Effects of gas composition in headspace and bicarbonate concentrations in media on gas and methane production, degradability, and rumen fermentation using in vitro gas production techniques

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Headspace gas composition and bicarbonate concentrations in media can affect methane production and other characteristics of rumen fermentation in in vitro gas production systems, but these 2 important factors have not been evaluated systematically. In this study, these 2 factors were investigated with respect to gas and methane production, in vitro digestibility of feed substrate, and volatile fatty acid (VFA) profile using in vitro gas production techniques. Three headspace gas compositions (N2+ CO2+ H2 in the ratio of 90:5:5, CO2, and N2) with 2 substrate types (alfalfa hay only, and alfalfa hay and a concentrate mixture in a 50:50 ratio) in a 3×2 factorial design (experiment 1) and 3 headspace compositions (N2, N2 + CO2 in a 50:50 ratio, and CO2) with 3 bicarbonate concentrations (80, 100, and 120 mM) in a 3×3 factorial design (experiment 2) were evaluated. In experiment 1, total gas production (TGP) and net gas production (NGP) was the lowest for CO2, followed by N2, and then the gas mixture. Methane concentration in headspace gas after fermentation was greater for CO2 than for N2 and the gas mixture, whereas total methane production (TMP) and net methane production (NMP) were the greatest for CO2, followed by the gas mixture, and then N2. Headspace composition did not affect in vitro digestibility or the VFA profile, except molar percentages of propionate, which were greater for CO2 and N2 than for the gas mixture. Methane concentration in headspace gas, TGP, and NGP were affected by the interaction of headspace gas composition and substrate type. In experiment 2, increasing concentrations of CO2 in the headspace decreased TGP and NGP quadratically, but increased the concentrations of methane, NMP, and in vitro fiber digestibility linearly, and TMP quadratically. Fiber digestibility, TGP, and NGP increased linearly with increasing bicarbonate concentrations in the medium. Concentrations of methane and NMP were unaffected by bicarbonate concentration, but TMP tended to increase due to increasing bicarbonate concentration. Although total VFA concentration and molar percentage of butyrate were unchanged, the molar percentage of acetate, and acetate-to-propionate ratio decreased, whereas the molar percentage of propionate increased quadratically with increasing bicarbonate concentration. This study demonstrated for the first time that headspace composition, especially CO2 content, and bicarbonate concentration in media could significantly influence gas and methane production, and rumen fermentation in gas production techniques.