Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

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.     

Microbiology and ration digestibility in the hindgut of the ovine

Contents of the terminal ilea, ceca-proximal colons, and terminal recta were obtained from nine sheep, three of which were fed 100% orchardgrass hay, three of which were fed 60% cracked corn-40% orchardgrass hay, and three of which were fed 80% cracked corn-20% orchardgrass hay. The digestibility of dry matter in the ceca was greatest when the all-hay diet was fed, whereas the percentage of cellulose digestion in the ceca increased with increasing levels of concentrate. For all diets, the total volatile fatty acid concentrations were higher in the ceca than in the other two sites. The cecal pH levels decreased with increased corn levels in the diet. The total microbiol numbers per gram of ileal and cecal contents increased in response to feeding of concentrate; however, across all diets, the ileal counts were 8% or less of the cecal counts. In contrast, the cellulolytic microbial numbers in the ilea were 50% or more of those in the ceca and were highest with the all-hay diet. Both bacterial and end product concentrations in the ceca were equivalent to those occurring in rumina. A total of 16 cellulolytic cultures were isolated and characterized from ileal and cecal contents of animals fed all three diets. Seven gram-negative-rod-shaped organisms were identified as Butyrivibrio fibrisolvens, whose capacity to digest cellulose exceeded that of several rumen strains. The guanine-plus-cytosine content of the DNA of one strain of B. fibrisolvens was 38.8 mol%, compared with the only previously reported value for this species of 41.2 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbiology and ration digestibility in the hindgut of the ovine.

Contents of the terminal ilea, ceca-proximal colons, and terminal recta were obtained from nine sheep, three of which were fed 100% orchardgrass hay, three of which were fed 60% cracked corn-40% orchardgrass hay, and three of which were fed 80% cracked corn-20% orchardgrass hay. The digestibility of dry matter in the ceca was greatest when the all-hay diet was fed, whereas the percentage of cellulose digestion in the ceca increased with increasing levels of concentrate. For all diets, the total volatile fatty acid concentrations were higher in the ceca than in the other two sites. The cecal pH levels decreased with increased corn levels in the diet. The total microbiol numbers per gram of ileal and cecal contents increased in response to feeding of concentrate; however, across all diets, the ileal counts were 8% or less of the cecal counts. In contrast, the cellulolytic microbial numbers in the ilea were 50% or more of those in the ceca and were highest with the all-hay diet. Both bacterial and end product concentrations in the ceca were equivalent to those occurring in rumina. A total of 16 cellulolytic cultures were isolated and characterized from ileal and cecal contents of animals fed all three diets. Seven gram-negative-rod-shaped organisms were identified as Butyrivibrio fibrisolvens, whose capacity to digest cellulose exceeded that of several rumen strains. The guanine-plus-cytosine content of the DNA of one strain of B. fibrisolvens was 38.8 mol%, compared with the only previously reported value for this species of 41.2 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feeding behavior, rumination, reticulo-ruminal fermentation and plasma volatile fatty acids, compared in goats and sheep; influence of the diet

Two-year old Alpine she-goats (n = 3) and Texel ewes (n = 3) were compared as to eating behaviour, rumination and volatile fatty acids (VFA) in rumen and blood. The animals were fed once daily with two different proportions (20 and 80%) of barley and hay. Dry matter intake was fixed at 48 g D.M/kg P0.75 per day. In similar feeding and environmental conditions, eating behaviour and rumination of goats and sheep did not differ much: the goats tended to eat faster and there were more rumination periods in the sheep. Latency time, mean duration of rumination periods, daily rumination time and circadian pattern of rumination did not differ significantly between the two species (fig. 1). With both diets we observed a higher VFA concentration and a lower acetate/propionate ratio in the rumen of the ewes; however, rumen pH was lower only in those eating the 80% barley ration (fig. 2). Blood VFA in the jugular vein did not differ between sheep and goats (fig. 3). The proportion of cereals and hay in the diets affected rumen fermentation and rumination pattern in both species. With a higher concentrate/roughage ratio, rumen and plasma VFA increased, while the pH and acetate/propionate ratio in the lumen juice, the number of rumination periods and daily rumination time decreased. When the animals were fed the 80% barley ration, there was practically no rumination in the first 9 h after the single meal. During this time, rumen pH was minimal and VFA levels in the rumen and blood were maximal.     

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.     

Influence of intoxication with organophosphates on rumen bacteria and rumen protozoa and protective effect of clinoptilolite-rich zeolite on bacterial and protozoan concentration in rumen

The effect ofO-ethyl-S-(2-diisopropylaminoethyl) methylthiophosphonate on rumen bacteria and rumen protozoa was investigated in sheep (after premedication with clinoptilolite-rich zeolite and without that premedication). In control animals a decrease in the total concentration of rumen protozoa was observed 3–7 d after intoxication (particularly in small and large ones). In clinoptilolite-rich-zeolite-treated animals only a slight decrease in protozoan numbers occurred during the first hours after the intoxication. Similarly, in every category of rumen bacteria marked differences between the groups were recorded, particularly in concentration of lipolytic bacteria. The results suggest some protective effect of clinoptilolite-rich zeolite for rumen microbiota against the organophosphate poison.     

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.     

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.     

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.     

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.     

Basal medium for the selective enumeration of rumen bacteria utilizing specific energy sources.

A 40% rumen fluid basal medium has been developed that without added substrate will support growth of about 10% or less of the total colony count obtained with 40% rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA). The basal medium is prepared by anaerobic incubation of all ingredients in RGCSA medium except the carbohydrates, Na2CO3, and cysteine for 7 days at 38 degrees C. After incubation, substrate(s), Na2CO3 and cysteine are added and the medium is tubed and sterilized as in normal medium preparation. When xylose was included with glucose, cellobiose, and starch as added carbohydrates in the incubated medium, colony counts were comparable to those obtained with RGCSA medium. The addition of specific carbohydrates or other substrates as energy sources to the basal medium suggested that the percentage of the bacterial population capable of utilizing these energy sources was influenced by the ration of the animal; however, considerable animal variation and day-to-day variation in a given animal was observed. Comparison of the population in animals fed either orchardgrass hay or 60% corn-40% orchardgrass (60-40) indicated little or no difference for the percentage of bacteria utilizing glucose, pectin, xylan, or mannitol. Increases in the percentages of xylose-, cellobiose-, Glycerol-, and lactate-utilizing bacteria occurred with the orchardgrass hay ration, whereas the percentage of starch-digesting bacteria was increased significantly (P less than 0.01) in the animals fed the 60-40 ration. A limited number of bacterial strains were isolated from the basal medium without added substrate, most of which were atypical with respect to the predominant rumen bacteria. Growth of these strains, even in complex media, was very slow and limited. Based on these data with isolated strains and colony counts obtained in roll tube medium containing only minerals, resazurin, agar, Na2CO3, and cysteine, the selective medium overestimated the percentage of bacteria able to use a specific energy source. This overestimate was 6 to 7% of the total culturable count.     

Why does the establishment of the starch preferring<Emphasis Type="Italic">Entodinium caudatum</Emphasis> in the rumen decrease the numbers of the fibrolytic ciliate<Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.     

Evaluation of a Method of Measuring Fermentation Rates and Net Growth of Rumen Microorganisms

The method of el-Shazly and Hungate for measuring gas production in rumen contents was slightly modified and used throughout this investigation. The variation in the fermentation rates due to samples collected separately from a sheep fed on hay was less than 2%. When samples obtained through a stomach tube were compared with samples collected through the rumen fistula, the variation was about 3%. The rates of gas and acid production were approximately similar in samples obtained from the rumen at the same time when no sodium bicarbonate was added. During in vitro incubation of whole ruminal contents, there was a highly significant correlation between the net growth rate values (obtained by using fermentation capacity as an index) and the change in concentration of viable rumen bacteria or total ciliate protozoa.     

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.     

A Note on the Flora and Fauna in the Rumen of Steers Fed a Feedlot Bloat-provoking Ration and the Effect of Penicillin

A study was made of the predominant culturable bacteria and ciliate protozoa present in the rumen of two steers that were regularly bloating on a pelleted ratio containing 22% alfalfa meal, 16% soybean oil meal, 61% barley, and 1% common salt. The ruminal microorganisms in the two animals differed as indicated by a high total culture count of bacteria, an almost complete absence of ciliate protozoa, a low pH, and a difference in the proportions of presumptively identified predominant bacterial groups in one animal (steer 26) as compared with the other (steer 32). The first exposure of the animals to procaine penicillin (75 or 150 mg per day on 2 successive days) resulted in an abnormal ruminal flora 31 hr after the first treatment as indicated by drastic drops in total and cellulolytic bacterial counts and a change in the proportions of predominant bacterial groups. The animals refused feed for 32 to 48 hr after the first treatment. After feed consumption resumed, further treatment with 75 mg penicillin on 4 successive days did not appear to greatly alter the flora and did not result in feed refusal in animal 32. In animal 26, amounts of penicillin progressing from 50 to 200 mg per day did not result in feed refusals and observations on rumen ingesta samples during this period indicated a decrease in total bacterial count, a great increase in numbers of ciliate protozoa, a higher pH, and a change in the proportions of predominant bacterial groups so that the ruminal picture was much more similar to that of animal 32 than to its own during the pre-penicillin period. Bloat was not relieved except during the period of feed refusal.

The results indicate that the ruminal flora rapidly adapts to penicillin and that bloat of the feedlot type can occur in animals with widely differing numbers and kinds of bacteria and protozoa. Feedlot bloat does not appear to be correlated with the occurrence or numbers of any of the individual predominant groups of bacteria cultured.

     

Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods.

Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose-cellobiose-starch-agar medium (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non-rumen fluid medium (medium 10) of Caldwell and Bryant (1966), 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

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.