Broiler breast muscle myopathies: association with satellite cells

Function of Satellite Cells in the Regeneration of Myofibers

Satellite cell-mediated repair results in the regeneration of existing myofibers or the generation of new myofibers. The regeneration process is initiated when the sarcolemma (plasma membrane) of the myofiber is damaged and creatine kinase levels increase. With the degeneration of the myofiber, there is influx of calcium into the myofiber with the activation of calpain. Once the myofiber undergoes necrosis, the cellular debris is phagocytized with an influx of neutrophils and macrophages.

For satellite cell repair mechanisms to regenerate the damaged myofiber, the satellite cells must first be activated and return to the cell cycle from a state of quiescence. After the growth period, satellite cells, in general, will withdraw from the cell cycle and remain in a state of quiescence. Upon activation, the cells must then be able to proliferate and differentiate into muscle specific structures. The environment surrounding the satellite cells, satellite cell niche, will affect satellite cell activity including proliferation and differentiation through its composition of extracellular matrix proteins, growth factors, and vascular supply (Christov et al., 2007; Bi and Kuang, 2012). Satellite cells need to be within 21 µm of capillaries to proliferate and sufficient vascularization is needed for muscle to regenerate (Christov et al., 2007; Rhoads et al., 2009).

The p. major muscle is a fast twitch anaerobic muscle and has lower vascularization due to it being an anaerobic muscle compared to oxidative slow-twitch muscles (Velleman, 2015). As selection has increased breast muscle mass largely based on hypertrophic growth resulting in larger diameter muscle fibers, the connective tissue spacing needed to separate fiber bundles and for circulatory supply has been starved out. When myofibers and fiber bundles begin to touch one another, degeneration begins (Velleman et al., 2003). If the amount of circulatory supply is insufficient, satellite cell mediated repair will be impeded affecting the regeneration of myofibers back to their original state.

Another factor affecting satellite cell-mediated regeneration of muscle is their heterogeneity. Satellite cells are not a uniform population of cells but are a dynamic stem cell population with variable proliferation and differentiation rates (Kuang et al., 2007). Changes in the satellite cell population over time can include the expression of cell surface receptors (Xu et al., 2023) which will modify the interaction of the cell with its satellite cell niche environment. Although the heterogeneity of satellite cells is not completely understood, we do know that satellite cells are not one homogenous population even within a same fiber type muscle like the p. major muscle (McFarland et al., 1995).

Effect of Selection for Breast Muscle Mass Accretion on Satellite Cell Activity in Broilers

Wooden Breast affected breast muscle is characterized by myofiber necrosis, inflammatory cell infiltration, and fibrosis (Sihvo et al., 2014; Velleman and Clark, 2015). Breast meat quality is the direct result of the morphological structure and cell biology of the muscle. Faster growing heavy weight broilers have a higher degree of degenerating muscle fibers associated with the presence of excessively enlarged myofibers from satellite cell-mediated hypertrophic growth. In commercial poultry, selection for growth has largely been determined posthatch when growth is from the satellite cells not the myoblasts. Satellite cell growth is through the donation of their nuclei to existing fibers increasing protein synthesis capabilities of existing fibers resulting in fiber enlargement not the generation of new fibers. For heavy weight fast-growing broilers, the increase in breast muscle mass has been correlated with larger muscle fiber cross sectional area due to hypertrophic growth (Berri et al., 2007).

As cross-sectional area of the myofibers has increased both circulatory supply and available connective tissue spacing have been significantly reduced. In the case of Wooden Breast affected birds, oxidative stress has greatly increased (Abasht et al., 2016). With the increase in myofiber diameter, when fibers and fiber bundles begin to make contact, degeneration occurs evoking satellite cell-mediated repair mechanisms. To regenerate damaged and necrotic myofibers, the satellite cells will be activated to proliferate and differentiate forming a new fiber identical to the originating one. The regeneration process is well regulated. To reassemble a muscle fiber requires the formation of parallel array of microfibrils which are composed of repeating units of contractile sarcomeres. The primary components of the sarcomere are the Z disks, actin thin filaments and myosin thick filaments. The regeneration of the muscle fiber is completely dependent upon the activity of satellite cells. Thus, if satellite cell proliferation and differentiation is suppressed, muscle fiber regeneration will be negatively impacted affecting breast meat quality.

In the case of Wooden Breast, Velleman et al. (2018) showed in affected p. major muscle that the diameter of the myofibrils was significantly smaller with disorganization of sarcomeric structure. Figure 1 contains representative images illustrating the scoring of sarcomere structure as it relates to the organization of the sarcomere. The high degree of smaller diameter fibers with sarcomere disorganization is of significant concern regarding the ability of Wooden Breast affected muscle to regenerate muscle fibers back to their original state. Thus, in Wooden Breast, degenerating and necrotic muscle will lead to fibrosis with the replacement of muscle with extracellular matrix proteins including collagen and proteoglycans, and fat deposition. The structural stability of muscle including its ability to stretch and hold water is determined by the extracellular matrix composition. Fibrosis results in an overproduction of primarily the fibrillar collagens, Types I and III, but it is the organization especially the crosslinking of these collagens once they form collagen fibers that will determine the effect on meat tenderness.

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Figure 1. Representative transmission electron microscopy images for sarcomere organization scoring from 1 to 5. (A) Score of 5 shows sarcomere with normal structure; (B) score of 4 means mildly altered; (C) score of 3 means further disorganization from a score of 4 but still moderate; (D) score of 2 means severely altered sarcomere structure; and (E) score of 1 indicates complete absence of sarcomere structure. A = A zone; M = M line; Z = Z line. Bar = 1 µm. Reproduced from Velleman et al. (2018).

To further understand the impact of collagen organization on the broiler breast meat, a series of research studies were undertaken by the Velleman group. Collagen fiber structure was accessed in 3 fast-growing commercial broiler lines with varying detection of Wooden Breast by palpation. The lines are identified as Lines A, B, and C. Detection of Wooden Breast by palpation was high in Line A, intermediate in Line B, and not detectable in Line C. In Line A with Wooden Breast detectable by palpation, the collagen fibers were closely packed in parallel arrays whereas Line B had a diffuse structure of the collagen fibers (Figure 2; Velleman and Clark, 2015; Velleman et al., 2017). Interestingly, microscopic examination of the p. major muscle showed that 70% of the birds in the study had necrosis and fibrosis with only a small percentage scored as being Wooden Breast positive by hand palpation. Thus, hand palpation and scoring will only detect those birds with a stiffened and hard breast muscle, but not with microscopically detectable necrosis and fibrosis.

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Figure 2. Masson trichrome staining of collagen organization in Wooden Breast-unaffected and -affected pectoralis major muscle. (A, C, and E) are representative images of Wooden Breast-unaffected pectoralis major muscle from Lines A, B, and C, respectively. (B, D) are representative images of Wooden Breast-affected pectoralis major muscle from Lines A and B, respectively. The arrows highlight fibrillar collagen and the boxes contain enlargements of the fibrillar collagen. Scale bar = 100 µm. Reproduced from Velleman et al. (2017).

Effect of Selection for Growth on Satellite Cell Activity in Broilers

Growth selection strategies have been similar in commercial chickens and turkeys (Havenstein et al., 2003, 2004, 2007), but p. major muscle necrotic and fibrotic myopathies like Wooden Breast have not been reported to date in heavy weight fast-growing meat-type turkeys (Zampiga et al., 2020). Since the ability to regenerate muscle fiber structure is dependent on satellite cell activity and suppressed in current commercial broilers, the question arises as to how selection for growth has affected satellite cell function in heavy weight commercial chickens compared to the turkey. Thus, myopathies like Wooden Breast may be associated with differences in the myogenic and regenerative potential of p. major muscle satellite cells. As shown by Xu et al. (2021), satellite cells isolated from the p. major muscle of modern commercial turkeys have greater proliferation and differentiation compared to satellite cells isolated from historic slower growing turkeys representing older commercial lines.

To study how selection for growth has been potentially altered satellite cells isolated from a modern commercial broiler line was compared to a Cornish Rock and Randombred chicken lines from the 1990s (Xu and Velleman, 2023). Modern commercial broiler satellite cells had decreased proliferation and differentiation compared to the Cornish Rock and Randombred chicken lines. In contrast, modern heavy weight fast-growing p. major muscle cells from a meat-type turkey have significantly higher proliferation and differentiation compared to the historic slower growing commercial line turkey satellite cells (Xu et al., 2021). These data are strongly suggestive that modern commercial meat-type chickens have decreased myogenic activity of their satellite cells which would reflect in decreased regenerative potential that may be associated with the development of necrotic and fibrotic myopathies like Wooden Breast. As put forth by Bailey (2023), industry management strategies are now placing emphasis on maintaining optimal levels of satellite cell proliferation immediately after hatch to aid in the control of p. major muscle myopathies.

Effect of Selection for Breast Muscle Yield: Fast Compared to Slower Growing Broilers

In recent years, there has been a consumer move toward heritage type or slower growing meat-type broilers as their meat quality are perceived to be better. This perception is, in part, driven by meat quality issues like Wooden Breast, the phenotypic appearance being negatively impacted by White Stripping, and the large size of the p. major muscle. Despite conditions like Wooden Breast having a low heritability (Bailey et al., 2015), how the p. major muscle grows during hyperplasia and hypertrophic growth must be monitored as these growth periods will determine the morphological structure of the muscle. It is certainly attractive to believe that moving toward slower growing birds will address many of the breast muscle pathophysiological conditions affecting meat quality in broilers. These conditions like Wooden Breast are recent in occurrence and associated with heavy weight fast-growing broilers. Since meat quality is the result of the morphological structure of the muscle and the cellular growth of the muscle cells, selection practices need to begin to include more muscle cell biology. Inclusion of muscle cellular biology will likely reduce the incidence of detrimental myopathies negatively impacting meat quality. Thus, approaches utilized in selection programs should encompass both muscle growth from hyperplasia and hypertrophy, and the overall muscle fiber organization of the breast muscle.

The activation, proliferation, and differentiation of satellite cells is a critical area that must be evaluated in the selection of birds to reduce incidence of degenerative, necrotic and fibrotic myopathies. As shown by Xu and Velleman (2023), the proliferation and differentiation potential of satellite cells has significantly declined in modern commercial broilers compared to older lines from the 1990s. Wooden Breast, for example, is characterized by persistent damage to the muscle fibers and inflammation, instead of the fibers being able to regenerate back to their original state they are replaced with fat and connective tissue including highly cross-linked fibrillar collagen. The impact of reduced satellite cell activity is supported by Wooden Breast-affected muscle having a greater amount of small myofibers (Clark and Velleman, 2017) with disorganization of sarcomere structure (Velleman et al., 2018).