Impact of CO₂ flushing and headspace volume on gas and methane kinetics in two in vitro Rumen systems: A comparative study of AnkomRF and gas endeavour

In vitro gas-production (GP) techniques are widely used to evaluate Rumen fermentation, yet methodological factors such as CO2-flushing duration and headspace volume can bias gas and methane (CH4) measurements. This study examined the effects of three CO2-flushing times (60, 30 and 10 min) and two headspace volumes (72 and 172 mL) in two fermentation systems, Ankom(RF) and Gas Endeavour (GES). GP was recorded over 24 h; CH4 was quantified from headspace gas by gas chromatography (CH4GC), quantified in real time by GES (CH4GE), and computed from volatile fatty acid using stoichiometric equations (CH4VFA). Fermentation parameters (VFA, NH3-N, pH) were also analyzed. Flushing time significantly affected GP (P < 0.01), with lower yields at 10 min than 30-60 min, while methane responses were moderate. CH4GC averaged approximate to 38-40 mL g(-)& sup1; DM (approximate to 21% of GP) and remained stable across treatments; CH4 model produced similar estimates (approximate to 46 mL g(-)& sup1; DM, approximate to 21%), confirming the robustness of the stoichiometric approach. In contrast, real-time CH4GE expressed as % of total gas declined with longer flushing (14.3-11.4%; P < 0.01). This pattern is most likely attributable to a methodological artefact related to CO2 measurement, specifically incomplete CO2 equilibration and under-recovery of CO2 at shorter flushing times (denominator effect), rather than reflecting true biological changes in methanogenesis. Headspace volume configuration strongly influenced GP and CH4 (P < 0.01). The smaller headspace improved gas displacement and apparent CH4 recovery in GES, whereas the larger headspace buffered pressure changes and stabilized gas recovery in AnkomRF. InstRument responses were therefore configuration-dependent: GES performed best at 72 mL, whereas AnkomRF required 172 mL for stable gas recovery. In contrast, a 60 min CO2 flushing time consistently provided stable and reproducible gas measurements across both systems. These findings indicate that while headspace configuration requires instRument-specific optimization, adequate CO2 flushing appears broadly applicable and should be standardized or at minimum explicitly reported to improve reproducibility and comparability across laboratories.