Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

Why does the establishment of the starch preferring<Emphasis Type="Italic">Entodinium caudatum</Emphasis> in the rumen decrease the numbers of the fibrolytic ciliate<Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.     

Antibacterial Action of Essential Oils of Artemisia as an Ecological Factor: II. Antibacterial Action of the Volatile Oils of Artemisia tridentata (Big Sagebrush) on Bacteria from the Rumen of Mule Deer

Rumen microorganisms of wild and captive deer were subjected to increasing amounts of volatile oils. The oils had a marked antibacterial effect on the rumen bacteria when the concentration reached approximately 16 muliters of oil per 10 ml of rumen fluid nutrient broth. The gross reactions of rumen bacteria obtained from wild, as well as captive, deer to the volatile oils seemed to be of the same magnitude; thus no adaptation by the bacteria to the oils was apparent.     

Rumen fluid transplantation affects growth performance of weaned lambs by altering gastrointestinal microbiota,immune function and feed digestibility

Although rumen fluid transplantation (RT) has been developed to confer benefits for adult ruminants by altering gastrointestinal tract microbiota, the question remains whether RT can also benefit weaned lambs. Hence, in this study, thirty-eight pre-weaning lambs were randomly assigned to one of three treatment groups: control lambs (CON) received 25 ml of normal saline solution, and lambs in two RT groups received 25 ml of rumen fluid either from 3-month-old lambs (LT) or from one-year-old adult ewes (AT). The effects on their growth performance, nutrient digestibility, some blood parameters and gastrointestinal tract microbiota were monitored. There were differences (P < 0.05) in rumen bacterial composition between the groups at weaning, at 3 months and at 1 year. Rumen fluid transplantation decreased (P < 0.05) average daily feed intake, average daily gain in live weight and apparent digestibility of ether extract in the LT group, and it decreased (P < 0.05) apparent digestibility of NDF and ADF in the AT group. Rumen fluid transplantation also increased (P < 0.05) concentrations of serum immunoglobulin A in the AT group and increased (P < 0.05) serum concentrations of interleukin-6, interferon alpha and D-lactate in both LT and AT groups. Bacterial α-diversity in the rumen and rectum was not affected by RT (P > 0.05), but a bacterial community change was observed after RT, and the abundance of some dominant bacteria in both rumen and rectum changed after RT (P < 0.05). Analysis of correlations between the parameters indicated that the altered gastrointestinal microbiota and accelerated maturity of rumen microorganisms induced by RT caused some impairment of gastrointestinal integrity and immunity, which led to decreased feed intake, reduced feed digestibility and lower growth performance of the weaned lambs. In conclusion, rumen fluid transplantation altered the gastrointestinal microbiota causing adverse effects on feed intake, feed digestibility and growth performance of the weaned lambs.     

Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur.

Sheep fed the forage Digitaria pentzii fertilized with sulfur were compared with those fed unfertilized forage for the rumen microbial population involved with fiber degradation. No differences were detected in the bacterial population as determined by anaerobic cultures on a habitat-simulating medium, xylan, or pectin, by 35S labeling techniques for microbial protein, or by transmission electron microscopic studies of bacterium-fiber interactions. Rumen volume and water flow from the rumen were not different for sheep fed each of the forages. Rumen fungi were prevalent in sheep fed sulfur-fertilized D. pentzii as shown by sporangia adhering to forage fiber and by colonies developing from zoospores in roll tubes with cellobiose plus streptomycin and penicillin. Fungi were absent or in extremely small numbers in sheep fed unfertilized forage. Nylon bag digestibility studies showed that the fungi preferentially colonized the lignified cells of blade sclerenchyma by 6 h and caused extensive degradation by 24 h. In the absence of bacteria in in vitro studies, extensive hyphal development occurred; other lignified tissues in blades (i.e., mestome sheath and xylem) were attacked, resulting in a residue with partially degraded and weakened cell walls. Nonlignified tissues were also degraded. Breaking force tests of leaf blades incubated in vitro with penicillin and streptomycin and rumen fluid from sheep fed sulfur-fertilized forage or within nylon bags in such sheep showed a residue at least twice as fragile as that from sheep fed unfertilized forage. In vitro tests for dry matter loss showed that rumen fungi, in the absence of actively growing bacteria, could remove about 62% of the forage material. The response of rumen fungi in sheep fed sulfur-fertilized D. pentzii afforded a useful in vivo test to study the role of these microbes in fiber degradation. Our data establish that rumen fungi can be significant degraders of fiber and further establish a unique role for them in attacking and weakening lignocellulosic tissues. The more fragile residues resulting from attack by fungi could explain the greater intake consistently observed by sheep eating sulfur-fertilized compared with unfertilized D. pentzii forage.     

Rumen Bacterial and Protozoal Responses to Insecticide Substrates

Insecticides containing organophosphate, chlorinated hydrocarbon, and carbamate were tested with bovine ruminal ingesta fractions. Rumen bacteria exposed to insecticide levels of 0 to 500 ppm in rumen fluid for 4 hr were inoculated into rumen fluid-starch feed extract medium. No apparent significant bacterial count inhibitions were noted. Also, when insecticides were used as carbon sources at concentrations of 500 ppm in carbohydrate-limited media, no increases in bacterial counts were indicated. Warburg manometric data showed that paraffin oil-Triton X-155 preparations of dimethoate, Diazinon, lindane, Thiodan and Sevin stimulated gas production in holotrich protozoa. Entodinium simplex, an oligotrich, produced less gas with insecticide substrates per unit of dry weight than did an Isotricha sp. Rumen bacteria and plant debris fractions from ruminal ingesta provided with insecticides did not give increased manometric responses over the endogenous control vessels. Washed suspensions of I. intestinalis produced volatile fatty acids in excess of the endogenous suspensions when provided insecticide substrates. Thiodan dissimilation by I. intestinalis was followed colorimetrically with 15% loss in substrate in 1 hr of incubation at 39 C. Diazinon-C¹⁴ substrate uptake was demonstrated with suspensions of E. simplex and I. intestinalis. Rumen ciliates are suggested as a possible means for screening out useful insecticides susceptible to microbial dissimilation for use on forage and other cattle-feed crops.     

Cultivation of Rumen Protozoa. I. Factors Influencing the Cultivation of Rumen Oligotrich Protozoa "In Vitro"

SYNOPSIS. Factors influencing the cultivation of entodinia in vitro have been studied. It was found that removal of particulate material, culture division, or a combination of both resulted in similar maximum protozoal concentrations. At the same time, untreated culture concentrations declined rapidly after reaching a maximum concentration at about the tenth day.
At concentrations of streptomycin greater than 25 μg per ml of media, increasing the level of streptomycin extended the time required for a culture to attain a maximum protozoal concentration. A significant relationship (P>.01) was demonstrated between the starch concentration and the protozoal concentration, and it was found that various combinations of starch and streptomycin produced different relative protozoal concentrations in initial and established cultures. Implications arising from these results are discussed.     

Rumen Ciliate Protozoa in Ohio White-tailed Deer (Odocoileus virginianus)

ABSTRACT. Rumen contents were obtained from 23 white-tailed deer (Odocoileus virginianus), located in the eastern portion of central Ohio. Samples were taken during late fall and winter over a 4-yr period, 1986–1990. Protozoan numbers ranged from 0.002 to 7.25 × 10⁶ per ml of rumen contents, with a mean of 2.96 × 10⁶. Six deer had protozoan concentrations higher than any values previously reported in ruminants. In all 23 animals, Entodinium dubardi was the only ciliate protozoan species present.     

Metabolism of Glycine by Rumen Microorganisms

Rumen microorganisms rapidly metabolized glycine at rates varying from 0.014 to 0.241 mumole of glycine per ml per min. The main metabolic products were carbon dioxide, acetic acid, and ammonia; little glycine was incorporated into bacterial protein. Use of carboxyl or methylene-labeled glycine showed that the carbon dioxide came mainly from the carboxyl of glycine, whereas both carbons of acetic acid were derived partly from the methylene carbon of glycine and partly from carbon dioxide. The ratio of carbon-14 to nitrogen-15 in glycine isolated from the protein of rumen bacteria incubated in the presence of N(15)- and C(14)-labeled glycine indicated that most of the extracellular glycine incorporated into protein was incorporated without intervening deamination.     

Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Lyophilization of rumen fluid for use in culture media

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Factors Influencing Agnotobiotic Cultures of the Rumen Ciliate, Entodinium simplex

The nutrition of Entodinium simplex was studied, with foliage of bluegrass (Poa pratense), perennial ryegrass (Lolium perenne), and grains of wheat (Triticum vulgare) as substrates in agnotobiotic cultures. Entodinium grew poorly when the substrates were autoclaved; better growth was obtained when the substrates were sterilized with ethylene oxide vapor. The concentration of ethylene oxide and the amount of moisture influenced the sterility and nutritional adequacy of the treated substrate. Autolysates and hydrolysates of mixed rumen protozoa stimulated growth. Protozoal extracts did not replace factors destroyed by autoclaving. Clarified rumen fluid assisted the cultivation of entodinia from small inocula but was detrimental to established cultures. Success of cultures was influenced by the medium used to grow the inoculum as well as by the medium inoculated. The results indicated that the composition of the bacterial population influences the growth of E. simplex.     

Long-Term Monensin Supplementation Does Not Significantly Affect the Quantity or Diversity of Methanogens in the Rumen of the Lactating Dairy Cow

A long-term monensin supplementation trial involving lactating dairy cattle was conducted to determine the effect of monensin on the quantity and diversity of rumen methanogens in vivo. Fourteen cows were paired on the basis of days in milk and parity and allocated to one of two treatment groups, receiving (i) a control total mixed ration (TMR) or (ii) a TMR with 24 mg of monensin premix/kg of diet dry matter. Rumen fluid was obtained using an ororuminal probe on day −15 (baseline) and days 20, 90, and 180 following treatment. Throughout the 6-month experiment, the quantity of rumen methanogens was not significantly affected by monensin supplementation, as measured by quantitative real-time PCR. The diversity of the rumen methanogen population was investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone gene libraries. DGGE analysis at each sampling point indicated that the molecular diversity of rumen methanogens from monensin-treated cattle was not significantly different from that of rumen methanogens from control cattle. 16S rRNA gene libraries were constructed from samples obtained from the rumen fluids of five cows, with a total of 166 clones examined. Eleven unique 16S rRNA sequences or phylotypes were identified, five of which have not been recognized previously. The majority of clones (98.2%) belonged to the genus Methanobrevibacter, with all libraries containing Methanobrevibacter strains M6 and SM9 and a novel phylotype, UG3322.2. Overall, long-term monensin supplementation was not found to significantly alter the quantity or diversity of methanogens in the rumens of lactating dairy cattle in the present study.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

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.     

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.