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

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 (CH 4 /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.