P-1

The difference of fit characteristics among N95/DS2 respirators ○Tatsuya Sugimoto1, Masako Araki2, Masanori Kanda3, Mari Ando4, Takeo Oshima1 1 SHIONOGI & CO., LTD., 2Shionogi General Service Co., Ltd., 3Shionogi TechnoAdvance Research Co., Ltd., 4SHIGEMATSU WORKS CO., LTD.

[Background] The animal research personnel are obligated to wear N95 or DS2 respirator to prevent the exposure to laboratory animal allergens and infectious agents. The instruction of mask wearing and the qualitative mask fit test are conducted to new animal research personnel. Since a new animal research person did not fit to any existing masks in our facility, the new mask was considered to adopt, and a large-scale quantitative mask fit test was performed. [Method] Using the instrument for measuring the intra-mask pressure and the leakage rate, the quantitative mask fit test was conducted on 135 personnel when they wore the DD02-N95-1 (SHIGEMATSU WORKS) or the existing masks (provisional designation as N95A, N95B, N95C, or DS2A). The test pass criterion was when the average leakage rate at normal breathing, deep breathing, turning head side to side, moving head up and down, and talking was below 5%. [Result] The test pass rate at DD02-N95-01 was 80% (108/135). The rate at N95A, N95B, N95C, and DS2A were 7% (1/14), 66% (50/76), 6% (1/18), and 43% (10/23), respectively. [Discussion] The great difference of test pass rate was observed among the N95/DS2 products. It was considered that the preparation of N95/DS2 products with fine fit, and the selection of the optimal N95/DS2 product through the mask fit test were of importance for the health and safety in the animal research personnel.

P-2

A novel decontamination method using a peracetic acid-based disinfectant alternative to formalin Wakako Kumita1, ○Akio Sakaki2, Ken-ya Sato1, Norio Okahara1, Erika Sasaki1,3 1

The Center of Applied Developmental Biology, Central Institute for Experimental Animals, Kanagawa, Japan, 2PharmaBio Instrument Co. Ltd., Tokyo, Japan, 3Keio University, Tokyo, Japan

Decontamination using formalin is common to eliminate microbiological pollution of laboratory equipment in many research facilities; however, the required time is a significant burden and the residual toxicity of formalin has become a major issue. Peracetic acid, an alternative disinfectant used in decontamination is devoid of the safety issues because the chemical decomposes spontaneously, with no residual toxicity, enabling rapid, reliable, and safe daily decontamination. In this work, we used FOG ACT® and/or FOG WORKS®, which automatically nebulize material to the atmosphere, to decontaminate a safety cabinet and a breeding room via complete atomization of PBio Actril® or Minncare® (peracetic acid-based disinfectants). We evaluated the efficacies of the procedures by culturing the biological indicator containing spore-forming bacteria. As a result, in the safety cabinet, both the work area and the HEPA filter were decontaminated. The animal room was also completely and rapidly decontaminated without affecting the stainless steel cages, or brass water nozzles. Thus, space decontamination using this system is very effective; researchers and experimental animals are protected from residual toxicity, strongly supporting the new decontamination method as an alternative to formalin fumigation.

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P-3

A study on the properties of chlorine dioxide gas as a disinfectant ○Yasufumi Shirasaki1, Ayumi Matsuura2, Kaori Ito2, Masashi Uekusa2, Yoshihiro Ito2, Toshiaki Hayashi2 1

Biological Research Department, Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan, 2Hamri Co., Ltd.

Objective: Microbial decontamination is one of the practical issues in animal facilities. The present study was undertaken to evaluate the properties of gaseous chlorine dioxide (ClO2) as a disinfectant. Methods: ClO2 gas was generated by mixing 2% sodium chlorite and phosphoric acid with a 10:1 ratio (v/v). We set up a test room (87 m3), and experiments were carried out using various amounts of sodium chlorite ranging from 0.25 to 4 ml/m 3. Staphylococcus aureus and Escherichia coli were chosen as the model microbes. Paper discs (diameter, 10 mm) with each bacterium were placed at various sites, including the ceiling, walls and floor. Results and Discussion: The gas concentration increased in a sodium chlorite volume-dependent manner and reached peak values at 2 to 3 h, ranging from 0.8 to 10 ppm, followed by a decline. Gas levels became nearly undetectable at any volume 24 h after gas generation. No differences in gas concentrations were observed between 0.1 m and 2.5 m above the floor, suggesting that gas is equally distributed throughout the entire test room. The colony formation of both bacteria was completely inhibited by exposure to ClO2 gas at 1 to 4 ml/m3. In addition, ClO2 gas inactivated bacteria in the covered petri dishes, indicating excellent diffusibility and penetrability. Thus, ClO 2 gas is expected to serve as an alternative method for cleaning and disinfecting the animal facilities.

P-4

The occurrence of Psocoptera and the extermination in the animal facility ○Ojima S.1, Y. Abe2, R. Ando1 1

Center Labo. Anim. Sci., Tohoku Pharm. Univ., Sendai, Japan, 2JAC, Tokyo, Japan

The occurrence of Psocoptera and the extermination in the animal facility. S. Ojima, Y. Abe, R. Ando. Center for Lab. Anim. Sci., Tohoku Pharm. Univ., Sendai, Japan, JAC, Tokyo, Japan. Occurrence of Psocoptera have been confirmed from breeding rooms at our facility. Psocopter which are nonvirulent microinsect become an index for the microbial govern quality of the facility of cleanliness-rearing environment. We report on the occurrence situation and the extermination methods of Psocoptera at our facility. In our facility perform the first type ventilation sysetem with clean air through the HEPA filter, and the room temperature and humidity are kept constantly in whole year. The onsite, after dust removal in a clean room for the vacuum cleaner, wiping disinfection by slightly acidic HClO water and a conventional method, breeding management, users have done. We carried out the extermination of Psocoptera with a smoking insecticide and high temperature steam or an insecticide mist. After Psocoptera extermination, although observing the implementation passed the stamp test using a cellophane tape habitat was not confirmed.Presumably, Psocoptera attached to various kinds of articles and invaded it from the outside and inhabited. It is the most important to perform the breeding management by an appropriate method, cleaning disinfectant work to maintain the cleanliness degree of facility and microbiological control of the breeding environment.

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P-5

Current status of laboratory animal facility in pharmacological services at DCB ○Shih-Ching Ho, Yung-Jen Tsai, Pei-Yi Tsai, Fang-Ju Chou, Jia-Ming Chang Department of Pharmacology, Institute for Drug Evaluation Platform, Development Center for Biotechnology, New Taipei City, Taiwan

Laboratory Animal Facility of DCB is a non-profit institute funded by the Ministry of Economic Affairs, Taiwan, ROC. Pharmacological services of DCB provide highly customizable in vivo drug evaluation to help researchers and clients in academic and industry. We can strengthen the drug discovery research program by partnering with our in vivo pharmacology services. Our main research experience is in the fields of oncology, metabolism, and immunology, combined with animal models of human disease. We have been accredited for the ISO/IEC 17025 certification, including the cancer pharmacology testing lab in 2013 (the certificate number is 2718), the metabolic pharmacology testing lab in 2013 (the certificate number is 2835) and the immunity pharmacology testing lab in 2014 (the certificate number is 2981). Furthermore, accredited animal facility including P2 grade animal rooms in the Laboratory Animal Facility (accredited by Department of Health, ROC in 2010 and 2013) supports the in vivo pharmacology study. These laboratories based on these accreditations, the Laboratory Animal Facility at DCB provides high qualified and reliable pharmacological tests, especially in drug development, which benefit to users in high quality and reliable pharmacological tests.

P-6

Enhancing animal welfare and quality control at laboratory animal breeders by IACUC and QA surveys ○Hironori Yamagishi1,2, Hirokazu Sudo1, Misae Ito1, Toshihiko Watanabe1, Keiji Mizoguchi1, Ryo Takai1 1

Chugai Phamaceutical Co., Ltd., 2Chugai Research Insititute for Medical Science, Inc.

Introduction: Laboratory animal breeders have been surveyed by our institutional animal care and use committee (IACUC) since 2006 and by our quality assurance department (QA) since 2013. Here we review the effect of IACUC and QA collaboration on enhancing animal welfare and quality control.Methods: Breeders were surveyed by document reviews and facility visits. We discussed points raised by the surveys in a meeting with persons in charge and later sent a written report of the survey results, good points, and suggestions for improvement.Results: We surveyed a total of 9 facilities from Jan 2013 to Oct 2014 and they all met our standards for animal welfare and quality control. We suggested further improvements related to animal welfare (51%), quality control (29%), occupational health and safety (18%), and education and training (2%).Discussion: The survey system has merits for both users and breeders. Users obtain data from experiments using animals of high quality and can show a strong commitment to animal welfare. Breeders benefit from varied opinions on animal welfare from an outside source and can build a system that supplies animals of a high standard required by pharmaceutical science. The involvement of QA made the system more accurate and efficient and developed breeders’ understanding of quality management. We will continue our effort to make a better survey system by reviewing our past surveys.

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P-7

Application of a database server to manage mouse bank and animal care services at CARD ○Shuuji Tsuchiyama1, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

A database server is a useful means of effectively managing a great amount of data within an animal facility. In our center, we provide a mouse bank service and an animal care service. To efficiently manage the service, all data and the status of each operation should be readily shared between staff. Until now, we have developed systems to manage data for all staff using a database server. In the server, we have established two systems: an IVF Support System and a Mouse Cage Sum System using Java and Oracle. Using the systems, all data can be handled via a web browser. The IVF Support System has accumulated about 11,000 items of data regarding IVF, and about 25,000 items of data regarding frozen embryo tubes since 1998. In the system, all data can be easily browsed for any category and can be extracted to form a list. Meanwhile, our Mouse Cage Sum System has accumulated about 59,000 items data regarding increases and decreases in mouse cages since 2007. In the system, animal care fees based on the number of cages is automatically calculated at the end of the month, and the present occupation rate of animal rooms can be easily confirmed. The two systems have greatly contributed to CARD’s continued provision of high quality services to all users.

P-8

The influence of the material of rearing cage, paper chip, metal mesh or resinous board in rat ○Takahiro Yoshizawa1, Sho Takizawa1, Shin Shimada1, Norihisa Shiohara2, Keisuke Kojima2, Minoru Kaneko3, Kiyoshi Matsumoto1 1

Division of Laboratory Animal Research, Research center for human and environmental sciences, Shinshu University, Nagano, Japan, 2A-TEC Co., Ltd., Hyogo, Japan, 3Shin-toyo Seisakusho Co., Ltd., Saitama, Japan

So far, several flooring were used for laboratory animal cage. The metal mesh has been used in the safety study to avoid a modification effect by the coprophagy, however, it is thought that use of metal mesh is undesirable for animal welfare. In this study, we investigated the perforated polypropylene board (PPB) for flooring that may be able to be replacement of the metal mesh.Acclimated male F344/N Slc rats were divided into 3 groups of the flooring using paper chip (PC), metal mesh (MM) or PPB (PP). In 4 or 12 weeks after the grouping, blood and organ samples were collected for using CBC, serum biochemical profile and gene expression analysis.Decrease tendency of food consumption were seen in PC. NEUT and MONO were decreased and LYMPH was increased in PC. T-CHO of PP was middle level of other groups. In PP, gene expression of CRH in hypothalamus was middle level of other groups. Thus, these data suggest that different flooring might affect the experimental data. Some data showed middle level in PP compared with other groups suggesting it may be able to be replacement of the metal mesh.Several test results in PP were intermediate between PC and MM suggesting the possibility that the PPB flooring gain advantage for housing enrichment.

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P-9

Effect of living conditions on coprophagy in rats: solid bottomed cages vs. wire mesh cages ○Heijirou Ohnishi, Kazuo Terasawa, Takehiro Ochi, Hironari Koyama Astellas Research Technologies Co., Ltd Osaka,Japan

Background: The eighth edition of the Guide for the Care and Use of Laboratory Animals recommends housing rodents in solid floors with bedding and not on wire mesh floors. However, there is a main reason for housing rats in wire mesh floors: prevention of delayed absorption of administered compounds due to coprophagy in the pharmacokinetic study. Objective: We investigated the effect of cage type, i.e. solid type floors and wire mesh floors, on the coprophagic behavior in rats. Methods: Male and female Sprague-Dawley rats aged 5 weeks were used. Coprophagic behaviors were observed with the naked eye from 7:00 to 19:00 h constantly. Results: The frequency of coprophagic behavior was 7.8 times/12 h in rats housed with wire mesh floors and 12.3 in those on solid floors. Rats housed on wire mesh floors ate 3.1% of their feces, while rats on solid floors ate 2.6% of their feces. Further, no marked changes were noted under general observation of behavior and body weight gain in either living condition. Conclusion: These results suggest there is little difference in coprophagic behavior of rats housed in these two conditions.

P-11

Influence of LED on the general condition and ophthalmological function of long-term breeding rats ○Mie Wakamatsu1, Kazuo Terasawa1, Toshirou Kasuga2, Naomi Saitoh2, Hikaru Mitori2, Hironari Koyama1 1

Laboratory Animal Science Div., Astellas Research Technologies Co., Ltd., Osaka, Japan, 2Drug Safety Research Labs, Astellas Pharma Inc., Osaka, Japan

Objective: To clarify the impact of a light emitting diode (LED) on animals in our breeding facilities, we investigated the general condition and ophthalmological function of long-term breeding rats exposed to these diodes. Methods: Male and female Sprague-Dawley rats aged 6 weeks were used. Animals were housed for 5, 13 and 26 weeks under LED light with a luminous intensity of 590 lux at 75 cm above the floor. Optical media and eyeground were examined using a binocular indirect ophthalmoscope with an aspheric lens. Eyeballs and optic nerves were subjected to pathological examination. Results: Minor metaplasia was observed in the retina by pathological testing in 1 out of 10 rats of each sex at 5 weeks, 3 out of 10 male rats at 13 weeks, and 2 out of 20 male rats at 26 weeks of breeding under LED light. Increased reflectivity was observed in the unilateral retina of 2 out of 10 male rats at 13 weeks and 2 out of 20 male rats at 26 weeks of breeding under LED light. These changes did not correlate with breeding period under LED light. No other changes were observed in tested rats. Conclusion: These results suggest that the general condition and ophthalmological function of rats at 26 weeks of breeding is not substantially influenced by exposure to LED light.

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P-12

An environmental hygienic management using photocatalytic technology in the animal facility ○Noboru Ogiso1, Satomi Takano1, Kazumichi Yamaguchi2, Kohei Tomita2, Kaori Muguruma1 1 Laboratory of Research Animal , National Center for Geriatrics and Gerontology, Aichi, Japan, 2KAC , Kyoto, Japan

The photocatalytic technology have been introduced to many laboratory animal facilities in recent years. In the present study we examined the efficacy of the technology in the breeding equipment. A visible light - responsive photocatalyst coating (Sagan Coat TPX-HLXB, Showa Ceramics Co. Ltd.) was applied to the mice cages, and we conducted the following three experiments: (1) endurance tests of cages for washing and sterilization treatments, (2) cleanliness measurements in cage with living mice (C57BL/6NCrSlc, 12 months old) and (3) cleanliness measurements in washed cages. To investigate cleanliness, we measured ammonia concentration, adherent microorganisms and ATP. Our results show that application of the photocatalyst coating might be useful for maintaining cleanliness in laboratory animal facilities. Further experiment will be needed to investigate the effects of the coating on breeding aged animals.

P-13

Mouse hepatitis virus infection and clean-up in a laboratory animal facility ○Yuka Ishida1, Takashi Okubo1,2, Kaori Tateno1,2, MIzuki Iina1,2, Wataru Ueno1, Tatsuo Hayao1, Seiji Kito1, Toshiaki Kokubo1 1

Development and Support Center, National Institute of Radiological Sciences, Chiba, Japan, 2Science Service Inc., Chiba, Japan

There are some specific-pathogen free animal facilities and some conventional animal facilities in National Institute of Radiological Sciences. Animal Research Building has the facilities of semi-barrier system, which is one of the laboratory animal facilities in NIRS. This facilities is CV facilities, however, we were performed regular microbiological test and recognized Mouse hepatitis virus (MHV) infection in October, 2013. We immediately started discussions about this accident with facilities user and animal control section. It has been decided that we carried out clean-up of this facility and preventing the spread of infection as follows. I. The cleaning of this facility carries out each four breeding areas. II. The mice that were not able to sacrifice immediately put it into two infection breeding rooms or three non- infection breeding rooms. Then, the mice are sacrificed within a fixed period of time. The summary about this accident was reported in Japanese Conference for Laboratory Animal Science and Technology, Sapporo 2014. Then we took various measures about the microorganism infection and accomplished the re-operation of this facility in June, 2014. In this meeting, we report a process of concrete clean-up for this facility and the later correspondence.

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P-14

Action and whole story on anti-Sendai Virus antibody-positive case occurred in our facility ○Yoshihiro Inoue1, Takashi Ishibashi1, Yohei Kudo1, Teruko Sueta2, Yayoi Yoshida3, Yasuhisa Matsui1, Noriyuki Kasai2,3 1 Institute for Experimental Animals (Division of Tumor Animals), Institute of Development, Aging and Cancer, Sendai, Japan, 2Institute for Animal Experimentation Tohoku University Graduate School of Medicine, Sendai, Japan, 3Center for Laboratory Animal Research, Tohoku University, Sendai, Japan

On September of 2013, we have received a report indicating positive for anti-Sendai Virus antibody on our facility’s samples (one positive sample per whole 26 samples) checked by microbiological monitoring test at ICLAS Monitoring Center, Central Institute for Experimental Animals. Immediately, we performed the serological tests (ELISA and IFA) on a mouse room suspected of infection and also on all animal rooms of our facility. As a result, all samples were negative. >From this case, it was shown that there happen to occur such the non-specific reaction in the anti-Sendai Virus antibody test. This time, it was proved that Sendai Virus infection had actually not occurred in our facility, and this pseudo infection problem has settled. However, we have sacrificed not a little amount of animals in this matter. For a reflection to this experience, we have established a countermeasure manual as a new rule in our institute which describes a course of action to take when such same case occurred.

P-15

Isolation of Staphylcoccus aureus from hairless SCID mouse in laboratory animal facility ○Gwang-Hoon Lee1, Jae-Bum Ahn1,2, Euna Kwon2, Sun-Deok Lee2, Eun-Seok Oh2, Jun-Won Yun2, Byeong-Cheol Kang1,2 1

Graduate School of Translational Medicine, Seoul National University College of Medicine, Korea, Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Korea 2

Hygiene is very important for laboratory animal facility to assure reliability of animal experiments conducted. The aim of this report is to introduce procedure in case of infection of Staphylococcus aureus occurred in our SPF laboratory animal facility. The Gram positive bacterium S. aureus is a major pathogen responsible for a variety of diseases, including pyogenic infections of conjunctiva, adnexa of eye, skin and adnex, or genital tract in susceptible strains, immunocompromised or immunodeficient animals. Healthy 6 weeks-old hairless SCID mice were purchased from the Charles River Laboratories. At 8 weeks of age, nodules on the head of mice (75 of 90; 27 of 33 male and 48 of 57 female) were observed. The mice with prominent nodules were sacrificed and evaluated microbiological contamination with culture, serological, parasitological and histopathological tests, indicating that S. aureus was isolated from in a culture, and necrosis/inflammation were observed in skin as evidenced by histopathological examination. To prevent infection to other animals, we firstly isolated infected animals from other animals. Second, we also separated the administrator of infected from those of non-infected animals. Third, we rearranged personnel and materials flows in animal facility, starting from infected area to non-infected area. Finally, we treated Cefazolin, which is effective to Gram positive cocci, BID for 5 days, and disinfected the skin with 70% Ethanol. As a result, incidence rate was alleviated from 83.3% to 66.7%, and there was no more infection of S. aureus to other animals after euthanization of the rest of animals. For immunodeficiency animals, including hairless SCID mouse, caution is necessary to identify and prevent S. aureus infection. For these reasons, our report suggests that immediate isolation of the animal and microbiological test are required to prevent microorganism, including S. aureus, if an animal is found to be colonized by S. aureus and associated clinical signs.

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P-16

Combination of midazolam, butorphanol, and inhalant isoflurane anesthesia in rat ○Atsushi Tsukamoto, Kaho Uchida, Shizuka Maesato, Tomo Inomata Laboratory of Laboratory Animal Science, Azabu University, Kanagawa, Japan

Isoflurane is representative inhalant anesthetic in rat. However, isoflurane mediates relatively strong respiratory depression. Aim of the present study was to establish novel balanced anesthesia in rat that combines midazolam and butorphanol with inhalant isoflurane anesthesia (MBI). Male Sprague Dawley rats received either isoflurane monoanesthesia or isoflurane with midazolam (2.5 mg/kg, ip) and butorphanol (2.0 mg/kg, ip). Minimum alveolar concentration (MAC) in each group was firstly evaluated. Induction and recovery times were measured. Adverse reaction during induction was also recorded. In each group, vital signs were assessed for 1hr under 1.5 μ MAC of isoflurane. Compared with isoflurane monoanesthesia, MBI anesthesia achieved 32% MAC reduction (Isoflurane monoanaesthesia: 1.30 ± 0.09 %, MBI 0.87 ± 0.08 %). Administration of MB caused smooth sedating action with low incidence of adverse reaction such as urination or defecation. Isoflurane monoanesthsesia showed remarkable decreases of respiratory rate and SPO2. In contrast, MBI anesthesia showed relatively stable respiratory rate without decrease of SPO2 during the entire anesthetic period. In conclusion, MB premedication is effective for the attenuation of respiratory depression induced by isoflurane, with smooth induction. This anesthetic protocol serves as novel option for the accomplishment of appropriate anesthesia in rat, particularly in the case of highanesthetic risk.

P-17

Analysis of TRPV3 function in pain sensation using a gain-offunction mutant rat ○Motoyo Maruyama1,2, Atsushi Sakai2, Hidenori Suzuki2, Toshio Akimoto1 1

Division of Laboratory Animal Science, Nippon Medical School, Tokyo, Japan, 2Department of Pharmacology, Nippon Medical School, Tokyo, Japan

Transient receptor potential (TRP) channels have been revealed to play important roles in thermosensation and pain. TRPV3 is activated by temperature above 33°C and various natural products such as oregano, and is thought to convey warm sensation without pain. However, TRPV3 is also suggested to contribute to pain sensation because TRPV3 knockout mice showed impaired responses to noxious heat. However, no available agonists and antagonists specific for TRPV3 have limited the elucidation of physiological functions of TRPV3. In addition, the TRPV3 expression in the primary sensory neurons remains controversial, while skin keratinocytes are consistently shown to express TRPV3. In the present study, we examined the TRPV3 roles in the pain sensation using WBN/Kob-Ht rat, a gain-of-function mutant of TRPV3 gene. Von Frey test was performed to examine the pain-related behavior in response to mechanical stimuli. To hot or cold stimuli, plantar test and hot or cold plate test were performed to examine hind paw responses, while tail immersion test and tail flick test were performed to examine tail responses. The response to mechanical stimuli in TRPV3 mutant rats was comparable to that in wild type rats. TRPV3 gain-of-function mutants showed an increased sensitivity to noxious heat and cold stimuli. These results suggest that TRPV3 contributes to pain sensation to cold stimulus as well as heat.

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P-18

Differences in saccharin preference and alterations in Tas1r3 among senescence-accelerated mouse ○Kimie Niimi, Eiki Takahashi Support Unit for Animal Resources Development, RIKEN BSI, Saitama, Japan

Purpose: The senescence-accelerated mouse (SAM) is used as animal models of senescence and age-associated disorders. SAM is derived from unexpected crosses between AKR/J and unknown mice. Although there are nine senescence-prone (SAMP) and three senescence-resistant (SAMR) strains, the genes responsible for the strainspecific alterations in SAMP are not fully identified. In this study, we evaluated the sweet taste perception by two-bottle test and compared the genotypes of the related gene, Tas1r3. Methods: 4-month-old male SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), SAMP10 (P10), and AKR/J mice were used. Saccharin preference was determined with two-bottle tests with 0.5, 1.6, and 10.0 mM saccharin and water. Six primer pairs were designed to amplify fragments including 8 sites of Tas1r3 coding region reported as regions associated with saccharin preference. Genomic DNA from each strain was used as a template to generate PCR products, which were sequenced. Results and discussion: Two-bottle tests revealed that R1, P6, P8, and P10 were saccharin tasters, whereas AKR/ J did not prefer saccharin. All the genotypes of R1, P6, P8, and P10 at the polymorphic sites in Tas1r3 known to influence saccharin preference were identical to those of C57BL/6J, a well-known saccharin taster strain, and were completely different from those of their parental AKR/J. These genetic alterations of SAM strains seem to come from the unknown strain thought to have initially crossed with AKR/J.

P-19

Ferulic acid in combination with octyl gallate prevents behavioral impairment in Alzheimer’s mice ○Naoki Koyama, Takashi Mori Department of Biomedical Sciences, Saitama Medical Center, Saitama Medical University, Saitama, Japan

Alzheimer’s disease (AD) is the most common progressive age-related dementia and is a progressive neurodegenerative disorder that is characterized by cognitive and psychiatric disturbances. We have been screened in a class of naturally-occurring dietary compounds (‘nutraceuticals’) and found that two phenolic compounds (ferulic acid, FA as a β-secretase inhibitor and octyl gallate, OG as an α-secretase enhancer) can negatively modulate the amyloidogenic pathway. Here, we examined whether the combination therapy of amyloid precursor protein (APP) modulators (FA and OG) might synergistically improve behavioral impairment. We orally administered FA plus OG, FA, OG, or vehicle to the aged AD model mice (PSAPP mice) and evaluated behavioral function. Beginning at 12 months of age, animals were gavaged with FA (30 mg/kg) plus OG (30 mg/ kg), FA (30 mg/kg), OG (30 mg/kg), or vehicle once daily for 3 months. The combination therapy of FA and OG as well as FA or OG treatment alone significantly prevented transgene-associated behavioral impairment including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter non-transgenic mouse behavior. Together, if this model mouse mirrors the clinical syndrome, then long-term dietary supplementation could be a safe and promising disease-modifying therapy to advanced AD with no discernible side effects.

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P-20

Suppression of dopamine D1 receptor expression causes decreased motor activity in mice ○Toshikuni Sasaoka1, Asako Sato2, Tadashi Okubo2, Nobuyoshi Fujisawa1, Toshiya Sato1,2, Kanako Oda1, Yoshitaka Maeda1, Minoru Tanaka1, Yoshitaka Yamamoto1, Seiko Sakai1, Yukihiro Jinbo1, Saori Chiba1, Eriko Umakawa1, MInesuke Yokoyama1,3 1

Brain Res Inst. Niigata Univ. Japan, 2Kitasato Univ. Sch. Med., 3Cent Inst Exp Anim

Dopamine controls a wide variety of behavior related to motor activity, cognition, motivation and reward. Dopamine D1 (D1R) and D2 receptors (D2R) are found at high levels in the striatal projection neurons, which have important roles in motor control. Although D1R signal theoretically promotes motor activity, conventional D1R knockout (KO) mice are hyperactive. It is assumed that potential developmental adaptation could cause hyperactive phenotype in D1R KO mice. To circumvent developmental adaptation, we generated a transgenic mice harboring tetracycline-regulated expression of D1R gene with D1R KO background (D1R -/-;Tg +). In the absence of doxycycline (Dox) the transgenic mice showed high expression of D1R under the control of D1R promoter, which rescued hyperactive phenotype in D1R KO mice. To clarify the role of D1R in adult mice, we suppressed the D1R expression in adult D1R -/-;Tg + mice, and home cage activity was examined. When Dox was administered, D1R expression was decreased to levels undetectable. Simultaneously, home cage activity level and rotarod performance were significantly decreased. These results demonstrate that D1R-mediated signal is required to maintain normal motor activity. A study of function of D1R regarding mechanism of motor control will be discussed.

P-21

Age-dependent kainate sensitivity in heterozygous rolling Nagoya CaV2.1 channel mutant mice ○Tae Yeon Kim, Kimie Niimi, Eiki Takahashi Support Unit for Animal Resources Development, RRC, RIKEN BSI, Wako, Japan

CaV2.1α1 is involved in glutamate release and kainate-induced intensive firing of neurons via glutamate receptors causes seizure and neuronal damage, especially in the hippocampus. To examine the relationship between CaV2.1 function and neurological disease, we investigated how CaV2.1 is related to kainate-induced seizure and neuronal damage using 2- and 18-month-old rol/+ mice. The seizure scores of 18-monthold rol/+ mice that received 20 mg/kg kainate intraperitoneally were significantly lower than those of wild-type mice. As a consequence of seizure, kainate induced delayed neuronal damage along with astrocytic growth in the hippocampus in wild-type mice, with a moderate effect observed in rol/+ mice. In the hippocampus of 18-monthold rol/+ mice, increased levels of mutant-type CaV2.1α1 were detected whereas phosphorylation of p38, a mitogen-activated protein kinase (MAPK) activated by kainate, was not increased after kainate injection. No difference was observed between 2-month-old rol/+ and wild-type mice intraperitoneally injected with 20 mg/ kg kainate in these analyses. The findings suggest that rol/+ mice experience age-related changes in sensitivity to kainate due to changesin the p38 MAPK signaling pathway via a mutant CaV2.1 channel. Hence, rol/+ mice may represent a novel model to delineate the association between CaV2.1 function, synaptic transmission, and the postsynaptic signaling cascade.

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P-22

Effects of herbal essential oil on physical and psychological stress Hyo Young Jung1, Dae Young Yoo1, Sung Min Nam1, Jong Whi Kim1, Jung Hoon Choi2, Jung Bo Hyun3, Yeo Sung Yoon1, ○In Koo Hwang1 1 Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, South Korea, 2Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, South Korea, 3Department of Oral Anatomy, College of Dentistry, Gangneung-Wonju National University, Gangneung 210-702, South Korea

In a previous study, we demonstrated that a Valeriana officinalis extract could attenuate increase of serum corticosterone levels in a mouse model for physical and psychological stress. In addition, our results showed that the extract could modulate serotonin (5-HT) and norepinephrine (NE) turnover in the hippocampus and amygdala region. In the present study, we intended to investigate the effects of valerenic acid (VA), the main component of V. officinalis extract, on corticosterone level in serum in normal mice and monoamine turnover in hippocampus-amygdala homogenates in a mouse model of physical and psychological stress. To determine the minimum dose of VA for anti-anxiety effect, eight-week-old ICR mice received oral administration of VA (0.2, 0.5, and 1.0 mg/kg/0.3 ml) once daily for 3 weeks to probe for immobility time and serum corticosterone levels. At a VA dose of 0.5 and 1.0 mg/kg, animals showed a decrease in the duration of immobility time and serum corticosterone levels. To confirm the anti-anxiety effect of VA, eight-week-old ICR mice received VA at a dose of 0.5 mg/kg, orally, once daily for 3 weeks, prior to being subjected to physical or psychological stress for 3 days, in a specially designed communication box, and followed by estimation of levels of monoamines and their metabolites in the hippocampus-amygdala region. VA administration at 0.5 mg/kg was strongly associated with reduction in the stress-induced increase of 5-HT and NE turnover in the hippocampus-amygdala homogenates. These results indicate that VA can mitigate the stress response by decreasing the turnover of 5-HT and NE in the hippocampus and amygdala.

P-23

Selegiline enhances cell proliferation and neuroblasts differentiation in the subgranular zone of the mouse hippocampus ○Dae Young Yoo1, Hyo Young Jung1, Sung Min Nam1, Jong Whi Kim1, Ki-Yeon Yoo2, Jung Hoon Choi3, Yeo Sung Yoon1, In Koo Hwang1 1

Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, South Korea, 2Department of Oral Anatomy, College of Dentistry, Gangneung-Wonju National University, Gangneung 210-702, South Korea, 3Department of Anatomy, College of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, South Korea

Selegiline (L-deprenyl) is an active monoamine oxidase B (MAO-B) inhibitor and inhibition of MAO-B reduces degradation of phenethylamine and dopamine. Dopamine is a neurotransmitter which plays numerous important roles and it has been reported that dopamine and its receptors are abundantly shown in the germinal regions deeply associated with neurogenesis. In this study, we investigated the effects of Selegiline on cell proliferation and neuroblast differentiation in the mouse dentate gyrus using anti-Ki67 and anti-doublecortin (DCX) antibodies. Mice were randomly divided into 4 groups: vehicle (0.9% saline)-treated group, 1, 3, and 10 mg/kg Selegiline-treated groups (n=10 in each group). Vehicle or Selegiline were orally administered to mice using a feeding needle once a day for 21 days from 7 weeks of age. Treatment of Selegiline significantly increased the number of Ki67-positive nuclei and DCX-immunoreactive neuroblasts compared to the vehicletreated group. In addition, we confirmed that treatment of Selegiline increased the levels of brain-derived neurotrophic factor (BDNF) in the western blot analysis. This result suggests that Selegiline significantly promotes cell proliferation and neuroblasts differentiation through enhancing BDNF levels in the mouse hippocampal dentate gyrus.

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P-24

Perinatal exposure of ethynyl estradiol affects expression of ERα and β in the brain of female rats ○Naoyuki Tachikawa1, Shiga Tatsuomi1, Takahiro Nakamura1, Chiaki Komine1, Yasuyuki Horii2, Gen Watanabe3,4, Kazuyoshi Taya1,3, Yasushi Mizoguchi1, Midori Yoshida5, Maiko Kawaguchi1 1

Sch of Agri, Meiji Univ JPN, 2Natl Inst Genetics, JPN, 3Tokyo Univ of Agri and Tech, JPN, 4United Graduate Sch of Veterinary Sci, Gifu Univ, JPN, 5National Inst of Health Sci, JPN

We evaluated how single exposure to ethynyl estradiol (EE) within 24 h of birth affects estrogen receptor ( ER) α and ERβ expression levels in juvenile female rats (Exp.1) and female adult rats (Exp.2). Wistar-Imamichi female rats were s.c. injected with 0.02 mg/kg EE (LEE), 2 mg/kg EE (HEE) or vehicle (Oil) within 24 h of birth. In Exp.1, 15 days old rats were sacrificed, and hippocampus (HP) and cortex (CT) were collected. In Exp.2, rats were ovariectomized at 10 wks old, and then at 16-18 wks old half the rats received 5 μg/0.1 ml Estradiol benzoate (EB). Next day, the ERα and ERβ expression levels were analyzed. (Exp.1) ERα and ERβ protein levels in the HP of both LEE and HEE were lower than in oil. (Exp. 2) In the non EB-injected rats, ERα protein levels in the CT of both LEE and HEE were lower than oil. In the EB-injected rats, ERα protein levels in LEE and HEE were lower than in oil .While, in non EB- injected rats ERβ protein levels in LEE and HEE were lower than in oil. These results suggest that neonatal exposure to EE exerts permanent, site-specific influences on ERα and ERβ expression, causing changes in estrogen sensitivity. This study was supported by a Great-in-aid by the Ministry of Health,Labour and Welfare,Japan.

P-25

Effects of neonatal female rat exposure to etynyl estradiol on passive avoidance learning ○Ikuya Tanabe1, Toru Okawara1, Chiaki Komine1, Midori Yoshida2, Maiko Kawaguchi1 1

School of Agriculture, Meiji University, Kanagawa, Japan, 2National Institute of Health Sciences, Tokyo, Japan

We investigated the effects of neonatal exposure to ethinyl estradiol (EE) on passive avoidance learning (PAL) in female rats. The general activity and anxiety-like behavior were also tested using the open field test (OF). Female Wistar-Imamichi rats were S.C. administered with either sesame oil (Oil), 0.02 mg/kg EE (LEE), 2 mg/ kg EE (HEE), or 20 mg/kg 17β-estradiol (E2) within 24 h after birth. Then the rats were tested for OF at 5 wks old [Exp. 1], PAL at 6 wks old [Exp. 2]. The other group of rats were ovariectomized at 7-10 wks old, and tested to OF at 12 wks old [Exp. 3], PAL at 16-18 wks old [Exp.4]. The half of rats in Exp. 3 and 4 were S.C. administered with estradiol benzoate (EB) 24 h before the respective tests. In Exp. 2, the learning behavior of LEE was significantly lower than with Oil. In Exp.4, the learning behavior shows tend to low in LEE than Oil in the presence of EB. In Exp. 1, there was no difference between treatments in anxiety-like behavior or the activity observed. In Exp. 3, E2 in activity or without EB was significantly low than the other groups. In conclusion, these results suggest that neonatal LEE exposure reduces learning ability through changes in the estrogensensitive brain. This study was supported by a Grant-in-aid from the Ministry of Health, Labour and Welfare, Japan (H25-KAGAKU-IPPANN-003).

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P-26

Effects of cross-fostering on passive avoidance learning in male Hatano high and low avoidance rats ○Toru Okawara1, Kosuke Satou1, Shingo Nakajima1, Ryo Ohta2, Yasuyuki Horii3, Maiko Kawaguchi1 1 3

Sch of Agri, Meiji Uni,kanagawa, JPN, 2Hatano Research Inst, Food and Drug Safety Center, JPN, National inst of Genetics, JPN

Development of learning ability is influenced by both genetic and environmental factors. We investigated whether passive avoidance learning (PA) ability of HATANO rats bred in accordance with their performance in the shuttle-box active avoidance task is influenced by maternal character, one of the environmental factor, or not. The two-way rat inbred strains of Hatano high- (HAA) and low avoidance (LAA) animals were originally selected from SD rats and bred in accordance with their high or low performance respectively in the shuttle-box active avoidance task. We previously demonstrated that the PA was significantly greater in HAA rats than LAA. In this experiment, we performed cross-fostering on the day after birth, and HAA male offspring reared by HAA dam (HH), HAA male offspring reared by LAA dam (HL), LAA male offspring reared by HAA dam (LH), and LAA male offspring reared by LAA dam (LL) underwent the PA task at 7-week-old. The present results show that HH offspring showed significantly high performance as compared with HL and LL offspring in the PA task. On the other hand, there was no significant difference between HL and LAA offspring. In conclusion, our study showed that the PA ability of HATANO rat is partly influenced by the environmental factor like maternal character. Part of this experiment was conducted by grant of meiji university priority research.

P-27

Behavioral analyses in rat by use of triaxial acceleration sensor ○Tatsunori Kobayashi1, Yoshiaki Shinozawa1, Atsushi Tsukamoto1, Yasushi Chida2,3, Tomo Inomata1 1

Department of Laboratory Animal Science, Azabu Univ., Kanagawa, 2Bycen Co., Ltd., 3Konan Univ.

The behavioral analysis of experimental animals is required in many experiments including toxicological evaluation. Present study was attempted to extract basic behavior in rat by use of a wireless triaxial acceleration sensor, which can detect the acceleration, ± 4 G in three directions (1 G= 9.8035m/s2). Five male Wistar rats were used in this study. Rats were anesthetized. A sensor module was fixed to each rat scull with stainless steel screw and a piezoelectric temperature sensor was inserted and fixed under the skin. After surgery, rats were allowed to recover for seven days. Open field (180 × 90 × 30 cm) test was performed for ten minutes. Data (sampling frequency; 400 Hz) were automatically sent to PC wirelessly and recorded. At the same time moving images of behaviors were also recorded. The acceleration data were offset processed and a scalar product was obtained, by each sampling interval (2.56 seconds=1024 points). The resultant vector and histogram were prepared and were examined by partial differentiation. In each type of behavior, there were significant differences in the median and total amount of movement in the histogram distribution of the resultant vector product. Grooming behavior was the only vector variation accompanied with rotation. From the above, the identification of each behavior in rat might be possible via the comparison of the scalar product of the acceleration data and vector analysis.

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P-28

Training courses on mouse reproductive techniques by CARD, Kumamoto University ○Yukie Haruguchi1,2, Kiyoko Fukumoto1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

We have provided training courses for mouse reproductive techniques since 2000 with the aim of supporting research using genetically engineered mice. The courses included lectures, demonstrations and practical training pertaining to the latest mouse reproductive techniques. Until now, we have held a total of forty-seven such courses, of which eight were held overseas. We have trained a total of 456 participants on these courses. In this conference, we will introduce a summary of mouse reproductive techniques acquired by trainees on our courses, including in vitro fertilization, embryo cryopreservation, oviduct flushing and cold-storage of embryos.

P-29

Application of mouse reproductive techniques at CARD, Kumamoto University —In vitro fertilization— ○Yuko Nakamuta1, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

In vitro fertilization (IVF) is a useful technique for producing many embryos for the storage or production of genetically engineered mice via embryo cryopreservation or embryo transfer. Since the establishment of our center, we have carried out IVF on more than 2,800 occasions, using 2,100 different strains of genetically engineered mice. During this time, we have improved our IVF technique to stabilize and enhance fertilization rate. In our IVF method we use FERTIUP® Sperm Preincubation Medium and CARD MEDIUM, both of which we developed ourselves. In this conference, we will explain our IVF techniques and introduce data regarding IVF accumulated in our center.

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P-30

Application of mouse reproductive techniques at CARD, Kumamoto University —Embryo transfer— ○Yumi Takeshita1,2, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

Embryo transfer is a useful technique for efficiently producing genetically engineered mice. In addition, the obtained pups are free from various pathogens derived from oocytes or sperm donors. In general, embryo transfer is performed by transferring embryos to oviducts of pseudopregnant female mice. In our center, we have conducted embryo transfer using around 4,300 different strains of genetically modified mice. We transferred a total of 211,438 embryos and obtained an average birth rate of 39.6%. In this conference, we will explain the embryo transfer technique and introduce data accumulated in our center.

P-31

Application of mouse reproductive techniques at CARD, Kumamoto University —Cesarean Section— ○Ai Miyagawa1,2, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

The Center for Animal Resources and Development (CARD), Kumamoto University, 2Kyudo Co., Ltd., Tosu, Japan

A cesarean section is performed when a recipient mouse has not given birth by Day 19 after two-cell embryo transfer (Day 0 is the day on which the two-cell embryos were transferred to recipients). Delivery dysfunction is frequently observed in pregnant females with few fetuses. We have performed cesarean section using 429 recipients until now. In 89% of recipients, all pups were successfully recovered after cesarean section. In this conference, we will explain our protocol for cesarean sections and introduce related data accumulated in our center.

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P-32

Application of mouse reproductive techniques at CARD, Kumamoto University —Embryo vitrification— ○Tomoko Kondo1,2, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

Embryo cryopreservation is useful for the efficient storage of genetically engineered mice. Since 1998, we have provided a useful mouse embryo/sperm bank service for managing a great number of mouse strains as genetic resources. During that time we have archived many cryopreserved embryos derived from 2,500 strains of genetically engineered mice. The embryos were cryopreserved via a simple vitrification method using the cryoprotectant that we developed (DAP213: 2 M DMSO, 1 M acetamide and 3 M propylene glycol). The average recovery and survival rates of vitrified-warmed two-cell embryos were 97 ± 4% and 86 ± 11% respectively. In this conference, we will explain our embryo cryopreservation method and introduce data regarding embryo cryopreservation accumulated in our center.

P-33

Application of mouse reproductive techniques at CARD, Kumamoto University —Sperm cryopreservation— ○Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

Our center provides a mouse bank service to promote research using genetically engineered mice. Through our service we carry out sperm cryopreservation for the efficient archiving of genetically engineered mice. Sperm cryopreservation offers many benefits, such as quick and simple operations, low storage cost and efficient embryo production via in vitro fertilization (IVF). Now, sperm cryopreservation is the preferred method for the efficient preservation of genetically engineered mice in transgenic labs. In our center, we have frequently performed IVF using cryopreserved sperm prepared using various methods. We recently developed a novel IVF method for legacy stock of cryopreserved sperm. In addition, we have conducted cryopreservation of sperm derived from the cauda epididymides of genetically engineered mice after shipment under refrigerated temperatures, and subsequently carried out IVF using the sperm. In this conference, we will explain the IVF methods that we have developed and introduce relevant data accumulated in our center.

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P-34

Application of mouse reproductive techniques at CARD, Kumamoto University —Storage of epididymides— ○Tomoko Umeno1,2, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

Storing cauda epididymides at refrigerated temperatures is extremely useful for the transportation of genetically engineered mice between research facilities. There are various benefits to transporting cauda epididymides in this manner, including user-friendliness, low shipment costs, a reduced risk of spreading infectious disease and no risk of losing animals due to death or escape. The transported epididymides are immediately available for IVF or sperm cryopreservation for the production or storage of genetically engineered mice. Until now, we have continuously improved our transport system for cauda epididymides and have established a robust system for shipping genetically engineered mice using the CARD Cold Transport Kit. In our center, we have overseen the transportation of cauda epididymides taken from genetically engineered mice on 262 occasions. All samples arrived at our center within 72 hours of shipment, and embryos were obtained via IVF using sperm collected from the samples (average fertilization rate = 80.6%). The obtained embryos were transferred to recipients before or after cryopreservation, and offspring were subsequently obtained from the embryos (average birth rate: fresh embryos = 46.6%, cryopreserved embryos = 40.3%).

P-35

In vitro fertilization using frozen-thawed sperm taken from a C57BL/6 mouse over twenty years ago ○Wataru Sakamoto1, Kiyoko Fukumoto1,2, Yukie Haruguchi1,2, Tomoko Kondo1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Ai Miyagawa1,2, Mari Iwamoto1, Fumi Takahashi1, Shuuji Tsuchiyama1, Toru Takeo1, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Kyudo Co., Ltd., Tosu, Japan

In this study, we examined the quality of cryopreserved sperm taken from a C57BL/6 mouse over twenty years ago. To assess sperm quality, we evaluated the fertility of the frozen-thawed sperm via in vitro fertilization (IVF) as well as the developmental ability of 2-cell embryos derived from the sperm via embryo transfer to pseudopregnant females. For IVF, we used FERTIUP® Mouse Sperm Preincubation Medium and CARD MEDIUM Mouse Fertilization Medium, both of which we developed ourselves. The fertilization rate was 94.6% (140/148), while the developmental rate of embryos to live pups was 58% (35/60). This study demonstrates that the quality of cryopreserved sperm can be well maintained for over twenty years.

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P-36

Effective production of offspring from vitrified embryos in three species of wild derived mice ○Keiji Mochida, Ayumi Hasegawa, Daiki Hama, Naoki Otaka, Maiko Ijuin, Kyuich Taguma, Michiko Hashimoto, Rika Takashima, Noriko Hiraiwa, Kazuyuki Mekada, Atsushi Yoshiki, Atsuo Ogura RIKEN BioResource Center, Ibaraki, Japan

We have succeeded in obtaining offspring by inter-subspecies and inter-species transfer of vitrified embryos from Mus musculus (BOR;2012, PLoS ONE;2014) and M. spretus (61th meeting) mice, respectively. Here we report successful production of M. spicilegus offspring by inter-species embryo transfer. In MSM strain (M. m.), a larger number of embryos per female (18.5 vs 6.1) were obtained by IVF after injection of anti-inhibin serum (AIS) than eCG. In SPR2 strain (M. spretus), the number of embryos was dramatically increased by natural mating than IVF (16.4 vs 0.8) after AIS injection. In Mus hortulanus and ZBN strains (M. spicilegus), 20.5 and 21.3 embryos were obtained by IVF after eCG injection, respectively. After transfer of MSM embryos, 39% of vitrified embryos developed into offspring by co-transfer of ICR embryos and injection of cyclosporine A to recipients. In SPR2 strain, the birth rates improved from 10% to 63% when the recipients were changed from ICR to B6C3F1 and embryos were changed from in vitro-fertilized to in vivo-fertilized. In M. hortulanus and ZBN strains, 12% and 5% of vitrified 2-cell embryos developed into term by 2nd embryo transfer after recovery from oviducts of recipients which transferred fresh embryos at the pronuclear stage. Thus, assisted reproductive techniques for these interspecific strains were successfully devised.

P-37

Fertilizing and developmental abilities in vacuum-drying sperm treated with methyl-beta-cyclodextrin ○Eri Nakamura, Norihiro Tada Res. Inst. for Diseases of Old Age, Juntendo Univ. Grad. Sch. of Med.

Spermatozoa treated with Methyl-beta-cyclodextrin(MBCD) are enhanced capacitation by depleting cholesterol from their plasma membrane. Mouse spermatozoa were treated with MBCD and then vacuum-dried with TrisEGTA solution containing trehalose, epicatechin (for green tea) and ascorbic acid in microtube. They were then stored for 1-4 week at RT in the vacuumed desiccator. After rehydration, spermatozoa were injected into oocytes by ICSI and embryonic development was followed. The percentages of fertilizing ability of oocytes injected with spermatozoa vacuum-dried and stored for 1 week or 4weeks after treating with 0mM, 0.75mM, 7.5mM or 75mM MBCD were 95.7%, 91.7%, 87.2% and 86.0% or 97.1%, 95.7%, 90.9% and 86.1%, respectively. There were no significantly differences among several MBCD concentrations in fertilization rates. On the other hand, the percentages of blastocyst development of oocytes injected with spermatozoa vacuum-dried and stored for 1week or 4weeks after treating with 0mM, 0.75mM, 7.5mM or 75mM MBCD were 17.8%, 29.5%, 0% and 5.4% or 14.7%, 27.3%, 0% and 0%, respectively. These results indicate that developmental ability to blatocyst stage was definitely inhibited by treating with high concentration of MBCD before being vacuum-dried and stored at RT in spermatozoa. Our data suggested that the depletion of cholesterol by MBCD treatment from sperm plasma membrane had negative effect for the developmental abilities of oocytes fertilized with vacuum-dried spermatozoa.

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P-38

Examination of in vitro growth using early secondary follicles in C57BL/6J mouse ovary ○Masayuki Anzai1, Megumi Sakita2, Rika Azuma3, Manami Nishimura4, Takao Nakagawa5, Tatsuya Inoue2, Mizuki Kazimoto2, Yoshihiko Hosoi1,2,3 1 Inst. Adv. Tech., Kinki Univ, 2B.O.S.T., Kinki Univ, 3Grand Sch of Biol-Ori Sci Tech., Kinki Univ, 4Kansai Med. Univ, 5Kiwa Laboratory Animals Co., Ltd

Objective: Recently, Mochida reported early secondary follicles from immature mouse were developed growth oocytes by the two-step follicle culture method. Further, it was obtained live pups. However, the twostep method was difficult and takes long time. In addition, these follicles were exposed to many factors. In this study, we assessed that early secondary follicles from C57BL/6J adult mouse were developed maturation oocytes and embryo by in vitro growth method using basement membrane matrix. Methods: After 46 hours the PMSG dosage to C57BL/6J Kwl adult female mouse, early secondary follicles (DIA: 60-100μm) were collected from the mouse ovaries. Collected follicles were embedded in matrigel and cultured in vitro growth medium (0.2AU/ mL FSH, 5% FBS in αMEM). The medium change was every 2 days. GV stage oocytes developed for 4-10 days were denuded from cumulus cells and cultured in mTaM medium for 16 hours. Subsequently, zona-drilled M2 stage oocytes were performed in vitro fertilization. Results: The present study reported the two-step culture for 17 days, but our results show in vitro growth method using matrigel takes 4-10 days and the in vitro growth rate was 80%(65/81). GV stage oocytes were developed to M2 stage oocytes (38%: 20/52). When performed in vitro fertilization rate of 64% (9/14).

P-39

Effect of reactive oxygen species (ROS) by the in vitro maturation medium with L-carnitine ○Tatsuya Inoue1, Rika Azuma2, Megumi Sakita2, Yoshihiro Noda3, Mizuki Kajimoto1, Takao Nakagawa4, Yoshihiko Hosoi1,2,5, Masayuki Anzai5 1

B.O.S.T., Kinki Univ, 2Grand Sch of Biol-Ori Sci Tech., Kinki Univ, 3Tokyo Metropolitan Institute of Gerontology, 4Kiwa Laboratory Animals Co., Ltd, 5Inst. Adv. Tech., Kinki Univ

Objects: Previously, it has been pointed out that qualitative dysfunction of endogenous oocytes caused by generation of reactive oxygen species (ROS) and declining in mitochondrial function with aging of mammals. We added L-carnitine to the in vitro maturation medium placed in the GV-stage oocytes. Furthermore, admitted the occurrence of normal offspring and the improvement of the blastocyst stage embryos. This report, we examined whether ROS affects the oocyte cytoplasm by adding L-carnitine to the in vitro maturation medium. Methods: Ovaries were extirpated after administration of PMSG into ICR female mouse. Extirpation ovaries were isolated, and COCs were freed from ovaries. Then, GV-stage oocytes were transferred to mTaM medium containing 0-10 mM L-carnitine and 300μM H2O2. Next, after 16 hours cultured, maturated oocytes were treated with mKSOM containing 5 μM CM-H2DCFDA for 10 min. Intracellular ROS yield was detected using a Fluorescence microscope. Results: These results were in vitro maturation rate of 94%. In the mTaM medium added 300 μM H2O2, ROS was detected. Then, each L-carnitine was added to this mTaM medium, as a result of in vitro maturation rate were respectively 67-86%. In addition, the results suggests that in vitro maturation medium with L-carnitine inhibited ROS detection.

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P-40

Effect of in vitro culture of an early embryo on development of a mouse ○Kanako Oda1, Toshiya Sato1,2, Yoshitaka Maeda1, Seiko Sakai1, Yukihiro Jimbo1, Nobuyoshi Fujisawa1, Minesuke Yokoyama1,3, Toshikuni Sasaoka1 1 Department of Comparative and Experimental Medicine Center for Bioresource-baced Researches Brain Research Institute, University of Niigata, Niigata, Japan, 2Department of Laboratory Animal Science Kitasato University School of Medicine, Kanagawa, Japan, 3Central Institute for Experimental Animals, Kanagawa, Japan

Reproductive engineering technology is widely used for production of mice. However, it remains unknown whether characteristics of in vitro cultured mouse embryos are the same as those of in vivo cultured mouse embryos. To clarify effects of the in vitro culture on mouse development, we examined in vitro cultured embryos with respect to morphology, developmental rate, cell numbers and amount of gene expression at blastocyst stage and measured body weight of mice derived from in vitro cultured embryos and in vivo cultured embryos. In vitro cultured embryos were subjected to be cultured in KSOMaa supplemented with human recombinant albumin (rHA) or modified Whitten’s medium (MW) with rHA. In vivo cultured embryos were transferred into the oviducts of pseudopregnant females and harvested.We found that number of pups from the in vitro cultured embryos decreased at birth and body weights of mice derived from the in vitro cultured embryos increased. These results suggest the in vitro culture of embryos has an influence on several aspects of mouse development. Further progress of the study will be discussed.

P-41

Large scale eradication of mouse hepatitis virus by assisted reproductive technologies in NIRS ○Seiji Kito1, Ayako Wada2, Megumi Ibayashi2, Masato Ito2, Mami Hayashi1, Kagari Kameda1, Yuka Ishida1, Shintaro Tateno3, Satoshi Tsukamoto1, Toshiaki Kokubo1 1

National Institute of Radiological Sciences, 2Science Service Inc., 3Neos Tech Inc.

In Japanese Conference for Laboratory Animal Science and Technology, Sapporo 2014, we have reported the mouse hepatitis virus infection at Animal Research Building, which is the largest conventional animal facility in National Institute of Radiological Sciences, and its cleaning procedures since October 2013. In this meeting we will report microbiological cleaning of mice which were carried out as parts of recovery process of the facility. At first, we temporarily cryopreserved sperm or embryos because of the limited animal rooms. Then, we introduced a bioBubble clean room with negative pressure enclosures in our SPF facility, and in combination with preinstalled capsule unit we established a large scale system which enables us to clean about 10 strains at a time with >10 SPF mice production of each strain by means of embryo transfer and Cesarean section.We have cryopreserved 7500 embryos and 245 sperm-straws using mice from the infected facility. Then, we transferred 2200 embryos and provided 643 mice. Using cryopreserved sperm, we obtained and transferred 340 embryos and provided 48 mice. We successfully cryopreserved embryos and sperm of all requested strains, except one which had very poor semen quality. After embryo transfer and Cesarean section, all mice were found hepatitis virus-free and were provided for research.

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P-42

Distribution of murine norovirus in the persistent infected cells ○Ken-Ichi Hanaki1, Seii Ohka1, Eri Matsuura2, Katsutoshi Ogasawara2, Tomohito Hanasaka2, Kinji Ishida2, Norio Hirano1 1 Department of Laboratory Animal Medicine, Iwate Medical University, Iwate, Japan, 2Center for Electron Microscopy and Bio-Imaging Research, Iwate Medical University, Iwate, Japan

Murine norovirus (MNV) is known to be able to establish subclinical chronic infection in immunocompetent mice. However, the cell culture system of MNV is transient infection with cell lysis and cytopathic effect. We therefore tried to develop persistent cell culture system of MNV as a model of chronic MNV infection in vitro. Then, distribution of MNV in persistently infected cells was investigated by microscopic analyses. Distribution of MNV antigen in persistently infected cells was confirmed by immunofluorescence double staining using mouse anti-MNV serum, rabbit anti-endosome or lysosome marker (EEA1, Rab7, Rab9, and LAMP1) antibodies, and fluorophore conjugated goat anti-mouse IgG or anti-rabbit IgG antibodies. Distribution of MNV particles in persistently infected cells was confirmed by a combination of transmission electron microscopy (TEM) and electron tomography. MNV antigen in persistently infected cells was distributed granularly in cytoplasm. Confocal laser scanning microscopy revealed that the distribution of viral antigen was matched to that of Rab7. TEM tomography analysis revealed that MNV particles were accumulated in cytoplasmic vesicles. These results demonstrated MNV particles in persistently infected cells were accumulated in maturing endosomes without cell lysis and cytopathic effect.

P-43

Study of murine norovirus and murine astrovirus contamination in deposited mice ○Ayako Kajita, Chiimi Ogawa, Hiromi Sakata, Shinobu Yuuki, Atsushi Yoshiki, Fumio Ike Experimental Animal Division, RIKEN BRC, Tsukuba, Japan

It was said that murine norovirus (MNV) and murine astrovirus (MuAstV) were prevalently observed in mouse colonies. At JALAS meeting 2014, we reported contamination of MNV and MuAstV in mice housed at conventional (CV) and barrier (SPF) facilities in RIKEN BioRecourse Center (BRC). The deposited mice housed in CV area showed contamination of these viruses but the mice housed in barrier area after rederivation showed no contamination. Since then, we have been monitoring MNV and MuAstV against the deposited mice. [Materials and methods] cDNA was synthesized from RNA extracted from fecal samples. RT-PCR assays were conducted using MNV and MuAstV specific primers. [Results and discussion] Eight MNV positive samples (3 facilities, 7 strains) and 29 MuAstV positive samples (4 facilities, 12 strains) were detected from 39 SPF derived mouse samples (6 facilities, 15 strains). Eleven MNV positive samples (1 facility, 6 strains) and 3 MuAstV positive samples (1 facility, 2 strains) were detected from 20 CV derived mouse samples (2 facilities, 10 strains). Both viruses were found from 6 SPF derived mouse strains. Although MuAstV seems non-pathogenic, we think MuAstV can be used as a SPF status indicator because this virus is easily eliminated by rederivation procedure. From the data showing MNV and MuAstV is prevalently contaminated in SPF facilities in Japan, monitoring these viruses will be required for a while.

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P-44

Epidemiological survey of Pneumocystis carinii infection in immunocompetent laboratory rats in Japan ○Masahiko Yasuda1, Rituki Uchida2,3, Youko Kamai1, Mika Yagoto1, Hanako Morita2, Tomoko Ishida2, Kenji Kawai1, Nobuhito Hayashimoto2 1

Pathological Analysis Center, Central Institute for Experimental Animals (CIEA), Kawasaki, Japan, ICLAS Monitoring Center, Central Institute for Experimental Animals (CIEA), Kawasaki, Japan, 3JAC Inc., Tokyo, Japan

2

Recently, in immunocompetent rats, Pneumocystis carinii (P. carinii) has been proven to cause interstitial pneumonia with perivascular lymphoid cuffs, that previously have been attributed to Rat Respiratory Virus. In this study, to reveal the current prevailing state of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan, an epidemiological survey of this agent was performed by a microscopic test using toluidine blue O stain and PCR assay for a total of 954 rats from 271 facilities in Japan. They were sent to the Central Institute for Experimental Animals (CIEA) for microbiologic monitoring from November, 2013 to October, 2014. In the results, 6 out of 954 immunocompetent rats (0.63%) from 4 out of 272 facilities (1.47%) were positive for P. carinii. Gross pulmonary lesions were found in three rats. The lungs of the rats were slightly interspersed with multiple dark-red foci. Histopathologically, in the lungs of the rats, interstitial pneumonia with lymphoid perivascular cuffs were observed, and the presence of Pneumocystis cysts were shown using Grocott’s methenamine silver stain. To our knowledge, this is the first report to reveal the prevalence of Pneumocystis carinii in immunocompetent laboratory rats in Japan.

P-45

Adaption of influenza A (H1N1 and H3N2) and influenza B virus in mice ○Tsubasa Nakano, Ryo Tanaka, Hayato Kakinuma, Shigemi Fukami, Ikumi Kamata, Akiyoshi Ishikawa, Hirofumi Komatsubara Tsukuba Research Center,HAMRI Co., Ltd,2638-2 Osaki,Koga-shi,Ibaraki-ken 306-0101,Japan

Background and aim: Experimental model animals such as mice have low sensitivity to human influenza virus. In order to evaluate the efficiency of anti-influenza drugs or vaccines in mice studies, a mice model of human influenza virus has been required. Therefore, we examined a production of highly sensitive influenza virus. Method: Female 5 week-old BALB/c mice were inoculated intranasally with 103 PFU/50μL of A/PR/8/34 (H1N1), A/Port Chalmers/1/73 (H3N2), or B/Lee/40. The mice were euthanized 2 or 3 days after the inoculation and their lungs were taken, homogenized, and inoculated into another group of mice. This passage process was repeated 8 times. The virus present in the lung homogenate after 8th passage was amplified by MDCK cell and the adaption of purified virus was tested with mice. Results and discussion: By inoculation of each human virus strain, which was repeatedly subcultured in mice lung, body weight of mice was decreased. This suggests that human virus had been mutated during passages in mice. Since more than 10% of body weight decreases were shown after 1 th, 4th, or 7th passage of A/PR/8/34, A/Port Chalmers/1/73, or B/Lee/40, respectively, the mutation ability varied among virus strains. The mice infected virus, purified after 8th passage were dead within 5 to 9 days of inoculation. These results suggest that highly sensitive influenza virus was produced by eight-time-passage in mice.

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P-46

Relationship between helper T-cell and the symptoms on particulate-induced respiratory allergy ○Risako Nishino, Tomoki Fukuyama, Yuko Watanabe, Yoshimi Kurosawa, Hideo Ueda, Tadashi Kosaka, Takanori Harada Institute of Environmental Toxicology

Exposure to environmental particulate matter is known as a leading cause of respiratory allergy, and we previously investigated pathogenic mechanisms of these symptoms (Nishino et al., 2014). We report here that the effects of helper T-cell on environmental particle-induced respiratory allergy. BALB/c and NC/Nga mice were sensitized dermally followed by inhaled challenge with pulverized (approximately 2 μm) chemical respiratory allergen, trimellitic anhydride (TMA). On the day after last challenge, all mice were examined for serum IgE levels, immunocyte counts and cytokine/chemokine levels in the hilar lymph node and bronchoalveolar lavage fluids (BALF). Type 2 helper T-cell reactions, involved in respiratory allergy, were significantly increased in BALB/c mice compared with NC/Nga mice. On the other hand, pro-inflammatory cytokine productions (IL-9 and -17) which might be produced by type 9 and 17 helper T-cell in NC/Nga mice are significantly higher than those of BALB/c mice. BALF analysis revealed that, mast cells and histamine production in NC/Nga mice significantly higher than those of BALB/c mice. Therefore, marked inflammation of lung tissue and respiratory allergic symptoms were noted only in NC/Nga mice. According to above results, it has been suggested that classification of helper T-cell might be involved in the aggravation of airway allergic symptoms induced by environmental particulate matter.

P-47

Long-term maintenance of human mature NK cells in human interleukin-15 transgenic NOG mice ○Ikumi Katano1,2, Takeshi Takahashi1, Ryoij Ito1, Asami Hanazawa1, Riichi Takahashi1, Hiroshi Suemizu1, Yutaka Kawakami1, Mamoru Ito1 1

Central Institute for Experimental Animals, Kawasaki, Japan, 2Keio University, School of Medicine, Institute for Advanced Medical Research, Tokyo, Japan

We newly generated NOG-interleukin-15 (hIL-15) mouse by introducing human hIL-15 gene, which is an activation and proliferation factor for natural killer (NK) cells. We investigated how human NK cells maintain in this NOG-hIL-15 strain after transplantation of human peripheral blood (PB)-derived mature CD56 + NK cells (huNK). Adult NOG-hIL-15 mice were irradiated with 2.5 Gy and then intravenously inoculated with 1-2 x 10 6 huNK cells (huNK-NOG-IL-15 mice). Kinetics of human NK cells in PB of these hu-NK-NOG-hIL-15 mice was analyzed using flow cytometry. After transfer, the number of human NK cells rapidly increased in PB of huNKNOG-hIL-15 mice for 4 weeks and gradually decreased until 24 weeks, whereas human NK cells disappeared up to 2 weeks after transfer in conventional NOG mice. Human dominant CD56+CD16+ cytotoxic NK cells were detected in spleen, liver and lung of huNK-NOG-hIL-15 mice. Expression of granzyme A and production of interferon-γ in human NK cells of huNK-NOG-hIL-15 mice was observed. Tumor growth in huNK-NOGhIL-15 mice subcutaneously transplanted human myeloma cell line K562 was significantly suppressed. These data suggest that NOG-hIL-15 mice might become a useful tool for analysis of human mature NK cell function in vivo.

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P-48

Analysis of background date on mouse model for dystrophy: C57BL/10Sc-mdx (the second report) ○Takayuki Gotoh1, Yasuda Masahiko2, Shimomura Chie1, Megumi Nishiwaki1, Miho Itoh1, Takuma Mizusawa2, Yuyo Ka2, Nao Yoneda2, Tomoyuki Ogura2, Riichi Takahashi2, Hideki Shinohara1 1

Technical Service Dept, CLEA Japan Inc, Shizuoka, Japan, 2Central Institute for Experimental Animals (CIEA)

CIEA has maintained the model mouse strains of muscular dystrophy, and has supplied them to research institutions. This time, in order to conribute to muscular dystrophy research, we analyzed background data on the Duchenne muscular dystrophy mouse model strain from C57BL/10Sc-mdx (mdx) mice against those from C57BL/6JJcl (B6) mice (20 animals/group/sex, 20 weeks of age). Background data included body weights and serum/plasma levels of creatine kinase (CK), LDH, AST, ALT, creatinine, total protein, glucose, Potassium, Calcium, ALP, Triglyceride, Blood urea nitrogen, Chloride and Total cholesterol. Serum/plasma levels of CK, LDH, AST, ALT, and creatinine from mdx mice, were much higher than those from B6 mice. Therefore, mdx mouse is an extremely useful animal model for Duchenne muscular dystrophy. This report has C57BL/10Sc-mdx mice, showed again characterized as muscular dystrophy model mice.

P-49

Construction of transgenic mice producing human neuropathy target esterase Nami Motosugi1, Yuki Yamamura1, Hiromi Miura1, Masato Ohtsuka1, Tomoichi Ohkubo2, Michiyo Yoshino2, Masafumi Tanaka1, Tomomi Hatanaka1, Kou Sakabe1, ○Minoru Kimura1 1

Department of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan, 2Support Center for Medical Research and Education, Tokai University, Kanagawa, Japan

Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP-induced delayed neuropathy (OPIDN) and nerve axon degeneration. We found that the enzymatic activity of NTE was significantly higher (P