Different concentrations (3, 30, 300, and 3000 mg/L of culture fluid) of garlic oil (GAR), diallyl sulfide (DAS), diallyl disulfide (DAD), allicin (ALL), and allyl mercaptan (ALM) were incubated for 24 h in diluted ruminal fluid with a 50:50 forage:concentrate diet (17.7% crude protein; 30.7% neutral detergent fiber) to evaluate their effects on rumen microbial fermentation. Garlic oil (30 and 300 mg/L), DAD (30 and 300 mg/L), and ALM (300 mg/L) resulted in lower molar proportion of acetate and higher proportions of propionate and butyrate. In contrast, at 300 mg/L, DAS only increased the proportion of butyrate, and ALL had no effects on volatile fatty acid proportions. In a dual-flow continuous culture of rumen fluid fed the same 50:50 forage:concentrate diet, addition of GAR (312 mg/L), DAD (31.2 and 312 mg/L), and ALM (31.2 and 312 mg/L) resulted in similar changes to those observed in batch culture, with the exception of the lack of effect of DAD on the proportion of propionate. In a third in vitro study, the potential of GAR (300 mg/L), DAD (300 mg/L), and ALM (300 mg/L) to decrease methane production was evaluated. Treatments GAR, DAD, and ALM resulted in a decrease in methane production of 73.6, 68.5, and 19.5%, respectively, compared with the control. These results confirm the ability of GAR, DAD, and ALM to decrease methane production, which may help to improve the efficiency of energy use in the rumen.

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.

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The effects of disodium fumarate on microbial growth, CH 4 production and fermentation of three diets differing in their forage content (800, 500 and 200 g/kg DM) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from four Merino sheep. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mM-fumarate, and 15 N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation. Fumarate did not affect (P.0·05) any of the measured gas production parameters. In 17 h incubations, the final pH and the production of acetate and propionate were increased linearly (P,0·001) by the addition of fumarate. Fumarate tended to increase (P¼0·076) the organic matter disappearance of the diets and to decrease (P¼0·079) the amount of NH 3 -N in the cultures. Adding fumarate to batch cultures tended (P¼ 0·099) to decrease CH 4 production, the mean values of the decrease being 5·4 %, 2·9 % and 3·8 % for the high-, medium-and low-forage diet, respectively. Fumarate tended to increase (P¼0·082) rumen microbial growth for the high-forage diet, but no differences (P.0·05) were observed for the other two diets. These results indicate that the effects of fumarate on rumen fermentation depend on the nature of the incubated substrate, the high-forage diet showing the greatest response.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato w ; Norel & Nature S.A., Barcelona, Spain; composed of disodium malate -calcium malate (0·16:0·84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato w was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-malate. Gas production was measured at regular intervals up to 120 h. Malate increased (P, 0·01) the average fermentation rate of all substrates, and the lag time decreased (P,0·05) linearly with increasing concentrations of malate for all substrates, with the exception of sorghum. In 17 h incubations, the final pH and total volatile fatty acid production increased (P,0·001) linearly for all substrates as malate concentration increased from 0 to 10 mM. Propionate and butyrate production increased (P,0·05), while the value of the acetate : propionate ratio and L-lactate concentrations decreased (P, 0·05) linearly with increasing doses of malate. Malate treatment increased (P,0·05) the CO 2 production and decreased the production of CH 4 , although this effect was not significant (P.0·05) for maize. Malate at 4 and 7 mM increased (P, 0·05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH 4 production and L-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mM-malate were observed.

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