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Fiber and large bowel energy absorption: validation of the integrated ileostomy-fermentation model using pigs.

Controversy exists over the contribution of dietary fiber and large bowel fermentation to human energy balance. It has been suggested that large bowel energy absorption, in the form of short-chain fatty acids, can be predicted from in vitro fermentation of human ileal digesta. Because it is difficult to obtain ileal digesta from humans with a functional large bowel to validate this approach, four normal pigs and four pigs fitted with a simple T-cannula at the distal ileum were fed a basal diet supplemented with 0, 5, 10 or 15 g pea fiber per 100 g basal diet in two parallel experiments (Latin-square design). Energy contents of diets, urine, ileal digesta and feces were determined by bomb calorimetry. Apparent total tract energy digestibility did not differ (P > 0.05) between normal and cannulated pigs. Apparent energy digestibility at the ileum decreased with increasing pea fiber intake (P < 0.05). The difference between ileal and fecal gross energy excretions in cannulated pigs was defined as large bowel energy absorption. Aliquots (0.2 g) of freeze-dried ileal digesta were fermented in vitro for 24 h with human fecal inoculum from three donors. In vitro short-chain fatty acid production was correlated with large bowel energy absorption (r = 0.90, P < 0.0001). The metabolizable energy content of pea fiber was determined to be 9.2 kJ/g pea fiber (r = 0.90, P < 0.0001) by regressing metabolizable energy retention against pea fiber intake. The integrated ileostomy-in vitro fermentation model seems to provide accurate estimates of large bowel energy absorption.

Animals↗

[Effect of moldy grass on intraluminal fermentation and thiamine metabolism in cattle (in vitro)].

The influence of mouldy grass (kind of moulds: Fusarium sp., Epicoccum oryzae, Ulocladium spp., Mucor spp.) on the in-vitro-fermentation especially the thiamine-metabolism of bovine rumen fluid was investigated using the longterm rumen simulation technique (RUSITEC). Four investigation periods keeping 23 days each were carried out. After a control period of nine days (normal hay) an eight days lasting testphase followed. During this time two reaction vessels (KF) were charged with normal hay, two vessels (VF II) with mouldy grass and two (VF I) with a 50:50-mixture of normal hay and mouldy grass. During the last five days of the testperiod 0.3 mg thiamine/reaction vessel were added daily. In the last six days all reaction vessels were fed with normal hay. The following effects of mouldy grass during the investigation period on the rumen fluid and rumen gas, respectively, could be noted: changes of short volatile fatty acids concentrations (acetate -9.9% [p < 0.05] propionate up to -12.2% [ns], i-butyrate up to +39.9% [p < 0.001], n-butyrate up to +25% [p < 0.01]); increase of ammonia concentrations up to 68% (p < 0.01); small increase of pH for 0.08 units (p < 0.01); decrease of the methane production up to 16.6% (p < 0.001); increase of thiamine input due to the mouldy grass up to 310% (p < 0.05); increased breakdown of substituted thiamine for 44%.

Animal Feed↗

Effect and stability of gliotoxin, an Aspergillus fumigatus toxin, on in vitro rumen fermentation.

Aspergillus fumigatus is a toxicogenic fungus usually found in contaminated animal feeds, especially in conserved forages where it can produce several mycotoxins. Gliotoxin, one of the most important toxic metabolites produced by this fungus, has antibacterial, immunosuppressive and apoptotic effects. Ruminants due to the high proportion of forages they receive in the ration would be particularly exposed to gliotoxin. The objective of this work was (1) to assess the effect of gliotoxin on in vitro rumen fermentation and (2) to determine the effect of fermentation on gliotoxin stability. Gliotoxin did not affect rumen fermentation at concentrations found in naturally contaminated feeds. No effects were observed up to a concentration of 20 microg toxin ml(-1) and an extremely high toxin concentration (80 microg ml(-1)) was necessary to affect dry matter degradation, gas and total volatile fatty acids production by 24, 37 and 18%, respectively (p < 0.01). In addition, the toxin was unstable in the rumen environment with 90% disappearance at 6 h of incubation (p < 0.05). In contrast, extracts of A. fumigatus cultures containing gliotoxin at concentrations several times lower than that used for experiments with pure toxin had a negative effect on fermentations indicating the toxicity and possible synergism of other metabolites produced by this fungus. Extracts containing 8.8 microg gliotoxin ml(-1) decreased dry matter degradation, gas and volatile fatty acids production by 28, 46 and 35%, respectively (p < 0.01). Identification of these toxic metabolites and assessment of the rate of passage of gliotoxin to the lower intestinal tract is necessary to evaluate the potential risk of these toxins to ruminants.

Animal Feed↗

Effect of Japanese horseradish oil on methane production and ruminal fermentation in vitro and in steers.

The effects of alpha-cyclodextrin-horseradish oil complex (CD-HR) on methane production and ruminal fermentation were studied in vitro and in steers. In the in vitro study, diluted ruminal fluid (30 mL) was incubated anaerobically at 38 degrees C for 6 h with or without CD-HR, using cornstarch as substrate. The CD-HR was added at various concentrations (0, 0.17, 0.85 and 1.7 g/L). Treatment affected neither the pH of the medium nor the number of protozoa. Total VFA increased in a linear manner (P = 0.02), and NH3-N decreased quadratically (P = 0.04) as the concentration of CD-HR increased from 0.17 g/L to 1.7 g/L. Molar proportions of acetate decreased in a linear manner (P = 0.03), and propionate increased linearly (P = 0.008) with increasing concentrations of CD-HR. Production of methane was inhibited up to 90%, whereas accumulation of dihydrogen was increased 36-fold by 1.7 g/L of CD-HR supplementation relative to controls. The effect of CD-HR on methane production, ruminal fermentation and microbes, and digestibility was further investigated in vivo using four Holstein steers in a crossover design. The CD-HR supplement was mixed into the concentrate portion of a (1.5:1) Sudangrass hay plus concentrate mixture that was fed twice daily to the steers. Ruminal samples were collected 0, 2, and 5 h after the morning feeding. No effects of CD-HR supplementation on ruminal pH (P = 0.63) or protozoal numbers (P = 0.44) were observed. Molar proportion of acetate was decreased (P = 0.04) and propionate was increased (P = 0.005) by CD-HR treatment. Molar proportion of butyrate was increased (P = 0.05) in CD-HR-supplemented steers. Ruminal NH3-N was decreased (P = 0.05) by treatment. Blood plasma glucose concentration was increased (P = 0.02) and urea-N was decreased (P = 0.04) with CD-HR supplementation. Daily DMI was decreased (P = 0.04), and apparent digestibility of DM (P = 0.13), NDF (P = 0.14), and CP tended (P = 0.14) to be increased by treatment. Methane production was decreased (P = 0.03) by 19%, and the number of methanogens was also decreased (P = 0.03). Although N retention (P = 0.11), total viable bacteria (P = 0.15), and sulfate-reducing bacteria (P = 0.17) were not significantly altered by treatment, tendencies for increases were noted with CD-HR supplementation. The number of cellulolytic (P = 0.38) and acetogenic bacteria (P = 0.32) remained unchanged by treatment. These results indicate that CD-HR supplementation can be used to decrease methane production in steers.

Animals↗

Short-chain fatty acids produced in vitro from fibre residues obtained from mixed diets containing different breads and in human faeces during the ingestion of the diets.

It was studied whether the type of bread (i.e. a low-fibre wheat-rye mixed bread and coarse or fine wholemeal rye bread) either as part of a diet or alone, had an influence on the short-chain fatty acids (SCFA) produced during in vitro fermentation. Fermentation substrates were dietary fibre residues obtained from diets and breads. In addition, it was investigated whether the faecal SCFA pattern in the inoculum donors, who ingested the experimental diets, could be predicted by in vitro fermentation. Yields of SCFA in vitro were 0.51-0.62 g/g fermented polysaccharide. In vitro, the molar ratios of butyrate were higher for the two high-fibre diets containing coarse or fine wholemeal bread than for the low fibre diet containing wheat-rye mixed bread; the difference was significant for the coarse (P < 0.01), but not for the fine bread diet (P = 0.0678). The coarse wholemeal bread alone produced a higher molar ratio of butyrate than the fine wholemeal bread (P < 0.05) and the wheat-rye mixed bread (P < 0.01). Ingestion by the inoculum donors of the diets containing wholemeal bread led to higher faecal butyrate ratios (molar ratios: coarse bread diet 19.6, fine bread diet 17.7) compared with the wheat-rye mixed bread-containing diet (14.9), but the differences between the diets were not significant. For the diets investigated, there were no significant differences between faecal and in vitro SCFA patterns.

Acetates↗

Comparison of fermentation of selected fructooligosaccharides and other fiber substrates by canine colonic microflora.

OBJECTIVE: To compare fermentation characteristics of fructooligosaccharides (FOS) and other fiber substrates that are commonly found in canine diets. SAMPLE POPULATION: Fecal samples from 3 adult dogs. PROCEDURE: The ability of fiber substrates to be used in microbial fermentation reactions was assessed by use of an in vitro fermentation system. Dogs were fed a commercially available food, and feces were collected for use as the microbial inoculum. Substrates used were beet pulp, cellulose, soy fiber, mannanoligosaccharides (MOS), FOS, and 4 inulin products (inulin 1, 2, 3, and 4). Each substrate was incubated anaerobically with fecal inoculum and growth media for 6, 12, and 24 hours, and production of short-chain fatty acids (SCFA) was measured. RESULTS: Total production of SCFA was higher for fermentation of the 4 inulin products and FOS, whereas fermentation of beet pulp, MOS, and soy fiber resulted in moderate concentrations of SCFA. Fermentation of cellulose produced the lowest concentrations of total SCFA without detection of butyrate or lactate. Butyrate production was greatest for fermentation of the 4 inulin products and FOS. Total lactate production was greatest for FOS and inulin 4. As expected, production of SCFA increased for all substrates as fermentation time increased. CONCLUSIONS AND CLINICAL RELEVANCE: Canine fecal microflora ferment FOS-containing substrates in a similar manner, with little fermentation of cellulose-based carbohydrates. Furthermore, results of an in vitro fermentation system indicate that fiber type affects the metabolic activity of microorganisms, thus influencing the amount and nature of the end products of fermentation.

Animals↗

Immobilization of infant fecal microbiota and utilization in an in vitro colonic fermentation model.

Bacteria isolated from infant feces were immobilized in polysaccharide gel beads (2.5% gellan gum, 0.25% xanthan gum) using a two-phase dispersion process. A 52-day continuous culture was carried out in a single-stage chemostat containing precolonized beads and fed with a medium formulated to approximate the composition of infant chyme. Different dilution rates and pH conditions were tested to simulate the proximal (PCS), transverse (TCS), and distal (DCS) colons. Immobilization preserved all nine bacterial groups tested with survival rates between 3 and 56%. After 1 week fermentation, beads were highly colonized with all populations tested (excepted Staphylococcus spp. present in low numbers), which remained stable throughout the 7.5 weeks of fermentation, with variations below 1 log unit. However, free-cell populations in the circulating liquid medium, produced by immobilized cell growth, cell-release activity from gel beads, and free-cell growth, were altered considerably by culture conditions. Compared to the stabilization period, PCS was characterized by a considerable and rapid increase in Bifidobacterium spp. concentrations (7.4 to 9.6 log CFU/mL), whereas Bifidobacterium spp., Lactobacillus spp., and Clostridium spp. concentrations decreased and Staphylococcus spp. and coliforms increased during TCS and DCS. Under pseudo-steady-state conditions, the community structure developed in the chemostat reflected the relative proportions of viable bacterial numbers and metabolites generally encountered in infant feces. This work showed that a complex microbiota such as infant fecal bacteria can be immobilized and used in a continuous in vitro intestinal fermentation model to reproduce the high bacterial concentration and bacterial diversity of the feces inoculum, at least at the genera level, with a high stability during long-term experiment.

Analysis of Variance↗

Effect of the combination of monensin and isoacids on rumen fermentation in vitro.

Effects of isoacids, monensin, or a combination of them on fermentation by mixed rumen bacteria were investigated using a continuous culture technique. The culture was allowed to stabilize for 4 d before treatments were imposed. Comparisons between treatments were made on d 11 and 12 of the culture. Isoacids (equal proportions of isobutyric, 2-M-butyric, isovaleric, and valeric acids) at 15 mg/dl of culture media increased acetate (6.17 vs. 5.48 meq/dl) and total VFA production (8.93 vs. 7.87 meq/dl) compared with that of controls. Monensin at 150 micrograms/dl reduced acetate (3.74 vs. 6.02 meq/dl) and VFA (6.84 vs. 8.54 meq/dl) but increased propionate (2.28 vs. 1.74 meq/dl) relative to control. The combination of isoacids and monensin increased acetate relative to monensin alone (5.24 vs. 3.74 meq/dl) but did not alter the effect of monensin on propionate concentration (2.32 vs. 2.28 meq/dl). It is concluded that monensin decreases acetate production by 35% and when isoacids are added to the cultures containing monensin, acetate production is restored.

Animals↗

Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another gram-positive antibiotic.

Mixed ruminal bacteria (n = 4) were incubated in anaerobic media for 24 h in vitro with either hay, corn meal, protein hydrolyzate or hydrogen gas as the substrate. The ionophore monensin and the polypeptide antibiotic bacitracin were added to the incubation flasks at concentrations ranging from 0 to 10 or 40 mg/liter. As was expected, monensin decreased methane production, increased the ratio of propionate to acetate and decreased the deamination of amino acids. Monensin had little effect on methane production, however, if hydrogen gas was the fermentation substrate. Bacitracin, another gram-positive antibiotic with a distinctly different cellular target, was somewhat less potent than monensin, but it produced strikingly similar responses. This similarity of fermentation patterns suggested that monensin action in the rumen is probably due to its activity as a gram-positive antibiotic, and that any gram-positive antibiotic not suppressed by resistance may produce fermentation effects similar to those of monensin. The cellular action of monensin as an ionophore in membranes is probably little more than a means of inhibiting sensitive species. Many gram-positive antibiotics have little affect on ciliate protozoa or coccidia.

Animals↗

[The effect of defaunation on fermentation in vitro].

An experiment was conducted to investigate the effects of defaunation on the fermentation of two different diets consisting of hay (100%) and hay+barley (80% + +20%) in a rumen pouch (RUSITEC). The survival and composition of protozoa were investigated in an in vitro system in control vessels where no defaunation was carried out. Defaunation decreased the production of total volatile fatty acids (P < < 0.01), acetate, butyrate (P < 0.001)--Tab. II; dry matter digestibility (P < 0.05) and detergent fibre digestibility: acid detergent fibre (ADF), neutral detergent fibre (NDF), cellulose (P < 0.001), hemicellulose (P < 0.01 and P < 0.001, respectively) in our experiment (Tab. I). These parameters also decreased: production of CH4 (P < 0.001), CO2 (P < 0.01 for the barley diet), total production of gases (P < 0.001 for the barley diet), total utilization (P < 0.01 and P < 0.001, respectively) and recovery of H2 (P < 0.05 and P < 0.001, respectively), NH3-N in effluent (P < 0.001 and P < 0.01, respectively)--Tab. III; and production of ATP (P < 0.01)--Tab. IV, as a result of fermentation in our experiment. On the other hand, there was a significant increase in the following parameters: production of propionate (P < 0.001 for the barley diet)--Tab. II; microbial nitrogen/kg of organic matter fermented--OMF (P < 0.001), Tab. III; energy yield--E of volatile fatty acids (VFA), proportion of VFA energy with respect to fermented hexose energy--E1, proportion of energy in bacterial cells with respect to fermented hexose energy--E3 (P < 0.001) and the effectiveness of microbial proteosynthesis--YATP (P < 0.001), Tab. IV, as a result of defaunation. The total number of protozoa and the number of some species, especially the number of big entodinio-morphid protozoa, were markedly higher (p < 0.001) for the barley diet--Tab. V. The number of protozoa decreased rapidly within three days after the system initiation. Later on, the values were more or less steady.

Animals↗

Effect of nicotinic acid on rumen fermentation in vitro and in vivo.

A series of in vitro studies tested effects of nicotinic acid on rumen fermentation. Urea was the nitrogen supplement for all substances. As expected, a substrate of corn produced the most gas and microbial protein followed by a substrate of corn plus brome hay (1:1). A substrate of brome hay produced the least amount of gas and microbial protein. Niacin had no effect on gas production but significantly increased synthesis of microbial protein. The most protein was synthesized with the corn substrate, but the greatest percentage increase due to niacin was with the substance of corn plus hay. Six rumen-fistulated cows were fed a ration of grain and grass hay (1:1) with 2.3% urea. The rations were supplemented with 0 or 200 ppm niacin. Niacin had no important effect on the concentration of rumen dry matter or lactic acid, or the molar proportions of acetic, butyric, or valeric acids. Niacin increased the concentration of rumen bacterial protein, ammonia, and propionic acid. Niacin reduced the rumen concentration of urea nitrogen. There were no differences in the amino acid composition of rumen bacteria due to niacin.

Amino Acids↗

Rumen fermentation in vitro as influenced by long chain fatty acids.

Responses of rumen microbes to fatty acids were evaluated by production of total volatile fatty acid and ratio of acetate to propionate. Fermentations were under carbon dioxide for 20 h in 50-ml Erlenmeyer flasks in a Dubnoff metabolic shaking incubator. Flasks contained 20 ml medium, 1 ml reducing solution, 750 mg substrate (450 mg hay plus 300 mg grain), and varying amounts of long-chain fatty acids supplied as free acids, as calcium salts, or as triglycerides. They were inoculated with 5 ml rumen fluid obtained from a cow fed 3.6 kg grass hay, 2.3 kg grain, and .2 kg tallow daily. Volatile fatty acid production was decreased by long-chain fatty acids that contained less than 18 carbon atoms and by unsaturated long-chain fatty acids with 18 carbon atoms. Lauric acid decreased volatile fatty acid production by 69% and induced unusual acetate/propionate ratio (40:1). Stearic acid, however, did not affect volatile fatty acid production or acetate/propionate ratio. Within two series of long chain fatty acids (myristic, palmitic, stearic, arachidic; stearic, oleic, linolenic), melting point accounted for 93 to 95% of the variation of volatile fatty acid production and acetate/propionate. As calcium salts, long chain fatty acids caused small changes of fermentation. Our data support the proposition that hard fats and calcium salts of long-chain fatty acids do not interfere with ruminal fermentation.

Acetates↗

In vitro study of the influence of virginiamycin and spiramycin on the composition and biochemical activities of the gastrointestinal flora of piglets. I. influence on the composition of the flora.

In vitro fermentations with the gastro-intestinal flora of 18 treated piglets (Control, Virginiamycin, Spiramycin) were carried out to study the influence of Nutritional doses of Virginiamycin and Spiramycin on the composition of the microflora. Each total flora was used for three different incubations: Control, 50 ppm Virginiamycin, 50 ppm Spiramycin. To obtain comparable results with experiments in vivo, an artificial fermentation medium was made on the basis of several chemical analyses of intestinal contents. The treatments with nutritional doses in vivo had little influence on the different in vitro fermentations. Addition of Spiramycin during incubation was of little influence on the growth and the composition of the flora. Virginiamycin on the other hand caused marked changes in the gastro-intestinal microflora, such as a decrease of the total growth and a decrease in the number of Lactobacilli and Enterococci, combined with a slight increase of the Gram negative aerobic population.

Anaerobiosis↗

Effect of plant oils and organic acids on rumen fermentation in vitro.

We determined the effect of plant oils (rapeseed, sunflower, linseed) and organic acids (aspartic and malic) on the fermentation of diet consisting of hay, barley and sugar beet molasses. Rumen fluid was collected from two sheep (Slovak Merino) fed with the same diet twice daily. Mixed rumen microorganisms were incubated in fermentation fluid, which contained rumen fluid and Mc Dougall's buffer. All supplemented diets significantly increased pH, molar proportion of propionate, and numerically decreased methane production. Lactate production was also decreased significantly (except with malate). Incorporation of plant oils into aspartate- and malate-treated incubations negated the decrease of butyrate, lactate and the increase of pH and ammonia with malate treatment, as well as in vitro dry matter digestibility and pH with aspartate treatment. The effect of combined additives on methane production and molar proportion of propionate was lower compared with additives supplemented separately. Combination of additives had no additive effect on rumen fermentation. All additives decreased total protozoan counts in rumen fluid.

Animal Feed↗

Effects of Moringa oleifera seed extract on rumen fermentation in vitro.

Moringa oleifera is a pantropical tree of the family Moringaceae. A previously undescribed property of an aqueous extract from the seeds of this plant is the modulation of ruminal fermentation patterns, especially protein degradation, as demonstrated in a short-term batch incubation system. Gas, short chain fatty acids (SCFA) and cellulolytic enzyme activities were determined as general fermentation parameters. A dot blot assay able to directly detect true protein in rumen fluid samples was used to quantify protein degradation. For complex substrates the interpretation of protein degradation profiles was amended by polyacrylamide gel electrophoresis (PAGE) of the samples. When incubated with pure carbohydrates at a concentration of 1 mg ml(-1), the extract reduced microbial degradation of the model protein, bovine serum albumin (BSA), such that its concentration was at least 40% above the control after 12 h of incubation. Total protein degradation was thus delayed by approximately 9 h. When fermented along with wheat straw, leaf protein (Rubisco) was almost entirely protected during 12 h of fermentation. The degradation of soy proteins was retarded by at least 4-6 h, depending on the protein band. There were strong side effects on the fermentation of pure cellulose (SCFA yield-60% after 12 h), whereas cellobiose and starch fermentation were less affected (-18 and -8%, respectively). When the complex substrates were fermented, SCFA yield was reduced by approximately 30% after 12 h. In our work we clearly demonstrate the efficacy of the new substance, which is neither a tannin nor a saponin, in an in vitro system, using pure as well as complex substrates. The properties shown in vitro for the crude extract suggest that it could have a positive effect on the protein metabolism of ruminants under intensive management and that negative side effects can be overcome by an optimized dosage. If the chemical nature of the active substance and its mechanism of action can be clarified, it may provide an alternative to replace critical synthetic feed additives (such as antibiotics) for high yielding dairy cows.

Animals↗

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus.

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.

Animals↗

Effect of forage to concentrate ratio on disappearance of vitamins A and E during in vitro ruminal fermentation.

The effects of forage to concentrate ratio and the commercial form of vitamins A and E on in vitro ruminal disappearance of retinol and alpha-tocopherol were studied. Ruminally fistulated cows were fed diets with either 80 or 50% forage. In vitro substrates that were similar to those fed to the donor cows were incubated with buffered ruminal fluid for 24 h. Different commercial forms of vitamin E (spray-dried, silicic acid adsorbate, and lipid-encased forms) and vitamin A (gelatin beadlet and lipid-encased forms) were added to the flasks. The vitamin E was all-rac-alpha-tocopheryl acetate, and the vitamin A was all-trans-retinyl acetate. The amount of alpha-tocopherol in the flasks was not affected by diet or form of vitamin E and did not change over the 24-h incubation. Retinol disappearance was not affected by form of vitamin A but was substantially higher for the 50% forage diet than for the 80% forage diet (72 vs. 20% at 24 h). These data suggest that ruminal metabolism of vitamin E is minimal and not affected by forage to concentrate ratio. Additionally, vitamin A destruction in the rumen was much higher when cows were fed a typical lactation diet than when fed a typical dry cow diet.

Animal Feed↗