Fermentation of methanol in the sheep rumen.

Sheep fed a hay-concentrate diet were adapted to pectin administration and ruminal infusion of methanol. Both treatments resulted in a strong increase in the rate of methanogenesis from methanol. Quantitative data show that methanol was exclusively converted into methane. Treatments did not influence ruminal volatile fatty acid percentages.     

Ribotyping to compare Fusobacterium necrophorum isolates from bovine liver abscesses, ruminal walls, and ruminal contents.

Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal. Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter. F. necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples. Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli. EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison. Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI. The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples. None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates. The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F. necrophorum isolates of liver abscesses originate from the rumen.     

The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics

Beef cattle have been fed ionophores and other antibiotics for more than 20 years to decrease ruminal fermentation losses (e.g methane and ammonia) and increase feed efficiency, and these improvements have been explained by an inhibition of gram-positive ruminal bacteria. Ionophores are not used to treat human disease, but there has been an increased perception that antibiotics should not be used as feed additives. Some bacteria produce small peptides (bacteriocins) that inhibit gram-positive bacteria. In vitro experiments indicated that the bacteriocin, nisin, and the ionophore, monensin, had similar effects on ruminal fermentation. However, preliminary results indicated that mixed ruminal bacteria degraded nisin, and the ruminal bacterium, Streptococcus bovis, became highly nisin-resistant. A variety of ruminal bacteria produce bacteriocins, and bacteriocin production has, in some cases, been correlated with changes in ruminal ecology. Some ruminal bacteriocins are as potent as nisin in vitro, and resistance can be circumvented. Based on these results, ruminal bacteriocins may provide an alternative to antibiotics in cattle rations.     

Mixed fungal populations and lignocellulosic tissue degradation in the bovine rumen

Anaerobic fungi in ruminal fluid from cows eating Bermuda grass hay plus a grain and minerals supplement were evaluated for diversity in sporangial morphotypes and colony growth patterns and for the degradation of various lignocelluloses. In selective cultures containing streptomycin and penicillin, an active population of ruminal fungi colonized leaf blades and degraded fiber at rates and extents almost equal to that of the total ruminal population. Three major sporangial morphotypes were consistently observed on leaf blades: oval, globose, and fusiform. Fungal colonies representing three distinct growth types consistently developed in anaerobic roll tubes inoculated with strained ruminal fluid. Sporangial morphotypes could not be matched to colony types due to multiple sporangial forms within a colony. Under identical growth conditions, one type exhibited a monocentric growth pattern, while two types exhibited polycentric growth patterns previously unreported in ruminal fungi. Mixed ruminal fungi in selective cultures or in digesta taken directly from the rumen produced a massive clearing of the sclerenchyma. Quantitation of tissue areas in cross sections by light microscopic techniques showed that fungal incubations resulted in significant (P = 0.05) increases in sclerenchyma degradation compared to whole ruminal fluid incubations. The mestome cell wall was at times penetrated and partially degraded by fungi; the colonization was less frequent and to a lesser degree than with the sclerenchyma. Conversely, ruminal bacteria were not observed to degrade the mestome sheath. Phenolic monomers at 1 mM concentrations did not stimulate to a significant (P = 0.05) extent the dry weight loss or fungal colonization of leaf blades; at 10 mM concentrations cinnamic and benzoic acids were toxic to ruminal fungi.     

Mixed fungal populations and lignocellulosic tissue degradation in the bovine rumen.

Anaerobic fungi in ruminal fluid from cows eating Bermuda grass hay plus a grain and minerals supplement were evaluated for diversity in sporangial morphotypes and colony growth patterns and for the degradation of various lignocelluloses. In selective cultures containing streptomycin and penicillin, an active population of ruminal fungi colonized leaf blades and degraded fiber at rates and extents almost equal to that of the total ruminal population. Three major sporangial morphotypes were consistently observed on leaf blades: oval, globose, and fusiform. Fungal colonies representing three distinct growth types consistently developed in anaerobic roll tubes inoculated with strained ruminal fluid. Sporangial morphotypes could not be matched to colony types due to multiple sporangial forms within a colony. Under identical growth conditions, one type exhibited a monocentric growth pattern, while two types exhibited polycentric growth patterns previously unreported in ruminal fungi. Mixed ruminal fungi in selective cultures or in digesta taken directly from the rumen produced a massive clearing of the sclerenchyma. Quantitation of tissue areas in cross sections by light microscopic techniques showed that fungal incubations resulted in significant (P = 0.05) increases in sclerenchyma degradation compared to whole ruminal fluid incubations. The mestome cell wall was at times penetrated and partially degraded by fungi; the colonization was less frequent and to a lesser degree than with the sclerenchyma. Conversely, ruminal bacteria were not observed to degrade the mestome sheath. Phenolic monomers at 1 mM concentrations did not stimulate to a significant (P = 0.05) extent the dry weight loss or fungal colonization of leaf blades; at 10 mM concentrations cinnamic and benzoic acids were toxic to ruminal fungi.     

The effect of pH on ruminal methanogenesis

Abstract: When a fistulated cow was fed an all forage diet, ruminal pH remained more or less constant (6.7 to 6.9). The ruminal pH of a concentrate-fed cow decreased dramatically in the period soon after feeding, and the pH was as low as 5.45. Mixed ruminal bacteria from the forage-fed cow converted CO₂ and H₂ to methane, but the ruminal fluid from the concentrate-fed cow did not produce methane. When the pH of the ruminal fluid from the concentrate-fed cow was adjusted to pH 7.0, methane was eventually detected, and the absolute rate constant of methane production was as high as the one observed with ruminal fluid from the forage fed cow (0.32 h⁻¹). Based on the zero-time intercepts of methane production, it appeared that the concentrate-fed cow had fewer methanogens than the forage-fed cow. When the mixed ruminal bacteria were incubated in a basal medium containing 100 mM acetate, methanogenesis was pH-dependent, and no methane was detected at pH values less than 6.0. Because the removal of acetic acid completely reversed the inhibition of methanogenesis, it appeared that volatile fatty acids were causing the pH-dependent inhibition. Based on these results, concentrate diets that lower ruminal pH may provide a practical means of decreasing ruminal methane production.     

Establishment and development of ruminal hydrogenotrophs in methanogen-free lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10(7) to 10(8) cells g(-1)) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H(2) utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H(2) utilization was similar to that in conventional lambs. H(2) utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H(2) utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H(2) in the rumen.     

分子生物学技术在瘤胃原虫研究中的应用

瘤胃原虫是瘤胃微生物的重要组成部分,它们直接影响饲料中碳水化合物、含氮物质、矿物质和维生素的消化和利用。分子标记和基于小亚基核糖雄RNA的分子生物学技术可以在基因和分子水平上为研究瘤胃原虫提供可靠而丰富的依据。有必要应用此技术,深入研究瘤胃原虫功能,及其与其他瘤胃微生物的互作。     

Establishment and Development of Ruminal Hydrogenotrophs in Methanogen-Free Lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10⁷ to 10⁸ cells g⁻¹) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H₂ utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H₂ utilization was similar to that in conventional lambs. H₂ utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H₂ utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H₂ in the rumen.     

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.     

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen.

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.     

Sensitivity of ruminal microorganisms to pentachlorophenol.

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.     

Inducible bacteriophages from ruminal bacteria

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Effects of seasonal changes in food quality and food intake on the transport of sodium and butyrate across ruminal epithelium of reindeer

Transport of 22Na and 14C-butyrate across the ruminal epithelium of captive reindeer fed a concentrate diet in summer (n=5) and in winter (n=5) and from free-ranging reindeer taken from summer (n=3) and winter pasture (n=5) was measured in vitro in Ussing chambers. Significant amounts of both Na+ and butyrate were transported across the isolated epithelium without any external driving force. The ruminal transport of Na+ and butyrate were interacting, as evidenced by both the observed amiloride-induced reduction of net butyrate-transport and by the positive correlation between net transport of butyrate and Na+. Amiloride also reduced the net transport of Na+ without significantly affecting the short-circuit current, indicating the presence of an apical Na+/H+ exchanger in the ruminal epithelium of reindeer. The captive reindeer increased the dry matter intake of a constant quality concentrate from winter to summer, but this neither affected their ruminal transport capacity nor their ruminal surface enlargement factor (SEF). Free-ranging reindeer increased their ruminal transport capacity for Na+ and butyrate from summer to winter but simultaneously reduced their ruminal SEF. The present data indicate that this food-induced increase in transport capacity was attributed to changes in the nutrient composition of the diet.     

Use of potassium depletion to assess adaptation of ruminal bacteria to ionophores.

When mixed ruminal bacteria from cattle fed timothy hay were suspended in a medium containing a low concentration of potassium, monensin and lasalocid catalyzed a rapid depletion of potassium from cells. The ionophore-mediated potassium depletion was concentration dependent, and it was possible to describe the relationship with saturation constants. Mixed ruminal bacteria never lost more than 50% of their potassium (Kmax = 46%), and the concentrations of monensin and lasalocid needed to cause half-maximal potassium depletion (Kd) were 178 and 141 nM, respectively. When cattle were fed 350 mg of monensin per day, the ratio of ruminal acetate to propionate decreased from 4.2 to 2.9, and the Kd of monensin was eightfold greater than the value for mixed ruminal bacteria from control animals. Monensin supplementation also caused a twofold increase in the Kd of lasalocid. Lasalocid supplementation (350 mg per day) had no effect on the ruminal acetate-to-propionate ratio, but it caused a twofold increase in the Kd values of monensin and lasalocid. Increases in Kd occurred almost immediately after ionophore was added to the ration, and the Kd values returned to their prefeeding values within 14 days of withdrawal. Ionophore supplementation had no effect on the Kmax values, and approximately 50% of the population was always highly ionophore resistant. Because the Kd values of even adapted ruminal bacteria were low (< 1.5 microM), it appears that a large proportion of the ruminal ionophore is bound nonselectively to feed particles or ionophore-resistant bacteria.     

Oral Samples as Non-Invasive Proxies for Assessing the Composition of the Rumen Microbial Community

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.     

Sensitivity of ruminal microorganisms to pentachlorophenol

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.     

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme

Summary Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trupsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.     

Transport of ketone bodies and lactate in the sheep ruminal epithelium by monocarboxylate transporter 1

Due to intensive intracellular metabolism of short-chain fatty acids, ruminal epithelial cells generate large amounts of D-beta-hydroxybutyric acid, acetoacetic acid, and lactic acid. These acids have to be extruded from the cytosol to avoid disturbances of intracellular pH (pH(i)). To evaluate acid extrusion, pH(i) was studied in cultured ruminal epithelial cells of sheep using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Extracellular addition of D-beta-hydroxybutyrate, acetoacetate, or lactate (20 mM) resulted in intracellular acidification. Vice versa, removing extracellular D-beta-hydroxybutyrate, acetoacetate, or lactate after preincubation with the respective monocarboxylate induced an increase of pH(i). Initial rate of pH(i) decrease as well as of pH(i) recovery was strongly inhibited by pCMBS (400 microM) and phloretin (20 microM). Both cultured cells and intact ruminal epithelium were tested for the possible presence of proton-linked monocarboxylate transporter (MCT) on both the mRNA and protein levels. With the use of RT-PCR, mRNA encoding for MCT1 isoform was demonstrated in cultured ruminal epithelial cells and the ruminal epithelium. Immunostaining with MCT1 antibodies intensively labeled cultured ruminal epithelial cells and cells located in the stratum basale of the ruminal epithelium. In conclusion, our data indicate that MCT1 is expressed in the stratum basale of the ruminal epithelium and may function as a main mechanism for removing ketone bodies and lactate together with H+ from the cytosol into the blood.