Influence of inoculum preparation method on in vitro methane production by ruminal microorganisms

Abstract

The characteristics of the inoculum are recognized as one of the most relevant factors infiuencing the results o fin vitro fennentations in batch cultures of ruminal microorganisms. Four rumen-fistulated sheep fed a 66:34 alfalfa hay:concentrate diet were used as donors to investigate the effect ofrumen contents' processing on in vitro methane (CH4) and volatile fatty acid (VFA) production from 3 substrates ofvariable composition. Rumen contents were sampled from each individual sheep and subjected to the following treatments: SQ) squeezed through 4 layers of cheesecloth; FIL) SQ treatment and further filtration through a 100-µm nylon cloth; STO) treated with a Stomacher for 3 min at 230 rev min-1 and followed by SQ. The resulting fiuids were used as inoculum for batch cultures containing alfalfa hay, concentrate, or a 50:50 mixtnre ofboth feeds. Cultures were incubated at 39°C for 8 and 24 h, and CH4 and VF A production was measured. There were no treatment x substrate interactions (P > 0.05) for any variable at any incubation time, excepting for the molar proportion of acetate at 24 h (P = 0.019). The method of processing the rumen contents did not affect (P > 0.05) eithertotal VFA and CH4 production or molar proportions of individual VF A at any time. At both incubation times, increasing the amount of concentrate in the substrate increased CH4 production (P < 0.001, quadratic) and molar proportion ofbutyrate (P < 0.001, linear), but decreased acetate proportion (P < 0.001, quadratic) without affecting (P > 0.05) proportions of propionate. Whereas total VFA production was linearly decreased (P = 0.007) by increased amounts of concentrate in the substrate at 8 h ofincubation, it was quadratically increased (P < 0.001) after 24 h of incubation. There were clear differences in CH4 and VFA production among inocula from different sheep, which persisted across substrates. The results show that the tested methods ofprocessing rumen contents did not affect in vitro fermentation characteristics of good quality substrates, but studies ana!yzing their possible infiuence on fermentation of!ow-quality substrates are required