Effect of release rate of the SF(6) tracer on methane emission estimates based on ruminal and breath gas samples.

The release rate (RR) of sulphur hexafluoride (SF(6)) gas from permeation tube in the rumen appears to be positively related with methane (CH(4)) emissions calculated using the SF(6) tracer technique. Gas samples of breath and ruminal headspace were collected simultaneously in order to evaluate the hypothesis that transactions of SF(6) in the rumen are the source for this relationship. Six non-lactating dairy cows fitted with rumen cannulae were subdivided into two groups and randomly assigned to a two-period crossover design to permeation tubes with low RR (LRR = 1.577 mg/day) or two-times higher RR (HRR = 3.147 mg/day) RR. The cows were fed limited amounts of maize silage (80% ad libitum) split into two meals (40% at 0800 h and 60% at 1600 h). Each period consisted of 3-day gas sampling. Immediately before the morning feed and then each hour over 8 h, ruminal gas samples (50 ml) were withdrawn through the cannula fitted with stoppers to prevent opening. Simultaneously, 8-h integrated breath gas samples were collected over the same period. Ratios of concentration of CH(4)/SF(6), CO(2)/SF(6) and CO(2)/CH(4) and emission estimates of CH(4) and CO(2) were calculated for each sample source using the SF(6) tracer technique principles. The LRR treatment yielded higher (P < 0.001) ruminal CH(4)/SF(6) (by 1.79 times) and CO(2)/SF(6) (by 1.90 times) ratios than the HRR treatment; however, these differences were lower than the 2.0 times difference expected from the RR between the LRR and HRR. Consequently, the LRR treatment was associated with lower (P < 0.01) ruminal emissions of CH(4) over the 8-h collection period than with the HRR treatment (+11%), a difference also confirmed by the breath samples (+11%). RR treatments did not differ (P = 0.53) in ruminal or breath CO(2) emissions; however, our results confirm that the SF(6) tracer seems inappropriate for CO(2) emissions estimation in ruminants. Irrespective of the RR treatment, breath samples yielded 8% to 9% higher CH(4) emission estimates than the ruminal samples (P = 0.01). The relationship between rumen and breath sources for CH(4) emissions was better for LRR than for HRR treatment, suggesting that tracer performance decreases with the highest RR of SF(6) tested in our study (3.1 mg/day). A hypothesis is discussed with regard to the mechanism responsible for the relationship between RR and CH(4) emission estimates. The use of permeation tubes with small range in RR is recommended in animal experiments to decrease variability in CH(4) emission estimates using the SF(6) tracer technique.

Modification of aflatoxin B1 and ochratoxin A toxicokinetics in rats administered a yeast cell wall preparation.

The cell wall of Saccharomyces cerevisiae can bind mycotoxins in vitro, but there is scarce information on whether this property decreases the absorption of mycotoxins in vivo. The effect of a yeast cell wall preparation (YCW) on toxicokinetics and balance excretion (urine and faeces) of aflatoxin B(1) (AFB1) and ochratoxin A (OTA) was tested in rats after oral administration of each toxin. The (3)H-labelled mycotoxins were used at low doses. Co-administration of YCW with AFB1 decreased the extent, but not the rate, of absorption. Concurrently, radioactivity excreted in faeces increased by up to 55% when compared with controls, whilst the excretion in urine decreased (p < 0.05). The effect of YCW on OTA was less marked, although it increased radioactivity excretion in faeces (up to 16%; p < 0.05) it did not result in changes in urine and toxicokinetic parameters. The in vivo effect is in agreement with the reported in vitro binding ability for these toxins (AFB1 > OTA). In conclusion, these results indicate that YCW could be used to protect monogastric animals against exposure to low dietary levels of selected mycotoxins.

Effect of live yeast culture supplementation on hindgut microbial communities and their polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber or high-starch diet.

Four cecum and right ventral colon-fistulated horses were assigned in a 4 x 4 Latin square design and fed a high-fiber (HF) or a high-starch (HS) diet with or without 10 g of Saccharomyces cerevisiae (SC; CBS 493.94) containing 4.5 x 10(9) cfu/g. The HF and HS diets consisted of pelleted feeds and long wheat straw (18.0 and 3.5 g of DM.kg(-1) of BW.d(-1), respectively) given in 2 equal meals to provide an NDF:starch ratio of 3.5 and 1.0, respectively. After a 21-d adaptation period intestinal contents were collected 4 h after the morning meal on d 23 and 25 to determine bacterial and SC concentrations. Polysaccharidase activities (CMCase, xylanase, amylase) and activities of glycoside hydrolases (alpha-l-arabinosidase, beta-d-cellobiosidase, beta-d-glucosidase, beta-d-xylosidase) were determined in liquid-associated bacteria (LAB) and solid-adherent bacteria (SAB) isolated from both compartments. Lactobacilli were increased in the cecum (P = 0.012) and colon (P = 0.086) when starch intake increased, whereas total anaerobes, cellulolytics, and streptococci did not change in either compartment. In yeast-supplemented horses, SC concentrations were greater in cecum (4.4 x 10(6) cfu/mL) than in right-ventral colon (5.6 x 10(4) cfu/mL) and did no change with diet. Concentrations of lactobacilli and lactic-acid utilizers were greater (P = 0.099 and 0.067, respectively) in the cecum but remained similar in the colon of SC-supplemented horses. The CMCase activities of SAB were not affected by diet. Colonic xylanase activities of SAB were reduced (P = 0.046) by starch addition, but no change was seen in the cecum. All SAB glucoside hydrolase activities in the cecum and colon, except beta-d-xylosidase in the cecum, were decreased when starch intake was increased. The LAB CMCase (P = 0.049 in the colon) and xylanase (P = 0.021 in the cecum; P < 0.001 in the colon) activities decreased with starch intake. No effect of starch on LAB or SAB amylase activity was observed. Addition of SC improved SAB CMCase in the cecum (P = 0.019) and colon (P = 0.037) as well as beta-d-cellobiosidase (P = 0.002) and beta-d-glucosidase (P = 0.041) in the cecum. Only xylanase in the cecum (P = 0.015) and beta-d-xylosidase in the cecum (P = 0.028) were improved with SC, whereas colonic LAB alpha-amylase activity was significantly decreased (P = 0.046). Most enzymes involved in plant cell wall digestion were increased after SC addition. This fact may contribute to explain a better digestion of fiber that has been previously reported in SC-supplemented horses.

Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid.

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 µg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.

Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil.

This experiment studied the effect of 3 forms of presentation of linseed fatty acids (FA) on methane output using the sulfur hexafluoride tracer technique, total tract digestibility, and performance of dairy cows. Eight multiparous lactating Holstein cows (initial milk yield 23.4 +/- 2.2 kg/d) were assigned to 4 dietary treatments in a replicated 4 x 4 Latin square design: a control diet (C) consisting of corn silage (59%), grass hay (6%), and concentrate (35%) and the same diet with crude linseed (CLS), extruded linseed (ELS), or linseed oil (LSO) at the same FA level (5.7% of dietary DM). Each experimental period lasted 4 wk. All the forms of linseed FA significantly decreased daily CH(4) emissions (P < 0.001) but to different extents (-12% with CLS, -38% with ELS, -64% with LSO) compared with C. The same ranking among diets was observed for CH(4) output expressed as a percentage of energy intake (P < 0.001) or in grams per kilogram of OM intake (P < 0.001). Methane production per unit of digested NDF was similar for C, CLS, and ELS but was less for LSO (138 vs. 68 g/kg of digested NDF, respectively; P < 0.001). Measured as grams per kilogram of milk or fat-corrected milk yield, methane emission was similar for C and CLS and was less for ELS and LSO (P < 0.001), LSO being less than ELS (P < 0.01). Total tract NDF digestibility was significantly less (P < 0.001) for the 3 supplemented diets than for C (-6.8% on average; P < 0.001). Starch digestibility was similar for all diets (mean 93.5%). Compared with C, DMI was not modified with CLS (P > 0.05) but was decreased with ELS and LSO (-3.1 and -5.1 kg/d, respectively; P < 0.001). Milk yield and milk fat content were similar for LSO and ELS but less than for C and CLS (19.9 vs. 22.3 kg/d and 33.8 vs. 43.2 g/kg, on average, respectively; P < 0.01 and P < 0.001). Linseed FA offer a promising dietary means to depress ruminal methanogenesis. The form of presentation of linseed FA greatly influences methane output from dairy cows. The negative effects of linseed on milk production will need to be overcome if it is to be considered as a methane mitigation agent. Optimal conditions for the utilization of linseed FA in ruminant diets need to be determined before recommending its use for the dairy industry.

Effect of live yeast culture supplementation on apparent digestibility and rate of passage in horses fed a high-fiber or high-starch diet.

Eight crossbred male horses aged 12 +/- 5 yr and with BW of 305 +/- 18 kg were used in pairs in a 4 x 4 Latin square design with 4 ground and pelleted diets. Each pair included a cecum and right ventral colon-fistulated animal and a cecal-fistulated animal. The 4 horse diets were a high-fiber diet (HF+0) based on dehydrated alfalfa, a high-starch diet based on barley and wheat bran (HS+0), and the HF or HS diets supplemented with Saccharomyces cerevisiae (SC) CBS 493.94 (HF+SC and HS+SC). The probiotic preparation contained 4.5 x 10(9) cfu/g of live yeast mixed with the culture medium, and was top-dressed onto the feed pellets at a rate of 10 g/d, equally distributed between the 2 daily meals. All 4 diets were offered in the same quantities (18.0 g of pelleted feed DM + 3.5 g of long wheat straw/kg of BW per d). Each of the 4 experimental treatments was divided into a 21-d period of diet adaptation followed by a 10-d period of total fecal collection for digesta flow rate and apparent digestibility measurements. Three markers were used to measure mean retention time (MRT) of the feed particles: Yb bound to the pelleted feeds for MRT in the whole digestive tract (MRT(Yb)), Eu bound to the pelleted feeds, and Dy bound to the fecal particles for MRT in the hindgut (MRT(Eu) and MRT(Dy)). Apparent digestibilities of DM, OM, and CP were greater (P < 0.001) in the HS than HF diet, independently of SC supplementation, whereas ADF digestibility was greatest in the HF diet (P = 0.035). Cellulolytic activity estimated through the in vitro disappearance rate of the dietary ADF fraction (IVAD(ADF)) was less (P < 0.001) in the HS than the HF diet. There was no dietary effect on NDF digestibility due to the longer MRT(Eu) of small particles in the hindgut (P = 0.036), which compensated for the lower fibrolytic activity expressed per unit of time in the HS compared with the HF diet. Supplementation with SC improved ADF digestibility (P = 0.038) and stimulated DM (P = 0.030) and NDF (P = 0.038) intakes, but had no effect on the MRT of solid digesta. The absence of any significant diet x SC interaction supports the strategy of using SC to stimulate cellulose digestion and improve the nutritional status of horses under both HF and HS diets.

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro.

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.

Use of 'natural' products as alternatives to antibiotic feed additives in ruminant production.

The banning in 2006 of the use of antibiotics as animal growth promoters in the European Union has increased demand from producers for alternative feed additives that can be used to improve animal production. This review gives an overview of the most common non-antibiotic feed additives already being used or that could potentially be used in ruminant nutrition. Probiotics, dicarboxylic acids, enzymes and plant-derived products including saponins, tannins and essential oils are presented. The known modes of action and effects of these additives on feed digestion and more especially on rumen fermentations are described. Their utility and limitations in field conditions for modern ruminant production systems and their compliance with the current legislation are also discussed.

Optimizing rumen functions in the close-up transition period and early lactation to drive dry matter intake and energy balance in cows.

The large deficit in energy intake in relation to energy requirements during the transition and early lactation periods means that high-producing cows need energy-dense rations. High-starch diets are intensively fermented by the microbial ecosystem in the rumen, giving rise to a high production of VFAs and resulting in a drop in pH and the accumulation of lactic acid, which exacerbates the decline in pH and is considered as the major cause of rumen acidosis. This rumen dysfunction affects rumen microbes and results in less efficient digestion, thereby decreasing feed intake and exacerbating the energy deficit in the cows. The main way to limit the risk of acidosis is by diet management. Thus, animals must be progressively adapted to grain ingredients during the 3-week period before calving. Furthermore, the diet management strategy will be to privilege low-degradable starch and include enough fiber in the diet to stimulate rumination and salivation. Chemical additives can be used to prevent rumen acidosis. Rumen pH can be controlled by direct addition of chemical buffers at doses of 1-2% of DM intake. Metabolic hydrogen can be mobilized through specific metabolic pathways such as propionogenesis to compete with the synthesis of lactic acid. This can be achieved by adding propionate precursors such as aspartate, malate or fumarate. However, these additives are not economically viable, and will not easily be accepted by consumers. Probiotics, which are mainly supplied as live yeasts, have to be regarded as a solution for preventing subacute acidosis but they are not considered as an efficient cure for acute acidosis. Although the mechanisms of action of probiotics are not fully understood, this paper proposes a plausible model of their mode of action. Recently, additives based on plant extracts have been proposed as a solution for optimizing rumen functions, but further efficacy studies and safety trials are required before these additives can be marketed. The addition of fibrolytic enzymes has been suggested to improve the digestion of the dietary fiber fraction during acidosis. Although the results obtained so far are encouraging, more ruminant-specific research needs to be carried out on these enzymes. Some antibiotics are potential inhibitors of Gram-positive bacteria, which are involved in rumen acidosis, but their use as feed additives has been banned from 01/01/2006 in the EU.

Adsorption of Zearalenone by beta-D-glucans in the Saccharomyces cerevisiae cell wall.

Cell walls of yeasts and bacteria are able to complex with mycotoxins and limit their bioavailability in the digestive tract when these yeasts and bacteria are given as feed additives to animals. To identify the component(s) of the yeast cell wall and the chemical interaction(s) involved in complex formation with zearalenone, four strains of Saccharomyces cerevisiae differing in their cell wall glucan and mannan content were tested. Laboratory strains wt292, fks1, and mnn9 were compared with industrial S. cerevisiae strain sc1026. The complex-forming capacity of the yeast cell walls was determined in vitro by modelling the plots of amount of toxin bound versus amount of toxin added using Hill's model. A cooperative relationship between toxin and adsorbent was shown, and a correlation between the amount of beta-D-glucans in cell walls and complex-forming efficacy was revealed (R2 = 0.889). Cell walls of strains wt292 and mnn9, which have higher levels of beta-D-glucans, were able to complex larger amounts of zearalenone, with higher association constants and higher affinity rates than those of the fks1 and sc1026 strains. The high chitin content in strains mnn9 and fks1 increased the alkali insolubility of beta-D-glucans from isolated cell walls and decreased the flexibility of these cell walls, which restricted access of zearalenone to the chemical sites of the beta-D-glucans involved in complex formation. The strains with high chitin content thus had a lower complex-forming capacity than expected based on their beta-D-glucans content. Cooperativity and the three-dimensional structure of beta-D-glucans indicate that weak noncovalent bonds are involved in the complex-forming mechanisms associated with zearalenone. The chemical interactions between beta-D-glucans and zearalenone are therefore more of an adsorption type than a binding type.

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.

Influence of pH on complexing of model beta-d-glucans with zearalenone.

Previous studies have shown that isolated beta-(1,3 and 1,6)-D-glucans and related alkali-extracted fractions from the cell wall of Saccharomyces cerevisiae are able to complex with zearalenone in vitro (affinity up to 50%) and thus may reduce the bioavailability of toxins in the digestive tract. The complexation mechanisms involve cooperative interaction between the two chemical entities that can be computed by Hill's model. Various linear or branched soluble or insoluble beta-D-glucans were evaluated to elucidate their roles in the adsorption mechanisms under three pH conditions (3.0, 6.0, and 8.0) found in the digestive tract. A constant quantity of each beta-D-glucans (1 mg/ml) was mixed at 39 degrees C with increasing amounts of zearalenone (2 to 100 microg/ml), and the amount of bound toxin was measured. Acidic and neutral conditions gave the highest affinity rates (64 to 77%) by beta-(1,3)-D-glucans, whereas alkaline conditions decreased adsorption except when beta-(1,6)-D-glucan side chains were branched on beta-(1,3)-D-glucans. Alkaline conditions appear to impede the active three dimensional conformation of beta-D-glucans and favor single helix and/or random coil structures. Study of the equilibrium between beta-D-glucan-bound and free toxins revealed that two types of chemical interactions occur during toxin complexation with beta-D-glucans, identified as weak chemical linkages such as hydrogen and van der Waals bonds.

Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank.

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria.

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on the digestion of the cell wall fraction of a mixed diet in defaunated and refaunated sheep rumen.

The objective of this study was to determine the effect of two probiotics, Saccharomyces cerevisiae (SC) and Aspergillus oryzae (AO), without their culture medium, on the digestion of plant cell wall components in sheep that had been successively defaunated and refaunated. Six sheep fitted with large rumen cannulae were used to study 1) defaunated sheep with no probiotic, 2) defaunated sheep with SC or AO, 3) refaunated sheep with no probiotic, 4) refaunated sheep with SC or AO. The apparent digestibility of the plant cell walls was not altered (P > 0.05) by the probiotics in defaunated sheep but was increased (P < 0.05) with SC (+16%) in refaunated sheep. Simultaneously, SC stimulated the growth of the protozoal population in the rumen. As noted in most previous experiments, the positive effect of the presence of protozoa on plant cell wall digestion (P < 0.001) was confirmed here. The effect (P > 0.05) of SC or AO on in situ ADF digestion was either not significant or negative in defaunated rumens, whereas it became positive in refaunated rumen after a residence time of 12 h. The improvement of in situ ADF digestion due to the presence of protozoa was significant (P < 0.05 for NDF; P < 0.001 for ADF). However, we could not determine whether this was a direct effect of protozoa or an indirect effect operating via bacteria. The limits of the nylon bag technique for evaluating the microbial activity are discussed in relation to the ability of protozoa to enter and grow in the bags, and the pH regulation inside the bags. The pH values below 5.5 noted inside the bags can significantly alter the bacterial and protozoal populations and limit the validity of the technique. SC increased (P < 0.05) the specific activity of CMCase and xylanase of the solid-adherent bacteria (SAB) isolated from the rumen digesta of refaunated sheep. Stimulation of both the bacterial activity and protozoa numbers could explain the positive effect of SC on cell wall digestion in the rumen since the modelling of marker excretion in faeces showed that the ruminal mean retention time of hay measured from the model of Danhoa et al. was not modified (P > 0.05) by either refaunation or the presence of probiotics. AO increased (P < 0.05) the total retention time of the solid particles in the whole digestive tract and increased the ruminal liquid volume in the refaunated animals but it had no effect on the protozoa population or on the polysaccharidase activity of the SAB, which could explain the absence of effect of AO on the total digestibility of plant cell walls. Both probiotics decreased the liquid turnover in defaunated rumens (P < 0.05) but neither had any effect (P > 0.05) on this parameter after refaunation. The improvement of plant cell wall degradation in the whole digestive tract is probably due to a stimulation of digestion at the rumen level as indicated by the higher activity of the SAB in rumen digesta and the growth of protozoa.

Effect of a Saccharomyces cerevisiae culture on nutrient digestion in lactating dairy cows.

The digestive effects of a Saccharomyces cerevisiae culture were investigated. Four cows in early lactation that were fitted with ruminal and duodenal cannulas were used in a crossover design. The diet, which consisted of 60% corn silage and 40% concentrates, was supplemented or not supplemented with a daily dose of 50 g of premix containing 0.5 g of S. cerevisiae (6 x 10(8) cfu/g of premix). Total and ruminal digestibilities of organic matter, duodenal flows of nonmicrobial and microbial N, solid and liquid turnover rates, ruminal protozoal numbers, pH, ammonia N and volatile fatty acid concentrations, and concentrations of some plasma metabolites were not modified by the addition of S. cerevisiae. Ruminal dry matter content increased when S. cerevisiae was supplemented to the diet. In situ ruminal degradabilities of dry matter and neutral detergent fiber from corn stalk and of N from soybean meal were not modified; degradability of acid detergent fiber from corn stalk increased (32.5% vs. 26.3%) with the addition of S. cerevisiae. A short-term increase in N degradation was observed after 4 and 8 h of incubation. This experiment showed no effect of S. cerevisiae on most quantitative digestive events; however, a positive transitory postprandial effect on some parameters of microbial activity was observed.

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid.

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble 14C from 14C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoa population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and this effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.

Methane production associated with rumen-ciliated protozoa and its effect on protozoan activity.

Methane production by methanogenic bacteria associated with ciliated protozoa in the rumen, and its effect on the metabolic activity of the protozoa, were measured in vitro. Apparent daily methane emission per protozoan cell ranged from a trace amount to 2 nmol. Enhanced substrate disappearance accompanIed methanogenesis.

Effect of rumen protozoa on nitrogen utilization by ruminants.

Results obtained during the past decade indicate clearly that protozoa are actively involved in the degradation of dietary and microbial proteins in the rumen. Because of the great ability of protozoa to ingest the particulate matter suspended in the rumen, protozoa are more active in degrading insoluble than soluble proteins. This indicates that studies carried out using lysed and sonicated protozoa are not appropriate for quantifying the actual contribution of protozoa to protein degradation in the rumen. In vivo trials have confirmed that duodenal flow of both undegraded dietary protein plus bacterial protein generally is increased by defaunation. The decrease in ruminal ammonia concentration consistently observed after defaunation accounts for the lower urinary nitrogen (N) excretion found in defaunated animals, whereas the increase in fecal N excretion in the same animals probably results from a shift of plant cell wall digestion from the rumen to the large intestine. Total N excretion is not altered significantly by defaunation. A summary of literature data indicates there are contradictory effects of defaunation on ruminant performance. This implies that animal response to defaunation may depend on the specific nutrient-limiting performance on the one hand and on the modifications of digestion and metabolism resulting from defaunation on the other. Different methods are proposed to either eliminate or decrease the numbers of ruminal protozoa or to alter their makeup. However, none of these approaches has been tested under practical feeding conditions.

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on fermentations in the rumen of faunated and defaunated sheep; protozoal and probiotic interactions.

We measured the effect of the direct addition to the rumen of Saccharomyces cerevisiae (SC 50 mg/day) and Aspergillus oryzae (AO 3 g/day) on the fermentation processes in fistulated sheep. The measurements were carried out on animals whose rumens were first defaunated and then refaunated. The animals received a ration composed of hay (600 g/day), barley (600 g/day) and soybean meal (150 g/day), fed twice daily in two equal meals. The number of fungi and total, viable or cellulolytic bacteria were lower after the inoculation of protozoa in defaunated rumens. The probiotics stimulated the development of total bacteria but reduced the population of cellulolytic bacteria. The addition of the probiotics and the presence of protozoa each incurred a decrease in the redox potential values. The association of both treatments had an additive effect on this parameter. The two probiotics and the protozoa stabilized the rumen pH after the meal, maintaining it above the value of 6 for a longer period of time. The positive effects on pH were accumulated in the refaunated animals receiving probiotics. The ammonia nitrogen concentration was considerably increased by the presence of the protozoa; the probiotics increased the ammonia concentration only in the refaunated sheep. The methane and hydrogen proportions in the fermentation gases were invariably higher in the refaunated animals. The probiotics had no clear effect either on the gas composition or the concentration and the composition of the mixture of volatile fatty acids; only the concentration of isovalerate was significantly increased by probiotics and only in refaunated animals. The protozoa did, however, considerably increase the concentrations of acetate, butyrate and isoacids and decreased the concentration of caproate.