PACHOL4

D.D. del MUR n. 104 del 02/02/2022

Codice progetto MUR: 2022EPWEPW

UE funding: 133.160,00 €

Duration: 12/10/2023 - 11/10/2025

Coordinator: DISTAL, University of Bologna

UNIBO Role: PI

Scientific Officer: Francesca Soglia

Research group: Francesca Soglia, Martiza Bordini, Federico Sirri, Martina Zappaterra

Research areas: Animal sciences

In the past decade, a group of growth-related abnormalities affecting the Pectoralis major muscles of fast-growing broiler chickens selected for meat production purposes has abruptly emerged all over the World with impressive incidence levels and severity. Among these spontaneous defects, the White Striping and Wooden Breast conditions are worth to be mentioned. These abnormalities often coexist within the same muscle, manifest with distinctive phenotypes and exhibit peculiar histological features which resemble those associated to myopathic conditions in humans. In addition, their occurrence remarkably affects the quality traits of the forthcoming breast meat (the most valuable portion of the chicken carcass) resulting in its downgrading and leading to economic losses at processing. Within this context, PACHOL4 project aimed at deepening the knowledge concerning the molecular processes and pathways involved in the occurrence of the growth-related abnormalities (with special reference to the Wooden Breast defect) affecting the Pectoralis major muscles of fast-growing broilers with high incidence rates. In detail, this proposal, built on a recently formulated hypothesis, aimed at evaluating whether an intracellular accumulation and/or an altered extracellular arrangement of collagen type IV (COL4) may represent the primary cause underlying the occurrence of growth-related defects.

Objectives and Purposes

The project aims to deepen the understanding about the etiology of specific meat quality defects, known as “white striping” (WS) and “wooden breast” (WB), affecting the breast muscle of broiler chickens selected for fast growth-rate and high breast-yield. Specifically, PACHOL4 intends to investigate the involvement of a particular type of collagen, Collagen type IV, in the biological and molecular processes underlying these abnormalities. Given that altered synthesis and/or composition of this protein has been hypothesized to be associated with the onset of WS and WB, the project includes analyses to assess potential differences in its syn-thesis and composition in affected and unaffected samples, as well as to identify genetic factors linked to their manifestation through transcriptomic studies, with the ultimate goal of identifying genes and regulatory elements involved in the development of such defects

Expected results

The results achieved by PACHOL4 will contribute to improve the knowledge concerning the white striping and wooden breast underlying mechanisms and might lead to the identification of potential biomarkers useful for excluding broilers more prone to the development of these conditions from breeding programs. Finally, considering the similarities between these defects and the poorly understood muscular disorders in humans, the potential use of Gallus gallus as an animal model may be explored to investigate the muscular defects associated with Collagen type IV abnormalities

Results achieved

The planned experimental activities have been completed and allowed the achievement of all the goals pursued by the project.

One of the major challenges commonly faced when studying the mechanisms underlying the onset and progression of the growth-related abnormalities in broilers is the likelihood of having the concurrent presence of more than one defect within the same Pectoralis major muscle that may affect the outcomes of the investigation resulting in a misleading interpretation of the results. Within this framework, multivariate analyses (i.e., Principal Component Analysis, PCA and Hierarchical Cluster Analysis, HCA) were performed with the data concerning meat quality traits and technological properties to identify those variables that better allow to discriminate between normal and abnormal samples.

Based on these results, further analyses at the protein level were conducted to characterize the collagen fraction of both normal and abnormal samples and quantify the expression of the alpha 1 and alpha 2 chains composing collagen type IV heterotrimer. The quantification of these proteins allowed to test the hypothesis of the existence of differences in their expression level that may be associated (or at least concur) to the development of the growth-related myopathies (with special reference to the wooden breast condition) in broiler chickens. In addition, immunohistochemistry highlighted that, on top of the differences in the amount of the translated proteins found between normal and abnormal muscles, even their localization differed thus corroborating the hypothesis of their involvement in the pathogenesis of these conditions.

Subsequently, transcriptomic analyses were performed to identify differentially expressed genes and miRNAs. Differential gene expression analysis evidenced a total of 193 differentially expressed genes, of which 174 up- and 19 down-regulated in abnormal muscles. Results coming from the functional enrichment analyses indicated an integrated cellular dysfunction in myopathic muscles, particularly involving the muscle structure and metabolism, as well as the neuromuscular transmission and response to cellular damage. In terms of differentially expressed microRNAs, a total of 18 miRNAs were found, four of which were significantly correlated with genes identified as differentially expressed in our samples. Among those, miR-21-5p and miR-21-3p are highly conserved miRNAs that have been extensively studied in human medicine due to their involvement in various pathological conditions. Specifically, in humans, miR-21–5p has been found associated with different metabolic diseases, such as diabetes, as well as with cardiac fibrosis. In pigs, miRNA-21 has been described as myogenic miRNA involved in skeletal muscle development by regulating PI3K/Akt/mTOR signaling pathways. Overall, despite years of efforts to mitigate growth-related myopathies, our results largely highlighted persisting alterations in terms of muscular and neuromuscular functions in abnormal muscles, suggesting the persistence of still unknown triggering factors underlying the observed gene expression patterns. Also, these results corroborate the evidence of a profound metabolic dysregulation as molecular hallmarks of this condition. Potential microRNA-mRNA regulatory mechanisms suggested epigenetic factors potentially involved in this metabolic dysregulation, with miRNA-21 having an important contribution to the alterations in abnormal muscles.

All that considered, the results achieved in PACHOL4 project allowed to shed new light on the knowledge about the causes underlying these breast meat defects thus ultimately paving the way to new strategies to control their onset and their negative consequences in poultry production.

Last but not least, considering the similarities existing between these abnormalities and muscular disorders in humans, Gallus gallus may be potentially proposed as a spontaneous alternative animal model for studying the pathogenesis of these conditions which currently requires the use of artificially induced laboratory animals.