Maintenance of a certain rumen protozoal population in a continuous in vitro fermentation system.

A continuous culture system suitable for maintaining certain rumen protozoa was developed by modifying the procedure of Walter and Pilgrim (R.A. Weller and A.F. Pilgrim, Br. J. Nutr. 32:341-351, 1974) to include a dialyzing system. The concentration of ciliate protozoa, the pH value, and concentrations of volatile fatty acid and ammonia-N could be maintained within normal rumen limits for more than 15 days by appropriate choice of mechanical agitation and of the amount of substrate and physical form of the substrate bags. The average concentration of protozoa in the free fluid around the substrate was about 10% that in the fluid squeezed from solid digesta residues. More than 10(6) protozoa per ml was present in the fresh substrates only 2 h after supplying the substrate. These facts suggest that sequestration of the protozoa among particulate digesta is an important factor in maintaining the concentration of protozoa.     

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.     

Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

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.     

The low availability of dietary choline for the nutrition of the sheep.

1. Choline, which is present in the diet of the sheep either in the non-esterified form or combined in phospholipids, is rapidly degraded in the rumen. The ultimate product formed from the N-methyl groups is methane. 2. Analysis of the non-esterified choline and the phosphatidylcholine in ruminal and abomasal digesta indicate that the phospholipid is the main vehicle for the passage of choline to the lower digestive tract. 3. The concentration of phosphatidylcholine in abomasal digesta is lower than that of ruminal digesta, which is in line with a selective retention of protozoa in the rumen as observed by others. 4. On defaunation of the rumen to remove ciliated protozoa the concentration of phosphatidylcholine in ruminal digesta falls markedly and becomes lower than that in abomasal digesta. 5. Calculation shows that the adult sheep obtains at most only about 20--25 mg of effective choline per day from its diet (0.002--0.0025% of dietary total dry-weight intake). This is some fifty times less than the minimum required to avoid pathological lesions and death in other species investigated (0.1%+ of dietary dry-weight intake). 6. Sheep liver can synthesize choline from [14C]ethanolamine both in vitro and in vivo, but the synthesis of choline per kg body weight is many times less than it is in the rat. 7. The intact sheep oxidizes an injected dose of [1,2-14C]choline to CO2 at a rate that is several times less than that observed for the rat. This could help to explain the apparent minimal requirement of sheep for dietary choline.     

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.     

Oral Samples as Non-Invasive Proxies for Assessing the Composition of the Rumen Microbial Community

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.     

Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach

A meta-analysis was conducted to evaluate the effects of protozoa concentration on methane emission from ruminants. A database was built from 59 publications reporting data from 76 in vivo experiments. The experiments included in the database recorded methane production and rumen protozoa concentration measured on the same groups of animals. Quantitative data such as diet chemical composition, rumen fermentation and microbial parameters, and qualitative information such as methane mitigation strategies were also collected. In the database, 31% of the experiments reported a concomitant reduction of both protozoa concentration and methane emission (g/kg dry matter intake). Nearly all of these experiments tested lipids as methane mitigation strategies. By contrast, 21% of the experiments reported a variation in methane emission without changes in protozoa numbers, indicating that methanogenesis is also regulated by other mechanisms not involving protozoa. Experiments that used chemical compounds as an antimethanogenic treatment belonged to this group. The relationship between methane emission and protozoa concentration was studied with a variance−covariance model, with experiment as a fixed effect. The experiments included in the analysis had a within-experiment variation of protozoa concentration higher than 5.3 log₁₀ cells/ml corresponding to the average s.e.m. of the database for this variable. To detect potential interfering factors for the relationship, the influence of several qualitative and quantitative secondary factors was tested. This meta-analysis showed a significant linear relationship between methane emission and protozoa concentration: methane (g/kg dry matter intake)=−30.7+8.14×protozoa (log₁₀ cells/ml) with 28 experiments (91 treatments), residual mean square error=1.94 and adjusted R²=0.90. The proportion of butyrate in the rumen positively influenced the least square means of this relationship.     

Evaluation of subsampling and fixation procedures used for counting rumen protozoa.

Total numbers of protozoa can be significantly lower in rumen fluid than in whole rumen contents, depending on the time of sampling and the procedure used to separate the fluid and solid fractions. Moreover, generic distribution in rumen fluid was significantly affected in all cases tested. The percentage of Entodinium spp. increased, whereas percentages of Diplodinium spp. and Ophryoscolex spp. decreased. Microscopic observation of fresh and fixed rumen contents did not indicate any marked attachment of protozoa to particulate matter. In addition, dilution of whole rumen contents with water, 5 mM sucrose, or 0.1% Tween 80 before fixation did not affect total numbers or generic composition of protozoa. It was thus concluded that attachment to feed particles is probably not a problem in counting procedures. Blending of whole rumen contents to facilitate subsampling caused a decrease in numbers of protozoa. The concentration of formaldehyde used for preservation of rumen contents, 4, 10, or 18.5%, did not affect the total count.     

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.     

Ruminal fermentation and fill change with season in an arctic grazer: responses to hyperphagia and hypophagia in muskoxen (Ovibos moschatus)

We studied castrated adult muskoxen fed a standard diet of grass hay and supplement throughout the year to determine seasonal changes in digesta passage, fill, and fermentation without the confounding effects of reproductive demands or changes in food quality. Although food intake increased by 74% between spring and autumn, mean retention times of fluid and particulate digesta markers were maintained between seasons in both the rumen (9-13 h) and the intestines (27-37 h). The rumen contained 84.5% of digesta and accounted for 79% of dry matter digestion in the whole digestive tract. Ruminal fluid space and whole-gut digesta fill increased by 31%-34%, while ruminal rates of in situ degradation increased by more than 100% between spring and autumn for cellulose and hemicellulose. Hyperphagia in autumn was accompanied by increased bacterial counts in ruminal fluid (30%), declines in ruminal pH, and increases in the concentration of fermentation acids (16%) when compared with spring hypophagia. Consumption of fresh hay and supplement increased the concentrations of acids most markedly during winter and spring when bacterial counts were low. Low food intakes in winter and spring may limit the microbial population, whereas hyperphagia in autumn may foster a much more active microflora that requires consistent supplies of substrate. Plasticity of fill and fermentation in muskoxen minimizes winter costs and maximizes nutrients and energy gained from coarse forages in small home ranges throughout the year.     

Digesta characteristics of dorsal, middle and ventral rumen of cows fed with different hay qualities and concentrate levels

The influence of fibre content of hay (H) and concentrate level (C) on local differences in the composition of ruminal digesta (ratio of solid to fluid digesta, DM, NDF, ADF and ADL content), particle size (MPL), specific gravity (SG) and fermentation (pH and concentrations of SCFA and bicarbonate) have been tested on two ruminally cannulated Friesian cows (520 kg BW) which were fed restricted, using individual cows as experimental units. Digesta samples were collected via cannula from three rumen layers: 5 to 10 cm (top) and 25-35 cm beneath the top of the particle mat (middle) and 5-10 cm above the rumen floor (bottom). For a main plot treatment (H x C), repeated samples were collected at four time intervals (1 h before and 2, 5 and 10 h after morning feeding) on each of two days. From top to bottom rumen the share of solid digesta mass (SM), DM and NDF contents of squeezed digesta fluid (SRF) and concentration of SCFA decreased (P < 0.05); pH and bicarbonate concentration increased (P < 0.05), while DM, NDF, ADF and ADL contents in SM, MPL and SG did not differ. Higher NDF content of hay (from 47-62%) increased SM, fibre fractions in SM, MPL, pH and concentration of bicarbonate in ruminal digesta, especially when 50% concentrate was given, while SG decreased. When the concentrate level was enhanced from 20 to 50%, digesta SM, MPL and the content of DM and NDF in SRF increased, while pH, concentrations of SCFA and acetate decreased when low-fibre hay was given. With longer time after feeding the digesta SM was reduced and fibre content in SM increased. The increase of the fibre content of hay reduced the possible negative effect of high concentrate level on the stratification of ruminal digesta. The decrease of the fibre content of hay promised better conditions for fibre digestion in the rumen when concentrate availability is limited.     

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.     

Investigation on the “In vitro” degradation of zearalenone in rumen fluid

Ruminal zearalenone (ZEA) degradation in “in vitro” digestion was examined in different variants using the Hohenheimer Gas Test. First, the mycotoxin degradation was measured using squeezed rumen fluid from solid digesta of the dorsal sack and free rumen fluid from the ventral sack, respectively. Then free rumen, fluid of the ventral sac was used with addition of concentrate and mixtures of concentrate with sun-flower oil and starch, respectively. Within 24 hours ZEA was degraded down to 63% and 49% of the initial concentration when incubated with solid and fluid rumen digesta, respectively. Using additives and rumen fluid concentrate for incubation, concentrate, concentrate with oil and concentrate with starch, respectively, a reduction of ZEA to 46, 56 and 37% of the initial toxin concentration was observed. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19 2004 Financial support: German Academic Exchange Service (approval of a grant) and Famy Foundation     

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.     

Research note: A method for studying local differences in ruminal fermentation in dairy cattle

A method was developed for studying local differences in ruminai fermentation. The developed sampler consisted of an acrylic glass container (460 cm³) with an aperture for digesta sampling, which could be opened and closed by the scaled “T”; rod. The scale was a reference for defined rumen layers: top, middle, 5 to 10 cm and 25 to 35 cm beneath the top of particles mat, respectively, and bottom 5 to 10 cm above the rumen floor. The repeatability of the method was proved in two rumen cannulated cows. Particle/fluid ratio, pH and sample amount were measured 2 to 2¹/₂ h after morning feeding in four replicates each day (over 5 days), rumen layer and animal. No significant differences between replicates were observed. The coefficients of variation (CV) of the particle/fluid ratio varied between 8.7% and 13.6%. Top layer had higher CV than middle and bottom layer. CV of pH ranged between 0.59% and 1.27%. The developed method of sampling showed satisfactory repeatability for investigation of digesta properties and fermentation in different rumen layers.     

Adenosine Triphosphate and Deoxyribonucleic Acid in the Alimentary Tract of Cattle Fed Different Nitrogen Sources

Total microbial cell counts and the content of deoxyribonucleic acid (DNA) and adenosine triphosphate (ATP) were determined in ruminal and abomasal digesta and in faeces from heifers fed a basal diet of barley and straw supplemented with urea, casein, soybean-protein concentrate or feather meal. The concentration of ATP in the rumen content varied independently of the DNA content and total cell count, but dependently upon the nitrogen sources, the highest ATP/DNA ratio being obtained with casein. The ATP/DNA ratio in faeces was only one-tenth of that found in the digesta of the rumen and abomasum, indicating either an extremely low level of activity of the microbial cells or more probably a very large number of dead organisms. It is suggested that DNA and ATP might be useful indices of the microbial status in terms of biomass and metabolic activity. The total cell count should still be included in routine studies to determine the proportion of protozoa to bacteria.     

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.