Development and Validation of a 500-mL In Vitro Fermentation Bottle System for Gas Production, Methane, and Nutrient...

A laboratory-scale in vitro fermentation system using modified 500-mL bottles was developed and validated for the measurement of gas, methane production, and nutrient degradability of ruminant feed substrates. The system was designed to accommodate a larger substrate volume than conventional small-volume in vitro systems while maintaining anaerobic conditions for gas sampling. Validation of the system was conducted using diets formulated at different roughage-to-concentrate ratios (80:20, 50:50, and 20:80), incubated with rumen fluid collected from Japanese Black cattle for 24 h at 39°C. In vitro dry matter and organic matter degradability and total gas production increased with a higher proportion of concentrate (p = 0.0002; p = 0.0002; and p < 0.0001, respectively), whereas methane production expressed per unit of degraded dry matter (CH₄/DMD) and pH after 24 h of incubation decreased (p = 0.0008 and p = 0.0118, respectively). The crude protein, neutral detergent fiber, and acid detergent fiber degradability and the amount of short-chain fatty acid produced were not affected by the dietary treatment (p > 0.05). The modified 500-mL bottle system enabled simultaneous assessment of gas and methane production and multiple degradability parameters within a single incubation, supporting its use in laboratory-scale in vitro rumen fermentation studies.