Bovine Rumen Contents Research Articles

Alkaloid Levels in a Species of Low Larkspur and Their Stability in Rumen Fluid

A survey on the levels of the neurotoxic diterpenoid alkaloid methyllycaconitine (MLA) in low larkspur (Delphinium nuttallianum Pritz.) was conducted at rangeland sites in southern British Columbia. Freeze-dried plant samples representing vegetative, flower bud, and bloom stages of growth over 4 growing seasons were analyzed for MLA by high pressure liquid chromatography (HPLC). Differences in MLA concentration were found between sites (P 0.2). The vegetative stages of growth yielded the highest levels of MLA, approaching 1% of the dry matter at 1 site. On average, the reproductive stages of growth yielded half the amount of MLA as the vegetative stages. Differences in MLA levels between sites could not be attributed to the elevation or the weather during the growing season. It is suggested that topoedaphic effects may have an impact on low larkspur growth and toxicity. Preliminary results are also reported on the stability of MLA in bovine rumen contents. The alkaloid is not readily hydrolyzed in rumen contents and therefore is probably not detoxified by this pathway.

Simulation in vitro des fermentations cæcales du lapin en fermenteur à flux semi-continu. II. Effet de la nature de l'inoculum

Two Rusitec fermentors were operated under identical conditions. One was seeded with an inoculum of rabbit caecal contents, and the other with bovine rumen contents. The fermentation substrate was rabbit feed that had been digested with amylase and pepsin. The substrate constituents (organic matter, OM and NDF) were lost in 48 h at a significantly higher rate in the presence of rumen inoculum (OM: +10%, NDF: +15%). The pHs of the 2 fermentors were similar at pH 6.6. The fermentors produced similar amounts of protein nitrogen per 24 h, after 6 d of adaptation. Volatile fatty acid production was slightly higher in the presence of rumen inoculum. The fermentor inoculated with rumen contents produced a higher percentage of propionic acid (25%) than of butyric acid (7%), while fermentation with rabbit caecal contents gave the opposite ratio (C3/C4 = 0.81). Consequently, only the rabbit caecal inoculum provided the fermentation profile characteristic of the species.

Production of benzodiazepine-like compounds in bovine rumen

The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found “in vivo” in samples obtained from a grazing cow with a rumen cannula.

Multiresidue Screen for Organophosphorus Insecticides Using Gel Permeation Chromatography—Silica Gel Cleanup

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.

Characteristics of methanogens isolated from bovine rumen.

Six strains of methanogens were isolated from 10(-8) and 10(-9) ml of bovine rumen contents. All strains had the morphologic and physiologic characteristics of Methanobrevibacter spp. Four strains required coenzyme M; two did not. Growth of all strains either depended on or was stimulated by a mixture of isobutyric, isovaleric, 2-methylbutyric, and valeric acids. None of the strains reacted with antiserum against the type strain of Methanobrevibacter ruminantium.

Thiomolybdates in rumen contents and rumen cultures

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants ot in vitro cultures of bovine rumen contents incubated with MoO 4 2− and potential sources ot S 2− and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates Only MoS 4 2− was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization ot thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS 4 2− rather than MoOS 3 2− was the predominant thiomolybdate species present in the aqueous phase Addition ot Cu 2+ either to rumen cultures before incubation or as a supplement to diets high in MoO 4 2− content inhibited the appearance ot MoS 4 2− in the aqueous phase. Evidence of the sequestration of MoS 4 2− and MoOS 3 2− by particulate or microbial tractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants

Isolation of proteolytic rumen bacteria by use of selective medium containing leaf fraction 1 protein (ribulosebisphosphate carboxylase).

The principle proteolytic bacteria isolated from bovine rumen contents by virtue of the ability to obtain nitrogen from the proteolysis of leaf fraction 1 protein (ribulosebisphosphate carboxylase, EC 4.1.1.39) were identified as Streptococcus bovis and Butyrivibrio spp. Substitution of fresh fodder, rich in soluble protein, for a hay-concentrates diet resulted in enhanced ruminal proteolytic activity and a significant increase in the number of bacteria able to use fraction 1 protein as the sole nitrogen source. Isolated proteolytic bacteria degraded fraction 1 protein and casein readily. Bovine serum albumin was attacked by Butyrivibrio spp. but was resistant to proteolysis by the streptococci.

Examination of the effect of copper and molybdenum on microbial protein synthesis in rumen contents using 35S

The influence of different concentrations of molybdenum and copper on microbial protein synthesis, volatile fatty acid production and other parameters has been investigated in bovine rumen contents incubated in vitro. The donor bull was fed a conventional diet based on a concentrate supplemented with urea and a hay. The concentrate contained Cu (6.5 mg/kg) and Mo (1.2 mg/kg). Firstly the concentration of molybdenum in the incubation mixture was raised from the basal level of 0.4 to 0.61 mg/L. It was found that volatile fatty acid production, net utilization of ammonia, sulphide concentration and overall protein synthesis were not significantly affected by the increase in molybdenum concentration. Secondly the concentration of copper was increased from 0.4 to 1.8 mg/L by adding copper acetate solution. The concentration of sulphide sulphur was significantly reduced when copper was added to the medium, but this appeared to have no significant effect on the utilization of sulphate sulphur for microbial protein synthesis. Ammonia utilization was not affected but volatile fatty acid production tended to be lower when copper concentrations were raised. Since the examined concentrations of copper and molybdenum were similar to those found in the rumen under normal conditions of animal production, it was concluded that these elements are unlikely to have a negative influence on the utilization of urea and sulphate for microbial protein synthesis when they are present in the diet in the amounts recommended in practice.

Degradation of maize protein in rumen contents Influence of ammonia concentration

1. The influence of ammonia concentration on the distribution of nitrogen derived from opaque-2 maize uniformly-labelled with 15N has been investigated during short-term in vitro incubation of bovine rumen contents. 2. Less 15N derived from maize was found in the non-protein-N (NPN) fraction during incubation without added NH3 than with added NH3, due entirely to differences in the amount of N derived from maize in the NH3 fraction. 3. From calculations based on the transfer of N derived from maize to the NPN pool and to a bacterial fraction, it was concluded that degradation of maize protein was not influenced by NH3 concentration within the examined limits. 4. The decrease in relative amount of N derived from maize in the NH3 fraction at low concentrations of NH3, together with evidence for an increased fractional turnover rate of NH3-N suggests that a deficient supply of NH3 is compensated for by increased catabolism of nitrogenous compounds derived from the rumen micro-organisms.

Isolation and characterization of large treponemes from the bovine rumen.

Ten strains of strictly anaerobic spiral organisms were isolated in pure culture from a 10(-7) dilution of bovine rumen contents. Three strains were studied in detail. These strains morphologically resembled previously isolated and described rumen treponemes except the new isolates were larger, 0.7 micrometer wide and 12 to 25 micrometer long. They rapidly fermented pectin and, less readily, L-arabinose, inulin, and sucrose. Acetic and formic acid were the main fermentation products from pectin; small amounts of succinic acid were also formed.

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.

Studies on rumen metabolism. VI. In vitro hydrolysis of triglyceride and isolation of a lipolytic fraction.

AbstractHydrolysis of groundnut oil and synthetic glyceryl tri(oleate‐1‐14C) by bovine rumen contents was demonstrated under in vitro conditions. Fractionation by differential centrifugation of total rumen contents obtained from a cow grazing on fresh pasture showed that the organisms responsible for hydrolysis of triglycerides were closely associated with the particulate material in the rumen and homogenisation released much of the lipolytic activity. Cell‐free extracts of mixed rumen bacteria were prepared by subjecting mixed rumen bacteria to high frequency sonication or osmotic shock followed by isolation of the released lipolytic enzymes by high‐speed centrifugation.A method for assaying lipase activity is described which uses glyceryl tri(oleate‐1‐14C) as substrate, and thin‐layer chromatography to separate the free fatty acids, mono‐, di‐ and tri‐glycerides prior to radio‐chemical determination. The method was developed using pancreatic lipase and was applied to the isolation of lipases from rumen micro‐organisms.

Formate as an intermediate in the bovine rumen fermentation.

An average of 11 (range, 2 to 47) mumoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k, times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K(m), for formate conversion to CH(4) was estimated at 30 nmoles/g. Also, the dissolved H(2) was measured in relation to methane production, and a K(m) of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K(m) of 1 nmole of H(2)/g, but the K(m) for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO(2) and H(2) are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.

Isolation and identification of rumen bacteria capable of anaerobic rutin degradation.

Fifteen strains of bacteria capable of degrading rutin anaerobically were isolated from bovine rumen contents and identified by morphological and biochemical evidence as strains of Butyrivibrio sp. Three cultures from a laboratory collection of 53 strains of rumen bacteria also used rutin anaerobically. Two, Butyrivibrio fibrisolvens D1 and Selenomonas ruminantium GA192, cleaved the glycosidic bond of rutin and fermented the sugar but did not degrade the insoluble aglycone produced; the third strain, Peptostreptococcus sp. B178, degraded the substrate to soluble products. Butyrivibrio sp. C3 degraded rutin, quercitrin, and naringin to water-soluble products, showing that the organism cleaved the heterocyclic ring of these compounds. Butyrivibrio sp. C3 fermented the sugar moiety of hesperidin but did not cleave the heterocyclic ring. It did not attack quercetin, taxifolin, protocatechuic acid, or phloroglucinol. In a medium containing rumen fluid, Butyrivibrio sp. C3 degraded rutin more than twice as fast as it did in a medium containing enzymatic casein hydrolyzate, volatile fatty acids, yeast extract, and hemin in place of rumen fluid.The observations reported in this paper are believed to represent the first recorded demonstration of degradation of the heterocyclic ring structure of rutin and other bioflavonoids in pure cultures of anaerobic bacteria.

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.

Pectin-fermenting bacteria isolated from the bovine rumen.

Thirty-two strains of pectin-fermenting rumen bacteria were isolated from bovine rumen contents in a rumen fluid medium which contained pectin as the only added energy source. Based on differences in morphology and the Gram stain, 10 of these strains were selected for characterization. Two strains were identified as Lachnospira multiparus, four strains were identified as Butyrivbrio fibrisolvens, and three strains were identified as Bacteroides ruminicola. Characteristics of the remaining strain did not correspond with any previously described species. It was a gram-positive anaerobic coccus, 1.0 to 1.2 mum in diameter, and occurred primarily as single cells or diplococci. The strain fermented pectin rapidly but showed little or no growth on any other energy sources tested. The only detectable end products were acetic acid and gas, a portion of which was identified as hydrogen. Although the physiological characteristics of this organism differ markedly from other described species, it has been placed in the genus Peptostreptococcus on the basis of morphology, Gram stain, relations to oxygen, and the occurrence of cell division in only one plane. End products of fermentation are somewhat similar to those of the cellulolytic ruminococci. Eight previously characterized strains of cellulolytic bacteria isolated in nonselective media were unable to ferment pectin, whereas ten strains of hemicellulolytic rumen bacteria, eight of which were isolated with a xylan medium, showed considerable variation in this characteristic.

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.

Characterization of Methanobacterium mobilis, sp. n., isolated from the bovine rumen.

A methanogenic bacterium, present in bovine rumen contents at concentrations of approximately 2 x 10(8) cells per ml, has been isolated in pure culture. The organism is a strictly anaerobic, weakly motile, nonsporeforming, gram-negative rod (0.7 mu x 1.5 to 2.0 mu) with rounded ends. There is a single polar flagellum. The organism grows at temperatures between 30 and 45 C, with an optimum at 40 C, and at pH values between 5.9 and 7.7, with optimal growth between pH 6.1 and 6.9. Of the 17 substrates tested, only formate and H(2) plus CO(2) supported growth. An unidentified, heat-stable factor(s) was required by the organism. The factor, which was not one of the common ones, was present in rumen fluid, mixed rumen bacteria, and yeast extract. On the basis of colony morphology, Gram reaction, and motility, the organism is classified as a new species of methanogenic bacterium, and the name Methanobacterium mobilis sp. n. is proposed.

Characterization of several bovine rumen bacteria isolated with a xylan medium.

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.

The characteristics of strains of Selenomonas isolated from bovine rumen contents.

The characteristics of strains of Selenomonas isolated from bovine rumen contents.