An in vitro study was conducted to investigate the effect of malate or fumarate on fermentation characteristics, and production of conjugated linoleic acid (CLA) and methane (CH 4 ) by rumen microbes when incubated with linolenic acid (-C 18:3 ). Sixty milligrams of -C 18:3 alone (LNA), or -C 18:3 with 24 mM malic acid (M-LNA) or -C 18:3 with 24 mM fumaric acid (F-LNA) were added to the 150 ml culture solution consisting of 75 ml strained rumen fluid and 75ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate and -C 18:3 (Control). Two grams of feed consisting of 70% concentrate and 30% ground alfalfa (DM basis) were also added to the culture solution of each treatment. In vitro incubation was made anaerobically in a shaking incubator up to 12 h at 39C. Supplementation of malate (M-LNA) or fumarate (F-LNA) increased pH at 6 h (p<0.01) and 12 h (p<0.001) incubation times compared to control and linolenic acid (LNA) treatments. Both malate and fumarate did not influence the ammonia-N concentration. Concentration of total VFA in culture solution was higher for M-LNA and F-LNA supplementation than for control and LNA treatments from 6 h (p<0.040) to 12 h (p<0.027) incubation times, but was not different between malate and fumarate for all incubation times. Molar proportion of C 3 was increased by F-LNA and M-LNA supplementation from 6 h (p<0.0001) to 12 h (p<0.004) incubation times compared to control and LNA treatments. No differences in C 3 proportion, however, were observed between M-LNA and F-LNA treatments. Accumulated total gas production for 12h incubation was increased (p<0.0002) by M-LNA or F-LNA compared to control or LNA treatment. Accumulated CH 4 production for 12 h incubation, however, was greatly reduced (p<0.0002) by supplementing malate or fumarate compared to the control, and its production from M-LNA or F-LNA treatment was smaller than that from LNA treatment. Methane production from LNA, M-LNA or F-LNA treatment was steadily lower (p<0.01 -p<0.001) from 3 h incubation time than that from the control, and was also lower for M-LNA or F-LNA treatment at incubation times of 6 h (p<0.01) and 9 h (p<0.001) than for LNA treatment. Methane production from LNA, however, was reduced (p<0.01 -p<0.001) from 3 h to 9 h incubation times compared to the control. Both malate and fumarate increased concentration of trans11-C 18:1 from 3 h to 12 h incubation (p<0.01), cis9,trans11-CLA up to 6 h incubation (p<0.01 -p<0.01), trans10,cis12-CLA at 3 h (p<0.05) and 12 h (p<0.01), and total CLA for all incubation times (p<0.05) compared to corresponding values for the -C 18:3 supplemented treatment (LNA). In conclusion, malate and fumarate rechanneled the metabolic H 2 pathway to production of propionate and CLA, and depressed the process of biohydrogenation and methane generation. Linolenic acid alone would also be one of the optimistic alternatives to suppress the CH 4 generation. (

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