Glutamate dehydrogenase and glutamine synthetase activity of the bacteria of the sheep's rumen after different nitrogen intake

In experiments on 18 sheep with a differentiated nitrogen intake (3.7, 6.2 and 21 g N/day), it was found that different enzyme activities--glutamate dehydrogenase (GDH) (NADH- and NADPH-dependent) and glutamine synthetase (GS)--of bacteria adhering to the rumen wall and to food particles and the rumen fluid bacteria altered in correlation to the nitrogen intake. With a nitrogen intake of 3.7-6.2 g/day there was a significant increase, and of 6.2-21 g/day a decrease, in NADH- and NADPH-dependent GDH activity in the three given bacterial fractions, with the exception of NADPH-dependent GDH activity of the rumen fluid bacteria of sheep given 3.7-6.2 g N/day, in which the difference was nonsignificant. GS activity was significantly higher only in adherent rumen wall bacteria in the presence of a nitrogen intake of 3.7-6.2-21 g/day. The results show that the effect of the nitrogen intake on the given enzyme activities is strongest in the case of bacteria adhering to the rumen wall. The high GS activity and low GDH activities in these bacteria during lower nitrogen intakes (3.7 g/day) as well as lower rumen ammonia concentration (2.39 +/- 0.98 mmol.l-1) indicate that bacteria adhering to the rumen wall utilize ammonia at an increased rate by means of CS catalyzed reactions. Reduced GDH activity in the presence of a high nitrogen intake (21 g/day) and the relatively high rumen ammonia concentration (36.63 +/- 5.28 mmol.l-1) indicate that ammonia inhibits this enzyme in the rumen bacteria in question.     

Urease activity of adherent bacteria in the sheep rumen

In experiments on six sheep fed on a low protein diet (6.2 g N/day), it was found that the urease activity of the rumen fluid did not change significantly in the first 6 hours after feeding and that it ranged from 45 to 75 nkat.ml-1. The major portion was bound to the bacterial fraction and formed about 70% of total rumen fluid activity. Urease activity determined in food particles with adherent bacteria removed from the rumen before and 3 and 6 hours after feeding ranged from 20 to 26 nkat.g-1 food (wet weight), and on rumen wall samples with adherent bacteria from 30 to 800 nkat per 2.5 cm2 tissue. Again, no significant changes correlated to the time after feeding were found. The results show that urease activity in the sheep rumen is localized on food particles and on rumen wall epithelium with adherent bacteria, as well as in the rumen fluid.     

Ammonia-utilizing enzymes of adherent bacteria in the sheep's rumen

In experiments on 6 sheep the authors found the following enzyme activities in bacteria in the rumen fluid, bacteria adhering to the epithelium of the rumen wall and bacteria adhering to food particles in the rumen (given in nkat X g-1 bacterial dry weight): GDH (NADH): 725 +/- 165, 558 +/- 127, 661 +/- 153; GDH (NADPH): 558 +/- 338, 255 +/- 88, 565 +/- 139; GOAT (NADH): 46 +/- 23, 67 +/- 31, 66 +/- 14; GOGAT/NADPH: 58 +/- 27, 56 +/- 15, 65 +/- 29; GS: 153 +/- 65, 69 +/- 35, 71 +/- 32; ALT: 71 +/- 25, 43 +/- 20, 52 +/- 11; AST: 52 +/- 12, 33 +/- 16, 28 +/- 15. The results show that, except for GDH (NADPH), there were no significant differences between the given enzyme activities in the rumen fluid and in bacteria adhering to the rumen wall and to food. Adherent rumen bacteria have the same potential possibilities as the rumen fluid bacteria for the utilization of ammonia, particularly for the synthesis of glutamic acid, glutamine, alanine and aspartic acid, with the above enzymes as catalysts. By means of the GS/GOGAT system, adherent rumen bacteria can probably synthesize glutamic acid in the presence of a limited NH3 concentration in the rumen.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep

Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine. However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood. The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne)-white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins). The bacterial populations were quantified using a competitive polymerase chain reaction. Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined. When sheep fed on ryegrass-white clover, populations of Clostridium proteoclasticum B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-1), respectively. When the diet was changed to Lotus corniculatus, the average populations (after 8-120 h) of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis decreased (P < 0.001) to 2.4 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-1), respectively. When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis were higher (P < 0.01-0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8-120 h) of 4.9 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(6), respectively. Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05-0.001) than sheep that were fed Lotus corniculatus plus PEG. The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum. The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus. Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen.     

Evidence for existence of lectins on the ruminal bacteria from steers fed roughage and concentrate diets

Mixed rumen bacteria, isolated by centrifugation from the rumen of steers fed a roughage (R) or concentrate (C) diet, were used to determine if lectins are present on rumen bacteria, based on hemagglutination (HA) and HA inhibition assays in vitro. Rumen bacteria from steers fed either diet agglutinated erythrocytes from cattle, sheep, pigs, and rats, suggested that lectins exist on rumen bacteria. Bacterial HA titers from steers receiving the R diet were much higher (p<0.001) than those from steers fed the C diet, depending on the erythrocyte source used. Centrifugation at 20,000xg for 30 min fractionated the rumen bacteria into upper (U) and lower (L) layers. The HA titers of the U bacterial fractions were significantly higher (p<0.001) than those of the L fractions. A remarkable reduction or complete disappearance of HA titers following treatment of rumen bacteria with protease, trypsin, sodium dodecyl sulfate (SDS) or sodium periodate indicates that rumen bacterial lectins are probably glycoproteins. Lectin specificity for saccharides (galactose, lactose, N-acetyl-D-galactosamine, methyl-alpha-D-galactopyranoside and methyl-beta-D-galactopyranoside) and glycoproteins (mucin, fetuin, and thyroglobulin) was found in the RU, RL, and CU bacterial fractions; no specific binding was determined in the CL fractions. The potential role of lectins in mediating the attachment of rumen bacteria to feed particles, rumen epithelia and other microorganisms is discussed.     

Nucleic acid synthesis from blood urea 15N in the sheep

In experiments on 4 sheep fed on a low protein diet [6.2 g N/day] and given a single i.v. dose of 15N-labelled urea [15 mg 15N/kg body mass], the authors found that, from 0.5 to 6 h, mean 15N incorporation rose progressively in the total rumen fluid nitrogen from 0.23 to 0.44 at. % 15N and in the rumen bacterial nitrogen from 0.11 to 0.51 at. % 15N. Up to 3 h, total nitrogen enrichment was greater (0.5 at. % 15N) than enrichment of bacterial nitrogen (0.28 at. % 15N), but from 3 to 6 h there was little difference between them. The mean 15N values in the nucleic acids isolated from rumen fluid bacteria in samples collected 3 and 6 hours after injecting labelled urea into the blood were 0.15 and 0.19 at. % 15N respectively, in nucleic acids isolated from the liver 0.042 and 0.04 at. % 15N, in the total rumen bacterial nitrogen 0.28 and 0.51 at. % 15N and in the total liver nitrogen 0.11 and 0.11 at. % 15N. It is concluded from the results that blood urea nitrogen is utilized for synthesis of the total nitrogenous substances of the sheep's rumen bacteria and liver far more intensively than for synthesis of the nucleic acids isolated from them. At the same time, it is utilized more intensively for nucleic acid synthesis in the rumen bacteria than in the liver.     

Association of methanogenic bacteria with rumen protozoa

Methanogenic bacteria superficially associated with rumen entodiniomorphid protozoa were observed by fluorescence microscopy. A protozoal suspension separated from strained rumen fluid (SRF) by gravity sedimentation exhibited a rate of methane production six times greater (per millilitre) than SRF. The number of protozoa (per millilitre) in the protozoal suspension was three times greater than that of SRF; however, the urease activity of this fraction was half that of SRF. The methanogenic activity of SRF and the discrete fractions obtained by sedimentation of protozoa correlated with the numbers of protozoa per millilitre in each fraction. Gravity-sedimented protozoa, washed four times with cell-free rumen fluid, retained 67-71% of the recoverable methanogenic activity. Thus it is evident from our observations that many methanogens adhere to protozoa and that the protozoa support methanogenic activity of the attached methanogens. When protozoa-free sheep were inoculated with rumen contents containing a complex population of protozoa, methanogenic activity of the microflora in SRF samples was not significantly enhanced.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Digestion of epithelial tissue of the rumen wall by adherent bacteria in infused and conventionally fed sheep.

Comparisons were made, by light and electron microscopy, of the rumen epithelium of sheep fed conventionally and fed by infusion of volatile fatty acids and buffer into the rumen and casein into the abomasum. Similar bacterial colonization of the epithelium was observed in each case. The mitotic index of epithelial cells in infused sheep was high, as it was in barley-fed animals, while the mitotic index of cells from animals receiving roughage was low. The bacterial flora appeared to be actively digesting the epithelial cells. The fate of sloughed epithelial cells in the rumen fluid of sheep fed by infusion was also studied. The sloughed cells were rapidly digested, probably by their attached flora of facultatively anaerobic, highly proteolytic bacteria, leaving abundant highly keratinized remnants in rumen fluid. The importance of epithelial cell turnover and of proteolysis by partially facultative bacteria in the rumen is discussed.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

The renal response of sheep to a low dietary nitrogen intake

Renal functions were tested in sheep fed on a low nitrogen diet (LN sheep), with a daily N intake of 4.7 g (gross energy 17.76 . 10(6) J). Sheep given a high nitrogen diet (HN sheep) with 21.2 g N (24.12 . 10(6) J) acted as the control. The functions of the left kidney were measured in anaesthetized animals by the standard clearance technique. A comparison of HN and LN sheep showed that a low nitrogen intake led to a drop in the plasma urea level (from 5.91 +/- 0.35 to 2.87 +/- 0.36 mmol.1-1, (P less than 0.001), the glomerular filtration rate (GFR, from 36.6 +/- 3.6 to 20.7 +/- 2.4 ml.min-1, P less than 0.005), amount of urea excreted (from 106.7 +/- 18.1 to 15.7 +/- 3.3 mumol.min-1, P less than 0.001), fractional urea excretion (from 51.0 +/- 3.0 to 24.6 +/- 3.1 %, P less than 0.001) and the absolute tubular reabsorption of urea (Reaburea/GFR (from 3.06 +/- 0.27 to 2.12 +/- 0.28 mumol.ml-1, P less than 0.05), without a significant change in the effective renal plasma flow (182.6 +/- 20.0 and 138.5 +/- 21.0 ml.min-1, non-significant - N.S.) and in sodium and potassium excretory function. Free water clearance rose in LN sheep (from -0.53 +/- 0.11 to -0.19 +/- 0.06 ml.min-1, P less than 0.05) owing to inhibited urea excretion. A regression analysis of the relationship of the tubular reabsorption of urea to the amount of filtered urea (both normalized to the GFR) showed that the urea transport capacity of the tubules of LN sheep was significantly higher.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

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.     

Effect of rumen ciliates on the digestion of different carbohydrates in sheep. I.--Utilization of cell wall carbohydrates (cellulose and hemicellulose) and of starch

Two diets rich in cell-wall carbohydrates or starch were given to 10 rumen-fistulated sheep; two sheep were defaunated and the others were inoculated with Polyplastron multivesiculatum (P) or Entodinium sp. (E), or both (P + E), or with conventional fauna. Ciliate biomass was greater when the animals were fed a high starch diet than when the diet was rich in cell-wall carbohydrates (table 2). With both diets, the Entodinium genus in the mixed fauna sampling predominated. We showed that Polyplastron was directly involved in cell-wall carbohydrate breakdown, while Entodinium capacity to digest cellulose remained low. We noted that with a diet rich in cellulose and hemicellulose, bacterial cellulolytic activity was improved by the presence of ciliates in the rumen but was decreased with the "starch" diet (table 3). The greater VFA concentration observed in the faunated animals expressed ciliate effect on the fermentations as well as activation of bacterial metabolism. With a high starch diet, the Entodinium sp. ciliates may have a buffering effect on the pH values in the rumen by limiting bacterial fermentation after food intake and by prolonging starch digestion during the day (table 4). The composition of the VFA mixture was modified by ciliate inoculation. The molar proportion of butyric acid always increased, while that of acetic and propionic acids evolved differently according to the diets and the ciliates (table 4). The higher ammonia concentration in the rumen liquor observed in faunated animals (table 4) could be explained either by the breakdown of both feed and bacterial proteins ingested by ciliates or by a lower ammonia nitrogen incorporation by fewer bacteria. Statistical analyses were used to explain the specific effect of P and E and also the interactions between them and between each of them and the diets.     

The effect of replacing rice straw with dry wormwood (Artemisia sp.) on intake,digestibility, nitrogen balance and ruminal fermentation characteristics in sheep

This study examined the effect of substituting dry wormwood (Artemisia sp.) for rice straw in sheep diets on intake and apparent digestibility in vivo, nitrogen (N) balance and ruminal fermentation characteristics. Four Corriedale×Polwarth sheep (51.7 ± 1.3 kg) were individually housed in metabolism cages and fed diets (ad. libitum) with a 70:30 forage to concentrate ratio (DM basis), in which the basal rice straw was substituted with 0 (Control), 30 (LW), 50 (MW) or 100 (HW) g/kg of dry wormwood. The experimental design was a 4×4 latin square design in which 10 days of dietary adaptation was followed by 6 days of total feces and urine collection in each period. Rumen fluid was collected from a stomach tube at −0.5, 0.5, 1, 2, 4, and 8 h after the morning feed on day 6 of each collection period and analyzed for volatile fatty acids (VFA). The intakes of dry matter (DM), organic matter intake (OM), crude protein (CP), ether extract (EE), neutral detergent fibre (NDF) and acid detergent fibre (ADF) were higher (P<0.05) in sheep fed diets containing wormwood, than those fed the Control diet. Compared to the Control diet, CP digestibility was higher (P<0.05) in sheep fed MW and N intake, retained N, EE digestibility, urinary purine derivatives and microbial N yield were higher (P<0.05) in sheep fed diets, containing wormwood. Rumen pH was unaffected by treatment. Rumen NH₃-N and VFA concentrations were similar across treatments except that most values for diets containing wormwood were higher (P<0.05) than those for the control diet within the first 2 h of feeding. The non-glucogenic acid ratio was also similar across Control, LW and MW treatments, but it was generally lower in MW versus the Control treatment. In conclusion, substituting wormwood for rice straw in the sheep diets increased feed intake, rumen fermentation, in vivo digestibility, N retention and microbial N yield, particularly at the medium and high levels of wormwood inclusion.     

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

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

Effect of dietary energy intake on tubular reabsorption of urea in sheep

The aim of the experiment was to determine the effect of dietary energy intake on renal urea excretion in sheep with different nitrogen intakes. The control sheep, with a high nitrogen and energy intake, were given a daily feed dose of 21.18 g N and 15.2 MJ digestible energy (DE). The two experimental groups, with an equal, low nitrogen intake, were given diets with a different energy content. The high energy diet contained 3.63 g N and 14.18 MJ DE, the low energy diet 3.4 g N and 6.44 MJ DE. After nine weeks' adaptation to the diets, renal functions were measured by a standard clearance technique. It was found that, under stable urine flow conditions, both groups given the low nitrogen diet had a significantly lower glomerular filtration rate, fractional urea excretion and total urea excretion. A reciprocal comparison of these two groups showed that fractional urea excretion by the sheep with a high energy intake was significantly lower than in the group with a low energy intake. There were no differences in the glomerular filtration rate. A raised dietary energy intake in the presence of a low nitrogen intake caused marked natriuresis and kaliuresis. The results indicate that a raised dietary energy intake can be a significant factor in potentiating the renal effect of urea retention in sheep with a low nitrogen intake.     

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.