In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C4 tropical grass Andropogon gayanus grass ensiled at two maturities – vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 μl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P<0.05) was observed for NDFD and ADFD after 24 h and for DMD, NDFD and ADFD after 48 h of incubation of the VS diet. For the FS diet, a dosage effect was observed for GP and NDFD after 24 h and for GP, DMD, NDFD and ADFD after 48 h of incubation. There was no difference among enzyme mixtures nor was there an enzyme×dose interaction for the studied parameters. Because of the greatest numerical effect on NDF disappearance and the least cost price, enzyme mixture E2 at 4 µl/g of diet DM was selected for the Rusitec experiment. The enzyme did not impact (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH4 production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.

The objective was to investigate the effect of variation in forage source and feed particle size of a diet, including interactions, on the amount and the composition of microbial crude protein (CP) in a semi-continuous culture system (Rusitec). Different microbial CP fractions were compared. Five diets with mean forage proportion of 0.88 and different maize silage to grass silage ratios (100 : 0, 79 : 21, 52 : 48, 24 : 76 and 0 : 100) were used. Diets were ground through sieves with a pore size of either 1 or 4 mm, matching the particle size of fine (F) and coarse (C), respectively. Diets were characterised by increasing concentrations of CP and fibre fractions, and decreasing concentrations of starch with ascending inclusion rates of grass silage. Microbial mass was isolated from feed residues after incubation from the liquid phase of the fermenter and from the liquid effluent. The amount of synthesised microbial CP was determined on the basis of 15N balance. It increased quite linearly by the stepwise replacement of maize silage by grass silage, and was higher in C treatments compared to F treatments. Efficiency of microbial CP synthesis (EMPS) was improved from 29 to 43 mg microbial N/g degraded organic matter (OM) by increasing the proportion of grass silage in the diet, but was unaffected by particle size. The N content as well as the profiles of amino acids of the three microbial fractions was affected by diet composition and particle size. The ratio of solid- to liquid-associated microbes was affected by diet composition and feed particle size. The amount and EMPS seemed to be improved by degradation of OM from grass silage and an increasing availability of N. Moreover, the results of this study indicated a shift in the composition of the microbial community caused by variation in forage composition and feed particle size.

Mitigation of methanogenesis in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal methanogenesis, has a low palatability; therefore, in the present study the aim was to evaluate the mitigation efficacy of its esterified form (monolaurin). Further, 13C-isotope abundance (δ13C) and 13C–12C fractionation during methanogenesis and fermentation were determined to evaluate possible microbial C-isotope preferences. Using the rumen simulation technique, four basal diets, characterised either by the C3 plants grass (hay) and wheat (straw and grain), or the C4 plant (13C excess compared with C3 plants) maize (straw and grain), and a mixture of the latter two, were incubated with and without monolaurin (50 g/kg dietary DM). Added to hay, monolaurin did not significantly affect methanogenesis. When added to the other diets (P < 0·05 for the wheat-based diet) methane formation was lowered. Monolaurin decreased fibre disappearance (least effect with the hay diet), acetate:propionate ratio, and protozoal counts. Feed residues and SCFA showed the same δ13C as the diets. Methane was depleted in 13C while CO2 was enriched in 13C compared with the diets. Monolaurin addition resulted in 13C depletion of CO2 and enrichment in CH4 (the latter only in the hay diet). In conclusion, monolaurin proved to effectively decrease methanogenesis in the straw–grain diets although this effect might partly be explained by the concomitantly reduced fibre disappearance. The influence on 13C-isotope abundance and fractionation supports the hypothesis that ruminal microbes seem to differentiate to some extent between C-isotopes during methanogenesis and fermentation.

Eight Rusitec and eight single-flow continuous-culture fermenters (SFCCF) were used to compare the ruminal fermentation of two diets composed of alfalfa hay and concentrate in proportions of 80 : 20 (F80) and 20 : 80 (F20). Results were validated with those obtained previously in sheep fed the same diets. Rusitec fermenters were fed once daily and SFCCF twice, but liquid dilution rates were similar in both types of fermenters. Mean values of pH over the 12 h postfeeding were higher (P < 0.001) in Rusitec than in SFCCF, with diet F80 showing higher values (P < 0.001) in both types of fermenters. Concentrations of total volatile fatty acids (VFA) were higher (P < 0.001) in SFCCF than in Rusitec, and in both systems were higher (P = 0.002) for diet F20 than for diet F80. There were significant differences between systems in the proportions of the main VFA, and a fermentation system × diet interaction (P < 0.001) was detected for all VFA with the exception of valerate. No differences (P = 0.145) between the two types of fermenters were detected in dry matter (DM) digestibility, but NDF, microbial N flow and its efficiency were higher (P = 0.001) in SFCCF compared to Rusitec. Whereas pH values and VFA concentrations remained fairly stable through the day in both in vitro systems, pH dropped and VFA increased shortly after feeding in sheep rumen reaching the minimum and maximal values, respectively, about 4 h after feeding. Both in vitro systems detected differences between diets similar to those found in sheep for liquid dilution rate, pH values, DM digestibility, microbial N flow and growth efficiency. In contrast, acetate/propionate ratios were lower for diet F20 than for F80 in sheep rumen (2.73 and 3.97) and SFCCF (3.07 and 4.80), but were higher for diet F20 compared to F80 (4.29 and 3.40) in Rusitec, with values considered to be unphysiological for high-concentrate diets. In vivo NDF digestibility was affected (P = 0.017) by diet, but no differences between diets (P > 0.05) were found in any in vitro system. A more precise control of pH in both types of fermenters and a reduction of concentrate retention time in Rusitec could probably improve the simulation of in vivo fermentation.

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34·3 endoglucanase, 0·57 exoglucanase, 24·7 xylanase and 5·51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P < 0·05) daily production of acetate, butyrate and methane, substrate DM and fibre disappearance at 6 and 48 h incubation, daily flow of microbial-N, and the microbial colonisation of substrate at 6 h incubation. Compared to FE, MIX treatment increased (P < 0·05) propionate production by 28 % and decreased (P < 0·05) the acetate:propionate ratio, but no other differences between both treatments were found (P>0·05). Supplementing with FUM increased (P < 0·05) volatile fatty acid production by 11 % and decreased (P < 0·05) the acetate:propionate ratio, but did not affect (P>0·05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Recently, transgenic crops have been considered as possible donors of transgenes that could be taken up by micro-organisms under appropriate conditions. In an in vitro rumen simulation system, effects of ampicillin on microbial communities growing either on rumen contents with transgenic maize carrying a gene that confers resistance to ampicillin or its isogenic counterpart as substrates were examined continuously over 13 d. Rate of production of SCFA was measured to determine functional changes in the rumen model and single-strand conformational polymorphism was used to detect alterations in structure of the microbial community. Rumen contents treated with ampicillin displayed a marked decrease in the rate of production of SCFA and diversity of the microbial community was reduced severely. In the presence of transgenic maize, however, the patterns of change of rumen micro-organisms and their metabolic profiles were different from that of rumen fluid incorporating maize bred conventionally. Recovery of propionate production was observed both in the rumen fluid fed transgenic and conventional maize after a delay of several days but recovery occurred earlier in fermenters fed transgenic maize. Alterations in the microbial population structures resulting from the ampicillin challenge were not reversed during the experimental run although there was evidence of adaptation of the microbial communities over time in the presence of the antibiotic, showing that populations with different microbial structures could resume a pre-challenge metabolic profile following the introduction of ampicillin, irrespective of the source of the plant material in the growth medium.

Biotin is required by rumen microbes for efficient fermentation. To evaluate the role of protozoa in ruminal biotin metabolism, five diets composed of grass hay or of grass hay/cereal grain mixtures were supplied to faunated or defaunated RUSITEC fermenters. In the mixed diets, hay was replaced to 33:67 or 67:33 w/w on an air-dried basis by either wheat or maize grain in order to simulate different cellulolytic and amylolytic fermentation conditions. Defaunation increased SCFA production, whereas NH4 concentration and the release of CH4 were reduced. Biotin input declined when cereal grain was used to replace the hay. With the exception of the high-wheat treatment, defaunated fermenters yielded higher biotin outputs than faunated fermenters. The biotin balance, calculated as the difference between the total biotin output (biotin in the solid residue contained in the nylon bags after fermentation plus the biotin in the effluent) and the biotin input with the feed, was negative for all the dietary treatments apart from fermenters supplied with the high-maize diet. It was less negative or, in the case of the high-maize diets, more positive for defaunated compared with faunated fermenters. It was concluded that, under normal faunated conditions, protozoa directly utilise or indirectly affect the bacterial synthesis and/or utilisation of biotin. With diets of a high fermentation potential, as realised with the high-wheat diet, protozoa prevent the development of a bacterial population that would utilise high or synthesise low amounts of biotin.

Two incubation trials were carried out with the rumen-simulation technique (RUSITEC). In each trial, four vessels received a diet of grass hay and concentrate (600 and 400 g/kg DM, respectively; diet F), and the other four were fed a diet composed of concentrate and barley straw (900 and 100 g/kg DM, respectively; Diet C). Vessels were given 20 g of the corresponding diet daily, and half of them were supplemented with disodium malate to achieve a final concentration of 6.55 mM. There were no effects (P>0·05) of malate either on pH or on the daily production of NH3-N, but malate treatment increased (P<0·05) DM, neutral detergent and acid detergent fibre disappearance after 48 h incubation. The daily production of propionate and butyrate increased (P<0·001), and the ratio CH4:volatile fatty acids decreased (P<0·001) by supplementing both diets with malate. Whereas adding malate to the F diet produced an increase in acetate production (P=0·011) and the growth of solid-associated micro-organisms (P=0·037), no effects (P>0·05) were observed for diet C. For both diets, there were no differences (P>0·05) between treatments in the daily flow of liquid-associated micro-organisms measured using 15N as a microbial marker. These results indicate that malate stimulated the in vitro fermentation of both diets by increasing the apparent disappearance of the diet and decreasing the ratio of CH4:volatile fatty acids, but a greater response was observed with diet F. If these results are confirmed in vivo, malate could be used as a feed additive for ruminants fed diets containing medium proportions of forage (i.e. dairy animals) and not only in animals fed high-concentrate diets, as has so far been proposed.

The interactions of lauric (C12) and myristic acid (C14) in suppressing ruminal methanogenesis and methanogens were investigated with the rumen simulation technique (Rusitec) using bovine ruminal fluid. The fatty acids were added to basal substrates (grass hay:concentrate, 1:1.5) at a level of 48 g/kg DM, provided in C12:C14 ratios of 5:0, 4:1, 3:2, 2·5:2.5, 2:3, 1:4 and 0:5. Additionally, an unsupplemented control consisting of the basal substrates only was employed. Incubation periods lasted for 15 (n 4) and 25 (n 2) d. CH4 formation was depressed by any fatty acid mixture containing at least 40 % C12, and effects persisted over the complete incubation periods. The greatest depression (70 % relative to control) occurred with a C12:C14 ratio of 4:1, whereas the second most effective treatment in suppressing CH4 production (60 % relative to control) was found with a ratio of 3:2. Total methanogenic counts were decreased by those mixtures of C12 and C14 also successful in suppressing methanogenesis, the 4:1 treatment being most efficient (60 % decline). With this treatment in particular, the composition of the methanogenic population was altered in such a way that the proportion of Methanococcales increased and Methanobacteriales decreased. Initially, CH4 suppression was associated with a decreased fibre degradation, which, however, was reversed after 10 d of incubation. The present study demonstrated a clear synergistic effect of mixtures of C12 and C14 in suppressing methanogenesis, mediated probably by direct inhibitory effects of the fatty acids on the methanogens.

The first objective of this experiment was to investigate the effect of using different bacterial isolates on the estimation of microbial protein production in semi-continuous fermenters (RUSITEC) given four diets, and to test whether a ‘mixed’ bacterial pellet was representative of the whole bacterial population in the fermenters. A second objective was to compare two different microbial markers (nitrogen-fifteen (15N) and purine bases (PB)). Diets consisted of neutral-detergent fibre from grass hay (10 g/day) and sugar-beet pulp (2 g/day) and 280 mg/day of one of four N forms (isolated soya-bean protein, soya-bean peptides, amino acids blended to profile soya-bean protein and NH4Cl). Two 14-day incubation runs were carried out and in each run each of the four different diets were given to two vessels. On days 12 and 13, total digesta (effluent plus nylon bags residues) was collected for analyses of non-ammonia N, 15N enrichment and PB concentration, and for isolation of total mixed bacterial pellets (TB). On the last day of each incubation run, the system was stopped for isolation of liquid- (LAB) and solid-associated (SAB) bacteria. Microbial N flow was estimated from the 15N enrichment and PB concentration in both total digesta and in the three different bacterial pellets (TB, LAB, and SAB). For all diets, LAB presented a greater (P < 005) 15N enrichment and PB: N ratio than SAB, with TB having an intermediate value. For both markers, the use of LAB produced the lowest (P < 005) estimates of microbial N flow and the use of SAB produced the greatest (P < 005) estimates. The use of TB produced intermediate values with all diets, suggesting that TB consisted of SAB and LAB. For all bacterial pellets, PB produced greater (P 005) values of microbial N flow than 15N. However, there was a positive relationship (r = 0·883; P 0001; no. = 15) between the values of microbial N flow determined with the two markers when TB were used as reference.

The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation. The energy source was fibre prepared from sugar-beet pulp. N was added as NH3 continuously infused (AC) or peptides (Bacto® Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS). Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment. Treatments had no effect on DM degradation. Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment. Microbial N flow tended to be higher with the peptide additions (P<0·061), with no significant difference between the two peptides treatments. The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0·05) and the proportion of LAM derived from NH3 lower (P<0·05). However, LAM only accounted for 20–30 % total microbial population. Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply. This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

The effects of an exogenous enzyme preparation, the application method and feed type on ruminal fermentation and microbial protein synthesis were investigated using the rumen simulation technique (Rusitec). Steam-rolled barley grain and chopped alfalfa hay were sprayed with water (control, C), an enzyme preparation with a predominant xylanase activity (EF), or autoclaved enzyme (AEF) 24 h prior to feeding, or the enzyme was supplied in the buffer infused into the Rusitec (EI). Microbial N incorporation was measured using (15NH4)2SO4 in the buffer. Spent feed bags were pummelled mechanically in buffer to segregate the feed particle-associated (FPA) and feed particle-bound (FPB) bacterial fractions. Enzymes applied to feed reduced neutral-detergent fibre content, and increased the concentration of reducing sugars in barley grain, but not alfalfa hay. Ruminal cellulolytic bacteria were more numerous with EF than with C. Disappearance of DM from barley grain was higher with EF than with C, but alfalfa was unaffected by EF. Treatment EF increased incorporation of 15N into FPA and FPB fractions at 24 and 48 h. In contrast, AEF reduced the 24 h values, relative to C; AEF and C were similar at 48 h. Infused enzyme (EI) did not affect 15N incorporation. Xylanase activity in effluent was increased by EF and EI, compared to C, but not by AEF. Xylanase activity in FPA was higher at 48 h than at 24 h with all treatments; it was higher with EF than C at 24 and 48 h, but was not altered by AEF or EI. Applying enzymes onto feeds before feeding was more effective than dosing directly into the artificial rumen for increasing ruminal fibrolytic activity.

Incubation trials were carried out with the rumen simulation technique (RUSITEC) to study the effects of four forms of N on the growth of ruminal micro-organisms and the fermentation variables when an all-fibre basal diet was incubated. The basal diet consisted of 10 g neutral-detergent fibre (NDF) from grass hay plus 2 g NDF from sugarbeet pulp. N forms were isolated soyabean protein, soyabean peptides, amino acids blended to profile soyabean protein and NH3 as NH4Cl. Half of the daily N supply was infused as NH4Cl and the other half was infused as each of the four treatments described. Non-NH3 N (NAN) forms increased NDF (P = 0·006), acid-detergent fibre (P = 0·003) and cellulose (P = 0·015) disappearance after 48 h incubation, CO2 (P < 0·001), CH4 (P = 0·002) and total volatile fatty acids production (P < 0·001), as well as the molar percentages of isobutyrate, isovalerate and valerate, which reflected the fermentation of amino acid C skeletons. NAN treatments also increased microbial N flow (P < 0·001) compared with NH3, with peptides and protein supporting more (P = 0·036) than amino acids. The proportion of microbial N derived from NH3 decreased successively (P < 0·05) with NH3 > amino acids > peptides > protein treatments, indicating preferential uptake of peptides without passage through the NH3 pool. Microbial efficiency (g microbial N/kg organic matter apparent disappearance) was greater (P = 0·002) for the NAN forms than for the NH3 treatment, with peptides and protein treatments supporting higher (P = 0·009) values than amino acid treatment. These results indicate that N forms other than NH3 are required for optimal fibre digestion and microbial growth.

The influence of sodium fumarate on rumen fermentation was investigated in vitro using batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral–vitamin supplement (500, 299·5, 100, 91 and 9·5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 μmol, equivalent to 0, 200 and 400 g/kg feed. CH4 production at 24 h (360 μmol in the control cultures) fell (P < 0·05) by 18 and 22 μmol respectively (SED 7·5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 μmol; SED 26·7, P < 0·01) were accompanied by increases in propionate formation (212 and 396 μmol; SED 13·0, P < 0·001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4 production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6·25 mmol sodium fumarate was a decrease in CH4 production of 1·2 mmol (SED 0·39, P < 0·05), equivalent to the consumption of 4·8 mmol H2, and an increase in propionate production of 4·9 mmol (from 10·4 to 15·3 (SED 1·05) mmol/d, P < 0·01). The inhibition of CH4 production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4 corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P > 0·05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8·8 v. 23·9 (SED 2·49) × 105 per ml, P < 0·01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0·476 v. 0·508 (SED 0·0123), P < 0·05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2 from CH4 production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.