Some Chemical and Physical Properties of a Slime from theRumen of Cattle

J. GUTIERREZ, R. E. DAVIS, AND I. L. LINDAHL

Animal Husbandry Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland

Received for publication August 11, 1960

Previous work has shown the percentage of encapsu-

lated bacteria increased when cattle were placed on a

feed-lot bloat type diet (Jacobson et al., 1957) and a

recent investigation has shown Streptococcus bovis andPeptostreptococcus elsdenii increases in numbers as theonset of bloat symptoms occurred in cases of feed-lotbloat (Gutierrez et al., 1959). Slime produced by ruminalmicroorganisms in grain bloat may increase the vis-cosity of the rumen fluid and thereby contribute to theentrapment of the fermentation gases in a stable foam.The froth interferes with the animal's normal belchingmechanism (Dougherty, Habel, and Bond, 1958).Although bacterial polysaccharides have been sug-gested as possible sources of ruminal slime, quantitativedata along these lines are not available. The workreported here concerns an attempt to isolate slimefractions from grain bloaters, and to give some of themain chemical and biological characteristics of theresidues found.

MATERIALS AND METHODS

The methods for the maintenance and the feeding ofthe animals for the production of experimental bloathave been described (Gutierrez et al., 1959). All rumen

samples were centrifuged for 1 hr at 5200 RCF toremove debris, protozoa, and most of the bacteria.Measurements of relative viscosity of the differentsamples of intact rumen fluid after centrifugation were

made with an Ostwald viscosimeter at 25 C. The slimefraction from animals on the high grain diet was pre-cipitated by the addition of two volumes of absoluteethanol to 20 ml of the centrifuged rumen fluid. Theslimy fibrous residue rose to the surface of the liquidand could easily be harvested without contaminationfrom other nonviscid precipitates which settled to thebottom. The slime mat was washed twice by suspendingin distilled water, centrifuging, and discarding thesupernatant. The phase microscopic examination(1455X magnification) of the freshly harvested slimeshowed a fine granular appearance with very fewbacterial cells and no protozoa embedded in the slime.

The mat was dried overnight at 60 C and weighed.For further analysis of amino acids, carbohydrates, andnucleic acids, the dried residue was usually ground to a

fine powder with mortar and pestle. The slime fraction

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(100 mg) was autoclaved for 9 hr with 5 ml of 3 NH2SO4 at 120 C and 15 lb pressure. The hydrolyzatewas neutralized with 2 N Ba(OH)2, centrifuged toremove the precipitate, and the supernatant evaporatedat room temperature with a jet of air over the surface.The residue was taken up with 0.2 ml of water and 20,ug spotted on strips of Whatman no. 1 paper. Carbo-hydrates in the slime fraction were developed withbutanol:acetic acid:water (40:11:19) (Lesley andHochster, 1959) and sprayed with 1 per cent AgNO3 inacetone followed with 2 per cent NaOH in ethyl alcohol.Amino acids were analyzed by hydrolyzing 15 mg ofthe slime fraction in the same manner as the carbo-hydrate sample except that the final residue was takenup with 0.2 ml ethanol instead of water. The hydrol-yzate for amino acid identification was spotted onlarge sheets of Whatman no. 1 paper and developed inthe first direction with phenol: water (4:1) in anatmosphere of ammonia, and the organic layer of amixture of n-butanol: acetic acid: H20 (4: 1: 5) wasused in the second direction. In some of the experimentsfor the development of the amino acids, a mixture ofn-butanol:methyl ethyl ketone: 17 N ammonia:H20(5:3: 1: 1) was substituted for phenol: water in thefirst direction (Wolfe, 1957).

For the chromatographic analysis of nucleic acids,20 mg of the slime fraction were hydrolyzed in 5 ml of2 per cent H2S04 for 3 hr at 100 C. The hydrolyzatewas evaporated to dryness and taken up with 0.5 mlethanol and spotted on sheets of paper. The nucleicacid hydrolyzate was developed with n-butanol satu-rated with 10 per cent aqueous urea in the first directionfollowed by 5 per cent Na2HPO4 and isoamyl alcoholin equal layers (Carter, 1950). Nucleic acid derivativesand the free nitrogen bases were located by theirfluorescence and ultraviolet absorption with a min-eralight lamp' at 2537 A and were identified from theirRF values (Carter, 1950). Tests for deoxyribonucleicacid (DNA) were made using diphenylamine andcysteine hydrochloride methods described by Dische(1930, 1944).Residual carbohydrate in the crude slime fraction

was determined by the anthrone reagent and comparedcolorimetrically to a glucose standard curve. Analysis

I Ultra-Violet Products, Inc., San Gabriel, California.

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J. GUTIERREZ, R. E. DAVIS, AND I. L. LINDAHL

of soluble polysaccharide in centrifuged rumen fluidwas made after treatment with 5 per cent trichloroaceticacid by the method of Roe (1954).

Total nitrogen content of the slime fraction wasobtained by Kjeldahl determinations. Ashing of thesamples was made at 600 C for 21, hr. Total moisturewas determined by drying the slime fraction overnightat 60 C, followed by 5 hr at 100 C in a vacuum oveni.

Figure 1. A 1:10 dilution of rumen contents from bloatedanimal. Long chains of paired streptococei and other organ-isms. Gram stained. Original magnification 1091X.

Figure 2. A 1:10 dilution of rumen contents from bloated

Total phosphorus in the slime residue was measuredaccording to Munsey (1948).

RESULTSGram stained smears of 1:10 dilution of rumen

contents removed from animals upon the onset ofbloat symptoms were very similar to those reported inprevious experiments (Gutierrez et al., 1959) (figure 1).

animal. Paired streptococci illustrating capsule formation.White's stain. Original magnification 1091X.

Figure S. Spatula showing coiled slime fraction which wasisolated from a bloating animal by the ethanol precipitationprocedure.

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SLIME FROM CATTLE RUMEN

Capsule formation of the large streptococci chains was

demonstrated using the combined positive and negativestaining technique of White (1947) (figure 2). The slimeharvested at various times by ethanol precipitationfrom the rumen fluid of a bloating steer are presentedin table 1. The amount of slime collected from thesurface of the ethanol and rumen fluid mixture increasedwith the onset and severity of the bloat symptoms.The amount of residue harvested from 6 animals rangedfrom 4.2 to 5.5 per cent calculated on a wet weightbasis. The material was extremely viscid and thefibrous-like threads could be wrapped around a spatulawith ease (figure 3). A preliminary Biuret test on thedried slime gave a positive reaction, and Kjeldahldeterminations for three different samples showed theslime contained between 33 and 35 per cent crudeprotein. After acid hydrolysis (see Materials andMethods section) and paper chromatography thefollowing amino acids were shown to be present insignificant amounts: leucine, isoleucine, phenylalanine,valine, tyrosine, proline, methionine, glycine, histidine,serine, threonine, arginine, and glutamic acid plustraces of aspartic acid.A test for residual carbohydrate in the crude slime

fraction with the anthrone reagent was positive andquantitative experiments on the slime showed thematerial contained 18 per cent carbohydrate (Roe,1954). Paper chromatography of acid hydrolyzates ofthe slime which were sprayed with AgNO3 and NaOHshowed spots which had RF values similar to glucoseand D-ribose. Using single dimension strips withNaHPO4-isoamyl alcohol solvents and AgNO3 spray,

the RF for the latter pentose was 0.87 (published value,0.87; Carter, 1950). Chromatography of acid hydrol-yzates for nucleic acid derivatives showed fluorescentand ultraviolet absorbing compounds which had RFvalues of thymine, cytosine, adenine, and guanine. Onedimensional strips run with 5 per cent Na2HPO4-isoamylalcohol mixtures revealed a spot with RF similar tothymine deoxyriboside (Carter, 1950). Color tests forDNA in the slime fraction were positive. A suspensioncontaining 500 jig per ml slime material gave a bluecolor with diphenylamine as described by Dische(1930) and a pink color with cysteine hydrochloride andH2SO4 (Dische, 1944). The ash content of the driedslime samples from different animals varied between

TABLE 1

eight of ethanol-precipitable slime at differentstages of bloat

Bloat Symptoms Dry Weight per 20 ml CentrifugedRumen Fluid

mg

NoneSlightSevere

0

7290

23.4 and 28.9 per cent, and analysis of the phosphoruscontent of the dried residue indicated an average levelof 6.43 per cent. Ether extractable materials run ondifferent samples showed low values of 0.13 to 0.36 percent, whereas the total moisture content of the slimeresidue was 97.3 per cent.Measurements of the viscosity of the centrifuged

rumen fluid indicated the viscosity increased with theonset of bloat symptoms and continued to rise withseverity of bloat. Usually 30 ml of the centrifuged rumenfluid were placed in the viscosimeter and the timerequired for the flow through the capillary comparedwith water for centrifuged rumen fluid from hay-fedor nonbloating animals. If the viscid fluid being testedtook twice the time required for water, this was takento present a relative viscosity of 2.0. Rumen samplesfrom animals on a hay diet gave a relative viscositysimilar to water (1.0), whereas steers with early bloatsymptoms had a relative viscosity double that of hay-fed animals (2.1). Severely bloated animals had greaterrelative viscosities (3.3). A 5 per cent (wet weight)water suspension of the slime fraction had a relativeviscosity of 2.0 when compared to water.The content of soluble polysaccharide in the centri-

fuged rumen fluid of bloating and nonbloating steerswas analyzed by the anthrone method after precipita-tion of the protein with 5 per cent trichloroacetic acid.The amounts found in different animals are given intable 2. Although some of the bloating animals showedhigh values, a definite correlation between the amountof soluble polysaccharide and the appearance or severityof bloat symptoms was not evident in all cases.

DISCUSSION

The amino acids, purine and pyrimidine bases,pentoses, and phosphorus content in the slime fractionindicated the nucleic acids and nucleoproteins producedby the microorganisms in the rumen reached a highlevel with the carbohydrate-rich rations which werefed in the current experiments. Significant amounts ofthe fibrous, viscid slime could not be harvested fromanimals fed hay or pasture diets. Deoxyribonucleicacid and to a lesser extent ribonucleic acid solutionshave a viscous characteristic (Chargaff and Davidson,

TABLE 2Soluble polysaccharide in rumen fluid of cattle

Animal Bloat Symptoms Anmount of PolysaccharideAnimal Bloat ymts per 100 ml Rumen Fluid

mg

303 Severe 15488 Severe 1879 Severe 9

491 None 4.566 None 2.2143 None 4.5

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J. GUTIERREZ, R. E. DAVIS, AND I. L. LINDAHL

1955) and the increase in nucleic acids found in thepresent experiments probably contributed to the in-crease in the viscosity of the rumen fluid. Increasesin the viscosity of rumen fluid were well correlatedwith the onset of bloat symptoms. Bacterial popula-tions have been reported to increase as bloat occurredon grain rations (Gutierrez et al., 1959), but informa-tion is lacking on the rates at which cells of the ruminalpopulation lyse and release nucleic acids into the me-dium. Lysis of cells in cultures has been reported forruminal species Bacteroides succinogenes (Bryant,Robinson, and Chu, 1959). The quantitative evidenceat hand indicates bacterial slime production arisingfrom nucleic acids and nucleoprotein probably playsa significant role in altering the viscosity of the rumenfluid. Glucose-containing polysaccharides may alsobe a contributing factor in viscosity changes.The sequence of ruminal biochemical events which

occurred in bloating animals on grain rations is notlikely to be the same for animals bloating on legumes,although the general principle of foam production hasbeen found to occur in both (Davis, Gutierrez, andLindahl, 1960). The current findings in feed-lot bloatwould indicate searches for compounds, either of plantor microbial origin, which have the capacity of in-creasing viscosities of fluids may be worthwhile instudies of legume bloat. There are indications thatplant pectins, hemicelluloses and saponins may beimplicated in pasture bloat (Conrad et al., 1959).

SUMMARYThe amount of slime isolated from the rumen of

cattle increased with the onset and severity of symp-toms of bloat. Quantitative analysis showed the slimeto contain 18 per cent carbohydrate and 33 to 35 percent crude protein. Fourteen amino acids were identi-fied in acid hydrolyzates. Thymine, cytosine, adenine,and guanine were detected by chromatography. Cor-relation was not observed between amount of solublepolysaccharide and appearance of severity of bloat.

REFERENCES

BRYANT, M. P., ROBINSON, I. M. AND CHU, H. 1959 Ob-servations on the nutrition of Bacteroides succinogenes-a

ruminal cellulolytic bacterium. J. Dairy Sci., 42, 1831-1847.

CARTER, C. E. 1950 Paper chromatography of purine andpyrimidine derivatives of yeast ribonucleic acid. J.Am. Chem. Soc., 72, 1466-1471.

CHARGAFF, E. AND DAVlDSON, J. N. 1955 The nucleic acids,vol. I. Academic Press, Inc., New York, New York.

CONRAD, H. R., POUNDEN, W. D., FETTER, A. W., AND RAi1I-SEYER, R. D. 1960 Total pectic substances and uronicacids in pasture plants and their relationship to incidenceof bloat. U. S. Dept. Agr. Report of Fifth Conference onRumen Function, Chicago, p. 25.

DAVIS, R. E., GUTIERREZ, J., AND LINDAHL, I. L. 1960 Ob-servations on the ruminal microbial populations duringbloat. U. S. Dept. Agr. Report of Fifth Conference onRumen Function, Chicago, p. 25.

DISCHE, Z. 1930 Uber einige neue charakteristische Far-breaktionen der Thymonukleinsaure und eine Mikro-methode zur Bestimmung derselben in tieriochen Organenmit Hilfe dieser Reaktionen. Mikrochemie, 8, 4-32.

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DOUGHERTY, R. W., HABEL, R. E., AND BOND, H. E. 1958Esophageal innervation and the eructation reflex in sheep.Am. J. Vet. Research, 19, 115-128.

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LESLEY, S. M. AND HOCHSTER, R. M. 1959 The extracellularpolysaccharide of Xanthomonas phaseoli. Can. J. Bio-chem. and Physiol., 37, 513-529.

MUNSEY, V. E. 1948 Report on phosphorus in cereal prod-ucts. J. Assoc. Offic. Agr. Chemists, 31, 269-272.

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WOLFE, M. 1957 The quantitative determination of aminoacids by paper chromatography. A solvent to replacephenol. Biochim. et Biophys. Acta, 23, 186-191.

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