In Vitro Maturation and Development of Porcine Oocytes Cultured in a Straw or Dish Using a Portable Incubator with a CO2 Chamber

We investigated the effects of a portable incubator with a CO₂ chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus‐oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO₂ incubator, in the CO₂ chamber in an incubator, or in the CO₂ chamber in a portable incubator. The matured oocytes were fertilized with frozen‐thawed spermatozoa and then cultured in a dish in the standard CO₂ incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO₂ incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO₂ incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO₂ chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.     

Contribution of different proton sources to pedogenetic soil acidification in forested ecosystems in Japan

The contribution of different proton sources to pedogenetic soil acidification was evaluated for three Japanese forest soils, i.e. ando soil, podzolic soil and brown forest soil in relation to the respective soil forming processes. Soil acidification rate and net proton generation were quantified based on the theory of proton budget for the respective soil horizon compartments (mainly the O, A and B horizons) by measuring fluxes of solutes entering and leaving the soil horizon compartment and vegetation uptake. Protons were produced by the dissociation of organic acids and nitrification in the O horizon and then consumed by adsorption and decomposition of organic acids and nitrate uptake by vegetation in deeper soil horizons at all plots. Excess uptake of cation over anion by vegetation was highest among proton sources in the whole soil compartment at all plots. Pedogenetic soil acidification was considered to include cation leaching from surface soil horizons due to proton generation by the dissociation of organic acids and nitrification and subsequent cation excess accumulation in wood in the growth stage of forests. In ando soil, andosolization resulted from the low contribution of net proton generation by the dissociation of organic acids as well as a lower soil acidification rate and complete acid neutralization. Dissolved organic carbon (DOC) fluxes in ando soil were lower than those in podzolic soil and brown forest soil due to high adsorption capacity of amorphous materials. In podzolic soil, podzolization resulted from intensive acidification in the O horizon, which derived from net proton generation by the dissociation of organic acids and nitrification as well as cation excess uptake by vegetation due to concentrated fine root biomass in the O horizon, and subsequent high proton efflux to subsoil. The high fluxes of DOC and Al leached from surface soil horizons were considered to contribute to eluviation of Al from surface soil and illuviation in subsoil in podzolic soil. In brown forest soil, brunification resulted from a lower DOC flux from the O horizon due to high decomposition and adsorption by oxides, where podzolization was weakened by high acid neutralization. Thus, the three representative processes involved in the pedogenesis of Japanese forest soils were well characterized by quantification of the respective proton-generating and consuming processes in each soil horizon.     

Selection of antigenic variants of the S glycoprotein of feline infectious peritonitis virus and analysis of antigenic sites involved in neutralization.

The type II feline infectious peritonitis virus (FIPV) epitopes for neutralizing and enhancing antibodies are present on large spike glycoprotein (S) protein. In this study, we established monoclonal antibody-resistant mutant viruses resistant to three different monoclonal antibodies with neutralizing activity in Felis catus whole fetus cells and enhancing activity in feline macrophages, recognizing distinct epitopes on type II FIPV S protein. By comparing the nucleotide sequences of these mutant viruses with that of wild-type virus, we attempted to identify the neutralizing epitopes. The mutations were localized in the region of amino acid residues from 480 to 649 from the N terminal of the S protein.     

PCR-DGGE analysis of bacterial communities in <Emphasis Type="Italic">funazushi</Emphasis>, fermented crucian carp with rice,during fermentation

Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan. It is characterized by a unique cheese-like flavor and characteristic sour taste. We analyzed the changes in the microbial community during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCR-DGGE) and by plate counts. The plate counts showed that lactic acid bacteria reached 8.0 log₁₀ CFU/g within 7 days of fermentation initiation before decreasing slowly to 4.0 log₁₀ CFU/g during the remainder of 1-year study period. PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and 30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L. acetotolerans. This is the first identification of L. acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive. This is the first report of PCR-DGGE being used to assess the microbial community in funazushi. This technique was also found to be effective in profiling microbial diversity.     

Effect of tributyltin oxide exposure on the F<Subscript>0</Subscript> and F<Subscript>1</Subscript> generations of a marine teleost,the mummichog <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>

The effects of organotin compounds on marine organisms, especially on their reproduction, are a continuing source of concern. We performed full-life-cycle exposure tests of tributyltin oxide (TBTO) at sub-lethal concentrations (0.13, 0.25, 0.5 and 1.0 μg/l) using a marine fish, the mummichog Fundulus heteroclitus, and studied the toxic effects of exposure to TBTO at various life-cycle stages of the fish. The effect of TBTO was initially studied on sex differentiation and then on spermatogenesis in maturing fish. A mating experiment was performed to determine the effects of exposure on the fecundity of female fish, fertilization success, and the hatch of the F₁ generation. In all experimental groups, TBTO exposure resulted in a tendency toward a male-biased sex ratio and caused a significant increase in the frequency at which apoptotic cells appeared in the testes. Exposure to 1.0 μg/l TBTO resulted in a significant decrease in fecundity, but no significant effect was found on the fertilization ratio. Time to hatch and hatchability of the F₁ generation were markedly affected in some exposure groups. These results indicate that long-term exposure of the mummichog to TBTO affects not only gonadal sex differentiation and spermatogenesis but also spawning and egg quality.     

Quantitative structure–fungicidal activity relationships of N-(4-difluoromethoxybenzyl)­pyrimidin-4-amines against wheat and barley fungi

A set of N-(4-difluoromethoxybenzyl)pyrimidinamines with various substituents at the 4- and 5-positions of the pyrimidine ring and at the benzyl position were prepared, and their fungicidal activities against wheat brown rust, Puccinia recondita, and barley powdery mildew, Erisiphe graminis, were measured. Variations in each of these activities were quantitatively analysed by the use of physicochemical substituent parameters and a regression analysis. Each of these activities was parabolically correlated with the steric bulkiness of the pyrimidine substituents and with the bulkiness or the hydrophobicity of the benzylic substituents. © 1999 Society of Chemical Industry     

Biological effects of leptospiral lipopolysaccharide to mouse B, T and NK cells

Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovars copenhageni and hebdomadis were tested for the biological effect to mouse B, T and NK cells. Each leptospiral LPS was a potent mitogen for spleen B cells. Activation of the cells was also expressed by polyclonal B cell activation. In contrast, mitogenicity for T cells, induction of interleukin-2 (IL-2) secretion in T cells and increase of tumor-killing activity and chemiluminescence in NK cells were not observed after stimulation with leptospiral LPS. After intravenous injection of leptospiral LPS in mice, the spleen and lymphnodes were examined by histocytochemical technique. Increase of Ig-bearing lymphocytes was recognized while decrease of T cells was observed in the lymphoid organs. Mitogenic response to PHA, Con A and PWM decreased with relation to the T cell depletion. In conclusion, it is apparent that leptospiral LPS possess marked immunological potencies on B cells but not T and NK cells. The biological effects of leptospiral LPS were common ones as LPS but the level was considered to be different from classical LPS such as Escherichia coli LPS.     

Isolation of lymphocytic choriomeningitis virus from wild house mice (Mus musculus) in Osaka Port, Japan.

Lymphocytic choriomeningitis virus (LCMV) was isolated from 14 out of 35 wild house mouse samples captured on two piers of Osaka port in Japan. Four of them were isolated from were isolated from 18 antibody positive mice while 10 were from 17 antibody negative mice. This is the first report of the isolation of LCMV from wild mice in Japan.     

Effects of climatic and soil properties on cellulose decomposition rates in temperate and tropical forests

Cellulose decomposition experiments were conducted under field conditions to analyze the effects of climatic and soil properties on rates of organic matter decomposition in temperate and tropical forests. The mass loss rates of cellulose filter papers buried in the soil surface were measured to estimate the respiratory C fluxes caused by cellulose decomposition and mean residence time (MRT) of cellulose. The rates of cellulose decomposition increased with soil temperature, except for during the dry season, while rate constants of decomposition (normalized for temperature) decreased with decreasing pH because of lower cellulase activity. The estimated MRTs of soil cellulosic carbohydrates varied from 81 to 495 days for the temperate forests and from 31 to 61 days for the tropical forests. As a major organic substrate, the C fluxes from cellulose decomposition can account for a substantial fraction of heterotrophic (basal) soil respiration. However, the respiratory C fluxes can be limited by the low substrate availability and low pH in tropical soils, despite high microbial activity. The rate-regulating factors of cellulose decomposition, i.e., temperature, soil pH, and substrate availability, can accordingly influence the rates of heterotrophic soil respiration.