Establishment of primary chicken embryo myoblast cell culture, antigenic epitopes prediction and production of anti activin receptor type IIB polyclonal antibody in chicken.

The detection of activin receptor typeIIB (ACTRIIB) protein, a prominent negative muscle growth regulator has paramount value in augmenting growth traits through molecular breeding schemes in chicken. The study was formulated to establish primary chicken embryo myoblast culture (CEM) using 9th and 18th day chick embryos and to develop antibodies for immunodetection of ACTRIIB protein. The physicochemical and structural attributes of the ACTRIIB sequence were evaluated to identify substantial antigenic regions. The ACTRIIB sequence was transfected into CEM and expressed protein was injected subcutaneously into rats to produce hyperimmune serum. The average propensity of protein sequence for beta turns, surface accessibility, chain flexibility, antigenicity, hydrophilicity and linear epitopes was 0.978, 1.000, 0.991, 1.038, 1.258 and 0.512, respectively. The 9th day CEM exhibited confluency (80-90%) earlier than the 18th day. The expression of myogenic regulatory factors in 9th day myoblasts was higher than the 18th day by 7.28, 5.16, 6.28 and 6.93 folds for MYF5, MRF4, MYOG and MYOD, respectively. The ACTRIIB mRNA was downregulated by 2.54 folds on the 9th day compared to the 18th day myoblasts and protein varied significantly between 9th and 18th day myoblasts. The CEM culture can be harnessed unequivocally to investigate molecular mechanisms underlying muscle growth besides raising antibodies.

Genetic polymorphism in core promoter sequence of ACTRIIB gene and association analysis with growth traits in chicken.

Molecular breeding exploiting candidate genes is burgeoning reproductive approach to improve growth traits in poultry. The activin type IIB receptor (ACTRIIB) is a negative growth regulator, modulating action of many muscle growth regulators. PCR-single-strand conformation polymorphism was employed to unravel polymorphism in promoter region of the ACTRIIB gene and delineate its association with growth traits in Aseel and control broiler (CB). Analysis of 5' promoter region (1122bp) of ACTRIIB gene identified five SNPs, that is g. [56 G > C (SNP1), 352A > C (SNP2), 580G > A (SNP3), 625C > T (SNP4) and 962C > T (SNP5)] at SMAD, paired box 7 homeodomain binding motif, GC box and bHLH-PAS type transcription factors in CB and Aseel. CB had significantly higher body weight (BW) and average daily gain (ADG) at all SNP sites, except at SNP 1. The haplotype construction resulted 8 haplotypes in CB and Aseel population. The BW and ADG differed significantly (p < .05) at all ages in CB and Aseel. The diplotypes H1H8 and H1H4 manifested higher BW and ADG, while diplotypes H3H8 and H3H7 displayed BW and ADG at each age in both lines (p < .05). Aseel exhibited higher expression of ACTRIIB gene than CB by 70.17, 4.83, 1.41, 2.38, 5.13, 1.20, 2.90, 6.53 and 11.75 times for h1h2, h1h3, h1h4, h1h6, h1h7, h1h8 h3h4, h3h7 and h3h8, respectively. The H3H8 and H3H7 diplotypes exhibited higher level of mRNA and protein than H1H8 and H1H4. The regulatory upstream region of ACTRIIB gene demonstrates high degree of genetic diversity and can be harnessed as potential marker in genetic selection programmes for increasing meat production.

Molecular characterization and expression profiling of BMP 3 gene in broiler and layer chicken.

A study was carried out to characterize and explore the expression profile of BMP 3 gene in control broiler and control layer chicken. The total open reading frame of BMP 3 (1389 bp) was cloned and sequenced. The control broiler and control layer chicken showed variation at nucleotide and amino acid level with reference gene (Gallus gallus, NCBI Acc. No. NM_001034819). When compared to reference gene, the control broiler showed four nucleotide differences (c.192A>G, c.519C>T, 903G>A and 960C>G), while, control layer showed variation at c.33G>C, 192A>G, 858G>A, 904G>A, 960C>G and 1257C>T making six differences in total. However, between control broiler and control layer lines, nucleotide differences was observed at c.33G>C, 519T>C, 858G>A, 903A>G, 904G>A and 1257C>T. The change at amino acid level between reference and control broiler was p.D320N and with control layer chicken, it was p.D302N and p.D320N. On the other hand, a single amino acid difference (p.D302N) was observed between the control broiler and control layer chicken lines. The phylogenetic study displayed a close relationship between broiler and layer lines and reference gene and also with other avian species resulting in a cluster formation. These cluster in turn displayed a distant link with the mammalian species. The expression profile of BMP 3 gene exhibited a variation at different stages of embryonic development and also at post embryonic period among the lines with control layer showing higher expression than that of broiler chicken. The protein was also detected in bone marrow tissue of broiler and layer lines by western blotting. It is concluded that the BMP 3 gene sequence differed at nucleotide and amino acid level among the lines and the gene expressed differentially at different periods of embryonic development and also at post hatch period.