Injury and Fibrosis at the Myoaponeurotic Junction of Pectoralis Major and Supracoracoideus Muscles in Broiler Chickens

Abstract

In this study, we aimed to identify the mechanism responsible for tissue degeneration and fibrosis in pectoral and supracoracoideus muscles. Ten chickens fed ad libitum broiler feed (Bro) were compared to 10 chickens fed breeding feed (Adj), which has lower metabolizable energy. The median body weight of Bro and Adj birds at 48 days of age was 4.9 and 0.9 kg, respectively. In Bro birds, hind legs were farther apart and tended to abduct, whereas their standing posture was often tilted forward, making them unstable. The two Bro males were heavier than the average, markedly less stable when standing or walking, and often flapped their wings vigorously to maintain balance. Myofiber damage and fibrosis were observed at the myoaponeurotic junction of the pectoralis major and supracoracoideus muscles in Bro birds. Myofiber damage and fibrosis were detected also in areas distal to the myoaponeurotic junction in the two heavier males but were otherwise less evident. By contrast, in Adj birds, almost no degeneration or fibrosis of muscle tissue was observed at the myoaponeurotic junction. In addition, the supracoracoideus muscle of one of the Bro birds showed coagulative necrosis of muscle tissue, surrounded by prominent fibrous tissue. Numerous incompletely formed blood vessels with irregular shapes and prominent branching proliferated in the fibrous tissue. These findings suggest that injury at the myoaponeurotic junction and abnormal capillary proliferation may be closely related to the formation of lesions, along with prominent fibrosis in the pectoralis major and supracoracoideus muscles.

Introduction

The primary function of the pectoralis major and supracoracoideus muscles in flying birds is to generate and control flight forces by the wings. The pectoralis major muscles present an intramuscular aponeurosis that originates from the deltopectoral crest of the humerus and is composed of a sternobrachial (extending from the aponeurosis to the keel) and thoracobrachial (extending to the dorsal area of the ribs) portions[1,2]. The supracoracoideus muscle originates from the sternum, coracoid bone, and sternocoracoclavicular membrane to connect the dorsal tubercle of the humerus via the triosseal canal[2]. The wing downstroke is caused mainly by contraction of the pectoralis major muscle, in which the intramuscular aponeurosis acts as an axis. By contrast, wing elevation is initiated by supination of the humerus via contraction of the supracoracoideus muscle at the end of the wing downstroke[3]. Lifting the wing does not require a large force; therefore, supracoracoideus muscles are smaller than the pectoralis major muscles[2]. Indeed, the pectoralis major muscles account for 17% of body weight, whereas the supracoracoideus for 2%–4% across various species[4]. Chickens can only fly short distances at low altitudes; hence, their pectoralis major muscles account for a lower proportion of body weight (<10%), although it has risen to >20% in newer breeds of broiler chickens[5,6]. Pectoralis major muscles develop more significantly in broilers than in flying birds; however, this is not due to functional necessity but rather due to breeding selection for birds with hyperplasia and hypertrophy of muscle bundles and myofibers[5,7]. In recent years, frequent occurrences of myodegeneration, often accompanied by effusion and fibrosis, known as wooden breasts, have been reported in highly hypertrophied pectoralis major muscles of broilers. However, the core mechanism of this syndrome has not yet been elucidated, and no mechanism has been identified to mitigate its occurrence. Even though myopathy has been found in the anterior latissimus dorsi and hind leg muscles, its etiology and relationship to wooden breast remain poorly known[8,9,10]. As myofiber degeneration/necrosis and regeneration accompanied by inflammatory cell infiltration and fibrosis have been observed in wooden breast muscles, myofiber damage is likely to be an important trigger.

In this study, we aimed to clarify differences in the incidence of damage at the myoaponeurotic junction of the pectoralis major and supracoracoideus muscles in broilers reared at different growth rates. Particularly, it focused on the intramuscular aponeurosis, which is subject to the largest mechanical load during contraction, as well as on whether myofiber degeneration and fibrosis occurred in supracoracoideus muscles.

Materials and Methods

Protocols and procedures for animal handling and sampling were approved by the Rakuno Gakuen University Institutional Animal Care and Use Committee (No. DH24A3).

Rearing management

Ten ROSS308 birds were fed ad libitum commercial broiler diets (Bro group), and 10 birds were fed a breeder-grower diet (Adj group) in amounts adjusted based on the weight gain curve presented in the ROSS308 breeder manual[11]. Bro birds were fed a diet with crude protein (CP) ≥ 20% and metabolic energy (ME) ≥ 3000 kcal from 0 to 12 days of age, followed by a diet with CP ≥ 19% and ME ≥ 3250 kcal from 12 days of age onwards. Adj birds were fed a diet with ≥19% CP and ≥2800 kcal ME throughout the experiment. From 0 to 12 days pf age, both groups were reared in brooder boxes, with 0.55 m² of floor space, whereas from 12 to 49 days of age, they were reared in a 1.44 m² concrete-floored pen covered with clean, soft sawdust litter. Before sampling, final body weight was measured at 48 days of age.

Thoracic width/length ratio

The width and length of chicken breasts were measured using photographs of the skinned breasts, as shown in Fig. 1, and the width-to-length ratio was calculated.

Fig. 1.

Breast width and length measurements.

Sample collection and histological analysis

The birds were euthanized by exsanguination under anesthesia at 49 days of age, at which point the breasts were skinned and photographed along with pectoral muscles. Muscle tissue samples were excised from the cranial parts of pectoralis major and supracoracoideus muscles overlying the cranial area of the sternum, and immersed in 10% formalin. Formalin-fixed tissues were trimmed to include areas containing the myoaponeurotic junction and were embedded in paraffin. Paraffin-embedded tissues were sliced into 3–4-μm-thick sections using a microtome. After drying, the sections were deparaffinized, stained with AZAN trichrome solution using a standard protocol, and observed under an optical microscope. The von Kossa method was used, as required.

Results

Clinical findings

At 48 days of age, the median body weight of Bro and Adj birds was 4.2 and 0.9 kg, respectively, whereas the mean body weight was 4.5 and 0.9 kg, respectively. The corresponding coefficients of variation were 10.4% and 16.6%. Body weight ranged around 4.2–5.4 kg for Bro males, 4.1–4.2 kg for Bro females, 0.8–1.2 kg for Adj males, and 0.7–1.1 kg for Adj females. The hind legs were farther apart in Bro birds than in Adj birds and both hind legs tended to abduct. The standing postures of the birds were slightly tilted forward and unstable. While walking, the birds were prone to losing balance in the front, back, left, and right directions when raising their legs. When birds are staggered, they often perform balancing maneuvers by spreading and flapping their wings to maintain an upright walking position. Such movements were observed in all Bro birds but were most pronounced in two males weighing 4.8 and 5.4 kg (Fig. 2 and Fig. 3).

Fig. 2.

Photographs of a male Bro bird weighing 4.8 kg. The bird’s legs tended to abduct and were often left in a splayed position. (A–F) Successive steps during recovery from the split position. (A) Bird with abducted hind legs, without balance, and forced to sit down. (B) The bird stands up and spreads its wings. (C–E) The standing bird flaps its wings vigorously to adjust its position. (F) The bird sits down immediately again.

Fig. 3.

Photographs of a male Bro bird weighing 5.4 kg. The bird had difficulty standing and walking and remained motionless when it put one leg forward. (A) The bird in a standing position, leaning forward and unsteady. (B) The bird sitting with the right leg forward. The bird tended to sit in this position. (C) After putting its right leg forward, the bird cannot lift its left leg, resulting in a standing position. (D) As the bird lifts one leg, it becomes unsteady, immediately flapping its wings violently to regain balance.

Thoracic width/length ratio and macroscopic findings

The breasts of the Bro birds were broader and chunkier than those of the Adj birds (Fig. 4). The thoracic width/length ratios for Bro and Adj birds were 0.66–0.83 and 0.46–0.54, respectively. Although statistical analysis was not performed owing to the small sample size, the thoracic width/length ratio did not appear to differ between males and females. In Bro birds, prominent milky-white stripes were observed along myofibers on the cephalad surface of pectoralis major muscles. Similar stripes were observed also in the supracoracoideus muscle of Bro birds but not in the pectoral and supracoracoideus muscles of Adj birds.

Fig. 4.

Representative photographs of the skinned breast of (A) Bro birds and (B) Adj birds.

The Bro male weighing 5.1 kg developed a thick, complex, unclearly delimited, grayish-yellowish-white, elastic, gelatinous tissue that surrounded the entire supracoracoideus muscle on both sides and adhered to the pectoralis major muscles and adjoining tissues. A large amount of blood was pooled from the gelatinous tissue. The areas of the supracoracoideus muscles surrounded by gelatinous tissue appeared pale greenish-yellow or similar to boiled meat, and exhibited signs of necrosis (Fig. 5).

Fig. 5.

Macroscopic and histologic findings of the supracoracoideus muscle. (A) Considerable blood leakage occurred when the pectoralis major muscle (P) was separated from the keel and the supracoracoideus muscle (S) was exposed. This image was taken after the spilled blood was removed. The supracoracoideus muscle was surrounded by a complex, thick, grayish-yellowish-white gelatinous tissue adherent to the pectoralis major muscle and other surrounding tissues. (B) Supracoracoideus muscles (S) were pale greenish-yellow and adhered to pectoralis major muscles (P) via a gelatinous tissue (*).

Histological findings at myoaponeurotic junctions

Structural injuries to myofiber termini and fibrosis were observed at the myoaponeurotic junction of pectoralis major and supracoracoideus muscles in Bro birds (Fig. 6). Myofiber termini forming the myoaponeurotic junction were rounded, with some having multiple shallow depressions, deep fissures or branches. Fibroblasts accumulated at these points and, together with collagen fibers, intruded into the fissures and branching grooves. In addition, thin collagen fibers and spindle cells proliferated between the myofiber termini and connective tissue of the aponeurosis. These areas were characterized by myofiber termini disconnected from the aponeurosis, multinucleated regenerating myofibers along degenerated or necrotic ones, ruptured myofibers, and myofiber splitting. In pectoralis major and supracoracoideus muscle tissues of most Bro birds, proliferation of intermuscular collagen fibers was observed at the myoaponeurotic junction, and intermuscular fibrosis decreased with increasing distance from the junction (Fig. 7). Irregularly shaped venous vessels and capillaries developed in areas of progressive fibrosis, and sporadic nodular lymphoid mononuclear cell infiltration was observed beside venous vessels (Fig. 8).

Fig. 6.

Representative histological preparations of myoaponeurotic junctions from the pectoralis and supracoracoideus muscles. (A) Supracoracoideus muscle of an Adj male bird, with body weight of 1.2 kg at 48 days of age: intimate connection between the myoaponeurotic junction and aponeurotic connective tissues, few signs of fibrosis between dense collagen fiber bundles and the myoaponeurotic junction. Diamond symbol: collagen fiber bundles of aponeurosis. (B) Supracoracoideus muscle of a male Bro bird, with body weight of 4.2 kg at 48 days of age: the myofiber termini branched out like fingers, and thin collagen fibers and spindle cells intruded into the grooves. Arrowheads: groove of myofiber termini with thin collagen fiber intrusion. Diamond symbol: collagen fiber bundles of aponeurosis. (C) Myoaponeurotic junction in the pectoralis muscle of the Bro bird shown in Fig. 2 (body weight of 4.8 kg at 48 days of age): myofiber termini detached from junctional tissue (asterisk), and bizarre shaped regenerated myofibers (arrow). (D) Pectoralis major muscle of the Bro bird in Fig. 2: detachment area of myofiber termini from the myoaponeurotic junction in bundles. Arrow: splitting myofiber. Asterisks: ghosts of shed myofibers. Diamond symbol: collagen fiber bundles of aponeurosis. Scale bars: 50 μm (A–C) and 200 μm (D).

Fig. 7.

Representative histological images of the myoaponeurotic junction (top) and a site approximately 1 cm away from it (bottom). Pectoralis major muscles were obtained from Bro birds with (A) severe and (B) mild fibrosis. (C) Adj birds showing no fibrosis in the pectoralis major muscles. Supracoracoideus muscles of Bro birds with (D) severe and (E) mild fibrosis. (F) Adj birds showing no fibrosis in the supracoracoideus muscles. Scale bar: 200 μm.

Fig. 8.

Typical histological images of blood vessels in normal muscle tissue (pectoral muscle of an Adj bird) and fibrotic muscle tissue (pectoral muscle of a Bro bird). (A) Normal muscular tissue: very little fibrous tissue between myofibers and thin fibrous tissue of the perimysium. (B) Enlarged image of the perimysial area framed in panel A showing inconspicuous capillaries between myofiber bundles. Vn: venous vasculature; Lc: lymphoid mononuclear cells. (C) Fibrotic muscle tissue between myofibers and in the perimysium area. (D) Enlargement of the area framed in panel C: thicker Vn walls, infiltration of Lc beside the Vn, well developed capillaries (Cp) and adipocytes (Ac) in the fibrous area. Scale bars: 200 μm (A, C) and 20 μm (B, D).

In the two birds uncapable of maintaining stable posture while standing or walking (Fig. 2 and Fig. 3), fibrosis was apparent in the pectoralis major and supracoracoideus muscles. In addition, degenerative changes and fibrosis were observed even in areas distant from the myoaponeurotic junction in these muscles.

In Adj birds, the termini of myofibers at the myoaponeurotic junction in both the pectoralis major and supracoracoideus muscles were closely connected to the collagen fibers of the aponeurosis over the entire surface, and degeneration of myofibers and fibrosis in these areas was hardly observed. Rounded and branched myofiber termini were sparsely distributed at the myoaponeurotic junction.

No considerable diffuse inflammatory cell infiltration or regenerating myofibers with abnormal nuclear chains were observed in any of the sampled muscle tissues.

Histology of the necrotic supracoracoideus muscle and surrounding thick gelatinous tissue

In one of the Bro birds, the supracoracoideus muscle was surrounded by thick gelatinous tissue and underwent coagulative necrosis. The muscle was composed of normally shaped myofibers, but histological examination using AZAN staining revealed a uniform bluish-purple color, rather than the normal myofibers usually stained reddish-pink hue. However, nuclei were presented in these muscle fibers. Thin myofibers were sparsely distributed within the thickened fibrous tissue and were stained mostly reddish-pink. The areas extending from the aponeurosis to the fascia were significantly thicker and comprised disordered fibrous tissue. In these fibrous tissues, spindle-shaped or polymorphic epithelioid cells with coarse chromatin and large nuclei were scattered or clustered between collagen fibers, as well as numerous incomplete capillaries with irregular shapes and prominent branching. In the large blood vessels adjacent to necrotic muscle tissue, myxoid capillary proliferation was observed in the inner walls, and some blood vessels showed calcification in the vascular walls (Fig. 9). In the peripheral regions of muscle tissue undergoing coagulative necrosis, macrophages infiltrated the cytoplasm of necrotic myofibers or the polygonal giant cells surrounding them. Significant fibrosis was observed in the distal area of necrotic muscle tissue, with thin myofibers embedded and dispersed in fibrous tissue. In fibrotic areas, lymphoid mononuclear cells infiltrated and formed nodules along venous blood vessels, together with numerous incomplete capillaries with irregular branching (Fig. 10).

Fig. 9.

Histological preparations of the muscle tissue underwent coagulative necrosis. (A) Myofibers showing bluish-purple coloring with AZAN stain, along with coagulative necrotic muscle tissue (Fig. 5). Myofibers scattered through the fibrotic areas were reddish pink (arrows). Irregularly shaped capillaries proliferated between necrotic muscle tissue and fibrotic tissue (arrowhead). (B) Incompletely formed capillaries with irregular shapes and prominent branching proliferated in a myxoid background containing collagen fibers. Red blood cells had spilled into myxoid areas around capillaries. (C) Intravascular papillae with myxoid polymorphous capillaries proliferated along the inner walls of large blood vessels adjacent to areas of necrotic muscle tissue (enlarged picture). (D) Calcification of blood vessel walls. Photographs in the boxes show calcium deposits (von Kossa method). Scale bars: 200 μm (A–C) and 100 μm (D).

Fig. 10.

Same supracoracoideus muscle as in Fig. 5 and Fig. 9. (A) Area showing intense phagocytosis of coagulative necrotic muscle tissue (*). (B) Nodular lymphoid mononuclear cell infiltration beside venous blood vessels in fibrotic areas. (C) Multiple macrophages in the cytoplasm of necrotic myofibers (*) and surrounding multinucleated giant cells. (D) Numerous pleomorphic capillaries with multi-branched proliferated in fibrotic areas. Scale bars: 200 μm (A), 50 μm (B, C), and 20 μm (D).

Discussion

Even before attention was drawn to the so-called “wooden breast” in the pectoralis major muscles, rapid growth rate chickens tended to the formation of fatty deposits in the pectoralis major muscles, and degeneration occurred in the supracoracoideus muscles[12,13,14]. These facts suggest that in broilers, in which the rates of weight gain and breast yield continue to improve year after year, some physiological dysfunction in skeletal muscles was present even before the first recognition of the wooden breast.

The pectoralis major muscles originate from the deltopectoral crest of the humerus, the pars sternobranchialis terminates at the carina of the sternum and sternoclavicular ligament, whereas the pars costobrachialis terminates at the clavicle and coracoid bone. The supracoracoideus originates from the dorsal tubercle of the humerus and terminates at the sternum, coracoid bone, and sternocoracoclavicular membrane. Flapping is achieved by alternating the pectoralis major and supracoracoideus muscles. Flapping forces are generated when myofibers that form pinnate angles join the aponeurosis and contract, pulling it caudally with both sides. As a result, the pulling force generated by muscle contraction is concentrated at the myoaponeurotic junction. The myotendinous junction can be torn by excessive stretching force[15]. This junction is often damaged in the human hamstring muscle when suddenly and excessively stretched; such injuries are prone to recurrence and require long recovery[16]. In chickens, sudden violent exercise or muscle overload can damage muscles or cause aponeurosis. Broilers, which are particularly heavy and unstable when standing or walking, often stretch their wings wide and flap vigorously to maintain balance. This puts them at high risk of injury to pectoralis major and supracoracoideus muscles. Additionally, areas with longer myofibers may generate greater pulling forces and are more likely to damage myoaponeurotic junctions. Degenerative changes and muscle tissue fibrosis, which are associated with wooden breasts, tend to occur in cranial ventral (epidermal) areas[9,17]. In the present study, fibrosis and damage to myofiber termini were observed at the myoaponeurotic junction in the cranial ventral areas of the pectoralis major and supracoracoideus muscles of Bro birds, which were markedly heavier than those of Adj birds. By contrast, Adj birds displayed no such lesions. At myoaponeurotic junctions of Bro birds, myofiber termini were connected to collagen fiber bundles of the aponeurosis via sparse tissue composed of spindle cells, including fibroblasts and thin collagen fibers. This observation suggested that these myoaponeurotic junctions were mechanically weaker than those in Adj birds. As shown in Fig. 6D, the fascicular separation of myofiber termini from the aponeurosis was likely due to excessive loading between adjacent myofiber termini and the aponeurosis. Disruption of myoaponeurotic junctions and myofibers ruptured during myofiber fascicle dissection can also lead to widespread muscle injury.

Of the 20 birds studied herein, one Bro bird showed coagulative necrosis of the supracoracoideus muscle, which had a boiled meat-like appearance that, at first glance, resembled deep pectoral myopathy. However, the area surrounding the boiled meat-like muscle contained a large amount of blood and was covered with a grayish-yellowish-white gelatinous tissue that adhered intricately to the surrounding muscle, giving the primary impression macroscopically that its nature differed from that of deep pectoral myopathy.

Histologically, the area affected by coagulative necrosis maintained proper myofiber structure and the presence of nuclei. However, it was unique in that it stained almost uniformly blue-purple with AZAN stain. Phagocytosis by macrophages and other cells was observed at the edge of coagulative necrotic muscle tissue, and the fibrous tissue was highly developed in the distal region. These histological findings suggest that the lesion was primarily a chronic granulomatous inflammation caused by reaction to the coagulative necrotic muscle tissue.

Coagulative necrosis of the supracoracoideus muscles, also known as deep pectoral myopathy, green muscle disease, or Oregon disease, has been documented in turkeys and broilers since 1968[18]. In chronic lesions of deep pectoral myopathy, macroscopically, necrotic muscle is shrunken, uniformly green, dry, brittle, and surrounded by a fibrous capsule. Histologically, the nuclei of myofibers are lost and an inflammatory reaction involving heterophils, macrophages, and giant cells is observed around the necrotic tissue, forming a fibrous capsule[19].

Here, the presence of nuclei was confirmed in the necrotic myofibers, which contrasts with previous observations in deep pectoral myopathy[19]. This discrepancy could be explained by less efficient nuclear staining of necrotic cells when using other dyes. Macroscopic and histological features identified the lesion as deep pectoral myopathy. Although this study could not clarify whether extensive coagulative necrosis of the supracoracoideus muscle occurred centrally at the myoaponeurotic junction, we confirmed that the latter was within the coagulative necrotic muscle tissue area. Adjacent to this tissue were also large blood vessels, whose inner walls presented intravascular papillae with myxopolymorphic capillaries (Fig. 9C). However, it is unclear whether these lesions contributed directly to muscle tissue necrosis. Further studies on similar lesions should clarify their occurrence.

The mechanisms underlying abnormal fibrosis in parenchymal organs have been reported in the case of liver fibrosis and idiopathic pulmonary fibrosis. In liver fibrosis, capillarization associated with liver dysfunction in hepatic sinusoidal endothelial cells is thought to induce angiogenesis and persistent activation of hepatic stellate cells, thereby contributing to the progression of fibrosis and hepatocellular injury[20,21,22]. Moreover, in idiopathic pulmonary fibrosis, damaged alveolar capillary endothelium associated with alveolar destruction contributes to abnormal angiogenesis and promotion of fibrosis[23].

In the excessive fibrotic lesions associated with coagulative necrosis of the supracoracoideus muscle, incomplete and irregular capillary blood vessels proliferated markedly. Considering that capillaries were abundant in fibrotic areas of pectoral muscles (Fig. 8), this suggests that excessive proliferation of blood vessels may be closely related to the development of fibrotic lesions (i.e. wooden breast) in skeletal muscles. Liver fibrosis and idiopathic pulmonary fibrosis are also suggested to induce and progress secondary from damage to parenchymal tissue[21,23]. If skeletal muscle fibrosis progresses through a mechanism similar to that of hepatic fibrosis and idiopathic pulmonary fibrosis, physical damage to myofibers and secondary capillary proliferation may be important factors in fibrosis of pectoralis major and supracoracoideus muscles in broilers.

In conclusion, this study identified damage at the myoaponeurotic junction as a risk factor for the development of fibrosis in broiler muscle tissue. Importantly, it suggests that the occurrence of these injuries could be limited by adjusting body weight through dietary control. Future studies should evaluate other skeletal muscles, further exploring the factors that predispose them to disorders and fibrosis. This would advance our understanding of the mechanisms underlying the development of skeletal muscle abnormalities, as well as possible means to prevent muscle damage associated with fibrosis.

Acknowledgements

This study was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (#24K09214 to HY, #21K05942 to TW, and #24K01912 to TI), a Grant-in-Aid from the Cooperative Research Fund (2022–03) of Rakuno Gakuen University, and a research fund from Kawavet LLC, which owns the Research Office Concerning the Health of Humans and Birds.

Author Contributions

Supervision of rearing and poultry house environmental management: MY. Sampling: NM, TW, NT, YH, TK, TI, and RK. Specimen preparation: TK. Pathological examination: TI, and TK. Manuscript writing: TK. Manuscript discussion: TI, TW, NT, and YH. Review: TW, NT, YH, NM, RK, and TI.

Conflicts of Interest

The authors declare no conflict of interest.

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