Effects of pure plant secondary metabolites on methane production, rumen fermentation and rumen bacteria populations in vitro

In this study, the effects of seven pure plant secondary metabolites (PSMs) on rumen fermentation, methane (CH4) production and rumen bacterial community composition were determined. Two in vitro trials were conducted. In trial 1, nine concentrations of 8-hydroxyquinoline, -terpineol, camphor, bornyl acetate, -pinene, thymoquinone and thymol were incubated on separate days using in vitro 24-hr batch incubations. All compounds tested demonstrated the ability to alter rumen fermentation parameters and decrease CH4 production. However, effective concentrations differed among individual PSMs. The lowest concentrations that reduced (p<.05) CH4 production were as follows: 8mg/L of 8-hydroxyquinoline, 120mg/L of thymoquinone, 240mg/L of thymol and 480mg/L of -terpineol, camphor, bornyl acetate and -pinene. These concentrations were selected for use in trial 2. In trial 2, PSMs were incubated in one run. Methane was decreased (p<.05) by all PSMs at selected concentrations. However, only 8-hydroxyquinoline, bornyl acetate and thymoquinone decreased (p<.05) CH4 relative to volatile fatty acids (VFAs). Based on denaturing gradient gel electrophoresis analysis, different PSMs changed the composition of bacterial communities to different extents. As revealed by Ion Torrent sequencing, the effects of PSMs on relative abundance were most pronounced in the predominant families, especially in Lachnospiraceae, Succinivibrionaceae, Prevotellaceae, unclassified Clostridiales and Ruminococcaceae. The CH4 production was correlated negatively (-.72; p<.05) with relative abundance of Succinivibrionaceae and positively with relative abundance of Ruminococcaceae (.86; p<.05). In summary, this study identified three pure PSMs (8hydroxyquinoline, bornyl acetate and thymoquinone) with potentially promising effects on rumen CH4 production. The PSMs tested in this study demonstrated considerable impact on rumen bacterial communities even at the lowest concentrations that decreased CH4 production. The findings from this study may help to elucidate how PSMs affect rumen bacterial fermentation.