Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Woldeghebriel, Abraham


This study investigated the effects of feed particle size and nitrate or fumarate alone or in combination on in vitro fermentation, abundances of methanogens, and methane production. Rumen fluid was collected from a Holstein-Friesian dry cow and a steer (experiment 1) and a dry cow and two steers (experiment 2) averaging 625.4 kg in body weight (BW). The cattle were offered 11.4 kg/d per animal of concentrate diet containing equal amounts of soybean meal, whole cottonseed, and ground corn once a day with grass hay at ad libitum (experiment 1) and 15.9 kg/d per animal of total mixed ration (TMR) of silage, hay, corn, corn gluten, soybean meal, and minerals (experiment 2). Feed grab samples were collected, oven-dried, coarsely pulverized in a regular kitchen blender for one minute, and separated into three particle sizes (PS: 0.85, 1.4, and 2.36 mm). The feed additives used were nitrate, fumarate, and a nitrate-fumarate mixture. The fermentation parameters measured after 48h incubation periods were methane (CH4), pH, VFA, nitrate (NO3), and ammonia (NH3). Real-time PCR was used to quantify the relative abundances of total and specific methanogens. The results of the study revealed that the addition of fumarate had no effect on CH4 production. The addition of nitrate reduced CH4 production (p < 0.05) by 57% and 59% in experiments 1 and 2, respectively. The addition of the nitrate-fumarate combination also reduced (p < 0.05) CH4 production by 40 % (experiment 1) and 68% (experiment 2). Methane production was affected by feed particle size. In experiment 1, CH4 production for the medium PS was 31% and 39% lower (p < 0.05) than the small and large PS, respectively. However, in experiment 2 CH4 production was 17% and 16% higher (p < 0.05) for the large PS compared to the small and medium PS, respectively. The addition of feed additives had no effect on total methanogens, while fumarate decreased (p < 0.05) the abundance of Methanobrevibacter sp. AbM4 and nitrate 3 decreased (p < 0.05) the abundance of Methanosphaera stadtmanae. Nitrate reduced (p < 0.05) individual and total VFAs in experiments 1 and 2, while fumarate increased (p < 0.05) propionate and decreased (p < 0.05) butyrate, valerate, and iso-valerate compared to the control (experiment 2). Fumarate had no effect on total VFA production in experiments 1 and 2. In experiment 1, the nitrate-fumarate combination reduced (p< 0.05) butyrate and iso-butyrate compared to the control and had no effect on other individual VFAs. The addition of the nitrate-fumarate combination decreased (p< 0.05) the production of acetate, butyrate and total VFAs (experiment 2). In experiment 1, addition of fumarate increased (p < 0.05) pH, but there was no effect in experiment 2. The addition of nitrate and nitrate-fumarate combination increased (p < 0.05) pH in both experiments. The addition of nitrate also increased (p < 0.05) the concentration of nitrate and ammonia. On the other hand, addition of fumarate and the nitrate-fumarate combination had no effect on nitrate and ammonia concentrations. In summary, nitrate alone or in combination with fumarate was effective in reducing in vitro methane production. The use of nitrate also reduced VFA production but when combined with fumarate, the reduction in VFA production was lessened, indicating that addition of fumarate to nitrate not only reduces methane production but also seem to spur the reduction of VFAs by nitrate. The effects of the individual feed additives appear to be very specific but when used in combination they seem to be more effective in reducing in vitro methane production. In conclusion, the mechanism by which nitrate alone or in combination with fumarate reduces methane production is less clear and the most likely scenario could be due to the direct effect on the methanogens or through the reduction of organic matter fermentation that lowers availability of free H2 for methane production. Keywords: methane, VFA, nitrate, fumarate, in vitro, methanogens