scholarly journals Use of stable isotopes to measurede novosynthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumenin vitro

2004 ◽  
Vol 91 (2) ◽  
pp. 253-261 ◽  
Author(s):  
C. Atasoglu ◽  
A. Y. Guliye
Keyword(s):  
Stable Isotopes ◽  
Amino Acid ◽  
Protein Hydrolysate ◽  
Rumen Fluid ◽  
Deconvolution Analysis ◽  
C And N ◽  
N Incorporation ◽  
Isoleucine Metabolism ◽  
Micro Organisms

Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay–concentrate diet were strained and incubatedin vitrowith starch–cellobiose–xylose in the presence of NH3and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were15NH3, [15N]APH or [13C]APH. Total15N incorporation was calculated from separate incubations with15NH3and [15N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3·5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70v.50 % respectively,P=0·001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C + asparagine-C was incorporated, while 45 % aspartate-N+asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C2and C4intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.

scholarly journals Hydrolysis of14C-labelled proteins by rumen micro-organisms and by proteolytic enzymes prepared from rumen bacteria

1983 ◽  
Vol 50 (2) ◽  
pp. 345-355 ◽  
Author(s):  
R. J. Wallace
Keyword(s):  
Proteolytic Enzymes ◽  
Rumen Fluid ◽  
Performic Acid ◽  
Cross Linking ◽  
Acid Oxidation ◽  
Rumen Bacteria ◽  
Rate Of Hydrolysis ◽  
Micro Organisms ◽  
Hydrolysis Of

1. Proteins were labelled with14C in a limited reductive methylation using [14C]formaldehyde and sodium borohydride.2. The rate of hydrolysis of purified proteins was little (< 10%) affected by methylation and the14C-labelled digestion products were not incorporated into microbial protein during a 5 h incubation with rumen fluid in vitro. It was therefore concluded that proteins labelled with14C in this way are valid substrates for study with rumen micro-organisms.3. The patterns of digestion of14C-labelled fish meal, linseed meal and groundnut-protein meal by rumen micro-organisms in vitro were similar to those found in vivo.4. The rates of hydrolysis of a number of14C-labelled proteins, including glycoprotein II and lectin from kidney beans (Phaseolus vulgaris), were determined with mixed rumen micro-organisms and with proteases extracted from rumen bacteria. Different soluble proteins were digested at quite different rates, with casein being most readily hydrolysed.5. Proteins modified by performic acid oxidation, by cross-linking using 1,6-di-iso-cyanatohexane or by diazotization were labelled with14C. Performic acid treatment generally increased the susceptibility of proteins to digestion, so that the rates of hydrolysis of performic acid-treated proteins were more comparable than those of the unmodified proteins. Cross-linking resulted in a decreased rate of hydrolysis except with the insoluble proteins, hide powder azure and elastin congo red. Diazotization had little effect on the rate of hydrolysis of lactoglobulin and albumin, but inhibited casein hydrolysis and stimulated the breakdown of γ-globulin.

Repeatability of in vitro digestibility assays of forages when using fresh, frozen or freeze-dried cow faeces instead of sheep rumen liquor as sources of micro-organisms

1995 ◽  
Vol 1995 ◽  
pp. 110-110 ◽  
Author(s):  
S Akhter ◽  
E Owen ◽  
M K Theodorou ◽  
S L Tembo ◽  
E R Deaville
Keyword(s):  
Freeze Drying ◽  
Rumen Fluid ◽  
In Vitro Digestibility ◽  
Two Stage ◽  
Freeze Dried ◽  
Fresh Frozen ◽  
Sheep Rumen ◽  
Micro Organisms

Previous studies (El Shaer, Omed and Axford, 1987; Akhter, Owen, Fall, O'Donovan and Theodorou, 1994) with the two-stage in vitro procedure of Tilley and Terry (1963) have shown a high correlation between digestibilities of forages as determined using either sheep rumen liquor, sheep faeces or cow faeces as the microbial inoculum. In the first study of the of the present investigation one objective was to examine the repeatability of these digestibility measurements when made on different occasions. A second objective was to assess whether the correlations between faecal and rumen fluid based inocula could be improved if microorganisms were obtained from pairs rather than individual animals. The objective in the second study using forages of known in vivo digestibility, was to investigate the effect of freezing or freeze-drying of faeces on the repeatability of digestibilities of forages determined in vitro using micro-organisms from cow faeces.

scholarly journals A comparative study of ruminal activity in Churra and Merino sheep offered alfalfa hay

1997 ◽  
Vol 65 (1) ◽  
pp. 121-128 ◽  
Author(s):  
M. J. Ranilla ◽  
M. D. Carro ◽  
C. Valdés ◽  
F. J. Giráldez ◽  
S. López
Keyword(s):  
Cell Wall ◽  
Rumen Fluid ◽  
Merino Sheep ◽  
Rumen Ph ◽  
Sheep Rumen ◽  
Fermentation Parameters ◽  
Micro Organisms ◽  
Kinetics Of

AbstractA study was carried out to compare the fermentation parameters and kinetics of digestion of a range of different foods in the rumen of two breeds of sheep (Churra and Merino). Ten mature sheep (five Churra and five Merino), each fitted with a rumen cannula, were used in this study. In situ rumen degradability of both dry matter (DM) and cell wall was greater in Churra than in Merino sheep, the breed differences being significant for most of the foods used in the study (P < 0·05). These differences were greater when the foods had a higher cell wall concentration and this could be related to differences in the ruminal environment. However, when the foods were incubated with rumen fluid their in vitro organic matter (OM) degradability was similar in both breeds. Rumen pH was higher (P < 0·05) and ammonia concentrations were lower (P < 0·05) in Churra than in Merino sheep. Rumen volatile fatty acid concentrations tended to be higher in Merino than in Churra sheep, though differences were only significant just before feeding (P < 0·05). The ratio acetate: propionate was higher in the Churra than Merino breed before and 12 h after feeding (P < 0·05). Protozoa numbers in rumen liquid were similar for both genotypes. The greater degradation of forages in the rumen of Churra sheep is discussed in relation to the possible higher activity of fibre-degrading micro-organisms and the greater buffering capacity of the rumen contents against fermentation acids, which could result in more favourable conditions for the microbial degradation of foods in the rumen.

scholarly journals Excretion of purine derivatives by ruminants: recycling of allantoin into the rumen via saliva and its fate in the gut

1990 ◽  
Vol 63 (2) ◽  
pp. 197-205 ◽  
Author(s):  
X. B. Chen ◽  
F. D. DeB. Hovell ◽  
E. R. ØRskov
Keyword(s):  
Fatty Acids ◽  
Uric Acid ◽  
Urinary Excretion ◽  
Volatile Fatty Acids ◽  
Rumen Fluid ◽  
Purine Derivatives ◽  
Complete Destruction ◽  
Series Of Experiments ◽  
Micro Organisms

The saliva of sheep was shown to contain significant concentrations of uric acid (16 (sd) 4.5) μmol/l) and allantoin (120 (sd 16.4) μmol/l), sufficient to recycle purine derivatives equivalent to about 0.10 of the normal urinary excretion. When allantoin was incubated in vitro in rumen fluid, it was degraded at a rate sufficient to ensure complete destruction of recycled allantoin. In a series of experiments in which allantoin was infused into the rumen of sheep fed normally, or into the rumen or abomasum of sheep and the rumen of cattle completely nourished by intragastric infusion of volatile fatty acids and casein, no additional allantoin was recovered in the urine. These losses were probably due to the degradation of allantoin by micro-organisms associated with the digestive tract. It is concluded that all allantoin and uric acid recycled to the rumen via saliva will be similarly degraded. Therefore, the use of urinary excretion of purine derivatives as an estimator of the rumen microbial biomass available to ruminants will need to be corrected for such losses.

An in vitro cultured rumen inoculum improves nitrogen digestion in mulga-fed sheep

1997 ◽  
Vol 48 (4) ◽  
pp. 403 ◽  
Author(s):  
S. M. Miller ◽  
A. V. Klieve ◽  
J. J. Plumb ◽  
R. Aisthorpe ◽  
L. L. Blackall
Keyword(s):  
Rumen Fluid ◽  
Mixed Cultures ◽  
Microbial Composition ◽  
Widespread Adoption ◽  
The Difference ◽  
Micro Organisms ◽  
Laboratory Fermentor ◽  
Feral Goat ◽  
Goat Rumen

Mixed cultures of anaerobic micro-organisms were derived from feral goat rumen fluid (FGRF) using a laboratory fermentor to selectively culture microbes actively degrading mulga, and were evaluated as rumen inocula in digestion and liveweight studies with mulga-fed sheep. When placed in the rumen of sheep, FGRF enhances mulga digestion; however, limited supplies of feral goats, the labour involved in locating and mustering goats, and likely variations in the microbial composition of FGRF between animals and localities make the production of an in vitro cultured inoculum a desirable alternative to enable widespread adoption. The cultured inoculum significantly (P < 0·05) improved nitrogen digestion and retention in mulga-fed sheep by 16 and 76%, respectively. Inocula consisting of simplified mixtures of bacteria isolated from sheep, feral goats, and native marsupials did not affect mulga digestion. In the first of 2 liveweight studies, sheep inoculated with the fermentor inoculum lost significantly less weight than uninoculated sheep for the first 57 days (0·3 v. 4·6 kg); however, after 83 days the difference in the rate of liveweight loss between the fermentor inoculum group and the uninoculated sheep was not significant (53 v. 95 g/day). In the second study, liveweight loss was not significantly reduced by the fermentor inoculum. An inoculum based on FGRF, and produced in vitro using a fermentor, is potentially valuable to grazing enterprises reliant on mulga-fed sheep. However, problems in generating a consistent inoculum need to be addressed before such an inoculum can be generally considered.

A comparison between equine faeces and caecal digesta as sources of inoculum for in vitro fermentation studies using the pressure transducer technique

1996 ◽  
Vol 1996 ◽  
pp. 225-225
Author(s):  
R.S. Lowman ◽  
M.K. Theodorou ◽  
A.C. Longland ◽  
D. Cuddeford
Keyword(s):  
Pressure Transducer ◽  
Rumen Fluid ◽  
In Vitro Fermentation ◽  
Faecal Material ◽  
Micro Organisms

Several studies have shown high correlations between in vtvo and in vitro degradation of fibrous feeds when preparations from either rumen fluid or ruminant faeces have been used as the inocula for the in vitro studies (El Shaer et al., 1987; Akhter et al., 1994 & 1995; Harris et al., 1995). Use of an inoculum prepared from faecal material is attractive, for unlike that obtained from rumen fluid, it precludes the need to prepare and maintain fistulated donor animals. This study investigated the use of pony faeces, as an alternative to pony caecal digesta, as a source of micro-organisms for in vitro feed degradability studies.

A comparison of methods for determining the concentration of extracellular peptides in rumen fluid of sheep

1990 ◽  
Vol 114 (1) ◽  
pp. 101-105 ◽  
Author(s):  
R. J. Wallace ◽  
N. McKain
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Rumen Fluid ◽  
Amino Acid Content ◽  
Reliable Estimate ◽  
Precipitate Protein ◽  
Micro Organisms ◽  
Hydrolysis Of ◽  
Amino Acid Concentrations

SUMMARYSamples of rumen fluid were removed from pairs of sheep on four grass-hay-based diets 7 h after feeding. Micro-organisms were sedimented by centrifugation and the cell-free supernatant was treated with perchloric acid (PCA) to precipitate protein. The remaining fluid was analysed for peptides by several methods to determine how much peptide escaped degradation. Ammonia interfered with analysis by amino group reagents, especially ninhydrin. In this respect,o-phthalaldehyde and trinitrobenzene sulphonic acid were more specific and more useful than ninhydrin. Use of all these reagents showed that significant quantities of amino groups (equivalent to up to 153 mg amino acid N/1 of rumen fluid) were released by hydrolysis of the PCA extract with 6 M-HCI for 24 h. However, fluorescamine analysis indicated that the peptide content of the unhydrolysed PCA extract was < 3 mg N/1. The true amino acid content of different extracts was resolved by chromatographic amino acid analysis: the sum of individual amino acid concentrations in acid-hydrolysed PCA extracts of extracellular rumen fluid ranged from 7·8 to 14·5 mg N/1. Thus most of the free amino N released by hydrolysis of the PCA extract was not from amino acids, and most of the amino acids that were released were originally present in a form that did not react with fluorescamine. Although none of the methods gave a reliable estimate of peptide concentrations, amino acid analysis provided an upper limit. It was therefore concluded that peptide concentrations in extracellular rumen fluid are much lower than indicated by previous ninhydrin estimations, and that little dietary peptide escapes degradation for a prolonged period in the rumen.

scholarly journals Inhibitory effects of sulphur compounds, copper and tungsten on nitrate reduction by mixed rumen micro-organisms

1989 ◽  
Vol 61 (3) ◽  
pp. 741-748 ◽  
Author(s):  
Junichi Takahashi ◽  
Nobuyuki Johchi ◽  
Hiroshi Fujita
Keyword(s):  
Nitrate Reduction ◽  
Rumen Fluid ◽  
Sodium Sulphate ◽  
Microbial Reduction ◽  
Inhibitory Effects ◽  
Sulphur Compounds ◽  
The Media ◽  
Micro Organisms ◽  
And Tungsten

1. The inhibitory effects of inorganic and organic sulphur-containing compounds, copper and tungsten on nitrate reduction by mixed rumen micro-organisms were investigated in two in vitro studies.2. Coarsely strained rumen fluid from nitrate-adapted (Expt 1) or non-adapted (Expt 2) Suffolk Down wethers maintained on lucerne (Medicago sativa) cubes was used as an inoculum. In Expt 1, anaerobic incubation was carried out for 24 h for each medium supplemented with 10 mM-sodium nitrate and the following chemicals: 0, 1, 2, 3, 5, 8 and 10 mM-sodium sulphide, 1 and 10 mM-sodium sulphite, 1 and 10 mM-sodium sulphate, 1 and 10 mM-L-cysteine, 1 and 10 mM-DL-methionine, 1 mM-sodium tungstate and I mM-copper sulphate. In Expt 2, 1 and 10 mM-Na2S, 1 and 10 mM-L-cysteine, 1 mM-Na2WO4, and 1 mM-CuSO4were added to incubation media to test for chemical inhibition of microbial reduction of nitrate.3. In Expt 1, the amount of nitrite formed decreased with increasing concentration of sulphide-S added. The additions of L-cysteine, W and Cu suppressed nitrite formation in media from both nitrate-adapted and non-adapted sheep.4. In contrast to the effects of sulphide, L-cysteine and W counteracted, to some degree, nitrate-induced reduction of volatile fatty acid (VFA) production. Addition of Cu to the media resulted in a further depression of VFA production.

Determination of rumen microbial growth in vitro from32P-labelled phosphate incorporation

1977 ◽  
Vol 38 (1) ◽  
pp. 101-114 ◽  
Author(s):  
C. J. Van Nevel ◽  
D. I. Demeyer
Keyword(s):  
Microbial Growth ◽  
Specific Activity ◽  
Rumen Fluid ◽  
List Type ◽  
Substrate Protein ◽  
Isotope Method ◽  
Total Growth ◽  
N Incorporation

1.The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth.2.Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80 % of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells.3.Incorporation of32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth.4.When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18.3 and 44.6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed.5.The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.

Utilization of urea by sheep

1964 ◽  
Vol 63 (3) ◽  
pp. 289-296 ◽  
Author(s):  
H. M. Schwartz ◽  
C. A. Schoeman ◽  
M. S. Färber
Keyword(s):  
Rumen Fluid ◽  
Soluble Starch ◽  
Large Numbers ◽  
Food Particles ◽  
Freely Suspended ◽  
Micro Organisms ◽  
Strained Material

1.The breakdown of urea, soluble starch and glucose was followed in the rumen of fistulated sheep and in artificial rumens containing rumen ingesta or strained rumen fluid.2. Carbohydrate remained in the rumen two to three times as long when starch was dosed in vivo as when glucose was given.3. The relative rates of breakdown of urea and carbohydrate by strained rumen fluid in vitro differed markedly from those in vivo. This was due to the fact that straining removed large numbers of starch- and glucose-utilizing micro-organisms which were attached to the larger food particles, while the concentration of the urea-splitting organisms which were freely suspended in the strained liquor was apparently the same as that obtained in the strained material. Strained rumen fluid should therefore not be used for studies of the utilization of urea in vitro.

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