Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.
