Microbial profiles of liquid and solid fraction associated biomaterial in buffalo rumen fed green and dry roughage diets by tagged 16S rRNA gene pyrosequencing.

The microbiome of buffalo rumen plays an important role in animal health and productivity. The rumen bacterial composition of both liquid and solid fraction was surveyed using pyrosequencing of the 16S rRNA gene. Sequences were analyzed using taxonomy-dependent clustering methods and revealed that the dominant ruminal bacteria shared by samples belonged to phyla Bacteroidetes, Firmicutes, Fibrobacteres and Proteobacteria. The core rumen microbiome of the rumen consisted of 10 phyla, 19 classes, 22 orders and 25 families. However, the relative abundance of these bacterial groups was markedly affected by diet composition as well as in type of biomaterial. In animals fed with a green and dry roughage diet, the cellulolytic bacteria, Ruminococcaceae, and Fibrobacteraceae was found in highest abundance in all biomaterials which reflected the need for enhanced fiber-digesting capacity in buffalo. The polysaccharide-degrading Prevotellaceae bacteria were most abundant in buffalo rumen. In taxonomic comparison of rumen bacteria, about 26 genera were differentially abundant among liquid and solid fraction of ruminal fluid. These results highlight the buffalo ruminal microbiome's ability to adapt to feed with different composition.

High through put 16S rRNA gene-based pyrosequencing analysis of the fecal microbiota of high FCR and low FCR broiler growers.

The performance of birds appears to vary among the flock of growing broilers which may in part be due to variation in their gut microbiota. In the view of poultry industry, it is desirable to minimise such variation. We investigated metagenomic profile of fecal bacteria in birds with high and low feed conversion ratio (FCR) to identify microbial community linked to low and high FCR by employing high throughput pyrosequencing of 16S rRNA genomic targets. Therefore feeding trial was investigated in order to identify fecal bacteria consistently linked with better feed conversion ratio in bird performance as measured by body weight gain. High-throughput 16S rRNA gene based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. The fecal microbial community of birds was predominated by Proteobacteria (48.04 % in high FCR and 49.98 % in low FCR), Firmicutes (26.17 % in high FCR and 36.23 % in low FCR), Bacteroidetes (18.62 % in high FCR and 11.66 % in low FCR), as well as unclassified bacteria (15.77 % in high FCR and 14.29 % in low FCR), suggesting that a large portion of fecal microbiota is novel and could be involved in currently unknown functions. The most prevalent bacterial classes in high FCR and low FCR were Gammaproteobacteria, Clostridia and Bacteroidia. However in low FCR birds Phascolarctobacterium, Faecalibacterium and Clostridium predominated among the Clostridia. In FCR comparison of fecal bacteria, about 36 genera were differentially abundant between high and low FCR birds. This information could be used to formulate effective strategies to improve feed efficiency and feed formulation for optimal gut health.

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.

Association of heat stress protein 90 and 70 gene polymorphism with adaptability traits in Indian sheep (Ovis aries).

Heat stress proteins assist cellular proteins in the acquisition of native structure. The present research was conducted to study how thermo-tolerance is modulated by HSP90 and HSP70 gene polymorphism and its association with hemato-physio-biochemical parameters, supported by their expression profiles in Chokla, Magra, Marwari, and Madras Red sheep breeds. Least square analysis revealed significant effect (P < 0.05) of season and breed on all the physiological parameters, i.e., temperature, respiratory rate, and pulse rate (a.m. and p.m.), as well as hematological parameters like Hb, packed cell volume, total erythrocyte count (TEC), neutrophil/lymphocyte (N/L) ratio, and total leukocyte count (TLC). There was a significant influence (P < 0.05) of breed on biochemical parameters such as glucose, SGOT, phosphorous, triglyceride, and cholesterol. Eight fragments were amplified and sequenced in HSP90, and 70 genes and 13 single-nucleotide polymorphisms (SNPs) were identified. Tetra-primer amplification refractory mutation system PCR, PCR-RFLP, and allele-specific PCR genotyping protocols were developed for large-scale genotyping of five SNPs. A significant difference (P < 0.05) of rectal temperature (a.m.), respiratory rate (p.m.), triglyceride, and total protein was observed at SNP01; albumin at SNP2; pulse rate (p.m.) at SNP3; and rectal temperature (p.m.), pulse rate (p.m.), Hb (g/dL), and N/L ratio at SNP4 and TLC at SNP5. Gene expression analysis revealed higher expression in less adapted animals with Madras Red < Magra < Chokla < Marwari expression pattern [corrected]. Predominant allele was found to be superior in most of the SNPs (SNP1-4) indicating the selection acting in directional manner (positive selection). Finally, it is concluded that TACCA haplotype combination of SNP1-SNP2-SNP3-SNP4-SNP5 might be of some selection advantage for the identification of animals more adaptable to heat stress.

Novel cytogenetic finding: an unusual X;X-translocation in Mehsana buffalo (Bubalus bubalis).

Cytogenetic investigations of a phenotypically normal Mehsana river buffalo calf (Bubalusbubalis) revealed an XXY chromosome complement due to X;X-translocation in all screened metaphase plates. The chromosomal anomaly was identified by GTG-banding while CBG- and RBG-banding revealed two heterochromatin blocks and that one of the two X chromosomes was late replicating, respectively. The normal cytogenetic profiles of sire, dam and relatives of the calf suggest that the anomaly could have arisen spontaneously during oogenesis. This is the first report on a male river buffalo calf having an XXY chromosome complement with translocation between the two X chromosomes.

Ultrastructural appearances of interstitial tissue of testis in vasectomised individuals.

PIP: In order to study the light and electron microscopic features of the interstitial tissue of the testis in vasectomized men, testicular biopsies were obtained using local anesthesia from 5 men ages 20-32 who had reported for vasovasostomy. Time interval between vasectomy and biopsy ranged from 1.5-5 years. Light microscopy showed increase in interstitial tissue in 3 of the 5 cases due to proliferation of loose fibrous tissue which was uniformly distributed between the seminiferous tubules in 2 cases while in the other cases it was aggregated focally. The seminiferous tubules in all the cases were distended, tortuous, and hypocellular. The lamina propria was thickened and the blood capillaries and Leydig cells did not reveal any alterations. These findings show that vasectomy does not produce any significant morphological alterations in the Leydig cells; this confirms the reports that both in man and rhesus monkeys no significant changes in the levels of testosterone or luteinizing hormone occur after vasectomy. It is also found that Leydig cells may degenerate and release their crystalloids and other inclusions into the extracellular space of the interstitial tissue in normal individuals as well.^ieng

Confirmation of the correct placement of lower end of DJ stent during open surgery: point of technique.

A simple technique to confirm the correct placement of the double J (DJ) stent at the time of antegrade insertion during open surgery is described. At the time of antegrade DJ stent placement about 30-50 ml of sterile methylene blue is instilled into the bladder. Appearance of methylene blue from the upper end and side holes of the DJ stent confirms the correct placement of the lower end of the stent.

Wilms' tumour : a clinicopathologic study with special reference to its morphologic variants.

Twenty five cases of Wilms' tumour were studied with special reference to their morphological variations. Wilms' tumour constituted 7.6% of all the malignant tumours of childhood and 50% of renal malignancies. Male/female ratio was 4:1; the mean age being 3.5 years. Left side was commonly involved (60.9%) center dot bilateral involvement in one case (4.3%). Morphologic study revealed triphasic pattern in 60% cases, biphasic (epithelial + stromal) in 12% and monophasic pattern in 28%. Other features observed were myxomatous degeneration, rhabdomyoblasts, calcification and squamous epithelial pearls seen in 20%, 12%, 8% and 4% respectively.

Changes in seminiferous tubules after vasectomy.

PIP: The histologic, immunohistochemical, and ultrastructural changes in the seminiferous tubules (ST) of 17 healthy adult males who had been vasectomized between 1 1/2 and 5 years are reported. There was minimal spermatogenesis in 4 cases. 8 of the cases underwent electron microscopy examination and 4 showed evidence of minimal histological spermatogenesis. The ST showed a thickening of the basement membrane and heavy deposits of lipofuchsin in the Sertoli cells (SC) seen as lipid infiltration. The spermatogenic cells presented variable changes characterized by the disorientation of cells, maturation arrest, and premature sloughing. No immune complex deposits were seen. Of the 9 cases followed, pregnancy occurred in 2 following vas reanastomosis; a regenerative capacity of the ST was seen in 6 cases. The follow-up studies also show that the morphological alterations initially produced by the vasectomy have little affect on the appearance of spermatogenesis after vas reanastomosis. Thus, we see that the vasectomy exerts its major effect on the SC and that the damage to these cells alters the testicular environment. However, these changes are apparently reversible following vasovasostomy.^ieng

Taxonomic and gene-centric metagenomics of the fecal microbiome of low and high feed conversion ratio (FCR) broilers.

Individual weight gain in broiler growers appears to vary, which may in part be due to variation in their gut microbiota. In this paper we analyse the fecal microbiota of low and high feed conversion ratio (FCR) broilers. After shotgun sequencing of the fecal microbiome, we used the SEED database to identify the microbial diversity and metabolic potential in low and high FCR birds. The domain-level breakdown of our samples was bacteria (>95 %), eukaryotes (>2 %), archaea (>0.2 %), and viruses (>0.2 %). At the phylum level, Proteobacteria (78.83 % in low and 52.04 % in high FCR), Firmicutes (11.97 % in low and 27.53 % in high FCR) and Bacteroidetes (7.10 % in low FCR and 17.53 % in high FCR) predominated in the fecal microbial community. Poultry fecal metagenomes revealed the sequences related to 33 genera in both low and high FCR with significantly different proportion. Functional analysis revealed that genes for the metabolism of carbohydrates, amino acids and derivatives and protein metabolism were most abundant in SEED subsystem in both samples. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Indeed, genes associated with sulphur assimilation, flagellum and flagellar motility were over represented in low FCR birds. This information could help in developing strategies to improve feed efficiency and feed formulation for broiler chickens.

High potential source for biomass degradation enzyme discovery and environmental aspects revealed through metagenomics of Indian buffalo rumen.

The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

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.

Metagenomic analysis of buffalo rumen microbiome: Effect of roughage diet on Dormancy and Sporulation genes.

Buffalo rumen microbiome experiences a variety of diet stress and represents reservoir of Dormancy and Sporulation genes. However, the information on genomic responses to such conditions is very limited. The Ion Torrent PGM next generation sequencing technology was used to characterize general microbial diversity and the repertoire of microbial genes present, including genes associated with Dormancy and Sporulation in Mehsani buffalo rumen metagenome. The research findings revealed the abundance of bacteria at the domain level and presence of Dormancy and Sporulation genes which were predominantly associated with the Clostridia and Bacilli taxa belonging to the phyla Firmicutes. Genes associated with Sporulation cluster and Sporulation orphans were increased from 50% to 100% roughage treatment, thereby promoting sporulation all along the treatments. The spore germination is observed to be the highest in the 75% roughage treatment both in the liquid and solid rumen fraction samples with respect to the decrease in the values of the genes associated with spore core dehydration, thereby facilitating spore core hydration which is necessary for spore germination.

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.

Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis).

A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.

Seminal gold content in healthy fertile men in India.

OBJECTIVE: Since centuries Ayurveda, mentions the role of gold in the treatment of male infertility 'Swarna Bhasma' (Ash of gold) has been used with good results by Ayurvedic practitioners in the treatment of infertility. Hence, a study was planned to estimate gold in whole semen by atomic absorption spectrophotometry. MATERIALS AND METHODS: Whole semen from 11 healthy males of proved fertility from Lucknow (India) was analyzed for gold content by Atomic Absorption spectrophotometry at wavelength 242.8 nm with Hollow Cathode Gold Lamp. Prior to analysis, all the samples were subjected to digestion procedure, achieved by treating them with mixture of concentrated Nitric acid and concentrated Perchloric acid in 6: 1 ratio. OBSERVATION: On analysis all semen samples were found to contain gold ranging from 0.36 to 1.98 µg/ml with a mean value of 0.88 µg/ml and a standard deviation of 0.51 µg/ml. CONCLUSION: In the present study, gold was estimated after complete digestion (oxidation of organic matters; hence, whatever amount of gold detected, denotes the levels in seminal plasma as well as the sperm itself) in whole semen (seminal plasma and sperm). It seems that the hypothesis made for presence of gold in sperm might be true. However, the literature available in this connection is very scanty and further studies are needed for scientific documentation of gold in male infertility.

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.