PATHOMORPHOLOGICAL AND MEAT QUALITY ALTERATIONS CONNECTED WITH WOODEN BREAST IN BROILER CHICKENS OF DIFFERENT GENOTYPES AND SLAUGHTER AGES

This study exa mined pathomorphological changes and meat quality alterations associated with Wooden Breast Myopathy (WB) in total of 192 broiler chickens divided into Ross 308 (n=96) and Cobb 500 (n=96) heavy hybrids at ages 42, 60, and 70 days. WB occurrence remained consistently high (>73%) across periods, peaking on day 70 (83% for Ross, 90% for Cobb). Cobb broilers had better production results and carcass traits parameters after day 42 and day 60 of the experiment (p ≤ 0.05). Genotype did not affect WB occurrence or severity, while slaughter age in ﬂ uenced severe cases WB occurrence, increasing from 11.67% on day 42 to 36.67% on day 70 (p=0.003). The presence of WB was associated with higher ultimate pH, lightness (L*), redness (a*), and yellowness (b*) of the muscle (p<0.0001), except on day 70. Physicochemical and color parameters were also in ﬂ uenced by slaughter age (p<0.0001). On day 42, drip loss (p<0.0001), cooking loss (p ≤ 0.05) and shear force (p<0.0001) were affected by genotype. On days 60 and 70, the differences in water retention capacity were observed only between normal and severely affected breasts (p<0.0001). For each slaughter age severely affected WB had higher shear force compared to normal breasts (p<0.0001). Additionally, with the increasing slaughter age of broilers, drip loss, cooking loss, and shear force of the breast meat were increased (p<0.0001). The results obtained regarding the occurrence and severity of WB and its consequent meat quality alterations suggest that extended fattening is not recommended for poultry production


INTRODUCTION
Selection of broilers for fast growth and high yield led to rapid muscle hypertrophy coupled with inadequate vascularization and an increase in white striping (WS), wooden breasts (WB), and spaghetti meat (SM) myopathies [1,2].These muscle abnormalities affect the pectoralis major (p.major) muscle, the most valuable chicken part in the broiler industry, resulting in the downgrading of broiler breasts and enormous economic losses to the poultry industry worldwide [3].Macroscopically, a WB is characterized by palpably hard, pale ridge-like bulges, accompanied by a clear, viscous fl uid, small hemorrhages, and white striping, which may manifest individually or concurrently.The underlying histological lesions of WB are polyphasic myodegeneration characterized by degenerative and atrophic fi bers, vacuolar degeneration, occasional regeneration, mononuclear cell infi ltration, interstitial infl ammation, and accompanying fi brosis and lipidosis [4].Wooden breast myopathy impairs not only breast meat appearance, but also its quality.Due to visual defects and unappealing texture, consumers exhibit low acceptance of WB fi llets as raw meat products.Furthermore, the compromised functional properties resulting from increased fat and connective tissue, along with the loss of functional proteins, restrict further processing and decrease the nutritional value of abnormal meat [1,[5][6][7].The occurrence of WB myopathy is most frequent in fast-growing, high-breast-yielding strains of broilers.The onset and severity of WB also seem to be higher in broilers that are male, fed high-energy diets, or slaughtered at older ages and heavier weights [8][9][10][11][12].
Despite the fact that WB myopathy is a worldwide problem, to author's knowledge, there are no available reports on the occurrence of WB in broilers in Serbia.Given that the broiler rearing system in Serbian households relies on an extended fattening period for broilers, with some birds reaching up to 10 weeks of age, the objective of this study was to compare the occurrence and severity of wooden breast myopathy in the two most common fast-growing broiler lines in Serbia, Ross 308 and Cobb 500, at 42, 60 and 70 days (d) of age.Furthermore, histological features and meat quality of normal broilers and broilers affected by WB were compared.

MATERIALS AND METHODS
The study was approved by the Ethical Committee for Animal Experimentation (Faculty of Veterinary Medicine) of the University of Belgrade, Serbia (Approval number: 10-5/2022).

Animals, housing and diets
The experiment was conducted at the registered animal breeding facility, during the period April -June 2023, Obrenovac, Ljubinić (44°32′17″N 20°03′19″E).A total of 192 one-day-old chickens of both sexes were taken from the same incubator station (Agrovet d.o.o., Žička 8, Beograd) by authorized transport means to the farm.Half of the chicks (96) belonged to a Ross 308 hybrid; the other half were from Cobb 500 genotype.On arrival, chicks were individually weighed and 16 chicks were housed in each of 12 pens made of solid wood and a fence (stocking density 6 birds/m 2 ), randomly allocated to experimental groups, i.e., 2 genotypes (Ross 308 vs. Cobb 500) × 3 slaughtering ages (42 d, 60 d and 70 d).Each pen had a concrete fl oor covered with 5 cm wood shaving.The temperature in the room was 32°C from days 1 to 5, then gradually lowered to 22°C on day 21.This temperature was maintained until the end of the study.The artifi cial lighting program 16 hours of light, 8 hours of dark during all experimental periods was used.To enable broilers to reach their maximum production potential, water and feed were available ad libitum.The feeder and drinker were circular with manual fi lling.During the experiment, the birds were fed standard commercial diets based on corn and soybean meal (Table 1) in a mash and granulated form of the rearing phases: starter (1-21 days) and fi nisher (22-70 days).Health conditions and mortality were monitored throughout the experiment.Individual weights were recorded at different ages, i.e., days 0, 21, 42, 60, and 70 and weight gain was calculated for each period.

Slaughter procedure
A total of 30 birds per genotype at 42, 60 and 70 d of age (180 in total) were slaughtered in a commercial slaughterhouse, after about 8h of feed withdrawal and about 4h of water withdrawal.Immediately before slaughter, individual measurements were performed and after electric stunning, broilers were slaughtered by cutting the jugular veins.After 24h chilling, the carcasses were measured and subjected to gross examination to evaluate the occurrence and degree of WB in the p. major muscles.Then, the carcasses were cut into basic parts and deboned breast meat (fi llets) was taken for further histology and meat quality assessment.

Macroscopic scoring
Immediately after chilling, the separated pectoral musculature was categorized based on the degree of expression of WB changes according to Tijare et al. [6] by bilateral palpation of the breast meat (p.major) in the craniocaudal direction.Depending on the degree of hardness and the area affected by the change, breast fi llets were divided into four categories (Figure 1): grade 0 (normal) = fi llets of elastic consistency, without any changes in color or consistency; grade 1 (mild) = normal fi llets with the subtle focal changes that were harder in consistency and pale in color, mostly in the cranial part compared to rest of the muscle; grade 2 (moderate) = changes in the cranial part of the muscle more pronounced than in the previous category, covering the entire cranial part of p. major which is much harder in consistency and pale in color.The middle and caudal part of the muscle without changes; grade 3 (severe) = extremely hard and rigid fi llets with diffuse changes, affecting the entire muscle; the presence of transparent, mucous content on the surface of the fi llet can also be noticed.After scoring, each fi llet was individually weighed, photographed on top of a white sheet labeled with an individual case identifi cation number and subjected to histology evaluation.

Histology
For histology evaluation of WB myopathy, samples of pectoral musculature were taken from all macroscopically examined fi llets.As WB is considered to be a bilateral disease and predominantly manifest in the cranial section of the fi llet, samples for histological analysis were taken from the cranial part of the right m. pectoralis major superfi cialis, size 1×1×1 cm.All tissue samples were fi xed in 10% buffered neutral formalin, standard processed and embedded in paraffi n blocks, then cut at a 4 μm thickness for evaluation of the muscle fi bers morphology, presence of myodegeneration and regeneration using hematoxylin and eosin (H&E) staining.Evaluation of the perimysial and endomysial distribution of connective tissue, specifi cally collagen was achieved using Masson's Trichrome staining.Immunohistochemical staining was conducted using EnVision FLEX Mini Kit based on Peroxidase/DAB method [13] with anti-CD3 antibody (monoclonal mouse anti-human F.7.2.38; DAKO, Glostrup, Denmark) and anti-CD21 antibody (monoclonal mouse anti-human Clone 1F8 ready to use; DAKO Glostrup, Denmark).
The histological scoring was conducted with a light microscope (BX51, Olympus Optical, Japan), and pictures were taken with Olympus Color View III ® digital camera.
Myopathic lesions were scored using four scale system by Trocino et al. [11]: grade 0 (normal) = presented samples with typical polygonal shape and noticeable cross striation of fi ber on cross-section, normal or central nuclei, without necrotic fi bers or infi ltration of connective tissues; grade 1 (mild) = represents the morphology of fi bers as in score 0, but can be detected some fi bers with hyaline cytoplasm and central nuclei or necrotic fi bers, connective tissue infi ltration normally distributed in endomysium and perimysium; grade 2 (moderate) = samples with diffusely organized necrotic fi bers, evident thickening of interstitial connective tissue, detection of infl ammatory cells, and aggregates of adipocytes; the highest score, grade 3 (severe) = samples that exhibited a signifi cant amount of interstitial connective tissue, myofi brillar degeneration represented with hyalinized fi bers that have lost cross striation, as well necrotic and fragmented fi bers of various size with surrounding macrophages or heterophilic granulocytes, and great amount of adipose tissue).

Meat Quality Analysis
The L*a*b* color indexes were measured on the ventral side of the fi llet (L* -lightness, a*-redness, and b*-yellowness) using a Minolta CM-2600d spectrophotometer (Konica Minolta, Ramsey, NJ, USA).For each sample, the meat color result was determined as the average of three measurements taken from parts of the fi llets that were devoid of obvious color defects such as bruises, discolorations, hemorrhages, or any other conditions that might have interfered with uniform color readings.The meat pH was measured in triplicate on the cranial parts of fi llets using a portable pH meter (Testo 205, Testo AG, Lenzkirch, Germany).Drip loss in meat samples from the p. major muscle was assessed using the "bag" drip loss method [14].Briefl y, each meat sample was weighed and stored for 24h at 4°C in a container.After storage, the meat samples were reweighed, and the percentage of drip loss was calculated.Cooking loss was assessed by initially weighing each sample individually.Subsequently, the samples were wrapped in aluminum foil and placed in a convection air oven once the temperature reached the predetermined value of 175°C.The cooking temperature was maintained for a duration of 45 minutes.After completion of the cooking process, the samples were allowed to cool at room temperature for one hour before being subjected to analysis for cooking loss.This was expressed as the percentage difference in meat weight after cooking, with the initial weight before cooking considered as 100%.Shear force evaluation was conducted on cylinder samples taken from the center of each fi llet, longitudinal to the muscle fi bers (1.27 cm in diameter).A Warner-Bratzler blade, using testing machine Texture Analyser TA XP (Stable Micro System, Godalming, England), was used to shear the samples across to the muscle fi bers.Ten measurements were performed on each sample to obtain mean values expressed as shear force (N).

Statistical Analysis
Data are presented as the mean ± SEM and analyzed using SPSS software package version 20.0 (SPSS Inc., Chicago, USA).Physicochemical parameters and meat quality data were evaluated using a General Linear Mixed Model (GLMM), with genotype, myopathy degree, and slaughter age as fi xed effects and the pen as a random effect.For productive performance characteristics and carcass traits, the independent samples t-test was used, while the intergroup comparisons for all data were appraised by one-way Analysis of Variance (ANOVA) followed by Tukey's multiple comparison tests.Differences were considered signifi cant at p≤0.05.In the regression models, genotype and slaughter age were assigned as independent variables.Binary logistic regression was employed to identify associations between the independent variables and the development of WB (non-WB = 1, WB = 2).Multinomial logistic regression described the effects of independent variables on WB degree (normal = 1, mild WB = 2, moderate WB = 3 and severe WB = 4).The results from the logistic regression analysis were reported as odds ratios including 95% CI and the respective p-values for each variable.The odds ratio >1 indicates an increased chance whereas <1 denotes a decreased chance of a dependent category as a result of an increase in the independent variable by one unit.

Macroscopic Findings
Macroscopically, p. major muscles affected by WB appeared pale, outbulging, and hardened.The affected areas exhibited petechiae or slightly larger hemorrhagic foci and were often covered with a clear viscous fl uid, predominantly localized to the cranial end of the breast muscles.Upon examination, a signifi cant disparity was noted between samples categorized as having moderate and severe cases of WB compared to unaffected samples.Within both the Ross and Cobb groups, all cases classifi ed as severe WB exhibited diffuse lesions.Conversely, samples categorized as mild to moderate WB displayed more focal lesions, primarily distributed in the cranial parts of the fi llets (Figure 1).

The occurrence and degree of wooden breast in relation to genotype and slaughter age
The occurrence and degree of wooden breasts in different genotypes and slaughter age are shown in Graph 1, Graph 2, Supplementary Table 1, 2 and 3.The genotype did not affect (p>0.05) the occurrence and degree of wooden breast (Graph 1, Suppl.Table 2).On the contrary, slaughter age affected the occurrence of wooden breasts, whereby the highest (p=0.003)percentage of severe degree was recorded on day 70 (11.67 vs. 36.67%,Graph 2, Suppl.Table 3).
At day 42 of experiment, the WB occurrence was approx.73% in both genotypes (Supplementary table 1).Both genotypes on day 70 had more than 85% affected cases of WB, without signifi cant differences between groups (p>0.05).The occurrence of WB in Cobb broilers on day 70 was 90% from which 40% were classifi ed as severe.In Ross broilers on day 70, 33% of p. major were evaluated as WB severe category.Marked discrepancy between the right and left p. major muscles in the presence and degree of the WB lesion was not noted.

Histologic Findings
On a histological level, p. major muscle macroscopically classifi ed without myopathy, revealed a typical polygonal shape and uniform size of myofi bers on cross section, with nuclei mostly peripherally located, with no presence of myopathic lesion, fi brosis or lipidosis (Figure 2 a, b).Samples classifi ed as mild on gross appearance showed myofi bers in some areas surrounded by a sparse amount of connective tissue within endomysium, scattered infl ammatory cells and rare areas of adipose tissue accumulation (Figure 2 c, d).All samples categorized as moderate and severe WB in both broiler groups, as determined by macroscopic examination, were confi rmed to exhibit severe myodegeneration and fi brosis with high number of hyalinized and necrotic fi bers, accompanied with regeneration (Figure 2 e, f, g, h).Conversely, some of the fi llets classifi ed as unaffected based on macroscopy revealed only mild myodegeneration and infi ltration without evident fi brosis.Some adipose tissue was present in all histologically evaluated cases, with evident increased amount of perivascular adipose tissue and myodegeneration in WB of moderate and severely affected samples.
Furthermore, a diffuse thickening of the perimysial network was observed, accompanied by variable amounts of loose connective tissue, granulation tissue, or collagen-rich connective tissue, indicative of fi brosis, which separated the muscle fi bers (Figure 3).
In all cases of wooden breast, perivascular infl ammatory cell infi ltration, primarily around the veins, was consistently observed.This infi ltration exhibited an irregular pattern and was predominantly composed of lymphocytes, occasionally resulting in disruption of the vascular wall.Immunohistochemical staining with the anti-CD3 antibody showed strong positivity, primarily in the cytoplasm, of perivascular cells (Figure 4).Additionally, occasional cells within the interstitium showed positivity for the anti-CD21 antibody.

Productive performance and carcas traits of broiler chicken in relation to genotype and slaughter age
The effects of genotype and slaughter age on the productive performance of broiler chickens are shown in Table 2. Genotype affected the productive performance in broiler chickens, whereby Cobb had higher (p<0.0001)body weight on all sampling days, except (p>0.05) on day 70.In addition, Cobb had higher (p<0.0001)daily weight gain during the fi rst and second periods.
Effects of genotype and slaughter age on carcass traits of broiler chickens are shown in Table 3.Most of the carcass traits differed (p≤0.05) between genotypes, except (p>0.05) for cold carcass weight and breast weight on day 70.Cobb had higher cold carcass weight (p<0.0001 and p=0.0040, respectively), dressing percentage (p=0.0189and p=0.0007, respectively), and breast yield (p<0.0001) on days 42 and 60.In contrast, Ross had higher breast yield (p<0.0001) on day 42 and dressing percentage (p<0.0001)on day 70.

DISCUSSION
The objective of the present study was to evaluate the effect of three slaughtering ages and genotype on the rates and degree of WB, changes in breast muscle at the histological level, and meat quality in two widely used fast-growing strains of broiler chickens.
Concerning the WB, in the present study the incidence of this myopathy in both genotypes was high (between 73% on day 42 and 83-90% on day 70), as reported by Cruz et al. [15] (89.2%).Contrarily, under experimental conditions, Trocino et al. [11] reported a lower WB occurrence of approximately 24% (8.0% in females and 16.3% in males) in broilers slaughtered at 46 d of age.As for the slaughter age, continuous fattening increased the fi nal body weight of chickens and WB rates.In fact, the occurrence of WB has been reported to increase as the birds became older and heavier [16], as observed in the present study.Moreover, Che et al. [17] reported increased live weight as a risk factor for WB occurrence due to fi ber hypertrophy and reduced capillary density, ultimately leading to elevated WB rates in heavier birds.Thus, similarly to the present fi ndings, Tijare et al. [6] found 96.1% of breasts showing WB in broilers slaughtered at 61 d of age.Indeed, the results from the combined database of 4,332 broilers pooled from 7 research experiments conducted at Texas A&M University showed that the occurrence of WB in broilers of 8 weeks of age could be as high as 86% [18].The generally high rates of myopathies reported in the studies conducted in controlled environmental conditions, such as in our study, could be due to the ideal growing conditions [3,6] favoring the development of these muscles abnormalities.Due to the relatively small number of broilers used in the present trial, a larger-scale study is required to verify the high WB rates reported herein.Even in industrial conditions, during gross examination of 1,920 breasts from 9-week-old broilers, approximately 85% were found to display signs of WB, of which, similarly to our fi ndings, more than 42% were severe or very severe [16].Moreover, Xing et al. [19] reported a relatively high WB rate (61.9%) under commercial conditions in China.Regarding myopathy scores, prolonged fattening resulted in decreased rates of normal and mild scored WB and increased occurrence of moderate and severe WB in both genotypes.The prolonged fattening increases the probability of severe WB in both genotypes.The impact of extended fattening on carcass traits is usually consistent with the results of fi nal live weight, as found in the present study.Thus, the increase in the rate and degree of myopathies during the experiment was likely related to the increased weight and yield of p. major, as previously reported [12,20].
Regarding genotype, Cobb had a higher body weight and weight gain during the fi rst 42 days of the experiment.The better productive performance for Cobb than Ross broiler chickens reported in the present study corroborates previous trials [21,22] reporting Cobb broilers to exhibit superior body weight.However, these differences between genotypes disappeared during fattening at the end of the experiment (day 70).Despite the higher breast weight, Cobb chickens had lower breast yield than Ross birds on day 42 of age.In contrast, on days 60 and 70 of the study, Cobb broiler chickens had a higher breast yield than Ross ones.Differences in the production results are likely due to genetic potential factors, as suggested by Olanrewaju et al. [23].Nevertheless, despite the differences in the body weight and breast yield between the two genotypes, no effect of the genotype on WB was found on day 42, as previously reported by other authors [11,24], nor later during fattening.Contrarily, Hammemi et al. [22] reported a higher rate of WB myopathy in Cobb than in Ross chickens, with Ross 308 chickens having a higher rate of mild WB (22.5% vs.15.62%) and Cobb 500 higher occurrence of moderate (4.38% vs. 12.5%) and severe (1.88% vs. 3.12%) WB.
Although the results of the WB occurrence among the studies are inconstant, likely due to different factors such as age [16], sex [11,12], genotype [22], diet and feeding strategies [11,25], fi nal weight [18], etc., the histological fi ndings reveal damaged muscle fi bers in over 97% of breasts examined [4,7,26].In agreement, several macroscopically normal p. major muscles from the present study exhibited mild myodegeneration and infi ltration without evident fi brosis at the microscopic level.Microscopic lesions reported in p. major muscle affected by WB from the present study corroborate previous histological fi ndings [7,11,26].Some studies reported initial microscopic features of WB in the p. major muscles could be found in broilers as early as two weeks of age [27,28].Papah et al. [29] reported phlebitis to appear in p. major muscle as early as in the fi rst week of life, before other WB microscopic lesions.Namely, these authors proposed that impaired venous drainage may lead to local accumulation of metabolic waste and ROS, triggering myofi brillar degeneration and necrosis.As in the present study, vasculitis and perivascular infi ltrations of infl ammatory cells (mostly CD3 T lymphocytes) have been observed in the breasts of market-age birds affected by WB [4,11,28,29].However, it is worth noting that multiple authors [27][28][29] reported the presence of robust lymphocytic phlebitis in p. major without other characteristics lesions for WB and vice versa.The etiology and pathogenesis of WB and other myopathies onset are under extensive investigation.Available data suggest that genetic selection of broilers for the hypertrophic growth of the muscle fi bers and inadequate vascular support lead to the impaired oxygen and nutrient supply to the muscle tissue on one side, and poor drainage and accumulation of metabolic by-products on the other [2].This mechanism is supported by transcriptomic data showing altered expression of genes involved in carbohydrates and lipid metabolism, remodeling of the extracellular matrix, infl ammation/immune response, fatty acid beta oxidation, and oxidative phosphorylation [30][31][32].
In agreement with the present fi ndings, other authors [7,27,33] reported a reduction in muscle fi bers in p. major with WB, loss of typical polygonal shape, an abnormally rounded fi bers with nuclear internalization, loss of cross striations, vacuolar degeneration and necrosis of fi bers coupled with fi brotic response in form of proliferation and diffuse thickening of endomysium and perimysium network, variable amount of loose connective tissue and granulation tissue.The severity of microscopic lesions increased as the degree of WB increased from normal to severe, reaching the highest levels of myodegeneration in p. major affected by severe WB.Microscopic fi ndings of myodegeneration, accompanied by regeneration, have been previously associated with the WB [27].In agreement, in the present study, necrotic fi bers detected in the myofi bers at all defective degrees of WB were surrounded by regenerating fi bers.Namely, the disruption of the sarcolemma due to myofi ber degeneration initiates necrosis from the infl ux of calcium from the sarcoplasmic reticulum, triggering an immune response [34], as confi rmed in the present study where infi ltration of CD3immunoreactive cells was found.Following tissue damage, activated satellite cells undergo proliferation and differentiation and engage in fusion with existing myofi bers or new fi bers synthesis, thus facilitating muscle regeneration [35][36][37].Depending on the niche environment, these multipotent cells can follow myogenic or adipogenic pathways [35,37,38].Considering adequate vascularization to be imperative during muscle regeneration, hypoxic conditions in WB cause transdifferentiation of satellite cells to adipocytes, which might explain, at least partially, the deposition of fat in WBaffected muscles, as demonstrated by Emami et al. [37].Other authors also reported adipocyte infi ltration in p. major muscle with WB [7,11].Muscle fi brosis, the structural hallmark of the WB, found in previous and the present study, is a secondary process generated as a response to muscle necrosis resulting in the progressive overproduction and deposition of fi brillar collagens Types I and III in the perymisial and endomysial connective tissue spaces [19,34].
Alterations to the morphological structure of the muscle has been linked to changes in meat chemical composition, decreased nutritional value and impaired meat quality [1,7,39].
The higher ultimate pH of p. major affected by WB (regardless of the degree of WB severity), compared with the normal muscles, is in line with previous reports [5,7,16,40].In this regard, proteomic data suggest the downregulation of carbohydrate metabolic pathways related to reduced glycolysis, gluconeogenesis, tricarboxylic acid cycle, glycogen degradation, and pyruvate fermentation to lactate to reduce glycolytic potential and elevate ultimate pH in defected muscles [16].In addition, previous fi ndings found that the degree of the myopathy can infl uence meat pH [41], as observed in the present study for day 42, with no differences between degrees of WB at later ages.As for the age, although it would be expected for older birds with greater breast weight and higher fi ber diameter to have a reduced in vivo glycogen storage and, thus, exhibit higher ultimate pH as previously reported [42], in the present study the decline in ultimate pH of p. major was reported.Nevertheless, in agreement with present fi ndings, Schneider et al. [43] [11,24].
Regarding color, meat lightness increased in abnormal p. major muscles compared to the normal counterparts, as seen before [41].However, the color did not differ between breasts affected by different degrees of WB.Contrarily, the yellowness and redness increased in breasts affected by WB, as found by other authors [5,16,41].
Corroborating previous fi ndings, the degree of WB increased b* values [16].These alterations in color indices are likely due to the accumulation of fat (along with liposoluble pigments from the feed) in abnormal breasts and the altered light refl ection caused by edema and changed muscle tissue structure [5,41].As for redness, increased redness in WB-affected muscles could be due to the high expression of myoglobin genes [46].Overall, redness and yellowness of breast meat decreased with age of the birds, whereas lightness increased, regardless of the myopathy degree.It is worth noting that changes in color of p. major muscle were evident between birds of 42 and 60 days of age, whereas minor color differences were reported between broilers slaughtered on days 60 and 70.The literature data on age-related color changes in breast meat are inconsistent.Opposite to present fi ndings, previous reports showed that the older chicken muscles were darker [42,47,48] and redder [47,48] than the chicken muscles of younger birds.However, corroborating present fi ndings few authors [47][48][49] reported the tendency of the breast meat to show a lower yellowness and redness due to the decrease in the heme pigment content, as birds get older.As for genotype, although the broiler line was found to be a relevant factor in color indices of breasts, as previously reported by other authors [49,50] when considering birds' age, there was no difference in color between genotypes.Similarly, Hammemi et al. [22] and Gratta et al. [24] did not fi nd a color difference between the breasts of two genotypes slaughtered at 35 and 45 days of age, respectively.
Considering fl uid losses, contrary to the expectation that muscles affected by WB may increase water-holding capacity due to the higher ultimate pH, in comparison to normal breast, abnormal muscles had higher drip and cooking loss, in agreement with available literature data [7,16,51,52].These losses could be attributed to the alteration in the structure of the p. major, mainly degeneration of muscle fi bers, thus decreased myofi brillar proteins, accompanied by accumulation of interstitial connective tissue seen at the microscopic level [7].Indeed, the present study found that the fl uid losses were generally higher in breasts with severe WB, whereas the moderate and mild degrees of WB did not signifi cantly increase drip loss except for day 42, nor cooking loss except for day 60, confi rming that ability to retain liquids depends on the severity of lesions in the muscle tissue.The imparted ability of WB muscles to bind water infl uences their marinade uptake limiting further processing of abnormal meat [5].
Regarding slaughter age, drip and cooking losses increased over time and were higher in older birds, as expected due to the increased muscle damage and occurrence of myopathies.Overall, the genotype did not infl uence drip losses, except on day 70 when the muscles of Cobb exhibited lower drip loss than Ross chickens.
Finally, the shear force of cooked p. major was affected by the presence of severe WB, associated with the high levels of collagen crosslinking due to fi brosis of WB-affected muscles [34] and higher cooking losses in such meat.The absence of the myopathy effect on breast toughness in mild and moderate affected WB breasts could be due to the cooking effect on shear values [53].Other authors [7,54,55] reported the same tenderness in cooked normal and WB breasts, regardless of the condition degree.Indeed, corroborating the present study, some authors found WB-affected meat to exhibit a higher hardness than the normal fi lets [12,39].Furthermore, the difference in shear force and cooking loss were observed between genotypes for day 42, with Ross exhibiting higher p. major toughness and lower ability to retain liquid compared to Cobb, likely due to a higher occurrence of severe WB in former compared to later.From this moment onward, the differences in meat cooking loss between genotypes were not evident as severe WB rates in Cobb increased during prolonged fattening, whereas the differences in hardness disappeared after day 60 of the experiment.As for slaughter age, the shear force of muscles increased with age, probably as a result of greater fi ber size [56] and increased collagen cross-links in older birds [57].

CONCLUSION
As for production performances and carcass traits, Cobb 500 genotype had higher body weight and breast weight, except for day 70, and greater breast yield during all trials than Ross 308 birds.The occurrence of WB was high (73-90%) but was not signifi cantly different between genotypes.As for birds' age, the occurrence of severe WB increases with age.The presence of WB, especially severe WB, impaired meat quality in terms of altered color, decreased ability to retain liquid, and increased shear force compared to normal chicken breasts, which can have repercussions for the processing of such meat.Moreover, gross examination proves to be effective as the primary method for detecting WB in moderate and severe cases, while mild cases may require subtler confi rmation methods such as microscopic evaluation.In the present study, both macroscopy classifi ed as severe and moderately affected WB were scored as severe at the histological level, sharing microscopic lessons including nuclear internalization, hyaline and vacuolar degeneration, infi ltration and marked fi brosis.In conclusion, considering myopathy occurrence and severity, as well as meat quality alterations, extended fattening is not recommended for poultry production.

Figure 1 .
Figure 1.WB categorization based on macroscopic fi ndings.Grade WB0 (normal)unaffected muscle of normal color and consistency; Grade WB1 (mild) -subtle presentation of focal myodegenerative lesions in cranial area of the muscle surrounded by unaffected muscle; Grade WB2 (moderate) -the entire cranial part of the muscle exhibits diffuse involvement characterized by hardened consistency and pale coloration; Grade WB3 (severe) -the fi llets exhibit extreme hardness and rigidity with diffuse changes, impacting the entire muscle (bilateral in presentation).

Graph 1 .
Occurrence (%) of chickens showing wooden breast (total, mild, moderate, severe): effect of genotype (the reference category is normal score) Graph 2. Occurrence (%) of chickens showing wooden breast (total, mild, moderate, severe): effect of slaughter age (the reference category is normal score)

Figure 4 .
Figure 4. a -P.major, broiler.Accumulation of mononuclear cells within the perivenular region, exhibiting mild infi ltration into the vein wall, with no discernible alterations observed in the arteria.HE. b -P.major, broiler.The perivenular mononuclear cells stain intensely with CD3 antibody, primarily within the cytoplasm mononuclear cells cytoplasm.Immunohistochemistry for CD3.

Table 2 .
Productive performance (Mean±SE) of broiler chickens in relation to genotype and slaughter age.

Table 3 .
Carcass traits (Mean±SE) in broiler chickens in relation to genotype and slaughter age Different superscripts within the same row indicate a significant difference (p < 0.05).

quality of broiler chickens in relation to genotype, WB degree and slaughter age
Effects of genotype, WB degree, slaughter age, and their interactions on physicochemical parameters of the p. major muscle of broiler chickens are displayed in Table4.The effect of genotype on meat color traits (L* and a* values, p=0.016 and p=0.014, respectively) was found.However, observing these parameters at separate slaughtering ages did not reveal a difference between Ross and Cobb broilers (p>0.05).The presence of WB, regardless of the degree of severity, was associated (p<0.0001) with higher ultimate pH, lightness (L*), redness (a*), and yellowness (b*) of p. major muscle, except on day 70 when no differences in pH and lightness were observed between meat affected by WB and normal breasts.Further, slaughter age affected (p<0.0001)physicochemical and color parameters, whereby ultimate pH, redness (a*), and yellowness (b*) decreased, while lightness (L*) increased with broiler age from day 42 to 60.Additionally, the differences between days 60 and 70 were less notable and limited to meat yellowness and pH.Effects of genotype, WB degree, slaughter age, and their interactions on water-holding capacity and textural traits of the p. major muscle of broiler chickens are depicted in Table5.Genotype affected the water-holding capacity and textural traits; Cobb had lower drip loss (p<0.0001) on day 70, cooking loss (p=0.020) on day 42, and shear force (p<0.0001) on day 42 and 60 than Ross chickens.Cooking loss and shear force were higher in severely affected WB than in normal breasts, whereas mild and moderate WB exhibited intermediate values.In addition, slaughter age affected (p<0.0001)waterholding capacity and textural traits, whereby drip loss, cooking loss, and shear force increased with broiler age, with noticeable differences observed between day 42 and two later periods of the experiment.

Table 4 .
P hysicochemical and color parameters of pectoralis major muscle in broiler chickens in relation to genotype, wooden breast degree and slaughter age

Table 5 .
Water-holding capacity and texture traits in broiler chickens in relation to genotype, wooden breast degree and slaughter age [45]rted higher pH in the breast meat of broilers slaughtered at 49 d than at 56 d, although no difference was found for day 42.Accordingly, Schneider et al.[43], Coban et al.[44], and Glamočlija et al.[45]also reported an age-related decline in pH in the p. major muscles (from 42 d to 63 d, 42 d to 56 d, and 42 d to 50 d, respectively) likely as the consequence of a greater degree of postmortem glycolytic metabolism in more mature muscles.As in the previous studies, genotype did not infl uence pH