Development of an in vitro method for determination of methane production kinetics using a fully automated in vitro gas system—A modelling approach

Abstract

The objective of the present study was to estimate methane production using the kinetic parameters from an automated in vitro gas production (GP) system in a mechanistic, dynamic rumen model. Four sample sizes [300mg (5g/l), 600mg (10g/l), 900mg (15g/l) and 1200mg (20g/l)] of timothy hay were incubated in 60ml buffered rumen fluid in an automated in vitro system to determine methane and total gas production. A logarithmic model (R2>0.99) was fitted to methane data to estimate methane concentrations at time intervals of 0.2h. The first-order gas production rates were not different (P=0.18) with values of 0.072, 0.061, 0.061 and 0.059/h for the sample size of 300, 600, 900 and 1200mg, respectively. The methane production rates were 0.052, 0.046, 0.046 and 0.045/h, respectively. Predicted methane production decreased linearly (PLIN<0.01) as the sample size increased from 300 (36.9ml/g dry matter, DM) to 1200mg (28.2ml/g DM). After 48h of incubation total volatile fatty acids (VFA) production decreased (PLIN<0.01) as the sample size increased from 300 to 1200mg (4.41mmol/g DM and 3.82mmol/g DM, respectively). Neutral detergent fibre digestibility (aNDFomD), apparent organic matter digestibility (AOMD) and true organic matter digestibility (TOMD) decreased (PLIN=0.01) as the sample size increased (0.479, 0.433 and 0.681 for 300mg, and 0.369, 0.379 and 0.614 for 1200mg of sample size, respectively). Actual methane production (24 and 48h of incubation) was strongly correlated (R2=0.97) with the methane production predicted from VFA stoichiometry (VFA measured at 24 and 48h of incubation). It is concluded that in vitro GP measurements can be successfully used to estimate kinetic parameters of methane production and consequently to predict methane production. It seems that sample size did not affect the first-order production rate of methane and therefore, it is possible to use greater amounts of substrate in the in vitro GP system up to 1000mg (16.6g/l).