Milk somatic cell DNA isolation and characterization of κ-casein gene in Halari donkey milk.

Halari donkey breed is one of the indigenous breeds of India and its population is rapidly decreasing. The Jenny milk is more similar to human milk, exhibits a wide range of probiotic diversity and hypo-allergenicity, especially among infants suffering from cow and buffalo milk protein allergy. Some studies indicated low levels of κ-casein fraction of casein protein in donkey milk. The k-casein gene was not amplified from the DNA derived from the milk somatic cells of Halari donkeys. The Halari donkey milk has low somatic cells count. We report the first isolation of DNA from milk somatic cells of Halari donkeys with subsequent characterization of k-casein gene. Through our work, we showed that the milk somatic cells can be used as a non-invasive source for DNA isolation towards molecular studies. It also proved the presence of k-casein gene in Halari donkey milk by its amplification from isolated DNA.

Indian Red Jungle fowl reveals a genetic relationship with South East Asian Red Jungle fowl and Indian native chicken breeds as evidenced through whole mitochondrial genome sequences.

Background: Native chickens are dispersed in a wide geographical range and have hereditary assets that are kept by farmers for various purposes. Mitochondrial DNA (mtDNA) is a widely utilized marker in molecular studies because of its quick advancement, matrilineal legacy, and simple molecular structure. Method and Results: We performed NGS sequencing to investigate mitochondrial genomes and to evaluate the hereditary connections, diversity, and measure of gene stream estimation in Indian native chicken breeds and Red Jungle fowl. The chicken breeds were genotyped using the D-loop region and 23 haplotypes were identified. When compared to Indian native breeds, more haplotypes were identified in the NADH dehydrogenase subunits, Cytochrome c oxidase, Cytochrome b, ATP synthase subunit 6, and Ribosomal RNA genes. The phylogenetic examination indicated that the analyzed chicken breeds were divided into six significant clades, namely A, B, C, D, E, and F, of which the F clade indicated the domestication of chicken breeds in India. Additionally, our work affirmed that the Indian Red Jungle Fowl is the origin for both reference Red Jungle Fowl as well as all Indian breeds, which is reflected in the dendrogram as well as network analysis based on the whole mtDNA and D-loop region. Indian Red Jungle Fowl is distributed as an outgroup, suggesting that this ancestry was reciprocally monophyletic. Conclusion: The mtDNA sequences of Indian native chickens provided novel insights into adaptation mechanisms and the significance of important mtDNA variations in understanding the maternal lineages of native birds.

Embryonic transcriptome unravels mechanisms and pathways underlying embryonic development with respect to muscle growth, egg production, and plumage formation in native and broiler chickens.

Background: Muscle development, egg production, and plumage colors are different between native and broiler chickens. The study was designed to investigate why improved Aseel (PD4) is colorful, stronger, and grew slowly compared with the control broiler (CB). Methods: A microarray was conducted using the 7th-day embryo (7EB) and 18th-day thigh muscle (18TM) of improved Aseel and broiler, respectively. Also, we have selected 24 Gallus gallus candidate reference genes from NCBI, and total RNA was isolated from the broiler, improved Aseel embryo tissues, and their expression profiles were studied by real-time quantitative PCR (qPCR). Furthermore, microarray data were validated with qPCR using improved Aseel and broiler embryo tissues. Results: In the differential transcripts screening, all the transcripts obtained by microarray of slow and fast growth groups were screened by fold change ≥ 1 and false discovery rate (FDR) ≤ 0.05. In total, 8,069 transcripts were differentially expressed between the 7EB and 18TM of PD4 compared to the CB. A further analysis showed that a high number of transcripts are differentially regulated in the 7EB of PD4 (6,896) and fewer transcripts are differentially regulated (1,173) in the 18TM of PD4 compared to the CB. On the 7th- and 18th-day PD4 embryos, 3,890, 3,006, 745, and 428 transcripts were up- and downregulated, respectively. The commonly up- and downregulated transcripts are 91 and 44 between the 7th- and 18th-day of embryos. In addition, the best housekeeping gene was identified. Furthermore, we validated the differentially expressed genes (DEGs) related to muscle growth, myostatin signaling and development, and fatty acid metabolism genes in PD4 and CB embryo tissues by qPCR, and the results correlated with microarray expression data. Conclusion: Our study identified DEGs that regulate the myostatin signaling and differentiation pathway; glycolysis and gluconeogenesis; fatty acid metabolism; Jak-STAT, mTOR, and TGF-ß signaling pathways; tryptophan metabolism; and PI3K-Akt signaling pathways in PD4. The results revealed that the gene expression architecture is present in the improved Aseel exhibiting embryo growth that will help improve muscle development, differentiation, egg production, protein synthesis, and plumage formation in PD4 native chickens. Our findings may be used as a model for improving the growth in Aseel as well as optimizing the growth in the broiler.

Silencing acetyl-CoA carboxylase A and sterol regulatory element-binding protein 1 genes through RNAi reduce serum and egg cholesterol in chicken.

Cholesterol is synthesized in chicken through de novo lipid biosynthetic pathway where two most important genes viz. SREBP1 and ACACA play immense role. To minimize cholesterol synthesis, RNAi approach was adopted and accordingly, we developed transgenic chicken possessing ACACA and SREBP1 shRNA constructs, which showed lower level of ACACA and SREBP1 in serum. The serum total cholesterol, triglycerides, HDL and LDL cholesterol was significantly lower by 23.8, 35.6, 26.6 and 20.9%, respectively in SREBP1 transgenic birds compared to the control. The egg total cholesterol and LDL cholesterol content was numerically lower in both ACACA and SREBP1 transgenic birds by 14.3 and 13.2%, and 10.4 and 13.7%, respectively compared to the control. It is concluded that the protocol was perfected to develop transgenic chicken through RNAi for knocking down the expression of ACACA and SREBP1 proteins, which minimized the cholesterol and triglycerides contents in serum and eggs.

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.

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.

Effect of Supplementation of Fermented Yeast Culture on Hormones and Their Receptors on Exposure to Higher Temperature and on Production Performance after Exposure in Nicobari Chickens.

Heat stress (HS) affects the production performance in chickens and causes economic loss to the producers. Most of the studies have been conducted on and for the welfare of broilers. We still lack information on the physiological parameters being affected during chronic heat stress in layers. To fill this gap, the present study evaluated the effect of heat stress (induced in the chamber) during the prelaying period (21-23 weeks) on plasma levels of the hormones leptin and ghrelin and GH and expression of the respective receptors and heat stress markers. Three groups were considered, one at room temperature (CR) and the other two groups (SH and CH) subjected to heat stress at 39°C for four hours for three weeks (21-23 weeks of age). The SH group (SH) feed was supplemented with fermented yeast culture (FYC, 700 mg/kg), whereas the CH group was devoid of it. After that, all the groups were shifted to shed under natural ambient conditions till 31 weeks of age. Studies were restricted to production performance only. Feed offered without yeast culture (CH group) had a smaller concentration of plasma hormones (P < 0.01) and increased expression fold of the hormone receptors (P < 0.01). Further, the group also presented higher liver AMP kinase enzyme, plasma MDA (malondialdehyde), and cholesterol concentrations. These changes likely explained the decrease in feed intake and the CH group's body weight and further reduced the production performance during the laying period. Supplementation with FYC to birds had an opposite effect on the above-mentioned parameters, reducing HS effects. In summary, supplementation with FYC (700 mg/kg) maintained physiological parameters as in the CR group under HS conditions and negated adverse effects on parameters both before and during laying periods.

Cloning, characterisation and expression of the SERPINB14 gene, and association of promoter polymorphisms with egg quality traits in layer chicken.

1. Ovalbumin (SERPINB14) is the most abundant protein present in egg white contributing about 54% of the total egg protein. In this study, the objectives were to clone and characterise the coding sequence of the SERPINB14 gene, to explore its expression profile, identify polymorphisms in the promoter of the gene and explore any association with egg quality traits in White Leghorn chickens.2. SNPs and mRNA expression of SERPINB14 in White Leghorn chicken lines were detected by PCR-single strand conformation polymorphism (SSCP) along with sequencing and qPCR. The open reading frame (ORF) was cloned in an expression plasmid vector and sequenced.3. The ORF of this gene was 1161 bp encoding a peptide of 386 amino acids. There were three SNPs observed in the coding region of the gene, one of which was of the mis-sense type, having c562G>A transition which resulted in substitution of alanine to threonine at position 188 in the protein sequence. In both the lines, an increase in expression of the gene was observed after onset of egg production with peak expression at the 40th week of age compared to before onset of lay. The SERPINB14 gene was expressed in the magnum, but not in ovary and infundibulum, tissues of each White Leghorn line. The promoter region of the gene showed SNPs with three haplotypes; H1, H2, and H3. The haplo groups were associated with the egg weight and age at sexual maturity in the IWI line and Haugh unit and albumin index in the IWK line.4. It was concluded that the ORF of SERPINB14 gene in White Leghorn chicken lines is polymorphic. The promoter region of the gene is also polymorphic and has significant (P < 0.05) association with Haugh unit and egg weight in IWK and IWI chicken lines, respectively.

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.

Comparative analysis of silencing expression of myostatin (MSTN) and its two receptors (ACVR2A and ACVR2B) genes affecting growth traits in knock down chicken.

Myostatin (MSTN), a growth differentiation factor-8 regulates muscular development through its receptors, ACVR2A (Activin receptor type IIA) and ACVR2B (Activin receptor type IIB) by inhibiting cellular differentiation of developing somites during embryonic stage and diminishing myofibriller growth during post-embryonic period. The objective of this study was to compare the effect of knockdown of expression of myostatin, ACVR2A and ACVR2B genes on growth traits in chicken. The shRNAs for Myostatin, ACVR2A and ACVR2B genes were designed, synthesized and cloned in DEST vector. The recombinant molecules were transfected into the spermatozoa and transfected spermatozoa were inseminated artificially to the hens to obtain fertile eggs. The fertile eggs were collected, incubated in the incubator and hatched to chicks. Silencing of ACVR2B gene showed significantly higher body weight than other single, double and triple knock down of genes in transgenic birds. The carcass traits such as dressing%, leg muscle%, and breast muscle% were found with the highest magnitudes in birds with silencing of the ACVR2B gene as compared to the birds with that of other genes and control group. The lowest serum cholesterol and HDL content was found in ACVR2B silencing birds. The total RBC count was the highest in this group though the differential counts did not differ significantly among various silencing and control groups of birds. It is concluded that silencing of only one receptor of MSTN particularly, ACVR2B may augment the highest growth in chicken during juvenile stage. Our findings may be used as model for improving growth in other food animals and repairing muscular degenerative disorders in human and other animals.

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.

Polymorphisms in GnRHI and GnRHII genes and their association with egg production and egg quality traits in chicken.

1. Two candidate genes, namely, Gonadotropin releasing hormone I (GnRHI) and Gonadotropin releasing hormone II (GnRHII) play pivotal roles in ovulation and egg production in chicken. The objective of this study was to explore polymorphism in these genes and to estimate the effects of polymorphism of these two genes on egg production and egg quality traits in White Leghorn laying hens. 2. Single strand conformation polymorphism followed by sequencing was performed to detect polymorphism in these genes. 3. The coding regions of the GnRHI and GnRHII genes were found to be polymorphic. In the GnRH1 gene, 12 haplotypes were determined, of which the h1 haplotype was predominant and the h5, h9 and h11 haplotypes were the least frequent ones. In the GnRHII gene, eight haplotypes were found, of which the h1 haplotype was the most frequent and the h6 was the least frequent haplotype in the White Leghorn population. 4. The haplogroups of GnRHI had a significant effect on body weight and egg production up to 64 weeks of age, yolk content, Haugh units and egg shell parameters. The h1h2 haplogroup of the GnRHI gene showed the highest egg production, with 211.0 ± 24.3 eggs up to 64 weeks of age, while the highest yolk content and Haugh unit was found in h3h10 haplogrouped birds. The haplogroups of GnRHII had a significant effect on age at sexual maturity (ASM) where the shortest ASM was found in the h1h4 birds (147.3 ± 5.9 d) and the longest ASM was observed in the h1h3 birds (160.6 ± 23.4 d). 5. It was concluded that GnRHI and GnRHII genes are polymorphic and have a significant effect on body weight, egg production and egg quality traits in White Leghorn laying hens.

In vivo cell-mediated immune, hemagglutination inhibition response, hematological and biochemical values in native vs. exotic chicken breeds.

Birds (364) of both sexes, 11-wk-old, belonging to 2 native (Brown Nicobari and Ghagus) breeds and 1 exotic breed (Dahlem Red) were evaluated for cell-mediated immune response (CMI) by phytohemagglutinin-P (PHA-P), hemagglutination inhibition (HI) assay against Newcastle disease virus (NDV) antigen (LaSota stock virus), flow cytometric analysis of CD8+ cytotoxic T lymphocytes (CTLs), and hematology and biochemical assays. The cutaneous basophil hypersensitivity response PHA-P% increase in wattle thickness (mm) was highest in Ghagus (431.14 ± 22.56) which differed significantly with that of Brown Nicobari (269.1 ± 22.66) and Dahlem Red (218.42 ± 22.30). Sex-wise observation showed that females are having significantly higher response than males. Hemagglutination inhibition test was performed to determine the serum antibodies against Newcastle disease (ND) virus. Brown Nicobari showed highest HI antibody titer than Ghagus and Dahlem Red to similar vaccination program after booster NDV dose. Flow cytometry assay revealed significantly higher CTLs proliferation in Brown Nicobari than Ghagus and Dahlem Red. Moreover, CTLs were found to be higher in control group than the treatment group. Other hematological parameters (103/µL) significant difference was found in white blood cell count between Dahlem Red (38.41 ± 1.03) with that of Brown Nicobari (35.28 ± 1.04) and Ghagus (34.57 ± 1.04) in treatment groups. Same trend was observed in the Lymphocyte treatment group. However, in Granulocyte treatment group, Brown Nicobari (11.04 ± 0.35) was found to be significantly different from Dahlem Red (8.68 ± 0.34) and Ghagus (9.27 ± 0.35). Correlations between body weight at 11 wk of age and CMI, HI, cytotoxic T cell were -0.093, 0.047, and -0.036, respectively. Egg weight was found to be positively correlated with that of chick weight. Serum biochemical values showed that Dahlem Red was having significantly higher creatinine levels compared to Ghagus. Triglycerides level was also significantly higher in Ghagus compared to Dahlem Red. No significant breed effect was observed for alkaline phosphate, aspartate transaminase, and alanine transaminase. Cholesterol and total serum protein levels were significantly higher in Dahlem Red compared to Brown Nicobari.

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.

Polymorphism of the myostatin gene and its association with growth traits in chicken.

An experiment was carried out on myostatin gene with the objectives of identification of polymorphism in the myostatin gene and estimation of the effect of polymorphism on growth traits in chickens. Single-stranded conformation polymorphism followed by sequencing was performed to reveal polymorphism of the gene. A total of 13 haplotypes were observed across 3 chicken lines (PB-1 and CB as broiler lines and IWI as the layer line). Myostatin haplogroups had a significant effect on BW at 28, 42, and 49 d of age in the PB-1 line. The significant association of haplogroups was observed with BW at d 14 and 49 in the CB line. In the IWI layer line, the myostatin gene was polymorphic but had no significant association with growth traits. It is concluded that the myostatin gene was polymorphic and had a significant effect on growth traits in broiler chickens.

Chronic D-amphetamine administered from childhood to adulthood dose-dependently increases the survival of new neurons in the hippocampus of male C57BL/6J mice.

Adderall is widely prescribed for attention deficit hyperactivity disorder (ADHD) though long term neurological effects of the main ingredient d-amphetamine are not well understood. The purpose of this study was to examine effects of clinically prescribed doses of d-amphetamine and one abuse dose administered from childhood to adulthood on adult hippocampal neurogenesis and activation of the granule layer of the dentate gyrus. Beginning in early adolescence (age 28 days) to adulthood (age 71), male C57BL/6J mice were administered twice daily i.p. injections of vehicle, 0.25, 0.5 or 2mg/kg d-amphetamine. Locomotor activity was measured in home cages by video tracking. At age 53-56, mice received bromodeoxyuridine (BrdU) injections to label dividing cells. Immunohistochemical detection of BrdU, neuronal nuclear protein (NeuN), doublecortin (DCX) and Ki67 was used to measure neurogenesis and cell proliferation at age 71. ΔFosB was measured as an indicator of repeated neuronal activation. An additional cohort of mice was treated similarly except euthanized at age 58 to measure activation of granule neurons from d-amphetamine (by detection of c-Fos) and cell proliferation (Ki67) at a time when the fate of BrdU cells would have been determined in the first cohort. d-Amphetamine dose-dependently increased survival and differentiation of BrdU cells into neurons and increased number of DCX cells without affecting the number of Ki67 cells. Low doses of d-amphetamine decreased c-Fos and ΔFosB in the granule layer. Only the high dose induced substantial locomotor stimulation and sensitization. Results suggest both therapeutic and abuse doses of d-amphetamine increase the number of new neurons in the hippocampus when administered from adolescence to adulthood by increasing survival and differentiation of cells into neurons not by increasing progenitor cell proliferation. Mechanisms for amphetamine-induced neurogenesis are unknown but appear activity independent. Results suggest part of the beneficial effects of therapeutic doses of d-amphetamine for ADHD could be via increased hippocampal neurogenesis.