Adhesion of cellulolytic ruminal bacteria to barley straw

Adhesion of the cellulolytic ruminal bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes to barley straw was measured by incubating bacterial suspensions with hammer-milled straw for 30 min, filtering the mixtures through sintered glass filters, and measuring the optical densities of the filtrates. Maximum adhesion of both species occurred at pH 6.0 and during mid- to late-exponential phase. Adhesion was saturable at 33 and 23 mg (dry weight) g of straw for R. flavefaciens and F. succinogenes, respectively. Methyl cellulose and carboxymethyl cellulose inhibited adhesion by 24 to 33%. Competition between species was determined by measuring characteristic cell-associated enzyme activities in filtrates of mixtures incubated with straw; p-nitrophenyl-beta-d-lactopyranoside hydrolysis was used as a marker for F. succinogenes, while either beta-xylosidase or carboxymethyl cellulase was used for R. flavefaciens, depending on the other species present. R. flavefaciens had no influence on F. succinogenes adhesion, and F. succinogenes had only a minor (<20%) effect on R. flavefaciens adhesion. The noncellulolytic ruminal bacteria Bacteroides ruminicola and Selenomonas ruminantium had no influence on adhesion of either cellulolytic species, although these organisms also adhered to the straw. We concluded that R. flavefaciens and F. succinogenes have separate, specific adhesion sites on barley straw that are not obscured by competition with non-cellulolytic species.     

Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.     

Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum.

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

COMBINATION OF DITHIOTHREITOL AND SULFIDE AS REDUCTANTS IN NITROGEN-FREE MINIMAL MEDIA FOR GROWTH OF ANAEROBIC RUMINAL BACTERIA AND PREVOTELLA RUMINICOLA STRAIN B14 ON AMMONIA

Cysteine is commonly employed as the medium reductant for ruminal bacteria, but many ruminal bacteria can use cysteine as a source of nitrogen as well as sulfur. The objective of the present study was to test a combination of dithiothreitol and sulfide as possible reductant substitutes for cysteine in anaerobic media containing ammonia as the nitrogen source. The type of reductant (cysteine versus dithiothreitol-sulfide) and ammonia concentration did not alter growth rates of Prevotella ruminicola strain B,4 (P>0.15). However, growth rates in dithiothreitol-sulfide reduced media varied tremendously between individual organisms ranging from 0.10 h⁻¹ for Ruminococcus flavefaciens to 1.6 h⁻¹ for Streptococcus bovis grown in 1 mM NH₃-N. At both 1 and 11 mM NH₄Cl, Str. bovis strain JB1 exhibited the greatest growth rate followed by Str. bovis strain C277. Megasphaera elsdenii strain T81 and Ruminococcus flavefaciens strain FD1 had the lowest growth rates at both NH₄Cl concentrations. Increasing NH₄Cl concentration from 1 to 11 mM resulted in increased growth rates for Ruminobacter amylophilus strains H18 and 70 and Str. bovis strain C277 (P<0.05), and decreased growth rates for S. ruminantium subsp. lactilytica strain HD₄ and Str. bovis strain JB1 (P<0.01). These results indicate that dithiothreitol and sulfide can be combined as reductants in nitrogen-free basal media for most ruminal bacterial species.     

Gas-liquid chromatography for evaluating polysaccharide degradation by Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85.

Two predominant rumen cellulolytic bacteria, Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85, were incubated with ground filter paper (Whatman no. 1), cattle manure fiber, wheat straw, Kentucky bluegrass, alfalfa, and corn silage as substrates. Analyses of the initial substrate and the recovered residue after 48 h of static incubation showed that R. flavefaciens C94 was quantitatively more effective than B. succinogenes S85 in degrading total dry matter (32.3% versus 16.1%). However, B. succinogenes S85 demonstrated a qualitative advantage in degrading the hemicellulose and hemicellulosic sugars of particular substrates. R. flavefaciens degraded a mean 29.7% of the cellulose and 35.6% of the hemicellulose in the various substrates, whereas B. succinogenes degraded a mean 17.9 and 31.6% of these fractions, respectively. Gas-liquid chromatography was an important aid in characterizing the polysaccharide-degrading capabilities of these rumen species.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Improvement in Isolation of Gliding Bacteria by the Elimination of Soluble Sugars

The selectivity and efficiency for the isolation of cellulolytic gliding bacteria were improved by eliminating soluble sugars from the usual cellulose agar medium in which the cellulolytic bacteria were often overgrown by fungi. The new selective medium did not support the growth of non-cellulolytic fungi.     

Ruminal cellulolytic bacteria and protozoa from bison, cattle-bison hybrids, and cattle fed three alfalfa-corn diets

Ruminal cellulolytic bacteria and protozoa and in vitro digestibility of alfalfa fiber fractions were compared among bison, bison hybrids, and crossbed cattle (five each) when they were fed alfalfa and corn in a ratio of 100:0, 75:25, and 50:50, respectively. The total number of viable bacteria (2.16 x 10(9) to 5.44 x 10(9)/ml of ruminal fluid) and the number of cellulolytic bacteria (3.74 x 10(7) to 10.9 x 10(7)/ml) were not different among groups of animals fed each diet. The genera of protozoa in all of the animal groups were similar; however, when either the 100:0 or 50:50 diet was used the percentage of Entodinium sp. was lower and the percentage of Diplodiniinae was higher (P less than 0.05) in bison than in bison hybrids or cattle. Bacteroides succinogenes made up the largest number of cellulolytic isolates from bison (58 and 36%, respectively, on the 100:0 and 75:25 diets), which were more numerous (P less than 0.05) than those from bison hybrids (36 and 12%) and cattle (33 and 18%). This was offset by a lower number of cellulolytic Butyrivibrio isolates. The numbers of Ruminococcus albus and R. flavefaciens isolates, in general, were similar among the bovid species, although R. flavefaciens generally made up less than 10% of the cellulolytic isolates. In vitro digestibility coefficients were greater (P less than 0.05) for the bison when the 75:25 diet was used and similar for the other two diets. The concentration of ruminal volatile fatty acids was larger (P less than 0.05) in bison than in bison hybrids and cattle when the 50:50 diet was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Effect of phenolic monomers on ruminal bacteria

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.     

Ruminal cellulolytic bacteria and protozoa from bison, cattle-bison hybrids, and cattle fed three alfalfa-corn diets.

Ruminal cellulolytic bacteria and protozoa and in vitro digestibility of alfalfa fiber fractions were compared among bison, bison hybrids, and crossbed cattle (five each) when they were fed alfalfa and corn in a ratio of 100:0, 75:25, and 50:50, respectively. The total number of viable bacteria (2.16 x 10(9) to 5.44 x 10(9)/ml of ruminal fluid) and the number of cellulolytic bacteria (3.74 x 10(7) to 10.9 x 10(7)/ml) were not different among groups of animals fed each diet. The genera of protozoa in all of the animal groups were similar; however, when either the 100:0 or 50:50 diet was used the percentage of Entodinium sp. was lower and the percentage of Diplodiniinae was higher (P less than 0.05) in bison than in bison hybrids or cattle. Bacteroides succinogenes made up the largest number of cellulolytic isolates from bison (58 and 36%, respectively, on the 100:0 and 75:25 diets), which were more numerous (P less than 0.05) than those from bison hybrids (36 and 12%) and cattle (33 and 18%). This was offset by a lower number of cellulolytic Butyrivibrio isolates. The numbers of Ruminococcus albus and R. flavefaciens isolates, in general, were similar among the bovid species, although R. flavefaciens generally made up less than 10% of the cellulolytic isolates. In vitro digestibility coefficients were greater (P less than 0.05) for the bison when the 75:25 diet was used and similar for the other two diets. The concentration of ruminal volatile fatty acids was larger (P less than 0.05) in bison than in bison hybrids and cattle when the 50:50 diet was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

Effect of phenolic monomers on ruminal bacteria.

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.     

Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose

To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic Treponema bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the production of succinic acid, lactic acid, and ethanol. Morphological examination, by electron microscopy, showed that cells of T. bryantii associate with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.     

Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows

The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108 v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104 v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108 v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.