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Biomedical subjects

K Ushida

Publications and source records attributed to K Ushida.

14 recordsLinked to original sources
PubMed2000

Effects of animal or plant protein diets on cecal fermentation in guinea pigs (Cavia porcellus), rats (Rattus norvegicus) and chicks (Gallus gallus domesticus).

Monogastric herbivores such as the guinea pig depend on energy supply from enteric fermentation as short-chain fatty acids (SCFA) corresponding to 30-40% of their maintenance energy requirements. They evolved specific digestive system to adapt their indigenous microflora to plant polysaccharides fermentation. No information has been available about the adaptability of microbial fermentation in hindgut of the monogastric herbivorous to an animal protein diet. We investigated if the guinea pig can fully retrieve energy of an animal protein diet by hindgut fermentation compared with a plant protein diet. For comparison, we also studied two omnivores. End products of in vitro cecal fermentation (SCFA, ammonia and gases) were measured to judge how well an animal protein diet could be fermented. The animal protein diet resulted in the less intensive fermentation with increased feed intake and volume of cecal contents than the plant protein diet only in guinea pigs. This may be due to a limited capacity of the hindgut microflora to adapt to the substrate rich in animal protein. We also found that chick cecal contents produced methane at higher emission rate than ruminants.

Animals↗
PubMed2000

Volatile sulfur production by pig cecal bacteria in batch culture and screening inhibitors of sulfate reducing bacteria.

We studied the effects of specific inhibitors of methanogenesis (2-bromoethane sulfonate, BES) and sulfate reduction (sodium molybdate) on volatile sulfur production in batch cultures of pig cecal bacteria. The volatile sulfur concentration in headspace gas was determined by flame-photometric detector gas chromatography. BES stimulated production of hydrogen sulfide (H2S) and methanethiol, and sodium molybdate completely inhibited the production of these volatile sulfur compounds. The results indicated that dissimilate sulfate reduction is mainly responsible for volatile sulfur production in the hindgut. Therefore the extracts of herbs, food colors, and aroma chemicals were tested for their inhibitory effects on H2S production by a dissimilatory sulfate-reducing bacteria. Desulfovibrio desulfuricans DSM642. H2S was measured by the chromatography of the headspace gas, using a flame photometric detector. Of 306 herbal extracts tested, 69 extracts from 38 herbs inhibited H2S production at 1.0 mg/mL. Sisymbrium officinale (hedge mustard) was the most potent inhibitor. Six pigments inhibited H2S release. Erythrosine and rose bengal showed inhibitory effects at 0.01 mg/mL. Peppermint oil and 96 aroma chemicals were assayed for their effects on H2S release. Thirty-two aroma chemicals suppressed H2S production at 0.1 mg/mL, and camphene, 1-decanol, and 2-nonanone were effective at 0.01 mg/mL.

Alkanesulfonic Acids↗
PubMed1999

Phylogenetic study of methanogens associated with rumen ciliates.

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii.

Animals↗
PubMed1999

Probiotic preparations dose-dependently increase net production rates of organic acids and decrease that of ammonia by pig cecal bacteria in batch culture.

We tested probiotic preparations containing Bifidobacterium, Enterococcus, or Lactobacillus to see if they affect production of organic acids and ammonia by mixed cecal bacteria. Four preparations (Bibalance, Neorakuton, Yakult Seichoyaku, or Yakult Seichoyaku BL) were digested with HC and pancreatin before adding to batch cultures of 50% pig cecal contents. Concentrations of organic acids and ammonia in the culture were quantified. Two preparations stimulated the net production of short-chain fatty acids (SCFA) during the first 8 hr of incubation. From 8 to 24 hr of incubation, all preparations accelerated the net production of SCFA, succinic and lactic acids, and three preparations slowed the net production of isovaleric acid and ammonia, all dose-dependently. The above results indicate that the preparations tested accelerated the breakdown of hard-to-degrade carbohydrate(s) and, possibly thereby, decreased the breakdown of proteinous materials or increased bacterial cell body synthesis in a mixed culture of cecal bacteria.

Acetic Acid↗
PubMed1998

Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3.

A cDNA clone encoding an endo-1,4-beta-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated. The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome. The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40 degrees C and pH 8.5.

Amino Acid Sequence↗
PubMed1996

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid.

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble 14C from 14C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoa population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and this effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.

Animals↗
PubMed1995

Sulphate reduction and methanogenesis in the ovine rumen and porcine caecum: a comparison of two microbial ecosystems.

A series of experiments was conducted to study the relationship between methanogenesis and sulphate reduction in ovine rumen and porcine caecum. Effect of 2-bromoethane sulphonate on hydrogen production by digesta suggested that the most important H2-disposal system in the rumen is methanogenesis and that methanogenesis is not predominant H2-disposal system in the porcine caecum. This inference was supported by the difference in predominant H2-utilizers in these two microbial ecosystems; Methanogenic bacteria (MB) were predominant in the rumen and sulphate reducing bacteria (SRB) were predominant in porcine caecum. Free sulphate levels in digesta appear to affect the relationship between MB and SRB. Sulphate levels in the rumen were likely to be insufficient for SRB to outcompete MB.

Animals↗
PubMed1990

Effect of defaunation on protein and fibre digestion in sheep fed on ammonia-treated straw-based diets with or without maize.

Using a defaunating method which preserved bacteria and fungi in the rumen, the effect of protozoa on protein and fibre digestion was studied in six adult wethers in relation to the nature of the diet. Sheep were given daily, 42 g dry matter (DM)/kg metabolic body-weight (W0.75), one of two isonitrogenous diets: one contained ammonia-treated wheat straw as the only energy source (diet S) and the other was supplemented with maize grain pellets (diet SM). Mean daily intakes (g/d) of nitrogen, neutral-detergent fibre and acid-detergent fibre were respectively 22, 573 and 373 for diet S and 23, 450 and 334 for diet SM. Elimination of protozoa increased duodenal non-ammonia-nitrogen flow. This result was mainly due to an increase in microbial protein flow and, to a lesser extent, to a higher dietary protein flow. Defaunation markedly increased the efficiency of microbial protein synthesis. Maize-grain supplementation had a net positive effect on this variable in defaunated sheep, but not in faunated sheep. Cell-wall carbohydrates were less well digested in the defaunated rumen, and the negative effect of defaunation was greatest with the diet SM. Intestinal fibre digestion increased in the defaunated sheep especially in those fed on diet SM, but not enough to compensate for the decrease in rumen digestion.

Ammonia↗
PubMed1986

Role of rumen protozoa in nitrogen digestion in sheep given two isonitrogenous diets.

1. The effect of protozoa on digestion in the rumen was studied using either defaunated or faunated sheep. 2. Six wethers, each fitted with rumen and simple duodenal cannulas, were given two isonitrogenous diets containing either lucerne (Medicago sativa) hay (diet L) or sodium hydroxide-treated wheat straw (diet S). The diets were given in eight equal portions per day at 3-h intervals. The mean intake of dry matter, 53 g/kg body-weight0.75 per d, was similar for the two diets and each diet had a similar digestible organic matter content. Diet L promoted a large protozoal population and was rich in nitrogen sources of low rumen-degradability, while diet S supported a smaller protozoal population and was rich in rumen-degradable N. 3. Digesta flow at the duodenum was estimated by means of a dual-marker technique using chromium-mordanted lucerne hay and polyethylene glycol as markers. The microbial flow at the duodenum was estimated using diaminopimelic acid (DAPA), nucleic-acid purine bases (PB) and 35S incorporation simultaneously. The different microbial markers were compared in the defaunated sheep. Protozoal N contribution was estimated in faunated sheep. 4. Defaunated sheep had lower rumen ammonia concentrations and molar proportions of butyric acid than faunated sheep, but they had higher molar proportions of propionic acid. 5. Rumen organic matter digestion was reduced by defaunation, but this decrease was compensated for by increased intestinal digestion. 6. There was a net increase of N flow (approximately 10 g/d) between mouth and duodenum in defaunated sheep. This was explained by increases in both microbial and dietary N flows from the rumen compared with faunated sheep. 7. The influence of protozoa on solid- and liquid-phase retention times in the rumen is discussed, as well as the protozoal contribution to microbial N flow in the duodenum of faunated sheep.

Animals↗
PubMed1985

Determination of assay parameters for RNA analysis in bacterial and duodenal samples by spectrophotometry. Influence of sample treatment and preservation.

A simple analytical procedure derived from that described by Zinn and Owens (1980), based on the determination of nucleic purine bases (RNA eq), was carried out to measure microbial nitrogen flow in the ruminant duodenum. Several procedures for sample preservation were tested; the efficiency of each step of the analytical method was also determined using high performance liquid chromatography (HPLC). The proposed method (RNA eq) was compared with two other methods considered as references (2-6 diaminopimelic acid and 35S incorporation) and microbial nitrogen flow was measured in defaunated sheep. The recovery of purine bases analysed by the Zinn and Owens method was generally good (101% pure bases; 90% when bases were added to bacterial samples; 96% when added to yeast RNA). The HPLC measurements allowed us to conclude that this spectrophotometric method is specific for purine bases, all pyrimidine bases being eliminated. Moreover, it was found that the method must be used on freeze-dried samples; storage at + 4 degrees C, defatting or freezing gave incorrect results. Using the described assay, we observed that microbial nitrogen flow at the duodenum of defaunated sheep was not significantly different from that obtained using more traditional markers such as sulphur-35 incorporation or diaminopimelic acid.

Adenine↗
PubMed1985

Effect of protozoa on rumen protein degradation in sheep.

We have studied the contribution of ciliate protozoa to the degradation of dietary protein, utilizing 5 defaunated and 6 faunated sheep. Rumen samples from these animals were used as inocular for in vitro determination of the degradability of different protein sources: lupine grain, peanut and soybean cake, and fish meal. We also applied the in sacco method to measure the digestibility of soybean proteins. Two pore sizes (50 and 100 microns) were used in the in sacco study. The nitrogen degradation curve was constructed according to the mathematical model: P(t) = a + b (1-e-ct), and the parameters a, b, and c were determined for both faunated and defaunated animals. The proteolytic activity of the rumen contents was also determined using azocasein. The in vitro degradability of proteins was significantly less in defaunated than in faunated animals: 17, 19, 28 and 64% with lupine grain, peanut cake, soybean cake and fish meal proteins, respectively. The protozoal effect was greater when protein solubility was low. The in sacco study showed that neither the protozoa nor bag pore size had any effect on the protein fraction (a) immediately soluble in the rumen juice. However, both factors increased the insoluble, potentially degradable fraction "b" (P less than or equal to .01). Protozoa had a positive effect on the rate of degradation (c) of the fraction b. This effect was most clear with the 100 micron bags. The significant interaction between protozoa and pore size on factor "c" showed that large ciliate protozoa (100 microns) were directly involved in determining this parameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗