PSV-4 Effect of diet and sampling method on ruminal methanogen populations.

Abstract Understanding ruminal methanogen populations is critical for advancing scalable methane-reduction strategies, including immunological interventions such as vaccines. Sampling from the rumen cannula is the primary collection method used to study the rumen microbiome and fermentation, but cannulation is costly and not feasible for large numbers of cattle. Esophageal tubing offers a more cost-effective method for collecting rumen fluid from larger numbers of cattle, but it has not been validated for analyzing ruminal methanogen populations. This study used 16S rRNA sequencing to compare rumen methanogen community samples collected by esophageal tubing versus rumen cannula. Seven crossbred beef steers (BW 429 ± 29 kg) were used in a cross-over design with two consecutive 42d periods with 12d for transition, 29d for microbial adaptation, and 1d for collection. In each period, steers had ad libitum access to a grower ration (0.97 Mcal NEg; 15.5% CP) or a finisher ration (1.1 Mcal NEg; 13.8% CP). On day 42, rumen samples from both rumen cannula (liquid and solid; CL and CS, respectively) and esophageal tubing (liquid only; EL) were collected. Ruminal pH and VFA concentration were measured. Diet had no effect on ruminal pH or total VFAs (P > 0.05), but sampling method did; ruminal pH and total VFAs were lower in CL samples compared to EL (P < 0.01), suggesting potential saliva contamination. Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii composed 95% of methanogen sequences in all samples regardless of diet or sampling method. There were no significant diet × sampling method interactions and diet had no effect on Methanobrevibacter spp. While diet had no effect on abundance of M. ruminantium or M. gottschalkii in differential abundance analysis, sample fraction (EL and CL vs. CS) affected their abundance. Relative abundance of M. ruminantium was significantly negatively associated with the liquid fraction versus the solid (P-adj < 0.02). In contrast, relative abundance of M. gottschalkii was positively associated with the liquid fraction (P-adj < 0.01) versus the solid. However, there was no effect of sampling method (tubing vs. cannulation) on M. ruminantium and M. gottschalkii abundance in the liquid samples. Both diet and sampling method had no effect on any measure of alpha diversity in any sample type (P > 0.05). Pielou’s evenness (P = 0.02) and Shannon entropy (P = 0.04) were higher in the liquid fraction compared to the solid fraction, suggesting greater methanogen diversity in liquid compared to solid samples. Overall, esophageal tubing is a viable alternative for measuring methanogen abundance in rumen liquid. However, due to observed differences between solid and liquid samples and the potential saliva contamination with esophageal tubing, ruminal cannulation provides a more robust and accurate description of ruminal methanogen populations, pH, and VFAs.