Effects of Addition Level and Chemical Type of Propionate Precursors in Dicarboxylic Acid Pathway on Fermentation Characteristics and Methane Production by Rumen Microbes In vitro

Two in vitro experiments were conducted to examine the effects of propionate precursors in the dicarboxylic acid pathway on ruminal fermentatation characteristics, $CH_4$ production and degradation of feed by rumen microbes. Fumarate or malate as sodium salts (Exp. 1) or acid type (Exp. 2) were added to the culture solution (150 ml, 50% strained rumen fluid and 50% artificial saliva) to achieve final concentrations of 0, 8, 16 and 24 mM, and incubated anaerobically for 0, 1, 3, 6, 9 and 12 h at $39^{\circ}C$. For both experiments, two grams of feed consisting of 70% concentrate and 30% ground alfalfa (DM basis) were prepared in a nylon bag, and were placed in a bottle containing the culture solution. Addition of fumarate or malate in both sodium salt and acid form increased (p<0.0001) pH of culture solution at 3, 6, 9 and 12 h incubations. The pH (p<0.0001) and total volatile fatty acids (VFA, p<0.05) were enhanced by these precursors as sodium salt at 3, 6 and 9 h incubations, and pH (p<0.001) and total VFA (p<0.01) from fumarate or malate in acid form were enhanced at a late stage of fermentation (9 h and 12 h) as the addition level increased. pH was higher (p<0.001) for fumarate than for malate as sodium salt at 3 h and 6 h incubations. Propionate ($C_3$) proportion was increased (p<0.0001) but those of $C_2$ (p<0.05) and $C_4$ (p<0.01 - p<0.001) were reduced by the addition of sodium salt precursors from 3 h to 12 incubation times while both precursors in acid form enhanced (p<0.011 - p<0.0001) proportion of $C_3$ from 6h but reduced (p<0.018 - p<0.0005) $C_4$ proportion at incubation times of 1, 3, 9 and 12 h. Proportion of $C_3$ was increased (p<0.05 - p<0.0001) at all incubation times by both precursors as sodium salt while that of $C_3$ was increased (p<0.001) from 6h but $C_4$ proportion was decreased by both precursors in acid form as the addition level increased. Proportion of $C_3$ was higher (p<0.01 - p<0.001) for fumarate than malate as sodium salt from 6 h incubation but was higher for malate than fumarate in acid form at 9 h (p<0.05) and 12 h (p<0.01) incubation times. Increased levels (16 and 24 mM) of fumarate or malate as sodium salt (p<0.017) and both precursors in acid form (p<0.028) increased the total gas production, but no differences were found between precursors in both chemical types. Propionate precursors in both chemical types clearly reduced (p<0.0001 - p<0.0002) $CH_4$ production, and the reduction (p<0.001 - p<0.0001) was dose dependent as the addition level of precursors increased. The $CH_4$ generated was smaller (p<0.01 - p<0.0001) for fumarate than for malate in both chemical types. Addition of fumarate or malate as sodium type reduced (p<0.004) dry matter degradation while both precursors in both chemical types slightly increased neutral detergent fiber degradability of feed in the nylon bag.