A new approach to measure methane production from in vitro rumen fermentation of concentrates

Analysis of methane (CH4) produced from in vitrorumen fermentation is usually conducted on gas samples collected from headspace of bottles where gas is not vented. However, especially when concentrates are incubated, this procedure can lead to overestimation of CH4 values, as no venting causes partial solubilization of carbon dioxide (CO2) in fermentation fluid and an increased concentration of CH4 in headspace of bottles. This study aimed to measure gas (GP) and CH4 production using bottles where gas was vented into tight bags. Bottles (310 ml), filled with 60 ml of buffered rumen fluid (headspace=250 ml) and 0.040 g of feed sample, were not vented (NV) or vented (VE) at threshold pressure of 6.8 kPa by a valve connected to tight bag. Three concentrates, different for fermentative properties (corn meal, beet pulp, and flaxseed expeller), were incubated for 24 h at 39 °C. The experimental design was: 3 feeds × 3 replicates × 2 venting systems, plus 4 blanks, for a total of 22 bottles. A PC wireless-connected to bottles monitored GP, and GP was adjusted for solubilized CO2 . Gas samples (2 μl) were collected from headspace of bottles or from tight bags by a syringe and analyzed for CH4 by gas-chromatography. Data were analyzed by ANOVA considering feed, venting system and their interaction as sources of variation. Compared to NV, VE system provided greater (P<0.01) GP values, for all feeds. Adjustment of GP values removed differences between systems for beet pulp and flaxseed expeller, but not for corn meal. Measured CH4 concentrations were greater (P<0.01) for NV compared to VE, for all feeds. When CH4 concentrations were adjusted for solubilized CO2, differences between two systems tended to decrease but did not disappear. Results evidence that gas venting plays a key role to achieve reliable measurements of in vitro CH4 production.