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.     

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 10⁵ cells/cm² (high‐density group) or 0.8 × 10⁴ cells/cm² (low‐density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high‐density group and with two DC pulses of 25 V/150 µm for 10 µs in the low‐density group. The fusion rate (37.5%) in the high‐density group was significantly (P < 0.005) lower than in the low‐density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high‐density and low‐density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.     

A Case of Feline Gastrointestinal Eosinophilic Sclerosing Fibroplasia

Feline gastrointestinal eosinophilic sclerosing fibroplasia was diagnosed in an 8-month-old Scottish fold that had a primary gastrointestinal mass involving the stomach, duodenum and mesenteric lymph nodes. Histopathologically, the most characteristic feature of this mass was granulation tissue with eosinophil infiltration and hyperplasia of sclerosing collagen fiber. Immunohistochemically, large spindle-shaped cells were positive for smooth muscle actin and vimentin. This case emphasizes the importance of feline gastrointestinal eosinophilic sclerosing fibroplasia as a differential diagnosis of gastrointestinal neoplastic lesions such as osteosarcoma and mast cell tumor in cats.     

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.     

X-ray fluorescence spectrometry of asteroid Itokawa by Hayabusa

X-ray fluorescence spectrometry of asteroid 25143 Itokawa was performed by the x-ray spectrometer onboard Hayabusa during the first touchdown on 19 November 2005. We selected those data observed during relatively enhanced solar activity and determined average elemental mass ratios of Mg/Si = 0.78 +/- 0.09 and Al/Si = 0.07 +/- 0.03. Our preliminary results suggest that Itokawa has a composition consistent with that of ordinary chondrites, but primitive achondrites cannot be ruled out. Among ordinary chondrites, LL- or L-chondrites appear to be more likely than H-chondrites. No substantial regional difference was found on the asteroid surface, indicating its homogeneity in composition.     

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.     

Severe nonpurulent encephalitis with mortality and feather lesions in call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with H5N1 highly pathogenic avian influenza virus

One-day-old, 2-wk-old, and 4-wk-old call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with the H5N1 highly pathogenic avian influenza virus A/chicken/Yamaguchi/7/2004 isolate (Ck/Yama/7/04) were examined clinically, pathologically, and virologically. Clinically, the birds exhibited mild-to-severe neurologic signs and corneal opacity. All birds in the 1-day-old group and one bird in the 4-wk-old group died within 4 days after the virus inoculation. Histologic changes were characterized by severe nonpurulent encephalitis and necrotic lesions of feather epithelium on day 3 postinoculation (PI) or later. Focal necrosis of myocardial cells, pancreatic acinar cells, skeletal myocytes, and corneal epithelial cells was observed. Viral antigens were detected in association with necrotic changes. Viruses were isolated from all examined organs including the skin with many feathers. Serum antibody against the virus was detected in all surviving birds on day 10 PI by hemagglutination-inhibition tests. These results suggest that Ck/Yama/7/04 has a pathogenicity that causes neurologic sign, nonpurulent encephalitis with mortality, and feather lesions for call ducks. Feather lesions with viral antigens and the virus isolation from the skin suggest that Ck/Yama/ 7/04 has a predilection for feathers in call ducks.     

Biogenic potassium salt particles as seeds for secondary organic aerosol in the Amazon

The fine particles serving as cloud condensation nuclei in pristine Amazonian rainforest air consist mostly of secondary organic aerosol. Their origin is enigmatic, however, because new particle formation in the atmosphere is not observed. Here, we show that the growth of organic aerosol particles can be initiated by potassium-salt-rich particles emitted by biota in the rainforest. These particles act as seeds for the condensation of low- or semi-volatile organic compounds from the atmospheric gas phase or multiphase oxidation of isoprene and terpenes. Our findings suggest that the primary emission of biogenic salt particles directly influences the number concentration of cloud condensation nuclei and affects the microphysics of cloud formation and precipitation over the rainforest.     

Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture

In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation. It was IFN-tau that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.     

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.