Hokkaido Animal Research Center

Hokkaido, Japan

Hokkaido Animal Research Center

Hokkaido, Japan
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Hiraoka H.,Kobe Regional Center | Yamamoto K.,Food and Agricultural Materials Inspection Center | Mori Y.,Sapporo Regional Center | Asao N.,Food and Agricultural Materials Inspection Center | And 5 more authors.
Mycotoxin Research | Year: 2013

The analysis of deoxynivalenol (DON) in silage samples using enzyme-linked immunosorbent assay (ELISA) often leads to an overestimation. To better analyze DON in rice and corn silages using a commercially available ELISA kit, a cleanup method using a MultiSep #226 column was developed. As a result, overestimation of DON by the influence of specific cross-reaction with acetyldeoxynivalenol (AcDON) was confirmed. In samples where AcDON was not detected by liquid chromatography with mass spectrometry (LC-MS), no samples showed a significant difference (P < 0.05) in DON amounts between ELISA with cleanup and LC-MS analysis. For the recovery study, blank silage was spiked with 0.5 or 1.0 mg/kg DON. The mean recoveries of DON determined by ELISA with cleanup and LC-MS analysis were 112 and 96%, respectively, and the relative standard deviation for the repeatability (RSDr) were 8.2 and 9.8%, respectively. No samples showed a significant difference (P < 0.05) in DON concentration determined by either ELISA or LC-MS analysis. A collaborative study to validate this rapid method was carried out using four samples, two rice and two corn silage, by 10 participating laboratories. Each sample was analyzed using blind duplicates. The mean values of DON detected were 1.5-2.3 mg/kg, RSDr and the relative standard deviation for the reproducibility (RSDR) were 4.1-12.7 and 7.6-23.4%, respectively, and the HorRat values were 0.5-1.6. Therefore, the overestimation of DON by the influence of nonspecific cross-reaction with sample matrix was reduced by the cleanup method using a MultiSep #226 column, and analysis of DON in silage was improved. This use of this method for estimation of DON contamination in silage allows rapid detection at the place of use that is likely to result in improved animal health. © Society for Mycotoxin Research and Springer-Verlag 2012.


Miyashita N.,Japan National Institute of Agrobiological Science | Kubo Y.,National Institute of Livestock and Grassland science | Yonai M.,Japan National Agricultural Research Center | Kaneyama K.,National Livestock Breeding Center | And 6 more authors.
Journal of Reproduction and Development | Year: 2011

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres. © 2011 by the Society for Reproduction and Development.


Sugimura S.,Tohoku University | Yokoo M.,Akita Prefectural University | Yamanaka K.-I.,Japan National Agricultural Research Center | Kawahara M.,Saga University | And 5 more authors.
Cellular Reprogramming | Year: 2010

Oxygen consumption reflects overall metabolic activity of mammalian embryos. We measured oxygen consumption in individual porcine somatic cell nuclear transfer (SCNT) and in vitro-fertilized (IVF) embryos by modified scanning electrochemical microscopy. Oxygen consumption in IVF embryos rapidly increased at day 5 of the blastocyst stage (D5BL). IVF embryos that consumed >0.81 ×1014/mol sec-1 of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence. © 2010, Mary Ann Liebert, Inc.


Fujii T.,Iwate University | Moriyasu S.,Hokkaido Animal Research Center | Hirayama H.,Hokkaido Animal Research Center | Hashizume T.,Iwate University | Sawai K.,Iwate University
Cellular Reprogramming | Year: 2010

High rates of embryonic, fetal, or placental abnormalities have consistently been observed in bovine cloning. Segregation of inner cell mass (ICM) and trophectoderm (TE) lineages in early embryos is an important process for fetal and placental formation. In mouse embryos, differentiation of ICM and TE is regulated by various transcription factors, such as OCT-4, CDX2, and TEAD4, but molecular mechanisms that regulate differentiation in bovine embryos remain unknown. To clarify gene transcripts involved in segregation of ICM and TE lineages in bovine embryos, we examined the relative abundances of OCT-4, CDX2, TEAD4, GATA3, NANOG, and FGF4 transcripts in blastocyst embryos derived from in vitro fertilization (IVF). Furthermore, transcript levels of such genes in bovine embryos derived from somatic cell nuclear transfer (NT-SC) and in vivo (Vivo) were also compared. OCT-4, NANOG, and FGF4 transcript levels in IVF embryos were significantly higher in ICM than in TE. In contrast, the CDX2 transcript level was lower in ICM than in TE. In NT-SC embryos at the blastocyst stage, transcript levels of all genes except CDX2 were lower than that in Vivo embryos. In the elongated stage, expression levels of the six genes did not differ between NT-SC and Vivo embryos. We observed aberrant expression patterns of various genes involved in segregation of ICM and TE lineages in bovine NT-SC embryos. These results raise the possibility that abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage. © 2010, Mary Ann Liebert, Inc.


Sawai K.,Iwate University | Takahashi M.,Japan National Agricultural Research Center | Moriyasu S.,Hokkaido Animal Research Center | Hirayama H.,Hokkaido Animal Research Center | And 3 more authors.
Cellular Reprogramming | Year: 2010

The epigenetic reprogramming of the donor cell nucleus is an important factor in the development of embryos and production of normal offspring derived by somatic cell nuclear transfer (NT-SC). During early development, a dramatic reduction in methylation levels occurs in mouse. In early embryos, this process makes it possible to erase gamete-specific methylation patterns and induce de novo methylation at defined developmental time-points. To clarify changes in DNA methylation in bovine NT-SC embryos, we examined satellite I sequences in bovine embryos derived in vivo (Vivo) and by NT-SC at the blastocyst (BC) and elongated (EL) stages. Because the EL stage embryo consists of the embryo disc (ED) and trophectoderm (TE), the methylation status of each part was analyzed with respect to the progress of differentiation. DNA methylation levels in Vivo embryos were increased during the elongation stage. In contrast, DNA methylation levels in NT-SC embryos remained unchanged in the ED and significantly decreased in the TE. Real-time PCR analysis showed that Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower than that in Vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. Our results suggest that the aberrant methylation level of bovine NT-SC embryos in the satellite I region is corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates. © 2010, Mary Ann Liebert, Inc.


Sawai K.,Iwate University | Takahashi M.,Japan National Agricultural Research Center | Fujii T.,Iwate University | Moriyasu S.,Hokkaido Animal Research Center | And 4 more authors.
Journal of Reproduction and Development | Year: 2011

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re- NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT. © 2011 by the Society for Reproduction and Development.


Sawai K.,Iwate University | Fujii T.,Iwate University | Fujii T.,Hokkaido Animal Research Center | Hirayama H.,Hokkaido Animal Research Center | And 2 more authors.
Journal of Reproduction and Development | Year: 2012

We examined the comprehensive epigenetic status, including histone H3 and H4 acetylation, DNA methylation and level of mRNA transcripts of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA), along with their full-term developmental efficacy. Treatment with 50 nM TSA enhanced early developmental competence; increased acetylation of two histones, H3K9K14 and H4K8, at the blastocyst stage; and maintained the DNA methylation status of the satellite I sequence in bovine SCNT embryos. The difference in IGFBP-3 transcript levels between in vivo and SCNT embryos disappeared in SCNT embryos after treatment with 50 nM TSA. Pregnancy, full-term developmental competence and body weight at birth of offspring did not differ between SCNT embryos treated with 50 nM TSA and untreated embryos. These results suggest that treatment with TSA improves preimplantation development and changes the epigenetic status but does not promote the full-term development competence in bovine SCNT embryos. © 2012 by the Society for Reproduction and Development.


Hara S.,Hokkaido Animal Research Center | Tanigawa T.,Hokkaido Animal Research Center
Animal Science Journal | Year: 2010

The effects of length of cut and mechanical processing on corn silage utilization by dairy cows were evaluated. Corn silage treatments were harvested at the black line stage of maturity and chopped at a theoretical length (TLC) of 9.5 mm without processing (Control) or at a TLC of 19 mm with processing at roller clearances of 1, 3, or 5 mm. Eight multiparous Holstein cows were assigned in a replicated 4 × 4 Latin square design with 21-day periods. Corn silage treatments were fed in diets containing 78.3% corn silage and 21.7% soybean meal (DM basis). Treatments had no significant effects on DMI, milk and 4% FCM production. The efficiency of converting DMI to FCM tended to be greater with processing at a roller clearance of 1 and 3 mm than at other clearances. Apparent total tract digestibility of starch tended to be lowest for cows fed control silage, and increased as roller clearance decreased. Ruminal ammonia concentrations in cows fed control silage were numerically higher than in cows fed proccesed silages. These results suggest that when corn silage is harvested at the black line of maturity, roller clearance should be 3 mm or less with a TLC of 19 mm. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Animal Science.


Fukuda S.,Hokkaido Animal Research Center | Okada H.,Japanese National Institute of Animal Health | Arai S.,Japanese National Institute of Animal Health | Yokoyama T.,Japanese National Institute of Animal Health | Mohri S.,Japanese National Institute of Animal Health
Journal of Comparative Pathology | Year: 2011

Bovine spongiform encephalopathy (BSE) is characterized by the appearance of spongy lesions in the brain, particularly in the brainstem nuclei. This study evaluated the degenerative changes observed in the central auditory brainstem of BSE-challenged cattle. The neuropathological changes in the auditory brainstem nuclei were assessed by determining the severity of vacuolation and the presence of disease-associated prion protein (PrP Sc). Sixteen female Holstein-Friesian calves, 2-4 months of age, were inoculated intracerebrally with BSE agent. BSE-challenged animals developed the characteristic clinical signs of BSE approximately 18 months post inoculation (mpi) and advanced neurological signs after 22mpi. Before the appearance of clinical signs (i.e. at 3, 10, 12 and 16mpi), vacuolar change was absent or mild and PrP Sc deposition was minimal in the auditory brainstem nuclei. The two cattle sacrificed at 18 and 19mpi had no clinical signs and showed mild vacuolar degeneration and moderate amounts of PrP Sc accumulation in the auditory brainstem pathway. In the animals challenged with BSE agent that developed clinical sings (i.e. after 20mpi), spongy changes were more prominent in the nucleus of the inferior colliculus compared with the other nuclei of the auditory brainstem and the medial geniculate body. Neuropathological changes characterized by spongy lesions accompanied by PrP Sc accumulation in the auditory brainstem nuclei of BSE-infected cattle may be associated with hyperacusia. © 2010 Elsevier Ltd.


Murayama Y.,Japanese National Institute of Animal Health | Yoshioka M.,Japanese National Institute of Animal Health | Masujin K.,Japanese National Institute of Animal Health | Okada H.,Japanese National Institute of Animal Health | And 7 more authors.
PLoS ONE | Year: 2010

Background: Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrPSc, a proteaseresistant misfolded isoform of the cellular prion protein PrPC. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrPSc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrPSc in BSE-affected cattle therefore remains unknown. Methodology/Principal Findings: We report here that PrPSc derived from BSE-affected cattle can be amplified ultraefficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrPSc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrPSc in blood was not substantiated in the BSE-affected cattle examined. Conclusions/Significance: The distribution of PrPSc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrPSc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials. © 2010 Murayama et al.

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