding tenure varied among individuals, but most tenures were within the range of 1 to 6 years. Human-caused mortality, mostly occurring near human residential areas, induced male turnover, indicating that the special wildlife protection area located in the inner part of the Shiretoko Peninsula is not free from human impact. The frequency of successful reproduction was highest in 10- to 14-year-old bears, which is consistent with the period when males reach physical maturity. These results contribute to an understanding of breeding systems in large solitary mammals and to appropriate conservation and management strategies for brown bear populations.
• Epidemiological study of sarcoptic mange in raccoon dogs (Nyctereutes procyonoides albus) in Hokkaido, Japan

  Mariko Sashika, Go Abe, Mariko Nakai, Aya Osaki, Ayako Fujimoto, Toshio Tsubota

  JAPANESE JOURNAL OF VETERINARY RESEARCH 67 4 253 - 261 2019年11月 

  Sarcoptic mange is a pruritic skin disease caused by Sarcoptes scabiei, and can impact wildlife populations. In Japan, sarcoptic mange has rapidly spread among medium-sized mammals, especially raccoon dogs, since the 1980s, and can cause regional extinction. We conducted an epidemiological survey on sarcoptic mange in raccoon dogs (Nyctereutes procyonoides albus) at two different sampling sites (Nopporo and Mukawa) in Hokkaido, Japan. We captured raccoon dogs using box traps in Nopporo from 2003 to 2018, and collected raccoon dog carcasses in Mukawa from 2005 to 2010; we investigated whether they were infested with S. scabiei. In Nopporo, sarcoptic mange incidences were detected from 2003 to 2004 and from 2016 to 2018, when the raccoon dog population density was high. Subsequently, the number of captured raccoon dogs decreased. Alternatively, when the raccoon dog population density was low, no S. scabiei-infested raccoon dogs were detected except in 2010. In 2010, three S. scabiei-infested raccoon dogs were captured at the southern end of the forest, and these individuals were subsequently confirmed to have died. Because they did not enter the central region of the forest, the incidence was localized. In Mukawa, 240 raccoon dogs were captured, of which 60 were infested with S. scabiei from 2005 to 2010, and a decrease in the number of captured raccoon dogs was confirmed after the sarcoptic mange epidemic. In conclusion, the increased population density might have resulted in the incidence of sarcoptic mange in raccoon dogs, and sarcoptic mange possibly impacted raccoon dog population density.
• Comparison of feeding habits and habitat use between invasive raccoons and native raccoon dogs in Hokkaido, Japan.

  Aya Osaki, Mariko Sashika, Go Abe, Kohei Shinjo, Ayako Fujimoto, Mariko Nakai, Michito Shimozuru, Toshio Tsubota

  BMC ecology 19 1 35 - 35 2019年09月11日 [査読有り][通常論文]
   

  BACKGROUND: In Japan, invasive raccoons cause severe ecological and social problems by transmitting pathogens to humans, livestock, and native species, causing substantial crop damage, and competing with native species. Possible competition between invasive raccoons and native raccoon dogs is of concern in Japan because Japanese raccoon dogs have a limited distribution and are native only to Japan and the two species have similar characteristics. We assessed potential competition between raccoons and raccoon dogs by comparing feeding habits and habitat use. RESULTS: Both species were captured in Hokkaido, Japan from 2004 to 2017. More raccoons were captured close to agricultural land at the forest periphery (70.1%, 358/511); conversely, more raccoon dogs were captured in the forest core (74.9%, 253/338). Feeding habits were then examined by fecal analysis and stable isotope analyses. Fecal analysis revealed both species to be opportunistic omnivores that consumed easily found food items. However, raccoon feces contained more crops, whereas raccoon dog feces contained more insects, reflecting the different locations in which the species were trapped. Moreover, stable isotope ratios were significantly higher in raccoons than raccoon dogs (Corn has the highest carbon stable isotope (δ13C) value, and amphibians and reptiles are high in nitrogen stable isotope (δ15N); forest resources such as insects and wild fruits are low in δ13C and δ15N). CONCLUSIONS: We conclude that both species ate similar food types, but their food preferences appeared to differ. Raccoon and raccoon dog habitat use also differed, possibly because the two species inhabited areas where they could easily obtain their preferred foods. Therefore, the current feeding habits and habitat use of raccoons do not appear to overlap sufficiently with those of raccoon dogs to impact the latter. The results of this study, particularly the stable isotope data, may provide a useful precedent for future studies of competition in medium-sized mammals, particularly canids.
• An immunohistochemical study on testicular steroidogenesis in the Sunda porcupine (Hystrix javanica).

  Nurliani A, Sasaki M, Budipitojo T, Tsubota T, Suzuki M, Kitamura N

  The Journal of veterinary medical science 81 9 1285 - 1290 2019年09月 [査読有り][通常論文]
   

  In the testes of the Sunda porcupine (Hystrix javanica), the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes, such as cytochrome P450 side chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom), was immunohistochemically examined to clarify the location of steroidogenesis. In this study, complete spermatogenesis (spermiogenesis) was observed in the testes of the examined Sunda porcupine, and spermatozoa of the Sunda porcupine had a spatulate sperm head unlike that of rats and mice which has an apical hook. On immunostaining of StAR, P450scc, 3 beta-HSD, P450c17 and P450arom, immunoreactivity for all proteins was only detected in the Leydig cells and not observed within the seminiferous tubules, suggesting that the Leydig cells can synthesize both androgen and estrogen from cholesterol in the Sunda porcupine testes.
• Human TB threat to wild elephants

  Sarad Paudel, Toshio Tsubota, Susan K. Mikota

  NATURE 571 7764 174 - 174 2019年07月
• Sex-biased dispersal and inbreeding avoidance in Hokkaido brown bears

  Shirane, Yuri, Shimozuru, Michito, Yamanaka, Masami, Tsuruga, Hifumi, Nakanishi, Masanao, Ishinazaka, Tsuyoshi, Nose, Takane, Kasai, Shinsuke, Shirayanagi, Masataka, Masuda, Yasushi, Fujimoto, Yasushi, Mano, Tsutomu, Sashika, Mariko, Tsubota, Toshio

  JOURNAL OF MAMMALOGY 100 4 1317 - 1326 2019年07月 [査読有り][通常論文]
   

  Natal dispersal likely plays an important role in avoiding inbreeding among large carnivores. We tested the hypothesis that male-biased dispersal reduces close inbreeding by limiting the spatial overlap of opposite-sex pairs of close relatives in brown bears (Ursus arctos) in the Shiretoko Peninsula, Hokkaido, Japan. We genotyped 837 individuals collected in 1998-2017 at 21 microsatellite loci and performed parentage analysis. To calculate natal dispersal distance, we considered the site where the mother was identified as the birthplace of her offspring, and the site where the offspring were identified as their dispersed place. As predicted, we found that dispersal distances were significantly greater for males (12.4 km +/- 1.0) than for females (7.7 km +/- 0.9), and those for males increased from 3 years old, indicating that males begin to disperse around the time sexual maturation begins. Relatedness decreased with distance among pairs of females, and the mean relatedness was significantly higher between pairs of females than between pairs of males or between female-male pairs within 3 km. Closely related female-male pairs rarely (5-6%) resided in close proximity (< 3 km), compared with pairs of closely related females. Our study revealed that the potential for close inbreeding was low in Hokkaido brown bears because males are effective dispersers.
• Incidence of Multiple Paternity and Inbreeding in High-Density Brown Bear Populations on the Shiretoko Peninsula, Hokkaido, Japan.

  Shimozuru M, Shirane Y, Tsuruga H, Yamanaka M, Nakanishi M, Ishinazaka T, Kasai S, Nose T, Masuda Y, Fujimoto Y, Mano T, Tsubota T

  The Journal of heredity 110 3 321 - 331 2019年05月 [査読有り][通常論文]
  
• Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.

  Paudel S, Nakajima C, Mikota SK, Gairhe KP, Maharjan B, Subedi S, Poudel A, Sashika M, Shimozuru M, Suzuki Y, Tsubota T

  Emerging infectious diseases 25 5 1031 - 1032 2019年05月 [査読有り][通常論文]
   

  Tuberculosis in elephants is primarily caused by Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African-Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.
• Heart rate during hyperphagia differs between two bear species

  Boris Fuchs, Koji Yamazaki, Alina L. Evans, Toshio Tsubota, Shinsuke Koike, Tomoko Naganuma, Jon M. Arnemo

  BIOLOGY LETTERS 15 1 2019年01月 

  Hyperphagia is a critical part of the yearly cycle of bears when they gain fat reserves before entering hibernation. We used heart rate as a proxy to compare the metabolic rate between the Asian black bear (Ursus thibetanus) in Japan and the Eurasian brown bear (Ursus arctos) in Sweden from summer into hibernation. In the hyperphagic period, black bears feed on fat- and carbohydrate-rich hard masts whereas brown bears feed on sugar-rich berries. Availability of hard masts has quantitative and spatial annual fluctuations, which might require increased activity and result in intraspecific stress. Using generalized additive mixed models we analysed the differences in heart rate between the two species. Black bears had decreased heart rates during summer but had doubled heart rate values throughout the hyperphagic period compared to brown bears. This letter illustrates the different physiological consequences of seasonal differences in food availability in two species of the same genus dealing with the same phenological challenge.
• Tuberculosis threat in Asian elephants

  Sarad Paudel, Susan K. Mikota, Toshio Tsubota

  SCIENCE 363 6425 356 - 356 2019年01月
• Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation.

  Mitsunori Miyazaki, Michito Shimozuru, Toshio Tsubota

  PloS one 14 4 e0215489  2019年 [査読有り][通常論文]
   

  Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to "muscle atrophy resistance" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization.
• Serodiagnosis of elephant tuberculosis: a useful tool for early identification of infected elephants at the captive-wild interface

  Sarad Paudel, Susan K. Mikota, Jeewan Thapa, Konstantin P. Lyashchenko, Kamal P. Gairhe, Ishwari Prasad Dhakal, Naresh Subedi, Bhagwan Maharjan, Suraj Subedi, Gretchen E. Kaufman, Toshio Tsubota

  EUROPEAN JOURNAL OF WILDLIFE RESEARCH 64 6 2018年12月 

  Tuberculosis (TB) is an emerging disease in elephants primarily caused by Mycobacterium tuberculosis (M. tb) and in some occassion by M. bovis. We performed culture and three serological teststhe Elephant TB STAT-PAK,(R) DPP VetTB (R) Assay, and MAPIA (multi-antigen print immunoassay)prospectively on samples from eight elephants in Nepal that died of suspected or confirmed tuberculosis (TB) between 2007 and 2013. Among them, all elephants were reactive to DPP VetTB (R) Assay, five to Elephant TB STAT-PAK,(R) and two were reactive to MAPIA. Similarly, six elephants were positive on culture on samples collected antemortem or postmortem. We observed antibody responses months to years before culture confirmation of TB which shows that serological tests can be highly useful for the early diagnosis of TB in elephants. Validated point-of-care serological tests are easily performed in the field and hold promise for improved TB surveillance in other non-domestic species.
• High genetic diversity and distinct ancient lineage of Asiatic black bears revealed by non-invasive surveys in the Annapurna Conservation Area, Nepal

  Kadariya, R, Shimozuru, M, Maldonado, J.E, Moustafa, M.A.M, Sashika, M, Tsubota, T

  PloS one in press 12 e0207662  2018年12月 [査読有り][通常論文]
   

  Asiatic black bears (Ursus thibetanus) have a widespread distribution in mountain landscapes, and are considered vulnerable globally, but are low-priority species for conservation in Nepal. Habitat fragmentation, illegal hunting, and human-bear conflict are the major threats to Asiatic black bears across their global range. Having an adequate level of genetic variation in a population helps with adapting to rapidly changing environments, and thus is important for the long-term health of bear populations. Accordingly, we conducted non-invasive surveys of bear populations in the Annapurna Conservation Area (ACA) to elucidate genetic diversity, genetic structure, and the phylogenetic relationship of Asiatic black bears from this region of Nepal to other subspecies. To assess levels of genetic diversity and population genetic structure, we genotyped eight microsatellite loci using 147 samples, identifying 60 individuals in an area of approximately 525 km(2). We found that the Asiatic black bear population in the ACA has maintained high levels of genetic diversity (H-E = 0.76) as compared to other bear populations from range countries. We did not detect a signature of population substructure among sampling localities and this suggests that animals are moving freely across the landscape within the ACA. We also detected a moderate population size that may increase with the availability of suitable habitat in the ACA, so bear-related conflict should be addressed to ensure the long-term viability of this expanding bear populations. Primers specific to bears were designed to amplify a 675 bp fragment of the mitochondrial control region from the collected samples. Three haplotypes were observed from the entire conservation area. The complete mitochondrial genome (16,771 bp), the first obtained from wild populations of the Himalayan black bear (U. t. laniger), was also sequenced to resolve the phylogenetic relationships of closely related subspecies of Asiatic black bears. The resulting phylogeny indicated that Himalayan black bear populations in Nepal are evolutionary distinct from other known subspecies of Asiatic black bears.
• Sex-biased natal dispersal in Hokkaido brown bears revealed through mitochondrial DNA analysis

  Shirane Yuri, Shimozuru Michito, Yamanaka Masami, Tsuruga Hifumi, Hirano Saiko, Nagano Natsuo, Moriwaki Jun, Nakanishi Masanao, Ishinazaka Tsuyoshi, Nose Takane, Kasai Shinsuke, Shirayanagi Masataka, Masuda Yasushi, Fujimoto Yasushi, Osada Masahiro, Akaishi Masao, Mano Tsutomu, Masuda Ryuichi, Sashika Mariko, Tsubota Toshio

  EUROPEAN JOURNAL OF WILDLIFE RESEARCH 64 6 65  2018年12月 [査読有り][通常論文]
  
• Differentiation of Japanese Black Bears' Adipose-Derived Stem Cells to Osteoblasts

  Alireza Nasoori, Yuko Okamatsu-Ogura, Woongchul Shin, Michito Shimozuru, Mohamed Abdallah Mohamed Moustafa, Toshio Tsubota

  JOURNAL OF BONE AND MINERAL RESEARCH 33 224 - 224 2018年11月
• Evaluation of body condition using body mass and chest girth in brown bears of Hokkaido, Japan (Ursus arctos yesoensis)

  Jun Moriwaki, Ryosuke Omori, Michito Shimozuru, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota

  Japanese Journal of Veterinary Research 66 2 71 - 81 2018年05月01日 [査読有り][通常論文]
   

  Chest girth and body mass of 3,576 brown bears (Ursus arctos yesoensis) harvested for conflict management in Hokkaido, Japan during 1991-2012 were used to establish methods to assess body condition and to compare the body condition of bears by sex, month, year, and reproductive status. The body mass was estimated based on the chest girth in cases with no measurements of the bear body mass. Using the measured and estimated body mass, a growth curve by age was demonstrated to ascertain the mean asymptotical body mass (245 kg for males, 114 kg for females) and ages at 95% asymptotic body mass (14.2 years for males, 7.1 years for females). The body condition value of bears was evaluated as body mass difference (kg) between the individual body mass and the standard body mass as estimated from the growth curve. Body condition value changed seasonally with a low in summer and the highest in the autumnal hyperphagic period. Female body condition value was higher than the males during September. Fluctuation in annual body condition value was found for females however, there was no difference between solitary adult females and females with offspring (cubs, yearlings, or offspring of unknown age). No significant fluctuation was found for males. Our body condition evaluation method using chest girth and body mass of brown bears is useful to elucidate different trends across sex, year, and season.
• Serological survey of severe fever with thrombocytopenia syndrome virus infection in Sika deer and rodents in Japan

  Tapiwa Lundu, Kentaro Yoshii, Shintaro Kobayashi, Shigeru Morikawa, Toshio Tsubota, Naoaki Misawa, Daisuke Hayasaka, Hiroaki Kariwa

  Japanese Journal of Veterinary Research 66 1 21 - 28 2018年 [査読有り][通常論文]
   

  Severe fever with thrombocytopenia syndrome (SFTS) is a newly recognized zoonosis that occurs in China, Japan, and South Korea and is caused by the SFTS virus (SFTSV), which is in the genus Phlebovirus, family Phenuiviridae. Since its discovery in Japan in 2013, SFTS has been reported in the western parts of the country. To elucidate the distribution of SFTSV, we conducted a serological survey of deer and rodents. Serum was screened using enzyme-linked immunosorbent assay (ELISA) and suspected cases were further tested with an indirect immunofluorescence antibody (IFA) assay. Serum samples from 315 deer from Hokkaido (non-endemic area), 41 deer from Miyazaki (endemic area), and 910 rodents from six locations in Japan were tested. Of the 41 deer from Miyazaki, 2 (4.9%) had high ELISA optical density (OD) values (0.1 < OD < 0.3) and a positive IFA result. All of the deer samples from Hokkaido were negative by ELISA (OD < 0.1). No SFTSV-positive rodents were found. Our results indicate that deer in Miyazaki were exposed to SFTSV, unlike deer from Hokkaido (P < 0.05).
• First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan

  Mohamed Abdallah Mohamed Moustafa, Michito Shimozuru, Wessam Mohamed, Kyle Rueben Taylor, Ryo Nakao, Mariko Sashika, Toshio Tsubota

  PARASITOLOGY RESEARCH 116 8 2321 - 2325 2017年08月 [査読有り][通常論文]
   

  Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.
• Reproductive parameters and cub survival of brown bears in the Rusha area of the Shiretoko Peninsula, Hokkaido, Japan

  Michito Shimozuru, Masami Yamanaka, Masanao Nakanishi, Jun Moriwaki, Fumihiko Mori, Masakatsu Tsujino, Yuri Shirane, Tsuyoshi Ishinazaka, Shinsuke Kasai, Takane Nose, Yasushi Masuda, Toshio Tsubota

  PLOS ONE 12 4 2017年04月 [査読有り][通常論文]
   

  servation and management of wildlife. In this study, we investigated the demographic parameters, including age of primiparity, litter size, inter-birth interval, reproductive rate, and cub survival rate, of Hokkaido brown bears (Ursus arctos yesoensis) in the Rusha area on the Shiretoko Peninsula, Hokkaido, Japan, based on a long-term, individual-based monitoring survey. A total of 15 philopatric females were observed nearly every year from 2006 to 2016, and these observations were used to estimate reproductive parameters. The mean age of primiparity was 5.3 +/- 0.2 (SE) years (n = 7, 95% CI = 5.0-5.6). We observed 81 cubs in 46 litters from 15 bears. Litter size ranged from one to three cubs, and averaged 1.76 +/- 0.08 (SE) cubs/litter (95% CI = 1.61-1.91). Inter-birth intervals ranged from 1 to 4 years, and the mean value was estimated as 2.43 (95% CI = 2.16-2.76) and 2.53 (95% CI = 2.26-2.85) years in all litters and in litters that survived at least their first year, respectively. The reproductive rate was estimated from 0.70 to 0.76 young born/year/reproductive adult female, depending on the method of calculation. The cub survival rate between 0.5 and 1.5 years ranged from 60 to 73%. Most cub disappearances occurred in July and August, suggesting that cub mortality is mainly due to poor nutrition in the summer. All reproductive parameters observed in the Rusha area on the Shiretoko Peninsula fell within the range reported in Europe and North America, and were among the lowest or shortest age of primiparity, litter size, and inter-birth intervals, and ranked at a high level for reproductive rate.
• Molecular identification and characterization of piroplasm species in Hokkaido sika deer (Cervus nippon yesoensis), Japan

  Elzahara Elbaz, Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Wessam Ahmed Mohamed Mohamed, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Emad Elsayed Ahmed Younis, Sabry Ahmed El-Khodery, Toshio Tsubota

  TICKS AND TICK-BORNE DISEASES 8 5 802 - 807 2017年 [査読有り][通常論文]
   

  Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido.
• Comparison of cortisol and thyroid hormones between tuberculosis-suspect and healthy elephants of Nepal

  Sarad Paudel, Janine L. Brown, Sharada Thapaliya, Ishwari P. Dhakal, Susan K. Mikota, Kamal P. Gairhe, Michito Shimozuru, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 78 11 1713 - 1716 2016年11月 [査読有り][通常論文]
   

  We compared cortisol and thyroid hormone (T3 and T4) concentrations between tuberculosis (TB)-suspected (n=10) and healthy (n=10) elephants of Nepal. Whole blood was collected from captive elephants throughout Nepal, and TB testing was performed using the ElephantTB STAT-PAK (R) and DPP VetTB (R) serological assays that detect antibodies against Mycobacterium tuberculosis and M bovis in elephant serum. Cortisol, T3 and T4 were quantified by competitive enzyme immunoassays, and the results showed no significant differences in hormone concentrations between TB-suspect and healthy elephants. These preliminary data suggest neither adrenal nor thyroid function is altered by TB disease status. However, more elephants, including those positively diagnosed for TB by trunk wash cultures, need to be evaluated over time to confirm results.
• Development and evaluation of an interferon-gamma release assay in Asian elephants (Elephas maximus)

  Sarad Paudel, Marvin A. Villanueva, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Suraj Subedi, Nabin Rayamajhi, Mariko Sashika, Michito Shimozuru, Takashi Matsuba, Yasuhiko Suzuki, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 78 7 1117 - 1121 2016年07月 [査読有り][通常論文]
   

  We developed an interferon-gamma release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-gamma (rEIFN-gamma) as well as native interferon-gamma from the Asian elephants was performed using anti-elephant IFN-gamma rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-gamma rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-gamma in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.
• Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears

  Michito Shimozuru, Akiko Nagashima, Jun Tanaka, Toshio Tsubota

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 196 38 - 47 2016年06月 [査読有り][通常論文]
   

  Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period. (C) 2016 Elsevier Inc. All rights reserved.
• AN EPIZOOTIC OF EMERGING NOVEL AVIAN POX IN CARRION CROWS (CORVUS CORONE) AND LARGE-BILLED CROWS (CORVUS MACRORHYNCHOS) IN JAPAN

  Daisuke Fukui, Makiko Nakamura, Tsuyoshi Yamaguchi, Makiko Takenaka, Mami Murakami, Tokuma Yanai, Hideto Fukushi, Kazumi Yanagida, Gen Bando, Keita Matsuno, Masashi Nagano, Toshio Tsubota

  JOURNAL OF WILDLIFE DISEASES 52 2 230 - 241 2016年04月 [査読有り][通常論文]
   

  In 2006-10, an epizootic of emerging avian pox occurred in Carrion Crows (Corvus corone) and Large-billed Crows (Corvus macrorhynchos), leading to mortality of juvenile crows in Hokkaido, the northernmost island of Japan. We diagnosed 27 crows with proliferative skin lesions (19 carcasses and eight biopsied cases [one in zoo captivity]) as avian pox clinically, histopathologically by detection of Avipoxvirus-specific 4b core protein (P4b) gene, and epidemiologically. The fatal cases demonstrated intensively severe infection and aggressive lesions with secondary bacterial infection. Since the first identification of avian pox in Sapporo, Japan, in 2006, the frequency of mortality events has increased, peaking in 2007-08. Mortalities have subsequently occurred in other areas, suggesting disease expansion. In Sapporo, prevalence of avian pox evaluated by field censuses during 2007-12 was 17.6% (6.6-27.2%), peaked during 2007-08 and 2008-09, and then decreased. All diseased crows were juveniles, except for one adult. The number of crows assembling in the winter roosts had been stable for >10 yr; however, it declined in 2007-08, decreased by about 50% in 2008-09, and recovered to the previous level in 2009-10, correlated with the avian pox outbreak. Thus, avian pox probably contributed to the unusual crow population decline. All P4b sequences detected in six specimens in Sapporo were identical and different from any previously reported sequences. The sequence detected in the zoo-kept crow was distinct from any reported clades, and interspecies transmission was suspected. This report demonstrates an emerging novel avian pox in the Japanese avifauna and in global populations of Carrion Crows and Large-billed Crows. Longitudinal monitoring is needed to evaluate its impact on the crow population.
• Mycobacterium orygis-Associated Tuberculosis in Free-Ranging Rhinoceros, Nepal, 2015

  Jeewan Thapa, Sarad Paudel, Amir Sadaula, Yogendra Shah, Bhagwan Maharjan, Gretchen E. Kaufman, Deborah McCauley, Kamal P. Gairhe, Toshio Tsubota, Yasuhiko Suzuki, Chie Nakajima

  EMERGING INFECTIOUS DISEASES 22 3 570 - 572 2016年03月 [査読有り][通常論文]
  
• Estimation of reproductive parameters and their annual variation in brown bears of Hokkaido, Japan

  Jun Moriwaki, Michito Shimozuru, Hifumi Tsuruga, Tsutomu Mano, Toshio Tsubota

  URSUS 27 2 99 - 109 2016年 [査読有り][通常論文]
   

  The reproductive tracts of 130 adult female brown bears (Ursus arctos) in Hokkaido, Japan, euthanized during 2010-2013 for conflict management, were examined for the presence and number of corpora lutea (CL) in the ovary and placental scars (PS) in the uterine horns. We scored PS on a 0-5 scale based on morphological features including their rim and color. They were characterized as representing either current-year parturition, as determined by accompanying cubs, or previous year parturition, as determined by accompanying yearlings. In total, 128 females were divided into 3 reproductive categories: 44 females with current-year parturition, 39 with previous-year parturition, and 45 solitary females that had not given birth for >2 years. The estimated ovulation rate was 2.21 +/- 0.55 (mean +/- SD), the implantation rate was 1.91 +/- 0.74, and the litter size was 1.54 +/- 0.51. Implantation rates were lower than ovulation rates and changed annually during 2010-2013. Systematic observation of PS and CL allowed us to evaluate the reproductive history and parameters in Hokkaido brown bears and ascertain their annual variation.
• Dynamics, co-infections and characteristics of zoonotic tick-borne pathogens in Hokkaido small mammals, Japan

  Mohamed Abdallah Mohamed Moustafa, Kyle Taylor, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Roberto Rosa, May June Thu, Annapaola Rizzoli, Toshio Tsubota

  TICKS AND TICK-BORNE DISEASES 7 5 922 - 928 2016年 [査読有り][通常論文]
   

  Many of the emerging infectious diseases originate in wildlife and many of them are caused by vector borne pathogens. In Japan, zoonotic tick-borne pathogens (TBPs) are frequently detected in both ticks and wildlife. Here, we studied the infection rates of potentially zoonotic species, including Anaplasma, Ehrlichia, Neoehrlichia and Babesia spp., in Hokkaido's most abundant small mammals as they relate to variable extrinsic factors that might affect the infection rates of these pathogens. A total of 412 small mammals including 64 Apodemus argenteus, 219 Apodemus speciosus, 78 Myodes rufocanus, 41 Myodes rutilus, 6 Myodes rex and 4 Sorex unguiculatus were collected from Furano and Shari sites in Hokkaido, Japan, in 2010 and 2011 and were examined by multiplex PCR for TBPs. A reverse line blot hybridization (RLB) was then developed for the specific detection of 13 potentially zoonotic TBPs. A total of 4 TBPs were detected: Anaplasma sp. AP-sd, Ehrlichia muris, Candidatus Neoehrlichia mikurensis and Babesia microti. The infection rates were 4.4% (18/412), 1.2% (5/412), 13.1% (54/412) and 17.2% (71/412), respectively. The infection rates of each of the detected TBPs were significantly correlated with host small mammal species. A total of 22 (two triple and 20 double) co-infection cases were detected (5.3%). The most frequent co-infection cases occurred between Candidatus N. mikurensis and B. microti 68.2% (15/22). Further studies are required to examine human exposure to these zoonotic TBPs in Hokkaido. (C) 2016 Elsevier GmbH. All rights reserved.
• Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan

  Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Kyle Taylor, Ryo Nakao, Mariko Sashika, Michito Shimozuru, Toshio Tsubota

  INFECTION GENETICS AND EVOLUTION 36 268 - 274 2015年12月 [査読有り][通常論文]
   

  A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan. (C) 2015 Elsevier B.V. All rights reserved.
• 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 2015年04月 [査読有り][通常論文]
  
• Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations

  Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Shin-ichi Saga, Hideto Okuyama, Daisuke Fukui, Gen Bando, Masashi Nagano, Seiji Katagiri, Yoshiyuki Takahashi, Toshio Tsubota

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 61 1 61 - 66 2015年02月 [査読有り][通常論文]
   

  Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17 beta and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17 beta, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17 beta after copulation.
• IDENTIFICATION OF INTERSPECIFIC DIFFERENCES IN PHASE II REACTIONS: DETERMINATION OF METABOLITES IN THE URINE OF 16 MAMMALIAN SPECIES EXPOSED TO ENVIRONMENTAL PYRENE

  Aksorn Saengtienchai, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayu Kakehi, Nesta Bortey-Sam, Wageh Sobhy Darwish, Toshio Tsubota, Masanori Terasaki, Amnart Poapolathep, Mayumi Ishizuka

  ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 33 9 2062 - 2069 2014年09月 [査読有り][通常論文]
   

  Interspecific differences in xenobiotic metabolism are a key to determining relative sensitivities of animals to xenobiotics. However, information on domesticated livestock, companion animals, and captive and free-ranging wildlife is incomplete. The present study evaluated interspecific differences in phase II conjugation using pyrene as a nondestructive biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Polycyclic aromatic hydrocarbons and their metabolites have carcinogenic and endocrine-disrupting effects in humans and wildlife and can have serious consequences. The authors collected urine from 16 mammalian species and analyzed pyrene metabolites. Interspecific differences in urinary pyrene metabolites, especially in the concentration and composition of phase II conjugated metabolites, were apparent. Glucuronide conjugates are dominant metabolites in the urine of many species, including deer, cattle, pigs, horses, and humans. However, they could not be detected in ferret urine even though the gene for ferret Uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) 1A6 is not a pseudogene. Sulfate conjugates were detected mainly in the urine of cats, ferrets, and rabbits. Interestingly, sulfate conjugates were detected in pig urine. Although pigs are known to have limited aryl sulfotransferase activity, the present study demonstrated that pig liver was active in 1-hydroxypyrene sulfation. The findings have some application for biomonitoring environmental pollution. (c) 2014 SETAC
• A Genetic Method for Sex Identification of Raccoons (Procyon lotor) with Using the ZFX and ZFY Genes

  Minami W. Okuyama, Michito Shimozuru, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 76 5 773 - 775 2014年05月 [査読有り][通常論文]
   

  A genetic method for sex determination in raccoons was developed based on nucleotide differences of the zinc finger protein genes ZFX and ZFY. Four novel internal primers specific for ZFX or ZFY were designed. PCR amplification using two primer sets followed by agarose gel electrophoresis enabled sex determination. 141-bp and 447-bp bands were in both sex, and 346-bp band was specific only in male with primer set I. 345-bp and 447-bp bands were in both sex, and 141-bp band was specific only in male with primer set II, which could distinguish raccoon's electrophoresis pattern from three native carnivores in Hokkaido. This method will be useful for conservation genetics studies or biological analyses of raccoons.
• Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal

  Sarad Paudel, Susan K. Mikota, Chie Nakajima, Kamal P. Gairhe, Bhagwan Maharjan, Jeewan Thapa, Ajay Poudel, Michito Shimozuru, Yasuhiko Suzuki, Toshio Tsubota

  TUBERCULOSIS 94 3 287 - 292 2014年05月 [査読有り][通常論文]
   

  Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. (C) 2014 Elsevier Ltd. All rights reserved.
• Changes in the Immunolocalization of Steroidogenic Enzymes and the Androgen Receptor in Raccoon (Procyon lotor) Testes in Association with the Seasons and Spermatogenesis

  Minami W. Okuyama, Michito Shimozuru, Yojiro Yanagawa, Toshio Tsubota

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 2 155 - 161 2014年04月 [査読有り][通常論文]
   

  The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3 beta HSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3 beta HSD expression, suggesting that 3 beta HSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.
• 2-1.全国のクマ分布の概要，過去との比較

  佐藤喜和, 中下留美子, 坪田敏男, 中島亜美

  日本クマネットワーク（編）「ツキノワグマおよびヒグマの分布域拡縮の現況把握と軋轢防止および危機個体群回復のための支援事業」報告書 4 - 13 2014年03月 [査読無し][通常論文]
  
• Areolae of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

  Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 1 62 - 67 2014年02月 [査読有り][通常論文]
   

  In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.
• A relapsing fever group Borrelia sp similar to Borrelia lonestari found among wild sika deer (Cervus nippon yesoensis) and Haemaphysalis spp. ticks in Hokkaido, Japan

  Kyunglee Lee, Ai Takano, Kyle Taylor, Mariko Sashika, Michito Shimozuru, Satoru Konnai, Hiroki Kawabata, Toshio Tsubota

  TICKS AND TICK-BORNE DISEASES 5 6 841 - 847 2014年 [査読有り][通常論文]
   

  A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks. (C) 2014 Elsevier GmbH. All rights reserved.
• Timing of Puberty and Its Relationship with Body Growth and Season in Male Raccoons (Procyon lotor) in Hokkaido

  Minami W. Okuyama, Michito Shimozuru, Go Abe, Mariko Nakai, Mariko Sashika, Ken-Ichiro Shimada, Nobuhiro Takahashi, Daisuke Fukui, Ryohei Nakamura, Toshio Tsubota

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 4 361 - 367 2013年08月 [査読有り][通常論文]
   

  The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.
• Hematologic and biochemical parameters of free-ranging female Nile monitors in Egypt

  Mohamed Abdallah Mohamed Moustafa, Mohamed Nour-Eldin Ismail, Adel El Sayed Ahmed Mohamed, Alsagher Omran Ali, Toshio Tsubota

  Journal of Wildlife Diseases 49 3 750 - 754 2013年07月01日 [査読有り][通常論文]
   

  We report blood parameters for 18 adult and 12 juvenile, free-ranging, female Nile monitors (Varanus niloticus) in Egypt. We report differences between adult and young monitors and compare parameters to those of other reptile species. © Wildlife Disease Association 2013.
• Differential Tick Burdens May Explain Differential Borrelia afzelii and Borrelia garinii Infection Rates among Four, Wild, Rodent Species in Hokkaido, Japan

  Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 785 - 790 2013年06月 [査読有り][通常論文]
   

  The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
• Pregnancy during hibernation in Japanese black bears: effects on body temperature and blood biochemical profiles

  Michito Shimozuru, Ruriko Iibuchi, Takuro Yoshimoto, Akiko Nagashima, Jun Tanaka, Toshio Tsubota

  JOURNAL OF MAMMALOGY 94 3 618 - 627 2013年06月 [査読有り][通常論文]
   

  Bears from the family Ursidae are the only terrestrial mammals that go through gestation, parturition, and lactation during hibernation. This is the 1st study to examine the influence of reproductive status (i.e., nonpregnant, pseudopregnant, or pregnant) on body temperature and blood biochemical profiles in hibernating black bears. Pregnant bears appeared to have higher and more stable body temperatures (37-38 degrees C) than nonpregnant ones (34-36 degrees C) during pregnancy, which was followed by a rapid drop to levels comparable to those of nonpregnant individuals after parturition. In midpregnancy (i.e., January), pregnant bears had higher blood glucose and lower triglyceride concentrations than did nonpregnant ones, whereas blood concentrations of free fatty acids, glycerol, and ketone bodies did not differ significantly. Plasma urea, creatinine, and urea/creatinine levels were significantly lower in pregnant bears than in nonpregnant ones. Pseudopregnant bears showed similar changes in body temperature and blood profiles to pregnant ones, but blood glucose levels in January were significantly lower than those in pregnant bears. These results suggest that pregnant bears maintain homeothermic conditions and supply nutrients to the fetus by stimulating thermogenesis, gluconeogenesis, and urea recycling during hibernation. In addition, these physiological changes appear to be mainly caused by corpus luteum-derived factors (e.g., progesterone) but also were facilitated by placental factors.
• Differential Tick Burdens May Explain Differential Borrelia afzelii and Borrelia garinii Infection Rates among Four, Wild, Rodent Species in Hokkaido, Japan

  Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 785 - 790 2013年06月 [査読有り][通常論文]
   

  The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
• Structure and Steroidogenesis of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

  Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 2 159 - 167 2013年04月 [査読有り][通常論文]
   

  There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.
• Prevalence of Lyme Borrelia in Ixodes persulcatus Ticks from an Area with a Confirmed Case of Lyme Disease

  Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 2 215 - 218 2013年02月 [査読有り][通常論文]
   

  In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038).
• Borrelia miyamotoi Infections among Wild Rodents Show Age and Month Independence and Correlation with Ixodes persulcatus Larval Attachment in Hokkaido, Japan

  Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota

  VECTOR-BORNE AND ZOONOTIC DISEASES 13 2 92 - 97 2013年02月 [査読有り][通常論文]
   

  To clarify how Borrelia miyamotoi is maintained in the environment in Hokkaido, we examined Ixodes persulcatus for its prevalence among wild rodents and its tick vector by detecting a portion of the borrelial flaB gene in rodent urinary bladder and blood samples, and from whole ticks. We compared B. miyamotoi infection rates to Borrelia garinii and Borrelia afzelii, which are human Lyme disease pathogens also carried by wild rodents, and which are transmitted by the same vector tick. Whereas B. garinii and B. afzelii showed age dependence of infection rates among wild rodents (18.4% and 9.9% among adults and 6.0% and 3.4% among sub-adults, respectively) when looking at urinary bladder samples, B. miyamotoi infection rates were not age dependent for either blood (4.2% among adults, and 7.9% among sub-adults) or urinary bladder samples (1.0% among adults, and 1.7% among sub-adults). Moreover, while B. garinii and B. afzelii infection rates showed increases across months (June, July [p < 0.05] and August [p < 0.01] had higher rates than in May for adult rodents with B. garinii, and July and August had higher rates than in May [p < 0.01] for adult rodents with B. afzelii), B. miyamotoi infection rates did not show significant month dependence. These differences in month and age dependence led us to suspect that B. miyamotoi may not develop persistent infections in wild rodents, as B. garinii and B. afzelii are thought to. Furthermore, we examined the extent of rodent exposure to I. persulcatus nymphs and larvae throughout most of the tick's active season (May through September), and determined that B. miyamotoi infection rates in sub-adult rodents were correlated with larval burden (p < 0.01), suggesting that larvae may be very important in transmission of B. miyamotoi to wild rodents.
• Prevalence of Lyme Borrelia in Ixodes persulcatus Ticks from an Area with a Confirmed Case of Lyme Disease

  Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 2 215 - 218 2013年02月 [査読有り][通常論文]
   

  In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038).
• Epidemiological study of zoonoses derived from humans in captive chimpanzees

  Takanori Kooriyama, Michiko Okamoto, Tomoyuki Yoshida, Toshisada Nishida, Toshio Tsubota, Akatsuki Saito, Masaki Tomonaga, Tetsuro Matsuzawa, Hirofumi Akari, Hidekazu Nishimura, Takako Miyabe-Nishiwaki

  PRIMATES 54 1 89 - 98 2013年01月 [査読有り][通常論文]
   

  Emerging infectious diseases (EIDs) in wildlife are major threats both to human health and to biodiversity conservation. An estimated 71.8 % of zoonotic EID events are caused by pathogens in wildlife and the incidence of such diseases is increasing significantly in humans. In addition, human diseases are starting to infect wildlife, especially non-human primates. The chimpanzee is an endangered species that is threatened by human activity such as deforestation, poaching, and human disease transmission. Recently, several respiratory disease outbreaks that are suspected of having been transmitted by humans have been reported in wild chimpanzees. Therefore, we need to study zoonotic pathogens that can threaten captive chimpanzees in primate research institutes. Serological surveillance is one of several methods used to reveal infection history. We examined serum from 14 captive chimpanzees in Japanese primate research institutes for antibodies against 62 human pathogens and 1 chimpanzee-borne infectious disease. Antibodies tested positive against 29 pathogens at high or low prevalence in the chimpanzees. These results suggest that the proportions of human-borne infections may reflect the chimpanzee's history, management system in the institute, or regional epidemics. Furthermore, captive chimpanzees are highly susceptible to human pathogens, and their induced antibodies reveal not only their history of infection, but also the possibility of protection against human pathogens.
• Structure and Steroidogenesis of the placenta in the Antarctic Minke whale (Balaenoptera bonaerensis)

  Motoki Sasaki, Yoko Amano, Daisuke Hayakawa, Toshio Tsubota, Hajime Ishikawa, Toshihiro Mogoe, Seiji Ohsumi, Masafumi Tetsuka, Akio Miyamoto, Yutaka Fukui, Teguh Budipitojo, Nobuo Kitamura

  Journal of Reproduction and Development 59 2 159 - 167 2013年 [査読有り][通常論文]
   

  There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale. © 2013 by the Society for Reproduction and Development.
• 中部地方の野生鳥類の消化管内から得られた寄生蠕虫類

  寺口 知子, 天木 亮太, 齊藤 ゆうこ, 横畑 泰志, 井澗 美希, 坪田 敏男, 須藤 明子, 矢田 新平

  日本野生動物医学会誌 18 1 33 - 39 Japanese Society of Zoo and Wildlife Medicine 2013年 [査読有り][通常論文]
   

  1999年から2007年にかけて，中部地方産の野鳥19種21個体から吸虫，条虫各1種，線虫23種，鉤頭虫2種を得た．8種の線虫と2種の鉤頭虫を種まで同定した。トラツグミから得られたPorrocaecum ensicaudatumは新宿主報告および国内初報告となった。オオコノハズクからのThominx tenuissima，トラツグミのDispharynx emberizaeも新宿主報告となった。
• Parasitology of five primates in Mahale Mountains National Park, Tanzania

  Takanori Kooriyama, Hideo Hasegawa, Michito Shimozuru, Toshio Tsubota, Toshisada Nishida, Takashi Iwaki

  PRIMATES 53 4 365 - 375 2012年10月 [査読有り][通常論文]
   

  Parasitological surveillance in primates has been performed using coprological observation and identification of specimens from chimpanzees (Pan troglodytes schweinfurthii) in Mahale Mountains National Park, Tanzania (Mahale). In this study, we conducted coprological surveillance to identify the fauna of parasite infection in five primate species in Mahale: red colobus (Procolobus badius tephrosceles), red-tailed monkeys (Cercopithecus ascanius schmidti), vervet monkeys (Cercopithecus aethiops pygerythrus), yellow baboons (Papio cynocephalus), and chimpanzees. Fecal samples were examined microscopically, and parasite identification was based on the morphology of cysts, eggs, larvae, and adult worms. Three nematodes (Oesophagostomum spp., Strongyloides sp., and Trichuris sp.), Entamoeba coli, and Entamoeba spp. were found in all five primate species. The following infections were identified: Bertiella studeri was found in chimpanzees and yellow baboons; Balantidium coli was found in yellow baboons; three nematodes (Streptopharagus, Primasubulura, an undetermined genus of Spirurina) and Dicrocoeliidae gen. sp. were found in red-tailed monkeys, vervet monkeys, and yellow baboons; Chitwoodspirura sp. was newly identified in red colobus and red-tailed monkeys; Probstmayria gombensis and Troglocorys cava were newly identified in chimpanzees, together with Troglodytella abrassarti; and Enterobius sp. was newly identified in red colobus. The parasitological data reported for red colobus, vervet monkeys, and yellow baboons in Mahale are the first reports for these species.
• Changes in expression of hepatic genes involved in energy metabolism during hibernation in captive, adult, female Japanese black bears (Ursus thibetanus japonicus)

  Michito Shimozuru, Akari Kamine, Toshio Tsubota

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 163 2 254 - 261 2012年10月 [査読有り][通常論文]
   

  Hibernating bears survive up to 6 months without feeding by utilizing stored body fat as fuel. To investigate how bears maintain energy homeostasis during hibernation, we analyzed changes in mRNA expression of hepatic genes involved in energy metabolism throughout the hibernation period in captive, adult, female Japanese black bears (Ursus thibetanus japonicus). Real-time PCR analysis revealed down-regulation of glycolysis(e.g.. glucokinase). amino acid catabolism- (e.g., alanine aminotransferase) and de novo lipogenesis-related genes (e.g., acetyl-CoA carboxylase 1), and up-regulation of gluconeogensis- (e.g., pyruvate carboxylase), beta-oxidation- (i.e., uncoupling protein 2) and ketogenesis-related genes (i.e., 3-hydroxy-3-methylglutary-CoA synthase 2), during hibernation, compared to the active period (June). In addition, we found that glycolysis-related genes (i.e., glucokinase and pyruvate kinase) were more suppressed in the early phase of hibernation (January) compared to the late phase (March). One week after the commencement of feeding in April, expression levels of most genes returned to levels comparable to those seen in June, but beta-oxidation-related genes were still up-regulated during this period. These results suggest that the modulation of gene expression is not static, but changes throughout the hibernation period. The transcriptional modulation during hibernation represents a unique physiological adaptation to prolonged fasting in bears. (C) 2012 Elsevier Inc. All rights reserved.
• Effects of intramuscular administration of tiletamine-zolazepam with and without sedative pretreatment on plasma and serum biochemical values and glucose tolerance test results in Japanese black bears (Ursus thibetanus japonicus)

  Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota

  AMERICAN JOURNAL OF VETERINARY RESEARCH 73 8 1282 - 1289 2012年08月 [査読有り][通常論文]
   

  Objective-To establish a safe anesthetic protocol with little effect on blood biochemical values and IV glucose tolerance test (IVGTT) results in Japanese black bears (Ursus thibetanus japonicus). Animals-16 captive female Japanese black bears (5 to 17 years of age). Procedures-Bears were randomly assigned to 4 treatment groups (4 bears/group) in which various treatment combinations were administered via blow dart: tiletamine HCI and zolazepam HCI (9 mg/kg) alone (TZ), TZ (6 mg/kg) and acepromazine maleate (0.1 mg/kg), TZ (6 mg/kg) and butorphanol tartrate (0.3 mg/kg), or TZ (3 mg/kg) and medetomidine HCI (40 mu g/kg). Glucose injection for the IVGTT was started 130 minutes after TZ administration. Blood samples were obtained before, at, and intermittently after glucose injection for measurement of biochemical variables as well as plasma glucose and serum insulin concentrations during the IVGTT Rectal temperature, pulse rate, and respiratory rate were assessed every 15 minutes during the experiment. Results-Induction and maintenance of anesthesia were safely achieved with little adverse effect on cardiopulmonary function when each of the 4 anesthetic regimens was used, although mild hypothermia was induced. No difference was evident between treatment groups in blood biochemical values. Blood glucose and insulin concentration profiles during the IVGTT were similar among the bears given TZ, with or without acepromazine or butorphanol, but hyperglycemia and hypoinsulinemia developed in bears given TZ with medetomidine. Conclusions and Clinical Relevance-All 4 anesthetic regimens yielded chemical restraint without affecting clinical and biochemical values in bears, but medetomidine appeared to affect IVGTT results. For this reason, medetomidine should not be used when anesthetizing bears for IVGTTs. (Am J Vet Res 2012;73:1282-1289)
• Changes in expression of hepatic genes involved in lipid metabolism during prehibernation period in captive adult female Japanese black bears (Ursus thibetanus japonicus)

  Michito Shimozuru, Kamine Akari, Toshio Tsubota

  CANADIAN JOURNAL OF ZOOLOGY 90 8 945 - 954 2012年08月 [査読有り][通常論文]
   

  Body fat accumulation in the prehibernation period is crucial for survival and reproduction during hibernation for bears. Bear body mass increases rapidly during their autumnal hyperphagia phase, which is attributed not only to an increase in food availability, but also to physiological changes in lipid metabolism. To test this hypothesis, we investigated changes in blood biochemical values and mRNA expression levels of hepatic genes involved in lipid metabolism during the active period (June, August, October, and November) in Japanese black bears (Ursus thibetanus japonicus Schlegel, 1857), which were fed a constant ration throughout this period. Blood biochemical analysis revealed that plasma triglyceride concentrations decreased in October and November, implying that peripheral triglyceride uptake was accelerated in autumn. The liver was sampled by needle biopsy. Real-time polymerase chain reaction (PCR) analysis revealed that mRNA expressions of enzymes involved in glycolysis (glucokinase), as well as fatty acid and triglyceride synthesis (ATP-citrate lyase, acetylCoA carboxykinase 1, fatty acid synthase, and diacylglycerol O-acyltransferase 2), increased in November, which suggests that hepatic lipogenesis becomes accelerated during the hyperphagia phase. These results suggest that lipid metabolism is seasonally controlled even without changes in food intake. These physiological changes seen in the prehibernation period would contribute to the rapid mass gain necessary for hibernation.
• Effects of intramuscular administration of tiletamine-zolazepam with and without sedative pretreatment on plasma and serum biochemical values and glucose tolerance test results in Japanese black bears (Ursus thibetanus japonicus)

  Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota

  AMERICAN JOURNAL OF VETERINARY RESEARCH 73 8 1282 - 1289 2012年08月 [査読有り][通常論文]
   

  Objective-To establish a safe anesthetic protocol with little effect on blood biochemical values and IV glucose tolerance test (IVGTT) results in Japanese black bears (Ursus thibetanus japonicus). Animals-16 captive female Japanese black bears (5 to 17 years of age). Procedures-Bears were randomly assigned to 4 treatment groups (4 bears/group) in which various treatment combinations were administered via blow dart: tiletamine HCI and zolazepam HCI (9 mg/kg) alone (TZ), TZ (6 mg/kg) and acepromazine maleate (0.1 mg/kg), TZ (6 mg/kg) and butorphanol tartrate (0.3 mg/kg), or TZ (3 mg/kg) and medetomidine HCI (40 mu g/kg). Glucose injection for the IVGTT was started 130 minutes after TZ administration. Blood samples were obtained before, at, and intermittently after glucose injection for measurement of biochemical variables as well as plasma glucose and serum insulin concentrations during the IVGTT Rectal temperature, pulse rate, and respiratory rate were assessed every 15 minutes during the experiment. Results-Induction and maintenance of anesthesia were safely achieved with little adverse effect on cardiopulmonary function when each of the 4 anesthetic regimens was used, although mild hypothermia was induced. No difference was evident between treatment groups in blood biochemical values. Blood glucose and insulin concentration profiles during the IVGTT were similar among the bears given TZ, with or without acepromazine or butorphanol, but hyperglycemia and hypoinsulinemia developed in bears given TZ with medetomidine. Conclusions and Clinical Relevance-All 4 anesthetic regimens yielded chemical restraint without affecting clinical and biochemical values in bears, but medetomidine appeared to affect IVGTT results. For this reason, medetomidine should not be used when anesthetizing bears for IVGTTs. (Am J Vet Res 2012;73:1282-1289)
• Seasonal Changes in Spermatogenesis and Peripheral Testosterone Concentration in Raccoons (Procyon lotor) in Hokkaido

  Minami W. Okuyama, Michito Shimozuru, Nobuhiro Takahashi, Daisuke Fukui, Ryohei Nakamura, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 74 6 727 - 732 2012年06月 [査読有り][通常論文]
   

  Feral raccoons (Procyon lotor) have been increasing in number since 1979 and are currently subject to pest control in Hokkaido. One of the reasons for the increase in numbers is thought to be the high reproductive potential of raccoons, but little is known about their reproduction. The main aim of this study was to clarify seasonal changes in spermatogenesis and peripheral testosterone concentration of raccoons in Hokkaido. In the present study, external characteristics and histology of the testis and epididymis and the plasma testosterone concentration were investigated in 68 feral, male raccoons culled for pest control and once a month in one live, captive male. The feral males exhibited seasonal changes in spermatogenesis, showing active spermatogenesis in autumn, winter and spring (October June) with noted spermatogenesis and inactive spermatogenesis in summer (July September) with lower mean levels of spermatozoa in the cauda epididymis. Even in the inactive period, spermatozoa were observed in about half of the individuals (14/26); therefore, individuals producing spermatozoa existed every month throughout the year. Testosterone concentrations were significantly high in the winter mating season. In the captive male, the testosterone concentrations were low from June to August, and spermatozoa could not be observed from July to September. These results suggest that raccoons exhibit seasonality of reproduction, but the time and duration of spermatogenetic decline varies widely among individuals. This individual variation in the inactive period is a feature of male raccoon reproduction and is unique among seasonally breeding mammals.
• Immunolocalization of steroidogenic enzymes and their expression during the breeding season in the testes of wild raccoon dogs (Nyctereutes procyonoides)

  Qiang Weng, Toshio Tsubota, Mingdao Dai, Jiaju Weng, Yang Tian, Meiyu Xu, Gen Watanabe, Kazuyoshi Taya

  ANIMAL SCIENCE JOURNAL 83 7 535 - 542 2012年 [査読有り][通常論文]
   

  The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3 beta HSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3 beta HSD, P450c17 and P450arom was measured in the testes during the breeding season. 3 beta HSD was found in Leydig cells during the breeding and non-breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non-breeding season. The localization of P450arom changed seasonally: no immunostaining in the non-breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3 beta HSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process.
• A novel relapsing fever Borrelia sp infects the salivary glands of the molted hard tick, Amblyomma geoemydae

  Takano, Ai, Sugimori, Chieko, Fujita, Hiromi, Kadosaka, Teruki, Taylor, Kyle R., Tsubota, Toshio, Konnai, Satoru, Tajima, Tomoko, Sato, Kozue, Watanabe, Haruo, Ohnishi, Makoto, Kawabata, Hiroki

  TICKS AND TICK-BORNE DISEASES 3 4 259 - 261 2012年 [査読有り][通常論文]
   

  A novel relapsing fever Borrelia sp. was found in Amblyomma geoemydae in Japan. The novel Borrelia sp. was phylogenetically related to the hard (ixodid) tick-borne relapsing fever Borrelia app. Borrelia miyamotoi and B. lonestari. The novel relapsing fever Borrelia sp. was detected in 39 A. geoemydae (39/274: 14.2%), of which 14(14/274: 5.1%) were co-infected with the novel relapsing fever Borrelia sp. and Borrelia sp. tAG, one of the reptile-associated borreliae. Transstadial transmission of the novel relapsing fever Borrelia sp. occurred in the tick midgut and the salivary glands, although Borrelia sp. tAG was only detected in the tick midgut. The difference of the borrelial niche in molted ticks might be associated with borrelial characterization. (c) 2012 Published by Elsevier GmbH.
• The study of polycyclic aromatic hydrocarbons (PAHs) and its metabolite as a biomarker for PAH exposures in wildlife animals

  SAENGTIENCAHI Aksorn, IKENAKA Yoshinori, LEE Kyunglee, TSUBOTA Toshio, ISHIZUKA Mayumi

  日本毒性学会学術年会 39 P - 125 日本毒性学会 2012年 

  [Introduction] Polycyclic aromatic hydrocarbons (PAHs) distribute ubiquitously as complex mixtures in the environment. Pyrene is one of PAHs that is widely used as model of PAH contamination in environment. Pyrene is metabolized by cytochrome P450s in phase I reaction then conjugated in phase II metabolism, and excreted into urine and feces. Although their metabolites are available for use in human as indicator for PAH exposure, there are still less information in wildlife. In present study, we focus on the elucidation of pyrene metabolites in wild animals. [Materials and Methods] Urine of 10 male sika deer (Cervus nippon), 1 to 5 year olds were collected from Hokkaido, Japan. All samples were extracted by Oasis WAX cartridge and they were analyzed by High performance liquid chromatography with fluorescence detector. [Results and Discussions] We could detect pyrene-1-glucuronide largely in urine. Moreover, pyrene-1-sulfate, and 1-hydroxypyrene also detected. When we compared this results with other ruminant as cattle, pyrene-1-glucuronide was main metabolites same as sika deer. Pyrene conjugate compounds were easily detected in urine. Due to observe the amount of polluted chemical as PAHs in environment, we could suggest that not only elucidate PAH concentration, but also its metabolite as 1-hydroxypyrene, pyrene 1-glucuronide, and sulfate conjugates were important to establish the appropriate biomarker for the assessment for PAH exposures in various wildlife animal species in environment.
• Multilocus Sequence Typing Implicates Rodents as the Main Reservoir Host of Human-Pathogenic Borrelia garinii in Japan

  Ai Takano, Minoru Nakao, Toshiyuki Masuzawa, Nobuhiro Takada, Yasuhiro Yano, Fubito Ishiguro, Hiromi Fujita, Takuya Ito, Xiaohang Ma, Yozaburo Oikawa, Fumihiko Kawamori, Kunihiko Kumagai, Toshiyuki Mikami, Nozomu Hanaoka, Shuji Ando, Naoko Honda, Kyle Taylor, Toshio Tsubota, Satoru Konnai, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata

  JOURNAL OF CLINICAL MICROBIOLOGY 49 5 2035 - 2039 2011年05月 [査読有り][通常論文]
   

  Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
• GENETIC CHARACTERISTICS AND ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI FROM JAPANESE MACAQUES (MACACA FUSCATA) IN RURAL JAPAN

  Keiko Ogawa, Keiji Yamaguchi, Masatsugu Suzuki, Toshio Tsubota, Kenji Ohya, Hideto Fukushi

  JOURNAL OF WILDLIFE DISEASES 47 2 261 - 270 2011年04月 [査読有り][通常論文]
   

  Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to irvestigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the Wild macaque population in Shimokita Peninsula, a rural area of japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PEG E), and microbial sensitivity tests. In total, 9,18 F., colt strains were isolated from 159 fecal samples from wild macaques, and 42 E. cell were isolated from 17 samples from captive macaques. None of the virulence genes cue, six, cif, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates hail no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. cell Was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.
• Evaluation of Stored Body Fat in Nuisance-killed Japanese Black Bears (Ursus thibetanus japonicus)

  Atsushi Yamanaka, Makoto Asano, Masatsugu Suzuki, Toshio Mizoguchi, Toru Oi, Michito Shimozuru, Toshio Tsubota

  ZOOLOGICAL SCIENCE 28 2 105 - 111 2011年02月 [査読有り][通常論文]
   

  We evaluated the stored body fat of Japanese black bears (Ursus thibetanus japonicus) killed as nuisances in Gifu and Fukushima prefectures, Japan, during 2005-2007. We employed femur marrow fat (FMF), modified kidney fat index (mKFI), and abdominal subcutaneous fat (ASF) as indices for quantitative evaluation. We examined the basic characteristics of these indices, such as seasonality, age and sex dependency, and the quantitative relationship among them. mKFI and ASF increased towards the beginning of the denning period (December), while FMF was relatively stable throughout the sampling period (July-December). In cubs, all indices showed significantly lower values than in the older age classes. There seemed to be a catabolizing order between FMF and mKFI, but not between mKFI and ASF. We also evaluated the yearly change in the indices, and discussed its relevance to the incidence of bear intrusion into human residential areas. Bears nuisance-killed in summer (July-September) 2006 had a significantly larger amount of stored body fat than those killed in summer 2007, although the number of nuisance kills was larger in 2006 than in 2007. This suggests that poor nutritional condition is not a direct cause of bear intrusion.
• 獣医学における保全医学の展開－生物多様性と野生動物感染症－（緒言）

  坪田敏男, 村田浩一

  獣医畜産新報 64 1 7-8 - 32 文永堂出版 2011年01月01日 [査読無し][通常論文]
  
• エゾヒグマ（Ursus arctos yesoensis）におけるHepatozoon ursiの疫学調査

  久保正仁, 千葉依里, 飯渕るり子, 坪田敏男, 佐々木基樹, 酒井洋樹, 柳井徳磨

  Jpn.J.Zoo Wildl.Med. 15 2 111 - 113 2010年12月 [査読有り][通常論文]
  
• Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

  I. Adachi, S. Kusuda, E. Nagao, Y. Taira, M. Asano, T. Tsubota, O. Doi

  Theriogenology 74 8 1499 - 1503 2010年11月 [査読無し][通常論文]
   

  Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy. © 2010 Elsevier Inc.
• Localization of Five Steroidogenic Enzyme mRNAs in Japanese Black Bear (Ursus thibetanus japonicus) Testes During the Mating Season by In Situ Hybridization

  Ruriko Iibuchi, Michito Shimozuru, Akari Kamine, Junko Nio-Kobayashi, Toshihiko Iwanaga, Toshio Tsubota

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 2 236 - 242 2010年04月 [査読有り][通常論文]
   

  The Japanese black bear (Ursus thibetanus japonicus) is a typical seasonal breeder that has a mating season in early summer. Spermatogenesis and testicular steroidogenesis are known to develop and regress annually; however, its molecular mechanism has not yet been investigated. In the present study, we clarified the mRNA sequence of 5 steroidogenic enzymes (P450scc, 3 beta HSD, P450c17, 17 beta HSD3 and P450arom) using RT-PCR and RACE methods and the localization of these gene expressions in the bear testis using an in situ hybridization technique. The amino acid sequence deduced from each mRNA sequence had high homology with the corresponding sequences of other species and possessed a motif typical of the P450 family or short chain alcohol dehydrogenase family. Expression of P450scc, 3 beta HSD and P450c17 mRNA in interstitial tissue indicated that conversion from cholesterol to androstenedione occurs in Leydig cells. On the other hand, the mRNA of 17 beta HSD3, which plays a central role in synthesizing testosterone, was detected not only in the interstitium but also inside the seminiferous tubules, along the basement membrane. P450arom mRNAs were distributed in the seminiferous tubules. These results suggest the possibility of testosterone and estradiol-17 beta synthesis inside the seminiferous tubules in the bear testis. We expect that the results of this study will be useful for further investigation of the molecular mechanism of steroidogenic seasonality in the bear testis.
• Genetic Variation in the Harbor Seal (Phoca vitulina) and Spotted Seal (Phoca largha) Around Hokkaido, Japan, Based on Mitochondrial Cytochrome b Sequences

  Emiko Nakagawa, Mari Kobayashi, Masatsugu Suzuki, Toshio Tsubota

  ZOOLOGICAL SCIENCE 27 3 263 - 268 2010年03月 [査読有り][通常論文]
   

  The harbor seal (Phoca vitulina) and spotted seal (Phoca largha) are the main seal species around Hokkaido, Japan. While some investigations have been conducted on the ecology and morphology of these two species, there is a lack of genetic information. We studied variation in mitochondrial cytochrome b sequences in the two species. Fifteen haplotypes were observed in 39 harbor seals from Erimo, Akkeshi, and Nosappu, and 23 were observed in 31 spotted seals from Erimo, Akkeshi, Nosappu, Rausu, Yagishiri Island, and Hamamasu. Phylogenetic trees showed two harbor seal lineages: Group I contained primarily haplotypes from Erimo, and Group II contained haplotypes from Akkeshi and Nosappu. Because the Erimo population had fewer haplotypes and less nucleotide diversity than the Akkeshi and Nosappu populations, we considered it to be isolated from the others. In contrast, genetic variance within populations of spotted seals (97.3%) was far higher than that among populations (2.7%), determined by analysis of molecular variance. There were no significant difference among the spotted seal populations, indicating the absence of distinct lineages around Hokkaido. The differences in the genetic population structure between the two species could have been generated by their ecological differences. This study provides basic genetic information on these seal species and will contribute to the conservation and management of fisheries and seals throughout Hokkaido.
• Immunohistochemical Localization of Steroidogenic Enzymes in the Testis of the Sika Deer (Cervus nippon) During Developmental and Seasonal Changes

  Daisuke Hayakawa, Motoki Sasaki, Masatsugu Suzuki, Toshio Tsubota, Hiromasa Igota, Koichi Kaji, Nobuo Kitamura

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 1 117 - 123 2010年02月 [査読有り][通常論文]
   

  Testicular steroidogenesis and spermiatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P45007, 3 beta HSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3 beta HSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.
• Muscular sarcocystosis in wild carnivores in Honshu, Japan

  Masahito Kubo, Tsukasa Okano, Keiko Ito, Toshio Tsubota, Hiroki Sakai, Tokuma Yanai

  PARASITOLOGY RESEARCH 106 1 213 - 219 2009年12月 [査読有り][通常論文]
   

  A total of 65 free-living carnivores collected on Honshu Island, Japan were examined for muscular Sarcocystis species infections. Among them, 12 Japanese raccoon dogs (Nyctereutes procyonoides viverrinus), one Japanese red fox (Vulpes vulpes japonica), three Japanese martens (Martes melampus melampus), and two Japanese badgers (Meles meles anakuma) were found to have sarcocysts in their muscles. No inflammatory reactions associated with sarcocysts were observed. Ultrastructurally, the sarcocysts detected in the Japanese raccoon dogs, Japanese red fox, and Japanese martens were similar to each other, with the sarcocyst wall being thin and exhibiting minute undulations. On the other hand, the sarcocysts detected in the Japanese badgers had a thick cyst wall with numerous finger-like protrusions which contained microtubules. The species of Sarcocystis in Japanese carnivores remain to be determined. This is the first published report on muscular sarcocystosis in Japanese carnivores.
• Normal Sperm Morphology and Changes of Semen Characteristics and Abnormal Morphological Spermatozoa among Peri-mating Seasons in Captive Japanese Black Bears (Ursus thibetanus japonicus)

  Tsukasa Okano, Tetsuma Murase, Sachiko Nakamura, Takeshi Komatsu, Toshio Tsubota, Makoto Asano

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 194 - 199 2009年04月 [査読有り][通常論文]
   

  The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 +/- 0.4, 4.5 +/- 0.3, 10.4 +/- 0.7 and 69.6 +/- 3.1 mu m (mean +/- SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 +/- 0.36 ml, 7.3 +/- 0.4, 659 +/- 644 x 10(6)/ml, 214 +/- 208 x 10(6) +/- 82.9 +/- 9.6%, 89.3 +/- 9.5% and 97.0 +/- 3.2% (mean +/- SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean +/- SD%; n=23) were simply bent tail (19.9 +/- 22.6), distal droplets (13.5 +/- 11.7), proximal droplets (9.6 +/- 7.8), teratoid spermatozoa (6.7 +/- 10.7), knobbed acrosome (4.9 +/- 8.6), acrosome damage (3.7 +/- 2.8) and bent midpiece (3.7 +/- 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.
• Leptin Receptor (Ob-R) Expression in the Ovary and Uterus of the Wild Japanese Black Bear (Ursus thibetanus japonicus)

  Sachiko Nakamura, Naohito Nishii, Atsushi Yamanaka, Hitoshi Kitagawa, Makoto Asano, Toshio Tsubota, Masatsugu Suzuki

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 110 - 115 2009年04月 [査読有り][通常論文]
   

  To verify target organ(s) of leptin in the reproductive system of the Japanese black bear, we examined the expression of leptin receptor (Ob-R) protein in ovaries and uteri collected from July to December 2006 by immunohistochemical techniques. Eleven of 22 female Japanese black bears examined had corpora lutea (CLs) in their ovaries and were thought to have undergone or to have terminated delayed implantation in the early pregnancy stage. The CLs were classified into 3 types based on morphological features. The maximum diameters of Type 1 CLs ranged 3 to 7 mm, and the luteal cells contained numerous vacuoles in the cytoplasm, which suggests that this type of CL was funcional. The maximum diameters of Type 2 CLs were approximately 7 mm, and the luteal cells contained fewer vacuoles in the cytoplasm, which suggests that this type of CL was in the early stage of regression. Finally, the maximum diameters of Type 3 CLs were approximately 1 mm, and these CLs contained collagen fibers among their luteal cells, which suggests that this type of CL had regressed. The 3 types of CLs showed different reactions to Ob-R, with positive staining in Type 1, much less positive staining in Type 2 and nearly negative staining in Type 3. In bears having CLs with functional and regressive features (Type I and 2 CLs), Ob-R was also immunolocalized in the developed glandular and ductal endometrial epithelium. In contrast, the Ob-R was absent in the undeveloped endometrial epithelium in bears with regressed (Type 3 CLs) or no CLs. These findings suggested that leptin directly targets CLs and the endometrium so as to develop and maintain them during the delayed implantation period in Japanese black bears.
• Fetal Age Estimation of Hokkaido Sika Deer (Cervus nippon yesoensis) Using Ultrasonography During Early Pregnancy

  Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Shin-ichi Saga, Hideto Okuyama, Daisuke Fukui, Gen Bandou, Seiji Katagiri, Yoshiyuki Takahashi, Toshio Tsubota

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 2 143 - 148 2009年04月 [査読有り][通常論文]
   

  In sika deer, the normal method of estimating fetal age, based on fetal weight, is not applicable during the early pregnancy period. The objective of the present study was to describe the growth and development of sika deer fetuses and to establish a method for fetal age estimation during early pregnancy using ultrasonography. Five captive female Hokkaido sika deer (Cervus nippon yesoensis) were observed for estrus and mated (day 0) with an intact male. At two- or three-day intervals, fetuses were observed by rectal ultrasonographic scans until 59-61 days of gestation. The straight crown-rump length (SCRL), curved crown-rump length (CCRL), head length (HL), trunk depth (TD) and heart rate (HR) of the fetuses were measured. Linear regression equations were computed for each measurement together with fetal age. Analyses were conducted after transformation to a natural logarithm for SCRL and CCRL. All equations were significant (P<0.001), with SCRL becoming measurable earlier (day 20) than the others and yielding the best correlation (Days = -2.08+14.15 LnX: X=SCRL, Ln=natural logarithm). Therefore, we concluded that a precise estimation of fetal age in early gestation is best performed using SCRL measurements.
• Annual Changes in Serum Leptin Concentration in the Adult Female Japanese Black Bear (Ursus thibetanus japonicus)

  Toshio Tsubota, Miho Sato, Tsukasa Okano, Sachiko Nakamura, Makoto Asano, Takeshi Komatsu, Haruki Shibata, Masayuki Saito

  JOURNAL OF VETERINARY MEDICAL SCIENCE 70 12 1399 - 1403 2008年12月 [査読有り][通常論文]
   

  In the present study, assay of the serum leptin concentration of the Japanese black bear (Ursus thibetanus japonicus) was attempted using a canine-leptin-specific sandwich enzyme-linked immunosorbent assay (ELISA). The dose-response Curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September, gradually increased from September or October, and then remarkably increased in late November. We conclude that this method may be useful for measuring bear serum leptin concentration and that the serum leptin concentration changes annually with a peak in late November.
• Immunohistochemical localization of the estrogen receptor alpha (ER alpha) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) during pregnancy

  Yojiro Yanagawa, Yukiko Matsuura, Masatsugu Suzuki, Seiji Katagiri, Toshio Tsubota

  JAPANESE JOURNAL OF VETERINARY RESEARCH 56 3 139 - 149 2008年11月 [査読有り][通常論文]
   

  Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ER alpha) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestational status was unknown: one female had only one developing corpus luteum: <= Day 4 (metestrus) and two females had two corpora lutea, one of which was at the developing stage equivalent to diestrus or early pregnancy: > Day 7 (diestrus). Staining of ER(x and PR was clear in all cell types during metestrus. During diestrus, the presence of ERa was also clear in deep glandular epithelium, stroma and myometrium., whereas it was suppressed in luminal epithelium and shallow glandular epithelium. Staining of PR was suppressed in luminal epithelium but was detectable in other cell types. Staining of ERa in all cell types and PR in luminal epithelium and glandular epithelium became undetectable by Day 28. PR was presented in stroma and myometrium. throughout pregnancy. The distribution pattern of ERa and PR was different during diestrus from that in a ruminant. This could be attributed to estrogen secretion from the maturing and ovulating follicles in the presence of developed corpus luteum.
• Relationships among changes of serum leptin concentration, leptin mRNA expression in white adipose tissue (WAT), and WAT fat-cell size in female Japanese black bears (Ursus thibetanus japonicus)

  S. Nakamura, T. Okano, H. Shibata, M. Saito, T. Komatsu, M. Asano, M. Sugiyama, T. Tsubota, M. Suzuki

  CANADIAN JOURNAL OF ZOOLOGY-REVUE CANADIENNE DE ZOOLOGIE 86 9 1042 - 1049 2008年09月 [査読無し][通常論文]
   

  As a first step to study the relationship between fat accumulation and reproductive success in Japanese black bears (Ursus thibetanus japonicus Schlegel, 1857) with the focus on leptin, we determined leptin cDNA sequences in the bears. Next, we studied the possibility of white adipose tissue (WAT) as a leptin secretion source by observing the changes of leptin mRNA expression in WAT by semiquantitative real-time reverse transcript-polymerase chain reaction, the index of WAT fat-cell size, and serum leptin concentration in pregnant bears. Then, based on our results, we discussed roles of leptin in those bears. The amino acid sequences of leptin from the bears were highly identical to that of other carnivores. The expression of leptin mRNA in WAT was detected from September to January, with a tendency to increase in late November and January; the relationship between changes in the index of WAT fat-cell size and those in serum leptin concentration was high (r = 0.55, P < 0.01), with an increase in both in mid-November. These results suggested that leptin was mainly secreted from WAT in bears and that serum leptin concentrations might reflect their nutritional condition. Moreover, leptin might serve as an indicator of their fat mass, which would affect their survival during hibernation and their reproductive success.
• Regional trend and tissue distribution of brominated flame retardants and persistent organochlorines in raccoon dogs (Nyctereutes procyonoides) from Japan

  Tatsuya Kunisue, Nozomi Takayanagi, Tomohiko Isobe, Shin Takahashi, Susumu Nakatsu, Toshio Tsubota, Keisuke Okumoto, Sumio Bushisue, Kazuyuki Shindo, Shinsuke Tanabe

  ENVIRONMENTAL SCIENCE & TECHNOLOGY 42 3 685 - 691 2008年02月 [査読有り][通常論文]
   

  The present study investigated concentrations and patterns of brominated flame retardants, such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs), and persistent organochlorines (OCs) in liver and adipose tissues of raccoon dogs (RD: Nyctereutes procyonoides) collected from two metropolises and a local prefecture in Japan during 2001-2006. Relatively high concentrations of PBDEs were found in RD livers, while HBCD levels were the lowest among the measured organohalogen compounds. Among PBDE congeners, BDE 209 was predominant in RDs from all the regions, indicating that pollution derived from the technical decaBDE product is extensive across Japan. On the other hand, concentrations of tetra- to nona-BDE congeners in RDs from a metropolis were significantly higher than those from the other two regions, implying that there were regional differences in the past usage of the technical tetraBDE and octaBDE products. Such a regional difference was also observed for HBCD levels. Lipid-normalized concentration ratios of liver to adipose tissue L/A ratio) for tri to hepta-BDE congeners were lower than 1.0 in the investigated eight RDs, suggesting lipid-dependent accumulation. However, the L/A ratios of BDE 209 exceeded 1.0 in all the specimens, suggesting hepatic retention of this compound. In addition, lipid-dependent accumulation of alpha-HBCD was observed, but the L/A ratios of gamma-HBCD were greater than 1.0 in some specimens. These results indicate that Japanese RDs have been recently exposed to BDE 209 and gamma-HBCD and accumulated both these compounds preferentially in blood-rich organs, probably due to their binding to proteins and/or rapid biotransformation, as reported in experimental rodents.
• 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 2008年 [査読有り][通常論文]
  
• A case of chondrosarcoma in a free-flying Great Egret

  Masahito Kubo, Keiya Kobayashi, Toshiaki Masegi, Hiroki Sakai, Toshio Tsubota, Makoto Asano, Miki Itani, Tokuma Yanai

  JOURNAL OF WILDLIFE DISEASES 43 3 542 - 544 2007年07月 [査読有り][通常論文]
   

  A free-flying Great Egret (Ardea alba) captured in Gifu, central Japan, in May 2006 had a large mass on the right carpal joint. The tumor was diagnosed as chondrosarcoma by histopathologic examination.
• Immobilization of Japanese black bears (Ursus thibetanus japonicus) with tiletamine hydrochloride and zolazepam hydrochloride

  Makoto Asano, Toshio Tsubota, Takeshi Komatsu, Atsushi Katayama, Tsukasa Okano, Sachiko Nakamura

  JOURNAL OF VETERINARY MEDICAL SCIENCE 69 4 433 - 435 2007年04月 [査読有り][通常論文]
   

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

  Okano, T, Nakamura, S, Nakashita, R, Komatsu, T, Murase, T, Asano, M, Tsubota, T

  Jpn. J. Zoo Wildl. Med. 12 61 - 70 2007年 [査読無し][通常論文]
  
• Persistent organochlorines in raccoon dogs (Nyctereutes procyonoides) from Japan: Hepatic sequestration of oxy-chlordane

  Tatsuya Kunisue, Nozonu Takayanagi, Toshio Tsubota, Shinsuke Tanabe

  CHEMOSPHERE 66 2 203 - 211 2007年01月 [査読有り][通常論文]
   

  The present study determined the accumulation features of persistent organochlorines (OCs) such as polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB), and chlordane compounds (CHLs) in wild raccoon dogs (RDs; Nyclereutes procyonoides) collected from Kanagawa prefecture in Japan during 2001. In livers of RDs, CHLs were remarkably dominant (20 times higher than PCBs) followed by PCBs > DDTs > HCHs > HCB, whereas the chemicals in muscles were in the order of CHLs > PCBs > HCHs > DDTs > HCB. The accumulation pattern of OCs in RDs was different from those in Japanese humans and avian species reported previously, which generally accumulate higher levels of DDTs and PCBs than CHLs. This result indicates that RDs have been exposed to relatively high levels of CHLs and have high metabolic and elimination capacity for DDTs. In fact, CHL levels in RDs were higher than those in humans and some avian species, while DDT levels in RDs were much lower than other animals. In particular, extremely high accumulation levels of oxychlordane, which is a metabolite from chlordanes and nonachlors, were observed in RD livers. The higher toxic potency of oxychlordane than parent compounds may suggest that RDs are at high risk by this metabolite. On lipid weight basis, PCBs, HCHs and HCB levels were almost similar in livers and Muscles, suggesting that the tissue distribution of these compounds principally followed the lipid-dependent accumulation. However, accumulation levels of oxychlordane and p,p'-DDD in livers were significantly higher than those in muscles, and concentration ratios of liver to muscle (L/M ratios) of these Compounds were greater than 1.0 in all the specimens. This phenomenon was similar to PCDD/DF congener accumulation patterns observed previously in RDs. When relationships between hepatic TEQs and L/M ratios were examined for oxychlordane and p,p'-DDD, L/M ratios for these compounds significantly increased with hepatic TEQ levels, suggesting their hepatic sequestration in TEQs-dependent manner.
• Incidence of ovulation without coital stimuli in captive Japanese black bears (Ursus thibetanus japonicus) based on serum progesterone profiles

  Tsukasa Okano, Sachiko Nakamura, Rumiko Nakashita, Takeshi Komatsu, Tetsuma Murase, Makoto Asano, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 10 1133 - 1137 2006年10月 [査読有り][通常論文]
   

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

  Tsukasa Okano, Sachiko Nakamura, Takeshi Komatsu, Tetsuma Murase, Kiyoshi Miyazawa, Makoto Asano, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 10 1101 - 1104 2006年10月 [査読有り][通常論文]
   

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

  T. Okano, T. Murase, C. Yayota, T. Komatsu, K. Miyazawa, M. Asano, T. Tsubota

  Animal Reproduction Science 95 1-2 134 - 143 2006年09月 [査読有り][通常論文]
   

  The Japanese black bear (Ursus thibetanus japonicus) is an endangered species in some areas of Japan, and semen collection and cryopreservation are important means to preserve genetic resources and prevent extinction. The aim of the present study was to improve the methods for electroejaculation and cryopreservation in these bears. We collected the semen from captive mature Japanese black bears during the mating season by the electroejaculation method using maximum stimulus voltages of 10 V (n = 9) and 7 V (n = 15), and compared the characteristics of fresh semen after collection. None of the variables examined (i.e. total sperm count, motility, viability, morphology, and rate of urine contamination) were statistically different between the two electro-stimulus voltages. After the semen obtained was cryopreserved, the effects of three different diluents (egg yolk-TRIS-citrate-glucose, egg yolk-TRIS-citrate-fructose, and egg yolk-TRIS-citrate-fructose-lactose-raffinose n = 10) and two different incubation times (1-1 and 3-0 h of cooling-glycerol equilibration times n = 12) on frozen-thawed spermatozoa. None of the variables examined (i.e. motility, viability and morphology) were statistically different among the three diluents and between the two incubation times. The results indicate that, in the collection and cryopreservation of Japanese black bear semen: (1) a maximum voltage of 7 V may be enough to obtain ejaculates (2) the three diluents examined were useful and (3) glycerol equilibration time may be omitted with prior cooling for 3 h. © 2005 Elsevier B.V. All rights reserved.
• Immunolocalization of steroidogenic enzymes P450scc, 3 beta HSD, and P450c17 in the ovaries of wild raccoon dogs (Nyctereutes procyonoides)

  Qiang Weng, Tetsuma Murase, Makoto Asano, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 9 999 - 1002 2006年09月 [査読有り][通常論文]
   

  We investigated the distribution of 3 types of steroidogenic enzymes, P450scc, 3 beta HSD, and P450c17, in wild raccoon dog ovaries by immunohistochemistry. Six pairs of ovaries were obtained from wild raccoon dogs between 2001 and 2003, with 3 of the 6 pairs of ovaries containing corpora lutea. P450scc, 3 beta HSD, and P450c17 were localized in the granulosa and theca cells of these raccoon dogs. Furthermore, lutein cells were stained positively for P450sce and 3 beta HSD in the pregnant and non-pregnant raccoon dogs. These results suggest that granulosa and theca cells may synthesize progesterone and androgens, which may play an important role in follicular development, and that lutein cells are a major source of progesterone in wild raccoon dogs.
• 妊娠ニホンツキノワグマ(Urusus thibetanus japonicus)における体脂肪量とレプチンの繁殖への関わりの検討

  中村幸子, 岡野司, 柴田治樹, 斉藤昌之, 小松武志, 淺野玄, 坪田敏男

  J Reprod Dev 52 Supplement J115 - 142 日本繁殖生物学会 2006年08月 [査読有り][通常論文]
   

  ニホンツキノワグマ（Ursus thibetanus japonicus）（以下、クマ）は冬眠中に出産・哺育を行うため、冬眠前時期の脂肪蓄積が繁殖に影響するとされる。レプチンは主に白色脂肪組織（WAT）より分泌される抗肥満ホルモンとして知られるが、ヒトや齧歯類では着床や妊娠に関わる働きを持つとされる。演者らはこのレプチンに注目し、妊娠雌グマにおける着床時期（11月下旬～12月上旬）前後の体脂肪量とレプチン産生量を検討した。秋田県阿仁クマ牧場における妊娠雌グマ5頭を用いた。9月から翌1月までおよそ1ヶ月に1回、麻酔下で体重・体脂肪率測定、採血および脂肪採取を行った。体脂肪率はRJL Systems Quantum Xを用いて測定し、アメリカクロクマに対する換算式を適用した。ELISAにより血中レプチン濃度を測定した。WATからtotal RNAを抽出し、ワンステップリアルタイムRT-PCRでレプチンmRNA発現を定量した。体重は10月から、体脂肪量は9月下旬から有意に上昇し、ともに11月下旬に最高値を示した後、冬眠に入ってからは徐々に減少した。両者の増減の相関は0.98であった。血中レプチン濃度は、10月まで緩やかに増加し、11月中旬・下旬に有意に上昇した(p<0.05)後、減少した。WATにおけるレプチンmRNA発現量は11月下旬および1月中旬に上昇したが、有意差は認められなかった。体重増減量は体脂肪量を反映していると考えられた。血中レプチン濃度とWATレプチンmRNA発現量に明確な関連はなかったが、共に11月に上昇が認められ、WATがこの時期のレプチン産生源の一つである可能性は高い。また、脂肪蓄積時期にわたって血中レプチン濃度が上昇したことから、産生されたレプチンは摂食抑制やエネルギー代謝亢進作用ではなく、むしろ着床の誘起など、繁殖に関与していると考えられた。今後レセプターの発現など、作用機序について検討する必要がある。
• Seasonal changes in immunolocalization of inhibin/activin subunits and testicular activity in wild male raccoon dogs (Nyctereutes procyonoides)

  Qiang Weng, Mohamed S. Medan, Meiyu Xu, Toshio Tsubota, Gen Watanabe, Kazuyoshi Taya

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 52 4 503 - 510 2006年08月 [査読有り][通常論文]
   

  Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin a and inhibin/activin (beta(A) and beta(B)) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidinbiotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin u., inhibin/activin beta(A) and inhibin/activin beta(B). The inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, beta(A), and beta(B) subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and nonbreeding seasons.
• PCDDs, PCDFs, and coplanar PCBs in wild terrestrial mammals from Japan: Congener specific accumulation and hepatic sequestration

  Tatsuya Kunisue, Michio X. Watanabe, Hisato Iwata, Toshio Tsubota, Fumio Yamada, Masatoshi Yasuda, Shinsuke Tanabe

  Environmental Pollution 140 3 525 - 535 2006年04月 [査読無し][通常論文]
   

  The present study determined the contamination levels and congener-specific accumulation features of dioxins and related compounds (DRCs) in wild terrestrial mammals such as large Japanese field mice (LJFM), lesser Japanese moles (LJMs), and raccoon dogs (RDs) collected from Kanto region in Japan during 2001. The toxic equivalent quantity (TEQ) levels in the carcasses or adipose tissues were in the order of RDs ≥ LJMs > LJFM. Comparison of DRC congener profiles in the three species and principal component analysis (PCA) demonstrated a higher contribution of OCDD, T4CB77, and P 5CB118 in LJMs. Analysis of liver-adipose distribution of DRC congeners in RDs showed that livers contained significantly higher TEQs than adipose tissues, indicating that liver is a depository organ and critical for determining the toxicokinetics of DRCs. As for most T4, P 5, H6CDD/DFs and for P5CB126, H 6CB169 and mono-ortho PCB congeners, their liver/adipose concentration ratios in RDs revealed a tendency to increase with hepatic TEQ levels, suggesting TEQ-dependent hepatic sequestration. © 2005 Elsevier Ltd. All rights reserved.
• Changes in serum inhibin levels and immunolocalization of inhibin/activin subunits during the breeding season in the wild male Japanese black bear (Ursus thibetanus japonicus)

  Qiang Weng, Mohamed S. Medan, Tsukasa Okano, Tetsuma Murase, Toshio Tsubota, Meiyu Xu, Gen Watanabe, Kazuyoshi Taya

  ENDOCRINE 29 2 345 - 350 2006年04月 [査読有り][通常論文]
   

  The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of inhibin a and inhibin/activin (beta(A) and beta(B)) subunits during the breeding season in the wild male Japanese black bear. Histological observations of testes were performed and seminiferous tubule diameters were measured. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin a, inhibin/activin beta(A), and inhibin/activin beta(B) during the breeding season. Serum concentrations of immunoreactive (ir-)inhibin, testosterone, and luteinizing hormone (LH) were measured by radioimmunoassay. Higher values of seminiferous tubule diameters and all types of spermatogenic cells including mature-phase spermatozoa were found during the breeding season. There were seasonal changes in serum concentrations of ir-inhibin, testosterone, and LH. Ir-inhibin was positively correlated with testosterone, and LH. in addition, immunoreactivity of inhibin alpha, beta(A), and beta(B) subunits were also detected in Sertoli and Leydig cells during the breeding season. These results suggest that Japanese black bear testes may secrete bioactive inhibins during the breeding season and that the circulating inhibin may be a useful indicator of the testicular function in wild male Japanese black bears.
• Reproductive characteristics of brown bears on the Oshima Peninsula, Hokkaido, Japan

  T Mano, T Tsubota

  JOURNAL OF MAMMALOGY 83 4 1026 - 1034 2002年11月 [査読有り][通常論文]
   

  We investigated reproductive characteristics of brown bears (Ursus arctos yesoensis) in the Oshima Peninsula, Hokkaido, Japan, based on the study of individuals harvested from 1983 to 1987. Analyses were based on age-class and reproductive status data determined for 196 females killed during the study period. The minimum age at 1st parturition was 4 years, but frequency of reproductive success among females <6 years old was notably lower than that among females greater than or equal to6 years of age. Females <6 years old were more apt to produce single offspring and lose them during the 1st year. There appeared to be few changes in frequency of reproducing cubs among females 6-20 years of age. Females typically separated from young when young were 15-27 months old. For mature adult females, mean litter size was 1.8, and interval between births was 2.3-3.0 years. Embryo loss and neonatal mortality were uncommon.
• Sequence comparison of the dopamine receptor D4 Exon III repetitive region in several species of the order Carnivora

  M Inoue-Murayama, N Matsuura, Y Murayama, T Tsubota, T Iwasaki, H Kitagawa, S Ito

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 8 747 - 749 2002年08月 [査読有り][通常論文]
   

  It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.
• 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 1 35 - 43 日本哺乳類学会 2002年 [査読有り][通常論文]
   

  本研究は，ニホンツキノワグマ（Ursus thibetanus japonicus，以下ツキノワグマと略す）による針葉樹幹の摂食量と，他の食物の摂食量との関係ならびにツキノワグマの栄養状態の年次変動を把握することにより，クマハギ被害の発生原因を食物環境面から解明し，さらに被害防除に向けた施策を提案することを目的とした．ツキノワグマの糞内容物分析の結果，クマハギ被害の指標となる針葉樹幹の重量割合は，1998年より1999年および2000年の方が有意に高く（p<0.05)，逆にツキノワグマの重要な食物種の一つであるウワミズザクラ（Prunus grayana）の果実の重量割合は，1999年および2000年より1998年の方が有意に高かった（p<0.05)．また，ツキノワグマの血液学的検査の結果，1999年および2000年より1998年の方が，血中尿素濃度は低い傾向にあり，血中ヘモグロビン濃度は有意に高かった（p<0.05）ことより，1998年はツキノワグマの栄養状態がよかったと考えられる．以上のことから，クマハギ被害は，ツキノワグマの食物量が少なく低栄養の年に発生しやすく，ウワミズザクラの果実の豊凶が被害の発生の指標となる可能性が示唆された．したがって，クマハギ被害は，被害発生時期にツキノワグマの食物量を十分に確保する食物環境を整えることにより，その発生を抑えられる可能性があると考えられた．
• 飼育条件下におけるニホンツキノワグマ (Ursus thibetanus japonicus) の性行動の観察

  山本 かおり, 坪田 敏男, 喜多 功

  The Journal of reproduction and development 44 5 j13 - j18 THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 1998年06月01日 

  秋田県阿仁クマ牧場において1996年6月22日から7月31日まで,ニホンツキノワグマ Ursus thibetanus japonicus(成獣雄22頭, 雌17頭)の性行動を観察した．本観察場所でのニホンツキノワグマの交尾期は6月中旬から8月上旬であると考えられた．乗駕行動は不特定多数の雌雄間で見られ，交尾形態としては乱交型であった．雌はある一定の期間に集中して雄の乗駕を許容(これを発情とみなした)し，発情期間は12～35日間と大きな個体差がみられた．また，発情期間と非発情期間が不規則に繰り返される傾向があり，発情様式にも大きな個体差がみられた．雄の性行動は，雌の発情に同調して発現すると推測された．乗駕が観察された総日数は，4歳および5歳の雄において有意に少なく(p<0.05)，6歳以上の雄に関しては体重の重い雄(80kg以上)において多い傾向があった(p<0.1)．このことから，本観察場所のような高密度な飼育条件下においては，年齢や体重による優劣関係の社会構造が発達し，生理的に性成熟に達した後も，社会的要因によって交尾の機会を持たない雄が存在することが推測された．
  
• 超音波断層診断および脱脂乳中プロジェステロン測定による乳牛分娩後の生殖器の修復と受胎成績

  三宅 浩文, 大島 由美, 坪田 敏男

  日本獣医師会雑誌 46 1 12 - 18 (公社)日本獣医師会 1993年01月
• Growth, Sexual Dimorphism, and Geographical Variation of Skull Dimensions of the Brown Bear Ursus arctos in Hokkaido

  Ohdachi, S, T. Aoi, T. Mano, T. Tsubota

  Journal of the Mammalogical Society of Japan 17 1 27 - 47 一般社団法人 日本哺乳類学会 1992年 [査読有り][通常論文]
   

  The growth, sexual dimorphism, and geographical variation in skulls of 596 brown bears (Ursus arctos) hunted in Hokkaido, Japan from 1971 to 1986 were investigated. Most cranial traits continued to increase after the age of sexual maturity, and in particular skulls became progressively wider with age. Skulls of males, even those of cubs and yearlings, were larger than those of females, and the difference became significant after two years of age. Skull dimensions tended to increase from south to north, in Hokkaido, and bears from the north-eastern region possessed higher brain cases and longer mandibles relative to skull length and width, than those in the southern region.
• 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年 [査読有り][通常論文]
  

書籍

• カリブー

  Aglukark, Dorothy, Aglukark, David, Sandland, Amanda, 坪田, 敏男 
  丸善出版 2020年01月 (ISBN: 9784621304488) 21p
• ホッキョククジラ

  Karpik, Joanasie, Uehara, Sho, 坪田, 敏男 
  丸善出版 2020年01月 (ISBN: 9784621304501) 21p
• ジャコウウシ

  Niptanatiak, Allen, McLeod, Kagan, 坪田, 敏男 
  丸善出版 2020年01月 (ISBN: 9784621304495) 21p
• 生理

  坪田, 敏男 
  東京大学出版会 2020年01月 (ISBN: 9784130642637) v, 125p
• ホッキョクオオカミ

  Flaherty, William, Bigham, Sean, 坪田, 敏男 
  丸善出版 2019年12月 (ISBN: 9784621304464) 21p
• セイウチ

  Paniaq, Herve, Shannon, Ben, 坪田, 敏男 
  丸善出版 2019年12月 (ISBN: 9784621304471) 21p
• ホッキョクグマ

  Flaherty, William, Christopher, Danny, 坪田, 敏男 
  丸善出版 2019年11月 (ISBN: 9784621304440) 19p
• イッカク

  Awa, Solomon, Lim, Hwei, 坪田, 敏男 
  丸善出版 2019年11月 (ISBN: 9784621304457) 21p
• ホッキョクグマ : 生態と行動の完全ガイド

  Derocher, Andrew E., Lynch, Wayne, 坪田, 敏男, 山中, 淳史, 中下, 留美子, 中島, 亜美, Taylor, Kyle 
  東京大学出版会 2014年10月 (ISBN: 9784130602266) 280p
• 獣医学・応用動物科学系学生のための野生動物学

  村田浩一, 坪田敏男 (担当:共著)
  文永堂出版 2013年02月 (ISBN: 4830032448) 341
• 日本のクマ―ヒグマとツキノワグマの生物学

  坪田 敏男, 山崎 晃司 (担当:共編者(共編著者))
  東京大学出版会 2011年02月 (ISBN: 4130602209) 370

  
• 野生動物を指標とした環境モニタリングプロジェクト

  坪田 敏男, 淺野 玄, 岐阜大学大学院連合獣医学研究科 
  [岐阜大学大学院連合獣医学研究科] 2007年
• JBN緊急クマシンポジウム&ワークショップ報告書 : 2006年ツキノワグマ大量出没の総括とJBNからの提言

  坪田 敏男, 日本クマネットワーク(JB 
  日本クマネットワーク 2007年 (ISBN: 4990323025)
• ツキノワグマとイヌワシの棲む森プロジェクト

  坪田 敏男, 岐阜大学大学院連合獣医学研究科 
  [岐阜大学大学院連合獣医学研究科] 2007年
• 冬眠する哺乳類(共著)

  東京大学出版会 2000年
• 小動物の繁殖と新生子マニュアル(Small Animal Reproduction and Neonatologyの翻訳本)(共著)

  学窓社 2000年
• 小動物の繁殖と新生子マニュアル

  Simpson Gillian M, England, Gary C. W, Harvey Mike, 稲葉 俊夫, 坪田 敏男, 村瀬 哲磨, 津曲 茂久 
  学窓社 2000年 (ISBN: 487362097X)
• 季節繁殖(共著)

  哺乳類の生殖生物学,学窓社 1999年
• ヒグマの妊娠・出生・子育て(共著)

  動物にみる妊娠・出産・子育て,メディカ出版 1999年
• 生理 (哺乳類の生物学)

  坪田 敏男, 高槻 成紀, 粕谷 俊雄 (担当:単著)
  東京大学出版会 1998年03月 (ISBN: 4130642332) 125

  
• 小さくうんで,大きく育てる(共著)

  ヒグマの原野,フレーベル館 1998年
• 哺乳類の生物学(3)生理(単著)

  東京大学出版会 1998年
• 哺乳類の生物学 (3)生理

  東京大学出版会 1998年
• ヒグマの冬眠と繁殖生理(共著)

  日本動物大百科1哺乳類I 1996年
• クマ

  獣医繁殖学 1995年
• 野生哺乳動物の性行動:エゾヒグマの観察(共著)

  哺乳動物の生殖行動 1992年
• Sexual behavior of wild mammals : Hokkaido brown bear

  1992年

作品等

• ツキノワグマ生息実態調査報告書(共著)、岐阜県根尾村

  2000年
• 訪問紀:サンディエゴ動物園希少種繁殖センター(CRES)、J. Reprod. Dev. 46/4:(]G0008[)-(]G0009[)

  2000年
• 欧米の野生動物医学事情-2ヵ月間の研修より-、Zoo and Wildlife News 10:9-12

  2000年
• 内分泌攪乱化学物質による野生生物影響実態調査マニュアル(共著)

  1999年
• 環境ホルモンと生殖異常(単著)環境保全(岐阜大学環境保全広報)創刊号:5-10

  1999年
• 第21回総会記念講演「環境ホルモンと生殖異常」(単著)自治研ぎふ(岐阜県地方自治研究センター広報)64:12-20

  1999年
• 野生生物のダイオキシン類汚染状況調査マニュアル(共著)

  1998年
• ジャイアントパンダの繁殖に関するワークショップに参加して,岐阜県獣医師会報37:77-79

  1997年
• ニホンジカ黄体退縮物の経産指標としての有効性,岐阜県獣医師会報36:20-25

  1996年
• クマ,畜産の研究50(I):145-150

  1996年
• 天然林におけるニホンツキノワグマの生息可能密度-ブナの豊凶に伴うツキノワグマの春と秋の食性の年次変動-、第4期プロ・ナトゥーラ・ファンド助成成果報告書17-25

  1995年
• 岐阜県ツキノワグマ個体群指標調査報告書(岐阜県林政部自然環境保全課編),35pp

  1995年
• クマの繁殖生理、はばたき(神戸市立王子動物園発行)36:9-10

  1994年
• ツキノワグマによる白山高山帯の利用について,天然林におけるツキノワグマの生態調査報告書(1989-1994年):19-26

  1994年
• アメリカでの研究生活を終えて、月輪:75-78

  1994年
• グリズリーの楽園-カトマイ国立公園-、Field Note40:3-6

  1993年
• ミネソタ州のクロクマ調査、月輪5:78-81

  1993年
• アメリカでのクマ研究事情Part1-イリノイでのクマ研究-、月輪4:83-85

  1992年
• SSRに参加して-とくにアメリカでの野生動物の繁殖生理学的研究、比較内分泌学会ニュース67:7-8

  1992年
• Denali National Parkでのクマの研究と保護管理、Field Note36:13-16

  1992年
• エゾヒグマの繁殖生理、遺伝45(7):77-81

  1991年
• クマ類の繁殖特性、日本科学者会議岐阜支部会報38:3-22

  1991年
• 日本産クマ類の研究概観、高教研会誌、20:96-99

  1991年
• 野生クマへの研究アプローチ-野生動物との共存への道-、岐阜県獣医師会報、28:9-14

  1991年
• 日本産クマ類の研究紹介、岐阜県高等学校教育研究会生物教育研究部会雑誌34:8-15

  1990年
• 1989年ツキノワグマ夏季白山高山帯調査報告、月輪2:14-20

  1990年
• 1989年白川村ツキノワグマ生態調査報告(共著)、月輪2:1-11

  1990年

その他活動・業績

• エゾシカに寄生するマダニ種構成の季節変化

  清水広太郎, 山中正実, 伊藤源太, 中尾亮, 佐鹿万里子, 下鶴倫人, 坪田敏男 日本生態学会大会講演要旨(Web) 70th 2023年
• 北海道のエゾシカとヒグマにおけるマダニ寄生状況の比較:寄生数に影響を与える要因は何か?

  清水広太郎, 中尾亮, 下鶴倫人, 釣賀一二三, 白根ゆり, 山中正実, 伊藤源太, 新庄康平, 梅村佳寛, 村上拓弥, 伊集院彩暮, 坪田敏男 日本哺乳類学会大会プログラム・講演要旨集 2023 2023年
• アライグマをセンチネルとしたダニ媒介性ウイルスのマダニからの伝播リスク評価

  伊藤萌林, 松野啓太, 南川未来, 南川未来, 重野麻子, 有泉拓馬, 伊藤哲治, KOVBA Anastasiia, 沼田栄花, 大森亮介, 尾針由真, 中尾亮, 下鶴輪人, 坪田敏男, 佐鹿万里子 日本野生動物医学会大会・講演要旨集 29th 2023年
• 北海道のエゾシカに寄生するシカシラミバエの病原体の保有状況

  清水広太郎, 下鶴倫人, 佐鹿万里子, 中尾亮, 坪田敏男 日本獣医学会学術集会講演要旨集 165th (CD-ROM) 2022年
• 知床半島におけるヒグマの食性の個体差と地域性

  神保美渚, 白根ゆり, 梅村佳寛, 新庄康平, 中西将尚, 石名坂豪, 宇野裕之, 佐鹿万里子, 坪田敏男, 下鶴倫人 日本生態学会大会講演要旨(Web) 68th 2021年
• 北海道斜里町においてエゾヒグマより採集されたタカサゴキララマダニの一例

  下鶴 倫人, 新庄 康平, 崎山 智樹, 小方 昌平, 草木迫 浩大, 木下 豪太, Mohamed Wessam Mohamed Ahmed, Moustafa Mohamed Abdallah Mohamed, 松野 啓太, 伊東 拓也, 野中 成晃, 佐鹿 万里子, 坪田 敏男, 中尾 亮 日本獣医学会学術集会講演要旨集 163回 327 -327 2020年10月
• ヒグマ体毛の伸長様式:野生ヒグマの生態研究への応用に向けて

  神保美渚, 松本直也, 坂元秀行, 石名坂豪, 柳川洋二郎, 鳥居佳子, 白根ゆり, 佐鹿万里子, 坪田敏男, 下鶴倫人 日本生態学会大会講演要旨(Web) 67th 2020年
• 冬眠中のツキノワグマはどのように骨格筋機能を維持しているのか？

  宮﨑 充功, 下鶴 倫人, 坪田 敏男 体力科学 69 (1) 89 -89 2020年
• 動物たちの特殊な生存戦略を科学する:冬眠・休眠動物を対象とした研究展開 冬眠中のクマにおけるエネルギー代謝および体温調節機構

  坪田 敏男 日本獣医学会学術集会講演要旨集 162回 282 -282 2019年08月
• 夏期の食物環境の変動がヒグマの栄養状態に与える影響の解明

  白根ゆり, 山中正実, 中西將尚, 石名坂豪, 神保美渚, 佐鹿万里子, 坪田敏男, 下鶴倫人 日本哺乳類学会大会プログラム・講演要旨集 2019 2019年
• 北海道立自然公園野幌森林公園におけるアライグマ(Procyon lotor)およびエゾタヌキ(Nyctereutes procyonoides albus)の食性と生息地比較に関する研究

  尾崎彩, 佐鹿万里子, 下鶴倫人, 坪田敏男 日本哺乳類学会大会プログラム・講演要旨集 2018 2018年
• 【グローバル化・温暖化と感染症対策】節足動物媒介感染症 ライム病

  坪田 敏男 小児科臨床 70 (増刊) 2215 -2219 2017年12月
• キツネ用駆虫薬ベイトを用いたエキノコックス症対策-小面積地域への適用(2)-

  浦口宏二, 入江隆夫, 孝口裕一, 八木欣平, 佐鹿万里子, 坪田敏男 日本哺乳類学会大会プログラム・講演要旨集 2017 2017年
• キツネ用駆虫薬ベイトを用いたエキノコックス症対策―小面積地域への適用―

  浦口宏二, 入江隆夫, 孝口裕一, 八木欣平, 稲森梓, 下鶴倫人, 坪田敏男 日本哺乳類学会大会プログラム・講演要旨集 2016 101 2016年09月23日 [査読無し][通常論文]
  
• 北海道の野生動物におけるマダニ媒介性感染症の宿主-ベクター-病原体の関係 (特集 野生動物医学の新展開"Disease Ecology"とはどのような学問分野か : 第21回日本野生動物医学会大会 日本野生動物医学会IWMC2015連携公開シンポジウム)

  坪田 敏男 日本野生動物医学会誌 = Japanese journal of zoo and wildlife medicine 21 (3) 47 -51 2016年09月
• Tuberculosis in Elephants : A Zoonotic Disease at the Human-Elephant Interface (特集 野生動物医学の新展開"Disease Ecology"とはどのような学問分野か : 第21回日本野生動物医学会大会 日本野生動物医学会IWMC2015連携公開シンポジウム)

  PAUDEL Sarad, 坪田 敏男 日本野生動物医学会誌 = Japanese journal of zoo and wildlife medicine 21 (3) 65 -69 2016年09月
• エキノコックス症対策におけるベイト(駆虫薬入りエサ)の小面積散布について

  浦口宏二, 孝口裕一, 入江隆夫, 稲森梓, 下鶴倫人, 坪田敏男, 八木欣平 日本獣医学会学術集会講演要旨集 159th 341 -341 2016年08月30日 [査読無し][通常論文]
  
• 北海道におけるアライグマの遺伝子型の地理的分布に関する研究

  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 -515 2016年08月30日 [査読無し][通常論文]
  
• 動物園獣医学の現状と課題 動物園動物の福祉向上を目指して 大学における動物園獣医学教育の現状と課題

  坪田 敏男 北海道獣医師会雑誌 60 (8) 439 -439 2016年08月
• クマの冬眠と繁殖の生理・内分泌メカニズム

  坪田 敏男 日本神経麻酔集中治療学会プログラム・抄録集 20回 22 -22 2016年07月
• 南アジアはMycobacterium orygisの風土病地域である(South Asia is an endemic region for Mycobacterium orygis)

  Thapa Jeewan, 中島 千絵, Rahim Zeaur, Paudel Sarad, Shah Yogendra, Maharian Bhagwan, Poudel Ajay, Gairhe Kamal Prasad, 坪田 敏男, 鈴木 定彦 日本細菌学雑誌 71 (1) 159 -159 2016年02月 [査読無し][通常論文]
  
• 知床半島におけるヒグマの移動分散様式の解明

  白根ゆり, 山中正実, 中西將尚, 石名坂豪, 能勢峰, 葛西真輔, 白柳正隆, 増田泰, 釣賀一二三, 間野勉, 藤本靖, 長田雅裕, 佐鹿万里子, 坪田敏男, 下鶴倫人 日本哺乳類学会大会プログラム・講演要旨集 2016 2016年
• 飼育下ツキノワグマにおける冬眠前および冬眠中の体温および心拍数の変化

  坪田敏男, 鈴木信吾, 宮城太輔, 山崎晃司, 佐鹿万里子, 下鶴倫人 日本野生動物医学会大会・講演要旨集 22nd 2016年
• ゾウにおける結核：ヒトとゾウの間を伝播する人獣共通感染症

  Sarad PAUDEL, 坪田 敏男 日本野生動物医学会誌 21 (3) 65 -69 2016年 

  ゾウにおける結核は，ヒト型結核の病原体であるMycobacterium tuberculosisによって主に引き起こされる再興感染症の1つである。ゾウからヒトへの伝播が，いくつかの動物園で報告されており，公衆衛生上大きな問題となっている。ゾウの鼻腔洗浄によって得られる呼吸器系サンプルの培養が，ゾウ結核診断の最も信頼できる手法である。しかしながら，この手法の適用はあまり現実的でない。世界の動物園やゾウ飼育施設では，ゾウの結核診断法として抗体検査が開発され，広く使われている。ゾウと象使いでの定期的な結核スクリーニング検査が実施されるべきである。結核陽性の象使いはすぐに隔離され，抗結核薬が処方されるべきである。スクリーニング検査，隔離および治療はゾウとヒトと間での結核伝播を防ぐのに有効であり，飼育ゾウ－野生ゾウ間の結核感染の広がりを抑えることが絶滅危惧種である野生ゾウの保全に貢献することにつながる。

• 【感染症 いま何が起きているのか 基礎研究、臨床から国際支援まで】(第2章)感染症研究のいま メカニズムから予防・治療まで マクロ生物学からのアプローチ 生物多様性と感染症 生物多様性保全による生態系での感染リスク低下

  坪田 敏男 実験医学 33 (17) 2815 -2820 2015年11月 

  近年、医学および獣医学から生物多様性保全に貢献する学問分野として「保全医学」が台頭してきている。感染症問題もこの保全医学の視点で捉えることが大事で、有効な感染症対策を考えるには、ヒトや家畜での病因学や疫学に関する情報に加えて、野生動物を含む自然界での病原体(微生物)の生態についての知識が必要である。本稿では、ライム病および回帰熱ボレリア、アナプラズマ症、バベシア症およびエーリキア症といったダニ媒介性感染症を例にとり、動物宿主-ベクター-病原体の関係を生態学的に眺める。(著者抄録)
• ツキノワグマにおける冬眠期の皮下体温、腹腔内体温および活動量測定の試み

  鈴木 信吾, 山崎 晃司, 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 158回 449 -449 2015年08月
• 北海道のエゾタヌキとアライグマにおけるイヌジステンパーウイルス感染に関する疫学調査

  佐鹿 万里子, 阿部 豪, 郡山 尚紀, 中井 真理子, 島田 健一郎, 前田 健, 下鶴 倫人, 坪田 敏男 日本獣医学会学術集会講演要旨集 158回 450 -450 2015年08月
• 「陸のクマ」と「海のクマ」

  坪田 敏男 UP 44 (2) 29 -34 2015年02月
• 北海道におけるヒグマの行動特性と遺伝構造

  坪田 敏男, 山中 正実 哺乳類科学 55 (1) 87 -88 2015年
• One Health と保全医学 －日本野生動物医学会20 年のあゆみ－

  坪田 敏男 日本野生動物医学会誌 20 (2) 27 -33 2015年 

  1995年に設立した日本野生動物医学会は20周年を迎え，毎年の大会開催や学会誌およびニュースレターの発行に加え，認定専門医協会の設立，スチューデントセミナーコースの開催，感染症専門家の現地派遣，アジア野生動物医学会のサポート，各種ガイドラインの制定など，活発な活動を続けている。この20年間に野生動物に関連する出来事は数多くみられ，例えば環境ホルモン（内分泌かく乱化学物質）の影響，ワシ類の鉛中毒の発生，ツキノワグマの人里への大量出没，トキやコウノトリの野生復帰，高病原性鳥インフルエンザの発生などは社会の関心も高かった。これらの問題に対応する学問として保全医学が台頭し，今やOne Healthと同じコンセプトとして重要な学際的分野として注目を集めている。今後もこの分野の重要性はますます高まるものと予想され，日本野生動物医学会の発展とともに他関連組織との連携を深めながら生物多様性の保全に貢献していくことが社会から求められている。

• 2007年以前の国内野生動物における重症熱性血小板減少症候群ウイルス(SFTSV)に対する抗体調査

  堀田明豊, 木村昌伸, 坪田敏男, 中村幸子, 片山敦司, 中下留美子, 猪島康雄, 鈴木道雄, 今岡浩一, 棚林清, 藤田修, 山本美江, 宇田晶彦, 森川茂 日本獣医学会学術集会講演要旨集 157th 394 -394 2014年08月11日 [査読無し][通常論文]
  
• ネコ目動物3種における尿道カテーテル挿入による精子採取

  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 -547 2014年08月11日 [査読無し][通常論文]
  
• 知床半島ルシャ地区におけるヒグマの繁殖様式に関する研究

  下鶴 倫人, 森脇 潤, 山中 正実, 中西 將尚, 石名坂 豪, 葛西 真輔, 能勢 峰, 増田 泰, 坪田 敏男 日本獣医学会学術集会講演要旨集 157回 548 -548 2014年08月
• 北海道のsika deerおよびHaemaphysalis属ダニより検出されたRF Borrelia属(A RF Borrelia sp. found among wild sika deer and Haemaphysalis ticks in Hokkaido)

  李 景利, 高野 愛, Taylor Kyle, 佐鹿 万里子, 下鶴 倫人, 今内 覚, 川端 寛樹, 坪田 敏男 日本獣医学会学術集会講演要旨集 157回 548 -548 2014年08月
• クマ類全国分布の最前線 (The JBN Times)

  佐藤 喜和, 中下 留美子, 坪田 敏男 Bears Japan 14 (3) 5 -10 2014年03月
• Egg Trapと箱わなによるアライグマの捕獲効率の比較

  佐鹿万里子, 阿部豪, 中井真理子, 島田健一郎, 坪田敏男 日本哺乳類学会大会プログラム・講演要旨集 2014 2014年
• 腎臓周囲脂肪および大腿骨骨髄内脂肪を用いたヒグマの栄養状態評価

  森真理子, 佐鹿万里子, 下鶴倫人, 坪田敏男, 間野勉 日本哺乳類学会大会プログラム・講演要旨集 2014 2014年
• 国内に潜在すると考えられる新興回帰熱に関する疫学調査研究

  高野 愛, 川端 寛樹, 大久保 梢[佐藤], 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 156回 304 -304 2013年08月 [査読無し][通常論文]
  
• 新しい技術による野生動物テレメトリーシステムの現状

  青井 俊樹, 山崎 晃司, 坪田 敏男 哺乳類科学 53 (1) 206 -208 2013年06月30日
• 北海道の野生下におけるボレリア種の維持、小型哺乳類とダニの関係について(On the maintenance of Borrelia spp. among wildlife in Hokkaido: the relationship between small mammals and ticks)

  カイル・テイラー, 川端 寛樹, 今内 覚, 下鶴 倫人, 高野 愛, 坪田 敏男 衛生動物 64 (2) 112 -112 2013年06月
• 回帰熱病原体Borrelia miyamotoiの検出法の確立・検証、およびIxodes属ダニからの病原体検出への応用

  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 -113 2013年06月 [査読無し][通常論文]
  
• 【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年04月 [査読無し][通常論文]
   

  ハンタウイルスはげっ歯類媒介性の人獣共通感染症の病原体で、腎症候性出血熱(HFRS)またはハンタウイルス肺症候群(HPS)を引き起こす。近年の日本におけるげっ歯類のハンタウイルス感染状況を明らかにするために、1994年から2010年にかけて国内の様々な地域で捕獲された1658匹のげっ歯類の血清について、抗ハンタウイルス抗体の検出を行った。840例のRattus属げっ歯類(ドブネズミとクマネズミ)は全て抗体陰性であった。北海道以外の地域で捕獲された野生げっ歯類113例はいずれも抗体陰性であったのに対し、北海道で捕獲された705例の野性げっ歯類のうち、エゾヤチネズミの7.4%(26/352)とアカネズミの1.2%(2/168)が抗体を保有していた。(著者抄録)
• クマの生息動向と最近の被害状況

  坪田 敏男 日本獣医師会雑誌 = Journal of the Japan Veterinary Medical Association 66 (2) 131 -137 2013年02月20日 

  近年，クマが人里に出没する問題が頻繁にマスコミに取り上げられるようになった。このようにクマが日本中を騒がすようになったのは，おそらく2004年のツキノワグマ大量出没が事の始まりだったと思われる。ときに，通学路で自転車に乗った中学生が襲われたり，ただしゃがんで庭仕事をしているところを背後から襲われたり，海辺で釣りをしていたら釣った魚をクマに持っていかれたなどといった事件が起きた。人里へのクマの出没は，すでに一種の社会問題になっており，市民の関心も非常に高い。クマに対する恐怖心が膨らむ一方で，駆除されるクマに同情を寄せる市民も少なくない。人間が山を荒らした結果，クマが人里に出てくるのだと主張する人たちは，何とかクマを救いたいと声をあげている。さまざまな感情や意見が交錯する中，科学的な情報に基づいた適切な保護管理の実現は，いまだほど遠い状況にある。北米で行われているような，行政主体の保護管理システムを早く日本にも確立・定着することが肝心である。そのためには，大学でクマ対応ができる人材を育成し，保護管理を実施するポジションに専門家を配置する自治体が増えていくことが望まれる。本稿では，最近のクマの生息動向と被害状況について，彼らの生態を交えて解説していきたい。
• アライグマ精巣組織における精子形成調節機構の解析：ステロイド代謝およびテストステロン感受性との関連性の解明

  奥山 みなみ, 下鶴 倫人, 坪田 敏男 日本繁殖生物学会 講演要旨集 106 P -6-P-6 2013年 

  【目的】一般に季節繁殖動物の雄では，非繁殖期には血中テストステロン(T)濃度が低く精子形成機能が低下する。しかし，冬に交尾期を持つアライグマ(Procyon lotor)では，非繁殖期である夏にT濃度が低いにも関わらず精子形成が活発な個体がいることが報告されている。このことは，T産生と精子形成が異なる機構により調節されている可能性を示している。本研究は，ステロイド代謝酵素(P450scc，P450c17，3βHSD，P450arom) およびアンドロジェン受容体(AR)発現の季節差と個体差を解析することにより，T産生および感受性と精子形成との関連性を明らかにすることを目的とした。【方法】冬期個体(n=4)，夏期に活発な精子形成がみられた個体(n=4)および夏期に精子形成がみられなかった個体(n=6)について，血漿中T濃度をELISA法にて測定した。また，精巣組織切片の免疫染色により上記4酵素とARの発現部位を特定し，3群間での発現強度を比較した。【結果・考察】精巣組織において，P450sccおよびP450c17はLeydig細胞，3βHSDはLeydig細胞およびSertoli細胞，P450aromはLeydig細胞，Sertoli細胞および一部の精子細胞で発現が確認された。夏期個体の平均T濃度は冬期個体に比べ低値であった(夏：0.2ng/ml，冬: 2.3ng/ml)。P450sccおよびP450c17は3群間で発現強度に差は見られなかったが，3βHSDは冬期個体に比べ夏期個体で発現が弱かったことから，T産生には3βHSDの発現変化が関係していることが示唆された。またP450aromおよびARは夏期に精子形成がみられた個体では冬期個体と同程度に発現したが，夏期に精子形成がみられない個体では極めて発現が弱かったことから，夏期の精子形成維持にはP450aromおよびARが関わっており，それぞれ精子の成熟機構および精巣組織におけるテストステロン感受性の維持に関係していることが示唆された。
• 北海道のエゾタヌキにおけるイヌジステンパーウイルス感染に関する疫学調査

  佐鹿 万里子, 阿部 豪, 郡山 尚紀, 前田 健, 坪田 敏男 霊長類研究 Supplement 29 256 -256 2013年 

  【背景】近年，エゾタヌキ Nyctereutes procyonoides albusの地域個体数減少が報告されており，この原因として疥癬やジステンパーなどの感染症や外来種アライグマの影響が考えられているが，その原因は明らかとなっていない．そこで本研究では，エゾタヌキ（以下，タヌキとする）を対象に，ホンドタヌキで集団感染死が報告されているイヌジステンパーウイルス（Canine distemper virus：以下，CDVとする）について疫学調査を行った．
  【材料と方法】調査地域は，2002～ 2004年に重度疥癬タヌキが捕獲され，さらにタヌキ個体数減少も確認されている北海道立野幌森林公園を選定した．2004～ 2012年に同公園内で捕獲されたタヌキ 111頭において麻酔処置下で採血を行うと同時に，マイクロチップの挿入と身体検査を行った．血液から血漿を分離し，CDVに対する中和抗体試験を行った．
  【結果】CDVに対する抗体保有率は 2004年：44.4％，2005年：8.3%，2006年：14.3%，2007年：11.1％，2008年：7.7％，2009年：54.5％，2010年：8.3%，2011年：0%，2012年： 0%であった．また，同公園内では 2003年に 26頭のタヌキが確認されたが，2004年には 9頭にまで激減し，その後，2010年までは 10頭前後で推移していた．しかし，2011年は 18頭，2012年には 16頭のタヌキが確認され，タヌキ個体数が回復傾向を示した．
  【考察】抗体保有率は 2004年および 2009年に顕著に高い値を示したことから，同公園内では 2004年と 2009年に CDVの流行が起きた可能性が示唆された．また，2002～ 2004年には，同公園内で疥癬が流行していたことが確認されているため，同公園内では CDVと疥癬が同時期に流行したことによってタヌキ個体数が減少したと考えられた．
• 知床半島ルシャ地域におけるヒグマの血縁関係解析

  森脇 潤, 下鶴 倫人, 山中 正実, 中西 將尚, 永野 夏生, 増田 泰, 藤本 靖, 坪田 敏男 霊長類研究 Supplement 29 242 -242 2013年 

   動物が高密度に生息する地域において，集団の血縁関係を明らかにすることは，その地域を利用する個体毎の繁殖，行動および分布様式を解明する上で重要である．そこで知床半島ルシャ地区におけるヒグマの繁殖，行動および移動分散様式を解明することを目的として，個体識別調査および集団遺伝学的解析により，個体間の血縁関係を解析した．材料は，同地区でヘアートラップ，ダートバイオプシー等により収集された遺伝子材料 51頭（雄 21頭，雌 30頭）分と，周辺地区（斜里，羅臼および標津地区）で学術捕獲あるいは捕殺個体 164頭分の遺伝子材料および各種メディアの情報を利用した．遺伝子解析には 22座位のマイクロサテライト領域を利用した．その結果，ルシャ地区には 15頭の成獣メスと，その子供からなる集団が生息しており，血縁は大きく２つの母系集団に分かれていた．また，最大で 3世代が共存していた．ルシャ地区で繁殖に関与する父親は，現在までに 5頭認められ，近親交配やマルチプルパタニティーが存在することが明らかになった．ルシャ地区を利用する個体の中で，5頭の亜成獣オスが斜里および羅臼地区側へ移動分散して捕殺されていることも明らかになった．このように，高密度に生息する知床半島ルシャ地区でのヒグマ集団の血縁関係を明らかにすることは，従来の野外調査では明らかには出来ないヒグマの繁殖システムの解明に寄与するだけではなく，繁殖個体の周辺地域への移動分散を明らかにすることができる．尚，本研究はダイキン工業寄附事業 「知床半島先端部地区におけるヒグマ個体群の保護管理，及び，羅臼町住民生活圏へ与える影響に関する研究」の一環として行われたものである．
• ヒグマにとって厳しかった夏 -2012年、知床半島での事例-

  下鶴 倫人, 坪田 敏男, 森脇 潤, 山中 正実, 西將 尚, 石名 坂豪, 葛西 真輔, 白柳 正隆, 能勢 峰, 増田 泰 霊長類研究 Supplement 29 92 -92 2013年 

   2012年知床半島では，夏期に極度に痩せたヒグマがメディアで報道され話題となるなど，夏期の食物不足の深刻さが窺える状況であった．斜里町・羅臼町におけるヒグマの目撃数・駆除数は例年の約2倍近くとなり，特に 8月に目撃数・駆除数がピークとなったことは例年にない特徴であった．また，親とはぐれたであろう当歳子や，餓死とみられる死体が夏期に複数確認されたことは，例年以上に夏期の死亡率が高かったことを推測させた．これらの原因として，カラフトマスの遡上が 8月末まで遅れたことや，ハイマツ等の実りが悪かったことが考えられるが，因果関係の有無については今後検証する必要がある．知床半島先端付近に位置するルシャ地区において実施しているヒグマ個体追跡調査においても同様の状況が見てとれた．同地区に定住している成獣メスの体型を追跡すると，7月初旬までは多くの個体が軽度に肥満していたが，8月に入ると急激に削痩し，そのうちの一頭は 8月末に死体で発見された．カラフトマスの遡上が始まった 9月以降は体型が回復し，10月には全個体が充分に肥満した状態となった．成獣メスの連れ子数を追跡した結果，当歳もしくは一歳齢の子を連れた個体（全 8頭）のすべてが 7月から 9月にかけて子の数を減らしたことが明らかとなった．2011年ではこのような事例は全 9頭中 1例のみしか認められなかったことから，2012年の連れ子の離別率は顕著に高かったといえる．さらに同地区ではこれまで多くの個体が 2年周期の繁殖サイクルを示す傾向にあったが，2012年は一歳齢を連れた成獣メスが例年に比べて多く，3年周期の繁殖サイクルを示す傾向にあった．このようなことから，知床半島先端部ではカラフトマスをはじめとした夏期の食物の豊凶が，ヒグマの生存率や繁殖率に大きく影響を与えていることが示唆された．尚，本研究はダイキン工業寄付事業の一環として行われたものである．
• 尿道カテーテル法を応用したニホンザルの精液採取

  高江洲昇, 柳川洋二郎, 永野昌志, 兼子明久, 今井啓雄, 岡本宗裕, 下鶴倫人, 坪田敏男 日本野生動物医学会大会・講演要旨集 19th 72 2013年 [査読無し][通常論文]
  
• エゾシカの発達および性成熟過程における卵胞形成と卵胞でのステロイド合成能

  小林すみよ, 柳川洋二郎, 坪田敏男, 永野昌志 日本野生動物医学会大会・講演要旨集 19th 95 2013年 [査読無し][通常論文]
  
• カメキララマダニより検出された新種回帰熱ボレリアの経ステージ感染様式 ボレリア属細菌の進化の軌跡を探る

  高野 愛, 杉森 千恵子, 藤田 博己, 角坂 照貴, Taylor Kyle, 坪田 敏男, 今内 覚, 田島 朋子, 佐藤 梢, 渡邉 治雄, 大西 真, 川端 寛樹 大原綜合病院年報 52 106 -107 2012年12月 [査読無し][通常論文]
  
• カメキララマダニより検出された回帰熱群ボレリアの経ステージ感染様式

  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 154回 270 -270 2012年08月 [査読無し][通常論文]
  
• 野生動物の感染症管理にどのように取り組むべきが

  高見 一利, 渡邊 有希子, 坪田 敏男, 福井 大祐, 大沼 学, 山本 麻衣, 村田 浩一 Japanese journal of zoo and wildlife medicine = 日本野生動物医学会誌 17 (2) 33 -42 2012年06月01日 [査読無し][通常論文]
  
• 胎子期におけるトド(Eumetopias jubatus)の成長様式と形態的特徴

  條野真奈美, 小林由美, 浅沼武敏, 浅沼武敏, 坪田敏男, 桜井泰憲 北海道大学水産科学研究い報 62 (1) 1 -7 2012年03月28日 [査読無し][通常論文]
   

  胎子が出生する際の発達段階は,進化の過程で変化し,種によって多様である。新生子はその成長形態によって,早成性(precocial)と晩成性(altricial)に区別される。海生哺乳類は,総じて早成性である。本研究では,トド胎子の成長様式および形態的特徴の知見を得ることを目的とし,成長および相対成長解析,外部形態の観察,ならびに内部器官観察を行った。そして,出生後の水中と陸上の生息環境への適応という観点から,胎子の成長について考察した。
• ダニによる寄生および感染症と生物多様性保全 北海道における齧歯類の動態とボレリア細菌種の感染率(Rodent dynamics and Borrelia spp. infection rates in Hokkaido)

  Taylor Kyle, 高野 愛, 川端 寛樹, 下鶴 倫人, 坪田 敏男 日本獣医学会学術集会講演要旨集 153回 181 -181 2012年03月
• Changes in blood glucose and insulin responses to intravenous glucose tolerance tests and blood biochemical values in adult female Japanese black bears (Ursus thibetanus japonicus)

  Akari Kamine, Michito Shimozuru, Haruki Shibata, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (1) 5 -13 2012年02月 [査読無し][通常論文]
   

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

  新井智, 浜田雅史, 多屋馨子, 岡部信彦, TAYLOR Kyle, 西澤次訓, 坪田敏男, 大館智志, 森川茂 現在、国内で分離・同定できないウイルス性出血熱等の診断等の対応方法に関する研究 平成23年度 総括・分担研究報告書 50 -55 2012年 [査読無し][通常論文]
  
• Ecological application of compound-specific stable nitrogen isotope analysis of amino acids: a case study of captive and wild bears (特集 有機化合物の安定同位体比を用いた有機地球化学的研究の発展とその応用(Part 2))

  Nakashita Rumiko, Suzuki Yaeko, Akamatsu Fumikazu, Naito Yuichi I, Sato-Hashimoto Miho, Tsubota Toshio Researches in organic geochemistry 27 (0) 73 -79 2011年09月30日 [査読無し][通常論文]
   

  Several recent studies have suggested an innovative method for estimating the food sources, feeding habits, and trophic position of consumers in food webs, based on the compound-specific stable isotope analysis (CSIA) of amino acids. In this study, we used CSIA to study terrestrial mammals in a controlled feeding experiment with captive Asiatic black bears (Ursus thibetanus). The trophic position is estimated to be 1.7-1.9 for the bears, which is consistent with their actual trophic position (approximately 2). We also investigated the feeding habits of six wild Asiatic black bears (the same...
• クロミンククジラ胎子精巣のステロイドホルモン調節機構

  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 -410 2011年08月10日 [査読無し][通常論文]
  
• 北海道のエゾタヌキにおける疥癬の疫学調査について

  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 -409 2011年08月10日 [査読無し][通常論文]
  
• 日本のげっ歯類のハンタウイルス感染状況に関する血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの遺伝的多様性

  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 -415 2011年08月 [査読無し][通常論文]
  
• 日本のげっ歯類のハンタウイルス感染状況に関する血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの遺伝的多様性

  尾崎 由佳, 萩谷 友洋, 真田 崇弘, 瀬戸 隆弘, Kyle Taylor, 吉川 佳佑, Leonid Ivanov, 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 152回 256 -256 2011年08月 [査読無し][通常論文]
  
• クマの大量出没の原因を明らかにして,人身被害を防ぐ

  大井徹, 中下留美子, 岡輝樹, 大西尚樹, 高橋裕史, 正木隆, 阿部真, 宮本麻子, 佐野真琴, 坪田敏男, 山崎晃司 森林総合研究所研究成果選集 2011 22 -23 2011年07月 [査読無し][通常論文]
  
• 道東・標津町における携帯電話を活用したリアルタイム通信によるヒグマ行動追跡

  坪田敏男, 平野沙衣子, 藤本靖, 上野洋一, 長田雅裕, 郡山尚紀, 下鶴倫人 日本生態学会大会講演要旨集 58th 211 2011年03月08日 [査読無し][通常論文]
  
• ツキノワグマからのCytauxzoon原虫の検出と分離

  陳内 理生, 前橋 克彦, 平田 晴之, 岩崎 いづみ, 中嶋 瑠衣, 藤澤 幸平, 寺田 裕, 下鶴 倫人, 坪田 敏男, 石原 智明 獣医寄生虫学会誌 9 (2) 168 -168 2011年03月
• Estimating the success rate of ovulation and early litter loss rate in the Japanese black bear (Ursus thibetanus japonicus) by examining the ovaries and uteri

  Atsushi Yamanaka, Kiyoshi Yamauchi, Tsunenori Tsujimoto, Toshio Mizoguchi, Toru Oi, Seigo Sawada, Michito Shimozuru, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 59 (1) 31 -39 2011年02月 [査読無し][通常論文]
   

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

  川端 寛樹, 及川 陽三郎, 川森 文彦, 熊谷 邦彦, 三上 稔之, 花岡 希, 安藤 秀二, 本田 尚子, Kyle Taylor, 坪田 敏男, 今内 覚, 高野 愛, 渡邉 治雄, 大西 真, 中尾 稔, 増沢 俊幸, 高田 伸弘, 矢野 泰弘, 石畝 史, 藤田 博己, 伊東 拓也 日本衛生動物学会全国大会要旨抄録集 63 (0) 69 -69 2011年 [査読無し][通常論文]
   

  Multi-locus sequence typing of <I>Borrelia garinii</I> isolates from humans in Japan, and comparison with environmental isolates was performed. All isolates could be divided into two groups, and an association was detected between clinical and rodent isolates, suggesting that Japanese clinical isolates of <I>B.garinii</I> come from rodents via ticks.
• 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 国内生態系におけるライム病ボレリアの維持伝播経路に関する研究

  川端寛樹, 石ぐろ史, 藤田博己, 高田伸弘, 矢野泰弘, 増澤俊幸, 中尾稔, 伊東拓也, 及川陽三郎, TAYLOR Kyle, 坪田敏男, 今内覚, 川森文彦, 三上稔之, 熊谷邦彦, 高野愛, 安藤秀二, 花岡希, 本田尚子, 渡邉治雄, 大西真 ワンヘルス理念に基づく動物由来感染症制御に関する研究 平成22年度 総括・分担研究報告書 17 -20 2011年 [査読無し][通常論文]
  
• エゾヒグマ(Ursus arctos yesoensis)におけるHepatozoon sp.寄生について

  久保 正仁, 千葉 依里, 飯渕 るり子, 坪田 敏男, 佐々木 基樹, 酒井 洋樹, 柳井 徳磨 日本野生動物医学会誌 15 (2) 111 -113 2010年09月
• ツキノワグマからのCytauxzoon原虫の検出と分離

  陳内 理生, 前橋 克彦, 平田 晴之, 岩崎 いづみ, 中嶋 瑠衣, 藤澤 幸平, 寺田 裕, 下鶴 倫人, 坪田 敏男, 石原 智明 日本獣医学会学術集会講演要旨集 150回 192 -192 2010年09月
• ヒグマ・トランクキットを用いた普及啓発活動(北海道) (This number 人里に出没するクマ対策の普及啓発および地域支援事業)

  坪田 敏男, 植木 玲一, 坪田 あゆみ Bears Japan 11 (1) 10 -13 2010年07月 [査読無し][通常論文]
  
• 【クマの保全医学の研究動向】ニホンツキノワグマにおける繁殖と母体栄養状態の関連性

  中村 幸子, 岡野 司, 柴田 治樹, 山中 淳史, 小松 武志, 淺野 玄, 坪田 敏男, 鈴木 正嗣 獣医畜産新報 63 (5) 361 -365 2010年05月 

  冬眠中に出産および授乳を行うツキノワグマ(以下、クマ)において、秋の脂肪蓄積量がクマの着床や繁殖率に影響を与えることが知られている。しかし、クマの栄養状態がどの様なメカニズムや要因、または作用により繁殖に影響を与えるかについては、未だ十分に解析されていない。本研究では、脂肪と繁殖の両者に関与するホルモンであるレプチンに着目した結果、クマにおいてレプチンは白色脂肪組織(WAT)より分泌され、クマの雌生殖器(機能黄体および子宮内膜)に直接作用することが明らかとなった。冬眠前の脂肪蓄積が十分ではなかった場合、クマの繁殖は抑制される生理学的メカニズムの存在が示唆された。(著者抄録)
• 【クマの保全医学の研究動向】ニホンツキノワグマにおける精子形成の季節変化の制御機序

  飯渕 るり子, 加味根 あかり, 下鶴 倫人, 坪田 敏男 獣医畜産新報 63 (5) 371 -374 2010年05月 

  本論は、筆頭著者が4年間の博士論文研究として行った、雄ツキノワグマの精子形成の季節性と、その季節性を形成する繁殖生理メカニズムについてとりまとめたものである。精子形成が開始される冬眠中を中心として、繁殖関連ホルモンの動態や精巣におけるテストステロン合成の変化に関与する因子について、先行研究なども含めて紹介する。(著者抄録)
• 【クマの保全医学の研究動向】Hepatozoon ursi ツキノワグマにおける新種の住血原虫

  久保 正仁, 宇仁 茂彦, 吾妻 健, 坪田 敏男, 酒井 洋樹, 柳井 徳磨 獣医畜産新報 63 (5) 379 -382 2010年05月 

  感染症のコントロールは野生動物の保全医学における重要課題の1つであり、そのためにはまず対象動物に感染する病原体について知る必要がある。クマ類における病原体の情報はまだまだ乏しいが、近年、ニホンツキノワグマ(Ursus thibetanus japonicus)から新種の住血原虫であるHepatozoon ursiが報告された。本稿では、今日までに明らかとなったH.ursiの寄生虫学的な特徴や病原性について紹介する。(著者抄録)
• ニホンツキノワグマにおける冬眠前の脂肪蓄積メカニズム (特集 クマの保全医学の研究動向)

  加味根 あかり, 下鶴 倫人, 坪田 敏男 獣医畜産新報 63 (5) 367 -370 2010年05月 [査読無し][通常論文]
  
• リス科動物精巣の機能形態学的研究 ミケリス(Callosciurus prevostii)、バナナリス(Callosciurus notatus)、オレンジクサビオモモンガ(Hylopetes Lepidus)

  段 麻優子, 佐々木 基樹, Budipitojo Teguh, 遠藤 秀紀, 木村 順平, 押田 龍夫, 坪田 敏男, 北村 延夫 日本獣医学会学術集会講演要旨集 149回 214 -214 2010年03月
• Changes in plasma gonadotropins, inhibin and testosterone concentrations and testicular gonadotropin receptor mRNA expression during testicular active, regressive and recrudescent phase in the captive Japanese black bear (Ursus thibetanus japonicus)

  Ruriko Iibuchi, Akari Kamine, Michito Shimozuru, Junko Nio-Kobayashi, Gen Watanabe, Kazuyoshi Taya, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (4) 185 -196 2010年02月 [査読無し][通常論文]
   

  Male Japanese black bears (Ursus thibetanus japonicus) have an explicit reproductive cycle. The objective of this study was to clarify the variation of plasma testosterone, FSH, inhibin, LH levels and testicular gonadotropin receptor mRNA expression of male bears associated with their testicular activity. Notably, this study investigated peripheral FSH concentration and localization of gonadotropin receptor mRNAs for the first time in male bears. Blood and testicular tissue samples were taken from captive, mature, male Japanese black bears during testicular active, regressive and recrudescent phases. Plasma hormone concentrations were measured by immunoassays, and gonadotropin receptor mRNA expression in the testis was investigated by in situ hybridization technique and also by real-time PCR. There were significant variations in plasma testosterone and inhibin concentrations. Changes in FSH concentration preceded these hormones with a similar tendency. Hormones started to increase during denning, and achieved the highest values at the end of the recrudescent phase for FSH and in the active phase for testosterone and inhibin. These changes in hormone concentrations were accompanied by testicular growth. In situ hybridization analysis revealed that FSH and LH receptor mRNA was possibly expressed in Sertoli cells and Leydig cells, respectively, as they are in other mammals. However, neither plasma LH concentration nor testicular gonadotropin receptor mRNA expression level varied significantly among the sampling months. These results suggest that FSH, inhibin and testosterone have roles in testicular activity in male bears. This study provides important endocrine information for comprehending seasonal reproductivity in male Japanese black bears.
• Immunohistochemical Localization of Steroidogenic Enzymes in the Testis of the Sika Deer (Cervus nippon) During Developmental and Seasonal Changes

  Daisuke Hayakawa, Motoki Sasaki, Masatsugu Suzuki, Toshio Tsubota, Hiromasa Igota, Koichi Kaji, Nobuo Kitamura JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 (1) 117 -123 2010年02月 [査読無し][通常論文]
   

  Testicular steroidogenesis and spermiatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P45007, 3 beta HSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3 beta HSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.
• 動物由来感染症の生態学的アプローチによるリスク評価等に関する研究 国内野生動物におけるFrancisella tularensis(野兎病菌)の保有状況

  棚林清, 堀田明豊, 山本美江, 藤田修, 宇田晶彦, 進藤順治, 朴天鎬, 小山田敏文, 畑井仁, 工藤上, 坪田敏男, 苅和宏明, 青木美樹子, 溝口俊夫, 中下留美子, 中村幸子, 片山敦司 動物由来感染症の生態学的アプローチによるリスク評価等に関する研究 平成21年度 総括・分担研究報告書 2010年
• 北海道における雄アライグマの性成熟時期の解明

  奥山 みなみ, 福井 大祐, 中村 亮平, 高橋 伸広, 下鶴 倫人, 坪田 敏男 日本繁殖生物学会 講演要旨集 103 43 -43 2010年 

  【目的】アライグマ(Procyon lotor)は、北海道において外来種として個体数を増やし分布域を広げてきている。その一因として高い繁殖力をもつ可能性が考えられるが、アライグマにおける繁殖生理には未だ解明されていない点が多い。特に、様々な種において個体の分散は若齢雄から始まるとされており、分布域拡大の要因を探る際に雄の性成熟時期の特定は不可欠といえる。そのため本研究では、北海道の雄アライグマがいつ性成熟を迎えるのかを明らかにすることを目的とした。【方法】2008年5月～2010年4月に北海道内で捕獲殺処分された234頭の野生雄アライグマを用いた。歯根の年齢線により区分された0、1、2および3歳以上の4グループにおいて、各月ごとに外見的特徴(精巣サイズ、陰茎露出の可否)、精巣組織所見(精細管径、精細胞発達ステージ、精巣上体管腔内精子存在率)を調べた。また、飼育下の0歳齢個体1例について、2008年12月～2010年3月までの毎月、精巣組織所見を調べた。【結果】野生の雄アライグマでは3～5月の出産期に生まれた後、体重増加と共に精巣重量が増加した。1歳の5月までは精細管内には精原細胞のみが認められた。その後、精巣重量は急増し、精細管径も大きく増大した。精原細胞は減数分裂を始め、10月には精子形成が認められた。また、ほとんどの個体で１歳の5～10月の間に陰茎が包皮から露出可能な状態になっていた。以上より、北海道では多くの個体で１歳の5月から春機発動が始まり、1歳の冬の交尾期には精子形成が完成すると考えられる。一方、個体によっては1歳に至る前に精子形成が観察されたものが数例存在した。これらの個体は農家の納屋などで捕獲されており、他の0歳齢個体と比べ体格が大きかったことからも、十分に餌資源を確保できる環境で生息していた可能性が高い。また、飼育下の0歳齢個体は生後1年足らずで精子形成が確認された。以上より、十分な餌資源のもとではより早期の精子形成が可能になる例があることも明らかになった。
• Growth variation in skull morphology of Kuril harbor seals (Phoca vitulina stejnegeri) and spotted seals (Phoca largha) in Hokkaido, Japan

  Emiko Nakagawa, Mari Kobayashi, Masatsugu Suzuki, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (3) 147 -162 2009年11月 [査読無し][通常論文]
   

  We examined morphological growth variations in skull features between the Kuril harbor seal and the spotted seal in Hokkaido, Japan. Skulls from 80 Kuril harbor seals and 41 spotted seals were collected, and we measured 29 metric and 6 non-metric cranial characteristics. Three growth classes were defined according to the postnatal developmental stage: pups (0 year), subadults (1-4 years old) and adults (more than 5 years old). We detected sexual dimorphism in Kuril harbor seal pups, subadults, and adults. Although interspecies differences were detected in each growth class, Kuril harbor seals were larger and more massive than spotted seals; this feature was already detectable in pups. We did not detect certain cranial characteristics with which to identify the two species, but it was possible to identify any unknown specimens to their species, sex, and growth class using the cranial data generated in this study. Using 6 non-metric cranial characteristics, we identified significant interspecies differences with regard to the shape of the temporozygomatic suture and the extent of the nasal-incisive suture; the shape of the temporozygomatic suture and the shape of the nares were indicators of growth class in Kuril harbor seals. Although non-metric cranial characteristics have a lower discriminating power than metric characteristics, they are easy to use in the field even by inexperienced researchers.
• 出没をめぐるツキノワグマの生理(<特集>クマ出没の生物学)

  山中 淳史, 坪田 敏男 森林科学 : 日本林学会会報 0 (57) 9 -12 2009年10月01日 [査読無し][通常論文]
  
• エゾシカにおけるPAGを用いた早期妊娠診断方法の検討

  柳川 洋二郎, 宮澤 千鶴, 松浦 友紀子, 下鶴 倫人, Noelita Melo de Sousa, Beckers Jean-Francis, 片桐 成二, 坪田 敏男 北海道獣医師会雑誌 53 (8) 109 -109 2009年08月
• キタキツネ(Vulpes vulpes schrencki)精巣の構造および機能に関する免疫組織化学的研究

  横山 知里, 佐々木 基樹, 坪田 敏男, 北村 延夫 北海道獣医師会雑誌 53 (8) 109 -109 2009年08月
• Change in body weight of mothers and neonates and in milk composition during denning period in captive Japanese black bears (Ursus thibetanus japonicus)

  Ruriko Iibuchi, Noriko Nakano, Tadashi Nakamura, Tadasu Urashima, Michito Shimozuru, Tetsuma Murase, Toshio Tsubota JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (1) 13 -22 2009年05月 [査読無し][通常論文]
   

  Japanese black bears, Ursus thibetanus japonicus, have been classified as a vulnerable species so that data on reproduction are needed to maintain and/or extend their population. They are known to have a peculiar style of reproduction, giving birth to their neonates and raising them during denning, a period of complete fasting. In this study, we investigated the metabolic rate and milk composition of mother bears raising neonates, and the changes in body weight of the neonates under captive conditions. Seven female bears kept in dens were weighed once a month, and the amount of energy they used was calculated. From birth, cubs were also weighed and their growth rate was determined. In addition, the milk composition was analyzed to investigate its characteristics. As a result, it was found that mother bears used 34% more energy than did solitary females. There was no significant difference in the energy used for nursing whether they had single or twin cubs. On the other hand, the body weight gain of single cubs was significantly higher than that of twin cubs, suggesting that the growth of the cubs was highly affected by the suppression of mother's energy consumption during the fasting period. The milk had high fat and low sugar concentrations. This indicates that mother bears used the fat accumulated prior to denning for their main energy source when raising cubs. Considering all results together, Japanese black bears showed remarkable efficiency in the use of energy for reproduction during the fasting period.
• 1998年以降の道内獣医大学における野生動物医学教育の動向

  浅川 満彦, 佐々木 基樹, 坪田 敏男 北海道獣医師会雑誌 53 (4) 179 -184 2009年04月
• 若手研究者によるクマの保全医学研究 雌の繁殖成功に関わる脂肪蓄積のメカニズムとその役割 ニホンツキノワグマにおける繁殖と母体栄養状態の関連性

  中村 幸子, 岡野 司, 柴田 治樹, 山中 淳史, 小松 武志, 淺野 玄, 坪田 敏男, 鈴木 正嗣 日本獣医学会学術集会講演要旨集 147回 193 -193 2009年03月
• 若手研究者によるクマの保全医学研究 雌の繁殖成功に関わる脂肪蓄積のメカニズムとその役割 ニホンツキノワグマにおける冬眠前の脂肪蓄積に関する代謝メカニズムの解明

  加味根 あかり, 中村 幸子, 飯渕 るり子, 山中 淳史, 柴田 治樹, 下鶴 倫人, 坪田 敏男 日本獣医学会学術集会講演要旨集 147回 193 -193 2009年03月
• 若手研究者によるクマの保全医学研究 雄の季節繁殖性のメカニズムと人工繁殖 ツキノワグマにおける精子形成の季節変化とその制御機構

  飯渕 るり子, 岩永 敏彦, 下鶴 倫人, 坪田 敏男 日本獣医学会学術集会講演要旨集 147回 194 -194 2009年03月
• 若手研究者によるクマの保全医学研究 クマに伝播する寄生虫感染症 野生ニホンツキノワグマ(Ursus thibetanus japonicus)におけるクマフィラリア(Dirofilaria ursi)及びクマ回虫(Baylisascaris transfuga)の病理学的及び分子生物学的検索

  辻 一, 柳井 徳磨, 坪田 敏男, 吾妻 健, 宇仁 茂彦, 酒井 洋樹, 柵木 利昭 日本獣医学会学術集会講演要旨集 147回 195 -195 2009年03月
• 若手研究者によるクマの保全医学研究 クマに伝播する寄生虫感染症 Hepatozoon ursi:ツキノワグマにおける新種の住血原虫

  久保 正仁, 宇仁 茂彦, 吾妻 健, 坪田 敏男, 佐々木 基樹, 酒井 洋樹, 柵木 利昭, 柳井 徳磨 日本獣医学会学術集会講演要旨集 147回 195 -195 2009年03月
• 地域支援事業作業部会報告 (This number 経過報告 人里に出没するクマ対策の普及啓発および地域支援事業)

  坪田 敏男 Bears Japan 9 (3) 64 -67 2009年02月 [査読無し][通常論文]
  
• ツシマヤマネコ（Prionailurus bengalensis euptilurus）の繁殖内分泌モニタリングのための糞中性ステロイドホルモン代謝物の定量と定性

  足立 樹, 楠田 哲士, 永尾 英史, 平良 由美子, 淺野 玄, 坪田 敏男, 土井 守 日本繁殖生物学会 講演要旨集 102 1023 -1023 2009年 

  【目的】ツシマヤマネコは国内で最も絶滅のおそれの高い哺乳類の一種であり、現在4施設で飼育下繁殖の取り組みが行われている。そこで本種の繁殖生理の解明を目的として、糞中の性ステロイドホルモン代謝物の測定による非侵襲的な内分泌動態のモニタリングを行った。さらにその測定法の有効性を検討するため、糞中の性ステロイドホルモン代謝物の定性分析を行った。【方法】福岡市動物園で飼育されていた雌のツシマヤマネコ1頭を対象として、2004～2006年の交尾期および妊娠期を含む時期に糞を採取した。糞は凍結乾燥後に80％メタノールで性ステロイドホルモン代謝物を抽出し、エストラジオール-17β（E₂）およびプロジェステロン（P₄）抗体を用いた酵素免疫測定法によりそれぞれの代謝物を定量した。また、代謝物を明らかにするため、E₂含量の高い期間および妊娠期間中の糞をメタノール抽出してSep-Pak C18カートリッジを用いて精製し、E₂およびP₄代謝物の分離を逆相HPLCにより行った。回収したフラクション中のE₂およびP₄代謝物の免疫反応性のピークを、E₂代謝物2種およびP₄代謝物6種の標準物質の溶出時間と比較した。【結果】糞中E₂代謝物含量は、交尾前に明確な上昇を示し、糞中P₄代謝物含量は、交尾日以降顕著な上昇を示した。糞中E₂代謝物の定性分析の結果、E₂およびエストロン（E₁）の溶出位置と一致する明確なピークが認められた。妊娠期の糞中にはP₄はわずかで、5α-pregnane-3α-ol-20-one、5α-pregnane-3β-ol-20-oneおよび5α-pregnane-3α、20-dioneの溶出位置と一致する明確なピークが認められた。以上の結果から、ツシマヤマネコの糞を用いた卵胞活動のモニタリングにはE₂またはE₁の測定が、また排卵や妊娠のモニタリングには5α系プレグナンに高い交叉性をもつ抗体を用いた測定が有効であることが示された。
• 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) 15th 2009年 [査読無し][通常論文]
  
• 成熟雌ニホンツキノワグマ (Ursus thibetanus japonicus) における血清レプチン濃度の周年変化

  坪田 敏男, 佐藤 美穂, 岡野 司, 中村 幸子, 淺野 玄, 小松 武志, 柴田 治樹, 斉藤 昌之 The journal of veterinary medical science 70 (12) 1399 -1403 2008年12月 [査読無し][通常論文]
   

  In the present study, assay of the serum leptin concentration of the Japanese black bear (Ursus thibetanus japonicus) was attempted using a canine-leptin-specific sandwich enzyme-linked immunosorbent assay (ELISA). The dose-response Curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September, gradually increased from September or October, and then remarkably increased in late November. We conclude that this method may be useful for measuring bear serum leptin concentration and that the serum leptin concentration changes annually with a peak in late November.
• Immunohistochemical localization of the estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) during pregnancy

  Yanagawa Yojiro, Matsuura Yukiko, Suzuki Masatsugu, Katagiri Seiji, Tsubota Toshio Japanese Journal of Veterinary Research 56 (3) 139 -149 2008年11月 [査読無し][通常論文]
   

  Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestatio...
• 第14回日本野生動物医学会神戸大会に参加しよう！

  浅川 満彦, 坪田 敏男 北海道獣医師会雑誌 52 (9) 460 -461 2008年09月
• Hepatozoon ursi n. spa (Apicomplexa : Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

  Masahito Kubo, Shigehiko Uni, Takeshi Agatsuma, Mitsuru Nagataki, Roger J. Panciera, Toshio Tsubota, Sachiko Nakamura, Hiroki Sakai, Toshiaki Masegi, Tokuma Yanai PARASITOLOGY INTERNATIONAL 57 (3) 287 -294 2008年09月 [査読無し][通常論文]
   

  Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. spa, in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7 +/- 4.6 x 42.7 +/- 4.5 mu m in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0 +/- 0.7 x 1.8 +/- 0.3 mu m in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9 +/- 0.3 x 3.3 +/- 0.2 mu m. The analyses of the 18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis fava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 mu m and 331.8 x 231.7 mu m, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2 +/- 2.5 x 27.0 +/- 2.9 mu m in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2 +/- 1.4 x 3.5 +/- 0.5 mu m in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
• ニホンツキノワグマ(Ursuus thibetanus japonicus)におけるBioelectrical impedance analysis(BIA)による体脂肪量測定(生理学)

  中村 幸子, 岡野 司, 吉田 洋, 松本 歩, 村瀬 豊, 加藤 春喜, 小松 武志, 淺野 玄, 鈴木 正嗣, 杉山 誠, 坪田 敏男 Japanese journal of zoo and wildlife medicine 13 (1) 15 -20 2008年03月 [査読無し][通常論文]
   

  Bioelectrical impedance analysis(BIA)によるニホンツキノワグマ(Ursus thibetanus japonicus)(以下,クマ)の体脂肪量FM測定法確立を試みた。クマを横臥位にし,前肢および後肢間の電気抵抗値を測定した。その値をアメリカクロクマに対する換算式に当てはめ,クマのFMを求めた。2005年9月から翌年の1月までの間,飼育下クマを用いて体重BMおよびFMを測定したところ,BMとFMの変動は高い相関(r=0.89)を示した。よって,秋のBM増加はFM増加を反映していること,ならびにBIAがクマのFM測定に応用可能であることが示された。飼育クマの体脂肪率FRは,9月初旬で最も低く(29.3±3.3%),12月に最も高い値(41.6±3.0%)を示した。彼らの冬眠開始期までの脂肪蓄積量(36.6kg)は約252,000kcalに相当し,冬眠中に1,900kcal/日消費していることが示唆された。一方,2006年6月から11月までの岐阜県および山梨県における野生個体13頭の体脂肪率は,6.9〜31.7%であった。野生個体のFRは飼育個体に比較して低かった。BIAを用いて,ニホンツキノワグマの栄養状態が評価でき,この方法は今後彼らの環境評価指標のツールとしても有用であると思われる。
• 下北半島のニホンザル（Macaca fuscata）の薬剤耐性大腸菌保有状況

  小川 恵子, 山口 敬治, 鈴木 正嗣, 坪田 敏男, 福士 秀人 霊長類研究 Supplement 24 63 -63 2008年 

  海外では、野生動物や生態系内の薬剤耐性菌が人や家畜への感染源になるのではないかという懸念から、あるいは水の糞便汚染の汚染源特定のための指標の一つとして、野生動物の薬剤耐性菌についての研究が近年行われている。国内でも、野生動物と人や人の居住地域との接触の程度を示す指標として、野生動物の耐性菌保有状況調査が行われるようになっているものの、いまだ十分な情報は得られていないのが現状である。そこで本研究では、遊動域などの生態学的データが蓄積されている青森県下北半島に生息するニホンザル（Macaca fuscata）を対象とし、薬剤耐性大腸菌の保有状況を調査した。2005年と2006年にニホンザルの糞便285検体（野生265検体、飼育20検体）を採取し、大腸菌の分離同定をした。一部の菌株に対しCLSI（Clinical and Laboratory Standards Institute）に従い、24種類の抗菌薬と抗生物質について1濃度ディスク法による薬剤感受性試験を実施した。糞便285検体中176検体から290株の大腸菌株を分離した。感受性試験を実施した糞便71検体（野生62検体、飼育9検体）における薬剤耐性大腸菌保有率は、それぞれ野生個体で79.0%（49/62）、飼育個体で44.4%（4/9）だった。セファロチンの単剤耐性株が17株、アンピシリンの単剤耐性株が13株、両剤耐性株が27株、ストレプトマイシンの単剤耐性株が1株分離された。今回試験を実施した全ての薬剤に対して感受性の菌株は25株だった。野生のサルの耐性菌保有率は、これまで報告されている他地域の野鳥などの保有率（0-40.0%）に比べて高かった。これは、過去の報告では人口密度の低い地域の希少種を中心に調査しているのに対し、本研究では農地や道路沿いなど人の居住地域を頻繁に利用するサルの群れを中心に調査したためだと考えられた。
• ニホンツキノワグマ(Ursus thibetanus japonicus) における精巣内ステロイド代謝酵素mRNA発現の季節変化

  飯渕 るり子, 岩永 敏彦, 下鶴 倫人, 坪田 敏男 日本繁殖生物学会 講演要旨集 101 203 -203 2008年 

  【目的】ニホンツキノワグマは長日季節繁殖動物であり、精子形成は顕著な季節性を示す。交尾期である初夏には精巣内で活発な精子形成が行われるが、秋から冬にかけて精細胞の分化が停止する。精細胞の分化が再開するのは冬眠後期にあたる2～3月頃である。この精子形成の季節変化に同調して、精巣における性ステロイド産生も季節変動することが知られている。しかし、このような性ステロイド産生および精子形成の季節変化を調節している機構は明らかではない。本研究では、ツキノワグマの精巣におけるステロイド代謝酵素mRNA発現の季節変化について検討した。【方法】交尾期（6月）および非交尾期（11月、1月、2月、3月）に成熟雄ツキノワグマ３頭より採血およびバイオプシーによる精巣組織の採取を行った。精巣組織の凍結切片を作製し、in situ hybridization法により性ステロイドの代謝・合成に関わる5種類の酵素（P450scc, 3βHSD, P450c17, 17βHSD3およびP450arom）のmRNAを検出した。【結果・考察】交尾期の精巣では精子の形成が行われていた。11および1月の精巣では精細胞の分化は見られなかったが、2～3月には分化の再開が確認された。P450scc, 3βHSDおよびP450c17 mRNAは、交尾期の精巣の間質組織に発現が認められた。3βHSDは、非交尾期である11～2月に発現が低下し、精子形成の再開が観察された3月頃に発現が再び上昇した。一方、P450sccおよびP450c17 mRNAは交尾期および非交尾期の全ての月で発現が認められ、季節変化は観察されなかった。17βHSD3 mRNAは精細管内の基底側に発現が見られ、季節変化は認められなかった。以上の結果より、ツキノワグマ精巣におけるアンドロジェン合成の鍵となる酵素は3βHSDであり、その発現の季節変化がアンドロジェン合成の季節変化に関与している可能性が示唆された。また、P450arom mRNAは主に交尾期の精細胞で発現が認められた。
• ニホンツキノワグマにおけるレプチンレセプター(Ob-R)の発現部位とその季節変化

  中村 幸子, 淺野 玄, 杉山 誠, 鈴木 正嗣, 坪田 敏男 日本獣医学会学術集会講演要旨集 144回 170 -170 2007年08月
• 回虫科におけるクマ回虫(Baylisascaris transfuga)の分子生物学的位置付けおよび病原性の検討

  辻 一, 柳井 徳磨, 柵木 利昭, 酒井 洋樹, 坪田 敏男, 宇仁 茂彦, 吾妻 健 日本獣医学会学術集会講演要旨集 143回 158 -158 2007年03月
• 食肉における家畜・家禽のウイルス疾病に関する研究 食肉に供される動物におけるA群ロタウイルスの感染状況調査

  杉山誠, 安部昌子, 川口まり子, 長谷部文子, 伊藤直人, 宮澤清志, 村瀬哲磨, 坪田敏男, 石黒直隆, 淺野玄, 伊藤雅, 山下照夫, 榮賢司 食肉における家畜・家禽のウイルス疾病に関する研究 平成18年度 総括・分担研究報告書 49 -58 2007年 [査読無し][通常論文]
  
• 第12回大会を終えて

  坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (23) 1 -2 2006年12月01日 [査読無し][通常論文]
  
• クロミンククジラ(Balaenoptera bonaerensis)における胎盤および非妊娠子宮の形態学的および免疫組織化学的研究

  天野 容子, 佐々木 基樹, 石川 創, 茂越 敏弘, 大隅 清治, 手塚 雅文, 宮本 明夫, 福井 豊, 坪田 敏男, 北村 延夫 日本獣医学会学術集会講演要旨集 142回 34 -34 2006年08月
• 牛精巣のステロイド産生機能は精巣温度上昇に伴い変化する(Steroidogenic functions of the bovine testis alters with increased testicular temperature)

  Devkota Bhuminand, 佐々木 基樹, 松井 基純, 坪田 敏男, 金子 悦史, 宮本 明夫, Montoya Carlos Amaya, 大澤 健司, 橋爪 力, 三宅 陽一 日本獣医学会学術集会講演要旨集 142回 129 -129 2006年08月
• ニホンツキノワグマ(Ursus thibetanus japonicus)における排卵様式の解明およびゴナドトロピン製剤による排卵の誘起

  岡野司, 中村幸子, 中下留美子, 小松武志, 村瀬哲磨, 村瀬哲磨, 淺野玄, 淺野玄, 坪田敏男, 坪田敏男 日本野生動物医学会大会・講演要旨集 12th 2006年
• 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 (4) 284 -285 2006年 [査読無し][通常論文]
  
• Immunolocalization of steroidogenic enzymes in the fetal, neonatal and adult testis of the Shiba goat

  Q Weng, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya EXPERIMENTAL ANIMALS 54 (5) 451 -454 2005年10月 [査読無し][通常論文]
   

  The localizations of steroidogenic enzymes (P450scc, 3 beta HSD, P450c17 and P450arom) in testes of Shiba goats were investigated by immunohistochemistry. P450scc, 3 beta HSD, P450c17 and P450arom were detected in all Leydig cells of adults. P450scc and P450c17 were observed in most Leydig cells in the fetus (90 days) and neonate (15 days). 3 beta HSD and P450arom were found in some Leydig cells of the fetus with weak immunostaining but the numbers of immunopositive Leydig cells and intense immunostaining were increased in Leydig cells of the neonate. These results suggest that Shiba goat testes have the ability to synthesize progestin, androgen and estrogen in the fetus, neonate and adult, and synthesis of these steroid hormones showed an age-related rise.
• 4 チンパンジーの繁殖特性に地域個体群変異をもたらす要因の検討(X.共同利用研究 2.研究成果)

  藤田 志歩, 坪田 敏男 霊長類研究所年報 35 (35) 103 -103 2005年08月31日
• Immunolocalization of steroidogenic enzymes P450scc, 3 beta HSD, P450c17, and P450arom in Gottingen miniature pig testes

  Q Weng, MS Medan, G Watanabe, T Tsubota, Y Tanioka, K Taya JOURNAL OF REPRODUCTION AND DEVELOPMENT 51 (3) 299 -304 2005年06月 [査読無し][通常論文]
   

  The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Gottingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3 beta HSD, P450c17 and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.
• Male pseudohermaphroditism in a raccoon dog (Nyctereutes procynoides)

  Q Weng, T Murase, M Asano, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (6) 603 -605 2005年06月 [査読無し][通常論文]
   

  A wild raccoon dog (Nyctereutes procynoides) which died due to a traffic accident on 18 October 2001, and was determined to be 4.5 years old, was examined. Visual appearance of the external genitalia in this animal showed to be female with a large penis-like clitoris protruding from the vulvar juncture. Visual examination of the internal genitalia revealed that the animal possessed both testes and uterus. Histological appearance of the removed gonads showed only Sertoli cells but no spermatogenesis. Using polymerase chain reaction with skin biopsy directed against the sex-determining region on the Y chromosome (SRY) gene, the genomic SRY gene was expressed as a single band and sequenced. Based on these findings, this raccoon dog was diagnosed as male pseudohermaphrodite.
• 雄ゲッチンゲンミニブタ精巣におけるステロイドホルモン合成酵素P450scc,P450c17,3βHSDおよびP450aromの免疫局在

  WENG Qiang, MEDAN Mohamed S, WATANABE Gen, TSUBOTA Toshio, TANIOKA Yoshikuni, TAYA Kazuyoshi The Journal of reproduction and development 51 (3) 299 -304 2005年06月 [査読無し][通常論文]
   

  9月（n＝2）、2月（n＝2）および6月（n＝2）に採取した6頭の雄ゲッチンゲンミニブタの精巣を用いて、ステロイドホルモン合成酵素の免疫局在を調べた。免疫組織化学染色には、choleserol side-chain cleavage cyochrome P450（P450scc）、3 β-hydroxy seroid dehydrogenase （3βHSD）、17α-hydroxylase cyochrome P450（P450cl7）およびaro-maase cyochrome P450（P450arom）に対するポリクローン抗体を用いた。9月、2月および6月のいずれの時期に採取した精巣でも、精細管内に精祖細胞から伸長精子細胞が確認され、採取時期による差は認められなかった。精巣組織中のライディッヒ細胞に、P450scc、P450cl7、3βHSDおよびP450aromの局在が認められた。以上の結果から、雄ゲッチンゲンミニブタにおいては、年間を通して精子形成が行われており、ライディッヒ細胞がプロジェステロン、アンドロジェンおよびエストロジェンの分泌細胞であろうと推察された。
• Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

  Q Weng, MS Medan, LQ Ren, G Watanabe, T Tsubota, K Taya JOURNAL OF REPRODUCTION AND DEVELOPMENT 51 (2) 247 -252 2005年04月 [査読無し][通常論文]
   

  In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome 17 alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3 beta HSD, P450c17 and P450arom. The distribution of P450scc, 3 beta HSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.
• Adaptation of the hindlimbs for climbing in bears

  M Sasaki, H Endo, O Wiig, AE Derocher, T Tsubota, H Taru, M Yamamoto, K Arishima, Y Hayashi, N Kitamura, J Yamada ANNALS OF ANATOMY-ANATOMISCHER ANZEIGER 187 (2) 153 -160 2005年04月 [査読無し][通常論文]
   

  The hindlimbs of the Malayan sun bear (Helarctos malayanus), the polar bear (Ursus maritimus), the brown bear (Ursus arctos) and the giant panda (Ailuropodo melanoleuca) have been anatomically and osteometrically studied. The Musculus tibialis cranialis of the Malayan sun bear and the giant panda possessed a well-developed rich fleshy portion until the distal end of the tibia. In the polar bear and the brown bear, however, the fleshy portion of the M. tibialis cranialis was not developed until the distal, end of the tibia. The tendon of the M. tibialis cranialis inserting on the proximal end of the Ossa metatarsalia was shorter in the Malayan sun bear and the giant panda than in the polar bear and the brown bear. In the Malayan sun bear and the giant panda, moreover, the M. popliteus was attached more distally to the tibia than in the polar bear and the brown bear. The stable dorsiflexion and supination of the foot and the efficient pronation of the crus are important for skillful tree climbing. The present study suggests that the Malayan sun bear and the giant panda have hindlimbs especially adapted to tree climbing by the well-developed fleshy portion of the A tibialis cranialis reaching the distal end of the tibia, its short tendon, and the A popliteus inserting near the distal end of the tibia. © 2005 Elsevier GmbH. All rights reserved.
• 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

  高木 愛香, 杉山 誠, 伊藤 直人, 淺野 玄, 坪田 敏男, 源 宣之 日本獣医学会学術集会講演要旨集 139回 185 -185 2005年03月
• ジャイアントパンダの自然史と保全生物学

  Durrant Barbara, 赤木 智香子, 坪田 敏男 Journal of Reproduction and Development 51 (1) 巻頭10 -12 2005年02月 [査読無し][通常論文]
  
• Rapid, easy and reliable sexing method for falconiformes by PCR

  伊藤 英之, 遠藤 千尋, 山地 明子, 阿部 素子, 村瀬 哲磨, 淺野 玄, 坪田 敏男 Jpn. J. Zoo Wildl. Med. 10: 79-84 (2) 79 -84 2005年 [査読無し][通常論文]
   

  タカ目の多くの種は他の多くの鳥類と同様に外部形態から性判別をすることが困難である。この問題は, タカ目に関する生態学の研究を妨げ, 保存のための計画を作製することを困難にする。そのため, タカ目における性判別方法の開発が望まれていた。我々は, CHD1WとCHD1Zの遺伝子間のイントロンの長さの違いを利用する方法を用いて, 日本に生息する8種類のタカ目において, 性判別を試みた。今回用いた方法は, これまでに開発された他の性判別方法よりも容易で迅速に行うことができる。また, 今回調査したすべての種において性判別が可能であった。さらに, わずかなサンプルから抽出したDNAからでも性判別が可能であり, この方法が野生個体/集団の研究に適用することが可能であることが示唆された。結論として, この研究において用いた方法は, タカ目の性判別に非常に有用であり, 希少なタカ目の将来の保全に大きな価値があると考えられた。
• ニホンツキノワグマ(Ursus thibetanus japonicus)における電気刺激射精法による精液採取-低刺激電圧およびぼうこう洗浄の尿混入抑制効果-

  岡野司, 中村幸子, 水上(中下)留美子, 小松武志, 村瀬哲磨, 村瀬哲磨, 浅野玄, 浅野玄, 坪田敏男, 坪田敏男 日本野生動物医学会大会・講演要旨集 11th 2005年
• 浮桟橋へのサンゴ移植の研究

  久高 將佑, 天 石文, 高山 百合子, 岩田 芳隆, 信田 浩司, 小倉 礁, 三富 聡, 大友 祥太, 渡辺 義公, 市村 靖光, 鈴木 健, 平安山 武雄, 大野 文良, 長谷 一矢, 多喜川 昇, 小林 仁, 岩崎 秀治, 篠原 伸和, 長 雄一, 高田 雅之, 大橋 和彦, 岡崎 克則, 阿出川 隆之, 金子 正美, 遠藤 大二, 浅川 満彦, 坪田 敏男, 淺野 玄, 赤松 里香, Buheaosier, Asai Miki, Kaneko Masami, Ohtaishi Noriyuki, 木原 一禎, 小野寺 収, 濱田 靖弘, 後藤 隆一郎, 窪田 英樹, 中村 真人, 桑原 浩平, 長田 勉, 三浦 二郎, 松島 健将, 中島 義晴, 太田 幸雄, 竹森 憲章, 樺澤 雅之, 渡辺 克実, 大山 成人, 久保 田学, 安保 芳久, 大束 淳一, 山形 定, 山崎 高, 北野 智也, 帆秋 利洋 地球環境シンポジウム講演論文集 13 (13) 341 -366 2005年
• 野生鳥類の大量死の原因となり得る病原体に関する情報収集_-_蓄積_-_解析_-_公開システムの試作について

  長 雄一, 赤松 里香, 高田 雅之, 大橋 和彦, 岡崎 克則, 金子 正美, 遠藤 大二, 浅川 満彦, 坪田 敏男, 淺野 玄 日本生態学会大会講演要旨集 52 (0) 432 -432 2005年 [査読無し][通常論文]
   

  生態学会２００４年大会において「野生鳥類の大量死リスク評価につながる病原体データベースの基本コンセプトについて」を発表し、病原体情報の収集分析のために、広域サンプリング_-_＞病原体タイプの同定_-_＞情報管理・蓄積_-_＞情報解析_-_＞情報公開及び活用といった基本的なコンセプト（設計図）を提示した。
  本発表においては、これらのコンセプトに基づいたシステムの実際的な運用例等を示すとともに、２００３年度に実施したサンプリングにより把握できた野生鳥類における病原体保有動態について中間的な報告を行う。また、実際に罹病あるいは死亡した個体に関する病原体（死因）情報のフォーマットや、「情報の共通化・共有化」等の具体的な運用面の問題についても述べる。さらに野生鳥類の移動_-_分散様式・病原体の伝播_-_発病様式・湖沼等の生息環境の空間構造・人間活動との相互作用を視野においた野生鳥類大量死発生予測モデリングについて手法検討を行う。
• Chemical characterization of sialyl oligosaccharides in milk of the Japanese black bear, Ursus thibetanus japonicus

  T Urashima, T Nakamura, K Teramoto, Arai, I, T Saito, T Komatsu, T Tsubota COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 139 (4) 587 -595 2004年12月 [査読無し][通常論文]
   

  Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by H-1-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following: Neu5Ac(alpha2-3)Gal(beta1-4)Glc Neu5 Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1 -3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Neu5Ac(alpha2-6)Gal(beta1-4)GIcNAc(beta1-6)]Gal(beta1-4)Glc (C) 2004 Elsevier Inc. All rights reserved.
• Ceramide enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in boar spermatozoa

  T Murase, N Imaeda, N Kondoh, T Tsubota JOURNAL OF REPRODUCTION AND DEVELOPMENT 50 (6) 667 -674 2004年12月 [査読無し][通常論文]
   

  Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17C for up to 3 days, were washed and preincubated for 10 min with C-2-ceramide, an analogue of endogenous ceramide, C-2-dihydroceramide (C-2-DH-ceramide), a negative control to C-2-ceramide, or with (1S,2R)-D-erythro-2(N-myristoylamino)- 1-phenyl-1-propanol (D-crythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 muM A23187 (Ca2+/A23187) at 37C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+ /A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C-2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+ /A23187 in a dose-dependent manner, whereas C-2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.
• Immunohistochemical localization of steroidogenic enzymes in the testis of Hokkaido Sika Deer (Cerpus nippon yesoensis)

  D Hayakawa, M Sasaki, C Akabane, N Kitamura, T Tsubota, M Suzuki, J Yamada JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1463 -1466 2004年11月 [査読無し][通常論文]
   

  The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.
• Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

  T Okano, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1371 -1376 2004年11月 [査読無し][通常論文]
   

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

  T Okano, T Murase, M Asano, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1359 -1364 2004年11月 [査読無し][通常論文]
   

  This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4degreesC (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4degreesC (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 X 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.
• 施設5 チンパンジーの繁殖特性に地域個体群変異をもたらす要因の検討(X.共同利用研究 2.共同利用研究成果)

  藤田 志歩, 坪田 敏男 霊長類研究所年報 34 (34) 151 -151 2004年09月30日
• タヌキ(Nyctereutes procynoides)の卵巣におけるP450aromの免疫局在およびmRNAの発現(繁殖学)

  翁 強, 村瀬 哲磨, 淺野 玄, 坪田 敏男 Japanese journal of zoo and wildlife medicine 9 (2) 65 -70 2004年09月 [査読無し][通常論文]
   

  2001年11月〜2003年4月の間に,神奈川県において野生雌タヌキ5頭(11月,2月,3月および4月)の死体を収集した。死体より卵巣を摘出した後,一方を液体窒素で急速凍結するとともに,残りをホルマリン固定し,パラフィン切片を作製した。免疫組織化学を実施し,P450aromの局在を確定した。また,凍結保存した卵巣からmRNAを抽出した後,RT-PCR法にてcDNA断片を増幅した。増幅したDNAのうちP450aromをコードすると予想されるサイズのバンドを確認し,増幅DNA断片のクローニングを行った。プラスミドにインサートされたことを確認した後,シークエンスを行った。免疫組織化学の結果,P450aromは内卵胞膜細胞および卵胞上皮細胞(顆粒層細胞)に認められた。また,3月における黄体細胞および4月における妊娠黄体を構成するほとんどの細胞の細胞質に陽性反応が認められた。PCRによりP450arom cDNAバンドの大きさは289bpであった。また,シークエンスにより得られたP450aromの塩基配列はヒト,ウシおよびラットのそれと76.3%,78.2%および72.6%の相同性を示した。以上の結果より,野生タヌキにおいて卵胞上皮細胞(顆粒層細胞),内卵胞膜細胞および黄体細胞がP450aromを発現し,エストロジェン合成能を有することが示唆された。
• 分化発達にともなうウシ精巣の細胞増殖能とステロイド合成酵素の局在

  佐々木 基樹, 松崎 重範, 高橋 健一, 坪田 敏男, 北村 延夫, 山田 純三 日本獣医学会学術集会講演要旨集 138回 41 -41 2004年08月
• ラテックス凝集試験を用いた数種の野生動物におけるA群ロタウイルスの血清疫学調査

  安部 昌子, 杉山 誠, 山吉 誠也, 伊藤 直人, 源 宣之, 淺野 玄, 坪田 敏男 日本獣医学会学術集会講演要旨集 138回 130 -130 2004年08月
• Evidence that bears are induced ovulators

  WR Boone, BB Keck, JC Catlin, KJ Casey, ET Boone, PS Dye, RJ Schuett, T Tsubota, JC Bahr THERIOGENOLOGY 61 (6) 1163 -1169 2004年04月 [査読無し][通常論文]
   

  The objective of this study was to determine if black bears are induced ovulators. We conducted a single experiment with two replicates; each replicate was divided into two arms: females exposed to male bears and females without male exposure. We used laparoscopy to examine ovaries for corpora lutea and measured serum progesterone, concentrations. Six of the seven isolated females failed to ovulate, while seven of the eight females exposed to males produced one to four corpora lutea. Furthermore, isolated females had significantly lower progesterone concentrations than females exposed to males. Thus, our data suggest that the American black bear is an induced ovulator. These results may aid biologists in their efforts to reproduce ursids in controlled environments. (C) 2003 Elsevier Inc. All rights reserved.
• 猛禽類間におけるマイクロサテライトマーカーの種間増輻の有用性(動物遺伝学)

  伊藤 英之, 須藤-山地 明子, 阿部 素子, 村瀬 哲麿, 坪田 敏男 Japanese journal of zoo and wildlife medicine 9 (1) 39 -43 2004年03月 [査読無し][通常論文]
   

  近年,イヌワシ(Aquila chrysaetos)やクマタカ(Spizaetus nipalensis)のような猛禽類は生息地の破壊等により減少しており,生息地や遺伝的多楡院の保全が重要となっている。しかしながら,猛禽類に開する遺伝的な情報はまだ少ない。本研究では,ハヤブサ(Falco peregrinus)とシロハヤブサ(Falco rusticalus)において開発された8つのマイクロサテライトマーカーを6種の猛禽類,トビ(Milvus migrans),オオタカ(Accipiter gentiles),チョウゲンボウ(Falco tinnunculus),イヌワシ,チュウヒ(Circus spilonotus),クマタカヘの応用を試みた。今回用いたマイクロサテライトマーカーは,1つのマイクロサテライトマーカーを除き,少なくとも1種以上の種において遺伝的多型を検出し,猛禽類における有用性を示した。我々は猛禽類間におけるマイクロサテライトマーカーの種間増幅の有用性を示した。この結果は今後猛禽類の保全や研究に有用であると考えられる。
• 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年02月 [査読無し][通常論文]
   

  We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 P HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.
• 岐阜大学にCOE野生動物救護センターを開設

  坪田 敏男, 淺野 玄 獣医畜産新報 57 (2) 142 -144 2004年02月 [査読無し][通常論文]
  
• 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年02月 [査読無し][通常論文]
   

  We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 P HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.
• Utility of cross-species amplification among reptors

  Hideyuki ITO, Akiko SUDO YAMAJI, Motoko ABE, Tetsuma MURASE, Toshio TSUBOTA Jpn. J. Zoo Wildl. Med. 9: 39-43 (1) 39 -43 2004年 [査読無し][通常論文]
  
• TR-FIA法によるニホンツキノワグマ糞中性ステロイドホルモン濃度測定法の確立およびその季節変動

  中村幸子, 岡野司, 八代田千鶴, 水上(中下)留美子, 小松武志, 村瀬哲磨, 村瀬哲磨, 浅野玄, 浅野玄, 坪田敏男, 坪田敏男 日本野生動物医学会大会・講演要旨集 10th 2004年
• 飼育下成熟雌 ニホンツキノワグマ(Ursus thibetanus japonicus)における血清中プロジェステロン濃度の変化-ツキノワグマは交尾排卵動物か?-

  岡野司, 八代田千鶴, 水上(中下)留美子, 小松武志, 村瀬哲磨, 村瀬哲磨, 坪田敏男, 坪田敏男 日本野生動物医学会大会・講演要旨集 10th 2004年
• 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年12月 [査読無し][通常論文]
   

  A study was conducted to determine the prevalence of bovine herpesvirus-1 (BHV-1), which causes infectious bovine rhinotracheitis, in cattle destined for market in Southern Province, Zambia. A total of 116 nasal secretion samples were tested using the direct fluorescent antibody test, while blood samples from the same cattle were examined by a commercial enzyme-linked immunosorbent assay kit. The prevalence of the BHV-1 antigens in cattle was 23.28% (27/116), while the mean prevalence of the BHV-1 antibodies was 48.28% (56/116). This study showed that cattle in transit to markets could easily spread the virus, which was reactivated by the stress of trekking for long distances under unfavourable conditions, to the other cattle with which they came into contact. Thus, these transit cattle posed a serious threat to other bovines. Systems of cattle trading where cattle must be transported a long way to market should be reviewed by the authorities to minimise the conditions that may exacerbate the spread of infection.
• Seasonal changes in spermatogenesis and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes procynoides)

  W Qiang, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1087 -1092 2003年10月 [査読無し][通常論文]
   

  Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3,3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.
• Spermatogenesis, serum testosterone levels and immunolocalization of steroidogenic enzymes in the wild male Japanese black bear (Ursus thibetanus japonicus)

  T Okano, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1093 -1099 2003年10月 [査読無し][通常論文]
   

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

  W Qiang, T Murase, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1087 -1092 2003年10月 [査読無し][通常論文]
   

  Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3,3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.
• 日本産鳥類におけるダイオキシン類の蓄積特性

  長谷川 淳, 松田 宗明, 河野 公栄, 須藤 明子, 坪田 敏男, 平岡 考, 脇本 忠明 環境化学 : journal of environmental chemistry 13 (3) 765 -779 2003年09月24日 [査読無し][通常論文]
   

  日本各地で採取された10種の野生鳥類中ダイオキシン類を測定した。各種における濃度及び組成から, 各々の生息環境や食性を反映した特有の蓄積特性が明らかとなった。PCDD/Fs, Co-PCBs共に各種の濃度レベルは, 穀類食≒雑食<肉食≒魚食<高次捕食者の傾向を示し, 栄養段階が高次のものほど残留性・毒性の高い異性体が選択的に蓄積されることを確認した。主に陸上生態系に依存する種ではPCDD/Fs, 水圏生態系に依存する種ではCo-PCBsの毒性寄与が顕著であり, 生息環境により汚染の形態が異なることが示唆された。これらの傾向を統計学的に類型化するため, PCDD/Fs残留組成比及びTEQ寄与率に基づくクラスター分析を行ったところ, 生態学的特性による分類とよく一致した結果が得られた。本研究で分析された日本産鳥類のTEQは, 欧米の近縁種が示すレベルの範囲内であったが, 特に陸域食物連鎖の頂点に立つ猛禽類が高い値を示し, 陸上生態系における汚染経路を解明する重要性を指摘した。
• 盲導犬における人工繁殖の試み

  白根 園絵, 村瀬 哲磨, 坪田 敏男, 古橋 博昭, 藤本 幸徳, 諏訪 義典 日本獣医学会学術集会講演要旨集 136回 191 -191 2003年09月
• スイムアップ法による犬精巣上体精子の回収と凍結保存

  茂崎 宇十沙, 村瀬 哲磨, 坪田 敏男 日本獣医学会学術集会講演要旨集 136回 191 -191 2003年09月
• Comparison of cation transport and regulatory volume decrease between red blood cells from the Japanese black bear and the dog

  H. Fujise, T. Nakayama, N. Iwase, T. Tsubota, T. Komatsu Comparative Clinical Pathology 12 (1) 33 -39 2003年05月 [査読無し][通常論文]
   

  Cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBC) of the Japanese black bear and compared with that of the normal dog (LK dog), which possesses RBC with high sodium (Na) and low potassium (K). Unlike the RBC of the American black bear, RBC from the Japanese black bear failed to exhibit K congener rubidium (Rb) influxes through Na-K pump and K-chloride (Cl) cotransport. In the RBC of the Japanese black bear, treatment with N-ethyl maleimide (NEM), calcium (Ca) ionophore A23187 and EGTA, and hyposmotic medium failed to elicit K-Cl cotransport, whereas these perturbations elicited K-Cl cotransport in the LK dog RBC. The Ca uptake was measured in RBC of both the Japanese black bear and the LK dog, and the fluxes were enhanced in hyposmotic medium and inhibited with quinidine, an inhibitor of Na/Ca exchange transporter. Extracellular Ca-dependent Na efflux was detected in RBC of both the Japanese black bear and the LK dog, and it was also inhibited with quinidine. The Ca uptake and Na efflux were probably mediated by Na/Ca exchange transporter. RBC of both the Japanese black bear and the LK dog were restored to their original volume after being swollen in hyposmotic medium with, but not without, Ca. Thus, Na/Ca exchange transporter causes regulatory volume decrease in the RBC of both the Japanese black bear and the LK dog.
• Comparison of cation transport and regulatory volume decrease between red blood cells from the Japanese black bear and the dog

  H. Fujise, T. Nakayama, N. Iwase, T. Tsubota, T. Komatsu Comparative Clinical Pathology 12 (1) 33 -39 2003年05月 [査読無し][通常論文]
   

  Cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBC) of the Japanese black bear and compared with that of the normal dog (LK dog), which possesses RBC with high sodium (Na) and low potassium (K). Unlike the RBC of the American black bear, RBC from the Japanese black bear failed to exhibit K congener rubidium (Rb) influxes through Na-K pump and K-chloride (Cl) cotransport. In the RBC of the Japanese black bear, treatment with N-ethyl maleimide (NEM), calcium (Ca) ionophore A23187 and EGTA, and hyposmotic medium failed to elicit K-Cl cotransport, whereas these perturbations elicited K-Cl cotransport in the LK dog RBC. The Ca uptake was measured in RBC of both the Japanese black bear and the LK dog, and the fluxes were enhanced in hyposmotic medium and inhibited with quinidine, an inhibitor of Na/Ca exchange transporter. Extracellular Ca-dependent Na efflux was detected in RBC of both the Japanese black bear and the LK dog, and it was also inhibited with quinidine. The Ca uptake and Na efflux were probably mediated by Na/Ca exchange transporter. RBC of both the Japanese black bear and the LK dog were restored to their original volume after being swollen in hyposmotic medium with, but not without, Ca. Thus, Na/Ca exchange transporter causes regulatory volume decrease in the RBC of both the Japanese black bear and the LK dog.
• 緒言 (特集 野生動物モニタリングと環境保護--第134回日本獣医学会学術集会 シンポジウムより)

  村田 浩一, 坪田 敏男 獣医畜産新報 56 (4) 281 -283 2003年04月 [査読無し][通常論文]
  
• Sex identification by alternative polymerase chain reaction methods in Falconiformes

  H Ito, A Sudo-Yamaji, M Abe, T Murase, T Tsubota ZOOLOGICAL SCIENCE 20 (3) 339 -344 2003年03月 [査読無し][通常論文]
   

  A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z From sequence analysis, a T-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the T-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.
• Sex identification by alternative polymerase chain reaction methods in Falconiformes

  Hideyuki Ito, Akiko Sudo-Yamaji, Motoko Abe, Tetsuma Murase, Toshio Tsubota Zoological Science 20 (3) 339 -344 2003年03月01日 [査読無し][通常論文]
   

  A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3′-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 3′-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.
• クマ類の繁殖に関する生理学的および内分泌学的研究

  坪田 敏男 日本獣医学会学術集会講演要旨集 135回 9 -9 2003年03月
• Sex identification by alternative polymerase chain reaction methods in Falconiformes

  H Ito, A Sudo-Yamaji, M Abe, T Murase, T Tsubota ZOOLOGICAL SCIENCE 20 (3) 339 -344 2003年03月 [査読無し][通常論文]
   

  A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z From sequence analysis, a T-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the T-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.
• 長谷川淳、松田宗明、河野公栄、須田明子、坪田敏男、平岡 考、脇本忠明／ 日本産鳥類におけるダイオキシン類の蓄積特性（共著）

  長谷川淳, 松田宗明, 河野公栄, 須藤明子, 坪田敏男, 平岡考, 脇本忠明 環境科学 13 (3) 765 -779 2003年 [査読無し][通常論文]
   

  日本各地で採取された10種の野生鳥類中ダイオキシン類を測定した。各種における濃度及び組成から, 各々の生息環境や食性を反映した特有の蓄積特性が明らかとなった。PCDD/Fs, Co-PCBs共に各種の濃度レベルは, 穀類食≒雑食<肉食≒魚食<高次捕食者の傾向を示し, 栄養段階が高次のものほど残留性・毒性の高い異性体が選択的に蓄積されることを確認した。主に陸上生態系に依存する種ではPCDD/Fs, 水圏生態系に依存する種ではCo-PCBsの毒性寄与が顕著であり, 生息環境により汚染の形態が異なることが示唆された。これらの傾向を統計学的に類型化するため, PCDD/Fs残留組成比及びTEQ寄与率に基づくクラスター分析を行ったところ, 生態学的特性による分類とよく一致した結果が得られた。本研究で分析された日本産鳥類のTEQは, 欧米の近縁種が示すレベルの範囲内であったが, 特に陸域食物連鎖の頂点に立つ猛禽類が高い値を示し, 陸上生態系における汚染経路を解明する重要性を指摘した。
• クマハギ激害地におけるニホンツキノワグマ (Ursus thibetanus japonicus) の食性

  吉田 洋, 林 進, 坪田 敏男, 村瀬 哲磨, 岡本 卓也, 白濱 直樹, 岡野 司, 尾崎 智子 日本生態学会大会講演要旨集 50 140 -140 2003年
• DNAフィンガープリント法による飼育下ニホンツキノワグマの父子判定(繁殖学)

  山本 かおり, 河村 篤紀, 坪田 敏男, 釣賀 一二三, 小松 武志, 村瀬 哲磨, 喜多 功, 工藤 忠明 Japanese journal of zoo and wildlife medicine 7 (2) 103 -108 2002年09月 [査読無し][通常論文]
   

  本研究では,飼育条件下のニホンツキノワグマにおいてDNAフィンガープリント法による父子判定の有用性を検討した。制限酵素Hinf Iおよび(GATA)_4プローブを用いたDNAフィンガープリントはニホンツキノワグマの個体識別および父子判定に有用であることが示された。1995年から1997年の間に11頭の母グマから生まれた13頭の子グマと22頭の父親候補の雄グマについて父子判定を行った結果,7頭の雄グマが父親と判定された。特に2頭の雄グマが8頭の子グマの父親と判定された。本研究では,飼育条件下において雌グマが多くの雄グマとの交尾の機会をもっても,ある特定の雄の繁殖成功が高くなることが示された。
• Sequence comparison of the dopamine receptor D4 exon 111 peptitive region in several species of the order carnivora （共著）

  Miho Inoue-Murayama, Naoto Matsuura, Yuichi Murayama, Toshio Tsubota, Toshiroh Iwasaki, Hitoshi Kitagawa, Shin'ichi Ito J. Vet. Med. Sci. 64 (8) 747 -749 2002年08月 [査読無し][通常論文]
   

  It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.
• Sequence comparison of the dopamine receptor D4 exon iii repetitive region in several species of the order Carnivora

  Miho Inoue-Murayama, Naoto Matsuura, Yuichi Murayama, Toshio Tsubota, Toshiroh Iwasaki, Hitoshi Kitagawa, Shin'ichi Ito Journal of Veterinary Medical Science 64 (8) 747 -749 2002年08月 [査読無し][通常論文]
   

  It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.
• 「野生動物の研究と管理技術」, 日本野生動物医学会・野生生物学会監修, 鈴木正嗣 編訳, 2001年11月, 文永堂出版株式会社 刊, A4判, 898ページ, 定価20,000円+税

  坪田 敏男 The journal of veterinary medical science 64 (6) s・1 2002年06月25日
• Sex identification of Japanese black bear, Ursus thibetanus japonicus, by PCR based on amelogenin gene （共著）

  K Yamamoto, T Tsubota, T Komatsu, A Katayama, T Murase, Kita, I, T Kudo J. Vet. Med.Sci. 64 (6) 505 -508 2002年06月 [査読無し][通常論文]
   

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

  K Yamamoto, T Tsubota, T Komatsu, A Katayama, T Murase, Kita, I, T Kudo JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (6) 505 -508 2002年06月 [査読無し][通常論文]
   

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

  坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (14) 7 -8 2002年06月01日 [査読無し][通常論文]
  
• ザンビア国のウシ及び野生レチュエ由来の肝蛭における形態学的及び分子遺伝学的解析

  板垣 匡, 志茂 順子, 杉本 千尋, 吉田 光敏, 小沼 操, 坪田 敏男, 福士 秀人, 中里 幸和, 安田 準 日本獣医学会学術集会講演要旨集 133回 71 -71 2002年03月
• ザンビア特産Kafue Lechwe(Kobus leche kafuensis)雄の生殖器形態と精子形成

  岩崎 絢子, 吉田 光敏, ムワンザ・AM, パテル・OV, バイヤット・IM, シャカリマ・M, 坪田 敏男, 福士 秀人, 板垣 匡, 杉本 千尋, 小沼 操, 中里 幸和, 安田 準 日本獣医学会学術集会講演要旨集 133回 138 -138 2002年03月
• 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(<特集>野生動物医学研究の実際)

  坪田 敏男, 瀧紫 珠子, 須藤 明子, 村瀬 哲磨, 野田 亜矢子, 柵木 利昭, 源 宣之 Japanese journal of zoo and wildlife medicine 7 (1) 69 -74 2002年03月 [査読無し][通常論文]
   

  人間の営みによって作り出された化学物質が,長期間分解されることなく環境に蓄積し,内分泌攪乱化学作用によって人や野生動物の生殖に異常をもたらすことが解明されつつある。内分泌攪乱化学物質には,DDTなどの農薬,PCB類などの工業化学物質,ダイオキシンなどの非意図的生成物,合成女性ホルモンとして使われたDESなどの医薬品などが含まれる。これまでに,アメリカ合衆国のアポプカ湖でのワニの個体数減少,ミンクやカワウソの繁殖率の低下,猛禽類の卵殻の薄化や孵化率の低下,イルカやアザラシの大量死,イボニシでのインポセックス,コイの雌雄同体化,ホッキョクグマの生殖能力の低下や間性といったさまざまな生殖異常が内分泌攪乱作用によって引き起こされている。日本においては平成10年度より内分泌攪乱化学物質およびダイオキシン類による野生生物への影響実態調査が開始され,さまざまな野生動物,とくに海獣類や猛禽類における内分泌攪乱化学物質の蓄積が認められた。今後さらに影響実態を究明し,内分泌攪乱化学物質問題を解決していく必要がある。
• Paternity determination using DNA fingerprinting in captive Japanese black .bears (Ursus thibetanusjaponicus) （共著）

  Jpn. J. Zoo Wildl. Med. 7, 103‐108 2002年 [査読無し][通常論文]
  
• 野生動物における内分泌攪乱化学物質の蓄積濃度と生殖への影響（共著） 2002

  Jpn. J. Zoo Wildl. Med. 7, 69‐74 2002年 [査読無し][通常論文]
  
• 黒毛和種種雄牛より得た凍結-融解精子の先体反応におけるジアシルグリセロールの生成

  村瀬 哲磨, 向島 幸司, 坂口 慎一, 大谷 健, 坪田 敏男 The Journal of Reproduction and Development 47 (Suppl.) a17 -a17 2001年12月
• Changes in sex steroids, gonadotropins, prolactin and inhibin in pregnant and nonpregnant black bears (Ursus thibetanus japonicus) （共著）

  M Sato, T Tsubota, T Komatsu, G Watanabe, T Taya, T Murase, Kita, I, T Kudo Biol. Reprod 65 (4) 1006 -1013 2001年10月 [査読無し][通常論文]
   

  We examined changes in the concentrations of serum progesterone (P-4), estradiol-17 beta (E-2), FSH, LH, prolactin (PRL), and inhibin to determine their interaction and their effect on the reproductive endocrine controls of pregnant and nonpregnant female Japanese black bears. Fourteen female bears were used in this study over a 2-yr period. In the first year, six of the bears were divided into two groups; a pseudopregnant group and a nonpregnant group. In the second year, the remaining eight bears were also divided into two groups; a pregnant group and a nonpregnant group. Pregnant and pseudopregnant bears had similar P-4 trends with both groups exhibiting a significant increase in December, which is the suspected time of implantation in pregnant bears. These trends correlated with an increase in PRL levels, whereas low levels of LH were maintained throughout the year. Nonpregnant bears maintained low concentrations of P-4 and compared with pregnant and pseudopregnant bears, they also exhibited a delayed elevation in PRL. Luteinizing hormone activity varied among individual animals, but regardless of reproductive status, fluctuation patterns of E-2 FSH, and inhibin did not differ among bears. Our results suggest that PRL may play a luteotropic role in both pregnant and pseudopregnant bears, and is possibly responsible for inducing reactivation of the dormant corpus luteum that precedes implantation in the Japanese black bear.
• Changes in sex steroids, gonadotropins, prolactin, and inhibin in pregnant and nonpregnant Japanese black bears (Ursus thibetanus japonicus)

  M Sato, T Tsubota, T Komatsu, G Watanabe, T Taya, T Murase, Kita, I, T Kudo BIOLOGY OF REPRODUCTION 65 (4) 1006 -1013 2001年10月 [査読無し][通常論文]
   

  We examined changes in the concentrations of serum progesterone (P-4), estradiol-17 beta (E-2), FSH, LH, prolactin (PRL), and inhibin to determine their interaction and their effect on the reproductive endocrine controls of pregnant and nonpregnant female Japanese black bears. Fourteen female bears were used in this study over a 2-yr period. In the first year, six of the bears were divided into two groups; a pseudopregnant group and a nonpregnant group. In the second year, the remaining eight bears were also divided into two groups; a pregnant group and a nonpregnant group. Pregnant and pseudopregnant bears had similar P-4 trends with both groups exhibiting a significant increase in December, which is the suspected time of implantation in pregnant bears. These trends correlated with an increase in PRL levels, whereas low levels of LH were maintained throughout the year. Nonpregnant bears maintained low concentrations of P-4 and compared with pregnant and pseudopregnant bears, they also exhibited a delayed elevation in PRL. Luteinizing hormone activity varied among individual animals, but regardless of reproductive status, fluctuation patterns of E-2 FSH, and inhibin did not differ among bears. Our results suggest that PRL may play a luteotropic role in both pregnant and pseudopregnant bears, and is possibly responsible for inducing reactivation of the dormant corpus luteum that precedes implantation in the Japanese black bear.
• Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats （共著）

  MS Syakalima, KC Choongo, P Chilonda, B Ahmadu, M Mwase, M Onuma, C Sugimoto, T Tsubota, H Fukushi, M Yoshida, T Itagaki, J Yasuda, Y Nakazato Bull. Environ. Contam. Toxicol. 67 (3) 438 -445 2001年09月 [査読無し][通常論文]
   

  2001
• Bioaccumulation of lead in wildlife dependent on the contaminated environment of the Kafue flats

  MS Syakalima, KC Choongo, P Chilonda, B Ahmadu, M Mwase, M Onuma, C Sugimoto, T Tsubota, H Fukushi, M Yoshida, T Itagaki, J Yasuda, Y Nakazato BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 67 (3) 438 -445 2001年09月 [査読無し][通常論文]
  
• 海外における動物疾病の動向 ザンビアにおけるKafue lechwe(ウシ科)及び畜牛における結核に関する研究 その公衆衛生的意義を含んで(STUDIES ON TUBERCULOSIS AMONG KAFUE LECHWE AND CATTLE INCLUDING ITS PUBLIC HEALTH SIGNIFICANCE IN ZAMBIA)

  パンディー・G.S., 野村 靖夫, 福士 秀人, 杉本 千尋, 小沼 操, 坪田 敏男, 吉田 光敏, 板垣 匡, 中里 幸和, 安田 準 日本獣医学会学術集会講演要旨集 132回 59 -59 2001年09月
• 海外における動物疾病の動向 ザンビアにおける家畜と野生動物の相互伝播性疾病に関する調査研究1.牛及びレチュエ由来の肝蛭における分子遺伝学的解析

  板垣 匡, 志茂 順子, 杉本 千尋, 吉田 光敏, 小沼 操, 坪田 敏男, 福士 秀人, 中里 幸和, 安田 準 日本獣医学会学術集会講演要旨集 132回 59 -59 2001年09月
• 犬精液の凍結において最終希釈倍率,最終精子濃度,一次平衡時間及び二次平衡時間が融解後の精液性状に及ぼす影響

  岡野 司, 村瀬 哲磨, 坪田 敏男 日本獣医学会学術集会講演要旨集 132回 222 -222 2001年09月
• Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese black bear, Ursus thibetanus japonicus, during pregnancy

  T Tsubota, S Taki, K Nakayama, JI Mason, S Kominami, N Harada, Kita, I REPRODUCTION 121 (4) 587 -594 2001年04月 [査読無し][通常論文]
   

  The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the 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) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3 beta HSD, P450c17 and P450arom. P450scc and SPHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3 beta HSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.
• An investigation of heavy metal exposure and risks to wildlife in the Kafue flats of Zambia

  M Syakalima, K Choongo, Y Nakazato, M Onuma, C Sugimoto, M Tsubota, H Fukushi, M Yoshida, T Itagaki, J Yasuda JOURNAL OF VETERINARY MEDICAL SCIENCE 63 (3) 315 -318 2001年03月 [査読無し][通常論文]
   

  Exposure and ecological risks to heavy metals (copper, zinc, manganese, iron) at Lochnivar and Blue Lagoon National Parks in wildlife dependent on the Kafue river contaminated with mining waste was evaluated. Samples included water, fish, grasses and Kafue Lechwe (Kobus leche kafuensis) liver. At both parks copper ranged from 0.03-0.04 mg/l; 3.0-6.0 mg/kg; 11.0-44.0 mg/kg; trace -199.0 mg/kg; while zinc was 0.01 mg/l; 32.0-82.0 mg/kg; 15.0-21.0 mg/kg; and 52.0-138.0 mg/kg; in water, fish, grasses and lechwe, respectively. Manganese ranges were 0.15-0.16 mg/l; 7.0-18.0 mg/kg; 51.0-145.0 mg/kg; and 40.0-53.0 mg/kg while iron ranges were 0.13-0.14 mg/l; 26.0-134.0 mg/kg; 1766.0-1797.0 mg/kg; and 131.D-856.0 mg/kg; in water, fish, grasses and lechwe, respectively. Levels in all samples except water were high indicating potential for adverse effects.
• ヒトと野生動物の関わり 新興感染症と有害化学物質汚染 日本の野生動物における内分泌撹乱化学物質汚染の現状

  坪田 敏男, 須藤 明子, 柵木 利昭, 源 宣之, 岩見 恭子 日本獣医学会学術集会講演要旨集 131回 112 -112 2001年03月
• Characterization of semen collected from beagles and captive Japanese black bears (Ursus thibetanus japonicus)

  E Kojima, H Tsuruga, T Komatsu, T Murase, T Tsubota, Kita, I THERIOGENOLOGY 55 (3) 717 -731 2001年02月 [査読無し][通常論文]
   

  This study characterized semen collected from the Japanese black bear, Ursus thibetanus japonicus, to provide information on semen cryopreservation for artificial breeding. Preliminary studies using a beagle dog as the model species showed that sperm concentration and total sperm count were lower in semen collected by electroejaculation than in semen collected by digital manipulation, but that sperm motility, viability and morphology were similar. Characterization of semen obtained from Japanese black bears by electroejaculation under general anesthesia revealed that semen volume and total number of spermatozoa collected were lower; but that sperm concentration, motility, viability and morphology were equivalent to those reported in other ursids. When semen was collected via a catheter inserted into the urethra during the stimulation for ejaculation, the sperm concentration, total sperm count and motility were relatively higher than when semen was collected directly in a test tube. Specific normal semen characteristics (mean +/- SEM) were pH, 7.6 +/- 0.0; volume, 0.212 +/- 0.038 mL; sperm concentration 361 +/- 100 x 10(6)/mL; total sperm count, 84.0 +/- 32.2 x 10(6); +++ motility, 30 +/- 5%; motility, 77 +/- 3%; viability 77 +/- 2%; and abnormal morphology, 11 +/- 2%. These results suggest that semen can be collected from Japanese black bears by electroejaculation. (C) 2001 by Elsevier Science Inc.
• Seasonal changes in testicular steroidogenesis Callorhinus ursinus （共著） and spermatogenesis in a Northern fur seal,

  Toshio Tsubota, Tomomi Nagashima, Kaoru Kohyama, Kenji Maejima, Tetsuma Murase, Isao Kita J. Reprod. Dev. 47 (6) 415 -420 2001年 [査読無し][通常論文]
   

  2001.
• Generation of diacylglycerol during the acrosome reaction induced by Ca2+ and Ca2+ ionophore A23187 in frozen-thawed spermatozoa from fertile and subfertile Japanese black bulls （共著）

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota J. Reprod. Dev. 47 (5) 311 -316 2001年 [査読無し][通常論文]
   

  This study investigated the activation of phosphoinositidase C (PIC) and phosphatidylcholine-specific phospholipase C (PLC) during the acrosome reaction of spermatozoa induced by Ca2+ and Ca2+ ionophore A23187 in a subfertile Japanese black bull. Sperm motility, viability, morphology and penetration of cervical mucus by spermatozoa in a subfertile bull were comparable to those in a fertile bull. When frozen-thawed spermatozoa were stimulated with 3 mM Ca2+ and l -4M A23187, both bulls' spermatozoa showed a time-dependent increase in percent acrosome reactions, but the increase was more rapid and higher in spermatozoa from the fertile bull. The amounts of 1,2-diacylglycerol (DAG) mass, a product of PIC and PLC, peaked at l min after a similar treatment with a gradual decline towards 10 min in both bulls' spermatozoa but were significantly higher throughout stimulation for the subfertile bull. These results suggest that active PIC/PLC may be present in spermatozoa of the subfertile bull, and that other mechanisms were possibly associated with the lowered induction of the acrosome reaction. However, it remains unclear whether the high levels of 1,2-DAG mass were responsible.
• Characterization of frozen-thawed Japanese black bull spermatozoa by standard semen analysis, mucus penetration test and the ability to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187 （共著）

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita J. Reprod. Dev. 47 (4) 237 -243 2001年 [査読無し][通常論文]
   

  2001
• Influence of the different batches of estrous cervical mucus mixed for homogenization on penetration by spermatozoa in cattle

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 47 (2) 109 -112 2001年 [査読無し][通常論文]
   

  This study examined differences in the penetration of various batches of mixed cervical mucus by bull spermatozoa. Mucus was collected from different numbers (2, 2, 3, 4 or 7) of cows or heifers in estrus and a part of the sample was individually, and the rest was mixed, and stored frozen. The distance traveled by the most advanced sperm cell in frozen-thawed mucus during incubation at 38 C for 10 min was measured (sperm penetration). Variation of sperm penetration in the individual mucus samples was reduced by mixing but the difference among the batches of mixed mucus was significant (p< 0.0002). Nevertheless, the number of animals from which mucus was collected and mixed did not seem to influence the sperm penetration of the different batches. Unexpectedly it was observed that some individual and mixed mucus samples had reduced viscosity after freezing and thawing and sperm penetration in the mixed mucus samples showed a significant, negative correlation with the proportion of mucus of reduced viscosity contained in the batch (r=-0.969, p< 0.01). These results suggest that the difference in the sperm penetration of mixed mucus may probably be rather independent of the number of mucus donors.
• Seasonal changes in testicular steroidogenesis and spermatogenesis in a northern fur seal, Callorhinus ursinus

  Toshio Tsubota, Tomomi Nagashima, Kaoru Kohyama, Kenji Maejima, Tetsuma Murase, Isao Kita Journal of Reproduction and Development 47 (6) 415 -420 2001年 [査読無し][通常論文]
   

  Seasonal changes in testicular steroidogenesis and spermatogenesis were studied in a captive male northern fur seal, Callorhinus ursinus, of Izu-Mito Sea Paradise. Under anesthesia, monthly blood samplings and testicular biopsies at intervals of 3 months were performed to measure serum testosterone concentrations by radioimmunoassay and to observe spermatogenic activity histologically and the immunolocalization of steroidogenic enzymes. Histological observation of testes revealed full spermatogenesis, including spermatogonia to spermatozoa in the seminiferous epithelium in June and a limited generation of germ cells such as spermatogonia and primary spermatocytes in September and December. The seminiferous tubule diameter and serum testosterone concentrations showed maximum fluctuations corresponding to full spermatogenic activity in June. Three kinds of steroidogenic enzymes, P450scc, 3βHSD and P450c17, which are necessary for androgen synthesis, were immunolocalized in Leydig cells, and P450arom, the key enzyme for estrogen synthesis, was immunolocalized in spermatogonia in the testes in all 3 seasons. These results indicate a definite seasonality in high-level testicular steroidogenic and spermatogenic activity of this northern fur seal during the breeding season in spite of there being no changes in the immunostaining of intratesticular steroidogenic enzymes throughout the year.
• Generation of diacylglycerol during the acrosome reaction induced by Ca2+ and Ca2+ lonophore A23187 in frozen-thawed spermatozoa from fertile and subfertile japanese black bulls

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota Journal of Reproduction and Development 47 (5) 311 -316 2001年 [査読無し][通常論文]
   

  This study investigated the activation of phosphoinositidase C (PIC) and phosphatidylcholine-specific phospholipase C (PLC) during the acrosome reaction of spermatozoa induced by Ca2+ and Ca2+ ionophore A23187 in a subfertile Japanese black bull. Sperm motility, viability, morphology and penetration of cervical mucus by spermatozoa in a subfertile bull were comparable to those in a fertile bull. When frozen-thawed spermatozoa were stimulated with 3 mM Ca2+ and l -4M A23187, both bulls' spermatozoa showed a time-dependent increase in percent acrosome reactions, but the increase was more rapid and higher in spermatozoa from the fertile bull. The amounts of 1,2-diacylglycerol (DAG) mass, a product of PIC and PLC, peaked at l min after a similar treatment with a gradual decline towards 10 min in both bulls' spermatozoa but were significantly higher throughout stimulation for the subfertile bull. These results suggest that active PIC/PLC may be present in spermatozoa of the subfertile bull, and that other mechanisms were possibly associated with the lowered induction of the acrosome reaction. However, it remains unclear whether the high levels of 1,2-DAG mass were responsible.
• Characterization of frozen-thawed Japanese black bull spermatozoa by standard semen analysis, mucus penetration test and the ability to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 47 (4) 237 -243 2001年 [査読無し][通常論文]
   

  To develop new tests to predict bull subfertility, a mucus penetration test, in which penetration of cervical mucus by spermatozoa is quantitated, was applied using frozen-thawed semen and a test to examine the ability of spermatozoa to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187 (A23187 stimulation test) was attempted. Frozen-thawed spermatozoa from 4 Japanese black bulls (2 fertile and the other 2 subfertile) were analyzed by standard semen analysis such as sperm concentration, motility, viability and morphology, the mucus penetration test and the A23187 stimulation test, and the relationship of the test results to fertility status was investigated retrospectively. The results of the standard semen analysis and the mucus penetration test did not reveal any remarkable differences among the bulls, showing no relationship to fertility status. However, when spermatozoa were stimulated with 3 mM Ca2+ and 1 μM A23187, the bulls all showed a time-dependent induction of the acrosome reaction but the subfertile bulls showed a slower and lower induction than the fertile bulls. These results suggest that A23187 stimulation test may be useful when abnormal sperm characteristics cannot be found by standard semen analysis or the mucus penetration test.
• Influence of the different batches of estrous cervical mucus mixed for homogenization on penetration by spermatozoa in cattle

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Tsuyoshi Ohtani, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 47 (2) 109 -112 2001年 [査読無し][通常論文]
   

  This study examined differences in the penetration of various batches of mixed cervical mucus by bull spermatozoa. Mucus was collected from different numbers (2, 2, 3, 4 or 7) of cows or heifers in estrus and a part of the sample was individually, and the rest was mixed, and stored frozen. The distance traveled by the most advanced sperm cell in frozen-thawed mucus during incubation at 38 C for 10 min was measured (sperm penetration). Variation of sperm penetration in the individual mucus samples was reduced by mixing but the difference among the batches of mixed mucus was significant (p< 0.0002). Nevertheless, the number of animals from which mucus was collected and mixed did not seem to influence the sperm penetration of the different batches. Unexpectedly it was observed that some individual and mixed mucus samples had reduced viscosity after freezing and thawing and sperm penetration in the mixed mucus samples showed a significant, negative correlation with the proportion of mucus of reduced viscosity contained in the batch (r=-0.969, p< 0.01). These results suggest that the difference in the sperm penetration of mixed mucus may probably be rather independent of the number of mucus donors.
• 黒毛和牛精子における子宮頸管粘液への侵入およびCa2+-イオノホアによる先体反応と受胎性との関連

  村瀬 哲磨, 向島 幸司, 坂口 慎一, 大谷 健, 坪田 敏男, 喜多 功 The Journal of Reproduction and Development 46 (Suppl.) a45 -a45 2000年12月
• ニホンツキノワグマにおける着床遅延中の卵巣のステロイドおよびインヒビン合成に関する免疫組織化学的研究

  佐藤 美穂, 坪田 敏男, 渡辺 元, 田谷 一善, 小松 武志, 村瀬 哲磨, 喜多 功, 工藤 忠明 The Journal of Reproduction and Development 46 (Suppl.) a41 -a41 2000年12月
• 授乳及び非授乳中のニホンツキノワグマ(Ursus thibetanus japonicus)における血清プロジェステロン,エストラジオール-17β,黄体形成ホルモン,プロラクチン濃度の変化

  佐藤 美穂, 中野 典子, 坪田 敏男, 小松 武志, 村瀬 哲磨, 喜多 功, 工藤 忠明 The Journal of Reproduction and Development 46 (5) 301 -308 2000年10月
• 欧米の野生動物医学事情 : 2カ月間の研修より

  坪田 敏男 Zoo and wildlife news = ズー・アンド・ワイルドライフニュース 0 (10) 9 -12 2000年06月01日 [査読無し][通常論文]
  
• Serum progesterone and estradiol-17 beta concentrations in captive and free-ranging adult female Japanese black bears (Ursus thibetanus japonicus)

  M Sato, T Tsubota, K Yamamoto, T Komatsu, Y Hashimoto, A Katayama, T Hazumi, Kita, I, T Kudo JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (4) 415 -420 2000年04月 [査読無し][通常論文]
   

  Progesterone(P-4) and estradiol-17 beta (E-2) concentrations were measured in serum samples obtained from 23 captive and 23 free-ranging adult female Japanese black bears. We then determined the relationship between changes in these sex steroid hormones and pregnancy. In all captive bears, which included animals of both known and unknown reproductive status, serum P-4 concentrations were low from April to July, then tended to become higher after August. The levels then became much higher still in November and December, but returned to low levels in March. Serum P-4 concentrations in eight captive pregnant bears, which had parturitions the following spring, increased gradually from August (0.5-2.4 ng/ml) to October (0.9-3.6 ng/ml), and achieved significantly higher maximum levels in December (7.2-18.0 ng/ml). Thereafter, serum P-4 concentrations tended to decrease (3.5-6.4 ng/ml in January and 0.3-0.7 ng/ml in March). In all captive bears, serum E-2 concentrations varied from April to October but showed low levels in November and December, and became high in January. Serum E-2 concentrations in the eight pregnant bears were high in May (95.6-191.4 pg/ml) and varied from August to October (35.6-143.3 pg/ml). Subsequently, serum E-2 concentrations in December dropped to significantly lower minimum levels (5.3-11.9 pg/ml) and increased again in January (67.6-153.1 pg/ml). Among the free-ranging bears, the data on serum P-4 concentrations in eight bears led to expectations of pregnancy, whereas serum E-2 concentrations showed no distinct evidence related to pregnancy. These results, particularly in captive pregnant bears, indicate that a marked increase of P-4 in December might be accompanied by reactivation of the corpus luteum preceding implantation. Furthermore, changes in E-2 concentrations suggested the possibility that a decline in December and an increase in January are associated with implantation and parturition, respectively.
• 糞分析からみたニホンツキノワグマ(┣DBUrsus(/)-┫DB ┣DBthibetanus(/)-┫DB ┣DBjaponicus(/)-┫DB)の食性-岐阜県本巣郡根尾村における事例-(共著)

  堀内みどり 中森研 48 149 -152 2000年 [査読無し][通常論文]
  
• Changes in serum progesterone, estradiol-17β, luteinizing hormone and prolactin in lactating and non-lactating Japanese black bears(┣DBUrsus thibetanus japonicus(/)-┫DB)(共著)

  J. Reprod. Dev. 46 (5) 301 -308 2000年 [査読無し][通常論文]
  
• 和牛精子が発情期の子宮頸管粘液へ侵入する能力を測定する方法の簡便化

  村瀬 哲磨, 向島 幸司, 坂口 慎一, 坪田 敏男, 喜多 功 The Journal of Reproduction and Development 45 (6) 425 -428 1999年12月
• 成熱雌ニホンツキノワグマにおける血清中FSH,LH,インヒビン,プロジェステロン及びエストラジオール-17β濃度の周年変化

  佐藤 美穂, 坪田 敏男, 小松 武志, 渡辺 元, 田谷 一善, 村瀬 哲磨, 喜多 功, 工藤 忠明 The Journal of Reproduction and Development 45 (Suppl.) a34 -a34 1999年12月
• Chemical characterization of milk oligosaccharides of the Japanese black bear, Ursus thibetanus japonicus

  T Urashima, W Sumiyoshi, T Nakamura, Arai, I, T Saito, T Komatsu, T Tsubota BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1472 (1-2) 290 -306 1999年10月 [査読無し][通常論文]
   

  Two trisaccharides, two tetrasaccharides, one penta-, one hexa-, two hepta-, one deca- and two undeca-saccharides were isolated from several Japanese black bear milk samples by chloroform/methanol extraction, gel filtration and preparative thin-layer chromatography. The oligosaccharides were characterized by H-1-NMR as follows: Gal(alpha 1-3)Gal(beta 1-4)Glc (alpha 3'-galactosyllactose), Fuc(alpha 1-2)Gal(beta 1-4)Glc (2'-fucosyllactose), Gal(alpha 1-3)(Fuc(alpha 1-2)]Gal(beta 1-4)Glc (B-tetrasaccharide), Gal(alpha 1-3)Gal(beta 1-4)(Fuc(alpha 1-3))Glc, Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (B-pentasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (monofucosylhexasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Gl (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3){Gal(alpha 1-3)Gal(beta 1-4)[Fuc(a 1-3)]GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (difucosyldecasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GIcNAc(beta 1-3){Gal-(alpha 1-3) Gal(beta 1-4)[Fuc(alpha 1-3)GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (trifucosylundecasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3){Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)}Gal(beta 1-4)Glc (trifucosylundecasaccharide). Lactose was present only in trace amounts. B-pentasaccharide was a dominant saccharide in early lactation milk, while alpha 3'-galactosyllactose was dominant in milk, later. The milk oligosaccharides of the Japanese black bear were compared with those of the Ezo brown bear. (C) 1999 Elsevier Science B.V. All rights reserved.
• 和牛精子が発情期子宮頸管粘液へ侵入する能力

  村瀬 哲磨, 向島 幸司, 坂口 慎一, 坪田 敏男, 喜多 功 日本獣医学会学術集会講演要旨集 128回 230 -230 1999年09月
• A simplified technique to determine the ability of Spermatozoa to penetrate the estrous cervical mucus in Japanese beef cattle

  Tetsuma Murase, Koji Mukohjima, Shin-Ichi Sakaguchi, Toshio Tsubota, Isao Kita Journal of Reproduction and Development 45 (6) 425 -428 1999年 [査読無し][通常論文]
   

  This study was carried out to simplify the previous method for the mucus penetration test in Japanese beef cattle. Frozen-thawed semen was brought into contact with frozen-thawed estrous cervical mucus loaded in a rectangular capillary tube under the following conditions for placement of the tube and temperature of incubation: 1) horizontal/38 C (the previous method, Horizontal 38 C), 2) upright/38 C (Upright 38 C), or 3) upright/room temperature (Upright RT). The previous method included fixing the capillary tube with a mixture of vaseline and paraffin and a cover of semen with paraffin oil and a cover slip on a glass slide. This procedure was simplified by use of a small vial, in which semen was placed and covered with paraffin oil in Upright 38 C and Upright RT. Mucus penetration, expressed as the distance travelled by the sperm cell that penetrated the mucus the farthest in 10 min, was similar to Horizontal 38 C and Upright 38 C but was significantly lower with Upright RT. Intra-assay and inter-assay coefficients of variation in Upright 38 C were below 5%. Thus this study simplified the technique of the mucus penetration test using Upright 38 C.
• Sex steroid and prolactin profiles in male American black bears (Ursus americanus) during denning

  T Tsubota, DL Garshelis, RA Nelson, JM Bahr JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (1) 81 -83 1999年01月 [査読無し][通常論文]
   

  Serum sex steroid and prolactin profiles were examined in the male American black bear, Ursus americanus during denning. Sera collected in December and the following March from 8 denning male black bears in Minnesota, U.S.A. were assayed for testosterone, estradiol-17 beta and prolactin. Eight bears were confirmed to be the denning mode based on a serum urea to creatinine ratio less than 10. Serum testosterone concentrations tended to increase from December to the subsequent March whereas serum estradiol-17 beta concentrations tended to decrease during this period. There were few changes in serum prolactin concentrations between December and March. These findings suggest that spermatogenesis and testicular steroidogenesis initiated during denning may be influenced by changes in serum sex steroid concentrations in the American black bear.
• 冬眠中のアメリカクロクマにおける性ステロイドホルモン及びプロラクチン濃度の変化

  坪田 敏男, Garshelis D.L., Nelson R.A., Bahr J.M. The Journal of Veterinary Medical Science 61 (1) 81 -83 1999年01月
• イヌの精巣におけるステロイド代謝酵素3βHSD及びP450aromのmRNAの発現

  坪田 敏男, Morrey Craig E., 長濱 嘉孝, 村瀬 哲磨, 喜多 功 The Journal of Reproduction and Development 44 (6) a48 -a48 1998年12月
• 飼育下及び野生下における成熟雌ニホンツキノワグマ(Ursus thibetanus japonicus)の血清プロジェステロン濃度の測定

  佐藤 美穂, 坪田 敏男, 山本 かおり, 小松 武志, 橋本 幸彦, 片山 敦司, 羽澄 俊裕, 喜多 功 The Journal of Reproduction and Development 44 (6) a55 -a55 1998年12月
• Serum progesterone, oestradiol, luteinizing hormone and prolactin profiles in the female black bear (Ursus americanus)

  T Tsubota, L Howell-Skalla, WR Boone, DL Garshelis, JM Bahr ANIMAL REPRODUCTION SCIENCE 53 (1-4) 107 -118 1998年10月 [査読無し][通常論文]
   

  Identifying steroid and pituitary hormone profiles in the female black bear (Ursus americanus) throughout pregnancy may provide a greater understanding of the reproductive cycle and indicate which hormones are required for implantation. Our objective was to assess endocrine activity in black bears oestrus onset, at oestrus, during pregnancy and after parturition. Serum samples were obtained from 12 captive, 16 uncollared and five radiocollared free-ranging female black bears from March through the end of December and assayed for serum progesterone, oestradiol, luteinizing hormone (LH) and prolactin (PRL). In captive bears, progesterone concentrations were low at days 0-10 after oestrus and increased significantly days 25-35 and 45-52 after oestrus, Oestradiol concentrations were high at oestrus (day 0) and days 4-10 after oestrus and then decreased days 25-35 and 45-52 after oestrus. LH concentrations were not significantly different throughout the sampling period. Changes in PRL concentrations pattern were similar to those of oestradiol, with elevated levels at oestrus and days 4-10 after oestrus, followed by a significant decrease 45-52 days after oestrus. In non-collared free-ranging bears, progesterone concentrations increased gradually after mating with a further significant increase in November-December. Oestradiol concentrations were highest in March (before mating) and in June (during mating) followed by a significant decrease in July (early delay period) and November-December (peri-implantation period). LH concentrations were low until November-December and then increased significantly. PRL concentrations were low in March (before mating), increased significantly during the mating season in June, decreased slightly in July, and were low in November-December (peri-implantation period). In radiocollared free-ranging bears, serum progesterone concentrations were elevated in pregnant bears in December and extremely low in lactating and non-lactating bears in March. Oestradiol levels were slightly higher in pregnant bears in December than in non-lactating or lactating bears in March. PRL concentrations were considerably higher in lactating bears in March than in pregnant bears in December. Our results suggest that: (1) serum progesterone concentrations are low, but detectable during the early delay implantation period and greatly elevated during the peri-implantation period; (2) serum oestradiol concentrations are elevated at oestrus and decline during the delay period; (3) LB may be involved in luteal activation; and (4) the decline of serum PRL concentrations during short days may be necessary for implantation to occur. (C) 1998 Elsevier Science B.V. All rights reserved.
• 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年06月 [査読無し][通常論文]
   

  The seasonal changes of the cytoskeletal protein expressions were immunohistochemically investigated in the testes of Japanese black bear, Ursus thibetanus japonicus. A strong immunoreaction for alpha-smooth muscle actin is restricted to the vascular smooth muscle cells and the peritubular cells which surround the seminiferous tubules by several layers throughout the year. Weak immunoreactions for B4 antigen and desmin were observed in the vascular smooth muscle cells and in a part of peritubular cells throughout the year. A strong immunoreaction for vimentin was also detected in the fibroblasts and Leydig cells, in addition to the vascular smooth muscle and epithelial cells and the peritubular cells throughout the year. A strong alpha-tubulin immunoreaction was detected in the elongating spermatids during the acrosome phase of spermiogenesis in May and June. The cytoplasm of several Sertoli cells was faintly immunoreacted for vimentin in the basal and lateral region, while an intense alpha-tubulin reaction was seen in the entire cytoplasm in May, April and June. In November, January and March, the immunoreactions for vimentin and alpha-tubulin strongly accumulate in a perinuclear region of Sertoli cells when developmental spermatids are not seen in the seminiferous tubules. These accumulations in the immunoreactions for vimentin and alpha-tubulin seem to be caused by the reduction in size of Sertoli cells cytoplasm with season. However, the seasonal changes of distributions in the cytoskeletal proteins are obscure in the bear testes. These results suggest that the contents of cytoskeletal proteins may not change in relation to the morphological differences with season in the testes of the seasonal breeders.
• Circumanal glands of the dog: A new classification and cell degeneration

  Y Atoji, Y Yamamoto, T Komatsu, Y Suzuki, T Tsubota ANATOMICAL RECORD 250 (3) 251 -267 1998年03月 [査読無し][通常論文]
   

  Background: The circumanal glands of the dog are thought to be a glandular tissue, but there is some controversy as to whether they should be classified as exocrine or endocrine. In this study, we examined the nature of the circumanal glands to determine whether they should be described as exocrine, endocrine, or something else altogether. In addition, we investigated the cell degeneration in lobules of the circumanal glands in relation to the apocrine glands. Methods: Light microscopic observations were made of paraffin sections stained with hematoxylin and eosin, and after immunohistochemical staining with antibodies against a-smooth muscle actin, keratin, filaggrin, and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD). Samples were also examined by electron microscopy after fixation by aldehyde perfusion. Results: The lobules of circumanal glands could be divided into two types on the basis of the presence or absence of cysts. Four layers (I-IV) were detected in the lobules with cysts. The outermost layer (layer I or the basal layer) consisted of flattened cells that contained bundles of tonofilaments and were stained immunohistochemically with the antibody against keratin. Layer II (the polyhedral or "spinous" layer) consisted of polyhedral cells that contained bundles of tonofilaments. These cells were connected to adjacent cells by desmosomes, interdigitations, and gap junctions, and they were immunopositive for keratin. A small number of polyhedral cells were immunopositive for 3 beta-HSD. Layer III (the granular layer) was composed of flattened cells that contained hematoxylin-stainable granules and were moderately immunopositive for filaggrin. The innermost layer (layer IV or the horny layer) consisted of keratin. Lobules without cysts consisted only of layer I (the basal layer) and layer II (the polyhedral layer). Lobules of the circumanal glands were not directly connected to apocrine glands. Polyhedral cells degenerated and were phagocytosed by basal cells at a periphery of lobules. Then, basal cells phagocytosing degenerated polyhedral cells escaped from lobules, moved into the walls of apocrine glands, and, finally, dropped into the lumen of apocrine glands. Conclusions: Lobules of the circumanal glands have many characteristics of epidermis (a basal layer, a polyhedral or "spinous layer," a granular layer, and a horny layer) and they should not be classified as glandular tissue. The cysts in lobules can be interpreted as "closed hair canals." We suggest that steroid metabolism might occur in the polyhedral cells of the lobules. (C) 1998 Wiley-Liss, Inc.
• 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(<特集>実践現場における野生動物医学 : その取り組みと成果の具体例)

  坪田 敏男, 溝口 紀泰, 喜多 功 Japanese journal of zoo and wildlife medicine 3 (1) 17 -24 1998年03月 [査読無し][通常論文]
   

  ニホンツキノワグマUrsus thibetanus japonicusは, 本州, 四国および九州に生息する大型哺乳動物の一種である。しかしながら, 最近では, 九州はほぼ絶滅状態となり, さらに四国山地, 西中国地域, 東中国山地および紀伊半島が絶滅のおそれのある地域となっている。1990年から1994年にかけて岐阜県白川村において直接観察, 痕跡調査(糞分析)およびラジオトラッキングといった生態調査が行われた。その結果, ツキノワグマの春と秋の食物種がブナ林という生息環境と密接に関係していることが示された。すなわち, ツキノワグマは, ブナ豊作年にはブナの花芽や種子を食べ, 一方ブナ不作年には他の食物を利用していた。また, 1992年から1993年のツキノワグマの行動圏が求められ, その平均値は雄で6.4km^2, 雌で3.4km^2であった。主に飼育下でのツキノワグマの繁殖生理学的研究により, 雄では季節繁殖性が顕著に認められること, また雌では着床遅延や冬眠中の出産といったクマ類特有の繁殖生理機構を有していることが解明された。これらの結果より, 将来にわたってツキノワグマを保護していくためには, 繁殖の成功につながる十分な食物環境を確保することが肝要であると結論づけることができる。
• ツキノワグマの生態と保護管理

  はくさん 25 (3) 7 1998年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
   

  We visually verified corpora lutea and measured serum progesterone concentrations during the estrous cycle in 8 semi-captive black bears (Ursus americanus) in South Dakota. Our data suggested that black bears ovulate after they mate, indicating that black bears are induced ovulators. These preliminary findings may aid scientists in their efforts to save endangered bear species through controlled breeding programs.
• クマの繁殖 (特集 動物・家畜の繁殖)

  坪田 敏男 畜産の研究 52 (1) 205 -210 1998年01月 [査読無し][通常論文]
  
• Seasonal changes in subcellular structures of Leydig and Sertoli cells in the Japanese black bear, Ursus thibetanus japonicus

  T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki ARCHIVES OF HISTOLOGY AND CYTOLOGY 60 (3) 225 -234 1997年08月 [査読無し][通常論文]
   

  The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year, Lipid inclusions of both electron-dense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity.
• Seasonal changes in subcellular structures of Leydig and Sertoli cells in the Japanese black bear, Ursus thibetanus japonicus

  T Komatsu, Y Yamamoto, Y Atoji, T Tsubota, Y Suzuki ARCHIVES OF HISTOLOGY AND CYTOLOGY 60 (3) 225 -234 1997年08月 [査読無し][通常論文]
   

  The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year, Lipid inclusions of both electron-dense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity.
• Seasonal changes in the immunolocalization of steroidogenic enzymes in the testes of the Japanese black bear (Ursus thibetanus japonicus)

  T Komatsu, T Tsubota, Y Yamamoto, Y Atoji, Y Suzuki JOURNAL OF VETERINARY MEDICAL SCIENCE 59 (7) 521 -529 1997年07月 [査読無し][通常論文]
   

  Seasonal changes in sites of immunostaining of steroidogenic enzymes were examined in testes of the Japanese black bear, Ursus thibetanus japonicus. In addition, serum concentrations of testosterone and estradiol-17 beta were investigated by radioimmunoassay, and the seasonal changes were compared with the results of immunostaining. On the basis of morphological observations of spermatogenic activity, the reproductive cycle was divided into five periods: an active period in May and June; a degenerative period in November; a resting period in January; an early-resumptive period in March; and a late-resumptive period in April. Serum concentrations of testosterone differed with season accompanied by differences in spermatogenic activity, with baseline levels in November and January, increasing levels in March and April, and high levels in May, and April and June of the next year. Immunoreactivities specific for cholesterol side-chain cleaving cytochrome P450, 17 alpha-hydroxylase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were observed in Leydig cells throughout the year. Only the percentages of Leydig cells immunopositive for 3 beta HSD exhibited seasonal differences that correlated with serum concentrations of testosterone. Aromatase cytochrome P450 (P450arom) was immunolocalized in Leydig and Sertoli cells throughout the year, in spermatids in May, and April and June of the next year and in myoid cells in January and March. The percentages of Leydig cells immunopositive for this enzyme increased in May, and January, March and June of the next year. On the other hand, no pattern of seasonal change in serum estradiol-17 beta concentration was observed. These results suggest that 3 beta HSD is a key enzyme in the regulation of the testosterone production in Leydig cells. Furthermore, estrogen derived from Leydig and myoid cells seems to play a role in the regulation of Leydig cells by negative feedback as a paracrine and/or autocrine mediator.
• 獣医野生動物学研究の最先端(5)雄の繁殖学

  坪田 敏男 獣医畜産新報 50 (5) 413 -416 1997年05月 [査読無し][通常論文]
  
• ブナの豊凶がツキノワグマの食性に与える影響-ブナとミズナラの種子落下量の年次変動に関連して-(共著)

  哺乳類科学 36 33 -44 1997年 [査読無し][通常論文]
  
• 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年01月 [査読無し][通常論文]
   

  American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March; 15 lune, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggest that aromatase and oestrogen may play a role in re-initiating spermatogenesis.
• Immunolocalization of steroidogenic enzymes in the corpus luteum and the placenta of Japanese black bears, Ursus thibetanus japonicus during pregnancy

  T Tsubota, S Taki, K Nakayama, JL Mason, S Kominami, N Harada, Kita, I BIOLOGY OF REPRODUCTION 56 30 -30 1997年 [査読無し][通常論文]
  
• Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido Brown bear, Ursus arctos yesoensis during the mating season

  H Araki, T Tsubota, N Maeda, N Harada, S Kominami, JJ Mason, Kita, I JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (8) 787 -790 1996年08月 [査読無し][通常論文]
   

  Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.
• ニホンカモシカCapricornis crispus子宮壁動脈の妊娠性硬変 : 経産歴との関連において

  喜多 功, 鈴木 法子, 丹羽 範郎, 坪田 敏男 Japanese journal of zoo and wildlife medicine 1 (2) 113 -117 1996年08月 [査読無し][通常論文]
   

  成熟雌ニホンカモシカCapricornis crispus30頭の子宮壁動脈を組織学的に検索した結果, 経産カモシカの子宮壁動脈に妊娠性硬変が見出された。すなわち, 未経産成雌の子宮壁動脈では, 内弾性板は薄く, その屈曲は単純であったが, 1〜3産例の子宮壁動脈では, 内弾性板は厚く明瞭で, 複雑な屈曲を示していた。5産以上の例では, 子宮壁動脈に2層以上の弾性板様構造物が出現し, 中膜における弾性線維の増加と平滑筋細胞の核の変性が観察された。8産以上になると子宮壁動脈の中膜に弾性線維の変性(エラストージス)および平滑筋細胞の核の変性・消失が起こった。以上のことから, 子宮壁の組織学的検査によって, 産歴不明のカモシカ個体における経産の判定および経産回数の推定が可能と思われた。
• 交尾期のエゾヒグマ卵巣におけるステロイド合成酵素の免疫局在

  荒城 寛, 坪田 敏男, 前田 菜穂子, 原田 信広, 小南 思郎, Mason J.I, 喜多 功 The journal of veterinary medical science 58 (8) 787 -790 1996年08月 [査読無し][通常論文]
   

  交尾期にあるエゾヒグマの卵巣を用いて4種のステロイド合成酵素の局在を免疫組織化学的に調べた. この卵巣は, 大型卵胞を有していたことより, おそらく卵胞期のものと考えられた. 大型卵胞においては, P450 sccおよび3βHSDは内卵胞膜細胞と顆粒層細胞で認められた. P450 c17は内卵胞膜細胞で認められたが, 顆粒層細胞では認められなかった. また, P450 aromは顆粒層細胞で認められた. しかし, 中型卵胞においては, P450 sccおよび3βHSDは内卵胞膜細胞にのみ認められ, P450 aromは内卵胞膜細胞と顆粒層細胞のいずれにも認められなかった. 間質細胞では, P450 aromを除く全ての酵素が認められた. 以上の結果は, エゾヒグマの卵胞におけるエストロジェンの産生機序は卵胞膜細胞と顆粒層細胞の両者の関連によるものであり, いわゆる2細胞説に従うこと, 大型卵胞の顆粒層細胞はプレグネノロンおよびプロジェステロンの産生能力を有すること, さらに間質がステロイド合成能力を有することを示唆している.
• Spermatogenic cycle in the testis of the Japanese black bear (Selenarctos thibetanus japonicus)

  T Komatsu, Y Yamamoto, T Tsubota, Y Atoji, Y Suzuki JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (4) 329 -335 1996年04月 [査読無し][通常論文]
   

  Spermatogenic cycle in the testis of the Japanese black bear (Selenarctos thibetanus japonicus) was studied by light and transmission electron microscopy. By light microscopy, spermatids were allocated into eleven steps based on morphological changes in the nucleus and the acrosome of spermatids. Cellular associations of the seminiferous epithelium were allocated into eight stages based on the changes in the nucleus and acrosome of spermatids, appearance of meiotic figures and time of spermiation. Cross-sections of the seminiferous tubule seldom contained more than one type of stage. Spermatids at steps 1-2 had the well-developed Golgi complex. The crescent-shaped Golgi complex was accompanied by the acrosome extending over the nucleus at steps 3-5. At step 6, spermatids faced the base, and the outer membrane of the acrosome converged upon the plasma membrane of spermatids. The acrosome projected into the cytoplasm of Sertoli cells at step 9. At step 11, most of the cytoplasm was phagocytosed by Sertoli cells, and spermatids were released in the lumen to become spermatozoa.
• ニホンツキノワグマ(Selenarctos thibetanus japonicus)の精巣における精子形成サイクル

  小松 武志, 山本 欣郎, 坪田 敏男, 阿閉 泰郎, 鈴木 義孝 The journal of veterinary medical science 58 (4) 329 -335 1996年04月 [査読無し][通常論文]
   

  野生個体1頭と飼育個体3頭から採取した精巣組織材料を用いて, ニホンツキノワグマ(Selenarctos thibetanus japonicus)における精子形成サイクルについて, 光学顕微鏡および電子顕微鏡による観察を行った. 光顕による観察から, 精子細胞の核およびアクロソームの形態学的変化に基づいて, 精子細胞を11ステップに分類した. さらに, この精子細胞の形態学的変化, 減数分裂像および精子細胞の管腔への遊離時期を指標にして, 精上皮サイクルを8ステージに区分した. 一つの精細管横断面は, 大抵単一のステージによって占められていた. ステップ1-2の精子細胞は, よく発達したゴルジ装置を持ち, ステップ3-5では核膜表面を被うアクロソームに沿って, 三日月状に観察された. ステップ6の精子細胞は先端を基底膜方向に向け, 精子細胞の細胞膜とアクロソーム外膜とが接着した. ステップ9においては, アクロソームがセルトリ細胞の細胞質ヘ突出している像が観察され, ステップ11になると, 精子細胞の細胞質のほとんどがセルトリ細胞に取り込まれ, 精子細胞そのものは精子として管腔へ遊離した.
• 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観察

  片山 敦司, 坪田 敏男, 山田 文雄, 喜多 功, 千葉 敏郎 Japanese journal of zoo and wildlife medicine 1 (1) 26 -32 1996年02月 [査読無し][通常論文]
   

  1991年3月から1993年8月までの間に, 岐阜県および京都府で捕殺された雌ニホンツキノワグマ(Selenarctos thibetanus japonicus)19頭の生殖器の肉眼的および組織学的観察により, 性成熟年齢, 排卵数, 着床数, 一腹産子数および繁殖歴などを推定した。卵巣の重量および大きさは加齢に伴って増加の傾向を示した。その傾向は未成熟個体で顕著であり, 性成熟個体で緩やかであった。黄体および黄体退縮物の存在を性成熟の基準とした場合, 4歳以上の全ての個体は性成熟に達していると判定された。しかし, 4歳未満でも性成熟に達する例も存在することが示唆され, 性成熟に達する年齢には個体差があることがうかがわれた。黄体, 黄体退縮物および胎盤痕の観察と連れ子の数から平均排卵数は1.89, 平均着床数は2.00, および平均連れ子頭数は, 1.86と算定された。さらに, 黄体退縮物の組織学的観察により, 捕殺時点における過去の総排卵数の推定を試みた。その結果, 黄体および黄体退縮物の数と交尾期経過回数には正の相関が認められた。しかし, 黄体およびその退縮物の数にはばらつきがあり, 交尾期経過回数との間に大きな差が認められる例もあった。
• Changes in steroidogenic sites during pregnancy in the domestic cat

  T Tsubota, H Araki, JI Matson, S Kominami, N Harada, Kita, I BIOLOGY OF REPRODUCTION 54 262 -262 1996年 [査読無し][通常論文]
  
• Validity of retrograde corpora lutea of pregnancy as an index of past gestation in sika deer, Cervus nippon

  Kita, I, M Eguchi, S Takatsuki, T Koizumi, T Tsubota JOURNAL OF VETERINARY MEDICAL SCIENCE 57 (6) 1029 -1033 1995年12月 [査読無し][通常論文]
   

  Corpora lutea of the sika deer (Cervus nippon, Cervidae) were histologically examined in order to elucidate their retrograding processes after parturition. They showed a remarkable regression within one month as luteal cells decreased in number and, by nine months after parturition, turned into masses of blood vessels containing many arterioles with a diameter of 30 mu m or less. In retrograde corpora lutea (RCL), older than 1.5 years after parturition, arterioles of these dimensions decreased markedly, while those measuring 50 mu m or more became dominant. These findings indicate that an examination of RCL can distinguish females less than nine months after parturition from those more than 1.5 years after parturition. Based on the findings of RCL from the ovaries of a 10.5-year-old female, it is suggested that RCL remain in the ovaries for at least 8.5 years after parturition. It was, however, difficult to distinguish RCL of pregnancy from retrograde accessory corpora lutea (RACL). In order to arrive at a complete count of RCL, the ovaries should be examined at thicknesses of 0.5 mm. To check the presence/absence of RCL of pregnancy within nine months or less after parturition, sectioning at thicknesses of 2 mm is sufficient.
• ニホンジカにおける経産指標としての黄体退縮物の有効性

  喜多 功, 江口 雅子, 高槻 成紀, 小泉 透, 坪田 敏男 The journal of veterinary medical science 57 (6) 1029 -1033 1995年12月 [査読無し][通常論文]
   

  分娩後におけるニホンジカ黄体の退縮過程について組織学的研究を行った. 黄体は分娩後1か月以内に黄体細胞の減少と共に急激にその大きさを減じ, 分娩後9か月迄に直径30μm以下の小動脈を多く含む血管の集積に変った. 分娩後1.5年以上の退縮物では直径30μm以下の小動脈が著しく減少して, 直径50μm以上の小動脈が多数を占めた. 従って, 黄体退縮物の検査によって分娩後9か月未満の個体と1.5年以上の個体とを区別することが可能である. 10.5歳の個体における退縮物の所見に基づき, 退縮物は分娩後少なくとも8.5年は卵巣中に残存すると推定された. 妊娠黄体退縮物と副黄体退縮物とを組織学的に区別することは困難であった. すべての黄体退縮物を洩れなく数えるためには, 卵巣を0.5mm間隔で観察する必要があるが, 分娩後9か月以内の黄体退縮物の有無を調べるだけならば, 卵巣を2mm間隔で観察すればよい.
• 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年07月 [査読無し][通常論文]
   

  Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig I-125-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4-14.8 ng ml(-1); t = 15-30 min, 18.4-28.7 ng ml(-1)). The sensitivity of the assay was 0.08 ng per tube. Intra- and interassay coefficients of variation were 5.5% (n = 6) and 5.7% (n = 6), respectively. Serum concentrations of prolactin changed seasonally, with the lowest concentrations in December (mean +/- SD = 1.1 +/- 0.1 ng ml(-1)); this was followed by a gradual increase between January (2.6 +/- 0.6 ng ml(-1)) and April (6.4 +/- 1.2 ng ml(-1)) and the highest concentrations in May (17.6 +/- 4.7 ng ml(-1)), preceding peak testosterone concentrations in June. The observation that prolactin secretion increased with increasing daylength suggests that photoperiod may be an external regulator. The presence of high concentrations of prolactin before peak testosterone concentrations suggests that prolactin may play a role in regulating seasonal changes in the testes.
• クマ類の冬眠を考える

  羽澄 俊裕, 間野 勉, 近藤 宣昭, 坪田 敏男 哺乳類科学 = Mammalian Science 34 (2) 158 -162 1995年03月30日
• 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年08月 [査読無し][通常論文]
   

  The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.
• 雄ニホンツキノワグマ(Selenarctos thibetanus japonicus)における性成熟と精子形成にかかわる幹細胞(共著)

  Takeshi Komatsu, Toshio Tsubota, Tosiro Tiba, Mayumi Kishimoto, Shin-Itiro Hamasaki Journal of Reproduction and Development 40 (6) j65-j71 1994年 [査読無し][通常論文]
   

  Testes of Japanese black bears (Selenarctos thibetanus japonicus) were collected from 27 bears shot by hunters and biopsied from 13 bears captured by traps. The size and weight of the testis, and the diameter of seminiferous tubules increased in 2 or 3 year-old bears. The percentage of sexually matured bears in each age group was 0% (1 year-old), 50% (2 year-old) and 100% (over 3 year-old), respectively. Therefore, puberty was assumed to be at 2 or 3 year of age. Only Sertoli and large round cells were the cell population in the seminiferous epithelium of prepuberal testis. The morphological characteristics of the large round cells were identified with that of gonocyte as reported in other species and were judged to be the stem cells in the testis of the prepuberal bear. The large cells resembling gonocyte were present in the seminiferous tubules of the puberal testis. In the present study, these cells were referred to as gonocyte-like cell. These cells increased in number during the non-breeding season. The gonocyte-like cells were morphologically similar to those of undifferentiated type A-spermatogonia. Therefore, these cells may be equivalent to undifferentiated type A-spermatogonia, that is, the stem cells in the testis of the puberal bear for resumption of spermatogenesis. © 1994, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
• Parturition and Postnatal Development in the Captive Hokkaido Brown Bear Ursus arctos yesoensis

  Tsubota Toshio, Maeda Naoko, Kanagawa Hiroshi Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ｍａｍｍａｌｏｇｉｃａｌ Ｓｏｃｉｅｔｙ ｏｆ Ｊａｐａｎ 19 (2) 75 -82 1994年 [査読無し][通常論文]
   

  This study was carried out to clarify the developmental biology of Hokkaido brown bears Ursus arctos yesoensis from birth to approximately 90-day-old. Though the birth date of 107 litters ranged from 11 January to 1 March, 91% of the cubs were born between 17 January and 5 February. The body weight of 40 neonates was 419±40 g (mean±SE) and did not differ significantly between the sexes (p>0.05). Mean litter size was 1.82±0.70 (SE). Mean body weight gain per day, an indicator of bear development, was calculated for 192 cubs from the date and body weight at birth and at about 90-day-old. The body weight gain varied greatly among individuals (range: 0.018-0.130 kg/day) and tended to be smaller as litter size increased. The body weight gain differed significantly (p<0.01) between the sexes when there was only one cub, but not when there were two or more cubs. The mean intake of calories per unit body weight gain from birth to 68 days after birth, was calculated at 4,000 kcal/kg for the hand-reared cubs.
  
• SERUM ESTRADIOL-17-BETA, PROGESTERONE, LUTEINIZING-HORMONE AND PROLACTIN PROFILES DURING THE LUTEAL-PHASE IN BLACK BEARS, URSUS-AMERICANUS

  T TSUBOTA, WR BOONE, DL GARSHELIS, RA NELSON, JM BAHR BIOLOGY OF REPRODUCTION 50 82 -82 1994年 [査読無し][通常論文]
  
• 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年12月 [査読無し][通常論文]
  
• 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年09月 [査読無し][通常論文]
   

  It was investigated in the domestic fowl, whether a mechanism for a synchronous stan of the proliferation of the differentiating spermatogonia in a transversely cut seminiferous tubule (called the ''co-ordination' between germ cell association) operates effectively in this species. A trapezoid-shaped cellular association demarcated by two perpendiculars from the tubular lumen to the basement membrane was the unit of observation (provisionally called a ''column'' Each column is composed of a bundle of elongate spermatids and the neighbouring, various types of germ cells). The extent of closeness of the co-ordination was examined in a total of 2,650 columns, based on the continuity of the spermiogenic sptep-number of the youngest generation of spermatids within adjacent columns. In no case did all columns in a tubular cross section show the same-step spermatids. In most cases (50.2 % of 2,650 columns), the same number was kept in only 2 to 6 adjacent columns. The arrangement of different but consecutive numbers (e. g., 1-2-3 or 1-2-1) was observed in a total of 882 columns. The co-ordination mechanism, therefore, is considered to work, albeit incomplete, among a limited number of adjacent columns in this species.
• 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年09月 [査読無し][通常論文]
   

  A cellular association demarcated by two perpendiculars which were drawn between adjacent bundles of elongate spermatids from the tubular lumen to the basement membrane, was made the unit of histometrical observation in this study (provisionally called a ''column''). Cell counting revealed that the average numbers per column of various types of germ cells do not show any significant differences among 5 fowls and between paired testes. The frequency of spermiogenic steps (numbered 1-8) was investigated in each column. A definite and common pattern was found in the frequency distribution in the 5 fowls observed. A relationship between spermiation and younger spermatid steps was also investigated in each column. The spermiation was found at different steps, but most frequently at step 2 (30.6 %). Based on these observations and referring to other author's information, an average time interval between two successive spermiations was calculated roughly at 3.3 +/- 1.2 days. Theoretically, this value is equal to an average length of one epithelial cycle. Such a variable cycle may have caused irregular cellular associations in this species.
• Morphological Characteristics of the Ovary, Uterus and Embryo during the Delayed Implantation Period in the Hokkaido Brown Bear (Ursus arctos yesoensis)

  Toshio Tsubota, Hiroshi Kanagawa Journal of Reproduction and Development 39 (4) 325 -331 1993年 [査読無し][通常論文]
   

  Ovaries having corpora lutea and uteri of 6 female Hokkaido brown bears (Ursus arctos yesoensis), which were legally killed by hunters from October to December in Hokkaido, Japan, were examined macroscopically and histologically. Four of the 6 female bears had unimplanted embryos in the uterine lumen during the period of delayed implantation between October 19 and November 20. Ovaries of bears which had unimplanted embryos contained the corpora lutea with extensive vacuolation or numerous granules in the cytoplasm of luteal cells. These bears had well-developed uterine glands which had tall epithelial cells with vacuoles in the cytoplasm of glandular cells and coagula in the glandular lumina. It is concluded that Hokkaido brown bears exhibit obligate delayed implantation and that this period of delayed implantation continues at least until late November. © 1993, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
• SERUM PROGESTERONE CONCENTRATIONS USING P-EIA KIT IN CAPTIVE AND FREE-RANGING HOKKAIDO BROWN BEARS, URSUS-ARCTOS-YESOENSIS

  T TSUBOTA, H KANAGAWA, K YAMAMOTO, T MANO, M YAMANAKA, KITA, I, T TIBA JOURNAL OF VETERINARY MEDICAL SCIENCE 54 (1) 1 -5 1992年02月 [査読無し][通常論文]
   

  Serum progesterone (P) concentrations using P-EIA kit (Ovucheck, Cambridge Life Science Co., Ltd.) were examined in 8 captive and 7 free-ranging female Hokkaido brown bears (Ursus arctos yesoensis). The intra- and inter-assay coefficients of variation were 8.9%, 12.6% and 16.6%, 22.7%, respectively, based on 2 serum samples. There was a significant correlation between EIA and radioimmunoassay results based on 64 serum samples (r = 0.725; p < 0.01). Serum P concentrations were examined in 5 pregnant, 2 solitary non-pregnant bears and a lactating non-pregnant bear in captivity. Annual changes of P levels in pregnant bears were observed as a small elevation during the mating season (May-June), a re-elevation in September-October and a sharp elevation in November-December. The sharp elevation was suspected to reflect changes when implantation occurred. Annual changes of P levels in solitary non-pregnant bears were similar to those in pregnant bears. An annual change of P levels in a lactating non-pregnant bear maintained levels under 5 ng/ml. Two of 7 free-ranging bears exhibited P levels over 1 ng/ml and the birth of cubs was confirmed in the following year in 1 of the 2 bears. P concentrations of other free-ranging bears exhibited less than 1 ng/ml, and these bears were considered to be non-pregnant. It was concluded that P-EIA kit was available for measuring P concentrations in Hokkaido brown bears.
• 飼育下および野生エゾヒグマUrsus arctos yesoensisにおけるP-EIAキットによる血清中プロジェステロン値の測定

  坪田 敏男, 金川 弘司, 山本 聖子, 間野 勉, 山中 正実, 喜多 功, 千葉 敏郎 日本獣医学雑誌 54 (1) 1 -5 1992年02月 [査読無し][通常論文]
   

  飼育下8頭および野生7頭の雌エゾヒグマについて, プロジェステロン(P)測定用エンザイムイムノアッセイ(EIA)キット(「オブチェック」ケンブリッジ・ライフ・サイエンス社)を用いて血清中P値を測定し, その有効性を検討した. 本キットによる2検体の測定内および測定間変動係数は, それぞれ8.9%, 12.6%および16.6%, 22.7%と比較的良好な成績であった. ラジオイムノアッセイ法との相関関係については, 64サンプルで相関係数r=0.725と高い相関が認められた(p<0.01). 飼育エゾヒグマでは, 妊娠個体5頭, 非妊娠単独個体2頭および非妊娠子連れ個体1頭についてP値が調べられた. 妊娠個体のP値は, 交尾期(5〜6月)後の小さな上昇, 9〜10月にかけての2回目の上昇, さらに11〜12月にかけての大きな上昇として観察された. この最後の大きなP値上昇は, 着床に伴う変化と推測される. 非妊娠単独個体のP値変化は, 妊娠個体のP値変化と類似した. 非妊娠子連れ個体のP値は, 6〜12月まで5 ng/ml以下の値を持続した. 野生エゾヒグマ7頭中2頭は, 1 ng/ml以上の値を示し, そのうちの1頭では出産が確認された. 他の5頭はいずれも1 ng/ml以下の低値であり, 非妊娠個体と考えられた. エゾヒグマでのP-EIAキットによるP値の測定は有効であると...
• クマ類の繁殖特性 (野生動物保護<特集>)

  坪田 敏男 生物科学 43 (4) p190 -194 1991年11月 [査読無し][通常論文]
  
• エゾヒグマの繁殖生理

  坪田 敏男 遺伝 45 (7) p77 -81 1991年07月 [査読無し][通常論文]
  
• IMMOBILIZATION OF THE FREE-RANGING HOKKAIDO BROWN BEAR, URSUS-ARCTOS-YESOENSIS WITH KETAMINE-HYDROCHLORIDE AND XYLAZINE HYDROCHLORIDE

  T TSUBOTA, K YAMAMOTO, T MANO, M YAMANAKA, H KANAGAWA JOURNAL OF VETERINARY MEDICAL SCIENCE 53 (2) 321 -322 1991年04月 [査読無し][通常論文]
  
• EMBRYO RECOVERY DURING DELAYED IMPLANTATION IN THE CAPTIVE HOKKAIDO BROWN BEAR, URSUS-ARCTOS-YESOENSIS

  T TSUBOTA, Y TAKAHASHI, H KANAGAWA, K GOHDA JOURNAL OF VETERINARY MEDICAL SCIENCE 53 (1) 141 -142 1991年02月 [査読無し][通常論文]
  
• Embryo Recovery during Delayed Implantation in the Captive Hokkaido Brown Bear, Ursus arctos yesoensis

  Toshio Tsubota, Yoshiyuki Takahashi, Hiroshi Kanagawa, Katsumi Gohda Journal of Veterinary Medical Science 53 (1) 141 -142 1991年 [査読無し][通常論文]
  
• ニホンジカの大量捕獲方法の検討

  梶 光一, 小泉 透, 大泰司 紀之, 坪田 敏男, 鈴木 正嗣 哺乳類科学 30 (2) 183 -190 1991年
• エゾヒグマの繁殖生理に関する研究(1990年度島村賞受賞講演)

  坪田 敏男 家畜繁殖学雑誌 36 (5) p1 -10 1990年12月 [査読無し][通常論文]
  
• 雄エゾヒグマにおける性成熟と精子形成の季節的変動

  坪田 敏男 日本獣医学会学術集会講演要旨集 110回 226 -226 1990年10月
• Studies on reproductive physiology of Hokkaido brown bear, Ursus arctos yesoensis

  Jpn. J. Anim. Reprod 36,1-10 1990年 [査読無し][通常論文]
  
• Annual Changes in Serum Testosterone Levels and Spermatogenesis in the Hokkaido Brown Bear,Ursus arctos yesoensis

  Tsubota Toshio, Kanagawa Hiroshi Journal of the Mammalogical Society of Japan 14 (1) 11 -17 1989年07月 [査読無し][通常論文]
  
• エゾヒグマにおける着床遅延期間の卵巣,子宮および胚の形態

  坪田 敏男 日本獣医学会学術集会講演要旨集 105回 261 -261 1988年04月
• エゾヒグマの胚,胎仔および新生仔の発育について

  坪田 敏男 日本獣医学会学術集会講演要旨集 104回 219 -219 1987年07月
• 少数飼育下におけるエゾヒグマの性行動

  坪田 敏男, 金川 弘司 家畜繁殖学雑誌 32 (4) 184 -187 1986年12月 [査読無し][通常論文]
  
• エゾヒグマの血清テストステロン値と精子形成の季節変化

  坪田 敏男 哺乳動物学雑誌 11 (1〜2) 82 -83 1986年05月
• エゾヒグマにおける白体,胎盤痕からの経産歴判定の試み

  坪田 敏男 日本獣医学会学術集会講演要旨集 101回 235 -235 1986年03月
• エゾヒグマの飼育条件下における性行動の観察

  坪田 敏男 家畜繁殖学雑誌 31 (4) 203 -210 1985年12月 [査読無し][通常論文]
  
• エゾヒグマの痕跡を求めて (動物が残したもの<特集>)

  坪田 敏男 動物と自然 15 (12) p20 -24 1985年11月 [査読無し][通常論文]
  
• REPRODUCTIVE PHYSIOLOGY OF EZO BROWN BEAR, URSUS ARCTOS YESOENSIS, WITH PARTICULAR REFERENCE TO REPRODUCTIVE CYCLE

  TSUBOTA Toshio Japanese journal of veterinary research 33 (1) 100 -100 1985年04月30日 [査読無し][通常論文]
  
• エゾヒグマ胎子の超音波断層像について

  椿 茂, 戸尾 祺明彦, 坪田 敏男, 高橋 芳幸, 金川 弘司 家畜繁殖学雑誌 31 (2) 90 -92 1985年 

  Pregnancy diagnosis by the ultrasonogram method was carried out in a captive 13 years old female Hokkaido brown bear. Two fetuses were detected in the left abdomen. The fetus transverse diameter was 5.2 cm and the heart rate was 180 beats/min. Further improvement is necessary to diagnose the number of fetuses by our method, for she gave birth to 3 cubs 4 days later.
• クマ牧場に集団飼育されているエゾヒグマの交尾行動の観察

  坪田 敏男 家畜繁殖学雑誌 29 (5) 50 -51 1983年12月

受賞

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

共同研究・競争的資金等の研究課題

• 冬眠動物の“衰えない筋肉”をヒントにした新たな骨格筋萎縮予防戦略の開発

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2022年04月 -2026年03月 

  代表者 : 宮崎 充功, 坪田 敏男, 山口 良文, 渡邊 正知, 下鶴 倫人
• オスグマの行動を決めるもの－個体レベルの行動・生理研究－

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 山崎 晃司, 坪田 敏男, 小池 伸介
• ネパールの希少種に致死感染症は侵淫しているか？生物多様性ホットスポットの保全科学

  日本学術振興会：科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))

  研究期間 : 2021年10月 -2024年03月 

  代表者 : 坪田 敏男, THAPA JEEWAN, 大沼 学, 鍋島 圭, 鈴木 定彦

   

  本年度は、新型コロナウイルス感染症のパンデミックにより研究対象国ネパールに行くことができなかったため、研究はほとんど進んでいない。しかしながら、海外渡航再開に向けて、日本でできる準備を進めた。 まず、ネパールの海外共同研究者であるDr. Naresh SubediおよびDr. Rabin Kadariyaとオンラインで打ち合わせ会議を実施した。研究の全体像、渡航可能となった際のおおよそのスケジュール、本研究プロジェクトに参加する大学院生、ならびに野生アジアゾウの捕獲とGPS首輪装着の必要性等について確認した。また、研究協力者の大学院生（ネパールからの留学生2名：Rajan PaudelとArjun Pandit）により、チトワン国立公園における野生動物（ナマケグマ）の生態学的研究ならびに野生アジアゾウからの結核菌の分離および遺伝子型別の試験的実験を進めることができた。具体的には、チトワン国立公園におけるナマケグマの分布状況を食物資源との関係で解析した。また、BCGに使われる結核菌を使って家畜の糞から効率的に結核菌DNAを検出する方法論を検討した。さらに、研究協力者であるDr. Amir Sadaulaが、チトワン国立公園において飼育下アジアゾウから血液および糞のサンプリングを行った。最後に、令和4年度にはネパールへの渡航が可能になると予想され、近い将来アジアゾウに装着するGPS首輪の入手について取扱業者と打合せを行った。
• ヒグマとシカを宿主とするマダニ媒介性感染症－感染率を決めるのは宿主かベクターか？

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 坪田 敏男, 佐鹿 万里子, 下鶴 倫人, 中尾 亮, MOHAMED MOUSTAFA
• 骨格筋萎縮耐性を獲得させる冬眠誘導性生理活性物質の探索

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  研究期間 : 2020年07月 -2023年03月 

  代表者 : 宮崎 充功, 坪田 敏男, 下鶴 倫人

   

  【本研究成果のポイント】●冬眠期において採取したツキノワグマ血清と共にヒト筋肉細胞を培養すると、筋肉細胞の総タンパク質量を増加させることを明らかにした。●この筋肉細胞の総タンパク質量の増加は、冬眠期クマ血清の添加が筋タンパク質分解系を抑制することで達成される可能性を示した。●これらの成果は、長期間の不活動・栄養不良を経験し、それでもなお筋肉が衰えないという冬眠動物の特徴を説明するものであり、将来的にはヒトの寝たきり防止や効果的なリハビリテーション手法の開発につながることが期待される。
  【研究成果の概要】本研究では、実験的に採取したツキノワグマ血清をヒト骨格筋培養細胞に添加するというin vitroの解析系を用いた検討を行ったところ、冬眠期クマ血清の添加により、骨格筋細胞における総タンパク質量が増加することを確認しました。また冬眠期クマ血清の添加は、タンパク質合成系の制御系であるAkt/mTOR系の制御に関与する可能性があること、またタンパク質分解系因子の一つであるMuRF1の発現量を転写因子FOXO3aの制御系を介して調節することなどを明らかにした。
  【今後の研究の展開】冬眠動物は、生命維持のために一定程度のエネルギー代謝を維持しながら長期間の不活動・栄養不良を経験し、それでもなお筋肉がほとんど衰えないという、不思議な形質を備えている。本研究により、冬眠期のクマ血清に存在する「何らかの因子」がヒト骨格筋培養細胞のタンパク質代謝を制御し、筋肉量維持に貢献する可能性が示された。しかしながら、この「何らかの因子」の特定には現在も至っていない。この因子の特定を含め、冬眠動物が有する「使わなくても衰えない筋肉」という未解明の仕組みを明らかにすることで、最終的にはヒトの寝たきり防止や効果的なリハビリテーション手法の開発などが期待される。
• それぞれのクマの春と夏の過ごし方－個体レベルの生態学的・生理学的研究－

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 山崎 晃司, 坪田 敏男, 森光 由樹, 小池 伸介

   

  食物が欠乏し，前年の秋期に蓄えた体脂肪が減少する春から夏はツキノワグマにとってのcritical seasonと言える。本来であれば，活動量を下げ，エネルギー消費を最小限に抑えたい時期である。異なる社会的ステータス（オス，メス，年齢，子の有無など）のクマが，どのようにこの状況に応答しているのかについて，最新の研究機材と手法を用いて調べた。その結果，子連れのメス，繁殖に参加するオス，そして特に亜成獣オスについては，活動を停めることは難しいことが示された。このような行動的特性が，クマの人里への出没に結び付いている可能性もあった。
• ツキノワグマにおける冬眠中の体温調節機構の解明－ふるえ産熱か非ふるえ産熱か－

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2017年04月 -2020年03月 

  代表者 : 坪田 敏男, 宮崎 充功, 佐鹿 万里子, 下鶴 倫人, MOHAMED MOUSTAFA, 岡松 優子

   

  冬眠中のツキノワグマにおける体温調節機構を解明することを目指して、とくに体脂肪を使った非ふるえ産熱に着目して研究を行った。飼育下（北秋田市阿仁クマ牧場）のツキノワグマに体温および心拍数測定用データロガーを埋め込み、冬眠期とその前後の期間の体温および心拍数をモニタリングした。その結果、11月下旬にツキノワグマを個室に移動させ、給餌を停止した直後に体温および心拍数が低下し、冬眠に入ったと判断された。その後は、雄と雌とで体温および心拍数の変化に違いが認められ、冬眠時期（雌では繁殖プロセス）によって体温と心拍数の調節機序が異なることが示唆された。非ふるえ産熱に使われる褐色脂肪の存在は認められなかった。
• ツキノワグマはいつ脂肪を蓄えどのように利用しているのか？－その行動生態学的研究－

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2016年04月 -2019年03月 

  代表者 : 山崎 晃司, 坪田 敏男, 森光 由樹, 小池 伸介

   

  ツキノワグマの秋期の生態は明らかになりつつあるが，春から夏の生態は未知である。そこで，生理ロガー，非侵襲式体重計測システム，首輪型カムの3種類の機材を用いてその解明を試みた。生理ロガーと体重計計測システムからは，クマの心拍や体重が，夏期に明瞭に落ち込むことが確認され，同属のヒグマとは大きく異なるメカニズムを示した。カムを用いた摂食量の推定は，レンズ部がクマの体によって遮蔽される割合が高く，十分な量のデータを得られなかったが，改善策に見込みをつけることができた。
• 絶食・低代謝状態における繁殖生理メカニズムの解明―冬眠するクマをモデルとして―

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 坪田 敏男, 佐鹿 万里子, 下鶴 倫人

   

  北秋田市阿仁クマ牧場（くまくま園）で飼育されている５頭の成熟雌ツキノワグマを対象にして、胸部皮下に心拍・体温測定用ロガー（DST milli-HRT、Star:Oddi）を埋め込み、冬眠前から冬眠後の約６ヶ月間、心拍数および皮下体温をモニタリングした。１月には超音波画像診断装置をを使って妊娠診断を行った。特筆すべき研究成果として、妊娠個体の体温と心拍数の関係において、冬眠前時期（着床遅延期間）には両者はおよそ24時間周期の変化を示したが、冬眠導入後の胎子発育時期には高体温を維持しながら心拍数は一過性のピークを示し、出産後には体温と心拍数が同調して周期性のない数日間隔での増減を繰り返した。
• 冬眠動物はなぜ寝たきりにならないのか？ －骨格筋萎縮耐性獲得の分子基盤の解明－

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 宮崎 充功, 坪田 敏男, 下鶴 倫人

   

  本研究では、「冬眠」を長期間の寝たきり状態と仮定して、冬眠動物（ツキノワグマ）がどのような仕組みを利用して骨格筋量を維持しているのか、筋肉細胞の中のタンパク質や遺伝子の働きに注目して解析しました。その結果、骨格筋量の維持に影響を与える因子のうち、筋タンパク質合成を促進する命令系統が冬眠後の筋肉で活性化されていることや、筋肉が大きくなり過ぎないようリミッターの役割をしている遺伝子の働きが冬眠中に抑えられていること、などがわかりました。
• 化学物質汚染に対する産業動物の生体防御機構の研究基盤

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2012年04月 -2016年03月 

  代表者 : 石塚 真由美, 池中 良徳, 坪田 敏男, 今内 覚

   

  産業動物について、第I相反応であるシトクロムP450、第II相反応であるグルクロン酸抱合および硫酸抱合酵素について種差を明らかにした。哺乳類の尿におけるグルクロン酸抱合と硫酸抱合体比を比較し、その特徴を明らかにした。また、国内外の産業動物に蓄積する環境化学物質を分析し、ウシなどについては、重金属蓄積性が比較的高いことを見出した。鳥類について家禽を中心にトランスクリプトーム解析を行い、肝臓に発現する第I相反応酵素および第II相反応酵素の網羅的解析を行った。以上の研究により、産業動物の化学物質に対する防御機構を、異物代謝酵素を中心に明らかにした。
• ツキノワグマは春から夏をどうしのいでいるのか？－その行動生態学的研究－

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2012年04月 -2015年03月 

  代表者 : 山崎 晃司, 坪田 敏男, 小池 伸介, 清水 慶子, 正木 隆, 郡 麻里, 小坂井 千夏, 中島 亜美, 根本 唯

   

  ツキノワグマの行動生態研究の多くは，秋の食欲亢進期に行われており，春から夏の行動はよく分かっていない。本研究では，各種新型機材を利用し，冬眠明け後の春から夏の野生グマの生理状態の把握と共に，その行動生態の解明を試みた。その結果，夏期には活動量，体温，心拍計共に低下することを確かめた。春は低繊維，高タンパクの新葉が利用できたが，その期間は極めて短かった。夏は食物の欠乏期として捉えられ，夏眠のような生理状態に入ることでエネルギー消費を防いでいたと考えられた。本研究は，秋の堅果結実の多寡だけでは説明できていない，本種の夏の人里への出没機構の解明にも役立つことが期待できる。
• ヒグマ個体群のアトラクティブ・シンク現象解明とモニタリングおよび被害管理への応用

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2011年04月 -2015年03月 

  代表者 : 佐藤 喜和, 高田 まゆら, 園原 和夏, 坪田 敏男, 間野 勉

   

  ヒグマの出没と駆除が増加している。その原因は生息数の増加ではなく，人里周辺が死亡リスクの高さ以上に魅力的であるためというアトラクティブ・シンク仮説を立て，北海道東部阿寒白糠地域のヒグマを対象にその検証を行った。人里周辺への移動が確認され，周辺は中心より好適でかつ死亡率が高かった。このような環境下で個体群保全と被害管理を両立するため，個体数動向を広域で継続的にモニタリングする手法を開発した。
• 冬眠動物における腸内細菌叢と腸粘膜バリア:新しい共生様式の解明

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2011年 -2012年 

  代表者 : 園山 慶, 坪田 敏男, 滝口 満喜

   

  冬眠動物における腸内細菌の制御と腸粘膜バリアについて解析した。シリアンハムスターの冬眠個体では小腸管腔内の分泌抗体濃度が高く、感染防御に貢献している可能性が考えられた。実際、冬眠ハムスターでは腸間膜リンパ節への細菌の移行は認められなかった。また免疫組織におけるリンパ球プロファイルの解析の結果、冬眠期においても消化管粘膜における感染防御機能および免疫制御機能が維持されていると推察された。さらに、冬眠ハムスターの小腸粘膜上皮における細胞更新が著しく遅滞しており、それにより細胞寿命の延長にともなう細胞老化が生じていることが推察された。
• なぜクマは肥満でも脂肪肝にならないのか?-冬眠前時期の体脂肪蓄積メカニズムの解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2009年 -2011年 

  代表者 : 坪田 敏男, 下鶴 倫人

   

  本研究では、なぜクマは体脂肪率30～40%の肥満状態でも脂肪肝や高脂肪血症を発症しないのか、その特徴的な体脂肪蓄積メカニズムを明らかにすることを目的にして次のような実験を行った。実験は、秋田県北秋田市マタギの里阿仁クマ牧場において、5～11月にかけて月1回行った。1回の実験に4頭のツキノワグマを用いた。塩酸チレタミンと塩酸ゾラゼパムの混合薬(Zoletil 100、Virbac、フランス)9mg/kgにより不動化を行い、麻酔状態下で以下のサンプリングおよび実験を行った。なお、使用したツキノワグマは前日の夕方5:00以降は絶食状態とした。1)基底レベルの血中グルコース、インスリン、中性脂肪、コレステロールおよび遊離脂肪酸濃度を測定した。血中グルコースおよびインスリン濃度には月別の変化は見られなかったが、血中中性脂肪、コレステロールおよび遊離脂肪酸濃度は冬眠前に減少する傾向にあった。2)グルコース投与後0～180分までおよそ30分間隔で血中グルコースおよびインスリン濃度を測定した。その結果、グルコース投与後血中グルコース濃度は一過性に上昇した後、徐々に減少し180分後にはおよそ基底値まで減少した。減少のスピードに月別変化はみられなかった。一方、血中インスリン濃度はグルコース投与直後から上昇し、およそ30分でピークに達した。この時のインスリン上昇度は11月で最も著しい傾向を示した(...
• 致死性人獣共通感染症および潜在的新興再興感染症のアフリカにおける動向調査

  文部科学省：科学研究費補助金(基盤研究(A))

  研究期間 : 2005年 -2007年 

  代表者 : 源 宣之, 福士 秀人, 福士 秀人, 杉本 千尋, 柵木 利昭, 坪田 敏男, 安田 準, 伊藤 直人, 淺野 玄

   

  近年,SARSやトリインフルエンザなどの新たな感染症が次々と発生し,社会をパニックに陥れている.これらの病原体の自然界における本来の住処は不明なことが多いが,各種野生動物の関与が示唆されている.したがって,環境破壊によるそれらの動物の生息分布域の変化が新たな感染症の出現の主な原因と考えられている.そこで,本課題では,各種動物が多数棲息し,毎年新たな感染症が突然出現しているアフリカの野生動物における種々の人獣共通病原体の保有状況を調べ,それと環境変化との因果関係を推定ることにより,新たな感染症の出現動向を予知すると共にその予防に役立てることを目的とした.また,これまでに東南アジアで行ってきた狂犬病に関する海外調査をアフリカ地域に拡大し,狂犬病ウイルスの世界規模の感染動態を検討した.1年次である2005年はザンビア共和国およびエジプトを対象国とし,検体収集ならびに現地協力機関との連携確認ならびに今後の協力体制を構築した.2年次である2006年はザンビア共和国,マラウイ共和国およびエジプトを対象国とし,前年に引き続き検体収集を行った.また,現地協力者の研究支援を行った.最終年度となった2007年はザンビア共和国およびエジプトに加え,タンザニア共和国を訪問し,検体収集ならびに現地協力者の研究支援および今後の研究連携の確認を行った.一連の研究により,アフリカにおける狂犬病ウイルスの生態...
• 希少野生動物における生殖周期モニタリングおよび人工繁殖の臨床応用

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2003年 -2006年 

  代表者 : 坪田 敏男, 村瀬 哲磨

   

  本研究では、希少野生動物から非侵襲的に得られる糞の中に含まれる性ステロイドホルモンの分子生物学的微量測定により各種希少野生動物の生殖周期をモニタリングすること、および希少野生動物の個体数回復を目指して飼育条件下において人工繁殖の臨床応用を実践することを目的とした。本研究期間の4年間に、希少野生動物であるツシマヤマネコとツキノワグマにおいて以下のような実験を行った。1.ツシマヤマネコおよびツキノワグマにおける糞中性ステロイドホルモン濃度の測定1)ツシマヤマネコのモデル動物としてイエネコにおいてTR-FIAキットを用いて糞中エストラジオールおよびプロジェステロン濃度測定法を確立した。2)ツシマヤマネコにおいてTR-FIA法での糞中性ステロイドホルモン濃度測定により生殖周期のモニタリングを行った。3)ツキノワグマの糞中性ステロイドホルモン濃度微量測定については、ER Cell linesを探索し、予備的に測定を行うに留まった。2.ツキノワグマにおける凍結精液を用いた人工授精に関する研究1)飼育下雄グマより麻酔下で電気刺激射精法により精液を採取した。2)精子の運動性、精子数および奇形率などの検査を行なった後、精液を凍結保存した。3)凍結時の希釈液および平衡時間の検討を行い、より精液性状の高い凍結保存法を確立した。4)雌グマの排卵様式を知るために、交尾期の血中プロジェステロン濃度測定を...
• 野生動物の救護に関する研究

  研究期間 : 2003年
• Wildlife rescue

  研究期間 : 2003年
• 極北生息海産獣の繁殖・泌乳生理、生態学的特徴ならびに体内汚染に関する調査

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2001年 -2002年 

  代表者 : 福井 豊, 佐々木 基樹, 手塚 雅文, 浦島 匡, 坪田 敏男, 中村 正

   

  事業報告:福井,坪田,浦島,手塚,佐々木はオスロ大学動物学博物館,ノルウェー極地研,スバールバル大学およびトロムソ大学を訪問し、アザラシの生息状況、体内汚染、クジラ,クマの繁殖生理、哺乳類の乳腺進化についての研究交流を行った。佐々木はオスロ大学においてホッキョクギツネ,ホッキョクグマの形態観察を行い、手塚はノルウェー近海域におけるホッキョクグマ,アザラシの体内におけるPCB汚染の実態調査を行った。スミソニアン動物学研究所のDr.O.oftedalを招聘し、"The origin and early evolution of lactation"と題する講演会を開催した。また、立教大学阿部教授による「環境教育の最前線」と題する講演会を開催した。「野生動物の生命と環境」に関するワークショッブを開催し、福井,坪田,浦島,手塚,佐々木,中村,阿部および日本鯨類研究所の石川が各々の研究成果を報告した。成果報告:福井は北大西洋のヒゲクジラ類の繁殖生理の解明を目的として、索餌期におけるミンククジラおよびニタリクジラの血清中性ホルモン濃度と精細管内組織像の関連性を検討した。坪田は野生動物の人工繁殖への応用を目的として、ビーグル犬ならびに飼育ツキノワグマにおける精液採集、凍結保存、糞中性ステロイドホルモンの測定を行った。佐々木はホッキョクギツネの咀嚼筋の形態および構成を肉眼解剖学的に検索し、さ...
• 内分泌撹乱物質による野生動物の生殖異常の解明とバイオマーカーの開発

  文部科学省：科学研究費補助金(特定領域研究(A))

  研究期間 : 2001年 -2001年 

  代表者 : 坪田 敏男, 村瀬 哲磨

   

  これまでに内分泌撹乱化学物質(EDs)が猛禽類に何らかの影響を与えているとする報告が数多くみられる。PCB類はハクトウワシにおいて生殖低下と関連付けられ、DDEはハヤブサやチョウゲンボウにおいて卵殻薄化との関連が示唆されている。わが国でも猛禽類の個体数減少及び繁殖状況の悪化が報じられている。その主たる要因は生息環境の悪化にあると思われるが、EDsも何らかの影響を与えていると推測されている。そこで本研究では、猛禽類において影響が大きいと考えられるダイオキシン類、PCB類およびDDT類の蓄積濃度とその影響を調べた。また、EDsの繁殖学的影響を明らかにするにあたり、猛禽類の繁殖生理解明の端緒としてトビの精巣の形態学的観察を行った。その結果、ダイオキシン類とP, P'-DDEに関して、何らかの影響が及んでいる可能性を否定できない蓄積が多くの猛禽類に認められた。繁殖期成鳥の検体が少なく、配偶子形成に関して十分な情報を得られなかったが、性腺に組織学的異常は認められず、これらの物質が発生学的影響を与えている可能性は低いと思われた。ダイオキシン類の蓄積傾向は種や生息地による食性の違いを反映していることが示唆され、今後食性を把握することにより猛禽類が地域の汚染状況のモニター生物として有用になり得ることが考えられた。しかし、影響や蓄積傾向を明らかにするには、食性や薬物代謝能、対象物質に対する感受...
• アフリカにおける家畜と野生動物の相互伝播性疾病に関する調査研究

  文部科学省：科学研究費補助金(基盤研究(A))

  研究期間 : 1999年 -2001年 

  代表者 : 安田 準, 杉本 千尋, 小沼 操, 坪田 敏男, 稲垣 匡, 福士 秀人, 吉田 光敏

   

  ウシのヘルペスウイルス1(BHV-1)は23.2%で抗原陽性、48.2%で抗体陽性であったことから、ザンビアでのBHV-1感染は既に広く拡散しているごとが示唆された。ザンビアにおける狂犬病発生率は3%〜28.8%で、イヌが75%ウシが13%を占めた。狂犬病ウィルス遺伝子解析からイヌとヌーの塩基配列は93〜99%の相同性があり、ヌーはイヌから感染を受けたと考えられた。ウシとレチュエに寄生する肝蛭は外部形態から野生動物由来のF.giganticaと同定した。遺伝子塩基配列はレチュエはC型のみ、ウシはA、B、C型であり、ウシのC型はレチュエからウシに伝播したと考えられた。レチュエでウシと同様なMycobacterium bovisによる泌尿生殖器結核感染症が確認され、尿が感染源となることが示唆された。DNA解析ではレチュエ株とウシ株は相同性があり、他の野生動物への感染の可能性を指摘した。レチュエは血液塗抹、PCRともタイレリア原虫は検出できなかった。ウシは血液塗抹で確認できなかったが、PCRでは7%でrRNA遺伝子が検出され、4種類のタイレリア種が同定された。アフリカ大陸に普遍的に存在し、バッファローでの感染も知られており、ウシとバッファローとの感染環が成立していると考えられた。シマウマで分離されたRhodococcus equiは病原性マーカータンパク質やプラスミドは全例保有せず...
• 内分泌かく乱物質による野生動物の生殖異常メカニズムの解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 1999年 -2000年 

  代表者 : 坪田 敏男, 村瀬 哲磨, 喜多 功

   

  1.野生動物における生殖異常の検出野外で死亡したタヌキ、クマ、ハクビシン、ネズミ、猛禽類の死体を収集し臓器材料を得た。また、飼育環境下にあったオットセイ、ラッコ、アシカ、イルカについても死体より臓器材料を採取した。これらの動物の肝臓および脂肪に含まれる内分泌かく乱化学物質(PCB、HCH、DDTおよびその異性体、有機スズ、各種農薬)の蓄積濃度を測定した(環境庁調査および基盤研究(A)による)。生殖器の組織標本を作製し病理学的検査を行った。これまでにタヌキ1例に精巣腫瘍が認められている。2.生殖異常をひき起こすメカニズムの解明性腺内でのステロイド合成を調節するステロイド合成酵素の発現量を調べる目的で、代表的なステロイド合成酵素である3βHSD、P450c17およびP450aromのmRNA断片の塩基配列をイヌおよびネコで決定した。今後これらをプローブとして使ってNorthern blottingやin situ hybridizationを行い、各ステロイド合成酵素の発現量および発現部位を調べる。その上で内分泌かく乱化学物質がこのステロイド合成にどのような影響を与えているか検討する。卵子の発育能および胎子の性分化への影響については引き続き実験中である。
• 野生動物の病態と生態からみた環境汚染に関する長期的モニタリングと環境保護

  文部科学省：科学研究費補助金(基盤研究(A))

  研究期間 : 1998年 -2000年 

  代表者 : 柵木 利昭, 坪田 敏男, 工藤 忠明, 源 宣之, 福士 秀人, 鈴木 義孝

   

  本研究によって得られた主な野生動物の病態と生態に関する調査結果は以下の通りである。1)再興感染症であるニホンカモシカから羊へのパラポックス感染症の伝搬の可能性を明らかにする目的で、カモシカ由来のパラポックスウイルスを用いて羊への接種実験を試みた。その結果、ニホンカモシカのパラポックス感染症が羊へ伝搬する可能性が示された。2)トムソンガゼルの集団斃死例から分離されたガゼルヘルペスウィルス1型は、新興感染症のウマヘルペスウイルスであることが判り、EHV-9として提唱した。EHV-9はこれまで検索したウマ、ヤギ、ウシ、ブタ、イヌ、ハムスター、マウス、モルモットのすべてに脳炎を引き起こすことが明らかとなった。この脳炎はウマ、ブタ及びウシを除き総て致死的であった。EHV-9は強い神経向性を有するウイルスであり広範囲の動物に強い毒力を持っていることが明らかになった。3)中部地区を中心とした各種野生動物における有機塩素系薬剤、PCBs,DDTs,HCHなどの残留濃度を測定したところ、陸生動物に比べて鳥類中でも猛禽類が各薬剤を高濃度保有していることが明らかになった。また、その生体への影響についても実験的に調査した。4)ツキノワグマおよびキタオットセイの繁殖に関わる生態学的および生理学的知見が得られた。また、希少野生動物の人工繁殖に向けてツキノワグマをモデルとした精液の凍結保存に関する技術開発...
• 精巣における性ステロイドホルモンを介した細胞間相互作用に関する研究

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1996年 -1996年 

  代表者 : 坪田 敏男

   

  本研究の目的は、各種雄動物における精巣でのステロイド産生能を検討することであった。そこで、イヌとツキノワグマの精巣を研究材料として、免疫組織化学および内分泌学的にステロイド産生能を検討した。方法は、イヌおよびツキノワグマの精巣を外科手術的に採取し、10%ホルマリン液で固定した。ツキノワグマについては各季節に採材を行なった。固定された材料は、常法にしたがってパラフィン切片とした。免疫染色は、キットを用いてABC法で行なった。使用した抗体は、4種類のステロイド合成酵素、P450scc、3βHSD、P450c17およびP450aromに対する抗血清であった。さらに、ツキノワグマについては血液を採取して、血液中のステロイド濃度をラジオイムノアッセイによって測定した。その結果、イヌでは、ライディッヒ細胞がアンドロジェン産生能を有し、また、精母細胞および精子細胞がエストロジェン産生能を有していた。ツキノワグマでは、交尾期にはやはりライディッヒ細胞がアンドロジェン産生能を有していたが、他の季節(非交尾期)でもP450sccおよびP450c17の染色性は変化しなかった。一方3βHSDについては、交尾期に比べて非交尾期に陽性細胞が顕著に減少した。また、交尾期には精子細胞がエストロジェン産生能を有していた。血中テストロン濃度は交尾期に高値を示し、非交尾期に底値を示した。
• 各種雌動物の卵巣および胎盤におけるステロイド産生能について

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1995年 -1995年 

  代表者 : 坪田 敏男

   

  本研究の目的は、各種雌動物における卵巣および胎盤でのステロイド産生能を検討することであった。そこで、ネコとツキノワグマの卵巣および胎盤を研究材料として免疫組織化学および内分泌学的にステロイド産生能の検討を行った。方法は、妊娠中のネコおよび妊娠していたと思われるツキノワグマの卵巣および胎盤を採集し、10%ホリマリン液で固定した。ネコについては生体より外科手術的に採取した。固定された材料は常法にしたがってパラフィン切片とした。免疫染色は、キットを用いてABC法で行った。使用した抗体は、4種類のステロイド合成酵素、P450scc、3βHSD、P450c17およびP450aromに対する抗血清であった。さらに、ツキノワグマについては妊娠雌より血液を採集して、血清中のステロイド濃度をラジオイムノアッセイによって測定した。その結果ネコでは、1)妊娠期のプロジェステロン産生母地は黄体から胎盤に移るが、妊娠後半期にも黄体はプレグネノロンの産生能を有する 2)胎盤の栄養膜合胞体層はエストロジェン(妊娠期間全体)およびプロジェステロン(妊娠後半期)の産生能を有し、加えて妊娠末期には脱落膜細胞がエストロジェンおよびプレグネノロンの産生能を有することが推察された。ツキノワグマでは、1)着床遅延初期には黄体が主要なプロジェステロン産生母地であるが、少量ながらエストロジェンも産生されている 2)黄体での...
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• 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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委員歴

• 2019年 - 現在   日本野生動物医学会   顧問
• 1997年 - 現在   日本獣医学会   評議員   日本獣医学会
• 1995年 - 現在   ヒトと動物の関係学会   評議員   ヒトと動物の関係学会
• 2017年 - 2020年   日本獣医学会野生動物学分科会   分科会長
• 2017年09月 - 2019年09月   日本獣医学会   理事
• 2013年 - 2018年   日本野生動物医学会   会長
• 2007年 - 2012年   日本野生動物医学会   事務局長
• 2001年 - 2006年   日本野生動物医学会   副会長
• 1997年 - 2000年   日本繁殖生物学会   編集委員   日本繁殖生物学会
• 1995年 - 2000年   日本野生動物医学会   事務局長   日本野生動物医学会
• ヒグマの会   会長
• 日本クマネットワーク   会長
• 日本哺乳類学会   評議委員
• 日本哺乳類学会   編集委員   日本哺乳類学会