An rRNA approach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acid deamination.

Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia. Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle. 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen. Clostridium aminophilum was monensin sensitive in vitro, but C. aminophilum persisted in the rumen after monensin was added to the diet. An in vitro culture system was developed to assess the competition of C. aminophilum, P. anaerobius, and C. sticklandii with predominant ruminal bacteria (PRB). PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates. PRB did not hybridize with the probes to C. aminophilum, P. anaerobius, or C. sticklandii. PRB deaminated Trypticase in continuous culture, but the addition of C. aminophilum, P. anaerobius, and C. sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia. C. aminophilum, P. anaerobius, and C. sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein. Monensin eliminated P. anaerobius and C. sticklandii from continuous cultures, but it could not inhibit C. aminophilum. The monensin resistance of C. aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture. On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.     

Effect of monensin and 2-bromoethanesulfonic acid on fatty acid metabolism and methane production from cattle manure

Summary The effect of monensin and 2-bromoethanesulfonic acid (BESA) on methane production from cattle manure and on volatile fatty acids metabolism was tested. At 10 days retention time 0.81 biogas per liter cattle manure and day were produced. Methanogenesis was inhibited 20% by 3 mM BESA per liter and 45% by 2–5 mg monensin per liter. When the digestion was inhibited with either of the both drugs, the acetate pool increased drastically. Like in untreated fermentations the propionate pool increased in BESA-inhibited fermentations for several hours after substrate addition. After 24 h however it did not decrease to the low level reached in non-inhibited fermentations. When monensin was the inhibitor, the propionate pool did not change for 15 h, but then decreased with the same rate as in the control experiment. Adaptation processes or detoxification may be responsible for the delayed degradation.The degradation of low concentrations of buty-rate to acetate and the turn over rates of the butyrate pool are almost identical in cattle manure containing BESA, monensin, or no inhibitor. The turn over of ¹⁴C-acetate from butyrate degradation is delayed in BESA and monensin inhibited fermentations.From the data presented it can be concluded, that BESA mainly inhibits the methanogens, while monensin seems to inhibit both, methanogenic and nonmethanogenic organisms. However, a fast adaptation to or detoxification of the antibiotic seems to occur.     

Cationomycin and monensin partition between serum proteins and erythrocyte membrane: consequences for Na+ and K+ transport and antimalarial activities

The ionophore properties of cationomycin and monensin were studied on human erythrocytes by measuring Na+ influx by 23Na NMR and concomitant K+ efflux by potentiometry in the presence of increasing amounts of serum. Both ion currents (Na+ or K+) decreased linearly with the reciprocal of serum amount. The serum effects on ion currents were stronger with cationomycin than with monensin. Assuming this decreased transport activity was due to drug binding to serum proteins, a partition coefficient between the protein and the membrane phase was determined for each ionophore by using a novel model. This partition coefficient is about 30 times higher for cationomycin than for monensin; the same result was obtained with purified human serum albumin, indicating that albumin may be the major ionophore binding protein of serum. In parallel, we also measured IC50 for 50% in vitro growth inhibition of Plasmodium falciparum, the agent of malaria. In the presence of increasing serum concentrations, the antimalarial activity was decreased for both ionophores. Serum effect was less severe for monensin than for cationomycin, in agreement with the weaker interaction of monensin with proteins as shown from the partition coefficient values. A correlation was established between the ion transport currents (sodium and potassium) and the IC50 measured on P. falciparum in the presence of the various concentrations of serum. The relative value of the ion transport currents (expressed as percentage of control in absence of serum) can be indicative of the ionophore unbound fraction in the medium.     

Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.     

In vitro method for determining the ruminal degradation rate of rapeseed meal protein using N isotope labelled ammonia nitrogen

An in vitro method based on observations of ¹⁴N and ¹⁵N isotope fluxes between ammonia N and non-ammonia (NAN) pools was established to study the ruminal degradation rate of rapeseed meal protein. Feed protein equal to 125 mg of N/l was incubated in the presence of rumen fluid, mineral buffer, and a carbohydrate mixture formulated to provide a constant supply of fermentable energy over the entire incubation period. The ammonia N was labelled with the ¹⁵N isotope, and the incubations were carried out for 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, and 10 h. A model with six pools was used to estimate the rate of protein degradation to ammonia N and the rate of microbial N synthesis from ammonia N. The parameter values were adjusted based on the sizes of the ammonia ¹⁴N, ammonia ¹⁵N, ¹⁴NAN, and ¹⁵NAN pools observed at different time points over the incubation period. The rate of rapeseed meal N degradation was 0.06/h (0.028 standard deviation between runs), and the predicted effective protein degradability was 0.38 (0.122 standard deviation between runs). The current approach seemed appropriate for determining microbial N synthesis from ammonia N, but measurement of the direct incorporation of amino acids into microbial N may be required to adequately characterize the metabolic events involved in ruminal protein degradation.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts.

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Effect of monensin on rumen metabolism in vitro.

The effect of Monensin (Rumensin, Eli Lilly & Co.) in incubations with mixed rumen microorganisms metabolizing carbohydrate or protein substrates was investigated. Monensin partly inhibited methanogenesis and increased propionate production, although the effect was not always statistically significant. Incubations with substrates specific for methane bacteria suggest that inhibition of methanogenesis by Monensin was not due to a specific toxic action on the methanogenic flora, but rather to an inhibition of hydrogen production from formate. Total and net microbial growth were considerably decreased by addition of Monensin, although the amount of substrate fermented was not altered, resulting in lowered values of microbial growth efficiency. In incubations with casein, Monensin lowered protein degradation in line with a lowered ammonia production, whereas a slight accumulation of alpha-amino nitrogen was observed. The results suggest that besides an influence of Monensin on the rumen carbohydrate fermentation pattern, another reason for the beneficial effects observed in vivo might be decreased food protein degradation in the rumen, altering the final site of protein digestion in the animal. Also, the possibility of a decrease in rumen microbial growth efficiency has to be considered when using Monensin as a food additive.     

Influence of dietary acetylated peptides on fermentation and peptidase activities in the sheep rumen

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro . An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples were taken during the last 7 d. Fermentation products and NH₃ concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala₂ also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala₂ hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides–this rate was 26% of that found with untreated peptides–and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.     

Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin

A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs. Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr. Calcium chloride inhibited the toxin-induced change in egg permeability. Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin. Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality. Exopeptidase activity in eggs of T. colubriformis was reduced significantly after exposure to the B. t. israelensis toxin. Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B. t. israelensis toxin for eggs. Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity. Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X). Microscopic measurement of T. colubriformis eggs after treatment with the microbial toxin revealed no significant size change.     

微生物介导反刍动物瘤胃氨生成及其对瘤胃功能的影响

反刍动物瘤胃中栖息着丰富多样的微生物,其在瘤胃内氨生成过程中发挥了重要的作用。微生物介导的氨基酸脱氨基作用和非蛋白氮水解作用是瘤胃内氨生成的主要途径。微生物介导了瘤胃内氨的生成,同时瘤胃内产生的氨也会反馈影响微生物菌群结构及瘤胃上皮功能,进而影响瘤胃发酵及宿主健康。本文主要综述了瘤胃微生物在介导氨生成中的作用和氨对瘤胃消化及瘤胃上皮功能的影响,以期对后续研究有所启发。     

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.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Monensin and dichloroacetamide influences on methane and volatile Fatty Acid production by rumen bacteria in vitro

The effect of monensin (0 or 33 mug/g of diet) upon rumen fermentation in the presence and absence of methanogenesis was determined in vitro by using mixed rumen organisms continuously cultured for 17 days. Methane was inhibited by dichloroacetamide (DCA; 32 mg/day) or by a pH of 5.1. Monensin effected a significant decrease in the ratio of acetic to propionic acid in the presence or absence of methanogenesis. In the absence of methanogenesis, the decrease in the ratio of acetic to propionic acid was entirely the result of increased propionic acid, whereas in the presence of methanogenesis the decrease in the ratio was the result of a combination of decreased acetic acid and increased propionic acid. There was a complementary interaction between monensin and DCA on volatile fatty acid production (expressed as millimoles of carbon per day). Addition of monensin to DCA-treated cultures resulted in the production of more acid; however, monensin and DCA had no beneficial effect on total carbon formed as acid and gases as compared with nonsupplemented control cultures. The monensin and DCA also resulted in greater digestion of neutral detergent fiber and less accumulation of formic acid and hydrogen as end products than did DCA alone. l-Lactic acid was produced in small but significantly greater amounts by the low-pH cultures, which also had less volatile fatty acid carbon formed from the fiber fraction of the forage supplied.     

Heat treatment of soybean meal and rapeseed meal suppresses rumen degradation of phytate phosphorus in sheep

The effect of heat treatment on rumen degradation of phytate in soybean meal and rapeseed meal was studied on three sheep fitted with rumen cannula. Soybean meal and rapeseed meal were roasted at 133°, 143° or 153°C for 3 h and the rumen degradation of phytate phosphorus in untreated and heat treated oilseed meals was examined using the nylon-bag technique. Effective degradability of phytate phosphorus in soybean and rapeseed meals, estimated at ruminal outflow rates of 0.02, 0.05 and 0.08 h⁻¹, was significantly (p < 0.05) reduced by heat treatment. The reduction was more marked in rapeseed meal than in soybean meal. These results suggest that heat processing of oilseed meals suppresses phytate degradation in the rumen and leads to a low availability of dietary phytate phosphorus.     

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.     

Effect of monensin on the specific activity of ammonia production by ruminal bacteria and disappearance of amino nitrogen from the rumen.

When unadapted mixed ruminal bacteria (312 mg of protein per liter) were treated with monensin (5 mM) in vitro, the rates of ammonia production from enzymatic digests of casein, gelatin, and soy protein (0.5 g of N per liter) were decreased from 46 +/- 2 to 24 +/- 1, 20 +/- 1 to 7 +/- 1, and 40 +/- 2 to 18 +/- 2 nmol/mg of protein per min, respectively. Monensin also caused a decrease in ammonia production in vivo. Nonlactating dairy cows which were fed 0.56 kg of timothy hay 12 times per day had a steady-state ruminal ammonia concentration of 2.7 +/- 0.1 mM, and the ammonia concentration decreased to 1.2 +/- 0.2 mM when monensin (350 mg/day) was added to the diet. The decrease in ammonia production was associated with a 10-fold reduction (4.1 x 10(6) versus 4.2 x 10(5)/ml) in the most probable number of ammonia-producing ruminal bacteria that could use protein hydrolysate as an energy source. Monensin had little effect on the most probable number of carbohydrate-utilizing ruminal bacteria (6.5 versus 7.0 x 10(8)/ml). The addition of protein hydrolysates (560 g) to the rumen caused a rapid increase in the ammonia concentration, but this increase was at least 30% lower when the animals were fed monensin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms.

The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.     

Establishment of cellulolytic bacteria and development of fermentative activities in the rumen of gnotobiotically-reared lambs receiving the microbial additive Saccharomyces cerevisiae CNCM I-1077

We studied the effects of a yeast additive used in ruminant nutrition on the establishment of cellulolytic bacteria, on plant cell wall degradation and on digestive functions in the rumen of gnotobiotically-reared lambs. Cellulolytic bacteria inoculated to the lambs tended to become established earlier in the presence of Saccharomyces cerevisiae CNCM I-1077 (SC). In addition, their population was maintained at a higher level, when the physico-chemical conditions of the biotope were altered. In these lambs, specific activities of fibrolytic enzymes were greater, and in sacco degradation of wheat straw tended to increase. In the presence of SC there was a decrease in ruminal ammonia concentration and a higher volatile fatty acid (VFA) concentration when lambs were 20 to 50 days old. These data suggest that this yeast strain may stimulate the development of cellulolytic microflora and enhance microbial activity in the rumen of young ruminants. Such activity could be beneficial in preventing microbial imbalance and a reduction of rumen function efficiency in the case of nutritional transitions. Further studies with conventional animals will soon be performed in order to verify these dings.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Abstract Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77 g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH3) and pH of rumen fluid were within the normal range for all dietary treatments (NH3: 9.1-32.3 mmol x l(- l), pH: 6.2-6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.