Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

Molecular identification of methanogenic archaea from surti buffaloes (bubalus bubalis), reveals more hydrogenotrophic methanogens phylotypes.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in breeds of buffaloes, as well as in response to geographical location and different diets, is required. Therefore, molecular diversity of rumen methanogens in Surti buffaloes was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from three Surti buffaloes. A total of 171 clones were identified revealing 23 different sequences (phylotypes). Of these 23 sequences, twelve sequences (12 OTUs, 83 clones) and 10 sequences (10 OTUs, 83 clones) were similar to methanogens belonging to the orders Methanomicrobiales and Methanobacteriales, and the remaining 1 phylotype (5 clones) were similar to Methanosarcina barkeri. These unique sequences clustered within a distinct and strongly supported phylogenetic group. Further studies and effective strategies can be made to inhibit the growth of Methanomicrobiales and Methanobacteriales phylotypes to reduce the methane emission from rumen and thus help in preventing global warming.

Study of rumen metagenome community using qPCR under different diets.

The aim of this study was to detect the major bacteria present in rumen microbiota. Here, we performed qPCR based absolute quantitation of selected rumen microbes in rumen fluid of river buffalo adapted to varying proportion of concentrate to roughage diets. Animals were adapted to roughage-to-concentrate ratio in the proportion of 100:00 (T1), 75:25 (T2), 50:50 (T3) and 25:75 (T4) respectively for 30 days. At the end of each treatment, rumen fluid was collected at 0 h and 2 h after feeding. It was found that among fibrolytic bacteria Ruminococcus flavefaciens (2.22 × 10(8) copies/ml) were highest in T2 group and followed by 1.11 × 10(8) copies/ml for Fibrobacter succinogenes (T2), 2.56 × 10(7) copies/ml for Prevotella ruminicola (T1) and 1.25 × 10(7) copies/ml for Ruminococcus albus (T4). In non-fibrolytic bacteria, the Selenomonas ruminantium (2.62 × 10(7) copies/ml) was predominant in group T3 and followed by Treponema bryantii (2.52 × 10(7)copies/ml) in group T1, Ruminobacter amylophilus (1.31 × 10(7)copies/ml) in group T1 and Anaerovibrio lipolytica (2.58 × 10(6) copies/ml) in group T4. It is most notable that R. flavefaciens were the highest in population in the rumen of Surti buffalo fed wheat straw as roughage source.

Molecular Genetic Diversity and Quantitation of Methanogen in Ruminal Fluid of Buffalo (Bubalus bubalis) Fed Ration (Wheat Straw and Concentrate Mixture Diet).

High roughage diet causes more methane emissions; however, the total methanogen abundance is not influenced by roughage proportion. Technologies to reduce methane emissions are lacking, and development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on present knowledge of the methanogens. In this work, we have investigated molecular diversity of rumen methanogens of Surti buffalo. DNA from rumen fluid was extracted, and 16S rRNA encoding genes were amplified using methanogen specific primer to generate 16S rDNA clone libraries. Seventy-six clones were randomly selected and analysed by RFLP resulting in 21 operational taxonomic units (OTUs). BLAST analysis with available sequences in database revealed sequences of 13 OTUs (55 clones) showing similarity with Methanomicrobium sp, 3 OTUs (15 clones) with Methanobrevibacter sp. The remaining 5 OTUs (6 clones) belonged to uncultured archaea. The phylogenetic analysis indicated that methanogenic communities found in the library were clustered in the order of Methanomicrobiales (18 OTUs) and Methanobacteriales (3 OTUs). The population of Methanomicrobiales, Methanobacteriales, and Methanococcales were also observed, accounting for 1.94%, 0.72%, and 0.47% of total archaea, respectively.

Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes.

Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%; the similarity was lower than 85% Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.

Molecular identification of methanogenic archaea from Surti buffaloes (bubalus bubalis), reveals more hydrogenotrophic methanogens phylotypes

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in breeds of buffaloes, as well as in response to geographical location and different diets, is required. Therefore, molecular diversity of rumen methanogens in Surti buffaloes was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from three Surti buffaloes. A total of 171 clones were identified revealing 23 different sequences (phylotypes). Of these 23 sequences, twelve sequences (12 OTUs, 83 clones) and 10 sequences (10 OTUs, 83 clones) were similar to methanogens belonging to the orders Methanomicrobiales and Methanobacteriales, and the remaining 1 phylotype (5 clones) were similar to Methanosarcina barkeri. These unique sequences clustered within a distinct and strongly supported phylogenetic group. Further studies and effective strategies can be made to inhibit the growth of Methanomicrobiales and Methanobacteriales phylotypes to reduce the methane emission from rumen and thus help in preventing global warming.