Ml Of Rumen Fluid Research Articles

Physiological diversity of rumen spirochetes.

Bovine rumen fluid contained relatively large numbers of spirochetes capable of fermenting polymers commonly present in plant materials. Polymers such as xylan, pectin, and arabinogalactan served as fermentable substrates for the spirochetes, whereas cellulose did not. Furthermore, spirochetes cultured from rumen fluid utilized as growth substrates hydrolysis products of plant polymers (e.g., D-xylose, L-arabinose, D-galacturonic acid, D-glucuronic acid, cellobiose), but did not ferment amino acids. Viable cell counts of spirochetes capable of fermenting individual plant polymers or their hydrolysis products yielded minimum values ranging from 0.2 X 10(6) to 4 X 10(6) cells per ml of rumen fluid. Thirteen strains of rumen spirochetes were characterized in terms of their fermentation products from glucose, the guanine plus cytosine content of their DNA, their ultrastructure, and their ability to ferment pectin, starch, or arabinogalactan. Of the 13 strains, 6 fermented glucose mainly to formate, acetate, and succinate, whereas the remaining 7 strains did not produce succinate, but instead formed ethanol, in addition to formate and acetate. The succinate-forming strains had two periplasmic (axial) fibrils per cell, measured 0.2 to 0.3 by 5 to 8 micrograms, had a guanine plus cytosine content of the DNA ranging from 36 to 38 mol%, and lacked the ability to ferment pectin, starch, or arabinogalactan. The ethanol-forming strains had from 8 to more than 32 periplasmic fibrils per cell, tended to be larger in cell size than the succinate-forming strains, and had a guanine plus cytosine content of the DNA ranging from 41 to 54 mol%. Some of the ethanol-forming strains fermented pectin, starch, or arabinogalactan. The results of this study indicate that the bovine rumen is inhabited by a physiologically and morphologically diverse population of spirochetes. It is likely that these spirochetes contribute significantly to the degradation of plant materials ingested by the ruminants.

Sequestration of Holotrich Protozoa in the Reticulo-Rumen of Cattle

Studies were carried out to determine the means by which holotrich protozoa can maintain their numbers within the rumen against the washout effect associated with the flow of ingesta. When a diet composed of 2 kg of concentrate and 1.5 kg of rice straw was fed to Holstein cows, about a fourfold increase in holotrich numbers per ml of rumen fluid was observed within 1 h after the commencement of feeding, and an abrupt decrease followed. This fluctuation in numbers was not related to the time of feeding. A sole feeding of 2 kg of concentrate had almost the same effect on the holotrichs as a sole feeding of 1.5 kg of rice straw. Administration of either 2 kg of concentrate or 1.5 kg of rice straw through the rumen fistula caused similar changes, though the extent of response to the former was greater than that to the latter. The administration of either 0.7 kg of starch or 0.2 kg of glucose through the fistula had a relatively minor effect on the holotrich population. Addition of rice straw to 0.5 kg of concentrate increased the change in numbers, but its addition had little, if any, effect when 1 kg of concentrate was fed. These results suggested that the fluctuation in holotrich numbers was related not only to the nature or component of feed but also to other factors such as the quantity or volume of a diet and the act of ingesting feed. Increasing the number of feedings up to eight times per day at 3-h intervals caused a decrease in the peak heights of holotrich numbers per milliliter of rumen fluid. A thick protozoal mass which primarily consisted of holotrichs was found on the wall of the reticulum of Holstein steers slaughtered after overnight starvation. These findings suggest that holotrichs would usually sequester on the reticulum wall and migrate into the rumen only for a few hours after feeding, and that this mode of behavior would be essential for holotrichs to maintain their population within the rumen of cattle. Possible mechanisms of the migration are also discussed.

Enumeration and selective isolation of rumen spirochetes

Enumeration by means of light microscopy showed that from 0.4 x 10(8) to 2.0 x 10(8) spirochetes were present per ml of bovine rumen fluid. Viable cell counts yielded slightly lower values, ranging from 0.1 x 10(8) to 1.2 x 10(8) spirochetes per ml of rumen fluid. The antibiotic rifampin, which served as a selective agent for rumen spirochetes, was added to agar media used in the estimation of viable spirochete numbers in rumen fluid. Morphologically diverse spirochetes were isolated from rumen fluid by means of a procedure involving the use of rifampin as a selective agent in agar media. The strains isolated represented seven morphological types of spirochetes differing in cell size, cell coiling pattern, and number of periplasmic fibrils per cell. Electron microscopy showed that the number of periplasmic fibrils present in the different morphological types of rumen spirochetes ranged from 2 to more than 20 per cell. The results of this study indicate that the bovine rumen is a highly favorable environment for a number of morphologically diverse spirochetes.

Effect of feeding frequency on the diurnal changes in microbial protein, volatile fatty acids and ammonia contents of the rumen of sheep

SUMMARYDiurnal variations in microbial protein nitrogen, total volatile fatty acids and ammonia were examined in two wethers fed once and twice daily with 0·4 kg of concentrateand 0·4 kg hay/day. The total protein nitrogen varied during the day from 76·9 to 130·2 mg/100 ml of rumen fluid. The greatest amount of protein nitrogen was noted immediately before feeding and the smallest 4 h after feeding. The protein nitrogen of rumen protozoa formed about 39·8–53·4% of total protein nitrogen in rumen fluid. Volatile fatty acids varied from 74·0 to 130·5 m-mol/1 of rumen fluid and ammonia from 187·0 to 305·6 mg/1. The higher concentration of total and protozoal protein nitrogen and of VFA was observed when animals were fed twice daily. The changing of feeding frequency from once to twice daily caused an increase of ammonia concentration in the rumen of only one wether. The results obtained are discussed.

Effect of Ruminal Ammonia Concentration on Nitrogen Utilization by Steers

The effect of ruminal ammonia concentration on nitrogen utilization was determined in eight rumen-fistulated steers weighing 183 to 226 kilograms. The steers received a basal 8.0% crude protein equivalent (CPE) diet that consisted of a forage-concentrate (30:70) mixture of 10.9% CPE which was fed plus cerelose that was infused. The animals were assigned to one of eight treatments in two 4 × 4 Latin squares. Ruminal ammonia concentration was varied by continuous intra-ruminal infusion of 15.3 liters/day of a tap water solution that contained cerelose, volatile fatty acids (VFA), magnesium sulfate, and between 0 and 140 g of urea. Dry matter digestibility was increased with the lowest level of urea infusion. Higher levels of urea infusion had no further effect on dry matter digestibility. Protein assayed as tungstic acid precipitable nitrogen (TAP-N) increased in ruminal digesta as urea input was increased to where the ammonia nitrogen (NH3-N) concentration was 2.2 mg/100 ml of rumen fluid. Increasing the NH3-N content beyond 2.2 mg/100 ml of rumen fluid had no effect on rumen digesta TAP-N. Increasing the NH3-N content beyond 4.5 mg/100 ml had no significant effect on total ruminal VFA concentration. Nitrogen retention increased as urea infusion increased until the ruminal NH3-N concentration was 4.5 mg/100 ml. Slight but nonsignificant further increases in nitrogen retention were observed as larger amounts of urea were infused, and ruminal NH3-N increased to 22.5 mg/100 ml. Possible explanations for this continued increase in nitrogen retention are discussed. Higher urea inputs increased blood plasma urea, arginine, and citrulline. The results of this study support the view that concentration of 2 to 5 mg NH3-N per 100 ml of rumen fluid is sufficient to allow maximum growth of rumen microbes.

A Colorimetric Method for the Determination of Proteolytic Degradation of Feed Proteins by Rumen Microorganisms

A new colorimetric method for the determination of the degradation of soybean meal by rumen microorganisms in vitro is described. Proteins in soybean meal were diazotized with 7-amino-1,3,naphthalene disulfonic acid. The diazotized meal was incubated at 37 C with rumen microbial suspension (approximately equivalent to 2.0 ml of rumen fluid) in 2.0 ml of. 1M potassium phosphate buffer, pH 7.6. The undigested diazo-protein was precipitated by the addition of perchloric acid and the precipitated protein was solubilized by incubating with crystalline bacterial protease. A portion of the solubilized protein was treated with .15N NaOH and the color intensity was measured at 440 nanometers. Application of the diazo method for the determination of the degradation of soybean meal when present as part of ruminant feed mixture, for evaluating the effectiveness of the protective treatment of soybean meal with formaldehyde and for the determination of solubility of soybean meal in boiled rumen fluid are described.

Transfer of R Factors from Escherichia Coli to Salmonellas in the Rumen of Sheep

Adult sheep were given into the rumen c. 10(8) cells each of donor strains of E. coli containing an R factor and prospective salmonella-recipient organisms and were maintained on a diet of lucerne chaff; the animals excreted the organisms, remained healthy, and no transfer of the R factor was detected. When the animals were starved for 48 h before inoculation, the ruminal environment was altered so that, on resumption of feeding, small numbers (c. 10(2)-10(4) cells--less than one cell per ml of rumen fluid) of the introduced organisms were able to multiply and reach sufficient numbers for the transfer of R factors to occur within the rumen. One animal, given 7-8 X 10(3) cells of recipient S. lomita after starvation for 48 h, became a carrier of this organism. A second animal, given 4-4 X 10(2) cells of S. typhimurium after starvation for 48 h, developed acute, fatal salmonellosis 5 days later; at the time of death, large numbers of salmonella organisms (c. 10(9) cells per g) were present in the faeces; these included many (c. 10(6) cells per g) that had received the R factor by transfer in vivo. These results indicate that short periods of starvation may enhance the transfer of R factors and possibly other plasmids between suitable micro-organisms in vivo, and may increase the susceptibility of animals to pathogenic micro-organisms.

Presence of bifidobacteria in the rumen of calves fed different rations

A study was made on the numbers and species of bifidobacteria present in the rumen of calves fed high-roughage and high-concentrate diets. With the roughage ration the bifidobacteria were not detectable in a 10(-3) dilution, whereas with the concentrate ration their number was high, usually in the order of 10(8) to 10(9)/ml of rumen fluid. The species most represented, identified by means of deoxyribonucleic acid-deoxyribonucleic acid hybridization tests, included Bifidobacterium ruminale, Bifidobacterium globosum, and an apparently new species.

Some morphological types of bacteriophages in bovine rumen contents.

Six morphological types of bacteriophage were found in bovine rumen contents. Minimal total phage count was 5 x 10(7) per ml of rumen fluid.

Some Morphological Types of Bacteriophages in Bovine Rumen Contents1

Six morphological types of bacteriophage were found in bovine rumen contents. Minimal total phage count was 5 x 10(7) per ml of rumen fluid.

In vitro lactate metabolism by ruminal ingesta.

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.

In Vitro Lactate Metabolism by Ruminal Ingesta1

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.