Can flushing gas distort the rumen in vitro experiment results?

To test whether the type of flushing gas could alter rumen in vitro experimental results, effects of three flushing gases (Ar, CO2, and N2) were compared with or without hypothetic experimental treatment (monensin supplementation) in a batch culture system. Rumen fluids obtained from three cannulated Holstein heifers were incubated for 3, 6, 12, 24, and 48 h according to 3 × 2 factorial arrangements. Total gas production was decreased, and CH4 proportion was increased by CO2 flushing regardless of incubation time. The effects of monensin on CH4 proportion were not shown in CO2 flushed group. After 3 h incubation, ammonia nitrogen was increased by N2 flushing, and interaction between flushing gas and monensin was observed in iso-butyrate. Total volatile fatty acids production was not influenced by the type of flushing gases regardless of incubation time. However, after 48 h incubation, by CO2 flushing, the valerate without monensin decreased while it increased with monensin, implying the interaction effect between flushing gas and monensin. The pH elevation effect of monensin was delayed by CO2 flushing compared to Ar and N2 flushing. This study demonstrated whether the type of flushing gas can confound experimental results. Since N2 flushing can influence rumen nitrogen parameters in early incubation and CO2 flushing affected pH results while Ar flushing did not show noticeable results, we suggest that Ar could be used as a new alternative flushing gas for rumen in vitro studies. •Nitrogen flushing influenced NH3-N and iso-butyrate after 3 h incubation.•Carbon dioxide flushing increased CH4 proportion and masked the effects of monensin on rumen CH4 proportion.•Carbon dioxide flushing delayed the effects of monensin on rumen pH.•Choosing flushing gas can confound rumen in vitro experimental results.•Argon flushing is suggested as a new alternative flushing gas for rumen in vitro batch culture system.