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.     

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Diurnal variation in ciliate protozoa in the rumen of black buck (Antilope cervicapra) fed green forage

The protozoa in the rumen of a black buck were a B-type population with numbers varying between 0·31 and 0·61 times 10⁶cells/ml rumen liquor, when the animal was fed either vegetative green oat or third cut berseem. The total protozoa, total holotrichs, Dasytricha, total spirotrichs and small spirotrichs were significantly higher ( P < 0·01) on berseem feeding than those on oat feeding, while the numbers of Isotricha and large spirotrichs were unaffected by change of diet. Numerically the most important group of protozoa was small spirotrichs (74·4–75·6% of total population) which accounted for only 9·85–13·61% of protozoal cell mass in the rumen.     

Assessment of protozoa in Yunnan Yellow Cattle rumen based on the 18S rRNA sequences

Library of ruminal protozoal 18S rRNA of Yunnan Yellow Cattle has been constructed in the present study. Phylogenic analysis of sequences was meanwhile employed to reveal the diversity of protozoa in the rumen of Yunnan Yellow Cattle. One Yellow Cattle was fed malt meal (YCRPB) and the other was fed wheat straw (YCRPS). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. The results showed as follows: A total of 121 clones were obtained and fell into four genera identified as Entodinium (66.9%), Dasytricha (5.8%), Isotricha (9.1%), and Diplodinium (18.2%). Within the genus Entidinium, 48 of the YCRPB sequences and 33 of the YCRPS sequences clustered with the Entodinium caudatum. 7 of the YCRPB sequences were phylogenetically placed within the genus Dasytricha. 11 of the YCRPB sequences were related with high confidence to Isotricha intestinalis. 22 of the YCRPS sequences were phylogenetically placed within the genus Diplodinium. The predominant protozoal genus identified in the rumen fluid belonged to the Entodinium group, and the divergences between two cattle may due to diet and individual differences.     

Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes

A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.     

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 protozoal 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 thzis 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.     

The effect of Aspergillus oryzae fermentation extract on the growth of fungi and ciliate protozoa in the rumen

When added to the diet of sheep, 2 g/d, Aspergillus oryzae fermentation extract (AO) stimulated total and cellulolytic bacterial numbers in rumen fluid by 34 and 90% respectively. AO had no effect on the numbers of protozoa or fungal zoospores. AO did not affect hydrogen production by the rumen fungi Neocallimastix frontalis (RE1), N. patriciarum (CX) or Piromonas communis (P) in pure culture or protozoal activity in vitro , estimated from the rate of breakdown of [¹⁴C] leucine-labelled Selenomonas ruminantium. It was concluded that increases in ruminal fibre digestion observed previously in animals fed AO, were most likely due to a stimulation of bacteria rather than eukaryotes in the rumen microbial population.     

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10(0) and 10(6) entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.     

Negative correlation between protozoal and bacterial levels in rumen samples and its relation to the determination of dietary effects on the rumen microbial population.

The bacterial protein content and protozoal protein content of unfractionated samples from the liquid-small particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 X 10(-11) mg of protein per bacterial cell and 5.9 X 10(-11) mg/micron 3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P less than 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P less than 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups.     

Interactions between rumen bacteria and ciliate protozoa in their attachment to barley straw

The attachment of ¹⁴C-choline-labelled mixed rumen protozoa to barley straw in vitro was not significantly affected when bacteria prepared from rumen fluid were added to the incubation mixture. There was similarly little effect on protozoal attachment when the straw had already been colonized by a bacterial population for 24 h. In contrast, it was deduced from measurements of enzyme activities associated with straw that bacterial attachment was reduced if protozoa were present. Bacteria that had colonized the straw for 25 h beforehand were less susceptible to predation by protozoa.     

Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.     

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.     

Effect of dietary sodium bicarbonate supplementation on fermentation characteristics and ciliate protozoal population in rumen of lambs

Effects of sodium bicarbonate (NaHCO₃) inclusion on rumen fermentation characteristics and ciliate protozoal population in high concentrate-fed lambs were studied. Twenty-four weaner (90 days old) Malpura lambs divided into four equal groups (G1, G2, G3 and G4) were fed basal (25:75 roughage and concentrate) diet (G1) or basal diet supplemented with 0.75% (G2), 1.50% (G3) and 2.25% (G4) sodium bicarbonate for 90 days. Daily dry matter (DM) intake and digestibility of organic matter (OM), crude protein (CP), gross energy and plane of nutrition were similar in all the groups while cellulose digestibility was higher in G4 than in G2 and G1. Ruminal pH increased (P<0.05) with increasing levels of dietary sodium bicarbonate. Concentrations of total volatile fatty acids (TVFA), total nitrogen and trichloroacetic acid-precipitable nitrogen (TCA-ppt.-N) were higher while ammonia nitrogen was lower in the rumen fluid of G4 and G3 than in G2 and G1. The number of total protozoa, Isotricha, Dasytricha, large and small spirotrichs were higher (P<0.01) in the rumen of G4 and G3 than in G2 and G1. Total live weight gain and average daily gain were also higher in lambs supplemented with sodium bicarbonate. It is concluded that sodium bicarbonate inclusion at the rate of 1.50% of total ration increased cellulose digestibility, ciliate protozoal number, ruminal pH and total nitrogen concentration resulting in improved growth of lambs maintained on high concentrate diet.     

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.     

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.