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Enya S.,Shizuoka Prefectural Research Institute of Animal Industry | Kawarasaki T.,Tokai University | Otake M.,Shizuoka Prefectural Research Institute of Animal Industry | Kangawa A.,Shizuoka Prefectural Research Institute of Animal Industry | And 5 more authors.
In Vivo | Year: 2016

Background/Aim: Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Materials and Methods: Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Results and Conclusion: Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.


Kangawa A.,Shizuoka Prefectural Research Institute of Animal Industry | Otake M.,Shizuoka Prefectural Research Institute of Animal Industry | Enya S.,Shizuoka Prefectural Research Institute of Animal Industry | Yoshida T.,Tokyo University of Agriculture and Technology | And 2 more authors.
Toxicologic Pathology | Year: 2016

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies. © The Author(s) 2016.


Nakamura T.,Tokyo Medical and Dental University | Sekiya I.,Tokyo Medical and Dental University | Muneta T.,Tokyo Medical and Dental University | Hatsushika D.,Tokyo Medical and Dental University | And 8 more authors.
Cytotherapy | Year: 2012

Background aims. Transplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig model. Methods. The chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10 min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10 min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 months. Results. Synovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control knees. Conclusions. Leaving a synovial MSC suspension in cartilage defects for 10 min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs. © 2012 Informa Healthcare.


Hiraizumi S.,Livestock Research Institute | Nishinomiya H.,Akita | Oikawa T.,Miyagi Prefectural Livestock Experiment Station | Sakagami N.,Kanagawa Prefectural Livestock Technology Center | And 7 more authors.
Theriogenology | Year: 2015

To reduce labor for superovulation treatment by twice-daily intramuscular (im) administration of FSH for more than 3 to 4days, we investigated the superovulatory responses of Japanese Black cows to porcine FSH (pFSH) used as a single subcutaneous (sc) administration at two different doses in two different volumes of saline. In experiment 1, 20 Armour units (AU) of pFSH dissolved in either 10mL (treatment A; n=14) or 50mL (treatment B; n=14) of saline was administered subcutaneously in the neck region. In experiment 2, 30AU of pFSH dissolved in either 10mL (treatment C; n=15) or 50mL (treatment D; n=15) of saline was administered subcutaneously in the neck region. The control animals in experiment 1 (n=14) and experiment 2 (n=15) received 20AU of pFSH administered intramuscularly twice daily in decreasing doses for more than 3days. In experiment 1, mean (±SEM) numbers of CL (15.4±2.5, 18.1±3.4, and 17.2±2.6), total number of ova and embryos (12.9±1.4, 15.9±3.5, and 16.2±2.8), and transferable embryos (7.5±2.0, 10.4±2.8, and 8.0±2.1) did not differ among treatments A, B, and control. In experiment 2, mean (±SEM) numbers of CL (20.5±4.3, 20.4±2.7, and 20.1±3.4), total number of ova and embryos (21.7±4.2, 17.3±3.4, and 16.5±3.2), and transferable embryos (8.1±1.6, 9.3±2.2, and 9.5±1.9) did not differ among treatments C, D, and control. Although there were no differences in serum pFSH concentrations among the three treatments at each of the time points in experiment 1, in experiment 2, the serum pFSH concentration at 6 and 8hours after pFSH administration in treatment C (3.1±0.8, 2.7±0.5ng/mL, mean±SEM) was significantly greater (P<0.05) than in the control (0.7±0.1, 1.1±0.2ng/mL). At 10hours after administration, the pFSH concentration had decreased and there were no differences among the three treatments at subsequent time points. These results suggest that increasing the volume of saline or the dose of pFSH does not affect the absorption pattern of pFSH administered as a single sc administration. In conclusions, single sc administration of pFSH at a dose of 20 or 30 AU dissolved in 10 or 50mL of saline is able to induce a superovulatory response comparable with that obtained by twice-daily im administration in Japanese Black cows. © 2015 Elsevier Inc.


Sato K.,University of Shizuoka | Sato K.,Shizuoka Prefectural Research Institute of Animal Industry | Saito T.,University of Shizuoka
Chemical Engineering Transactions | Year: 2013

Environmental regulations for dairy farms and milk-plants tend to be tightened in Asian countries, particularly in Japan, because the milk-including wastewater discharged from them causes serious regional environmental problems. For small and medium-sized dairy farms and milk plants, we propose a novel process of using ozone which has very strong oxidative power. This proposed process is different from conventional treatment processes with ozonation, and rapidly reduces BOD, TOC, total nitrogen (T-N) and chromaticity of the wastewater containing milk. This new ozonation process imposed partial polymerization of the unsaturated fatty acids including carbon-double bonds as well as simple decomposition of the above acids into low molecules. Whitish solid-like products formed from the polymerized fatty acids (i.e. ozonide) and the insoluble proteins were adsorbed to the ozone bubbles. The solidification and purification efficiency of this process depended on the initial condition of the milk-containing wastewater. Thus, we propose a two-stage process against this wastewater treatment method. Under an acid initial condition, the ozonation increases yield on the solid-like products which are compounded from the polymerized fatty acids and the insoluble proteins. When these solid-like products were removed from the wastewater, the pollutants (i.e. BOD, TOC, T-N and chromaticity) in the wastewater were decreased almost simultaneously. Accordingly the water quality was improved in a short time. The ozonation under an alkaline initial condition with a low temperature also improved the water quality, despite the fact that the yield of the solid-like products was small. In this condition, the solubility of the ozone was higher than that under the high temperature condition, hence the OH-radicals generated from the ozone were enhanced in the alkaline condition and decomposed the pollutants powerfully. These two reaction pathways depending on the pH and temperature were strongly related to the decomposition of the pollutants by ozone. Thus, the ozonation under an acid condition is more useful for small and medium-sized dairy farms and milk plants, because they do not need the control of the wastewater temperature under an acid condition. Copyright © 2013, AIDIC Servizi S.r.l.


Hirao A.,Jichi Medical University | Kawarasaki T.,Tokai University | Konno K.,Kyoto Sangyo University | Enya S.,Shizuoka Prefectural Research Institute of Animal Industry | And 3 more authors.
Acta Zoologica | Year: 2014

Domestic pigs possess a well-developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP-expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP-expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP-expressing cells had longitudinal dendrites. A few GFP-expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP-expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns. © 2013 The Royal Swedish Academy of Sciences.


PubMed | Ito Business Planning, Japan National Institute of Infectious Diseases, University of Wisconsin - Madison, Tokyo Medical University and Shizuoka Prefectural Research Institute of Animal Industry
Type: Journal Article | Journal: Journal of virology | Year: 2016

Pigs are considered a mixing vessel for the generation of novel pandemic influenza A viruses through reassortment because of their susceptibility to both avian and human influenza viruses. However, experiments to understand reassortment in pigs in detail have been limited because experiments with regular-sized pigs are difficult to perform. Miniature pigs have been used as an experimental animal model, but they are still large and require relatively large cages for housing. The microminipig is one of the smallest miniature pigs used for experiments. Introduced in 2010, microminipigs weigh around 10 kg at an early stage of maturity (6 to 7 months old) and are easy to handle. To evaluate the microminipig as an animal model for influenza A virus infection, we compared the receptor distribution of 10-week-old male pigs (Yorkshire Large White) and microminipigs. We found that both animals have SA2,3Gal and SA2,6Gal in their respiratory tracts, with similar distributions of both receptor types. We further found that the sensitivity of microminipigs to influenza A viruses was the same as that of larger miniature pigs. Our findings indicate that the microminipig could serve as a novel model animal for influenza A virus infection.The microminipig is one of the smallest miniature pigs in the world and is used as an experimental animal model for life science research. In this study, we evaluated the microminipig as a novel animal model for influenza A virus infection. The distribution of influenza virus receptors in the respiratory tract of the microminipig was similar to that of the pig, and the sensitivity of microminipigs to influenza A viruses was the same as that of miniature pigs. Our findings suggest that microminipigs represent a novel animal model for influenza A virus infection.


PubMed | Japan National Agriculture and Food Research Organization, Tokai University, Shizuoka Prefectural Research Institute of Animal Industry and Fuji Micra Inc.
Type: Journal Article | Journal: In vivo (Athens, Greece) | Year: 2016

Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology.Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded.Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.


PubMed | Tokyo University of Agriculture and Technology and Shizuoka Prefectural Research Institute of Animal Industry
Type: Journal Article | Journal: Toxicologic pathology | Year: 2016

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.


PubMed | Shizuoka Prefectural Research Institute of Animal Industry
Type: Journal Article | Journal: Journal of animal science | Year: 2013

In the present study, we investigated the influence of exogenous estrogen on embryo survival after transfer into prepubertal gilts in which estrus had been induced. In the first experiment, estrus was induced in prepubertal gilts by the administration of 1,000 IU of eCG and 750 IU of hCG every 72 h. Several blastocysts were recovered on d 6 (d 0 is the day of hCG administration), and 1 embryo was transferred to the tip of 1 side of the uterine horn on d 6 (Control). In treated groups, after embryo transfer, 5 mg of estradiol benzoate (EB) was administered on d 11 (EB5mg-1) or d 11, d 13, and d 15 (EB5mg-3) or d 11, 12, 13, 14, and 15 (EB5mg-5) or 20 mg of estradiol dipropionate (EDP) was administered on d 11 (EDP20mg-1) or d 11 and d 14 (EDP20mg-2). Autopsy examinations were performed on d 53 to 60. Although nontreated gilts did not become pregnant, gilts in each of the estradiol-treated groups became pregnant. The greatest pregnancy rate (77.8%, 7/9) was obtained with EDP20mg-2 (EDP20mg-2 > control: P < 0.05). In a second experiment, 1 blastocyst was transferred to prepubertal gilts and treated with EDP20mg-2. Pregnancy in recipient pigs was confirmed by ultrasonography, and pigs were allowed to farrow. Embryo survival rate was high on d 30 of pregnancy (75%, 9/12) but had a tendency (P = 0.0995) to decline from d 30 to delivery (33.3%, 4/12). In a third experiment, prepubertal gilts were administered 5 mg of EDP on d 11 (EDB5mg-1) and d 11 and d 14 (EDP5mg-2). Autopsy examinations were performed on d 53 to 58. Pseudopregnancy rate was high for EDP5mg-2 (63.6%, 7/11) compared with EDP5mg-1 (0%, 0/11; P < 0.05). In a fourth experiment, prepubertal gilts were transferred 1 blastocyst and treated with EDP5mg-2. Pregnancy was confirmed in recipient pigs by ultrasonography, and pigs were subsequently allowed to farrow. Embryo survival rate remained unchanged from d 30 of pregnancy to delivery (66.7%; 8/12). One piglet died from dystocia, and 1 suffered from deformity involving double-breasted hooves and died 6 d after birth. There was no difference (P > 0.05) in survival rate on d 30 of pregnancy and weaning (50%, 6/12). Body weight at birth and at weaning did not differ from that reported in previous studies. In conclusion, this study showed that EDP5mg-2 treatment during early pregnancy leads to full-term development of a single embryo.

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