Source: MISSISSIPPI STATE UNIV submitted to NRP
WHOLE PROTEOMIC ANALYSIS TO DETERMINE BIOCHEMICAL PATHWAY DIFFERENCES BETWEEN NORMAL, PALE, SOFT, AND EXUDATIVE AND WOODY BREAST MEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1011839
Grant No.
2017-67017-26473
Cumulative Award Amt.
$454,986.00
Proposal No.
2016-09106
Multistate No.
(N/A)
Project Start Date
May 1, 2017
Project End Date
Apr 30, 2022
Grant Year
2017
Program Code
[A1361]- Improving Food Quality
Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
Food Sci Nutrition Hlth Promo
Non Technical Summary
This Strengthening Standard Grant proposal addresses USDA AFRI program area priority Improving Food Quality (Program Area Priority Code - A1361). As of 2016, woody breast and pale, soft, and exudative (PSE) meat are quality defects in poultry that affect 30-50 % of broilers that are harvested in the United States. PSE meat has an unacceptable appearance at retail display and is extremely dry. Woody breast meat is characterized by a hard, tough texture at the cranial and/or caudal portions of the breast. Results from our previous USDA AFRI Seed Grant demonstrated that the muscle proteomes of PSE and normal broiler breast meat were different and the abundance of numerous proteins was correlated with meat quality. Therefore, the objectives of the current proposal are - (1 and 2 ) to characterize biochemical pathways in live muscle, pre-rigor meat and post-rigor meat to explain the biochemical pathways associated with PSE meat development and woody breast meat and to elucidate mechanisms for the variations in whole-muscle proteomes in PSE and woody broiler breast meat compared to normal breast; (3) To evaluate biochemical and proteomic differences in genetic strains that produce woody breast meat to characterize the genetics and biochemical pathways that are consistent with the production of woody breast meat. Successfully characterizing the proteome basis and biochemical pathways of woody breast meat and PSE meat will aid engineering strategies to prevent the incidence of PSE and woody and improve poultry meat quality, ultimately enhancing the competitiveness of the US poultry industry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5023260100025%
5023260106025%
5023260103025%
5023260102025%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
3260 - Poultry meat;

Field Of Science
1060 - Biology (whole systems); 1030 - Cellular biology; 1000 - Biochemistry and biophysics; 1020 - Physiology;
Goals / Objectives
Characterizing the relationships between the PSE condition and the biochemical pathways during muscle-to-meat conversion and determining the proteome components involved in these pathways will allow us to identify strategies to minimize the incidence of both the PSE condition and woody breast meat in broiler production. An additional objective of this grant is to continue to build on our research expertise in proteomics at Mississippi State University so that meat quality can be related to environmental stressors and genetic expression of live animals. Over the next 5-7 years, we envision having an established laboratory that has determined how the genetics of broilers can be modified to minimize the incidence of meat quality defects, including woody breast meat, PSE, green muscle disease, and lesions on wings and to determine the impact of genetic factors and environmental stress on these quality defects.Therefore, the specific objectives of this grant proposal are:1. To characterize biochemical pathways in live muscle, pre-rigor meat and post-rigor meat to explain the biochemical pathways associated with PSE meat development and mechanisms for the whole muscle proteomes in PSE breast meat;2. To characterize biochemical pathways in live muscle, pre-rigor meat and post-rigor meat to explain the biochemical pathways associated with woody breast meat development and mechanisms for the whole muscle proteomes in woody breast meat;3. To evaluate biochemical and proteomic differences in genetic strains that produce woody breast meat to characterize the genetics and biochemical pathways that are consistent with the production of woody breast meat.
Project Methods
Objective 1: To characterize biochemical pathways in live muscle, pre-rigor meat and post-rigor meat to explain the biochemical pathways associated with PSE meat development and mechanisms for the whole muscle proteomes in PSE breast meat; Broiler meat samples: Two genetic lines of broilers (Ross 708, Cobb 700) will be raised at the Mississippi State University Poultry Farm according to approved animal welfare protocol (IACUC pending). Prior to harvest, a sample of the broilers will be subjected to short-term stress. The short-term stress treatment will include catching broilers by hand, releasing the broilers to a lighted poultry house at 38°C with increased stocking density for 2 hr and then re-catching the broilers immediately prior to the slaughter process. This will cause the production of a minimum of 20% PSE meat. Control birds will be hand caught, placed in live haul crates at 21°C for 2 h and then slaughtered. In addition, this will allow us to obtain PSE meat samples that range in pH from 5.4-5.7 and CIEL* from 60-70 at 24 h postmortem and normal samples that range in pH from 5.8-6.2 and CIEL* from 50-60. The broilers will be harvested, and the meat will be deboned at 4 h postmortem. PSE and normal breast meat samples will be sampled (n = 36 per replication) and analyzed for pH, color, water-holding capacity, and tenderness at three separate sampling times. A total of at least 18 broiler breast samples that are normal and 18 samples that are PSE will be selected within each replication based on pH and color. Proteomic samples will be taken at 3 time points for each broiler that is harvested. Immediately prior to slaughter and after stunning, 5 g biopsies will be taken from the breast muscle, placed in liquid nitrogen to stop biochemical processes and then frozen at - 70 C. Each broiler will be tracked through the slaughter process. Five g samples will be taken from the breast of each broiler after going through the slaughter line (10-15 min postmortem) and 4 hr postmortem. All samples will be maintained at -70 C until proteomic analysis is conducted. Not all samples will be analyzed for proteomics. Only a sample of normal and PSE meat from each genetic strain will be subjected to proteomic analysis. However, since we cannot identify the condition until the meat is produced, samples will be taken for all broilers. The whole muscle proteome (n = 8 per replication) will be characterized using two-dimensional gel electrophoresis. Samples will be chosen from each replication so that they are representative of the range of pH and color of the L* groups for each replication. This will allow us to correlate the proteomes that differ in abundance to pH, color, and Warner-Bratzler shear force. Mass spectrometry will be used to identify and quantify proteins that are expressed in different concentrations.Objective 2: To characterize biochemical pathways in live muscle, pre-rigor meat and post-rigor meat to explain the biochemical pathways associated with woody breast meat development and mechanisms for the whole muscle proteomes in woody breast meat;Broiler meat samples: Four genetic lines of broilers (Ross 708, Cobb 700, Ross 308, Cobb 300) will be raised at the Mississippi State University Poultry Farm according to approved animal welfare protocol (IACUC pending). These broilers will be provided by the University of Georgia Poultry Science department (see attached letter). The broilers will be harvested at 7-9 weeks of age, and the meat will be deboned at 4 h postmortem. Woody and normal breast meat samples will be sampled (n = 36 per replication) for each breed and analyzed for pH, color, water-holding capacity, and tenderness at three separate sampling times. A total of at least 18 broiler breast samples that are normal and 18 samples that are woody will be selected within each replication based on appearance and touch (Owens, 2016). Proteomic samples will be taken at 3 time points for each broiler that is harvested. After stunning and immediately prior to slaughter, 5 g biopsies will be taken from the breast meat of broilers, placed in liquid nitrogen to stop biochemical reactions and then frozen and stored at - 70 C. Each broiler will be tracked through the slaughter process. Five g samples will be taken from the breast of each broiler after going through the slaughter line (10-15 min postmortem) and 4 hr postmortem as well. All of these samples will be maintained at -70 C until proteomic analysis is conducted. Only a subset of samples of normal and woody meat from each genetic strain will be subjected to proteomic analysis. However, since we cannot identify the condition until the meat is produced, samples will be taken for all broilers. The whole muscle proteome (n = 8 per replication) will be characterized using two-dimensional gel electrophoresis. Differences will be determined for proteomes between genetic strains (Ross 708, Cobb 700, Ross 308, Cobb 300) and woody and normal breast from the Ross 708 and Cobb 700 broilers since it is unlikely that the Ross 308 and Cobb 300 broilers will yield woody breast meat. However, if woody breast meat is yielded from Ross 308 and Cobb 300 broilers, the proteome of these samples will be determined. All measurements for breast meat quality, sensory analyses, and proteomic analyses will be the same as those conducted for Objective 1. Biochemical pathways and differences between genetic strains and normal and woody breast meat will be evaluated by comparing proteome differences between breast samples for individual broilers based on live muscle, prerigor, and postrigor breast samples. Objective 3: To evaluate biochemical and proteomic differences in genetic strains that produce woody breast meat to characterize the genetics and biochemical pathways that are consistent with the production of woody breast meat.Transcriptome profiling: The W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois will perform transcriptome profiling to determine differential expression of mRNA between woody and normal breast meat samples. This analysis will use a subset of the experimental population. Three animals from 4 breeds (Ross 708, Cobb 700, Ross 308, Ross 300) will be analyzed for the pre-rigor, post-rigor, and live muscle samples. In addition, both normal and woody breast samples will be evaluated for the Ross 708 and Cobb 700 genetic strains. The overall quality of the raw RNAseq data will be determined using FastQC v.0.11.3. Adapter sequences, low quality bases, and low quality reads will be trimmed using Trimmomatic v.0.33. Trimmed data will be aligned to the chicken reference genome available at the time of the analysis using the software STAR v.2.4.2a. Read summarization will be performed using the subread-featureCounts pipeline v1.4.6-p5. Normalization factors will be calculated using Trimmed Mean of M-values (TMM) normalization to account for differences in library size between samples. The R/Bioconductor package SVA v. 3.18.0 will be used to remove unrecognized batch effects. Samples will be fitted to a negative-binomial general linear model and tested for differential gene expression between the groups using a likelihood-ratio test. P-values of differential expression tests will be corrected for multiple-hypothesis testing using Benjamini-Hochberg false discovery rate (FDR) correction; threshold for significance will be set to FDR < 0.01. One-way ANOVA will be performed for the entire dataset (already adjusted for the surrogate variables) using edgeR. For the overall ANOVA test, raw P-values for each gene will be adjusted using the false discovery rate method. The genes passing the significant threshold (FDR P-value < 0.25) will be used to conduct a Weighted Gene Correlation Network Analysis (WGCNA).

Progress 05/01/17 to 04/30/22

Outputs
Target Audience:Poultry processors; Poultry Industry; General Public; Academics in Food Science, Animal and Poultry Sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students, one postdoctoral student, and one undergraduate have worked on this project and gained expertise in evaluating broiler meat quality, sensory analyses, and performing microbiome, transcriptomic, and proteomic techniques. An additional PhD student raised broilers and harvested them. This has helped them gain experience in poultry nutrition, genetics, and processing. This research has allowed students to interact with multiple poultry companies and present research at professional conferences. The postdoctoral student and graduate student have discussed the research with poultry companies in the southeastern United States. They have enhanced their ability to explain scientific research to people in the industry and have enhanced their professional network and networking skills. This research has also contributed to one student receiving an industry internship and another student to obtain an industry position. Two Brazillian visiting scholars were trained on proteomic studies on beef samples. In addition, 6 journal papers were published. These papers are completed by this team of PhD students, postdocs, and undergraduate students. How have the results been disseminated to communities of interest?An invited Keynote Address to an audience of approximately 800 was given on proteomics at the 2017 International Congress in Cork Ireland in Fall 2017. An Invited Conference Proceedings were published at the 2018 American Meat Science Association Annual Meeting (Reciprocal Meats Conference). There was also an Invited Symposium given at this conference to an audience of 200-300 people. Eight abstracts were presented at the 2018, 2019, and 2020 Reciprocal Meats Conference. Two papers were published in Carnetec. Results have been shared with multiple poultry companies in the Southeast as a written report. Six journal papers were published in Poultry Science (4), Meat and Muscle Biology (1), and Journal of Agricultural and Food Chemistry (1). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Woody breast meat incidence due to broiler genetic strains and diets. A total of 1,200 mixed-sex broilers (strains 1-5) were raised for 8 weeks at the MSU Poultry Research Farm at Mississippi State University and fed with commercial control and reduced essential amino acid diets (20% reduction of digestible lysine, total sulfur amino acids, and threonine).On d 56, AA reduction in the diet led to lower (P<0.05) absolute live BW, carcass, and breast weights in strains 4 and 5 but not (P>0.05) in strains 1, 2, or 3. AA reduction did not affect the incidence of WB within strains 1, 2, or 3 (P > 0.05) but led to less WB incidence within strains 4 and 5 (P < 0.05). Feeding an AA-reduced diet decreased (P < 0.05) the percentage of moderate and severe WB from 45% to 11% for strain 4 and from 84% to 35% for strain 5, as well as decreased the body and breast weights of strains 4 and 5 compared with the birds fed with a control diet. These results indicate that strains 4 and 5 are more susceptible to the dietary AA reduction than strains 1, 2 and 3.Reduced diets should be provided to broiler strain 1, 2 and 3 to reduce WB incidence without compromising meat yields. 2.Sensory evaluation of woody breast chicken meat. Normal, moderate and severe WB meat from strains 1-5 were either fried or baked and evaluated by a trained panel. Results indicated that WB severity had a greater impact on sensory attributes and consumer acceptability than genetic strain. Higher WB severity created an undesirable texture that negatively impacted the acceptability of baked meat. However, the increased crunchiness and cohesiveness due to woodiness had a positive impact on the fried chicken acceptability. Results indicated that a large percentage of consumers rated baked, fried, and fajita samples as acceptable regardless of whether normal (NB) or WB (moderate, severe) meat was used, but some consumers did not like baked or fajita meat that was made from SEV WB meat. 3. Proteomic differences between normal and woody breast samples. In total, 24 proteins were expressed differentially between NB and WB meat. Twenty-one of these proteins were overexpressed in WB meat and three proteins were underexpressed in WB meat. The differentially expressed proteins in each comparison were consistently upregulated or downregulated in WB tissue although the different protein profiles were noticed for each comparison. Strains 2 and 5 had more protein profile differences between WB and NB meat than strains 1, 3, and 4, which potentially indicates a stronger genetic component for strains 2 and 5 with respect to WB formation. Reducing AA intake resulted in minimizing the difference in protein profiles between normal and woody chicken breasts. The proteins that were overexpressed in WB are involved in carbohydrate metabolism, oxidative stress, cytoskeleton structure, and transport and signaling. Ingenuity Pathway Analysis (IPA) indicated that regulated pathways in WB were mainly related to carbohydrate metabolism, cellular repair, cellular organization and maintenance, and cell death and survival. The results support the potential causes of WB myopathy, including the presence of hypoxia, oxidative stress, increased apoptosis, misfolded proteins, as well as inflammation. 4. Proteomic differences between live muscle and rigor meat. Twenty out of 24 proteins changed exclusively in either NB or WB, indicating the differences in their post-mortem metabolism. Reduced glycolytic potential and a shorter extension of glycolysis was seen in post-mortem WB as glycolytic proteins declined faster in WB than in NB. WB condition affects the post-mortem metabolism, as indicated by changes in structural proteins, metabolic proteins, stress-related proteins, transport proteins as well as the degree of post-translational modifications due to the differences in antemortem conditions in NB and WB. These differences might indicate a faster and more oxidative muscle to meat conversion process for WB. Future research will be conducted on the characterization of PTM proteins in post-mortem WB to further investigate their responses to WB conditions and their impact on WB meat quality. These protein biomarkers and their post-translational modified products will improve our knowledge of the biochemical process in post-mortem WB meat and provide more insight on the development of WB and the potential strategies to reduce WB incidence. 5. Proteomic differences between different broiler genetic strains. It was observed that genetic variation causes phenotypic differences in five strains of broilers that are most commonly used in the broiler industry and produce NB and WB.Strain 5 breast proteomes were most different from other strains in both NB and WB groups. Strain 5 birds are genetically more prone to develop WB when birds were fed the control diet due to the evidence of increased apoptosis and protein synthesis, more intense contraction, and high oxidative stress in strain 5 WB muscle in comparison to other strains. In summary, the proteomic approach indicates that the WB myopathy in broilers is related to genetics, nutrition, and rigor mortis. The protein profiles of normal and woody chicken breast samples were different, which might help explain some of the changes in meat quality. 6. Metagenomic differences between broilers with normal and woody breast meat The bacterial diversity of cecal content was characterized in male broilers from two commercially available broiler genetic strains for birds with normal and woody breast fillets (phenotype effect), birds of two strains (strain effect), as well as birds fed a control diet and an amino acid (AA) reduced diet (digestible lysine, total sulfur amino acids, and threonine reduced by 20% as compared to the control diet) (diet effect). DNA of cecal samples was extracted and amplified using universal primers that target the V3~4 regions of bacterial 16S rRNA for sequencing in Illumina MiSeq following data processing with Quantitative Insights into Microbial Ecology (QIIME 2). The samples were clustered mainly based on the phenotype (P = 0.001) followed by diet (P = 0.024), revealing that the microbiota of each bird with normal breast was more similar to each other than the microbiota of birds with WB. Regardless of diet nutrition, unaffected broilers and WB affected broilers respond differently to the nutritional and environmental stimuli, which can be seen in the differences in their gut microbiota. Butyricicoccus pullicaecorumas greater than 2 times more abundant (false discovery rate, FDR = 0.01) in the ceca of non-affected birds than WB affected birds. Bacteroides plebeius (12.3%) and Selenomonas bovis (12.6%) were more abundant in WB affected than non-affected birds.In WB affected birds, the ceca were characterized with decreased glycolysis and urea cycling, increased TCA cycle and vitamin biosynthesis, andaltered amino acid and nucleotide metabolism.It was determined that differences in the microbiome may be associated with the development of WB. Further studies are needed to investigate the potential mechanism and how to reduce broiler WB incidence by regulating their gut microbiota. 7.Transcriptomic differences between normal and woody breast samples. The multidimensional scaling clustering indicated different gene expression pattern between WB status with some overlapping. Dimension 1 mainly explains the difference between normal and woody breast samples, which accounts for 11% of total variability. The difference of diet can be found at dimension 2, which accounts for 5% of total variability. In total, 14,342 genes were identified; among them 3,055 genes (21.3% of total genes identified) were differentially expressed when compared WB samples with normal breast samples. In comparison, 487 genes (3.4% of total genes identified) were differentially expressed when compared reduced diet samples with control diet samples.

Publications


    Progress 05/01/20 to 04/30/21

    Outputs
    Target Audience:Poultry processors; Poultry Industry; General Public; Academics in Food Science; Animal and Poultry Sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students, one undergraduate student, and one postdoctoral student gained expertise in proteomics and microbiome research. In addition, these students were involved in writing journal papers and grant proposals related to the results of the research. How have the results been disseminated to communities of interest?These results have been disseminated at multiple meat science and poultry science meetings over 2020 and 2021. In addition journal papers have been published in Poultry Science, Meat and Muscle Biology, andJournal of Agricultural and Food Chemistry. What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period, our research team will continue to evaluate transcriptomic data to determine how genetic expression differs for broilers that yield woody breast mean and normal breast meat.

    Impacts
    What was accomplished under these goals? The effects of woody breast (WB) myopathy on muscle metabolism during pre-rigot and rigor stages and the the impact on meat quality was evaluated. The majority of proteins changed exclusively in either normal breast (NB) or WB during early post-mortem, which indicates that there were differences in their post-mortem metabolim. A faster decline of glycolyic proteins in WB than in NB indicated a lower glycolytic potential and a shorter dutation of glycolysis in post-mortem WB. It is therefore confirmed that the antemortem WB condition imoacts post-mortem biochemical processes. Thedifferences in post-mortem metabolism of WB was demonstrated by greater changes in structual proteins, metabolic proteins, stree-related proteins, transport proteins as well as the degree of posttranslational modifications (PTMs) in WB muscle postmortem in comparison to NB postmortem. These differences are indicative of a faster and more oxidative muscle to meat conversion for WB meat. Future research will be conducted on the characterization of protein PTMs in postmortem WB to futher investigate their responses to WB conditions and their impact on WB meat quality. These protein biomarkers and their post-translational modified products will our knowledge of the the biochemical processes in postmorten WB and provide more insight on the development of WB and the potential strategies to reduce WB incidence.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, X., Antonelo, D.S., Hendrix, J.D., To, K.V., Campbell, Y.L., Von Staden, M.E., Li, S., Suman, S.P., Zhai, W., Schilling, M.W. 2020. Quantitative proteomic characterization associated with woody breast meat from broilers fed a standard or an amino acid-reduced diet. Meat and Muscle Biology. 4(1):9, 117.
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, B., Zhang, X., Schilling, M.W., Tabler, G.T., Peebles, E.D., Zhai, W. 2020. Effects of Broiler Genetic Strain and Dietary Amino Acid Reduction on (Part I): Growth Performance and Internal Organ Development. Poultry Science. 99(6): 3266-3279. https://doi.org/10.1016/j.psj.2020.03.024
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, X., Zhai, W., Li, S., Suman, S.P., Chen, J., Zhu, H., Antonelo, D.S., Schilling, M.W. 2020. Postmortem proteome changes in normal and woody broiler breast muscle. Journal of Agricultural and Food Chemistry. 68(39):11000-11010.
    • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, X., To, K.V., Jarvis, T.R., Campbell, Y.L., Hendrix, J.D., Suman, S,P., Li, S., Antonelo, D.S., Zhai, W., Chen, J., Zhu, H., Schilling, M.W. 2021. Broiler Genetics Influences Proteome Profiles of Normal and Woody Breast Muscle. Poultry Science. 100(4):100994. https://doi.org/10.1016/j.psj.2021.01.017.
    • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, B., Zhang, X., Schilling, M.W., Li, X., Tabler, G.T., Peebles, E.D., Zhai, W. 2021. Effects of Broiler Genetic Strain and Dietary Amino Acid Reduction on Meat Yield and Quality (Part II). Poultry Science. 100(4):101033. https://doi.org/10.1016/j.psj.2021.101033.
    • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, X., Zhang, L., Li, X., White, S.L., Campbell, Y.L., Ruiz, A.A., To, K.V., Zhai, W., Schilling, M.W. 2021. Cecal Microbiota Contribute to the Development of Woody Breast Myopathy. Poultry Science. 10.1016/j.psj.2021.101124


    Progress 05/01/19 to 04/30/20

    Outputs
    Target Audience:Poultry Processors; Poultry Industry; General Public; Academics in Food, Animal and Poultry Sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?TwoPhD students, one postdoctoral student, and one undergraduate have worked on this project and gained expertise in evaluating broiler meat quality, sensory analyses, and performing microbiome, transcriptomic, and proteomic techniques. An additional PhD student raised broilers and harvested them. This research has allowed students to interact with multiple poultry companies and present research at the 2019Reciprocal Meats Conference. In addition, 2 journal papers were published, 2 more were submitted, and 2 papers will be submitted this year. These papers are completed by this team of pHD students, postdocs, and undergraduate students. How have the results been disseminated to communities of interest?Twoabstracts have been accepted at the 2020International Congress of Meat Science and Technology. Two journal papers were published in peer reviewd journals. Two more journal papers are under review, and 2 journal papers are in progress. Six journal papers are in progress.Proteomic, microbiome, transcriptomic, and sensory analyses are completed. What do you plan to do during the next reporting period to accomplish the goals?Data will continue to be analyzed for microbiomeand transcriptomic results. The sensory research will be submitted to a journal. After data analysis is complete for the omic and microbiome data, manuscripts will be written and the data will be shared with the industry. Additional research will be planned that is based on the findings of this research and other woody breast meat research that has been conducted by researchers over the past 2-3 years.

    Impacts
    What was accomplished under these goals? Four proteins, including EH domain-containing protein 2, elongation factor 2, phosphoglycerate mutase 1 and, and T-complex protein 1 subunit gamma, were changed in both niormal breast (NB) and woody breast (WB) muscles during postmortem storage. Twenty proteins were uniquely changed in either NB (6 proteins) or WB (14 proteins) postmortem muscles, indicating the differences in their postmortem metabolism. In postmortem WB meat, the changes in protein degradation products were indicated by the presence of a desmin fragment, ovotransferrin chain A and troponin I chain I. In addition, a few glycolytic proteins in WB consisted of post-translational modified postmortem, including enolase, phosphoglucomutase-1, phosphoglycerate mutase 1 and pyruvate kinase. WB meat exhibited a greater number of changes in structural proteins, metabolic proteins, stress-related proteins, and transport proteins. In addition, WB had a greater rate of postmortem metabolism and a greater number of post-translational modifications postmortem due to the differences in antemortem conditions that exist in NB and WB. These differences indicate a faster and more oxidative muscle to meat conversion process for WB. These protein biomarkers and their post-translational modifications improve our knowledge on the biochemical processes in postmortem WB meat and provide novel insights on the development of WB and the potential strategies to reduce WB incidence, such as alleviating oxidative stress and regulating protein degradation and oxidation.

    Publications

    • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Zhang, X., Zhai, W., Li, S., Suman, S.P., Chen, J., Zhu, H., Antonelo, D.S., Schilling, M.W. 2020. Postmortem proteome changes in normal and woody broiler breast muscle. Journal of Agricultural and Food Chemistry.
    • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Zhang, B., Zhang, X., Schilling, M.W., Li, X., Tabler, G.T., Peebles, E.D., Zhai, W. 2020. Effects of Broiler Genetic Strain and Dietary Amino Acid Reduction on Meat Yield and Quality (Part II). Poultry Science.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Von Staden, M.E., Byron, M.D., Jarvis, T.R., Zhang, X., Williams, J.B., Crist, C.S., Schilling, M.W. 2019. Impact of woody breast severity on the sensory properties and acceptability of chicken. Reciprocal Meats Conference. Fort Collins, CO, June 2019. American Meat Science Association.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhang, X., Zhang, L., Hendrix, J.D., Zhai, W., Schilling, M.W. 2019. Characterization of caecal microbiota in broilers that differ in genetic strain, nutrition, and development of woody breast. Reciprocal Meats Conference. Fort Collins, CO, June 2019. American Meat Science Association.
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, X., Antonelo, D.S., Hendrix, J.D., To, K.V., Campbell, Y.L., Von Staden, M.E., Li, S., Suman, S.P., Zhai, W., Schilling, M.W. 2020. Quantitative proteomic characterization associated with woody breast meat from broilers fed a standard or an amino acid-reduced diet. Meat and Muscle Biology. 4(1):9, 117.
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, B., Zhang, X., Schilling, M.W., Tabler, G.T., Peebles, E.D., Zhai, W. 2020. Effects of Broiler Genetic Strain and Dietary Amino Acid Reduction on (Part I): Growth Performance and Internal Organ Development. Poultry Science. 99(6): 3266-3279. https://doi.org/10.1016/j.psj.2020.03.024
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhang, X., Hendrix, J.D., To, K.V., Campbell, Y.L. Von Staden, M.E., Suman, S.P., Li, S., Zhai, W. Schilling, M.W. 2019. Quantitative proteomic characterization associated with woody breast meat from broilers fed a standard or an amino acid-reduced diet. Reciprocal Meats Conference. Fort Collins, CO, June 2019. American Meat Science Association.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Schilling, M.W., Zhang, X., Von Staden, M.E. 2019. Effects of genetics and nutrition on the incidence of woody breast meat and use of proteome and microbiome analysis to characterize woody breast meat. Woody Breast Symposium. Perdue Foods. Salisbury, MD. July 30, 2019. Invited Presentation.


    Progress 05/01/18 to 04/30/19

    Outputs
    Target Audience:Poultry Processors; Poultry Industry; General Public; Academics in Food, Animal and Poultry Sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Twp PhD students, one postdoctoral student, and one undergraduate have worked on this project and gained expertise in evaluating broiler meat quality, sensory analyses, and performing microbiome, transcriptomic, and proteomic techniques. An additional PhD student raised broilers and harvested them. This research has allowed students to interact with multiple poultry companies and present research at the 2018 Reciprocal Meats Conference. In addition, 5 abstracts have been accepted for presentation at the 2019 Reciprocal Meats Conference. This research has also contributed to one student recieving an industry internship and another student to obtain an industry position. How have the results been disseminated to communities of interest?Five abstracts have been accepted at the 2019 Reciprocal Meats Conference. Two papers have been published in Carnetec. Results have been shared with multiple poultry companies in the Southeast as a written report. Six journal papers are in progress, and the plan is to have a minimum of 4 of those journal papers submitted in 2019. What do you plan to do during the next reporting period to accomplish the goals?Proteomic, microbiome, transcriptomic, and sensory analyses are completed. Data will continue to be analyzed for the proteomic, microbiome, and transcriptomic results. The sensory research will be submitted to a journal. After data analysis is complete for the omic and microbiome data, manuscripts will be written and the data will be shared with the industry. Additional research will be planned that is based on the findings of this research and other woody breast meat research that has been conducted by researchers over the past 2-3 years.

    Impacts
    What was accomplished under these goals? One-day-old mixed sex broilers from two strains (A2 and B2) were raised in 32 pens in a chicken house. Birds of each strain were randomly assigned to 16 pens (15 birds per pen) and 8 pens were fed a control diet and 8 pens were fed a reduced diet (20% reduction of digestible lysine, total sulfur amino acids, and threonine). After 8 weeks of growth, 4 male broilers with normal breast (1 chick per pen) and 4 male broilers with woody breast meat (WB,1 chick per pen) were selected for each treatment (breed × diet). The cecum samples were collected after birds were euthanized and bled to evaluate the gut microbiota. DNA was extracted, and amplified using universal primers that target the V3~4 regions of bacterial 16S rRNA for sequencing in Illumina MiSeq. These results revealed that the microbiota of each bird with normal breast was more similar to each other than the microbiota of birds with WB. In the ceca of WB birds Selenomonas bovis (12.6%) and Bacteroides plebeius (12.3%) were the top two predominant bacteria, however, the relative abundances of these two bacteria were only 5.1% and 1.2% in normal birds. It was determined that differences in the microbiome may be associated with the development of WB. Further studies are needed to investigate the potential mechanism and how to reduce broiler WB incidence by regulating their gut microbiota. Proteomics Apolipoprotein A-I, desmin, annexin A2, annexin A5, and ubiquitin carboxyl-terminal hydrolase were overexpressed (P<0.05) in WB. Peptidyl-prolyl cis-trans isomerase, four and a half LIM domains protein 1 isoform X3, and an uncharacterized protein were only present in WB muscle but not in normal chicken breast. There were two proteins, keratin, type II cytoskeletal 8 and alpha-1,4 glucan phosphorylase, that were overexpressed (P<0.05) in normal chicken breast. These differentially expressed proteins were involved in glycolytic metabolism, cell structure, cell defense, etc. The protein profiles of normal and woody chicken breast samples were different, which might help explain some of the changes in meat quality. Essential amino acid intake resulted in less difference in protein profiles between normal and woody chicken breast. Sensory Results indicated that WB severity had a greater impact on sensory attributes and consumer acceptability than genetic strain. Higher WB severity created an undesirable texture that negatively impacted the acceptability of baked meat. However, the increased crunchiness and cohesiveness due to woodiness had a positive impact on the fried chicken acceptability. Results indicated that a large percentage of consumers rated baked, fried, and fajita samples as acceptable regardless of whether NOR or WB (MOD, SEV) meat was used, but some consumers did not like baked or fajita meat that was made from SEV WB meat. Transcriptomics Transcriptomics research is complete and the data is being analyzed.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhang, X., Hendrix, J.D., Zhai, W., Suman, S.P., Schilling, M.W. 2018. The effects of genetics and nutrition on the incidence of woody breast meat and proteomic methods to characterize poultry meat quality defects. Pages 1-9. Conference Proceedings. Invited Concurrent Technical Session. Reciprocal Meats Conference. Kansas City, MO, June 24-27th. American Meat Science Association.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhang, X., Hendrix, J.D., Byron, M.D., Mukherjee, S., Campbell, Y.L. Zhai, W., Schilling, M.W. 2018. Broiler genetic strain and diet on the incidence of woody breast meat. Reciprocal Meats Conference. Kansas City, MO, June 2018. American Meat Science Association.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhang, X., Hendrix, J.D., Byron, M.D., Mukherjee, S., Campbell, Y.L. Zhai, W., Schilling, M.W. 2018. Broiler genetic strain and diet on the incidence of woody breast meat. USDA/NIFA AFRI A1341 and A1361, Program Directors Meeting. Boston, MA. June 2018.
    • Type: Other Status: Published Year Published: 2018 Citation: Hendrix, J.D., Zhang, X., Zhai, W., Schilling, M.W. 2018. The effect of genetic strain and diet on the incidence of meat quality and woody breast meat from broilers that are used for commercial production. CarneTec In Print. July, pp.1-5.
    • Type: Other Status: Published Year Published: 2019 Citation: Byron, M.D., Von Staden, M.E., Zhang, X., Dhowlaghar, N., Schilling, M.W. 2019. Impact of Refrigerated Storage Time on the Instrumental Quality Traits of Woody Broiler Breast Meat. CarneTec In Print. January, pp.1-6.


    Progress 05/01/17 to 04/30/18

    Outputs
    Target Audience:Poultry Processors; Poultry Industy, General Public; Academics in Food, Animal, and Poultry Sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One PhD graduate student and one postdoctoral student have worked on this project and gained expertise on evaluating muscle food quality, sensory analyses, and performing microbiome, transcriptomic, and proteomic techniques. An additional graduate studentrasied broilers and harvested them. This has helped themgain experience in poultry nutirion, genetics, and processing. This research will provide valuable information tothe broiler industry in the United States aswell as throughout the world. The postdoctoral student and graduate student presented this research at the 2018 Reciprocal Meats Conference and have discussed the research with poultry companies in the southeastern United States. They have enhanced their ability to explain scientific research to people in the industry and have enhanced their professional network and networking skills. How have the results been disseminated to communities of interest?One abstract and an Invited Conference Proceedings were published at the 2018 American Meat Science Association Annual Meeting (Reciprocal Meats Conference). There was also an Invited Symposium given at this conference to an audience of 200-300 people.An invited Keynote Address to an audience of approximately 800 was given on proteomics at the 2017 International Congress in Cork Ireland in Fall 2017. Two journal papers arealso in progressprogress and will be submitted during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?Two dimensional gel analyses will continue to be conducted. Mass spectrometric analysis will be conductedto identify and quantify proteins for the 2 dimensional gels. These gels are a protein profile of broiler breast meatfrom different broiler genetic strains that have been fed different diets.Transcriptomic analyses will also be conducted todetermine the expression of DNA in the breast muscle tissue, and microbiome data that had been taken for the different broiler strains will beanalyzed. Sensory descriptive analysis (n=8 panelists, replications=3) will also be conducted to determine the sensory descriptors and intensities forwoody and normal broiler breast meat. Consumer testing will be conductedto determine how the descriptive sensory attributes of normal and woody broiler breast meat impact the consumer acceptability of the breast meat.

    Impacts
    What was accomplished under these goals? Five strains of mixed-sex broilers (A1-3, B1-2; n = 240 per strain) were fed with commercial or reduced essential amino acid diets (20% reduction of digestible lysine, total sulfur amino acids, and threonine). A 5 (breeds) × 2 (diet) factorial analysis within a randomized complete block design with 8 blocks (10 pens per block) and 8 replicate pens (15 chicks per pen) of each treatment was utilized. At 8 weeks, 4 broilers from each pen were randomly selected and harvested in a lab scale commercial processing facility. A total of 32 broilers from each treatment were evaluated for live weight, carcass weight, breast weight, breast pH, and breast color. The WB of chicken breasts was evaluated where 0 = normal, 1 = slight, 2 = moderate, and 3 = severe. The pH was measured at 15 min and 24 h post processing, and color was measured 24 h post processing. Cook loss and shear force were also evaluated. B1 broilers that were fed the control diet expressed the WB condition with an average of 2.34, which was greater (P<0.05) than all other treatments with the exception of the A1 reduced diet treatment (P<0.05). Feeding broilers the reduced diet led to less WB incidence in B breeds (P<0.05) but had minimal impact (P>0.05) in the A breeds. This indicates that WB incidence wass impacted predominantly by genetics in both cases, but that diet had a greater impact on woody breast incidence in B in comparison to A genetic strains. There was greater than 25% severe WB in the A1 and B1 control diet treatments and the A1 reduced diet treatments. Feeding a reduced diet to B1 decreased incidence from 88 % to 28% for moderate and severe WB After 15 min of processing, A1 broilers had greater (P<0.05) pH (6.53-6.54) than B1 and B2 reduced diet treatments, but did not differ (P>0.05) from other treatments. The pH declined after 24 h of processing, and B1 fed with control diet had a greater (P<0.05) pH (5.96) than all other treatments (5.77-5.87). Differences existed among treatments (P<0.05) with respect to cooking loss and shear force, but all samples were tender (<45 N) and cooking loss was similar to values reported in literature. Feeding the reduced diet to B decreased (P<0.05) carcass weight when compared to control treatments; but feeding the reduced diet to A led to no difference (P>0.05) in carcass wight. For the control diet, B1 had a greater breast weight (P<0.05) than all other treatments. Feeding the reduced diet decreased (P<0.05) breast weight for all breeds with the exception of A1. Conclusion: A1 and B1 breeds had the greatest WB incidence. Feeding a reduced diet led to greater reductions in WB incidence and breast meat weight in B genetic strains when compared to A genetic strains. Proteomic, transcritome, and microbiota analyses are currently being conducted and data is being evaluated.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Schilling, M.W., Suman, S.P., Zhang, X. Nair, M.N., Desai, M.A., Kezhou, C., Ciaramella, M.A., Allen, P.J. 2017. Proteomic approach to characterize biochemistry of meat quality defects. Meat Science. 132:131-138.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kezhou, C., Shao, W., Chen, X., Campbell, Y.L., Nair, M.N., Suman, S.P., Beach, C.M., Guyton, M.C., Schilling, M.W. 2018. Meat quality traits and proteome profile of woody broiler breast Pectoralis major) meat. Poultry Science. 97(1):337-346.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Schilling, M.W., Suman, S.P., Zhang, X. Nair, M.N., Desai, M.A., Kezhou, C., Ciaramella, M.A., Allen, P.J. 2017. Proteomic approach to characterize biochemistry of meat quality defects. International Congress of Meat Science and Technology: Keynote Speaker Cork, Ireland. August, 2017.