Effect of the ionophore monensin and tannin extracts supplemented to grass silage on populations of ruminal cellulolytics and methanogens in vitro.

This study examined whether the methane-decreasing effect of monensin (∼21%) and different hydrolysable tannins (24%-65%) during in vitro fermentation of grass silage was accompanied by changes in abundances of cellulolytics and methanogens. Samples of liquid (LAM) and solid (SAM) associated microbes were obtained from two rumen simulation technique experiments in which grass silage was either tested in combination with monensin (0, 2 or 4 mg d-1 ) or with different tannin extracts from chestnut, valonea, sumac and grape seed (0 or 1.5 g d-1 ). Total prokaryotes were quantified by 4',6-diamidino-2-phenylindol (DAPI) staining of paraformaldehyde-ethanol-fixed cells and relative abundances of ruminal cellulolytic and methanogenic species were assessed by real time quantitative PCR. Results revealed no change in absolute numbers of prokaryotic cells with monensin treatment, neither in LAM nor in SAM. By contrast, supplementation of chestnut and grape seed tannins decreased total prokaryotic counts compared to control. However, relative abundances of total methanogens did not differ between tannin treatments. Thus, the decreased methane production by 65% and 24% observed for chestnut and grape seed tannins, respectively, may have been caused by a lower total number of methanogens, but methane production seemed to be also dependent on changes in the microbial community composition. While the relative abundance of F. succinogenes decreased with monensin addition, chestnut and valonea tannins inhibited R. albus. Moreover, a decline in relative abundances of Methanobrevibacter sp., especially M. ruminantium, and Methanosphaera stadtmanae was shown with supplementation of monensin or chestnut tannins. Proportions of Methanomicrobium mobile were decreased by monensin in LAM while chestnut and valonea had an increasing effect on this methanogenic species. Our results demonstrate a different impact of monensin and tannins on ruminal cellulolytics and gave indication that methane decrease by monensin and chestnut tannins was associated with decreased abundances of M. ruminantium and M. stadtmanae.