Factors stimulating migration of holotrich protozoa into the rumen.

The effects of feeding and various reticular infusions on ruminal holotrich concentrations were studied in an attempt to identify possible factors stimulating their migration into the rumen. It was concluded that glucose entering the reticulo-rumen shortly after feeding could stimulate migration of holotrich protozoa.     

Interactions between the methanogen Methanosarcina barkeri and rumen holotrich ciliate protozoa

Interspecies hydrogen transfer between rumen holotrich ciliate protoza and methanogenic bacteria has been demonstrated. As a result of the metabolic interaction with Methanosarcina barkeri , the metabolite profile of Isotricha spp. was altered and the production of butyrate and lactate was suppressed in the presence of the methanogen.
Use of membrane-inlet mass spectrometry confirmed that the presence of rumen holotrich ciliates reduced the apparent sensitivity of methanogenesis to the inhibitory effects of oxygen; a gas phase concentration of 7·4 kPa oxygen was required to inhibit methanogenesis in the presence of protozoa, while in pure cultures of M. barkeri , methanogenesis was inhibited by a gas phase oxygen concentration of 1·0 kPa.     

An improved technique for the isolation of holotrich protozoa from rumen contents by differential filtration with defined aperture textiles

An improved differential filtration method for the isolation of preparations of the holotrich protozoa Dasytricha ruminantium and mixed Isotricha prostoma and I. intestinalis from rumen contents is described. The technique utilizes combinations of defined aperture Nylon or polyester textiles, which are superior to the sintered glass filters previously used in the isolation of rumen protozoa. Data obtained in a comparative study of some aspects of the metabolic activity exhibited by cell preparations isolated by the two filtration procedures are presented.     

Electron microprobe analysis of zinc incorporation into rumen protozoa

With the aid of electron microprobe analysis on ciliate spreads, we detected zinc in ciliates and its accumulation in the endoplasm. A correlation was found between the amount of zinc accumulation and its concentration in the medium. By the same microprobe analysis of of ultrathin sections, we determined semiquantitatively the zinc accumulation in the intracytoplasmic granules and its presence in macronuclei and in intra- and extracellular bacteria.     

Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium) showed that both organisms have an efficient O2-scavenging capability (apparent Km values 2.3 and 0.3 microM, respectively). Reversible inhibition of H2 production increased almost linearly with increasing O2 up to 1.5 microM; higher levels of O2 gave irreversible inhibition. In situ determinations of H2, CH4, O2 and CO2 in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O2 was present before feeding at 1-1.5 microM and decreased to undetectable levels (less than 0.25 microM) within 25 min after feeding. A transient increase in O2 concentration after feeding occurred only in defaunated animals and resulted in suppression of CH4 and CO2 production. The presence of washed holotrich protozoa decreases the O2 sensitivity of CH4 production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri. It is concluded that holotrich protozoa play a role in ruminal O2 utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H2 utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O2 levels, and are themselves regulated by the low ambient O2 concentrations they experience in the rumen.     

Some factors controlling the attachment of the rumen holotrich protozoa Isotricha intestinalis and I. prostoma to plant particles in vitro.

The rumen holotrich protozoa Isotricha intestinalis and I. prostoma showed chemotaxis to sucrose, glucose and fructose. They attached themselves, by means of an organelle on the anterior cell surface, to particulate sources of these carbohydrates provided soluble protein was present in the medium. The concentration of protein eliciting attachment varied with the species and the state of nutrition of the cell, but was between 20 and 150 microgram ml-1. Attachment occurred only if the concentration of carbohydrate, at its source, exceeded the chemotaxis threshold concentration (50 micrometer for sucrose) and if it was less than 1 mM. At concentrations exceeding 1 mM, indiscriminate attachment to gas-liquid and solid-liquid interfaces occurred, provided the protein concentration was high enough to elicit attachment. In the rumen, soluble carbohydrates diffusing from food particles may attract the protozoa which attach themselves to the particles in the presence of soluble plant protein at less than 20 microgram ml-1; these conditions exist in the host animal soon after feeding when fed infrequently. The attachment mechanism may confer an ecological advantage on the Isotricha spp. over other rumen organisms dependent on soluble carbohydrates as energy and carbon sources.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

The aminoethylphosphonate-containing lipids of rumen protozoa

1. A method is presented for identifying and estimating the aminoethylphosphonate (ciliatine)-containing phospholipids in a complex mixture. 2. Evidence was obtained that the phospholipids of a pure culture of Entodinium caudatum and a mixed rumen protozoa sample contain diglyceride ciliatine, and a plasmalogen ciliatine was detected in the latter. 3. A ninhydrin-positive sphingolipid was isolated from rumen protozoa. Although chromatographically homogeneous on silica gel it contains two components, which were provisionally identified as ceramide ciliatine and ceramide phosphorylethanolamine. 4. A detailed phospholipid analysis of E. caudatum and rumen protozoa is presented. They contain no phosphatidylserine or cardiolipin, but an unidentified phosphoglyceride containing a zwitterionic amino acid is present.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Association of methanogenic bacteria with rumen protozoa

Methanogenic bacteria superficially associated with rumen entodiniomorphid protozoa were observed by fluorescence microscopy. A protozoal suspension separated from strained rumen fluid (SRF) by gravity sedimentation exhibited a rate of methane production six times greater (per millilitre) than SRF. The number of protozoa (per millilitre) in the protozoal suspension was three times greater than that of SRF; however, the urease activity of this fraction was half that of SRF. The methanogenic activity of SRF and the discrete fractions obtained by sedimentation of protozoa correlated with the numbers of protozoa per millilitre in each fraction. Gravity-sedimented protozoa, washed four times with cell-free rumen fluid, retained 67-71% of the recoverable methanogenic activity. Thus it is evident from our observations that many methanogens adhere to protozoa and that the protozoa support methanogenic activity of the attached methanogens. When protozoa-free sheep were inoculated with rumen contents containing a complex population of protozoa, methanogenic activity of the microflora in SRF samples was not significantly enhanced.