Rumen Microbial Ecology in Mule Deer

Mule deer rumen microbial populations from animals in the natural habitat in Utah and from captive deer fed various rations were studied. The microorganisms were characterized on the basis of morphology and Gram reaction. Rumen samples contained 13 identifiable types of bacteria and one genus of ciliate protozoa (Entodinium). Highest rumen bacterial populations were produced on rations containing barley. No differences in proportions of ruminal bacteria in the various morphological groups could be detected when animals were fed either natural browse plants or alfalfa hay. The total numbers of bacteria were similar for animals feeding on controlled diets of browse or hay and those in the natural habitat. Numbers of some bacterial types were directly related to ciliate protozoal numbers, whereas others were inversely related. Highest rumen ciliate protozoal populations were observed on rations containing barley. No differences in protozoal populations were noted between diets containing only browse or hay. Seasonal variations were noted in ciliate protozoal numbers from deer feeding in the natural habitat. The total number of ciliate protozoa decreased in the fall and winter and remained low until spring. There were indications that salt in the deer diet favorably affected rumen ciliate protozoa. Rather than revealing direct deer management applications, this study serves to stimulate and illuminate new approaches to research in range and wildlife nutrition.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Comparative Analysis of Microbial Profiles in Cow Rumen Fed with Different Dietary Fiber by Tagged 16S rRNA Gene Pyrosequencing

The ruminal microbiome of cattle plays an important role not only in animal health and productivity but also in food safety and environment. Microbial profiles of rumen fluid obtained from dairy cows fed on three different fiber/starch diet compositions were characterized. Tagged 16S rRNA gene pyrosequencing and statistical analysis revealed that the dominant ruminal bacteria shared by all three sample groups belonged to phyla Bacteroidetes, Firmicutes, and Proteobacteria. However, the relative abundance of these bacterial groups was markedly affected by diet composition. In animals fed with a high fiber diet, the fibrolytic and cellulolytic bacteria Lachnospiraceae, Ruminococcaceae, and Fibrobacteraceae were found in highest abundance compared with animals fed other diets with lower fiber content. The polysaccharide-degrading Prevotellaceae and Flavobacteriaceae bacteria were most abundant in the rumen of cows fed on diet with the highest starch content. These data highlight the ruminal microbiome’s ability to adapt to feed composition and also provide a basis for the development of feed formulation systems designed to improve livestock productivity.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

A lack of predatory interaction between rumen ciliate protozoa and Shiga-toxin producing Escherichia coli

AIMS: To investigate interactions between rumen protozoa and Shiga toxin-producing Escherichia coli (STEC) and to ascertain whether it is likely that rumen protozoa act as ruminant hosts for STEC. METHODS AND RESULTS: The presence of stx genes in different microbial fractions recovered from cattle and sheep rumen contents and faeces was examined using PCR. In animals shedding faecal STEC, stx genes were not detected in the rumen bacterial or rumen protozoal fractions. Direct interactions between ruminal protozoa and STEC were investigated by in vitro co-incubation. Rumen protozoa did not appear to ingest STEC, a STEC lysogen or non-STEC E. coli populations when co-incubated. CONCLUSIONS: The ruminal environment is unlikely to be a preferred habitat for STEC. Bacterial grazing by rumen protozoa appears to have little, if any, effect on STEC populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that ruminal protozoa are unlikely to be a major factor in the survival of STEC in ruminants. They appear as neither a host that protects STEC from the ruminal environment nor a predator that might reduce STEC numbers.     

Simple Method to Remove Completely Ciliate Protozoa of Adult Ruminants

Dioctyl sodium sulfosuccinate (Aerosol OT) has been used in a remarkably short period to obtain rumen ciliate-free cows. Two 30-g doses of Aerosol OT given on consecutive days appeared to effectively eliminate all types of rumen ciliate protozoa without harming the health of the host animal. Measurement of the rumen metabolic parameters of gas production, carbon dioxide to methane ratio, pH value, volatile fatty acids, ammonia, and in vitro cellulose digestion, along with total amylolytic streptococci counts of the rumen contents, showed that conditions in rumens of defaunated animals are normal 4 days after such treatment. It appears that such animals may be used in ruminal studies that require defaunated animals.     

High Molecular Weight Protease in Rumen Ciliate Protozoa

Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.     

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.     

Relation of rumen ATP concentration to bacterial and protozoal numbers.

Cultures of Streptococcus bovis and mixed populations of rumen bacteria were used to investigate the concentration of ATP and rumen bacterial numbers at various stages of growth. ATP, extracted with Tris buffer, was analyzed using the firefly luciferin-luciferase bioluminescent reaction. ATP concentrations of S. bovis and mixed cultures of rumen bacteria significantly correlated with live cell counts during the log phase of growth but not during the stationary phase. The average cellular ATP concentration of rumen bacteria was calculated to be 0.3 fg of ATP per cell. Studies done with in vivo artificial rumen apparatus revealed that the protozoal contribution to rumen fluid ATP pool size was much more substantial than was the bacterial contribution. The rumen fluid ATP concentration was greater in cattle with protozoa than in those that were defaunated. Differences in ATP concentration due to size differences of ciliate protozoa were observed. Due to the unbalanced distribution of ATP in rumen microbes, ATP appears to be an unsuitable indicator of rumen microbial biomass.     

Rumen ciliates: their metabolism and relationships with bacteria and their hosts

To explore the ruminal ecosystem, this paper is an attempt to illustrate the biochemistry and the metabolism of rumen protozoa. Different culture techniques (gnotobiotic, axenic, continuous culture) of ciliate protozoa are reviewed. The ability of ciliates to invade plant tissues, their association and the degradation of plant cell constituents (cellulose, hemicellulose, pectin, glycolipids), and the use of starch and soluble sugars for polysaccharide storage are determined. The interrelationships between ciliate protozoa and bacteria are discussed. These include the enzymic degradation of the engulfed plant particles, the bacterial nitrogen (amino acids, nucleic acids), and vitamins and trace element supplies as nutrient sources for protozoa. The composition of the rumen ciliate population, controlled by phylogenetic factors, geographical distribution, feeding habits and physiological status of the host, by competition between microorganisms and by the diet (nature of the diet, number of meals), is related. The transmission and establishment of the microfauna and its contribution to the microbial nitrogen available for the host are discussed.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

Rumen microbial diversity in Svalbard reindeer, with particular emphasis on methanogenic archaea

Ruminal methanogens, bacteria and ciliate protozoa of Svalbard reindeer grazing natural pastures in October (late fall) and April (late winter) were investigated using molecular-based approaches. The appetite of the Svalbard reindeer peaks in August (summer) and is at its lowest in March (winter). Microbial numbers, quantified by real-time PCR, did not change significantly between October and April, when food intakes are at similar levels, although the numbers of methanogens tended to be higher in October ( P =0.074), and ciliate numbers tended to be higher in April ( P= 0.055). Similarly, no change was detected in the bacterial and protozoal population composition by rRNA gene-based denaturing gradient gel electrophoresis analysis. Dominant methanogens were identified using a 16S rRNA gene library (97 clones) prepared from pooled PCR products from reindeer on October pasture ( n =5). Eleven of the 22 distinct operational taxonomic units (OTUs) generated exhibited a high degree of sequence similarity to methanogens affiliated with Methanobacteriales (eight OTUs), Methanomicrobiales (one OTU) and Methanosarcinales (two OTUs). The remaining 11 OTUs (53% of the clones) were associated with a cluster of uncultivated ruminal archaea. This study has provided important insights into the rumen microbiome of a high-arctic herbivorous animal living under harsh nutritional conditions, and evidence suggesting that host type affects the population size of ruminal methanogens.     

Variation in Numbers and Mass of Ciliate Protozoa in the Rumens of Sheep Fed Chaffed Alfalfa (Medicago sativa)

Masses and numbers of rumen ciliate protozoa were markedly different in individual sheep fed chaffed alfalfa hay under different feeding regimens. Studies on the ciliate contribution to specific aspects of rumen fermentation should take into account the size of members of each genus in individual animals as well as the numbers present.     

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.     

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.     

Ecological factors determining establishment of cellulolytic bacteria and protozoa in the rumens of meroxenic lambs

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B). The total number of bacteria established in the rumen was between 10(9) and 5 x 10(10) g-1. In lambs inoculated with dilutions (10(-6), 10(-7), 10(-8)) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10(-6) and 10(-7) dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10(-8) dilution. Similar results were obtained for the protozoan Entodinium sp. With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10(-6) and 10(-7) inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10(-6) dilution of pool B. Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.     

Observations on Bacterial Feeding by the Rumen Ciliate Isotricha prostoma

SYNOPSIS. Starvation of Isotricha prostoma for 72–96 hours decreased the cellular amylopectin granules and facilitated the microscopic search for bacterial feeding. I. prostoma selected and ingested only certain rods from among many types of rumen bacteria. In order to isolate the bacteria important as a food source for Isotricha , the starved protozoa were allowed to feed on mixed rumen bacteria, washed, and the crushed protozoan contents quickly cultured for bacteria. Several strains of bacteria were isolated in pure culture. Three of the rod strains isolated were rapidly ingested by I. prostoma when fed to the ciliate. In a monobacterial culture I. prostoma divided once before succumbing.     

Fermentation of plant cell walls by ruminal bacteria,protozoa and fungi and their interaction with fibre particle size

Abstract

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

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.