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Chicken Health: Preventing wooden breast by identifying and editing regulators of severe chicken myopathy

Alternative title: Kylling Helse: Reduksjon av wooden breast ved å identifisere og editere regulatorer av kyllingmyopati

Awarded: NOK 12.1 mill.

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2021 - 2025

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The main goal of Chicken-Health is to elucidate the role of relevant extracellular matrix (ECM) components, with a particular focus on the proteoglycan family syndecans in wooden breast (WB) severe chicken myopathy, and if they can serve as therapeutic targets. In addition, understanding syndecan molecular mechanisms in WB will help to establish reliable and appropriate strategies, not just to understand but to be able to treat the disease. A PhD student has been allocated in the project, and presentation of scientific results at MBE2022 extracellular matrix conference and international visit performed. A kick-off meeting together with 2 national WP-meeting and 1 international at Stony Brook University between consortium partners has been performed. A draft of a scientific paper for the PhD student is in the beginning. Planning for short-term research stay for the PhD-student regarding molecular mechanism is initiated. WP1: A chicken model with feeding-induced WB at 36-day growth has been successfully performed. Skeletal muscle, heart, blood, and primary cells were collected. NIR-spectroscopy and palpation of skeletal muscle samples were used for scoring the level of wooden breast. Furthermore, HE histology together with WGA and Picro-Sirius Red was used to confirm groups based on the level of fibrosis. NIR-spectroscopy and grouping by histology showed similar pattern regarding WB-detection, but with some discrepancy for some of the individuals. Comprehensive extracellular matrix remodeling was observed between normal and WB-affected samples. RNA- sequencing and bioinformatic cluster analysis pointed out syndecans to be located in clusters defining WB-affected samples. Proteomic analysis is on-going to verify these bioinformatic clusters at protein level. A distinct expression pattern of the syndecan family members has been verified at mRNA by real-time PCR and protein analysis by western in support of the RNA-seg data. The expression of syndecan family members differed in normal and WB-affected samples, both at mRNA and protein level. Altogether, these findings support the hypothesis of syndecan family as relevant candidates to be addressed in further in vitro cell cultures of primary cells isolated in the chicken trial. WP2: A syndecan-1,2,3 and-4 chicken specific ab tool-box has been developed by antibody production and specificity testing by peptide blocking, laying the foundation for in depth molecular analysis and investigation of this family group as potential regulators of WB-myopathy. Screening of commercial antibodies with specific staining for chicken has also been performed and still ongoing, laying the foundation for a toolbox for skeletal muscle fibrosis in chicken. WP3: Primary cells were isolated for further in vitro investigation of molecular mechanisms at the cellular level in the project period. Growth media and cell culture conditions have been optimized. WP4: Investigating the profile of promising biomarkers of wooden breast in blood samples has started. WP5: Combining NIR-spectroscopic analysis, transcriptomics and further molecular depth analysis at the individual level is initiated to increase knowledge of syndecan potential regulatory role in WB and fibrosis.

Intensive breeding and genetic manipulation have led to severe myopathic defects named Wooden breast (WB). Chicken suffering from these illnesses usually must be withdrawn from the food chain due to insufficient food quality and less nutritional value. The underlying molecular mechanisms of WB is still only partially understood. Morphological characteristics of WB demonstrate severe skeletal muscle fibrosis, necrosis and multifocal degeneration of muscle tissue. Fibrosis in general is characterized by extensive extracellular matrix (ECM) remodeling and is caused by a dysfunctional muscle satellite cell activity in combination with an imbalanced regulation of fibroblast/myofibroblast activity. A family of ECM receptors called syndecans are known to be involved in human skeletal muscle myopathies, they regulate fibrosis in cardiac muscle and have crucial functions in normal MuSC and fibroblast activity. These molecules have a wide spectrum of biological functions and are master regulators of tissue homeostasis, and gene expression of this family is up- regulated in WB chicken. However, no studies of syndecans and their possible role in WB exist. And more importantly, how can these complex molecules be modified to avoid developing the disease, and at the same time retaining functions important for key cellular processes? The main goal is to elucidate the role of syndecans and relevant ECM components in severe chicken myopathy and if they can serve as therapeutic targets. Understanding syndecan molecular mechanisms in WB will help establish reliable and relevant strategies, not just to understand but to be able to treat the disease. In addition, this project has a potential for a broader societal impact with benefits to the food industry, public sector and civil society in a longer term. Reducing WB will secure healthy chickens without suffering, will provide healthy meat for humans and will contribute with a solution to the increasing protein demand of the future.

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