Effects of plants containing secondary metabolites as feed additives on rumen metabolites and methanogen diversity of buffaloes

Four fistulated adult Murrah buffaloes were fed on a basal diet consisting of wheat straw and concentrate mixture in a 4 × 4 Latin square design to study the effects of feeding plants containing secondary metabolites on rumen metabolites and methanogen diversity. The four groups were Control (no additive), Mix-1 (ajwain oil and lemon grass oil in a 1 : 1 ratio @ 0.05% of dry matter intake), Mix-2 (garlic and soapnut in a 2 : 1 ratio @ 2% of dry matter intake) and Mix-3 (garlic, soapnut, harad and ajwain in a 2 : 1 : 1 : 1 ratio @ 1% of dry matter intake). In each phase of 30 days’ duration, after 19 days of feeding, rumen liquor was sampled for two consecutive days at 0, 2, 4, 6 and 8 h post-feeding, whereas rumen content was sampled at 0 h feeding. The pH of the rumen liquor was recorded at every collection and then the rumen liquor of every collection was pooled day-wise and animal-wise. These pooled samples were used for estimation of rumen metabolites like ammonia, lactic acid and volatile fatty acids. Microscopic counting of protozoa was done in both 0 h and pooled samples of rumen liquor. Rumen contents collected from different locations of rumen were processed for enzyme estimation. The rumen contents were squeezed and the liquid portion was used for DNA isolation, which was further processed to determine methanogen diversity. Daily intake of feed was similar (P > 0.05) in all the four groups. The ammonia-N concentration and ciliate protozoa population were reduced significantly in the treatment groups supplemented with additives. Rumen pH, lactic acid, volatile fatty acids and enzyme activities were not affected (P > 0.05) by feeding of any of these additives. Methanogenic diversity comparison was made between the Control and Mix-1 group. The basic local alignment search tool (BLAST) analysis of the 133 (44 from the Control group and 89 from the Mix-1 group) sequences showed similarity of the sequences of rumen archaea by up to 97% to the known sequences of rumen methanogens. The sequences with minimum length of 750 bp were selected for phylogenetic analysis. Per cent identity of these sequences with that of the available nearest neighbour as calculated by MEGA 5.03 software showed identity of the clones in the range of 88–97%. The clones were similar with Methanobrevibacter smithii ATCC 35061, uncultured Methanobrevibacter sp. clone MEME95 and M. ruminantium M1. Overall, feeding of any of these feed additives to fistulated buffaloes did not affect feed intake, rumen pH, or rumen metabolites except ammonia and enzyme profile. Methanogen diversity showed the possibility of Methanobrevibacter as the major methanogen in buffalo rumen liquor.