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The Research Center for Animal Science (RCAS) Hiroshima University’s Promising Research Initiatives (recognized in 2014) Education & Research for advanced technological developments contributing to safe and high quality animal productions Presented by Dr Yukinori Yoshimura (Graduate School of Biosphere Science) Where is Hiroshima University? Hiroshima is best known as the first city in history to be destroyed by a nuclear weapon on August 6, 1945, near the end of World War II. Peace Memorial Park is a famous place to promise and learn the peace of the world. However, Hiroshima is now one of the biggest city in Japan. The population is more than 1 million. MAZDA that is automotive manufacture is based on Hiroshima. Hiroshima University has two campuses. Faculties: Hiroshima University Graduate Schools (12) Faculties (11) Faculty of Integrated Arts and Sciences Faculty of Letters Faculty of Economics Faculty of Law Faculty of Education Faculty of Science Faculty of Engineering Faculty of Medicine Faculty of Dentistry Faculty of Pharmaceutical Sciences Faculty of Applied Biological Science Students Undergraduate:11,040 Master course:2,510 PhD course:1,750 Others:500 Foreign students:760 from 57 countries Graduate Schl of Integrated Arts and Sci Graduate Schl of Letters Graduate Schl of Education Graduate Schl of Social Sciences Graduate Schl of Science Graduate Schl of Engineering Graduate Schl of Biomedical Sciences Graduate Schl of Health Sciences Graduate School of Biosphere Science Graduate Schl for International Development and Cooperation Graduate Schl of Advanced Sciences of Matter Staff Academic : 1,830 Non-academic: 1.440 Departments DEPARTMENT OF BIORESOURCE SCIENCE Animal Science, Aquatic Biology, Food and Resource Economics, Field Science DEPARTMENT OF BIOFUNCTIONAL SCIENCE AND TECHNOLOGY Molecular and Applied Biosciences, Food Science and Biofunctions Int’l summer school AIMS program DEPARTMENT OF ENVIRONMENTAL DYNAMICS AND MANAGEMENT Modeling, Management and Assessment of Environmental Dynamics Int’l symposium Short visit Vet Tech students DEPARTMENT OF BIORESOURCE SCIENCE Animal Science (Academic staff: 11) Animal breeding, Reproduction, Nutrition and Feed, Management and Behavior, Physiology and Histology Aquatic Biology (Academic staff: 14) Aquatic Biochemistry, Ecology, Pathology, Neurobiology, Biology of Aquatic Resources, Aquaculture Food and Resource Economics (Academic staff: 4) Food Production, Management, Agricultural Marketing Field Science (Academic staff: 4) Animal Farm, Aquatic field studies gene Animal Science Division Graduate School of Biosphere Science Hiroshima University The goal of this division is to discover novel functions in animals and applying such discoveries for the technological advancement of domestic animal production. The research will contribute to sustainable and efficient production of safe food for humans derived from animal products. Current research fields are: breeding and genetics based on modern genomic analysis, reproductive biology and biotechnology for enhanced reproductive performance, applied nutritional physiology and feed evaluation for improved nutritional management, ecology and neurophysiology for better animal management and viability, and fundamental histology and molecular physiology to enhance veterinary production and health.. Animal Breeding and Genetics Animal Reproduction Animal Nutrition and Feeding Animal Behavior and Physiology Animal Histophysiology Animal Farm Goal of RCAS Contribution to development of technologies for safe and high quality animal products • Progression for animal nutrition, breeding and reproduction, and health • Contribution for food safety and security • Innovation for new agricultural science by crossdisciplinary research collaboration nutrition metabolism health productivity breeding reproduction well-being Members of The Research Center for Animal Science Members Leader Yukinori Yoshimura, PhD Institutes Grad Schl Biosphere Sci, HU Majors Immunodefense in poultry reproductive and digestive system Manager Masayuki Shimada, PhD Grad Schl Biosphere Sci, HU Reproductive Molecular Endocrinology Reproductive technology Manager Toshihisa Sugino, PhD Grad Schl Biosphere Sci, HU Ruminant metabolism and endocrinology Masaoki Tsudzuki, PhD Grad Schl Biosphere Sci, HU Genome of Japanese native chickens Masahide Nishibori, PhD Grad Schl Biosphere Sci, HU Genome and Polymorphism of Animals and Poultry Teruo Maeda, PhD Grad Schl Biosphere Sci, HU Manipulation of germ cells Yusuke Sotomaru, PhD Nat Sci Center Basic Res Dev, HU Animal developmental engineering Ikuo Miura, PhD Grad. Schl Sci, HU Sex determination and differentiation Taketo Obitsu, PhD Grad Schl Biosphere Sci, HU Ruminant nutrition and feed evaluation Takashi Bungo, PhD Grad Schl Biosphere Sci, HU Animal Behavior and Physiology Shin-ichi Kawakami, PhD Grad Schl Biosphere Sci, HU Animal management and neurology Naoki Isobe, PhD Grad Schl Biosphere Sci, HU Ruminant mastitis and mammary gland immune system Yuzo Kurokawa, PhD Grad Schl Biosphere Sci, HU Dairy milk production and grassland utilization Kensuke Kawamura, PhD IDEC, HU Grazing management and remote control system Soichi Tanabe, PhD Grad Schl Biosphere Sci, HU Gastro-protection by lactobacilli and bifidobacteria Noriyuki Yanaka, PhD Grad Schl Biosphere Sci, HU Nutritional factors and obesity Shiro Kushibiki, PhD Natl Agr Food Res Center Animal nutrition and metabolism Tetsuji Okazaki, PhD Oita Pref Animal Res Center Porcine reproductive technology JoAnne S. Richards, PhD Baylor College of Medicine, USA Animal reproduction Masahito Oba, PhD University of Alberta, Canada Animal nutrition 8 Research activities in RCAS Project 1. Hostdefense and genetic resources research (Example) Creation of livestock disease-resistance Natl. and prefectural institutes, Japan Kasetsart University (Thailand) Univ. Gadjah Mada (Indonesia) Domestic and international collaborations Innate immune function for Genome analysis and infection-resistance breeding in Japanese improvement native chickens Excellent genetic trait of Japanese native chickens. Sichuan Agricultural Univ.; North West Agriculture and Forestry Univ.(China) Fundamental studies on reproduction and development Finding of antimicrobial peptide in reproductive and digestive organs. Salmonella E Analysis of genetic locus for genome breeding New strategy for disease resistance improvement in chickens and other livestock. Avian β-defensin Development of new chicken breeds that are excellent in products quality, disease-resistance and reproductive performance. Contribution for sustainable development of poultry production in Asia and world. Fundamental studies on sex determination, developmental engineering, and reproductive endocrinology Research activities in RCAS Project 2. Reproductive biology, (Example) Breeding technology development North West Agriculture and Forestry Univ.(China) Identification of sperm motility suppressing factors in semen Fe3+ Ca2+ ATP LH (ovulation stimli) Granulosa/ cumulus cells NRG1 ovulation AREG, Oocyte maturation Fertilizing ability Ca2+ Fertilizable ovum Determination of the fertilization mechanism Determination of ovulation mechanism and sperm cryopreservation techniques in pigs day0 day4 SNP analysis for genetic close-relations in pigs DNA analysis for determination of parent-child relationship and identification of individual origins sire pigs day6-9 Pig No. 0001 Pig No. 0002 SNP SNP SNP No. Genotype Genotype SNP1001 A SNP1001 A SNP1002 T SNP1002 C ↓ ↓ ↓ ↓ SNP1023 G SNP1023 A SNP1024 A SNP1024 G ↓ ↓ ↓ ↓ SNP1192 T SNP1192 T Individual Identification using SNP genotypes SNP No. Identification of heat (0h) AI optimum AI optimum time time Novel artificial insemination program in pigs Term possible for fertilization Sperm survival time Establishment of the novel pig breeding technology Contribution for sustainable development of pig production in Asia and world. Research activities in RCAS Project 3. Endocrinology, Physiology of nutrition, Animal management technology (Example) Animal management technology development Kasetsart University (Thailand) Nutrient metabolism in ruminants University of Alberta(Canada) Nutritional factors causing lifestyle disease and obesity Gastro-intestinal health and protection by probiotics Natl. and prefectural institutes, Japan Hiroshima University (Japan) Improvement of Analysis of body productivity in dairy score by 3D image milk using advanced and remote sensing robot system Work-saving management, High performance, Health enhancement and Safe products in Animal production Production and Tissue regulatory 3D image health analysis factors analysis analysis Milk amount and Body shape and Metabolism regulation quality, estrus cycle obesity evaluation by blood factors and identification etc. etc. hormones etc. Expression of avian β-defensins, a member of antimicrobial peptides, in hen oviduct Yukinori Yoshimura Graduate School of Biosphere Science, Research Center for Animal Science, Hiroshima University, HigashiHiroshima 739-8528, Japan [email protected] Infection of reproductive organs ovary Reproductive disorder TE TI G Y Drop in egg production Disorder of egg-forming functions such as eggshell formation. Contamination of eggs oviduct Problems in food safety L E S Salmonella enteritidis infect the ovary and oviduct Increase in mortality of embryo and chicks The lower oviductal segments are the primary tissues that are infected by microorganisms colonizing in the cloaca. Innate immune system * First line of defense against infection by various pathogenic microbes including antibiotics-resistant bacteria and unforeseen microbes. Virus flagellin Lipoprotein Peptidoglycan (Gram +ve) Unmethylated CpG DNA ssRNA dsRNA LPS (Gram -ve) Lipoprotein, peptidoglycan (Gram positive bacteria ) TLR2 TLR1 lipopolysaccharide; LPS (Gram negative bacteria ) dsRNA type 1 & 2 TLR4 CD14 TLR3 Flagellin TLR5 ssRNA NF-kB/IkB MyD88 -dependent, NF-kB -independent pathway AP-1 type 1 & 2 Bacterial protease Receptors recognizing microbe-associated molecular patterns Proinflammatory cytokines Transcription TLR7 TLR15 Avian Toll-like receptors TLR21 Unmethylated CpG-DNA Chemokines Antimicrobial peptides (avian β-defensins: AvBDs) Avian β-defensins (AvBDs) + charge - charge * Cysteine-rich cationic antimicrobial peptides Bacterial cell membrane * Antimicrobial activity against a wide spectrum of microorganisms [Grampositive and Gram-negative bacteria, protozoa, fungi and enveloped viruses] Breaking down cell membrane and killing bacteria * Fourteen genes have been identified M 1 2 3 4 5 AvBDs 6 7 8 9 10 11 12 13 14 Escherichia coli AvBD9 Fourteen AvBDs genes have been identified in chicken. control AvBD9-AvBD8 Antimicrobial activity of recombinant AvBD (Gal)-9 and AvBD-9-AvBd-8 against Escherichia coli. (from Ma DY et al. Protein Expr Purif 58: 284-291. 2008. ) Goal of the study To determine the mechanism by which AvBDs expression is induced in response to microbial agents in hen oviduct Questions 1. Are TLRs and avian β-defensins (AvBDs) expressed in the oviduct? 2. Do microbial components induce the expression of AvBDs and cytokines? 3. How is the AvBDs expression refulated? LPS Poly (I:C) Flagellin CpG ODN TLRs pathogen LPS TLR4 NFκB/AP-1 Transcription IL receptor Proinflammatory cytokines AvBD cells NFκB/AP-1 Transcription AvBDs AvBDs pathogen Estimated pathway from TLR stimulation to synthesis of AvBDs and cytokines TLRs M 1-1 ligands 1-2 2-1 2-2 3 4 5 7 15 21 peptidoglycan Expression of TLR in vaginal cells M Expression of TLR adaptor molecules and transcription factors in vaginal cells Expression of Toll-like receptors and transcription factors in the vaginal cells cloaca vagina M 1 2 AvBD3 protein, isthmus L C) 3 4 5 6 AvBD3 protein, uterus L AvBDs 7 8 9 10 AvBD3 protein, vagina L 12 13 14 kDa E E 37 LP TG 11 E TG LP LP 25 20 15 AvBD3 mRNA, vagina Identification of avian β-defensins (AvBDs) in the vagina 10 Western blot of AvBD-3 in the uterus 3 AvBD1 8 1.5 4 1 2 0.5 0 102 10 103 104 0 0 2 AvBD4 1.5 1.5 1 1 0.5 0.5 0 0 0 10 102 103 104 Fold change 5 4 AvBD10 b ab 3 2 Fold change 6 2 25 AvBD3 2 0 Fold change 2.5 a a a 1 0 0 10 102 103 LPS (ng/mL), 3h 104 7 6 5 4 3 2 1 0 102 103 10 20 10 102 103 104 AvBD12 104 b ab ab ab a 0 10 102 103 104 LPS (ng/mL), 3h Effects of lipopolysaccharide on the expression of avian β-defensins (AvBDs) in the cultured vaginal cells c bc bc 10 5 0 AvBD5 0 IL1β 15 Fold change Fold change 10 6 5 4 3 2 1 0 ab a 0水 2 3 10 10 104 10 100 10 1000 10000 LPS (ng/mL) ab IL6 ab b a 00 a 4 3 2 10 10 10 100 10 1000 10 10000 LPS (ng/mL), 3h Fold change 4 AvBD1 b b 3 2 2 00 Fold change M ab 4 a 0 100 102 1000 103 AvBD4 a 0 00 2 0.8 100 102 1000 103 Expression of IL1 and IL-6 receptors in the vaginal cells AvBD5 1.5 0.6 1 0.4 0.5 0.2 0 00 Fold change b AvBD3 6 1 1 8 1.2 1 0.8 0.6 0.4 0.2 0 100 102 1000 103 AvBD10 0 102 103 0IL1β100 1000 (ng/mL), 3h 0 1.2 1 0.8 0.6 0.4 0.2 0 00 2 10 100 3 10 1000 AvBD12 00 102 100 103 1000 IL1β (ng/mL), 3h Effects of IL1β on the expression of AvBDs in the cultured vaginal cells Conclusions 2. The innate immune system may attack and prevent infections from a wide spectrum of pathogenic microbes including those to which vaccinations are not performed, those resistant to antibiotics, and other unforeseen microbes. AvBD8 TE Y AvBD12 1. Innate immune system formed by TLRs and avian β-defensins is expected to play the first line of host defense in the oviduct. That immunity is essential not only for the health of individuals but also for the production of hygienic eggs. Largest yellow follicle TI G 20 m TE TI G 20 m Y Immunohistochemistry For the future AvBD3 Outer layer AvBD3 L E Inner layer TG LP 20 μm AvBD12 kDa 50 37 L E 25 20 15 TG 20 μm 10 AvBD3 kDa 50 37 25 20 15 AvBD12 Non-specific Non-specific 10 LP AvBDs in the uterus Are AvBDs transmitted from oviduct to the eggs? AvBDs in the eggshell For the future AvBDs bp M 1 2 3 4 5 6 7 8 9 10 11 14 12 13 500 100 mucosal fold of the proventriculus E LP proventricular gland L F G GL For production of healthy chickens Do antimicrobial peptide play roles in the defense in the gastrointestinal mucosa? Are probiotics and other natural substances useful to enhance the synthesis of them?