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 (CH₄ GC ), quantified in real time by GES (CH₄ GE ), and computed from volatile fatty acid using stoichiometric equations (CH₄ VFA ). 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. CH₄ GC averaged ≈ 38–40 mL g⁻¹ DM (≈ 21% of GP) and remained stable across treatments; CH₄ model produced similar estimates (≈ 46 mL g⁻¹ DM, ≈ 21%), confirming the robustness of the stoichiometric approach. In contrast, real-time CH₄ GE 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.