Technical note: Evaluation of sampling methods for methane concentration from in vitro fermentation

The objectives of this multipart study were 1) to assess the efficacy of sampling methods of methane concentration ([CH4]) of headspace gas produced during in vitro gas production (IVGP) fermentation, 2) to verify whether headspace [CH4] sampled from an exetainer has the same [CH4] as the headspace of IVGP bottles, 3) to measure relative humidity (RH) within an IVGP bottle, and 4) to compare [CH4] on a dry-gas (DG) basis when accounting for water vapor pressure (Pw). The original IVGP protocol recommends placing bottles on ice (0 °C) for 30 min to stop fermentation (ICE). A laboratory protocol recommends placing the bottles in the refrigerator (4 to 6 °C) to slow fermentation for 48 h and subsequently allowing the bottles to return to ambient temperature before sampling (FRIDGE). This study evaluated the previous methods against a direct sampling of the headspace gas after incubation (DIRECT). Rumen inoculum from four rumen-cannulated beef steers was combined and homogenized before incubating the fermentable substrate of ground alfalfa hay. After 48 h of IVGP incubation, each bottle was randomly assigned to a treatment protocol. The pressure (P), volume (V), and temperature (T) of headspace gas in each bottle were recorded. Headspace gas was then thoroughly mixed, and 12 mL gas was removed into an evacuated exetainer for [CH4] sampling via gas chromatography (EXET; Objective 1). Eight bottles from ICE and FRIDGE were randomly selected to follow EXET, whereas the remaining bottles had [CH4] directly measured from their headspace (BOTT; Objective 2). Five diets of differing feed composition and nutrient densities were used with a blank to test the RH of the IVGP slurry (Objective 3). Using RH, [CH4] was transformed to a DG basis to account for Pw (Objective 4). Statistical analysis was completed using a random coefficients model. There were no differences between EXET and BOTT (P = 0.28). The RH of the IVGP slurry was 100% (P = 1.00), confirming that IVGP gas is saturated with water vapor. The P, V, and T differed among treatments (P < 0.01). The [CH4] of DIRECT, ICE, and FRIDGE were different (P < 0.01). Dry-gas P, V, and [CH4] differed among treatments (P < 0.01). As the methods differ in their assessment of [CH4], there is no clear recommendation. Instead, to present a more accurate [CH4], P, V, and T should be measured when sampling headspace gas and equations presented should be used to remove volume inflation due to water vapor and present [CH4] on a DG basis.