The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.     

The antimicrobial effects of hops (Humulus lupulus L.) on ruminal hyper ammonia-producing bacteria

Aim:  To assess the antimicrobial effects of hops ( Humulus lupulus L.) on hyper ammonia producing-bacteria (HAB), which catabolize amino acids and peptides in the bovine rumen.
Methods and Results:  When media were amended with dried hops or hops extract (30·7% lupulone), ammonia production by mixed rumen bacteria was inhibited. The growth and ammonia production of pure cultures ( Peptostreptococcus anaerobius, Clostridium aminophilum, or Clostridium sticklandii ) was inhibited by 30 ppm lupulone at pH 6·7, and bactericidal activity was observed at pH 5·6. When hops extract was added to energized cell suspensions, the intracellular pH rapidly decreased and intracellular potassium was lost.
Conclusions:  The three HAB species were sensitive to the antimicrobial components in hops, and the inhibition of ammonia production by mixed rumen bacteria indicates that similar effects could be expected in the rumen.
Significance and Impact of the Study:  As much as half of the amino acids consumed by ruminants can be lost due to microbial degradation in the rumen. This study supports the idea that biologically active plant metabolites can be used to mitigate this wasteful process.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

Total mixed ration pellets for light fattening lambs: effects on animal health

Fifty male Merino lambs (6 to 8 weeks, 14.1 kg; n=10 per group) were used to study the effect of feeding system: barley straw in long form and concentrate pellets in separate troughs (Control), ad libitum alfalfa supplemented with concentrate in separate troughs (Alfalfa) or including various levels of ground barley straw in concentrate pellets (B05, B15 and B25 for 50, 150 and 250 g barley straw/kg), on rumen characteristics, acid-base status, blood cell counts and lymphocyte stimulation. Alfalfa lambs had the heaviest digestive tract contents, highest rumen pH values, lowest volatile fatty acid concentration, highest papillae counts and best mucosa colour and the greatest blood pCO₂ values, lowest sodium and chloride and highest potassium concentrations (P<0.05). Including ground barley straw in the concentrate pellet or providing straw in long form separately from the concentrate reduces rumen pH and darkens ruminal mucosa as compared with alfalfa-fed lambs, thus affecting acid-base status.     

Influence of barley grain particle size and treatment with citric acid on digestibility,ruminal fermentation and microbial protein synthesis in Holstein calves

Chemical and physical treatments of barley grain increase ruminally resistant starch and can improve the rumen fermentation pattern. The objective of the present study was to evaluate the effects of chemical (addition of citric acid, CA) and physical (grinding to two different particle sizes, PS) treatment of barley grain on performance, rumen fermentation, microbial protein yield in the rumen and selected blood metabolites in growing calves. In all, 28 male Holstein calves (172±5.1 kg initial BW) were used in a complete randomised design with a factorial arrangement of 2 barley grain particle sizes×2 levels of citric acid. The diets were as follows: (i) small PS (average 1200 µm) barley grain soaked in water (no CA addition); (ii) small PS barley grain soaked in a CA solution (adding 20 g CA/kg barley); (iii) large PS (average 2400 µm) barley grain soaked in water (no citric acid addition) and (iv) large PS barley grain soaked in a citric acid solution (adding 20 g CA/kg barley). Barley grain was then incorporated at 35% in a total mixed ration and fed to the calves for 11 weeks. Feeding small PS barley decreased feed intake (P=0.02) and average daily weight gain (P=0.01). The addition of CA to barley grain did not affect intake but increased weight gain (P<0.01) and improved feed to gain ratio (P=0.03). Digestibility of organic matter and NDF tended (P<0.10) to increase, whereas faecal scoring was improved (P=0.03) and the presence of undigested grain particles in faeces was reduced (P<0.01) with CA-treated barley grain. Glucose and urea concentrations were increased (P<0.01) in the blood of calves fed the CA-treated barley grain. Ruminal pH tended (P=0.08) to be decreased with more finely ground barley and was increased when barley grain was treated with CA. Total volatile fatty acid concentrations in the rumen did not differ among treatments (P>0.05). However, the molar proportion of propionate was increased (P=0.03) when barley was more finely ground, and that of acetate was increased (P=0.04) when CA was added to barley grain. The ruminal concentration of ammonia nitrogen was increased (P<0.01) and microbial nitrogen synthesis in the rumen tended to decrease by adding CA to barley. Treating barley grain with citric acid increased fibre digestibility of total mixed rations, attenuated the decrease in ruminal pH, and improved weight gain and feed efficiency in male Holstein growing calves fed a high-cereal diet (550 g cereal grain/kg diet).     

Effect of Ammonia Concentration on the Composition, Hydrolytic Activity and Nitrogen Metabolism of the Microbial Flora of the Rumen

The mean NH₃ concentration in the rumen of sheep fed whole barley (08 kg/d) by continuous feeders was increased from 61 to 134 HIM by supplementing the feed with urea (30 g/kg). This change caused a 90% increase in the rate of degradation of rolled barley, and smaller increases in the rates of degradation of protein and plant fibre in the rumen. The total viable count and numbers of pectinolytic bacteria in rumen fluid increased with the urea supplement. Enzyme studies indicated that NAD-linked glutamate dehydrogenase was the main pathway of NH₃ assimilation by rumen bacteria at both NH₃ concentrations. Glutamate was the main free amino acid found in the rumen at low NH₃ but, despite the low activity of alanine dehydrogenase and glutamate-pyruvate aminotransferase, alanine was the principal amino acid at high NH₃ concentrations. Hydrolytic rumen bacteria may require the higher NH₃ concentration either for effective NH₃ assimilation by an unknown mechanism involving alanine or for full expression of enzyme activity.     

Growth and Cellulase Formation by Cellvibrio fulvus

S ummary : The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell-bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p -chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.     

Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens

When incubated in axenic culture, strains of anaerobic rumen fungi were more active than cellulolytic bacteria in solubilizing barley straw stem fragments 5 to 10 mm in length. Pretreatment with ammonia had little effect on microbial attack. Of three species of methanogens tested, Methanobrevibacter smithii strain PS formed the most stable and reproducible co-cultures with the fungi and with Ruminococcus albus , and the presence of this organism enhanced the extent of degradation of straw, although this effect was less marked than that previously observed when pure cellulose was used as substrate.     

The redox potential, rH and pH values in the gastrointestinal tract of small ruminants

Oxidation-reduction potentials (Eh), pH and rH in the gastrointestinal tract were measured in six goats and two sheep fed on ground barley and hay, with or without the addition of urea. Each ration was supplied for three weeks. The animals were slaughtered after morning feeding, the contents of relevant parts of the gastrointestinal tract were sampled, pH and Eh values were measured and rH values calculated. The range of the oxidation-reduction potential was rather extensive, from -300 to +186 mV. The variability of rH values was smaller, being between 4.6 and 12.9, except for three values. The following linear relationship holds for findings of Eh and pH: Eh (mV) = 292-69.9 pH with a correlation coefficient of r = 0.84. In those parts of the gastrointestinal tract, where the fermentation process occurs (the rumen, caecum and colon), the Eh and rH values are lower than those in the abomasum and duodenum. Urea addition has no effect on the oxidation-reduction equilibrium.     

Indigestion in Young Calves. II. The Influence Of Ground Barley,Coarse and Fine Hay

Four experiments comprising 86 calves have been carried out in order to examine the influence of ground barley, coarse and fine hay, when raising young calves on a high lactose milk replacer causing diarrhoea. The frequency of diarrhoea decreased when feeding barley (P < 0.01) or fine hay (P < 0.001), but increased when feeding coarse hay (P<0.01). Barley increased (P < 0.01) live weight gain and carcass weight, reduced (P < 0.001) rumen pH, favoured gram-positive cocci and rods in the rumen, caused hyperkeratosis and gave increased (P < 0.01) empty reticulo-rumen weights, the latter being supposed to be enhanced by hyperkeratosis. The calves ate more (P < 0.001) fine than coarse hay, fine hay being superior (P < 0.01) in promoting growth of the rumen. All hay maintained a predominantly gram-negative rumen flora, but somewhat different from that on the milk replacer, and counteracted (P < 0.01) low pH in the rumen and apparently also hyperkeratosis due to barley. The results with barley indicated no relationship between diarrhoea and hyperkeratosis.     

High Molecular Weight Protease in Rumen Ciliate Protozoa

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

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Ground,dry-rolled and steam-processed barley grain for midlactation Holstein cows

Fine grinding of barley grain has traditionally been considered to be a potential risk to rumen function, feed intake and milk yield. These concerns are thought to be reduced by steam-rolling or coarse dry rolling. We hypothesized that finely ground barley grain is as effective in stimulating feed intake and milk production as are dry- and steam-rolled barley grain, and so the objective was to determine effects of feeding either (1) finely ground, (2) steam-rolled, (3) finely dry-rolled, or (4) coarsely dry-rolled barley grain on rumen fermentation, digestibility and milk yield and composition. Eight multiparous midlactation Holstein cows were used in a replicated 4×4 Latin square design experiment with four periods of 21 d. Diets contained 256 g barley grain/kg on a dry matter (DM) basis. Processing method did not affect milk yield and composition, DM intake, rumen pH and volatile fatty acids, fecal and urine pH, and apparent total tract nutrient digestibility. Results suggest that finely ground barley grain is no different than dry-rolled and steam-rolled barley grains in stimulating feed intake and productivity of midlactation cows, when 256 g of dietary DM/kg is barley grain.     

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.     

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.     

Effect of pH on Population and Fermentation in a Continuously Cultured Rumen Ecosystem

The effect of pH on rumen fermentation and microbial population was studied in a continuously cultured rumen ecosystem. A marked decrease in the production of volatile fatty acids and methane from alfalfa hay occurred when the cultures were maintained at pH values below 6.0. The decrease in acetate and methane production was greater than that of propionate production. The culture maintained at pH 6.7 contained the types of bacteria often found in high concentration in the rumen, whereas the culture maintained at pH 5.0 had a high percentage of bacteria which could not be identified with the major rumen bacteria found in rumens of animals fed alfalfa hay. Replacement of the bicarbonate-phosphate buffer used to maintain fermentor pH at 6.7 with phosphate alone did not greatly alter the fermentation products produced from a hay-concentrate substrate.     

Factors affecting lysine degradation by ruminal fusobacteria

Fusobacterium necrophorum can readily be enriched from the rumen with lysine, and its deamination rate is very rapid. The addition of F. necrophorum JB2 to mixed ruminal bacteria significantly increased lysine degradation, but only if the ratio of ruminal fluid to basal medium was less than 25%. If more ruminal fluid (pH 6.1) was added, ammonia production decreased by as much as 80%. Clarified, autoclaved ruminal fluid was also inhibitory. When F. necrophorum JB2 was grown in a lysine-limited continuous culture (0.1 h(-1) dilution rate) and pH was decreased using HCl, optical density decreased linearly, and the culture washed out at pH 5.6. Batch cultures of F. necrophorum JB2 deaminated as much lysine at pH 6.1 as at pH 6.6, but only if fermentation acids were not present. Sodium acetate (100 mM) had little effect at pH 6.6, but the same concentration inhibited ammonia production by 80% at pH 6.1. The idea that fermentation acids could prevent the enrichment of fusobacteria in vivo was supported by the observation that dietary lysine supplementation did not enhance the lysine deamination rate of the mixed ruminal bacteria.