Relative gene expression of fatty acid synthesis genes at 60 days postpartum in bovine mammary epithelial cells of Surti and Jafarabadi buffaloes.

AIM: Aim of the study was to study the relative gene expression of genes associated with fatty acid synthesis at 60 days postpartum (pp) in bovine mammary epithelial cells (MECs) of Surti and Jafarabadi buffaloes. MATERIALS AND METHODS: A total of 10 healthy Surti and Jafarabadi buffaloes of each breed were selected at random from Livestock Research Station, Navsari and Cattle Breeding Farm, Junagadh, Gujarat, respectively, for this study. Milk sample was collected from each selected buffalo at day 60 pp from these two breeds to study relative gene expression of major milk fat genes using non-invasive approach of obtaining primary bovine MECs (pBMEC) from milk samples. RESULTS: In this study overall, the relative expression of the six major milk lipogenic genes butyrophilin subfamily 1 member A1 (BTN1A1), stearoyl-CoA desaturase (SCD), lipoprotein lipase (LPL), glycerol-3-phosphate acyltransferase mitochondrial (GPAM), acetyl-coenzyme A carboxylase alpha (ACACA), and lipin (LPIN) did not show changes in expression patterns at 60th day of lactation in both Surti and Jafarabadi buffaloes. CONCLUSION: The pBMEC can be successfully recovered from 1500 ml of milk of Surti and Jafarabadi buffaloes using antibody-mediated magnetic bead separation and can be further used for recovering RNA for down step quantification of major milk lipogenic gene expression. The relative expression of the six major milk lipogenic genes BTN1A1, SCD, LPL, GPAM, ACACA, and LPIN did not show changes in expression patterns in both Surti and Jafarabadi buffaloes, suggesting expression levels of lipogenic genes are maintained almost uniform till peak lactation without any significant difference.

Short hairpin RNA-induced myostatin gene silencing in caprine myoblast cells in vitro.

Myostatin (MSTN) belongs to the transforming growth factor (TGF)-ß superfamily and is a potent negative regulator of skeletal muscle development and growth. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the remarkable muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. We utilized three antisense RNA expressing vectors with six constructs to knockdown MSTN gene in in vitro caprine myoblast cell culture system. We observed that all six shRNA constructs were successful in MSTN silencing with efficiency ranging from 7 to 46 % by quantitative real-time PCR and up to 19 % by western blotting. The significant upregulation of interferon response gene OAS1 (5- to 11-fold) in cells transfected with shRNA constructs were indicative of induction of interferon response. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for improving the production traits and economic properties of livestock.

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells.

Myostatin (MSTN), a member of transforming growth factor-ß (TGF-ß) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-ß) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study.

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.

A preliminary sketch of horn cancer transcriptome in Indian zebu cattle.

Horn cancer, a type of squamous cell carcinoma, in zebu cattle is an expensive affair in Indian agriculture sector, which accounts for 83.34% of total tumors found. In general, cancer tissue confirms considerably different expression patterns when compared to a normal stage. This includes not only up/down regulation, but also, the aberrant gene expression, the presence of different non-coding RNAs (ncRNAs), pseudogenes expression and genes involved in unusual pathways. We employed Roche 454 next generation sequencing platform to sequence Bos indicus cancerous and normal horn tissue transcripts. This resulted into a total of 909,345 high-confidence deep sequencing reads and detected a range of unusual transcriptional events including tumor associated genes. We also validated expression of two of the four tested genes in five other similar tissue samples by RT-qPCR. Further, seven cancer specific non-coding transcripts were accessed and a few of them have been suggested as cancer specific markers. This study for the first time provides primary transcriptome sketch of Bos indicus horn cancer tissue, and also demonstrates the suitability of the 454 sequencer for transcriptome analysis, which supports the concept of varied gene expression in cancerous condition.

Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

Somatotropin-mediated gene expression profiling of differentially displayed ESTs during lactation in Indian buffalo (Bubalus bubalis).

The study of bovine mammary gland functional genomics requires appropriate cDNA library collections to access gene expression patterns from different developmental and physiological stages. The present study was undertaken with the objective to identify candidate genes involved in the process of increased milk synthesis following 0, 48 and 96 h of recombinant bovine somatotropin (rbST) treatment to Surti buffalo (Bubalus bubalis) through differential display reverse transcriptase PCR (DDRT-PCR). Of a total 50 sequenced DD bands, 64% of ESTs were differentially expressed (appeared only in post-treatment samples, i.e. 48 h and 96 h) and 36% were up-regulated after rbST treatment. Of the ESTs 32%were found to be located on Bos taurus chromosome 24 (equivalent to buffalo chromosome 22), whereas 16% of ESTs could not be mapped, indicating that they are specific to buffalo. Quantitative real time PCR assay of 15 ESTs revealed transcript level surge in 13 ESTs, and decline in one EST, while one showed up-regulation in expression level at 48 h while down-regulation at 96 h. This study indicates more than 30 novel transcripts, with unknown function, involved in increased milk synthesis and also the involvement of many more genes in the physiology of milk production than once thought.

Anti-obesity potential of Clerodendron glandulosum.Coleb leaf aqueous extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Clerodendron glandulosum.Coleb leaf aqueous extract (CG) is traditionally used by people of North-East India to alleviate symptoms of diabetes, obesity and hypertension. Previous study from our laboratory have documented anti-diabetic and anti-hypertensive properties of CG extract but, till date there are no pharmacological studies available on its anti-obesity potential. This inventory investigates the underlining molecular mechanism/s of CG induced regulation of in vivo HFD induced obesity and in vitro adipocyte differentiation. AIM: To evaluate effects of CG extract on (i) expression of genes regulating visceral adiposity and (ii) in vitro adipocyte differentiation and LEP release. MATERIALS AND METHODS: Body weight, lee index, plasma lipids and LEP, mRNA expression of PPARγ-2, SREBP1c, FAS, CPT-1 and LEP in epididymal adipose tissue of control and experimental groups were evaluated. Also, potential of CG extract on in vitro adipocyte differentiation and LEP release was assessed. RESULTS: Supplementation of CG extract to HFD fed mice significantly prevented HFD induced increment in bodyweight, lee index, plasma lipids and LEP, visceral adiposity and adipocyte hypertrophy. Also, CG extract supplementation resulted in down regulation of PPARγ-2, SREBP1c, FAS and LEP expression along with up-regulation of CPT-1 in epididymal adipose tissue compared to HFD fed mice. In vitro study recorded significant anti-adipogenic effect of CG extract that resulted in decreased adipogenesis, TG accumulation, LEP release, G3PDH activity along with higher glycerol release without significantly altering viability of 3T3L1 pre-adipocytes. CONCLUSIONS: Clerodendron glandulosum.Coleb extract prevents adipocyte differentiation and visceral adiposity by down regulation of PPARγ-2 related genes and Lep expression thus validating its traditional therapeutic use in controlling obesity.

Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse 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, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.

A modified enrichment protocol for adult caprine skeletal muscle stem cell.

To establish an adequate model to study the proliferation and differentiation of adult caprine skeletal muscle in response to bioactive compounds, a pool of satellite cells (SC) was derived from the rectus abdominis muscle of adult goat. Skeletal muscle contains a population of adult stem cells, named as satellite cells that reside beneath the basal lamina of skeletal muscle fiber and other populations of cells. These SC are multipotent stem cells, since cells cultured in the presence of specific cell lineage inducing cocktails can differentiate into several types of mesenchymal lineage, such as osteocytes and adipocytes. In the present study, we have developed a modified protocol for isolating satellite cells (>90%) and examined their myogenic and contractile properties in vitro.