Bacterial colonisation of the tanniferous forage lotus corniculatus in the bovine rumen

The tanniferous forage Lotus corniculatus generally has a beneficial effect on ruminal fermentation, protecting dietary protein from excessive degradation. In L. corniculatus, condensed tannins (CTs) are normally concentrated within plant cell vacuoles, in subsets of plant cells within the spongy and palisade mesophyll. However, the effect of these concentrated pockets of CT on the rumen bacteria that colonise L. corniculatus is not clear. In order to investigate this, two L. corniculatus varieties with differing CT content in leaf (0.64% v 2.3% dry matter) and stem (0.34% v 0.77% dry matter) were incubated in the rumen of two cows over a 72 h period. For each cow duplicate polyester bags containing fresh, chopped samples of each variety were incubated per time point (1, 2, 4, 8, 12, 24, 48 and 72 h), with 0 h bags processed directly. At the end of their incubation, bag residues were hand washed and then frozen. DNA was extracted from the residues and the population composition and size of bacteria colonising the plant material was assessed using 16S rDNA denaturing gradient gel electrophoresis and quantitative PCR. The size of the colonising bacterial population was significantly affected by ruminal incubation time (P<0.001), with no
effect of variety (P>0.05). The population size initially increased during 0–1 h of incubation, and then increased again during 2–4 h of incubation; thereafter the population size remained stable for the remainder of the incubation period. Bacterial population composition was also significantly affected by time, with no effect of variety. The bacterial population composition of the rumen incubated samples were distinct from the 0 h samples, and separated into two main clusters containing 1–8 h and 12–72 h time points. Within the latter cluster a further separation was evident after 24 h, which was characterised by a substantial increase in the intensity of one band. The sequence from the dominant band had 98% identity with Butyrivibrio hungatei. The dominance of this bacterium in latter time points is intriguing as its ecological niche is thought to be in the utilisation of oligo- and monosaccharides, as it does not possess proteolytic or fibrolytic activity. Further work is needed to clarify the basis of this observation, and the change in the colonising bacterial population composition after 8 h.