Solid-associated Bacteria Research Articles

Effect of phosphate solubility on phosphorus kinetics and ruminal fermentation activity in dairy goats

This study was carried out to determine the impact of phosphorus (P) solubility on its kinetics in liquid and bacterial ruminal phases and also on rumen fermentation activity. For the first time, the fractional disappearance rates of ruminal P were estimated and postprandial variations of bacterial P content were studied. Two groups were formed, each with four ruminal-canulated goats weighing 60 ± 1.4 kg, and were housed indoors within individual stalls. Their days in milk (DIM) ranged between 42 and 52. During adaptation and experimental periods, goats received twice a day (08:00 and 15:00 h) a ration consisting of 400, 300 and 300 g/kg of dry matter (DM) of sugar beet pulp silage, grass hay and experimental semi-purified concentrates, respectively. The concentrates supplied 0.71 g of P/g of dietary P either as soluble source (monocalcium phosphate, HSP group) or insoluble one (dicalcium phosphate, LSP group). Liquid and solid ruminal contents were obtained at time 0, 90, 180 and 360 min after morning feeding. Dry matter intake (DMI) and P intake (Pint) were recorded at each sampling time. Phosphorus in ruminal liquid, ruminal bacteria and volatile fatty acid (VFA) contents were quantified. DMI was 1.22 times higher for the HSP group. Available P concentration was 1.3 times higher for the same group at time 360. Fractional disappearance rates of P from rumen liquid content were not different between groups (−0.007 h −1). Postprandial changes (times 0–90) of P concentrations in liquid-associated bacteria (LAB) from LSP group suggested a relative P deficiency status (HSP = 1.6, LSP = 1.4, ±0.04 g/100 g of organic matter (OM)). Phosphorus content of solid-associated bacteria (P-SAB = 0.59 ± 0.02 g/100 g of SAB) presented less variation suggesting a capacity to use P from feedstuffs. The total and individual VFA kinetics were affected by P solubility. Under extreme conditions, where P supply derives mainly from inorganic sources, P solubility affects the kinetics of DMI and P concentrations in ruminal liquid, as well as fermentation activity. Phosphorus solubility significantly affects P content of LAB, whereas SAB appeared to adapt to variations of P concentrations in rumen liquid. However, further research is needed in these areas.

Effect of feed 15N incorporation into solid-associated bacteria on the in situ nitrogen degradability of 15N labelled Italian ryegrass

The objective of this study was to examine the effect of microbial contamination of the incubation residue on the in situ nitrogen degradability of Italian ryegrass. Microbial nitrogen in the bag residues was estimated by the feed 15N dilution procedure, and the incorporation of feed 15N into the adherent bacteria was assessed after isolating a bacterial pellet and determining its 15N enrichment. Isotopically labelled Italian ryegrass (fertilized with 15NH 4 15NO 3) was harvested either in spring (early cut) or in summer (late cut). Forages were incubated in the rumen of two steers for 6, 24 and 48 h. The proportion of microbial N per unit of total N in the bag residue increased with incubation time, ranging from 184 to 853 mg microbial N/g total N at 6 and 48 h, respectively. 15N enrichment in the bacterial pellet was highest after 6 h of incubation (3.6 mg 15N/g N) and then declined steadily (0.8 mg 15N/g N in 48-h residues). Microbial 15N represented up to 0.422 g/g total 15N in the bag residue, but the incorporation of feed 15N into bacterial N did not account for more than 57 mg/g 15N incubated, this incorporation rate decreasing progressively with incubation time. Correction of apparent N disappearance for microbial N resulted in higher values of N degradability, especially for the late cut grass. A further correction considering the amount of microbial 15N had little effect on the estimation of corrected N degradability values. Therefore, some of the feed 15N is incorporated into bacterial N, but this fraction has a minor effect on the estimation of total microbial N in the bag residue by the feed 15N dilution approach, and hence on the estimation of corrected degradability values.

Protein value for ruminants of a sample of whole cottonseed

AbstractThe effective ruminal degradability (ED) of dry matter (DM), crude protein (CP) and amino acids, and the effective intestinal digestibility (IED) of DM and CP of a sample of whole cottonseed was measured using in situ and rumen outflow rate techniques in three wethers cannulated in the rumen and duodenum. The microbial contamination of rumen incubated residues was corrected by a continuous rumen infusion of15NH3as microbial marker and rumen solid associated bacteria as reference sample. Microbial contamination resulted in an overestimation of the undegradable fraction of DM (0·291v. 0·275;P<0·05) and CP (0·071v. 0·037;P<0·01) and a small underestimation of ED of DM (0·500v. 0·512;P=0·09) and CP (0·755v. 0·779;P=0·052). A proportion of 0·1 of the ruminal undegraded CP was of microbial origin and for essential amino acids this proportion varied from 0·042 to 0·150. Differences in ED between amino acids modified the amino acid profile, with an important reduction (0·2;P<0·01) in the proportion of lysine. Apparent intestinal digestibility of the insoluble fraction of this food, measured with the mobile nylon bag technique, showed large reductions (P<0·001) with the increase of the ruminal incubation time between 0 and 72 h: from 0·392 to 0·026 for DM and from 0·851 to 0·099 for CP. These evolutions fitted an exponential function with a previous lag. The IED was estimated either by integration of these equations and those describing the ruminal degradation and rumen outflow or by incubation through the intestines of a sample pooled to be representative of rumen flow of the undegraded food. The two methods gave similar values for both DM (0·222v. 0·203) and CP (0·659v. 0·658).

Effects of the forage to concentrate ratio on B-vitamin concentrations in different ruminal fractions of dairy cows

Two studies were undertaken to verify the effect of the forage to concentrate ratio of the diet on B-vitamin concentrations in ruminal contents. In Study 1, eight primiparous and eight multiparous cows were used in a cross-over design, and concentrations of biotin, folates and vitamin B12 were determined in ruminal fluid and plasma of cows fed a high-forage (HF; 58:42 forage to concentrate ratio; DM basis) or a low-forage (LF; 37:63 forage to concentrate ratio; DM basis) diet. In Study 2, six ruminally cannulated lactating cows were used in a cross-over design to evaluate the effects of forage to concentrate ratio (HF = 60:40; LF = 40:60; DM basis) on concentrations of seven B-vitamins in the particle-free fluid and in both liquid- and solid-associated bacteria. Results showed that B-vitamins were present mainly in the bacterial fractions of the ruminal content, while only limited amounts were found in the surrounding fluid. A change in the forage to concentrate ratio had a greater effect on vitamin concentration in the bacteria associated with the solid fraction than in those present in the liquid portion of the rumen. The most noticeable effects of a low forage diet were an increase in riboflavin, but a decrease in true vitamin B12 concentrations in solid-associated bacteria as well as a decrease in biotin concentration in particle-free fluid. In conclusion, it appears that ruminal B-vitamin concentration is altered by changes in the forage to concentrate ratio, which suggests that the supply of vitamins to dairy cows is influenced by diet composition. Key words: B-vitamins, dairy cow, ruminal fractions, forage to concentrate ratio

Fatty Acid Profiles Associated with Microbial Colonization of Freshly Ingested Grass and Rumen Biohydrogenation

Two in situ studies were conducted to examine the use of odd-chain fatty acid profiles to study microbial colonization of freshly ingested herbage in the rumen as well as fatty acid biohydrogenation. In the first study, fresh perennial ryegrass was subjected to a range of sample preparation methods before incubation in the rumen for 2 or 7h. In the second study, fresh perennial ryegrass was chopped into 1-cm lengths and incubated in polyester bags in the rumen for 2, 8, and 24h. After removal of bags from the rumen, 4 different washing methods, ranging from manual squeezing to machine washing, were applied. Fatty acids were extracted from washed residues and determined, as methyl esters, by gas chromatography. The main odd-chain fatty acids (with the exception of anteiso C15:0) were not found in fresh grass and were useful markers of the effects of incubation time, sample preparation method, and washing method on microbial colonization/contamination. The concentration of these and other odd-chain fatty acids increased with incubation time in both studies. The results indicate rapid and continued microbial colonization of freshly ingested forages, although patterns of odd-chain fatty acids did not reveal any further information about the types of bacteria-colonizing herbage. Principal component, biplot analysis provided a useful overall description of the processes of microbial colonization and degradation of plant fatty acids on fresh herbage incubated in the rumen. Bolus formation during mastication and ingestion results in extensive damage to herbage; none of the techniques (cutting, crushing, and drying/grinding) investigated in this work was able to replicate the effects of bolus formation in the animal. The study provided further evidence of loss of unfermented feed particles through polyester bag pores, especially when feeds are dried and ground. Biohydrogenation of the polyunsaturated fatty acids of fresh herbage was used principally by solid-associated bacteria to enable them to take up high levels of trans-11C18:1 and C18:0 fatty acids. Although trans-11C18:1 was strongly associated with bacterial markers (odd- and branched-chain fatty acids), its precursor (cis-9, trans-11C18:2) was not associated with bacterial variation, suggesting that its production in the rumen under these conditions was mainly extracellular.

Effects of feed intake on composition of sheep rumen contents and their microbial population size.

The present study was conducted to determine the effect of feed intake on the composition of the rumen contents of sheep and on their bacterial densities. Whole rumen contents were sampled after a period of continuous inter-rumen infusion of 15NH3 from four rumen-cannulated wethers successively fed on a hay-concentrate diet (2:1, w/w on a DM basis) at two rates of feed intake: 40 and 80 g DM/kg body weight0.75. Total weight and chemical composition of rumen contents, as well as the distribution by size and chemical composition of particles, were determined. The populations of bacteria associated with the liquid (liquid-associated bacteria, LAB) and solid (solid-associated bacteria, SAB) fractions of rumen digesta and the distribution of SAB according to feed particle size were also examined. The greater feed intake caused an increase in the mass of the rumen contents, while its chemical composition did not change, except for a higher content of organic matter (P=0.023). The distribution of feed particles by size was similar at both levels of intake. The concentrations of neutral- and acid-detergent fibre in feed particles decreased and those of total, dietary, and microbial N increased, both with a quadratic response (P=0.001), as particle size decreased. The proportion of LAB in the microbial biomass of rumen digesta reached only 8.0 %. This proportion and the density of LAB were unaffected by the level of feed intake, whereas an apparent reduction (10.4 %) occurred with the SAB biomass in whole rumen contents. A systematic, but not significant, reduction (mean value 11.9 %) in the level of microbial colonisation in the different particle fractions with the increase of feed intake was also observed.

Comparison of microbial markers (15N and purine bases) and bacterial isolates for the estimation of rumen microbial protein synthesis

AbstractThe first objective of this experiment was to investigate the effect of using different bacterial isolates on the estimation of microbial protein production in semi-continuous fermenters (RUSITEC) given four diets, and to test whether a ‘mixed’ bacterial pellet was representative of the whole bacterial population in the fermenters. A second objective was to compare two different microbial markers (nitrogen-fifteen (15N) and purine bases (PB)). Diets consisted of neutral-detergent fibre from grass hay (10 g/day) and sugar-beet pulp (2 g/day) and 280 mg/day of one of four N forms (isolated soya-bean protein, soya-bean peptides, amino acids blended to profile soya-bean protein and NH4Cl). Two 14-day incubation runs were carried out and in each run each of the four different diets were given to two vessels. On days 12 and 13, total digesta (effluent plus nylon bags residues) was collected for analyses of non-ammonia N,15N enrichment and PB concentration, and for isolation of total mixed bacterial pellets (TB). On the last day of each incubation run, the system was stopped for isolation of liquid- (LAB) and solid-associated (SAB) bacteria. Microbial N flow was estimated from the15N enrichment and PB concentration in both total digesta and in the three different bacterial pellets (TB, LAB, and SAB). For all diets, LAB presented a greater (P < 005)15N enrichment and PB: N ratio than SAB, with TB having an intermediate value. For both markers, the use of LAB produced the lowest (P < 005) estimates of microbial N flow and the use of SAB produced the greatest (P < 005) estimates. The use of TB produced intermediate values with all diets, suggesting that TB consisted of SAB and LAB. For all bacterial pellets, PB produced greater (P 005) values of microbial N flow than15N. However, there was a positive relationship (r = 0·883; P 0001; no. = 15) between the values of microbial N flow determined with the two markers when TB were used as reference.

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta.

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.

In situ rumen degradation of amino acids from different feeds corrected for microbial contamination

Variations of the amino acid profile produced by rumen actions were established for fish meal (FM), corn grain (CG), corn gluten feed (GF), dehydrated beet pulp (DBP), and lucerne hay (LH), using the nylon bag technique. The effects of degradation of soluble or insoluble proteins were mea- sured by rumen incubations at 0 h (washout value) and 16 h, respectively. Rumen incubations were performed on four cannulated wethers and the microbial contamination of incubated residues was cor- rected using a continuous 15 N-intraruminal infusion and solid associated bacteria isolates as the ref- erence sample. The results showed that rumen degradation of both soluble and insoluble proteins pro- duced changes in the amino acid profile, which were not uniform among feeds. Differences between feed and insoluble protein amino acid composition were limited for FM and DBP, important for GF and LH, and intermediate for CG. Differences between insoluble and undegraded protein amino acid composition were also limited for FM, GF, DBP, and LH and important in CG. The results showed that the amino acid profile in the undegraded protein and in the whole feed should be close for FM, DBP and LH. On the contrary, in CG, important variations are expected, since both solubility and degradability effects produced changes with the same tendency for most of the essential amino acids. In particular, our results showed a great decrease in the lysine content of the undegraded protein, which increases the lysine deficiency of this feed. This decrease of lysine was also evidenced in GF. rumen degradation / amino acid profile / microbial contamination / 15 N

Effect of dietary factors on distribution and chemical composition of liquid- or solid-associated bacterial populations in the rumen of dairy cows.

Effects of dietary factors including kernel thickness of processed barley grain, ratio of forage to concentrate, and forage particle length on chemical composition of bacteria, bacterial colonization of feed particles and distribution in the rumen, and duodenal flow of bacteria in dairy cows were evaluated. The experiment was designed as a double 4 x 4 quasi-Latin square with a 2 x 2 x 2 factorial arrangement of treatments using eight lactating cows with ruminal and duodenal cannulas. Barley grain was steam-rolled to two thicknesses: coarse (1.60 mm) or flat (1.36 mm); ratio of forage:concentrate was low (35:65) or high (55:45) (DM basis); and forage particle length was long (7.59 mm) or short (6.08 mm). Cows were offered ad libitum access to a total mixed diet. Chemical composition was different (P < 0.01) between liquid-associated bacteria (LAB) and solid-associated bacteria (SAB). Reduced barley thickness increased (P < 0.05) N content and 15N enrichment, but a high ratio of forage:concentrate decreased (P < 0.01) 15N enrichment of both the LAB and SAB. Significant differences between AA composition of the LAB and SAB were observed for 15 out of 17 AA studied. Bacterial colonization was linearly increased (P < 0.01) from about 5 to 70% as particle length of rumen contents was reduced from 3.35 mm to 0.15 mm (sieve size). The degree of colonization on each fraction of the rumen particulate matter was only affected (P < 0.10) by the ratio of forage:concentrate, with consistently higher (P < 0.10) bacterial colonization noted for high than for low forage:concentrate diets. Of the total bacterial mass within the rumen, less than 20% was associated with the liquid and over 70% was associated with the small particles that passed through the 0.6-mm sieve. Although the bacterial pool in the rumen was lower (P < 0.04) when flatly rolled barley rather than coarsely rolled barley was fed, bacterial flow to the duodenum was greater (P < 0.10) with increasing ratio offorage:concentrate. The present results confirm the differences of chemical composition and biomass for LAB and SAB. Manipulation of dietary factors such as ratio of forage:concentrate have the potential to alter bacterial colonization of rumen particles and the relative proportion of LAB to SAB, which were positively correlated to bacterial flow to the duodenum.

Effects of fibre level and particle size on rumen microbial fermentation and protein metabolism using liquid and solid associated bacteria

The effects of fibre level (F) and forage particle size (S) on ruminal fermentation profile is often mediated through changes in feed intake, rates of digestion or passage, ruminal pH and/or bacteria population. Therefore, most in vivo studies have confounded the direct effect of F or S with changes in the rumen environment. In vitro systems allow to control several fermentation conditions independently (pH, flow rates, intake). Total, bacterial and dietary nitrogen (N) flows are generally calculated using liquid associated bacteria (LAB), although solid associated bacteria (SAB) represent about 80% of total bacterial population in the rumen (Olobobokun and Craig, 1988). The objective of this experiment was to study the effects of F and S on microbial fermentation and N metabolism using LAB or SAB values in a dual flow continuos culture system.

Rumen effective degradability of amino acids from soybean meal corrected for microbial contamination.

Rumen degradation kinetics and effective degradability of individual amino acids, total analysed amino acids (TAA) and crude protein (CP) of soybean meal were measured on four rumen-cannulated wethers using the nylon bag technique. Microbial contamination of the incubated residues was corrected using a continuous 15N intraruminal infusion and isolated solid associated bacteria as a reference sample. TAA showed a lower soluble fraction (14.9 vs. 20.8%; P < 0.01), a similar insoluble-degradable fraction (79.0 vs. 79.2%) and a higher degradation rate (11.5 vs. 8.4% x h(-1); P < 0.05) than CP. As a consequence, effective degradability was similar for TAA and CP (74.7 vs. 75.7%). Degradability values of individual amino acids varied moderately (range: +/-6% of TAA degradability). Valine, isoleucine, leucine, alanine, aspartic acid and tyrosine showed significantly lower degradability than TAA, while the opposite effect was observed for histidine, threonine and glutamic acid. Degradability of individual amino acids was related to their soluble fraction (r = 0.877; P<0.001).

Composition of bacteria harvested from the liquid and solid fractions of the rumen of sheep as influenced by feed intake

A study was conducted to determine the effect of the feed intake on the chemical composition of bacteria associated with the solid (solid-associated bacteria; SAB) and liquid (liquid-associated bacteria; LAB) fractions of rumen digesta, the digestive passage kinetics and their relationships. Whole rumen contents were sampled after a period of continuous infusion of 15NH3 from four ruminally-cannulated wethers provided successively with a hay-concentrate diet (2 : 1 w/w on a DM basis) at two rates of feed intake: 40 and 80 g DM/kg body weight 0.75. SAB had a higher content of organic matter and total lipids (P < 0.001) and a similar N content as compared with LAB. The concentration of purines and 15N was lower (P = 0.011 and P < 0.001 respectively) in SAB than LAB, whereas the opposite was observed for the concentration of amino acids (mg/g DM; P = 0.031). An increase in feed intake produced an increase in the N (P = 0.034) and purine (P = 0.066) concentrations in bacteria and a decrease (P = 0.033) in their amino acid concentrations. Significant increases of rumen outflow rates of liquid and particles were also observed with increased feed intake. Rates of rumen outflow showed positive and negative linear relationships (P < 0.001) with the purine : N ratio and the proportion of amino acid on total N of bacteria respectively. SAB contained significantly higher proportions of leucine, isoleucine, lysine and phenylalanine and lower proportions of alanine, methionine and valine than LAB. The increase in feed intake also induced significant changes in the amino acid profile of bacteria, increasing arginine and methionine and decreasing alanine and glycine proportions. Results show that the outflow rate of rumen contents is a major factor in determining the proportion of nucleic acids and protein in rumen bacteria and explains some of the differences observed between LAB and SAB.

Effects of source of bacterial isolate and microbial marker on the magnitude of absolute values of microbial nitrogen yield in sheep

Microbial protein is the most variable and uncertain element of current systems of evaluating protein requirements for ruminants. The variability is partly a result of the multiplicity of techniques used to quantify microbial nitrogen (MN) yield and partly due to unrepresentative sampling of microbial cells. In a number of studies in the literature it is tacitly assumed that marker:N ratios in rumen liquid associated bacteria (LAB), solid associated bacteria (SAB) and duodenal digesta bacteria (DDB) are identical. Under ordinary feeding conditions ruminal SAB are numerically predominant and may comprise 50-75% of the total microbiota (Mitsumori and Minato, 1997). This study highlights the variations in chemical composition in LAB, SAB and DDB and their implications in estimating microbial growth. The study also simultaneously compares four microbial markers in vivo.

Influence of sampling site on concentrations and carbohydrate-degrading enzyme activities of protozoa and bacteria in the rumen.

Four ruminally cannulated cows were used to assess the distribution of the concentrations and carbohydrate-degrading enzyme activities of the liquid-associated protozoa (LAP) and solid-associated bacteria (SAB) in the rumen. The cows were fed diets (7 kg of DM/d) of 100% hay (Diet F) or 60% barley plus 40% hay (Diet C) in a 2 x 2 crossover experimental design. Samples of ruminal digesta were collected successively from the dorsal (DS), ventral (VS), and anterior (AS) sacs 1h before and 3 h after the morning feeding on two sampling days with a 2-d interval. Irrespective of diet and sampling time, the greatest proportion of entodiniomorphs, representing the main population of protozoa, was found in the DS (40% mean; P < .05). Low pH values observed in the DS (P < .05) indicated higher fermentative activity in this site than in the other parts of the rumen. Protozoa may contribute more than previously thought to the high digestive potential present at the top of the rumen that has classically been attributed to bacteria. The specific activity of plant cell wall polysaccharide-degrading enzymes in the LAP was correspondingly greater (P < .05) for DS than for VS or AS. For the two diets and sampling times, specific activity of fibrolytic enzymes in the SAB tended to be less in the upper than in the lower parts of the rumen, and less in the VS than in the AS. This tendency became significant (P < .05) for total fibrolytic enzyme activity. Differences in bacterial colonization of particles among the three sampling sites may explain such differences in fibrolytic activity of the SAB. Data suggest a spatio-temporal complementary action of the bacteria and protozoa in ruminal plant cell wall degradation, at least with the barley diet, for which the number of protozoa was highest.

Rumen degradability and microbial contamination of fish meal and meat meal measured by the in situ technique

Rumen degradation characteristics and effective degradability of six fish meals (FM) and five meat meals (MM) were measured in two trials by using nylon bag and rumen outflow rate techniques in rumen cannulated sheep. In trial 1, apparent degradation of crude protein (CP) of five FM and four MM samples from different processing plants were studied with three animals. In trial 2, microbial contamination in the incubated residues of one additional FM and MM sample was determined by continuous 15 N intraruminal infusion and using isolated solid associated bacteria as reference sample, with four animals. These values were used to correct the estimates of apparent degradation of dry matter (DM) and CP. During both trials, animals were fed at the same intake level (40 g DM Kg −1 LW −0.75) with 2:1 hay to concentrate diets. Crude protein content (on DM) and buffer CP solubility of the samples ranged from 61.5% to 68.2% and 5.4% to 14.3% for FM and from 52.3% to 66.7% and 9.9% to 22.7% for MM, respectively. Apparent CP degradability ranged from 18.2% to 34.9% and 41.1% to 60.4% in FM and MM, respectively. Degradability values of CP in FM were conditioned by a lag phase (range 3.5–20.7 h) which was inversely correlated with the in situ ( r=−0.76) and buffer ( r=−0.52) CP solubilities. Buffer CP solubility explained 81% of the CP degradability variation and can be used as a predictor of it. Changes of microbial contamination with incubation time of residues, expressed as percentages of DM and N, fitted well to a simple mono-exponential model. Contamination levels were always very low, with higher values for MM than for FM. The fractional rate of microbial accumulation was markedly higher in the MM than in the FM sample. No correction for microbial contamination decreases CP degradability estimates ( P<0.001), but this error (0.82% and 1.11% for FM and MM) can be considered of low nutritional significance in both meals.

Composition of liquid-and particle-associated bacteria and their contribution to the rumen outflow

A method is described to estimate the composition and rumen outflow of microbes associated with liquid (LAB) and solid (SAB) digesta. Four rumen-cannulated Rasa Aragonesa ewes were given, in random order, the following 4 diets: (1) NaOH-treated barley straw, as a sole diet (700 g/day, TS); (2) NaOH-treated barley straw mixed (50 : 50) with 400 g/day of rolled barley grain (BS); (3) Diet 1 with addition of 8 g/day of urea; and (4) Diet 2 with addition of 16 g/day of urea. Co-EDTA was used as a marker for the liquid phase to estimate rumen outflow of liquid-associated purine bases (PB), and urinary purine derivatives were used as an indirect marker of total duodenal flow of PB. Solid-associated PB were calculated by the difference between both estimates. Urea infusion increased ammonia-N concentration in the rumen fluid from 4·8 to 15·9 mg/100 mL (P &lt; 0· 05) and enhanced dry matter intake of TS diets (from 343±63· 5 to 556±41·2 g/day, P &lt; 0·001). Significant differences were observed in the PB/N ratio of bacteria harvested from the liquid phase compared with that isolated from the solid phase (1·89±0·25 v. 1·66±0·32 mol/mg in LAB and SAB, respectively). Because of the differences observed between the liquid- and solid-associated bacteria, estimated values of bacterial N supply varied depending on which bacterial extract was used as reference. The fractional contribution of LAB and SAB to the postruminal bacteria was significantly influenced by the experimental diets, mainly through variations in the amount of LAB flowing out of the rumen.

Effect of microbial isolates on microbial yield estimation in RUSITEC system

The estimation of rumen microbial protein synthesis is one of the main points in the nitrogen (N)-rationing systems for ruminants, as microbial protein provides proportionately 0.4 to 0.9 of amino acids entering the small intestine in ruminants receiving conventional diets (Russell et al., 1992). Methods of estimating microbial protein synthesis rely on marker techniques in which a particular microbial constituent is related to the microbial N content. Marker : N values have generally been established in mixed bacteria isolated from the liquid fraction of rumen digesta and it has been assumed that the same relationship holds in the total population leaving the rumen (Merry and McAllan, 1983). However, several studies have demonstrated differences in composition between solid-associated (SAB) and fluid-associated bacteria in vivo (Legay-Carmier and Bauchart, 1989) and in vitro (Molina Alcaide et al, 1996), as well in marker : N values (Pérez et al., 1996). This problem could be more pronounced in the in vitro semi-continuous culture system RUSITEC, in which there are three well defined components (a free liquid phase, a liquid phase associated with the solid phase and a solid phase), each one having associated microbial populations.The objective of this experiment was to investigate the effect of using different bacterial isolates (BI) on the estimation of microbial production of four different diets in RUSITEC (Czerkawski and Breckenridge, 1977), using (15NH4)2 SO4 as microbial marker, and to assess what effects any differences would have on the comparison of microbial protein synthesis between diets.This study was conducted in conjunction with an in vitro experiment described by Carro and Miller (1997). Two 14-day incubation trials were carried out with the rumen simulation technique RUSITEC (Czerkawski and Breckenridge, 1977). The general incubation procedure was the one described by Czerkawski and Breckenridge (1977) and more details about the procedures of this experiment are given elsewhere (Carro and Miller, 1997).

The effect of high lipid sheep diets on fatty acids of liquid and solid associated rumen bacteria

The effect of high lipid sheep diets on fatty acids of liquid and solid associated rumen bacteria

Contribution of dietary purine bases to duodenal digesta in sheep. In situ studies of purine degradability corrected for microbial contamination

The dietary contribution of purine bases (PB) to duodenal flow was evaluated by the in situ method after correcting for microbial contamination using 15N as microbial marker and rumen solid associated bacteria as reference sample. Four ruminally fistulated sheep were offered at 4 h intervals a mixed diet 2:1 vetch-oat hay:concentrate, and 179 mg of (( 15NH 4) 2SO 4) were continuously infused in the rumen during a minimum of 5 days prior to incubation and rumen sampling. The following feeds were tested: meat meal (MM), fish meal (FM), gluten feed (GF), barley grain (BG), corn grain (CG), sunflower meal (SFM), soya bean meal (SBM), dry brewery distilled grain (DBDG), alfalfa hay (AH) and barley straw (BS). The largest residual proportion of microbial purine bases was found in BS followed by AH, CG, BG, DBDG, GF, SFM, MM, SBM and FM, in that order. For all feeds, the extent of degradation of dietary purine bases at the studied fractional outflow rates increased after the correction for microbial contamination. Apparent and true degradability were significantly different ( P < 0.05) in DBDG, CG, AH, GF and MM, ranging the feeds in that descending order; whereas they did not differ in BG, SBM, SFM and FM. Correction for contamination of feeds by microbial purine bases did not substantially affect the proportion of dietary purines escaping ruminal degradation ranging from 5 to 17% for AH, BG, GF, SFM, CG, from 11 to 23% for MM, SBM and from 20 to 40% for FM and DBDG when fractional rates of outflow from the rumen of 0.02–0.08 were assumed.