Diurnal changes in concentration of rumen ciliates and in occurrence of dividing forms in water buffalo (Bubalus bubalus) fed once daily.

When buffalo were fed once daily, significant diurnal variations in concentration of rumen ciliates and occurrence of dividing protozoa were found. Differences in proportions of dividing Entodinium- and Diplodinium-type ciliates were also observed. Results obtained suggest that the range of diurnal fluctuations in rumen protozoa concentration may be related to the percentage of dividing cells in populaitons of these organisms.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.     

Mureinolytic ability of the rumen ciliate <Emphasis Type="Italic">Diploplastron affine</Emphasis>

Rumen ciliate protozoa intensively engulf bacteria. However, their ability to utilize murein which is the main polysaccharide of bacterial cell wall has hardly been recognized. The present study concerns the ability of the rumen protozoa Diploplastron affine to digest and ferment murein. The ciliates were isolated from the rumen fluid and grown in vitro or inoculated into the rumen of defaunated sheep. The results of long-term cultivation of protozoa showed a positive correlation between their number and murein content in the culture medium. It was also found that bacteria-free D. affine ciliates incubated with or without murein produced volatile fatty acids at the rate of 12.3 and 8.7 pmol/h per protozoan, respectively, acetic, butyric and propionic acids being the three main acids released to the medium. Enzyme studies performed with the use of protozoan cell extract prepared from bacteria-free ciliates degraded murein at a rate of 25 U/mg protein per h; two mureinolytic enzymes were identified by zymographic technique in the examined preparation.     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.     

Diurnal changes and effect of ration on concentrations of the rumen ciliate Charon ventriculi.

Charon ventriculi comprised over 30% of the total protozoa observed in rumen contents of a Flamenga cow fed Rhodes grass hay (Chloris gayana). Both percentage of composition and concentration decreased markedly when concentrate was added to the ration or the animal was fed in pasture. Although C. ventriculi is classified as a holotrich, concentrations of this species in the rumen appear to follow a diurnal cycle more closely related to be entodiniomorph protozoa.     

Occurrence of the Ciliate Protozoa Bütschlia parva Schuberg in the Rumen of the Ovine

The holotrich ciliate protozoa, Bütschlia parva Schuberg, has been observed in the rumen of the ovine. Limited data suggest that the concentration of B. parva in the rumen follows a diurnal cycle similar to that of the other holotrichs.     

Establishment of ciliate protozoa in the rumen of conventional and conventionalized lambs: influence of diet and management conditions

The establishment of ciliate protozoa in the rumen was studied in conventional lambs reared under different conditions of management. The role of the microflora in the kinetics of this establishment was also investigated in conventionalized lambs. In lambs reared under farm conditions ciliate protozoa appeared in the following order: Entodinium (15-20 days), Polyplastron, Eudiplodinium, and Epidinium (20-25 days), and Isotricha (50 days). Entodinium was the most abundant (10(5)-10(6) ciliates mL-1). During the 3rd month, ciliates disappeared spontaneously in about 60% of the lambs during a period that varied from 1 to 4 weeks. In lambs fed only cow's milk Entodinium spp. and Polyplastron multivesiculatum became established at low levels. The results obtained with the conventionalized lambs demonstrate that the establishment of the ciliates in the rumen requires that the bacterial flora be well established beforehand.     

Formation of Lysine from, α, ε-Diaminopimelic Acid Contained in Rumen Bacterial Cell Walls by Rumen Ciliate Protozoa

Cell walls containing α,ε-diaminopimelate-l,7-¹⁴C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.

From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

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.     

Metabolism of Threonine,Methionine, and Related Compounds in Mixed Rumen Ciliate Protozoa1

Metabolic pathways of L-threonine and L-methionine in starved, bacteria-free, mixed rumen ciliate protozoa were examined. Rumen ciliates produced 2-oxobutanoate (2OB) from both L-threonine and L-methionine; 2OB was then converted to 2-aminobutanoate (2AB) and propionate. The 2AB appeared to be converted slowly to propionate. D-Threonine seemed to be hardly metabolized.     

Effects of Changes in Feed Level, Starvation, and Level of Feed After Starvation Upon the Concentration of Rumen Protozoa in the Ovine

Four rumen fistulated sheep were used in five experiments to investigate the effect of feed level upon the concentration of rumen ciliate protozoa. The sheep were fed once daily 650 g of a pelleted diet composed of corn cobs, 45%; alfalfa meal, 35%; oats, 12.5%; cane molasses, 5%; urea, 0.4%; and vitamins and minerals, 2%. The concentration of protozoa reached minimum and maximum values at 5 and 22.5 h after feeding, respectively. Thus, to estimate apparent generation rates, concentrations of protozoa were determined at 5 and 20 h postfeeding. Apparent generation rate/h = natural log of ([concentration of protozoa at 20 h divided by concentration at 5 h] divided by the time interval, [T20 to T5]). Alteration of the feed to protozoa ratio by starvation and by changing the level of feed (200 to 900 g/day) showed that as the ratio of feed to protozoa increased, generation rate increased. Measurements of liquid turnover rates in the rumen showed that turnover rate decreased as feed level decreased. Turnover rate was near zero when the sheep were starved. Small quantities of soluble substrates, added directly to the rumen of starved sheep, maintained the protozoal population when rumen turnover was minimal. Furthermore, as rumen turnover rate increased with increased levels of feed, the effect of substrate on maintaining the protozoal population was negated. Thus, at high feed levels, turnover rate may be the dominant factor controlling the establishment and concentration of protozoa in the rumen.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Influence of Host Diet on the Fatty Acid Composition and Content of Rumen Protozoa in Cattle

The fatty acid profiles and contents of protozoa from the rumen fluid of cattle varied according to the type of diet consumed by their host. Changing from a high-quality hay diet to a low-quality hay diet (DA) decreased the proportions of saturated acids and increased the proportions of the unsaturated acids 18:1 cis-9, 18:2 and 18:3 in neutral lipids (NL) and phospholipids (PL). Adding sucrose, urea and sulphur (SUS) to DA increased the proportions of branched chain acids in PL while addition of safflower oil increased polyunsaturated acids in PL and 18:1 trans-11 in NL. Diet did not alter the PL fatty acid content of protozoa but oil supplement of DA resulted in a 10-fold increase in the content of free fatty acids. The defaunating effect of oil supplement was partly reversed by SUS suggesting that factors other than the fatty acid content of cells are important in determining the toxicity of oil to rumen protozoa. The results indicate that the amounts of individual long-chain fatty acids taken up by rumen ciliates are largely determined by their concentrations in rumen digesta.     

Influence of host diet on the fatty acid composition and content of rumen protozoa in cattle

The fatty acid profiles and contents of protozoa from the rumen fluid of cattle varied according to the type of diet consumed by their host. Changing from a high-quality hay diet to a low-quality hay diet (DA) decreased the proportions of saturated acids and increased the proportions of the unsaturated acids 18:1 cis-9, 18:2 and 18:3 in neutral lipids (NL) and phospholipids (PL). Adding sucrose, urea and sulphur (SUS) to DA increased the proportions of branched chain acids in PL while addition of safflower oil increased polyunsaturated acids in PL and 18:1 trans-11 in NL. Diet did not alter the PL fatty acid content of protozoa but oil supplement of DA resulted in a 10-fold increase in the content of free fatty acids. The defaunating effect of oil supplement was partly reversed by SUS suggesting that factors other than the fatty acid content of cells are important in determining the toxicity of oil to rumen protozoa. The results indicate that the amounts of individual long-chain fatty acids taken up by rumen ciliates are largely determined by their concentrations in rumen digesta.     

Rumen ophryoscolecid protozoa in the hindgut of the capybara (Hydrochoerus hydrochaeris)

Rumen ophryoscolecid protozoa were observed in feces obtained from two capybara (Hydrochoerus hydrochaeris) housed at the Columbus Zoo, Columbus, Ohio. Total numbers were 58.1 X 10(4) and 19.0 X 10(4) per gram of wet feces in a male and female capybara, respectively. Four common rumen species of Entodinium were observed in the feces from both animals, with low numbers of Eudiplodinium maggii and Elytroplastron bubali also occurring in the male. Establishment of rumen ophryoscolecid ciliates in the intestinal tract of non-ruminant herbivores has not been reported previously.     

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; -l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.     

Hydrolysis of Fraction 1 leaf protein and casein by rumen entodiniomorphid protozoa

Cell-free extracts of fourteen individual species of rumen ciliate protozoa and of mixed rumen ciliates degraded Fraction 1 leaf protein. For Entodinium caudatum and Eudiplodinium maggii the optimum pH was 3·2. The maximum rates of proteolysis (in µmol acid soluble-tyrosine formed/mg protein/h) were 0·16 to 5·7 with protozoa grown in vivo and 0·38 to 6·4 with protozoa grown in vitro. The highest rates were obtained with Entodinium caudatum and E. simplex and the lowest with the cellulolytic species grown in vivo. K _m values (mg/ml) ranged from 0·42 to 19 with protozoa grown in vivo and 0·35 to 13·3 with protozoa grown in vitro. All single species (with one exception) whether grown in vivo or in vitro degraded Fraction 1 leaf protein faster (1·4 to 21 times) than casein. Partial inhibition of the activity of Entodinium caudatum was obtained with pepstatin and N-ethylmaleimide and almost complete inhibition with leupeptin suggesting the presence of 'carboxyl' and 'thiol' enzymes.     

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-D: -glucosamine and chitobiose. Three exochitinases, two endochitinases and two beta-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified beta-N-acetylglucosaminidases was 45 kDa.     

Xylanases and carboxymethylcellulases of the rumen protozoa Polyplastron multivesiculatum, Eudiplodinium maggii and Entodinium sp

Endoglucanase and xylanase activities of three rumen protozoa, Polyplastron multivesiculatum, Eudiplodinium maggii, and Entodinium sp. were compared qualitatively by zymograms and quantitatively by measuring specific activities against different polysaccharides. A set of carboxymethylcellulases and xylanases was produced by the large ciliates whereas no band of activity was observed for Entodinium sp. in zymograms. Specific activity of endoglucanases from P. multivesiculatum (1.3 micromol mg prot(-1) min(-1)) was twice that of E. maggii, whereas xylanase specific activity (4.5 micromol mg prot(-1) min(-1)) was only half. Very weak activities were observed for Entodinium sp. A new xylanase gene, xyn11D, from P. multivesiculatum was reported and its gene product compared to 33 other family 11 xylanases. Phylogenetic analysis showed that xylanase sequences from rumen protozoa are closely related to those of bacteria.