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 (P-w). The original IVGP protocol recommends placing bottles on ice (0 degrees C) for 30 min to stop fermentation (ICE). A laboratory protocol recommends placing the bottles in the refrigerator (4 to 6 degrees 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 P-w (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. Lay Summary Greenhouse gas emissions (GHG) from ruminant production equate to 81% of total global livestock supply chain emissions, with 51% originating from beef cattle production. Traditional in vivo estimation methods of methane (CH4), a highly scrutinized greenhouse gas, are timely and costly. In vitro gas production (IVGP) methods can accurately describe CH4 emission patterns from the rumen but tend to overestimate quantities. Additionally, in vivo estimation methods present CH4 on a dry-gas basis, whereas in vitro do not. In vitro methods utilize a gas chromatography machine to estimate CH4. Laboratory constraints can impose deviations to a strict IVGP protocol. This multi-objective study evaluates three treatment methods of IVGP bottles to understand whether discrepancies exist in CH4 estimation when deviating from the published protocol. To estimate CH4 from IVGP more accurately and provide a more comparable number to in vivo methods, this study also evaluates environmental conditions within an IVGP bottle to formulate a system of equations to calculate CH4 on a dry-gas basis. This study found that the treatment method of the IVGP bottle had an impact on CH4 estimation, and the developed equations should be utilized to produce more comparable estimates. This study shows that postincubation treatment of in vitro gas production fermentation bottles can cause discrepancies in the quantification of methane in the headspace gas. To reduce those discrepancies, this study suggests a system of equations to remove water vapor from the headspace gas to present methane estimates on a dry-gas basis.