Expression and polymorphism of Follistatin (FST) gene and its association with growth traits in native and exotic chicken.

Follistatin (FST), a member of the transforming growth factor beta super-family regulates body growth by inhibiting the binding of myostatin (an inhibitor of growth) with its receptor in chicken. An experiment was conducted to explore ontogenic expression of the follistatin gene, determine polymorphism at the coding region of the gene and estimate its effect on growth traits in native (Aseel) and exotic broiler (PD-1) and layer (White Leghorn) chicken. The significant differences of FST gene expression were observed among the breeds revealing significantly (p < 0.05) higher expression in PD-1 line followed by White Leghorn and Aseel breeds during both embryonic and post-hatch period. The polymorphism at the functional domain of the FST gene was identified with the presence of 4 haplotypes. The follistatin haplogroups had the significant effect on body weights (p < 0.05) at 42 days of age in the White Leghorn, PD-1 and Aseel breeds (h1h1 in PD-1, h1h4 in White Leghorn and h1h2 haplogroups in Aseel breeds had the highest body weights of 770.04 ± 12.96, 246.28 ± 7.60 and 270.00 ± 10.68 g, respectively). It is concluded that the follistatin gene expressed differently during the embryonic and post-embryonic period across the breeds and the coding region of the gene was polymorphic having significant effects on growth traits in chicken.

Molecular characterization and computational structure prediction of activin receptor type IIB in aseel and broiler chicken.

The present study was formulated to characterize and comprehend the molecular structural characteristics of ACTRIIB receptor in Aseel and control broiler (CB) populations. The full length coding sequence (1539 bp) of the receptor was amplified, cloned, sequenced and analyzed using bioinformatic tools. The physico chemical properties of protein and structural features like secondary structure, solvent accessibility and disorder regions were computed. The 3D structure was predicted by I-TASSER and evaluated by Ramachandran Plot and tools under Structural Analysis and Verification Server. The nucleotides differences between CB and Aseel were c. [156G > A; 210 T > C; 493C > T; c.520G > C; 665A > C; 686G > A; 937C > G; 1011A > C; 1130A > G; 1208 T > A; 1326 T > C; 1433 T > C]. The amino acid substitutions between CB and Aseel were p. [(Pro165Ser; Glu174Gln; Gln222Pro; Ser229Asn; His313Asp; Gln377Arg; Val403Asp; and Ile478Thr)]. While, the silent changes includes p. [(Lys53=; Glu71=; Leu337=; Asp442=)]. The molecular weight of mature protein was predicted to be 55.51 kDa and 57.80 kDa in Aseel and CB, respectively. The higher rank 3D model had a C-score of -1.60 in Aseel and - 1.41 in CB, while the estimated TM-score (0.54 ±â€¯0.14) and RMSD (5.8 ±â€¯1.2 Å) were found to be similar in Aseel and CB. Among the 512 residues, >90% were in favored region, 4.7% in allowed region and <1.5% in disallowed region in both Aseel and CB. The pattern of contact map was comparable in Aseel and CB. The Hydrogen bond plots of the Aseel and CB shared similar secondary structure pattern. The ACTRIIB protein was predicted to interact with ACVR1B, ACVR1C, INHBA, SMAD 1,2,5,7 & 9 and BMPR1A&B. Clustal and phylogenetic analysis implied that both the lines were closely related and formed a sub cluster with in avian cluster. The current research provides insights about structural and functional aspects of the receptor and also aids in understanding the evolutionary history of ACTRIIB.

In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference.

Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔGoverall, ΔGduplex, ΔGbreak-target, ΔGintra-oligomer, ΔGinter-oligomer and ΔΔGends). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.

Knock down of the myostatin gene by RNA interference increased body weight in chicken.

Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (P<0.05) between knock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken.

Gene expression and silencing of activin receptor type 2A (ACVR2A) in myoblast cells of chicken.

Activin receptor type 2A (ACVR2A) acts as receptor for myostatin (MSTN) protein involved in inhibiting satellite cell proliferation and differentiation. The importance of the ACVR2A gene during embryonic and post-hatch periods in broiler and layer chicken was studied in an in vitro cell culture system. The expression pattern of the ACVR2A gene during embryonic stages was similar in broiler and layer lines. Post-hatch expression of the ACVR2A gene varied significantly between broiler and layer lines. Five shRNA molecules were designed to knockdown expression of the ACVR2A gene in chicken myoblast cells. The silencing of the ACVR2A gene in a cell culture system varied from 60% to 82%. It is concluded that between broiler and layer lines, there were no significant changes in expression of the ACVR2A gene during embryonic stages but it varied significantly during the post-hatch period. The shRNA showed silencing of the ACVR2A gene under an in vitro cell culture system.

Gene Expression and Polymorphism of Myostatin Gene and its Association with Growth Traits in Chicken.

Myostatin is a member of TGF-ß super family and is directly involved in regulation of body growth through limiting muscular growth. A study was carried out in three chicken lines to identify the polymorphism in the coding region of the myostatin gene through SSCP and DNA sequencing. A total of 12 haplotypes were observed in myostatin coding region of chicken. Significant associations between haplogroups with body weight at day 1, 14, 28, and 42 days, and carcass traits at 42 days were observed across the lines. It is concluded that the coding region of myostatin gene was polymorphic, with varied levels of expression among lines and had significant effects on growth traits. The expression of MSTN gene varied during embryonic and post hatch development stage.

Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken.

1. The objectives of the study were to detect polymorphism in the coding region of the IGF1 gene, explore the expression profile and estimate association with growth traits in indigenous and exotic chickens. 2. A total of 12 haplotypes were found in Cornish, control layer and Aseel breeds of chicken in which the h1 haplotype was most frequent. 3. Nucleotide substitutions among haplotypes were found at 21 positions in the IGF1 gene in which 4 substitutions resulted in non-synonymous mutations in the receptor binding domain of the IGF1 protein. 4. The haplogroup showed a significant effect on body weight at 24 and 42 d of age in the control layer line, body weight at 42 d and daily weight gain between 29 and 42 d in the control broiler line, daily weight gain between 29 and 42 d in Cornish, and body weights at 42 d as well as daily weight gain between 29 and 42 d in Aseel birds. 5. IGF1 expression varied among the breeds during embryonic and post-hatch periods. The expression among the haplogroups varied in different chicken tissues. The effect of haplogroup on myofibre number in pectoral muscle was non-significant, although there was significant variation in numbers between d 1 and d 42, and between broiler and layer lines. 6. It was concluded that the coding region of the IGF1 gene was polymorphic, expressed differentially during the pre-hatch and post-hatch periods, and haplogroups showed significant association with growth traits in chicken.

Cloning, characterization and expression of myostatin (growth differentiating factor-8) gene in broiler and layer chicken (Gallus gallus).

A study was conducted to characterize myostatin gene in broiler and layer chicken and to explore mRNA expression profile in these two varieties of chicken. The myostatin cDNAs of broiler and layer varieties were cloned and sequenced. The total length of the cDNA was 1128 bp. The differences of nucleotides between PB-1 broiler and IWI layer were C > 65 > T, C > 306 > T and C > 1094 > T while those between CB broiler and IWI layer were C > 65 > T, C > 195 > G, G > 234 > A, C > 306 > T, T > 939 > C, C > 961 > T, G > 966T and C > 1094 > T. The amino acid differences of myostatin protein between PB-1 and IWI strains were alanine > 22 > valine and proline > 365 > leucine while those between CB and IWI strains were alanine > 22 > valine, histidine > 321 > tyrosine and proline > 365 > leucine. The phylogenetic study revealed closeness of PB-1 and control broiler forming a cluster, which was also closely related to IWI layer chicken formed a separate cluster. The gene was cloned and expressed in E. coli. The gene expression profile in muscle was different between broiler and layer strains. Between two broiler strains, the pattern of expression was similar. Between IWI layer and either PB-1 or CB broilers, differences in expression was found at different time points, particularly at second, fourth and seventh weeks of age. The myostatin expression was significantly associated with body weights in chicken. It is concluded that myostatin gene sequences varied at nucleotide as well as amino acid level between broiler and layer chicken varieties and the gene also expressed differentially in these two varieties.

A quantitative kinetic study of polysorbate autoxidation: the role of unsaturated fatty acid ester substituents.

PURPOSE: To study the role of unsaturated fatty acid ester substituents in the autoxidation of polysorbate 80 using quantitative kinetics. METHODS: Oxidation kinetics were monitored at 40 degrees C in aqueous solution by tracking head space oxygen consumption using a fiber optic oxygen sensor with phase shift fluorescence detection. Radical chain initiation was controlled using an azo-initiator and assessed by Hammond's inhibitor approach, allowing oxidizability constants (k(p)/(2k(t))(1/2)) to be isolated. Reaction orders were determined using modified van't Hoff plots and mixed polysorbate micelles. RESULTS: The oxidizability constant of polysorbate 80 ((1.07 +/- 0.19) x 10(-2) M(-1/2) s(-1/2)) was found to be 2.65 times greater than polysorbate 20 ((0.404 +/- 0.080) x 10(-2) M(-1/2) s(-1/2)). The additional reactivity of polysorbate 80 was isolated and was first-order in the unsaturated fatty acid ester substituents, indicating that the bulk of the autoxidative chain propagation is due to these groups. This data, and the observation of a half-order dependence on the azo-initiator, is consistent with the classical autoxidation rate law (-d[O(2)]/dt = k(p)[RH](R(i)/2k (t))(1/2)). CONCLUSIONS: Polysorbate 80 autoxidation follows the classical rate law and is largely dependent on the unsaturated fatty acid ester substituents. Clarification of the substituents' roles will aid formulators in the selection of appropriate polysorbates to minimize oxidative liabilities.