The influence of Lentinus edodes (Shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets.

Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of beta-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of beta-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50 mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1 cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1-2 log10 CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing beta-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet : 44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of beta-glucans with increased release of IgA onto the mucosa surface.

Effect of pH on biohydrogenation of polyunsaturated fatty acids and their Ca-salts by rumen microorganisms in vitro.

The influence of different pH values on the protection of Ca-salts of polyunsaturated fatty acids (PUFA) against ruminal biohydrogenation was investigated. Ca-salts were prepared from soya bean oil fatty acids (SOH) and incubated in vitro with rumen fluid at different pH values. Biohydrogenation of Ca-salts of the PUFA's and their corresponding free fatty acids in SOH was compared. Two series of incubations were carried out, a first series with pH varying between 6.9 and 5.5, while in the second series, a narrower pH range was studied (6.8-6.3). The experiments showed that between pH 6.9 and 6.3, Ca-salts of PUFA's are partly protected against biohydrogenation. Ca-salt of linoleic acid was protected to a greater extent than Ca-linolenate. Biohydrogenation of the salts is most probably due to dissociation of the solubilized Ca-salts. For efficient protection of Ca-salts of PUFA's against hydrogenation, maintenance of pH values above 6.3 will be necessary.

Influence of pH on lipolysis and biohydrogenation of soybean oil by rumen contents in vitro.

The effect of different pH values on rumen lipolysis and biohydrogenation was investigated during incubations of the rumen contents with 40 or 80 mg of soybean oil as the sole substrate. Mean pH values studied were 6.8, 6.3, 6.0, 5.6 and 5.2. Lipolysis was calculated from the decrease in fatty acids present in triacylglycerols (TAG), as well as from the accumulation of free fatty acids (FFA) during the incubation. At pH < or = 6.0 lipolysis was low, and the inhibition became greater with decreasing pH. At the same pH value, the inhibition in incubations with 80 mg of soybean oil was more pronounced than with 40 mg. Even at the lowest pH value, after incubation, no free linolenic acid could be detected because of biohydrogenation, whereas linoleic acid hydrogenation was only partially inhibited at pH 5.2. This means that lipolysis is much more sensitive to low pH values than biohydrogenation. Literature data indicate however that, besides pH, other factors must be involved in the decrease of both lipolysis and biohydrogenation in the rumen of animals fed highly concentrated diets.

Control of rumen methanogenesis.

During the last decades, considerable research on methane production in the rumen and its inhibition has been carried out. Initially, as methane production represents a significant loss of gross energy in the feed (2-15%), the ultimate goal of such intervention in rumen fermentation was an increase in feed efficiency. A second reason favouring research on methane inhibition is its role in the global warming phenomenon and in the destruction of the ozone layer. In this review, the authors describe briefly several interventions for reducing methane emission by ruminants. The objective can be reached by intervention at the dietary level by ration manipulation (composition, feeding level) or by the use of additives or supplements. Examples of additives are polyhalogenated compounds, ionophores and other antibiotics. Supplementation of the ration with lipids also lowered methanogenesis. More biotechnological interventions, e.g., defaunation, probiotics and introduction of reductive acetogenesis in the rumen, are also mentioned. It can be concluded that drastic inhibition of methane production is not unequivocally successful as a result of several factors, such as: instantaneous inhibition often followed by restoration of methanogenesis due to adaptation of the microbes or degradation of the additive, toxicity for the host animal, negative effects on overall digestion and productive performance. Therefore, methanogenesis and its inhibition cannot be considered as a separate part of rumen fermentation and its consequences on the animal should be taken into account.

Transformations and effects of lipids in the rumen: three decades of research at Gent University.

A survey is given of research results on ruminant lipid digestion obtained at the authors' laboratory. Results are presented in terms of lipid changes occurring in the rumen and in terms of effects on nature, extent and site of digestion. The rumen can be adapted to an extremely high capacity for triglyceride lipolysis, preferentially releasing polyunsaturated fatty acids that are then further hydrogenated with accumulation of oleic acid isomers in vitro only. Evidence was obtained for both microbial incorporation and synthesis of polyunsaturated acids. In vitro lipolysis is inhibited by pH values below 6.3 and by ionophores. Free fatty acids inhibit methanogenesis with associated increases in propionate production and decreases in acetate and butyrate productions; the latter being related to their defaunating effect. Both in the faunated and defaunated rumen, free fatty acids decrease fibre digestion, which is shifted to the hindgut, at least in sheep. Defaunation increases rumen microbial growth efficiency and may result in a higher duodenal flow of both feed and microbial protein, provided these increases are not overcome by a decreased apparent rumen OM digestibility. Considerable between animal variability exists for these effects, associated with variable effects on rumen particle and liquid volumes and outflow rates.

[Adaptation of rumen fermentation to monensin]. / Essai sur l'adaptation de la fermentation ruminale au monensin.

Adaptation of rumen fermentation to monensin feeding has been studied with rumen-fistulated sheep receiving a daily dose of 30 mg of monensin for a period of 21 d followed by a 28 d period during which 60 mg doses were administered. The ration consisted of 300 g of hay and 300 g of concentrates, fed at 9.00 h and 16.00 h. Monensin was placed in the rumen as an aqueous suspension, just prior to the morning feeding. Monensin infusion was preceded and followed by a period during which no monensin was infused. The following rumen fermentation parameters were determined: methane production, pH, volatile fatty acids (VFA) molar proportions, total volatile fatty acid concentration, lactate and ammonia concentrations and in sacco degradability of hay. Rumen gas expelled through the fistula was collected for 6 h per day and analysed. Total VFA concentration, molar proportions of individual VFA, pH, lactate and ammonia concentration were determined on rumen contents, sampled just prior the administration of monensin and 2 and 6 h later. In vitro incubations of 3 h were carried out with rumen fluid, sampled 1 h after feeding. In vivo and in vitro methane production was decreased by monensin feeding. The molar proportion of propionate in the rumen was increased, while acetate and butyrate percentages were lowered. The total VFA and ammonia concentrations were also decreased by monensin, but pH values were increased. In vitro production of propionate was stimulated by monensin administration and methanogenesis decreased. The organic matter in sacco degradability was not affected, probably because of the time difference between the introduction of bags and monensin in the rumen. These modifications of rumen fermentation persisted as long as monensin was given, indicating that in this experiment, there was no adaptation to the ionophore.

Incorporation of soya oil hydrolysate in the diet of defaunated or refaunated sheep: effect on rumen fermentation in vitro.

The effects of incorporation in the diet of 7% soya oil hydrolysate (SOH) on in vitro incubations of cellobiose + maltose, maize starch and casein by rumen microbes were studied using defaunated and refaunated sheep as rumen fluid donors. Feeding refaunated sheep the SOH supplemented diet lowered the protozoal numbers in the rumen from 1.61 10(6)/ml to 6.1 10(5)/ml. SOH addition reduced in vitro methane production, rather by a depletion of methanogens is than by a simple inhibition of their activity. This reduction seemed to be independent of protozoa depletion. With cellobiose-maltose and maize starch incubations, SOH supplementation increased molar proportion of propionate while acetate decreased. Both variations could be linked to the inhibition of methanogenesis. Volatile fatty acid production from casein was strongly reduced by SOH supplementation with or without protozoa in the rumen of the donors animals.

Effect of defaunation and refaunation of the rumen on rumen fermentation and N-flow in the duodenum of sheep.

In order to confirm earlier fragmentary results, the effect of defaunation and refaunation of the rumen on the fermentation pattern and flow of N-components in the proximal duodenum of two sheep was investigated. Defaunation had no effect on acetic acid as a proportion of the total volatile fatty acids in the rumen, while the proportions of propionic acid increased with a concomitant decrease in butyrate. Refaunation resulted in lower acetic acid and higher butyric acid proportions. The concentration of ammonia N in the rumen was clearly decreased after defaunation, already indicating an effect of the elimination of protozoa on nitrogen metabolism in the rumen. Defaunation also increased significantly the flow of total N, non ammonia N and individual and total amino acids in the proximal duodenum. Defaunation resulted in higher bacterial growth efficiency, significantly in one sheep, but the decrease after refaunation was statistically significant for both sheep. Determination of rumen digestibility of organic matter and acid detergent fibre (ADF) revealed lower values in the absence of the protozoa, while total digestibility was only influenced to a much lower extent. This indicated a shift of digestion from rumen to the lower digestive tract. Finally, earlier work is discussed in the light of the present findings.

Effect of defaunating the rumen on growth and carcass composition of lambs.

The effect of defaunating the rumen on growth performance and carcass composition of lambs fed a molasses-urea diet was investigated. Before the growth trial, all the animals were defaunated. Based on live weight and daily gain during a preliminar period, the animals were divided in two groups whereafter one group was refaunated. Defaunation caused a decrease in propionic acid percentage in the rumen. Daily gain and food conversion efficiency were better in the defaunated group, but only during the first five weeks. The response over the whole trial (0-9 weeks) remained positive however. There was a trend towards more meat and less fat in the carcass of defaunated lambs. The fact that two animals died during the defaunation procedure indicates the need for a completely harmless and effective defaunating agent.

Effect of monensin on fermentation pattern and soybean protein degradation in the rumen of sheep.

The effect of a daily dose of 30 mg Monensin on rumen fermentation pattern and degradation of the crude protein fraction of soybean meal was investigated with sheep. Degradation parameters for soybean meal were measured by the nylon bag technique. Monensin increased propionic acid percentages in the rumen, with a concomitant decrease in acetic- and butyric acid proportions, thus confirming earlier work. Ammonia N concentration in the rumen was also lowered, indicating an inhibitory effect on rumen protein breakdown. However, no effect on degradation rate, degradability and effective degradability (only with one sheep) could be observed. Possible reasons for these findings are discussed. Amino acid analysis on the residues in incubated bags revealed slight changes in amino acid composition, but these changes were considered to be nutritionally unimportant.

Effect of virginiamycin on carbohydrate and protein metabolism in the rumen in vitro.

The effect of virginiamycin in incubations of rumen fluid with carbohydrate or protein substrate was investigated. In carbohydrate incubations, methane production was partially inhibited while propionate proportions increased. Total microbial growth was slightly decreased, but net microbial growth was considerably lower. Protein degradation was slightly lowered after addition of virginiamycin. These effects were compared with results obtained with other rumen manipulating additives.

Effect of methane inhibitors on the metabolism of rumen microbes in vitro.

In incubations in vitro with rumen fluid, the effect of two methane inhibitors, linseed oil hydrolysate (LOH) and chloral hydrate (CH) on the efficiency of microbiol growth was investigated. Total and net microbial growth were determined from 32PO43- and NH3--N incorporation respectively and expressed as g N incorporated per kg organic matter fermented (gN/kgOMf). In a first series on incubations, it was found that LOH had no influence on overall microbial growth efficiency, while with CH, a small but significant decrease of total and net growth efficiency was measured. Further experiments showed that this was not due to accumulation of hydrogen gas in the CH incubations. Microscopic examination showed a toxic effect of LOH on protozoa, but with CH, no such effect was observed. This observation, together with earlier work where a considerable increase in microbial growth efficiency was found in vitro after defaunation of the rumen suggested the following hypothesis: both inhibitors lowered bacterial growth. In the case of LOH, this effect is marked by the defaunating action of LOH, the latter resulting in an increased growth efficiency of the bacterial fraction. This hypothesis was confirmed by incubations with washed cell suspensions (WCS) of mixed rumen bacteria, where growth efficiency was indeed decreased by both inhibitors. The possible mechanism explaining this phenomenon was discussed.

Effect of defaunation on the metabolism of rumen micro-organisms.

1. Rumen contents of a fasted fistulated wether, obtained in a faunated, defaunated and refaunated period were incubated in vitro with a mixture of cellobiose and maltose, in the presence of ammonium bicarbonate and 32PO43-. Total synthesis of microbial N (Nt) was calculated from 32P incorporation and N:P determined in microbial matter. The N:P value was not affected by defaunation. Net synthesis of microbial N (Nn) was calculated from ammonia-N incorporation. An estimate of degradation of microbial N was calculated as Nt-Nn. Energetic efficiency of synthesis was calculated from the volatile fatty acids produced during incubation, as g N incorporated per kg organic matter fermented (g N/kg OMf). 2. Defaunation decreased the proportions of acetate, butyrate and methane and increased those of propionate in fermentation end-products. Fermentation rate when expressed per mg microbial N was not affected by defaunation. 3. Expressed per unit volume of rumen contents, Nn was increased by defaunation whereas Nt remained unchanged. Thus, a decrease in degradation can be calculated. Energetic efficiences of total and net synthesis were increased from 35 and 13 to 47 and 30 g N/kg OMf respectively. 4. Specific rates of both total and net synthesis of microbial N were significantly increased by defaunation whereas the specific rate of degradation was not affected.

Effect of monensin on rumen metabolism in vitro.

The effect of Monensin (Rumensin, Eli Lilly & Co.) in incubations with mixed rumen microorganisms metabolizing carbohydrate or protein substrates was investigated. Monensin partly inhibited methanogenesis and increased propionate production, although the effect was not always statistically significant. Incubations with substrates specific for methane bacteria suggest that inhibition of methanogenesis by Monensin was not due to a specific toxic action on the methanogenic flora, but rather to an inhibition of hydrogen production from formate. Total and net microbial growth were considerably decreased by addition of Monensin, although the amount of substrate fermented was not altered, resulting in lowered values of microbial growth efficiency. In incubations with casein, Monensin lowered protein degradation in line with a lowered ammonia production, whereas a slight accumulation of alpha-amino nitrogen was observed. The results suggest that besides an influence of Monensin on the rumen carbohydrate fermentation pattern, another reason for the beneficial effects observed in vivo might be decreased food protein degradation in the rumen, altering the final site of protein digestion in the animal. Also, the possibility of a decrease in rumen microbial growth efficiency has to be considered when using Monensin as a food additive.

Determination of rumen microbial growth in vitro from 32P-labelled phosphate incorporation.

1. The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with 32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth. 2. Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80% of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells. 3. Incorporation of 32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth. 4. When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18-3 and 44-6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed. 5. The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.

Rumen microbial growth rates and yields: effect of amino acids and protein.

Effects of amino acids upon microbial growth, optimum ratio of nonprotein to amino acid nitrogen for microbial growth, and incorporation of amino acids into microbial cells were determined with washed cell suspension in vitro as were rumen microbial cells. Rumen microbial dry matter, nitrogen, ribonucleic acid, deoxyribonucleic acid, and substrate disappearance was greatest when a mixture of 18 amino acids was substituted for urea. Substitutions of mixtures of 10 essential amino acids, 8 nonessential amino acids, and sulfur containing amino acids and glutamate also stimulated microbial growth. Acid hydrolyzed casein markedly improved microbial growth. Branched amino acid addition did not affect growth. The optimum ratio of nonprotein to amino acid nitrogen for microbial growth was 75% urea nitrogen and 25% amino acid nitrogen. With this amount of amino acids, an average of 53% of added amino acid was incorporated into microbial cells, 14% was fermented to carbon dioxide and volatile fatty acids, and 33% remained in the supernatant. Both 100% urea and 100% amino acid in growth media were unfavorable for maximal microbial growth. With all carbohydrate substrates, 100% urea nitrogen supported the growth of 9 mg bacterial dry matter per 100 mg of substrate. Substitutions of amino acids for urea increased yields to over 20 mg/100 mg. Microbial growth yields in incubations under carbon dioxide were less than when flasks were flushed with nitrogen. However, yield of bacterial dry matter per unit of substrate was less under nitrogen than under carbon dioxide.

Some characteristics of Anaerovibrio lipolytica a rumen lipolytic organism.

Strains of Anaerovibrio lipolytica isolated from sheep- and cow-rumen contents on a linseed oil -- rumen fluid -- agar medium fermented ribose, glycerol and DL-lactate. Fermentation products from glycerol were propionate and succinate, while ribose, fructose and DL-lactate were fermented mainly to acetate, propionate and carbon dioxide. Propionate is formed in this organism by the dicarboxylic acid pathway similarly as in propionibacteria. Measurements of the rate of lipolysis by pure cultures suggest that the organism may play an important role in the lipolytic activity of rumen contents of sheep. The demonstrated fact that the cell-free lipase excreted in the culture medium can easily be adsorbed on particulate matter in autoclaved rumen fluid may explain the absence of free lipase in clarified rumen liquor.