Two experiments were conducted to investigate the effects of adding gallic acid (GA) to ruminant diets on long- and short-term in vitro rumen fermentation and methane (CH4) production, and to test possible interactions between GA and ethanol on fermentation. The first experiment was conducted using the rumen simulation technique (Rusitec), as a completely randomised block design with four replications and the following four doses of GA: 0, 5, 10 and 20 mg GA/g dry matter (DM). Ethanol was used in all treatments to increase the solubilisation of GA in rumen fluid. The experimental period lasted 16 days, of which the first 7 days were for adaptation and the subsequent 9 days were for sampling. The second experiment was a 48-h batch-culture incubation conducted as a completely randomised design with a 4 (GA dose; 0, 10, 20, and 40 mg GA/g DM) × 2 (with or without ethanol) arrangement of treatments. In the Rusitec experiment, addition of GA up to 20 mg/g DM did not affect DM disappearance (DMD), organic matter (OM) disappearance, neutral detergent-fibre disappearance (NDFD), acid detergent-fibre disappearance (ADFD) or starch disappearance (P > 0.05), but crude protein disappearance was linearly decreased (P = 0.04) from 78.3% to 72.0%. Daily gas production and CH4 production expressed as mL/g DM and mL/g DMD were not affected by addition of GA (P > 0.05). Addition of GA up to 20 mg/g DM increased butyrate and isovalerate production (P < 0.05) and tended to increase isobutyrate (P = 0.09) and decrease heptanoate production (P = 0.07). In the batch-culture experiment, adding GA up to 40 mg/g DM linearly increased 48-h DMD, NDFD and ADFD (P < 0.05) and decreased (P < 0.05) CH4 expressed as mL/g DMD, mL/g NDFD and mL/g ADFD. Methane production was decreased after 24 h and 48 h only when GA was added at 10 mg/g DM without ethanol. Fermentation liquid pH and concentration of ammonia-nitrogen (ammonia-N) were also reduced (P < 0.05) with an increasing concentration of GA. Treatments with ethanol notably enhanced 48-h DMD, NDFD, ADFD, gas production (mL/g DM, mL/g OM or mL/g DMD), CH4 production (mL/g DM, mL/g DMD or mL/g NDFD), total volatile fatty acid concentration, the acetate:propionate ratio, acetate, valerate, isovalerate and caproate molar proportions (P < 0.01) and decreased propionate, butyrate and isobutyrate molar proportions (P < 0.01). Significant dose of GA × ethanol interaction was observed only for acetate molar proportion (P = 0.03). In conclusion, our study suggests that the beneficial effects of GA on feed digestion and CH4 production may be short term, while improvements in N metabolism may be sustained over the long term. It may be useful to conduct long-term in vivo studies using a range of diets and doses to verify whether GA can be used as a feed additive to mitigate enteric CH4 production and improve N metabolism of ruminants.
Heat Stress Increases In Vitro Hindgut Fermentation of Distinct Substrates in Iberian Pigs
