Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.     

Sequence of the domestic duck (Anas platyrhynchos) growth hormone-encoding gene and genetic variation in the promoter region

The duck growth hormone encoding gene and its promoter region were amplified by polymerase chain reaction (PCR). A total of 5.25 kb were cloned and sequenced. Duck growth hormone (GH) consists of five exons and four introns and is structurally similar to mammalian and chicken GH gene. Although the distal region of duck GH promoter showed no similarity to chicken and turkey promoters, the proximal region of the promoter contained two putative Pit‐1 binding sequences, and showed similarity to chicken and turkey GH promoters. Genetic variation was detected at five positions of the promoter region. The results of this study indicate that the expression of duck GH is likely regulated in a similar manner to that of chicken GH via enhancer‐type cis‐acting elements and the presence of genetic variation in the duck GH gene may be applicable to marker‐assisted selection.     

Space-occupying leiomyoma in the pelvic canal of a dog

A large space-occupying mass was found in the pelvic canal of an 11-year-old, entire female shih tzu with rectal tenesmus and dyschezia. Computed tomography revealed an extraluminal mass located on the dorsal aspect of the rectum, causing complete rectal obstruction. Histopathological examination of biopsy tissue identified it as a leiomyoma. The tumour was successfully removed by blunt dissection, via a dorsal approach to the rectum, and there were no complications after surgery.     

Fixation to the canine bone of artificial implant with new surface structure

Screw and laser (SL) column by making screw threads and forming small holes using laser irradiation on the base metal and conventional beads coating (BC) columns were embedded into the shaft of canine femurs, and compared the implant fixation to the host bone. The interfacial strength in SL columns was almost equivalent as BC columns, and bone-column contact rate was higher than BC columns significantly at twelve weeks after implantation. The newly devised SL surface had almost equivalent bone fixation strength comparable to the conventional BC surface. Also, this surface should provide a useful porous surface for use in artificial joints since there is no risk of surface structure detachment.     

Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.     

Effects of different application timings of methane fermentation digested liquid to paddy plots on soil nitrogen and rice yield

Treatment of animal and food wastes using a methane fermentation technique is drawing considerable public attention as a suitable option for the utilization of biomass resources. The application of a fermentation byproduct (methane fermentation digested liquid) as an agricultural fertilizer has been investigated. Determining the appropriate timing required for applying digested liquid on a rice (Oryza sativa L.) paddy plot is important. The concentrations of soil nitrogen (N) components and rice yield should be considered because digested liquid contains both inorganic and organic N. This study compares the N transformation and the rice yield and growth at different application times over a period of 3 y. The effects of the timing of basal application on soil N were different and the timing that maximized the rice yield was different in each year. Days before ponding (DBP) affected soil N before mid-summer drainage, and rice growth rates at the panicle formation stage and the ear emergence stage. The effects of DBP disappeared before harvest. The results indicated that sufficient potentially mineralizable N existed regardless of DBP, and the effect of DBP lessened after the mid-summer drainage, which coincides with the period when N uptake is most active.     

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.     

Comparison of environmental conditions in two representative oyster farming areas: Hiroshima Bay,western Japan and Oginoham a Bay (a branch of Ishinomaki Bay), northern Japan

ABSTRACT:   Sea water environmental conditions over annual cycles were investigated and compared between two oyster farming areas, western Hiroshima Bay and Oginohama Bay (a branch of Ishinomaki Bay) in Miyagi Prefecture, to appropriately manage oyster culture or more efficiently utilize farming areas. The environmental parameters of temperature, salinity, nutrient concentrations (NO₂–N, NO₃–N, NH₄–N, PO₄–P, and SiO₂–Si) and size-fractionated chlorophyll- a (<0.2, 2–20, >20µm), and abundances of microzooplankton were measured in each bay at the surface, and 2 and 5 m depth layers. Differences in the annual mean values and results with monthly paired Student's t -tests showed that salinity was lower, and temperature, nutrient (especially PO₄–P) and chlorophyll- a concentrations, and abundance of microzooplankton, were higher in Hiroshima Bay than in Oginohama Bay. Differences in environmental conditions between inshore and offshore areas of each bay suggest that inflows of river water in western Hiroshima Bay and sea water from offshore had the most significant effects on the environmental conditions. It is concluded that such oceanographic and biological differences strongly affect the oyster farming system, especially regarding the optimum usage of offshore areas in Summer under clean, cold and stable seawater conditions, rather than food quantity in Hiroshima Bay, and under more abundant food conditions in Oginohama Bay.