The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

Aims:  To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin.
Methods and Results:  Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus , Butyrivibrio fibrisolvens , Prevotella bryantii and Selenomonas ruminantium . The in vitro production of l - and/or d -lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin.
Conclusion:  Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture.
Significance and Impact of the Study:  This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

Effects of dietary crude protein and tannic acid on rumen fermentation,rumen microbiota and nutrient digestion in beef cattle

The objectives of the trial were to study the effects of dietary crude protein (CP) and tannic acid (TA) on rumen fermentation, microbiota and nutrient digestion in beef cattle. Eight growing beef cattle (live weight 350 ± 25 kg) were allocated in a 2 × 2 crossover design using two levels of dietary CP [111 g/kg dry matter (DM) and 136 g/kg DM] and two levels of TA (0 and 16.9 g/kg DM) as experimental treatments. Each experimental period lasted 19 d, consisting of 14-d adaptation and 5-d sampling. The impacts of dietary CP and TA on ruminal microbiota were analysed using high-throughput sequencing of 16S rRNA gene. Results indicated that no interactions between dietary CP and TA were found on rumen fermentation and nutrient digestibility. Increasing dietary CP level from 111 to 136 g/kg DM increased the ruminal concentrations of ammonia nitrogen (NH₃-N) (p < 0.01) and improved the CP digestibility (p < 0.001). Adding TA at 16.9 g/kg DM inhibited rumen fermentation and decreased the digestibility of dietary CP (p < 0.001), DM (p < 0.05) and organic matter (p < 0.01). Increasing the dietary CP level or adding TA did not affect the relative abundances of the major bacteria Firmicutes and Proteobacteria at the phylum level and Prevotella_1 and Christensenellaceae_R-7_group at the genus level, even though adding TA increased the Shannon index of the ruminal bacterial community. TA was partly hydrolysed to pyrogallol, gallic acid and resorcinol in rumen fluid and the inhibitory effects of TA on rumen fermentation and nutrient digestibility could have been resulted from the TA metabolites including pyrogallol, gallic acid and resorcinol as well as the protein-binding effect.     

Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet

AIM: To examine the effect of sulfur-containing compounds on the growth of anaerobic rumen fungi and the fibrolytic rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in pure culture and within the cattle rumen. METHODS AND RESULTS: The effect of two reduced sulfur compounds, 3-mercaptopropionic acid (MPA) or 3-mercapto-1-propanesulfonic acid as the sole S source on growth of pure fibroyltic fungal and bacterial cultures showed that these compounds were capable of sustaining growth. An in vivo trial was then conducted to determine the effect of sulfur supplements (MPA and sodium sulfate) on microbial population dynamics in cattle fed the roughage Dichanthium aristatum. Real-time PCR showed significant increases in fibrolytic bacterial and fungal populations when cattle were supplemented with these compounds. Sulfate supplementation leads to an increase in dry matter intake without a change in whole tract dry matter digestibility. CONCLUSIONS: Supplementation of low S-containing diets with either sodium sulfate or MPA stimulates microbial growth with an increase in rumen microbial protein supply to the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of real-time PCR monitoring, a better understanding of the effect of S supplementation on discrete microbial populations within the rumen is provided.     

Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage

Aims: To determine the effects of the removal of forage in high‐concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture‐independent methods. Methods and Results: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson’s index of 16S PCR‐DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real‐time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet. Conclusions: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined. Significance and Impact of the Study: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.     

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.     

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

AIMS: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. METHODS AND RESULTS: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 10(10)R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. CONCLUSIONS: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.     

Supplementation of a clay mineral-based product modulates plasma metabolomic profile and liver enzymes in cattle fed grain-rich diets

Grain-rich diets often lead to subacute ruminal acidosis (SARA) impairing rumen and systemic cattle health. Recent data suggest beneficial effects of a clay mineral (CM)- based product on the rumen microbiome of cattle during SARA. This study sought to investigate whether the CM supplementation can counteract SARA-induced perturbations of the bovine systemic health. The study used an intermittent diet-induced SARA-model with eight dry Holstein cows receiving either no additive as control or CM via concentrates (n=8 per treatment). Cows received first a forage diet (Baseline) for 1 week, followed by a 1-week SARA-challenge (SARA 1), a 1-week recovery phase (Recovery) and finally a second SARA-challenge for 2 weeks (SARA 2). Cows were monitored for feed intake, reticular pH and chewing behavior. Blood samples were taken and analyzed for metabolites related to glucose and lipid metabolism as well as liver health biomarkers. In addition, a targeted electrospray ionization-liquid chromatography-MS-based metabolomics approach was carried out on the plasma samples obtained at the end of the Baseline and SARA 1 phase. Data showed that supplementing the cows’ diet with CM improved ruminating chews per regurgitated bolus by 16% in SARA 1 (P=0.01) and enhanced the dry matter intake during the Recovery phase (P=0.05). Moreover, the SARA-induced decreases in several amino acids and phosphatidylcholines were less pronounced in cows receiving CM (P≤0.10). The CM-supplemented cows also had lower concentrations of lactate (P=0.03) and biogenic amines such as histamine and spermine (P<0.01) in the blood. In contrast, the concentration of acylcarnitines with key metabolic functions was increased in the blood of treated cows (P≤0.05). In SARA 2, the CM-cows had lower concentrations of the liver enzymes aspartate aminotransferase and γ-glutamyltransferase (P<0.05). In conclusion, the data suggest that supplementation of CM holds the potential to alleviate the negative effects of high-grain feeding in cattle by counteracting multiple SARA-induced perturbations in the systemic metabolism and liver health.     

Parameters of fermentation and rumen microbiota of Nellore steers fed with different proportions of concentrate in fresh sugarcane containing diets

The objective of this study was to evaluate the effect of a fresh sugarcane-based diet and different roughage-to-concentrate ratios (70:30, 60:40, 40:60 and 20:80) on the rumen microbiota associated with rumen fermentation parameters and the intake and apparent digestibility of nutrients in Nellore steers. Eight rumen-cannulated Nellore steers (331 ± 8 kg BW) were distributed in a double 4 × 4 Latin square design balanced for the control of the residual effect. The ruminal pH decreased (p < 0.01) and the concentrations of N–NH₃, isovaleric and valeric acids increased linearly (p < 0.05) with an increase dietary concentrate level. Furthermore, an increased concentrate proportion reduced the population of Fibrobacter succinogenes and Ruminococus flavefaciens (p < 0.01) and increased the population of Selenomonas ruminantium and Megasphaera elsdenii (p < 0.01). The protozoa count revealed a predominance of the genus Entodinium. The synthesis of microbial N [g/d] and the efficiency of microbial synthesis [g of microbial N/kg of organic matter apparently digested in the rumen] increased as the proportion of concentrate was increased (p < 0.05). Therefore, it can be concluded that an increasing proportion of concentrate in sugarcane-containing diets enhances the synthesis of microbial protein and does not alter the fibre digestibility, although the population of fibre fermenting bacteria was reduced.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

The stimulatory effect of the organic sulfur supplement, mercaptopropane sulfonic acid on cellulolytic rumen microorganisms and microbial protein synthesis in cattle fed low sulfur roughages

Two metabolism trials (experiments 1 and 2) were conducted to examine the effect of the organic S compound, sodium 3-mercapto-1-propane sulfonic acid (MPS) on feed intake, fiber digestibility, rumen fermentation and abundance of cellulolytic rumen microorganisms in cattle fed low S (<0.11%) roughages. Urea was provided in all treatments to compensate for the N deficiency (<0.6%) in the roughages. In experiment 1, steers (333 ± 9.5 kg liveweight) were fed Angleton grass (Dicanthium aristatum) supplemented with S in equivalent amounts as either MPS (6.0 g/day) or sodium sulfate (9.56 g/day). Supplementation of Angelton grass with either sulfate or MPS resulted in an apparent increase in flow of rumen microbial protein from the rumen. Sulfur supplementation did not significantly change whole tract dry matter digestibility or intake, even though sulfate and MPS supplementation was associated with an increase in the relative abundance of the fibrolytic bacteria Fibrobacter succinogenes and anaerobic rumen fungi. Ruminal sulfide levels were significantly higher in the sulfate treatment, which indicated that the bioavailability of the two S atoms in the MPS molecule may be low in the rumen. Based on this observation, experiment 2 was conducted in which twice the amount of S was provided in the form of MPS (8.0 g/day) compared with sodium sulfate (6.6 g/day) to heifers (275 ± 9 kg liveweight) fed rice straw. Supplementation with MPS compared with sulfate in experiment 2 resulted in an increase in concentration of total volatile fatty acids, and ammonia utilization without a change in feed intake or whole tract fiber digestibility even though S and N were above requirement for growing cattle in both these treatment groups. In conclusion, supplementation of an S deficient low-quality roughage diet with either MPS or sodium sulfate, in conjunction with urea N, improved rumen fermentation, which was reflected in an increase in urinary purine excretion. However, MPS appeared to have a greater effect on stimulating short-chain fatty acid production and ammonia utilization when provided at higher concentrations than sulfate. Thus, the metabolism of MPS in the rumen needs to be investigated further in comparison with inorganic forms of S as it may prove to be more effective in stimulating fermentation of roughage diets.     

Magnesium absorption as influenced by the rumen passage kinetics in lactating dairy cows fed modified levels of fibre and protein

The potassium sensitive magnesium absorption through the rumen wall may be influenced by additional dietary properties, such as diet type, forage type or forage to concentrate ratio. These properties are likely associated to rumen passage kinetics modified by dietary fibre content. The study aimed to assess the effects of rumen passage kinetics on apparent Mg absorption and retention in lactating dairy cows fed modified levels of fibre. Six lactating Red-Holstein and Holstein cows, including four fitted with ruminal cannulas were randomly assigned to a 3 × 3 cross-over design. The experimental diets consisted of early harvested low NDF (341 g NDF/kg DM) and late harvested high NDF (572 g NDF/kg DM) grass silage (80% DM) and of concentrates (20% of DM). As the low-fibre diet was excessive in protein, a third high-fibre diet was formulated to be balanced in digestible protein with the low-fibre diet to avoid any eventual confounding effects of NDF and protein excess. All diets were formulated to contain iso-Ca, -P, -Mg, -K and -Na. Passage kinetics of solid and liquid phase of rumen digesta were evaluated using ruminal marker disappearance profiles. Cows fed the low-fibre diet had compared to the other diets, an up to 40% lower solid and 26% lower liquid phase volume of rumen digesta and a 10% numerically higher fractional rumen liquid passage rate. Rumen pH lost 0.6 units and Mg concentration in the rumen liquid phase tripled when cows were fed the low-fibre diet. Faecal Mg excretion was up to 14% higher in cows fed the low-fibre diet and Mg absorbability was 12% compared to up to 19% in other diets. Urinary Mg excretion in cows fed the low-fibre diet was half of the ones in the other treatments, but Mg retention was not affected. Dietary protein excess neither affected rumen passage kinetics nor Mg absorption and retention. Absorption of Mg was correlated with rumen liquid volume which both decreased with decreasing daily NDF intake (NDFi, 11.8 ± 2.4 l/kg NDFi). Consequently, daily Mg absorption decreased by 1.32 ± 0.28 g/kg decreasing NDFi. To conclude, in addition to the known antagonistic effect of dietary K, the present data indicate that Mg absorption was dependent from NDFi which modified rumen liquid volume, but was independent of dietary protein excess likely associated to low NDF herbages.     

Effects and modes of action of live yeasts in the rumen

Live yeasts (Saccharomyces cerevisiae) are more and more widely used as feed additives for ruminants. They are considered as allochtonous microorganisms in the rumen environment, however, distributed daily to dairy cows or beef cattle they can survive in the digestive tract and interact with autochtonous microbial populations. The positive effects of yeast cells have been mainly demonstrated on growth and activity of fibre-degrading bacteria and fungi, on stabilisation of rumen pH and prevention of lactate accumulation, on ruminal microbial colonization and on the set up of fermentative processes during the pre-weaning period. Modes of action of yeast probiotics depend on their viability and stability in the rumen ecosystem. Up to now, the main modes of action identified are the supply of growth factors to rumen microorganisms, oxygen scavenging inducing more favourable conditions for the anaerobic communities, and nutritional competition with autochtonous ruminal species. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Comparison of bacterial communities in faeces of beef cattle fed diets containing corn and wet distillers' grain with solubles

Aim: The mammalian intestinal microflora has been shown to impact host physiology. In cattle, intestinal bacteria are also associated with faecal contamination of environmental sources and human illness via foodborne pathogens. Use of wet distillers’ grains with solubles (WDGS) in cattle feed creates a gastrointestinal environment where some bacterial species are enriched. Here, we examine if a diet containing 40% WDGS results in fundamentally different microbial community structures. Methods and Results: The 20 002 16S r‐RNA gene sequences from 20 beef cattle were analysed using Sanger sequencing methods. At the genus level, Prevotella (Gram negative) and Anaerobacter (Gram positive) were the most frequently occurring bacteria in our beef cattle faecal samples. Diet‐associated differences in prevalence were noted for Prevotella but not Anaerobacter. Conclusions: Diet affects community structure. Faecal communities of co‐housed beef cattle are not identical. Significance and Impact of the Study: It is known that a diet of 40% corn‐based WDGS increases the generic Escherichia coli in the faeces and enriches E. coli O157:H7. The results from the current study suggest that in addition to previously observed changes in E. coli, the entire bacterial community structure is different for animals fed 40% corn‐based WDGS compared to a traditional corn‐finishing diet.