Progress 03/01/16 to 02/28/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for training and professional development of twoPhD students and threeMS studentsand over 10 undergraduate students. How have the results been disseminated to communities of interest?Results have been disseminated in the form of publications in scientific journals and presentationsatmeetings and invited talks What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Obj1.1. Genome-wide association study of Wooden Breast and White Striping. Our main objectives in this project were to characterize the genetic architecture of Wooden Breast (WB) and to identify and validate candidate genes for this muscle disease. The work involved phenotyping and restriction-enzyme-based genotyping (by sequencing) of 1,193 commercial broiler chickens and applying the state-of-science statistical methods for a genome-wide association study (GWAS) of WB and WS. Chickens were raised to market age at the University of Delaware and scored for the severity of WB and WS at 7 weeks of age. GWAS was conducted in Dr. Abasht's lab, in collaboration with co-PI Dr. Jack Dekkers at Iowa State University, and identified genomic regions significantly associated with WB and WS in commercial broiler chickens. Our GWAS detected 28 quantitative trait loci (QTL) for WB and 6 QTL for White Striping (WS). Notably, the majority of QTLs we identified for WB and all QTLs for WS were located in an 8.3-Mb region on chicken chromosome 5 where there is highly conserved synteny between the chicken chromosome 5 and human chromosome 11. This region on human chromosome 11 is associated with numerous lipid-metabolism related metabolic and growth disorders and various forms of diabetes mellitus. For example, KCNQ1 locus in this region is part of an imprinted gene cluster that when dysregulated can cause metabolic and overgrowth disorders including Beckwith-Wiedemann syndrome. This gene encodes a highly-studied voltage-gated potassium channel, which interacts with numerous other protein subunits and switches from voltage-dependent to constitutive activity. Some pleiotropic KCNQ1 variants in humans can simultaneously increase insulin secretion in the pancreas, reduce serum potassium upon oral glucose challenge, and cause long QT syndrome, putting individuals at risk of sudden, uncontrollable, arrhythmias which may lead to fainting or sudden death. This could represent a potential cause of sudden cardiac death reported in broilers, especially those with wooden breast. We re-examined data from our study of differential gene expression associated with wooden breast in 2-week-old female broilers and found that, although there was no differential expression of KCNQ1 in the pectoralis major of 2-week-old birds, expression levels of the gene were very low in all birds. It is therefore likely that genetic variation in KCNQ1 would primarily affect organs other than the pectoralis major, potentially including the pancreas. Please refer to (Lake et al. 2021. Scientific Reports. 24;11(1):6785) for full list of candidate genes and discussion on their clinical significance. Heritability was estimated at 0.5 for WB and WS, with high genetic correlation between them (0.88). Moderate heritability for wooden breast and white striping indicates that both genetic and environmental factors exert strong influences on phenotypic differences for these traits, which is consistent with studies that have demonstrated the ability to reduce severity and incidence of breast muscle myopathies through both genetic selection and manipulation of dietary energy. Overall, In addition to advancing our understanding of genetic basis of WB and WS, this study provided critical information for practical applications in broiler breeding programs. Obj1.2. Effect of candidate genes on pectoralis major muscle satellite cell function. To determine the effect of candidate genes on satellite cell growth characteristics, broiler satellite cells previously isolated and purified representing broilers prior to WB and current commercial broilers were used. Candidate genes were assayed by Dr. Velleman's lab at Ohio State University by knocking down their expression during both proliferation and differentiation by small interfering RNA (siRNA) technology. The effect of slow myofiber genes on proliferation and differentiation suggested the presence of aerobic Type I satellite cells in the pectoralis major muscle which contains anaerobic Type IIb cells. The findings from this research support that WB results from a dysregulation of lipid and glucose metabolism. Obj2.1. Metabolomics analysis of Wooden Breast and White Striping. To develop more quantitative and objective measures of disease severity, we utilized untargeted metabolomics alongside two statistical approaches to evaluate plasma metabolites as potential biomarkers of wooden breast and white striping in 250 male commercial broiler chickens. We constructed a support vector machine using stepwise feature selection to determine the smallest subset of metabolites with the highest prediction accuracy for wooden breast. The final support vector machine achieved a prediction accuracy of 94% using only 6 metabolites, showing great promise for future diagnostic applications. The metabolite 3-methylhistidine, which is often used as an index of myofibrillar breakdown in skeletal muscle, was the top metabolite for both wooden breast and white striping in our mixed linear model and was also the metabolite with highest marginal prediction accuracy (82%) for wooden breast in our support vector machine. To our knowledge, this is the first untargeted plasma metabolomics study of WB and WS, and our findings provides potential biomarkers for diagnostic applications. Obj2.2. Gene expression profiling of the early pathogenesis of Wooden Breast. We have conducted RNA-sequencing experiments to identify molecular mechanisms involved in the onset and progression of wooden breast over the growth period. Results from these studies suggested altered lipid metabolism in the early phase of WB. We identified a number of differentially expressed genes with key roles in lipid uptake, transport and deposition [fatty acid translocase (CD36), fatty acid binding protein 4 (FABP4), lipoprotein lipase(LPL)], as well as adipocyte differentiation [peroxisome proliferator-activated receptor gamma (PPARG)]. A prominent gene in this list is LPL, which has been intensively studied for its encoded protein role in lipoprotein metabolism, lipid uptake and utilization. The increased expression of LPL in the early phase of WB is consistent with microscopic evidence of lipid accumulation in the p. major in affected birds. Notably, wereported higher expression of PPARG in 2-week-old broilers that were later diagnosed with WB at 7 weeks of age. The protein encoded by this gene, PPARg, is a master regulator of lipid metabolism and plays a major role in stimulating lipid uptake and adipogenesis by increasing transcription of a number of genes involved in lipid metabolism including LPL. So, it is likely that LPL expression in the early phase of WB is induced by increased expression of PPARG, though the triggers for the increased expression of PPARG in the early phase of WB remain to be elucidated. Obj2.3. Localize the cellular expression of LPL in the p. major muscle tissue. This study has revealed for the first time that LPL is expressed in the vascular endothelial cells of capillaries and small-calibre veins in chickens. Importantly, LPL is expressed more highly in veins undergoing phlebitis, characterized as the circumferential cuffing of small calibre veins by immune cells in affected muscle. The increased expression of LPL in veins compared to arteries suggests enhanced lipoprotein hydrolysis activity in the venous end of affected chickens, increasing permeability of the venous endothelium to free fatty acids (FFA); lipoprotein remnants such as LDL; and inflammatory cells including monocytes, lymphocytes and heterophils. These infiltrating immune cells may contribute to the onset and progression of the lymphocytic phlebitis lesion. In fact, the role played by LPL in development of vascular diseases such as in atherogenesis has been shown in previous studies in humans, and we speculate the increased expression of LPL plays a similar role in the occurrence of phlebitis in WB.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Tomlinson MJ 4th, Polson SW, Qiu J, Lake JA, Lee W, Abasht B*. 2021. Investigation of allele specific expression in various tissues of broiler chickens using the detection tool VADT. Scientific Reports. 11(1):3968.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Lake JA, Dekkers JCM, Abasht B*. 2021. Genetic basis and identification of candidate genes for wooden breast and white striping in commercial broiler chickens. Scientific Reports. 24;11(1):6785.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Velleman SG, Coy CS, Abasht B. 2021. Effect of growth selection of broilers on breast muscle satellite cell function: Response of satellite cells to NOV, COMP, MYBP-C1, and CSRP3. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 255:110917.
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Le Bihan-Duval E, Alnahhas N, Pampouille E, Berri C, Abasht B. 2020. Advances in poultry genetics and genomics. Aggrey SE, Zhou H, Tixier-Boichard M, Rhoads, editors. Cambridge CB22 3HJ UK: Burleigh Dodds Series in Agricultural Science; Chapter 5, Genetics and genomics of meat quality traits in poultry species.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Lake JA, Yan Y, Dekkers JCM, Qiu J, Brannick EM, Abasht B. Identification of circulating metabolites associated with wooden breast and white striping.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Abasht B. Genetic basis of white striping and wooden breast in broilers. Invited talk. Belgian Branch of the World Veterinary Poultry Association. (virtual presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Abasht B. Identification of candidate genes and promising plasma biomarkers for Wooden Breast and White Striping. Invited talk at Poultry Breeder's Roundtable, St Louis. MO (To be held on Nov 30, 2021).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Abasht B. Lipoprotein Lipase and Myopathies of the P. Major Muscle in Chickens. Invited talk. Penn Muscle Institute Muscle Club, University of Pennsylvania. May 26, 2020.
|
Progress 03/01/19 to 02/29/20
Outputs
Target Audience:The general public has been reached extensively by my efforts through featuring highlight of my research in Udaily, Delaware Public Media--BBC World, and WATTAgNet: • METABOLIC MIX-UP. UDailyhttps://www.udel.edu/udaily/2019/november/poultry-wooden-breast-chicken-syndrome-human-health-diabetes/#.XdrkAPSJEro.twitter • UD researcher tackling Wooden Breast Syndrome in chicken Delaware Public Media--BBC World Service • Metabolic discovery provides insight into wooden breast syndrome WATTAgNet The Udaily story about my research was also featured in other media outlets: • The cause of chewy chicken meat Phys.org • The Cause of Chewy Chicken Meat Lab Manager Lab Manager • News story from Eurasia Review on Saturday 23 November 2019 Eurasia Review • The cause of chewy chicken meat Eurek Alert! • The cause of chewy chicken meat News wise • The cause of chewy chicken meat Science Daily • The cause of chewy chicken meat: Poultry industry will be encouraged by study that finds the root of wooden breast syndrome Daily Herald • The cause of chewy chicken meat Long Room • US researchers Discovers a link between enzyme and wooden breast syndrome in chickens efeedlink • Researchers investigate wooden breast in broilers WATTAgNet • UD researchers find clues to development of wooden breast syndrome in broiler chickens Morning Ag Clips • Poultry industry will be encouraged by study that finds the root of wooden breast syndrome Poultry Producer • Researchers find enzyme that may contribute to 'wooden breast' condition in broiler chickens Delaware Business Now • Why is Some Chicken So Chewy? Researchers Have an Answer Labroots • Researchers Discovered the Cause of Chewy Chicken Meat Sci Tech Daily • The Cause Of Chewy Chicken Meat Science Blog • The Cause Of Chewy Chicken Meat Scienmag Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Two papers were published in peer-reviewed scientific journals and 3 presentations in scintific meetings and oneinvited talk in the seminar series of the Department of Animal and Avian Sciences at the University of Maryland, College Park.Highlights of findings were featuredin popular press:Udaily, Delaware Public Media--BBC World,WATTAgNet and in over 17 other media outlets. Link to the Udaily article:https://www.udel.edu/udaily/2019/november/poultry-wooden-breast-chicken-syndrome-human-health-diabetes/#.XdrkAPSJEro.twitter What do you plan to do during the next reporting period to accomplish the goals? Complete GWA analysis and identify candidate genes for Wooden Breast and White Striping Complete the cell culture RNAi study ofcandidate genes for Wooden Breast and White Striping. Identify novel biomarkers for Wooden Breast and White Striping using metabolomics data
Impacts
What was accomplished under these goals?
The impact of the project:Results from genome-wide association study of wooden breast myopathy in commercial broilersidentified genomic regions associated with this muscle condition. Results are expected to help determine the cause of Wooden Breast myopathy in broiler chickens and provide path to identify the causal variants or variants in strong correlation with the causal variants. Such information is needed for selection against Wooden Breast in commercial broilers.Results from RNA in situ hybridization analysisrevealed for first time thatLPLis expressed in the vascular endothelial cells of capillaries and small-caliber veins in chickens (Papah and Abasht, 2019). This discovery has been central not only to understanding the development of WB but also to formulating a novel hypothesis implicating etiological similarities between WB in chickens and complications caused by type 2 diabetes in humans (Lake and Abasht, 2020). Genome-wide association study of WB.A total of 1,229 crossbred (Cobb 500) broilers from the same breeding population of 15 sires and 200 dams were raised as two separate hatches (n1= 686, n2= 543) to 7 weeks of age, at which time they were euthanized and the p. major muscle of each bird was evaluated for gross signs of wooden breast and white striping as described previously (Papah et al., 2017; Lake et al., 2019). Blood samples from 1,194 birds were selected for DNA extraction and genotyping. These represented all males (n=564) and females (n=630) that did not exhibit any noticeable health conditions. DNA was isolated from blood samples and used for restriction enzyme-based low-depth genotyping by sequencing (GBS). The GBS libraries were constructed according to the methods outlined in Elshire et al. (2011) using two restriction enzymes (MspIandApeKI). Single-end 1x100 sequencing of 96-plex libraries was performed on an Illumina HiSeq 2500. To control and detect lane bias and batch bias, three samples of control DNA were included in each library and each library was run on either 8 or 9 lanes. Fastq files were demultiplexed using GBSX (Herten et al., 2015) and mapped by sample and lane onto the GRCg6a reference genome (Ensembl release 95) using BWA-MEM (Li, 2013). Samtools v1.9 (Li, 2011) was used to remove reads with mapping quality below 30, convert the alignment file to BAM format, and merge all alignment files by sample. Variants were detected across all samples on a per chromosome basis using samtools and bcftools v1.9 with parameters set to limit variants to biallelic single nucleotide polymorphisms (SNPs) (Li, 2011). The results were then combined into a single variant call file (VCF) and filtered to only include SNPs with a minimum read depth of 5 for at least 50% of samples and a minor allele frequency of 5% or greater. Samples were also filtered to only include those with a minimum read depth of 5 for at least 50% of SNPs. A genomic relatedness matrix (GRM) was constructed from the resulting variant data using the R package KGD (Dodds et al., 2015), which implements a method developed to account for the low depth of coverage often associated with GBS. Genotype posterior probabilities were estimated using the Bayesian genotype caller polyRAD, with population structure and linkage disequilibrium as a prior, and were subsequently converted to allele dosages (i.e. posterior mean genotypes) for use in association analyses (Clark et al., 2019). Variance components and heritability of wooden breast were estimated from the GRM using ASReml 4 (Butler et al., 2018).Quantitative trait loci (QTL) were detected using two separate approaches - Bayesian multi-marker regression and single-SNP analysis. Bayesian models were implemented in GenSel (Fernando and Garrick, 2009) and involved first estimating the proportion of markers with null effect (π) usingBayesCπ followed by computation of the genetic variance explained by each 1 Mb window of SNPs in the genome with BayesB. The BayesCπ algorithm was run for 200,000 MCMC iterations with a burn-in of 150,000 iterations and used a starting value of 0.5 for π. BayesB was run for 60,000 iterations with a burn-in of 20,000 and used the posterior mean of π calculated by BayesCπ for each trait. analysis of WB found ten 1-Mb regions that each explained >1% of genetic variance. Together, these ten regions explained approximately 20% of the genetic variance for WB. Single-SNP analysis was performed with a mixed linear modelusing GCTA version1.26.0 (Yang et al., 2011) with software patches provided by van den Berg et al. (2019) that adapted the program to accept allele dosages. Eq. 1 was used for all single-SNP analyses with the addition of a SNP effect. Genomic regions containinga minimum of 2 significant SNPs (based on FDR-adjusted p-value) in a distance of less than 20 kb were considered as regions significantly associated with WB. Consequently, we identified 10 regions passing these criteria. Genomic regions detected by Bayesian multi-marker regression and single-SNP analysis were consistent, and as expected, regions detected by single-SNP analysis were narrower (~20 kb), reflecting rapid decay of LD in this broiler population.The heritability estimate for WB was 0.49, indicating the substantial role of genetic factors in the etiology of WB.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Papah, M. B., and Abasht, B. (2019). Dysregulation of lipid metabolism and appearance of slow myofiber- specific isoforms accompany the development of Wooden Breast myopathy in modern broiler chickens. Sci. Rep., 112. doi:10.1038/s41598-019-53728-8.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Lake, J.A. and Abasht, B., 2020. Glucolipotoxicity: A Proposed Etiology for Wooden Breast and Related Myopathies in Commercial Broiler Chickens. Frontiers in Physiology, 11, p.169.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Michael Babak and Behnam Abasht. Myofiber-type switching, and dysregulation of lipid metabolism accompanies development of wooden breast myopathy in commercial broiler chickens.
Poultry Science Association Annual Meeting, July 15-18, 2019, Montreal, Canada
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Juniper Lake, Jack Dekkers, Sandra Velleman, Erin Brannick, and Behnam Abasht. Preliminary results from genome wide association study of wooden breast in commercial broilers. Poultry Science Association Annual Meeting, July 15-18, 2019, Montreal, Canada
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Juniper A. Lake, M. Walugembe, Luke Kramer, Jack C.M. Dekkers, Behnam Abasht. Genetic Basis of Wooden Breast and White Striping in Commercial Broilers. Plant & Animal Genome, San Diego, CA. January 11-16, 2020
|
Progress 03/01/18 to 02/28/19
Outputs
Target Audience:Results were presented at Plant and Animal Genome meeting (Jan 2019) and Poultry Scince Assaociation meeting (July 2019) Three peer-reviewed reserach articles were published,one additional article is under review for publication, and threemanuscripts in preparation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project providedopportunities for graduate students to attend Plant and Animal Genome Meeting in Jan 2019. How have the results been disseminated to communities of interest?Three research articles were published in peer-reviewed scientific journalsand 5 presentations were given in scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Complete GWA analysis and identify candidate genes for Wooden Breast and White Striping ApplyVADT to identify allele-specific expression from RNA-seq data Determine the effects of candidate genes on pectoralis major muscle satellite cell function by manipulation of gene expression. Identifynovel biomarkers forWooden Breast and White Stripingusing metabolomics data
Impacts
What was accomplished under these goals?
Objective 1 1.1.Genome-Wide Association Study A genome-wide association study is currently being conducted in Dr. Behnam Abasht's lab at UDin collaboration with Dr. Jack Dekkers at Iowa State University.This study includes1,193 Cobb 500 broilers, which were raised to seven weeks of age and evaluated for wooden breast, white striping, and body weight. Genotyping was performed using restriction enzyme-based genotyping by sequencing on DNA extracted from whole blood. Heritability and genetic correlation was assessed for all traits and quantitative trait loci were detected using single-SNP analysis and Bayesian multi-marker regression (BayesB). Heritability was estimated to be moderate for all traits: 0.51±0.06 for wooden breast, 0.53±0.06 for white striping, and 0.41±0.06 for body weight. Genetic correlation between wooden breast and white striping was high (0.87±0.02), but genetic correlation between either wooden breast or white striping and body weight was low (0.24±0.03 and 0.18±0.03, respectively). Preliminary results of the multi-marker analysis reveal eight 1-Mb regions that each explain >1% of genetic variance. Together, these eight 1-Mb regions explain 18.5% of the genetic variance for wooden breast. Multi-marker analysis of white striping and body weight identified, respectively, five and four 1-Mb regions that each explained >1% of genetic variance. These results improve our understanding of the genetic architecture of these myopathies and suggest that wooden breast and white striping would respond to selection. Ongoing data exploration aims to examine the quantitative trait loci that have been discovered and identify candidate genes associated with wooden breast, white striping, and body weight. 1.2.Blood Gas Analysis This work has been conducted in Dr. Abasht's lab.The effect of wooden breast on venous blood parameters and body weight distribution was determined for 107 Cobb500 broilers were raised to 7 weeks of age. All chickens were sampled for 13 blood parameters and the relative weights of the pectoralis major muscle, pectoralis minor muscle, external oblique muscle, wing, heart, lungs, liver, and spleen between wooden breast affected and unaffected chickens. Blood analysis was performed on samples taken from the brachial vein of live birds and revealed significant differences in blood gases and electrolytes between affected and unaffected chickens. After correcting for sex and body weight, chickens with wooden breast exhibited significantly higher partial pressure of carbon dioxide (pCO2), total carbon dioxide (TCO2), bicarbonate (HCO3-), and potassium (K+), and lower partial pressure of oxygen (pO2), oxygen saturation (sO2), and pH. Wooden breast affected broilers also possessed a significantly larger pectoralis major muscle and whole feathered wing. Blood gas disturbances characterized by high pCO2, low pH, and high HCO3-levels are indicative of insufficient respiratory gas exchange, suggesting that wooden breast affected broilers have a relatively high metabolic rate that may also be inadequately compensated due to cardiovascular deficiencies such as poor venous return. These results further indicate tissue hypoxia and the buildup of metabolic wastes as major features of the wooden breast phenotype and suggest a more systemic effect of its pathology. 1.3.Differential Gene Expression Analysis A gene expression analysis was performed inDr. Abasht's labon the pectoralis major muscle of two-week-old birds that were dignosed with or withoutwooden breast by market age at 7 weeks to identify early molecular signals in the wooden breast transcriptional cascade. Biopsy samples of the left pectoralis major muscle were collected from 101 birds at 14 days of age. Birds were subsequently raised to 7 weeks of age to allow sample selection based on the wooden breast phenotype at market age. RNA sequencing was performed on 5 unaffected and 8 affected female chicken samples, selected based on wooden breast scores (0 to 4) assigned at necropsy where affected birds had scores of 2 or 3 (mildly or moderately affected) while unaffected birds had scores of 0 (no apparent gross lesions). Differential expression analysis identified 60 genes found to be significant at an FDR-adjusted p value of 0.05. Of these, 26 were previously demonstrated to exhibit altered expression or genetic polymorphisms related to glucose tolerance or diabetes mellitus in mammals. Additionally, 9 genes have functions directly related to lipid metabolism and 11 genes are associated with adiposity traits such as intramuscular fat and body mass index. This study suggests that wooden breast disease is first and foremost a metabolic disorder characterized primarily by ectopic lipid accumulation in the pectoralis major. 1.4. Effects of Candidate Genes on Pectoralis Major Muscle Satellite Cell Function Research in Dr. Sandra Velleman's laboratory at The Ohio State University has focused on how the top genes altered by the Wooden Breast myopathy affect the function of the adult myoblast stem cell population of cells.Wooden Breast is a serious disorder in the breast muscle of current commercial broilers affecting meat quality and resulting in breast meat condemnation. Dr. Abasht's research effort at University of Delaware has identified the primary genes affected by the Wooden Breast condition in whole breast muscle tissue.The adult myoblast stem cell or satellite cell is the only cell type responsible for the growth of muscle after the bird hatches and the repair of muscle with injury. Results from my laboratory have shown with the primary genes altered by the Wooden Breast myopathy are differentially regulated in breast muscle satellite cells isolated from a broiler line prior to the onset of Wooden Breast and a current commercial broiler line afflicted with Wooden Breast.These results demonstrate that Wooden Breast has altered the satellite cell population responsible for all posthatch muscle growth and the repair of muscle with injury.A peer reviewed manuscript is in preparation for submission for this first phase of our research. 1.5.Tool for detecting allele specific expression This work has been conducted in Dr. Abasht's lab.We developed a tool for detecting allele specific expression VADT (VCF ASE Detection Tool)Program takes in a raw VCF file, filters the data and then performs various statistical analysis for detection of allele specific expression (ASE) to identify highly confident occurrences of ASE.This will be applied to RNA-seq samples to determine ASE in the breast muscle and compare the results with top genes from GWAS. Objective 2 2.1. Blood Metabolomics Plasma samples collected at necropsy from 250 male chickens were utilized for metabolomics profiling. Frozen aliquots were shipped on dry-ice to Metabolon Inc. (Durham, NC) for metabolomics profiling using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on their DiscoveryHD4™ platform. A total of 615 biochemicals were identified and quantitated using this platform. Preliminary results have been received from Metabolon Inc. and data analysis is ongoing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lake, J.A., Papah, M.B. and Abasht, B., 2019. Increased Expression of Lipid Metabolism Genes in Early Stages of Wooden Breast Links Myopathy of Broilers to Metabolic Syndrome in Humans. Genes, 10(10), p.746.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Abasht, B., Zhou, N., Lee, W. R., Zhuo, Z. & Peripolli, E. 2019. The metabolic characteristics of susceptibility to wooden breast disease in chickens with high feed efficiency. Poultry Science doi:10.3382/ps/pez183
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Brothers, B.K., Zhuo, Z., Papah, M. and Abasht, B., 2019. RNA-seq analysis reveals spatial and sex differences in pectoralis major muscle of broiler chickens contributing to difference in susceptibility to wooden breast disease. Frontiers in Physiology, 10, p.764.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Behnam Abasht. Metabolic Risk Factors of Wooden Breast Disease in Commercial Broiler Chickens. Plant & Animal Genome, San Diego, CA. January 11-17, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Brilynn Brothers, Zhu Zhuo, Michael P. Babak, Behnam Abasht. Spatial and Sex Differences in Gene Expression and Metabolic Shift Phenomenon in Breast Muscles of Modern Broiler Chickens. Plant & Animal Genome, San Diego, CA. January 11-17, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Juniper Ada Lake, Sandra G Velleman, Erin M Brannick, Behnam Abasht. Blood Analysis and Proportional Muscle and Organ Weights in Broilers with Wooden Breast. Plant & Animal Genome, San Diego, CA. January 11-17, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Amelie Hoste and Behnam Abasht. RNA-Seq Analysis of Liver in Commercial Broiler Chickens with High and Low Feed Efficiencies Plant & Animal Genome, San Diego, CA. January 11-17, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lake JA, Tomlinson IV MJ, and Abasht B. FastqBLAST: A tool to quickly assess the organismal and genomic diversity present in a FASTQ file. Poster presented at: 26th Conference on Intelligent Systems for Molecular Biology; 2018 Jul 6-10; Chicago, IL.
|
Progress 03/01/17 to 02/28/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided hands-on research experience opportunities for15 undergraduate and 6 graduate students. They participated in live animal experiments, phenotypic data and sample collection in this project. This project provided data/samples/funding support for 2 undergrad senior thesis projects, 2 masters thesis projects and one PhD thesis project. How have the results been disseminated to communities of interest?Two posters from this project were presented at Plant and Animal Genome Conference in San Diego, Jan 13-17, 2018. What do you plan to do during the next reporting period to accomplish the goals? DNA isolation and genotyping of about 1000 samples collected in the current reporting period Genome-Wide Association Study of WBD using phenotypic records and genotype data Metabolomics analysis of blood samples from WBD-affected and unaffected samples collected in the current reporting period Blood parameter analysis of blood samples from WBD-affected and unaffected samples collected in the current reporting period Identification ofloss of function and missense mutations and allele-specific expression from RNA-seq data Functional validation of candidate genes using the siRNA technology.
Impacts
What was accomplished under these goals?
1. Main live animal experiment.After evaluating the suitability of a commercial broiler cross in our preliminary experiment (reported in the first reporting period), we conducted the main live animal experiment for this project in the current reporting period. Briefly,to collected phenotypic data and blood samples for the Genome-Wide Association Study, metabolomics analysis, blood parameters analysis of WBD, a large-scale live animal experiment of approximately 2,200 commercial broiler chickens was conducted in chicken houses at the University of Delaware. The chickens used in this study were from a high breast meat yield line of broiler chickens (the same commercial broiler cross that was evaluated in our preliminary experiment). There were three hatches of birds, the second hatch was added four weeks after the first, and the third hatch was added two weeks after the second hatch. Each day, the temperature of the chicken houses, bird condition, feed, and water supply were monitored to ensure proper growth. In addition, any sickly birds were humanely euthanized by cervical dislocation and removed from the study. Sickly birds were identified by qualities such as inactivity or physical abnormalities, for example, a broken wing or crippled walk. At market age, after measuring each bird's body weight and drawing blood from the wing vein, the birds were euthanized by cervical dislocation and tissue samples were collected in cryogenic tubes and flash-frozen in liquid nitrogen then stored at -80°C. 1.1. Main accomplishment: We were able to collected samples and information needed for GWAS study, metabolomics analysis, blood parameters analysis, which will be performed during the next reporting period. 1.2. Other accomplishments: We took advantage of the preliminary and main experiments and collectedsamples for two undergrad senior thesis projects: The first undergrad senior thesis project aimed to characterize biological differences in the p. major between regions of the muscle and sexes of birds. Samples were taken from the cranial and caudal aspects of p. major muscles of six 3-week-old unaffected male and female birds for a total of twelve tissue samples to be used for RNA sequencing. RNA was extracted using the mirVana miRNA Isolation kit (ThermoScientific), and complimentary DNA (cDNA) libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). Libraries were sequenced by the Delaware Biotechnology Institute (DBI) using HiSeq2500. Sequence reads were then aligned to the chicken reference genome with HISAT, and genes were analyzed for differential expression between regions of the breast muscle and sexes of birds using CuffDiff. Functional analysis was performed on differentially expressed genes (DEGs) between sex groups using DAVID and Ingenuity Pathway Analysis (IPA). There were 12 DEGs between cranial and caudal samples, and 260 between male and female birds. There was increased expression of genes involved fat metabolism and oxidative stress responses in the cranial region of the p. major, as well as increased expression of fat metabolism, oxidative stress response, inflammatory and immune response, antiangiogenesis, and connective tissue proliferation genes in male broilers. These results support a hypothesis that there are biological characteristics in male broilers and the cranial region of the breast muscle that make them more susceptible to WBD. The second undergrad senior thesis project aimedto characterize gene expression and histologic differences between lung tissue of affected and unaffected commercial broiler chickens. For this specific project, a total of twelve samples, 6 WBD-affected and 6 unaffected chickens, were used for RNA-seq and gene expression analysis. Of the six affected samples, three are from male chickens and three are from female. The same follows for the unaffected samples. RNA samples were isolated and RNA sequencing and histologic analysiswere completed. We are currently in the process of biological analysis and interpretation of the RNA-seq and histologic data. 3.Developing a bioinformatics pipeline for identification ofloss of function and missense mutations and allele-specific expression from RNA-seq data. This pipeline follows GATK's Best Practices for calling of variants and also corrects for reference allele bias. For detection of ASE SNPs custom python scrips were developed that test for ASE using the binomial test. This pipeline was used to analyze transcriptomic data from three tissues (breast muscle, abdominal fat and liver). At this time period, all data have been analyzed, variants called and ASE SNPs identified; however, further development of the ASE software needs to be done.
Publications
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Progress 03/01/16 to 02/28/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided hands-on research experience opportunities for3 undergraduate and 4graduate students. They participated in live animal experiments, phenotypic data and sample collection in this project. This project provided data/samples/funding support for 2 undergrad senior thesis projects How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Condyct main live animal experiment to collected phenotypic and blood samples for Genome-Wide Association study of WBD and metabolomics analysis of WBD Developea bioinformatics pipeline for identification ofloss of function and missense mutations and allele-specific expression from RNA-seq data.
Impacts
What was accomplished under these goals?
1. Preliminary live animal experiment: A preliminary live animal experiment was conducted in order to assess incidence rate of wooden breast disease (WBD) in a commercial cross. A total of 171 broilers were placed on the floor with wood shavings in chicken houses with automatic ventilation and a light cycle consisting of one hour of darkness followed by 23 hours of light per day. Houses were kept initially at 95°F and lowered by 5°F each week until they reached 70°F. Birds were fed standard commercial starter and grower diets, and were allowed free access to feed and water. At week 1, 2, and 3 weeks of age, 5, 15 and 24 birds were euthanized by cervical dislocation and necropsies were performed for gross and histopathologic evaluations of the pectoralis major muscle. Also, tissue samples were taken from the cranial and caudal aspects of the pectoralis major muscle of the selected bird, then immediately transferred to liquid nitrogen to be flash-frozen. All surviving birds euthanized at 56 days old and subjected to gross evaluation of the p major muscle for wooden breast disease. 1.1. Main accomplishment: This experiment revealed that the incidence rate of WBD is ~14% in the commercial cross that was tested, and therefore using this cross for the main experiment will be appropriate in terms ofproviding sufficient number of affected and unaffected chickens.
Publications
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