In Vitro Gas Production and Methane

On day 17 of each period, rumen fluid was collected to quantify in vitro total gas and methane production (Quinn et al., 2010). One litter of rumen fluid from each steer in each experimental period (n = 4) was collected 4 h after-feeding, filtered through 4 layers of cheesecloth, and placed in a pre-warmed thermos (39 °C). Samples were immediately transported to Ruminant Nutrition Laboratory to be used for in vitro procedure. A 50 mL aliquot of rumen fluid from each thermos (1 per steer) was incubated in a 160 mL serum vial (Wheaton Science Products, Millville, NJ); a total of 4 serum vials (quadruplicate) was used for each steer per incubation period. Results obtained from quadruplicate were averaged for data analysis. The serum bottles were then flushed with CO2 for 15 s (high-purity, analytical grade), sealed with a rubber stopper and aluminum ring, and incubated at 39 °C for 24 h with constant agitation at 125 rpm (Lab-Line Environ-Shaker, LabLine Instruments Inc., Melrose Park, IL). Following 24 h incubation, total gas production was measured by water displacement by piercing the rubber stopper in the serum vial with a needle attached to 250 mL inverted burette. The sample of headspace gas remaining in each bottle after total gas was retained for measuring methane concentration as outlined by Ponce et al. (2012) using gas chromatography.