Two trials were conducted to identify the optimal levels of essential oil active components (EOAC) and their combination with fumarate on in vitro rumen fermentation. In trial 1, eugenol, carvacrol, citral and cinnamaldehyde were mixed at ratios of 1:2:3:4, 2:1:4:3, 3:4:1:2, 4:3:2:1 and 1:1:1:1 to make up five combinations (EOAC1, EOAC2, EOAC3, EOAC4 and EOAC5 respectively). The mixtures were supplied at levels of 0, 50, 200 or 500 mg/l to identify the optimal combination for methane reduction. Methane production and ammonia nitrogen were decreased by adding EOAC, irrespective of component compounds, but the production of gas and total volatile fatty acids (VFA) were also decreased. Hydrogen balance analysis indicated that the ratio of hydrogen consumed via methane to hydrogen consumed via VFA was lowest at 200 mg/l of EOAC5 treatment, from which the proportional change in methane was more than the change in VFA, with 31.5% of methane reduction and 12.9% of VFA reduction. In trial 2, 200 mg/l of EOAC5 was added with 0, 5, 10 and 15 mm monosodium fumarate to see whether fumarate had a further effect on rumen fermentation. The addition of fumarate had no influence on gas production, but it further decreased methane and increased the total VFA in comparison with EOAC added solely, with the greatest decrease occurring in methane (78.1%) from 10 mm of fumarate. Quantification of the microbial populations in rumen fluids by RT-PCR showed that methanogen, protozoa, fungi, Fibrobacter succinogenes and Ruminococcus flavefaciens populations were significantly decreased by EOAC5, but were not influenced by fumarate. In summary, the addition of EOAC had consistent effects on rumen fermentation parameters, but high levels of EOAC would induce the inhibition of rumen fermentation. Adding fumarate can enhance the methane-inhibiting effect of EOAC, and the decrease was higher than that calculated stoichiometrically.

Two trials were conducted to investigate the effect of a combination of essential oils (CEO) along with fumarate on in vitro rumen fermentation. In trial 1, the essential oil (EO) from thyme, oregano, cinnamon and lemon were mixed at five different ratios. The CEO were applied at levels of 0-500 mg/l. Addition of CEO decreased gas, methane, total volatile fatty acid (VFA) production at 24 h incubation in a dose-dependent manner. Methane tended to decrease much more than total VFA and gas at the same EO level. The mixture of oils at an equal ratio at 500 mg/l that decreased methane much more than VFA was chosen as the optimal combination. In trial 2, the optimal combination was used with 0, 5, 10 or 15 mmol/l of monosodium fumarate. Addition of fumarate further decreased methane production, with 10 mmol/l fumarate resulting in the largest reduction (80.2%) and the smallest decrease in total VFA (5.7%) and gas production (16.7%). Quantification of several ruminal microbe populations by RT-PCR showed that the optimal combination sharply decreased ruminal protozoa; the populations of fungi and fibrolytic bacteria were also decreased. In summary, at an appropriate level, CEO can inhibit methane production. Inclusion of fumarate can further decrease it, which is attributed mainly to inhibition of protozoa and methanogens.