Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.     

Reproductive cycle observation of the Okinawa rail (Gallirallus okinawae) in the wild

The captive breeding program of the Okinawa rail started in 2008. For successful captive breeding, information related to reproduction, such as age at sexual maturity, testicular cycles and ovulatory cycles, is essential to predict when reproduction is possible and when certain reproductive behaviors are most likely to occur. We made gross and histological observations of the reproductive organs of Okinawa rails to gain understanding of sexual maturity, the testicular cycle and the ovulatory cycle. We found that the weight of the testis was smallest in December and largest in March. Changes in the diameter of the seminiferous tubule showed the same pattern. Mature sperm were observed from March to June. The heaviest ovary was observed in April. A single peak of reproduction, from March to April, was observed in males and females. Our observations suggested that the Okinawa rail is a seasonal breeder. Establishing suitable breeding pairs will be critical to ensure success of the Okinawa rail captive breeding program. Our results suggested that pairing must be started before March. If supportive breeding is used, semen should be collected from March to June and artificial insemination conducted in April.     

Prevalence of equine herpesvirus type 1 strains of neuropathogenic genotype in a major breeding area of Japan

A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were induced by ORF30 G(2254) strains. This prevalence is lower than those observed in the U.S.A. (10.8-19.4%), Argentina (7.4%), France (24%), and Germany (10.6%).     

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.     

Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells

This study attempted to establish a culture model to recreate the milk production pathway in bovine mammary epithelial cells (BMECs). BMECs were isolated from Holstein cows (nonlactating, nonpregnant, and parous) and were stored by cryopreservation. To separate the apical and basolateral compartments, BMECs were cultured on a cell culture insert with a collagen gel in the presence of bovine pituitary extract and dexamethasone to induce milk production and tight junction (TJ) formation. The culture model showed the secretion of the major milk components, such as β‐casein, lactose, and triglyceride, and formed less‐permeable TJs in BMECs. Moreover, the TJs were distinctly separated from the apical and basolateral membranes. Glucose transporter‐1, which transports glucose into the cytoplasm through the basolateral membrane, localized in the lateral membrane of BMECs. Toll‐like receptor‐4, which binds to lipopolysaccharide in the alveolar lumen in mastitis, localized in the apical membrane. Beta‐casein was mainly localized near the Golgi apparatus and the apical membrane. Moreover, milk components were almost secreted into the upper chamber of the cell culture insert. These findings indicate that this model has clear cell polarity as well as in vivo and is effective to study of milk production and the blood–milk barrier in lactating BMECs.     

Selective determination and formation of elemental selenium in soils

A selective method for the determination of elemental selenium in soil was developed and was applied to the study of elemental selenium in soil. (1) Elemental selenium extracted with carbon disulfide from soil was selectively transformed into selenocyanate ion by reacting with potassium cyanide in carbon disulfide. The selenocyanate ion formed was recovered into an aqueous solution and the amount of selenium in the aqueous solution was determined. This method was specific to elemental selenium and did not interfere with the other selenium compounds and soil components. The method was also highly sensitive and enable to determine more than 0.1 μg kg^-1 of elemental selenium in soil. (2) The formation of elemental selenium was confirmed, when a soil was submerged and the redox potential of the soil decreased. The amount of elemental selenium formed was proportional to the selenite content of the soil, indicating that elemental selenium is transformed from selenite upon its reduction.     

Biosafety assessment of transgenic poplars overexpressing xyloglucanase (AaXEG2) prior to field trials

We performed biosafety assessments of transgenic poplars prior to field trials. Constitutive expression of the Aspergillus aculeatus xyloglucanase in Populus alba increased the cellulose content and specific gravity of its stem, the leaves of which were visibly greener, thicker, and smaller than those of the wild-type plant. Although the young transgenic poplars grew faster than the wild type in a growth chamber, there was no distinguishable difference in growth between the poplars when they were placed in a special screened greenhouse. Allelopathic tests showed that the transgenic poplars do not produce harmful substances. Based on all the biosafety assessments and the scientific literature on poplar species, we came to the conclusion that transgenic poplars probably do not disturb the biological diversity of the surrounding environment, even when they are submitted to field trials.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples

The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high‐risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.