Effect of Ammonia Concentration on the Composition, Hydrolytic Activity and Nitrogen Metabolism of the Microbial Flora of the Rumen

The mean NH₃ concentration in the rumen of sheep fed whole barley (08 kg/d) by continuous feeders was increased from 61 to 134 HIM by supplementing the feed with urea (30 g/kg). This change caused a 90% increase in the rate of degradation of rolled barley, and smaller increases in the rates of degradation of protein and plant fibre in the rumen. The total viable count and numbers of pectinolytic bacteria in rumen fluid increased with the urea supplement. Enzyme studies indicated that NAD-linked glutamate dehydrogenase was the main pathway of NH₃ assimilation by rumen bacteria at both NH₃ concentrations. Glutamate was the main free amino acid found in the rumen at low NH₃ but, despite the low activity of alanine dehydrogenase and glutamate-pyruvate aminotransferase, alanine was the principal amino acid at high NH₃ concentrations. Hydrolytic rumen bacteria may require the higher NH₃ concentration either for effective NH₃ assimilation by an unknown mechanism involving alanine or for full expression of enzyme activity.     

Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Selective plating procedures were used to follow the fate of rifampicin-resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 10(7)/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (less than 10(3)/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell-free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo. In the case of S. ruminantium, introduced rifR strains persisted in the rumen at levels around 10(6) ml for at least 30 days.     

Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Selective plating procedures were used to follow the fate of rifampicin-resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 10⁷/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (10³/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell-free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo . In the case of S. ruminantium , introduced rif^R strains persisted in the rumen at levels around 10⁶ ml for at least 30 days.     

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l⁻¹). The ability of strain BTX to compete with other micro-organisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rumen fluid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. When added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron ), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10¹⁰ ml⁻¹). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.     

Diurnal variation in ciliate protozoa in the rumen of black buck (Antilope cervicapra) fed green forage

The protozoa in the rumen of a black buck were a B-type population with numbers varying between 0·31 and 0·61 times 10⁶cells/ml rumen liquor, when the animal was fed either vegetative green oat or third cut berseem. The total protozoa, total holotrichs, Dasytricha, total spirotrichs and small spirotrichs were significantly higher ( P < 0·01) on berseem feeding than those on oat feeding, while the numbers of Isotricha and large spirotrichs were unaffected by change of diet. Numerically the most important group of protozoa was small spirotrichs (74·4–75·6% of total population) which accounted for only 9·85–13·61% of protozoal cell mass in the rumen.     

A note on the antibacterial properties of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid in the ovine rumen

B rewer , D., P arkinson , V.O. & T aylor , A. 1990. A note on the antibacterial properties of 3-{3'-isocyanocyclopent-2'-enylidene)propionic acid in the ovine rumen. Journal of Applied Bacteriology 69 , 701–704.
The concentration of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid after introduction into the ovine rumen declined by 50% during 30 min. Its antibacterial activity in the rumen could be demonstrated by the decline in numbers of cellulo-lytic rumen bacteria from about 10⁶/ml to about 10²/ml.     

Correlation between composition of the bacterial community and concentration of volatile fatty acids in the rumen during the transition period and ketosis in dairy cows

The transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity of Veillonellaceae organisms was reduced and that of Streptococcaceae organisms was increased in rumen samples from the transition period and ketosis groups, with the number of Lactobacillaceae organisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes, Megasphaera elsdenii and Selenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium, Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number of Lactobacillus sp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers of Selenomonas ruminantium and Megasphaera elsdenii organisms in the rumen.     

Isolation and attempted introduction of sugar alcohol-utilizing bacteria in the sheep rumen

R.J. WALLACE AND N.D. WALKER. 1993. Bacteria that use sorbitol, xylitol, maltitol and dulcitol (galactitol) were isolated from the sheep rumen following enrichments in which bacteria were grown in rumen fluid medium where the sugar alcohol was the only added energy source. Only isolates obtained with sorbitol and maltitol grew sufficiently rapidly to be considered for enrichment by the sugar alcohol in vivo. Isolate SS2, a strain of Selenomonas ruminantium var. lactilytica which grew on sorbitol at 0.87 h^-1 was selected for further study and a rifampicin-resistant mutant, SS2/R5, was isolated to facilitate tracking in the mixed population. Despite an initial transient increase in numbers, a significant population of S. ruminantium SS2/R5 failed to establish in sheep which were dosed twice daily with 10 g of sorbitol. Continuous infusion of sorbitol increased numbers only slightly compared with twice-daily dosing. In vitro experiments indicated that strain SS2/R5 grew less well in the presence of other rumen organisms, particularly ciliate protozoa, than in pure culture. Furthermore, the concentration of sorbitol in vivo was lower than predicted from in vitro experiments, indicating that sorbitol was absorbed rapidly from the rumen. Similar observations were made with xylitol, dulcitol and maltitol. Proposed enrichment strategies that use sugar alcohols or other materials to support the growth of introduced bacteria will thus have to take account of the combined problems of microbe-microbe interactions and the loss of the compounds by absorption from the rumen.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

Abstract The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.     

A note on the antibacterial properties of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid in the ovine rumen

The concentration of 3-(3'-isocyanocyclopent-2'-enylidene)propionic acid after introduction into the ovine rumen declined by 50% during 30 min. Its antibacterial activity in the rumen could be demonstrated by the decline in numbers of cellulolytic rumen bacteria from about 10(6)/ml to about 10(2)/ml.     

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.     

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.     

Establishment of Ureolytic Staphylococci in the Rumen of Gnotobiotic Lambs

Six strains of ureolytic staphylococci isolated from rumen digesta and the rumen wall of conventionally reared sheep were inoculated per os into two germ-free lambs. High numbers of staphylococci established in rumen fluid, and urease activity and normal NH₃ and urea concentrations were maintained for approximately 4 weeks until slaughter. Staphylococci were found also to be associated with the rumen wall, conferring urease activity on this tissue.     

Effects of Food Intake on Numbers of Salmonellae and Escherichia coli in Rumen and Faeces of Sheep

When 10⁷–10⁸ Salmonella anatum or Salm. typhimurium were inoculated into the rumen of sheep consuming 1·3 kg of lucerne chaff daily, salmonellae were eliminated from the rumen in 2 days, and could not be detected in the faeces after c. 1 week. During starvation, both Escherichia coli and salmonellae grew in the rumen. Resumption of feeding after starvation for 3 days caused further multiplication of E. coli and salmonellae in the rumen. The organisms were subsequently eliminated with further feeding. Inoculation with as few as 400 salmonellae cells into a starved sheep led to large numbers of salmonellae appearing in the faeces and being excreted in varying numbers for at least 5 weeks after resumption of feeding.     

Effect of low-roughage diets on the microflora and lipid metabolism in the rumen

Changing the diet of five lactating cows and one nonlactating cow from high to low roughage induced milk fat depression in the lactating cows and altered the composition of the rumen microflora. While the numbers of lactic and propionic acid-producing bacteria increased, the numbers of Butyrivibrio spp. decreased. The numbers of lipolytic bacteria and the in vitro lipolytic activity of the rumen fluid were also decreased, as was the extent of hydrogenation of linoleic and linolenic acids combined in soybean oil incubated in vitro with rumen fluid. It is suggested that among the bacterial population in the rumen the vibrios, which were adversely affected by the low-roughage diets, may contribute significantly to both lipolysis and hydrogenation in the rumen.     

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.