C. Van Nevel

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.

Effect of anatomical location on the composition of fatty acids in double-muscled Belgian Blue cows

Double-muscled cows of the Belgian Blue breed, ranging from ca. 680 to 880 kg live weight were slaughtered and various fat depots sampled for lipid analysis. Subcutaneous fat (SCF), intermuscular fat in m. serratus (IMF1) and m. transversalis (IMF2), kidney fat (KF) and intramuscular fat in m. longissimus thoracis (IMF3) were sampled. In IMF3 samples, polar lipids were separated from other lipid classes by thin layer chromatography. Both the proportions (w w %) and gravimetric concentrations (mg g(-1) of sample) of long-chain fatty acids were determined in total lipids of SCF, IMF1, IMF2, KF and in lipid classes of IMF3 by gas chromatography. The greatest concentration of total fatty acids was found in KF (777.6 ± 82.6 mg g(-1)), followed by SCF (721.3 ± 92.2 mg g(-1)), IMF2 (709.8 ± 72.5 mg g(-1)) and IMF1 (682.1 ± 71.6 mg g(-1)). Triacylglycerol and polar lipid fatty acid content of m. longissimus thoracis (IMF3) were respectively 8.1 ± 3.3 and 3.1 ± 0.6 mg g(-1). Fatty acid content, particularly the triacylglycerol fatty acid content in IMF3, increased (p < 0.01) with increasing carcass fat content. Polar lipid fatty acids in IMF3 contained a higher proportion of polyunsaturated fatty acids (32.6 ± 4.8 %) and lower proportion of saturated fatty acids (27.4 ± 5.0%) compared to the triacylglycerol fatty acid fraction (p < 0.01), which may reflect a prerequisite for proper membrane functioning. Internal fat depots were more saturated (p < 0.01) compared to SCF. The proportion of monounsaturated fatty acids differed (p < 0.01) between IMF1 and IMF2, possibly reflecting differences in muscle activity and functioning. Oleic (C18:1) and stearic (C18:0) acids comprised more than 60% of the total fatty acids in all anatomical locations.

Effect of defaunation on straw digestion in sacco and on particle retention in the rumen

Abstract The effect of chemical defaunation on composition of rumen contents, rate of straw digestion in sacco and rumen fluid and particle retention was determined. Two rumen fistulated sheep were used during a faunated, defaunated and refaunated period. Chromium mordanted soya bean meal was used to determine particle retention in the rumen. Defaunation increased rumen propionate proportion and concentration of lactate and bacterial DM. Total volatile fatty acids and ammonia concentrations were depressed. Digestibility in sacco was lowered, although interpretation of data was difficult owing to invasion of bags by feed particles and/or bacteria. Rate of particle passage was nearly doubled by defaunation. It is concluded that the decrease in rumen fibre digestibility observed after defaunation in vivo is related to both a decrease in degrading activity of rumen organisms and a decrease in rumen retention time.

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 PUFAs 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 PUFAs 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 PUFAs against hydrogenation, maintenance of pH values above 6.3 will be necessary.

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 β-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of β-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 β-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 β-glucans with increased release of IgA onto the mucosa surface.

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.

Attempts to induce reductive acetogenesis into a sheep rumen

A rumen fistulated wether was used for continuous infusion of a 2-bromoethanesulfonic acid (BES) solution (2 g/d in 50 ml of water). The infusion was started after introduction of a pulse dose of BES (2 g) into the rumen. Immediately after introduction of the pulse dose, methane concentration in rumen gases was lowered from about 40 to less than 1%, with concomittant decreases and increases in the molar proportions of acetic and propionic acids respectively in the rumen volatile fatty acids. After 4 days of infusion however, and despite repeated pulse dosage of BES, methanogenesis adapted to BES and methane concentration in rumen gases reached 20%. Addition of BES to incubations of rumen contents with hay resulted in considerable inhibition of methanogenesis. Extra addition of methanol in such incubations increased both acetate and methane production, whereas addition of formate had no effect. In a second experiment using a second rumen fistulated whether, a 4 day control period was followed by 10 days of daily introduction of 11 of cattle cecal contents into the rumen. The cattle cecal contents were collected from slaughterhouse cattle, filtered and kept at -20 degrees C until use. Comparison of in vitro fermentation of thawed with fresh contents showed absence of methanogenesis but not of reductive acetogenesis after freezing and thawing. Evidence for the latter was sought by calculation of metabolic hydrogen recoveries from amounts of end products formed in incubations. In a similar way, evidence for induction of reductive acetogenesis was sought from incubations in vitro, carried out with rumen contents obtained before, during and after introduction of cecal contents into the rumen. No such evidence was obtained.

Effect of soya oil hydrolysate on rumen digestion in defaunated and refaunated sheep

The effects of soya oil hydrolysate (SOH) supplementation on the kinetics of rumen contents and on in sacco rumen degradabilities of various feeds were studied using two defaunated and refaunated sheep. This allowed us to investigate the relative importance of protozoa depletion in the overall effect of oil feeding on rumen metabolism. The addition of 7% SOH to the diet lowered protozoal numbers from 1.6 106 ml−1 to 6 105 ml−1. SOH supplementation significantly enhanced rumen fluid volume and rumen dry matter, and this effect was partly connected with protozoa depletion. The addition of SOH did not modify the amount of volatile fatty acids in the rumen, but shifted the fermentation patterns to a greater proportion of propionate, while decreases in butyrate and acetate were observed. SOH supplementation did not affect in sacco degradabilities of maize grains and soya-bean meal in the rumen of refaunated animals, but strongly enhanced them in defaunated sheep (respectively, 57% DM vs. 37% and 64% DM vs. 48% after 8 h incubation). SOH lowered in sacco degradabilities of straw (25% DM vs. 46% after 48 h) and cellulose (43% DM vs. 65% after 24 h) in refaunated animals, but only in sacco degradability of straw in defaunated animals (31% DM vs. 44% after 48 h). Coating of cellulose by SOH did not affect its in sacco degradability in the rumen. An estimation of SOH fatty acid kinetics in the rumen seemed to indicate that the variation of rumen content kinetics during the adaptation period to SOH favoured a decrease by 25% in the concentration of SOH fatty acids in the rumen.

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.