Histomorphometric study of the anterior latissimus dorsi muscle and evaluation of enzymatic markers of broilers affected with dorsal cranial myopathy

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1 Histomorphometric study of the anterior latissimus dorsi muscle and evaluation of enzymatic markers of broilers affected with dorsal cranial myopathy R. Sesterhenn, F. M. Siqueira,,1 A. C. Hamerski, D. Driemeier, S. F. Valle, S. L. Vieira, L. Kindlein, and V. P. Nascimento Department of Preventive Veterinary Medicine; Department of Clinical Pathology; and Department of Animal Science, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil ABSTRACT Dorsal cranial myopathy (DCM), which selected for histomorphometric evaluation and analysis affects the anterior latissimus dorsi (ALD) muscles of commercial broilers, is of unknown etiology, and it represents up to 6% of the partial condemnations in Brazilian slaughterhouses. This study was performed to achieve histomorphometric characterizations of the ALD muscles from male Cobb 500 broilers slaughtered at either 35 d or 42 d and to evaluate the effects of DCM on the enzymatic markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) and on uric acid and creatinine metabolites. Blood samples (1.5 to 3 ml) and ALD muscle fragments were collected from each carcass, all of which were processed in a commercial inline processing system. For each age, twelve macroscopically normal animals and twelve animals found to exhibit DCM were randomly of serologic profiles. Microscopic evaluations demon- strated that the muscle fibers of those with DCM exhibited a strong presence of multifocal regenerative myodegeneration as well as a substitution of muscle tissue with connective tissue (P < 0.001) through fibrosis, thus characterizing the chronicity and hardness of the affected muscle. It is suggested that DCM is a localized muscle lesion because the detected serum levels of CK (P < 0.001), AST (P < 0.001), ALT (P = 0.01), and LDH (P < 0.001) enzymes were strongly associated with the group affected by DCM. Additional studies are needed to gain an understanding of this myopathy because it is an emerging problem in the poultry industry. In addition, it is related to DCM lesions in fast-growing broilers with the greatest slaughter weights. Key words: broiler, morphometry, muscular fiber, myopathy, serum enzymes 2017 Poultry Science 0:1 7 INTRODUCTION In poultry production, rising economic pressures on the slaughter of broilers with the fastest weight gain in the shortest amount of time has allowed genetics to evolve very rapidly in the last decades with a special focus on improving muscular performance (Olivo and Shimokomaki, 2002; Havenstein et al., 2003; Velleman and Nestor, 2003; Vieira, 2008). Nevertheless, these increases in growth rates and muscle size, as the consequences of production intensification to meet market demands, have contributed to metabolic alterations that resulted in damage to the cell structure of skeletal muscle, both morphologically and biochemically, with consequential meaningful losses in the poultry chain (Dransfield and Sosnicki, 1999; MacRae et al., 2006; C 2017 Poultry Science Association Inc. Received February 23, Accepted August 17, Corresponding author: franmaboni@gmail.com MacRae et al., 2007). Among muscle changes present in chickens, dorsal cranial myopathy (DCM) stands out, evidenced in the anterior latissimus dorsi (ALD) muscle. The etiology and other factors that cause this muscular disorder have not yet been identified. However, this lesion affects male broilers of high performance lineages that are in good body condition with the greatest slaughter weights and no other apparent problems or infection (Zimermann et al., 2012). This pathology has been reported in several slaughterhouses in Brazil, initially detected in the southern region in the 2000s, reaching 6% of the partial condemnations of the country s slaughterhouses (Giacomin et al., 2011; Roso and Dickel, 2011; Ferreira et al., 2012; Zimermann et al., 2012; Sesterhenn et al., 2014; Amaral et al., 2015). Our objective was to analyze the histomorphometric characteristics of the ALD muscle from male Cobb 500 broilers slaughtered at 35 and 42 d and to evaluate the effects of DCM on the serum enzymatic markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), 1

2 2 SESTERHENN ET AL. γ-glutamyltransferase (GGT), and lactate dehydrogenase (LDH) and the metabolites of uric acid and creatinine. MATERIALS AND METHODS Poultry Management Male Cobb 500 commercial broiler chickens aged 35 and 42 d were obtained during two sequential slaughterings in a processing plant located in southern Brazil. Average body weights were 2.8 ± 0.2 kg and 3.2 ± 0.2 kg, respectively. Of 1,224 carcasses analyzed, 48 (3.52%) were selected and collected for further evaluation. Twenty-four birds of each age were randomly selected and classified as normal (NORM; n = 12) or DCM (n = 12) based on the degree of severity of the criteria established by Zimermann et al. (2012). The ALD muscles were classified based on the visual appearance of the skin covering this muscle, its coloration, shape, and consistency, as well as the presence and type of exudate and/or hemorrhages. Morphometry Measurements All muscle fragments collected were fixed in 10% buffered neutral formalin, dehydrated with ethanol in increasing concentrations, diaphanized in xylol, and embedded in paraffin. From each sample, 10 semiserial cross-sections and longitudinal sections (3 μm thick) were cut (Microtome Leica DM500; Leica Biosystems Nussloch GmbH, Germany) and stained with hematoxylin-eosin for analysis the myopathic lesion in the tissue. In addition, 4 semi-serial cross-sections and longitudinal were stained with Masson s Trichrome stains to confirm the presence collagen. Microscopy was performed using a light microscope (Leica DM500; Leica Biosystems Nussloch GmbH, Germany) with an image capture system (ICC50HD) and LAS EZ software for image capture and storage. Morphometric analysis of the density and partial volume (Vv, %) of the muscular and connective tissue of each sample was performed. In total, 672 images (480 of muscle tissue and 192 of connective tissue) were captured in cross-section and submitted to the multifunctional M42 test system using the superposition method (Weibel, 1979; Wilson et al., 1990; An et al., 2010). Serum Analysis Blood collection for serology (between 1.5 and 3 ml from each bird) was performed upon bleeding after electronarcosis. Samples were stored in disposable capped vacuum tubes (Vacutainer) without anticoagulant. Samples were kept at room temperature for 30 to 60 min then centrifuged (3,000 g for 10 min at 4 C), and the serum was stored at 20 C until analyzed. Biochemical tests were performed on thawed samples in duplicate. A high performance reading spectrophotometer with automatic calibration (CM 200; Wiener lab, Rosario, Argentina) was used at a wavelength of 340 nm (Konelab 20i; ThermoElectro Corporation, Espoo, Finland) by means of commercially diagnostic kits (Wiener lab, Rosario, Argentina) to estimate the serum enzyme activities of ALT, AST, CK, GGT, LDH, and the metabolites of uric acid and creatinine. The methodology used by UV-Kinetic (IFCC) method (Autopack, Bayer Diagnostics). Statistical Analysis Data were analyzed through the analysis of covariance considering the effects of myopathy (NORM/DCM) and age at slaughter (35 d and 42 d) on muscle tissue, connective tissue, enzyme activities of ALT, AST, CK, GGT, LDH, and the metabolites of uric acid and creatinine. The statistical analyses were performed using IBM SPSS 18.0, with the significance level set at 95%. In the presence of meaningful differences (P < 0.05), multiple comparisons were performed using the Bonferroni test. In addition, carcass weight was applied as a co-variable because previous studies showed it influenced DCM prevalence (Zimermann et al., 2012). RESULTS Of 1,224 carcasses analyzed, 90 (7.35%) broilers showed macroscopic DCM signs. This frequency was greater among those aged 35 d (n = 58, 4.73%) compared to those aged 42 d (n = 32, 2.61%). Macroscopically, the NORM group did not present lesions in the skin and muscle (Figures 1a and b). However, the DCM group presented yellowish coloration on the skin and greater subcutaneous volume (Figure 1c). After cutting the skin, one or both portions of the ALD muscle were remarkably hardened, pale, and thick. The surface was often covered with a thin layer of clear or slightly turbid viscous material as well as scattered petechiae or hemorrhages (Figure 1d). Upon histopathologic evaluation of the ALD muscle, important alterations were observed in both groups. Normal ALD muscle eventually presented hypereosinophilic and hypercontracted fibers, flocculus sarcoplasm, and macrophage and lymphocyte infiltrate in addition to a remarkable presence of connective and adipose tissue (Figure 2a). Nonetheless, the lesions were evidently more intense in muscle fragments of carcasses affected by DCM (degeneration, necrosis, fibrosis, and regeneration; Figures 2band 2c). Staining with Masson s trichrome showed a proliferation of connective tissue (Figure 2d). Altered muscle fibers were characterized by the presence of hypercontraction, hypereosinophilia, rounded edges (loss of polyhedral pattern), and sarcoplasmic homogenization, characterizing a hyaline degeneration. An abundance of interspersed necrotic cells in connective and adipose tissue was seen in addition to inflammatory infiltrate, which was predominantly mononuclear. Variations in

3 MACROSCOPIC ANALYSIS ON BROILER DORSAL CRANIAL MYOPATHY 3 Figure 1. Broiler carcasses and anterior latissimus dorsi (ALD) muscles. (a) Normal carcass showing no dorsal cranial myopathy (DCM); (b) normal ALD muscle showing no DCM; (c) carcass with DCM evidencing volume increase and yellow coloration (circles); (d) ALD muscle exhibiting DCM with scattered petechiae and hemorrhage on the surface and a greater consistency (arrows). the sizes of myofibers with cytoplasmic fragmentation, flocculus sarcoplasm (floccular necrosis), and regeneration were also observed. According to results obtained in the morphometric analysis of the relationship between the partial volume of muscle tissue (Vvm, %) and that of connective tissue (Vvc, %; Figure 3), meaningful differences were found (P < 0.001) with a strong influence of carcass weight (P = 0.001) on Vvc. Also, Vvm showed that broilers with DCM had less muscle tissue (34.63% ± 2.67%) when compared to those with normal muscles (47.28% ± 4.78%). Additionally, Vvc was higher in those with DCM (29.98% ± 2.79% vs % ± 5.13%). However, the relationships between Vvm and Vvc with age at slaughter (35 d or 42 d) were not significantly different between groups (P > 0.05). Enzyme and metabolite levels were estimated to assess tissue damage associated with DCM. In this way, serum levels for ALT (P = 0.01), AST, CK, and LDH (P < 0.001) were found to be greater in broilers affected by DCM (Table 1). Carcass weight strongly influenced ALT, AST (P 0.001), and LDH (P = 0.05) enzymes. A difference (P < 0.001) was found in GGT enzyme levels, which were lower in the DCM group. Moreover, no differences (P > 0.05) in uric acid and creatinine metabolites were found between the groups even though serum levels were lower in the DCM group. Greater serum levels of ALT, AST, CK, LDH, and creatinine were seen in broilers aged 42 d when compared to those aged 35 d (Table 2); however, differences were significant only in ALT, GGT, LDH (P < 0.001), and CK (P = 0.02) enzymes. Broilers aged 42 d showed

4 4 SESTERHENN ET AL. Figure 2. Photomicrographs of the anterior latissimus dorsi (ALD) muscles in broilers. Normal muscle with the presence of hypereosinophilia and primarily mononuclear inflammatory infiltrate (a); muscle with DCM presenting loss of the polyhedral pattern in the fibers and size variation at 42 d (b); and 35 d (c); multifocal areas of muscle fibers in hyaline degeneration (asterisk), floccular necrosis (arrow), and infiltration by inflammatory cells, mainly heterophils and macrophages (arrowhead tip; hematoxylin and eosin). Proliferation of connective tissue (fibrosis) loosely organized, colored in blue (Masson s trichrome) (d). lower serum levels of GGT (P < 0.001) and uric acid (P < 0.03) compared with those aged 35 d. DISCUSSION This study indicates that DCM is histopathologically characterized by chronic myopathic lesions with a notable presence of muscular polyphasic degeneration, regeneration, necrosis with a variable amount of interstitial connective tissue, fibrosis, and adipocytes. Similar results were reported in muscle myopathies of DCM (Zimermann et al., 2012), white striping (Kuttappan et al., 2012, 2013b), and wooden breast (Sihvo et al., 2013; Velleman and Clark, 2015). In addition, lesions found in these samples are very similar to those found with wooden breast: pallor, increased hardness and muscle thickness, and exudate on the muscular surface (Sihvo et al., 2013). According to Dubowitz (1985) the myopathic lesions are nonspecific and might be the result of any of several neuromuscular disturbances. Furthermore, genetic selection has led to greater muscle mass, tripling muscle size and rendering it vulnerable to oxidative damage and antioxidant deficiencies as a result of metabolic residue accumulation, leading to oxidative stress and tissue lesions (MacRae et al., 2006). In addition, under stress, animals antioxidant mechanisms could define the quality of the final food product. Indeed, stress has been associated with losses in the oxidative stability of tissues (Young et al., 2003). Until now, the precise etiology of DCM could not be confirmed. Previous studies suggest that high-performance lineages and the heaviest broilers could be associated with DCM (Zimermann et al., 2012) and white striping myopathy (Kuttappan et al., 2012, 2013a,b). Bauermeister et al. (2009) reported that the severity of white striping can increase between the ages of six and eight weeks, suggesting it correlates with age. In addition, literature data (Mudalal et al., 2014) show that wooden breast myopathy is also generally seen with greater weight and thickness. These results corroborate with studies suggesting that greater growth rates could predispose broilers to these myopathies (Dransfield and Sosnicki, 1999). The white fibers of breast muscle are more sensitive to

5 MACROSCOPIC ANALYSIS ON BROILER DORSAL CRANIAL MYOPATHY 5 Figure 3. Distribution of the partial volumes of muscle (Vvm, %) and connective tissue (Vvc, %) from samples of the anterior latissimus dorsi muscle of broilers macroscopically normal (NORM) and those affected by dorsal cranial myopathy (DCM). a, b Means within each morphometric analysis are significantly different (P < 0.001). NORM = normal (no dorsal cranial myopathy); DCM (dorsal cranial myopathy). Table 1. Serological profile of normal (NORM) broilers and those affected by dorsal cranial myopathy (DCM). Attribute NORM DCM P value Enzyme (U/L) ALT AST CK 15, , GGT ,60 LDH 4, , Metabolite (mg/dl) Uric acid NS Creatinine NS NS = P > 0.05; P 0.01; and P 0.001; or indicates a significant difference between NORM and DCM groups within each row. Table 2. Serological profile of broilers relative to age at slaughter. Attribute 35 d 42 d P value Enzyme (U/L) ALT AST NS CK 17, , GGT LDH 5, , Metabolite (mg/dl) Uric acid Creatinine NS NS = P > 0.05; P 0.05; and P or indicates a significant difference between NORM and DCM groups within each row. oxidation, as well as vitamin deficiencies, than red fibers, and they have a greater tendency to suffer injuries due to exercise (Page, 1995). On the other hand, red fibers, as in the ALD muscle, are more sensitive to changes in oxygen levels followed by reperfusion (Carmo-Araújo et al., 2007). Also, research has shown that fast-growing broilers have up to 20% more fibers in the ALD muscle, and the fiber size is larger compared to slow-growing broilers (Remignon et al., 1994, 1995). However, fiber size is of more importance in determining muscle size than the number of fibers (Prentis et al., 1984). Differences in growth rates among various muscle fiber types could contribute to differences in myopathic changes in these muscles (Ono et al., 1993). These results agree with those found by Gross et al. (2016), who verified that broilers affected by wooden breast myopathy exhibited lower Vvm and greater VVc compared to normal broilers. Similarly, Kuttappan et al. (2012) verified that with white striping, fat was increased and protein decreased relative to normal broilers. Thus, fibrosis could explain the characteristic hardness of the affected tissue (Sihvo et al., 2013). Increased levels of serum enzymes in broilers of greatest body weight indicate muscle damage along with myopathic changes (MacRae et al., 2006). Increased average concentrations of ALT, AST, CK, and LDH enzymes were observed. According to previous research, Kuttappan et al. (2013a) found that serum levels of ALT, AST, CK, and LDH were much greater in broilers affected by white striping compared to normal broilers. Research suggests that changes in plasmatic enzymes indicate hepatic damage or damage in the skeletal muscles of broilers (Mitchell and Sandercock, 1995; Yan et al., 2009). The enzyme CK is considered the primary organspecific serum enzyme indicative of muscle lesions (Kramer, 1989). Acute elevations in CK enzyme activity in the plasma were reported in several muscular pathological conditions in broilers, including other myopathies (Mitchell et al., 1992; Mitchell and Sandercock, 1994, 1995) and congenital muscular dystrophy in the skeletal muscle (Stewart et al., 1981). Comparing fast growing genetic lines with those exhibiting lower growth rates, higher levels of CK (Sandercock and Mitchell, 2003) were observed. High levels of AST, ALT, and LDH enzymes along with the muscle-specific enzyme CK confirms muscular lesions (Gonzáles and Silva, 2006; Schmidt et al., 2007; Hoffman and Solter, 2008). However, increases in AST, ALT, and LDH enzyme activities are associated not only with muscle damage, but also with hepatic damage (Hochleithner, 1994; Lumeij, 2008; Grunkemeyer, 2010; Capitelli and Crosta, 2013). According to Kramer and Hofmann (1997), muscle and hepatic lesions can occur concomitantly through the same pathological process or a distinct process. Nonetheless, the plasma activity of LDH increases considerably with hepatocellular disease or muscle injury and, in comparison to AST or ALT, its increase and decrease are faster (Capitelli and Crosta, 2013) and can provide information on the chronicity of the disease (Grunkemeyer, 2010). Normal AST activity in broilers is less than 275 IU/L (Thrall et al., 2004). In general, moderate AST activity (350 IU/L) and AST activity (from 800 IU/L) are highly suggestive of severe hepatic damage (Campbell, 2004; Capitelli and Crosta, 2013). Nevertheless, the enzymes are age dependent, varying between species and generating distinct interpretations (Lumeij, 1997). Mitchell (1999) reported that

6 6 SESTERHENN ET AL. increased muscle growth compromises the integrity of skeletal muscle membranes, thus causing alterations at the enzymatic level, for example, quantitative changes in CK, AST, and LDH enzyme activities. Similarly, ALT activities in broilers can increase as a result of damage to various tissues, making interpretation difficult (Grunkemeyer, 2010). Kuttappan et al. (2013a) suggest that when serological findings such as this are discovered, severe myopathy is associated with muscle damage and not with liver abnormalities. Although differences (P < 0.001) between the NORM and DCM groups were found in serum GGT levels, the enzyme GGT remained normal in the DCM group. This enzyme is an indicator of hepatocellular and renal damage, which is manifested in increased levels in serum or plasma and urine (Hochleithner, 1994). Kuttappan et al. (2013a) similarly showed that serum GGT levels were higher in normal broilers even though no difference was found (P > 0.05). Uric acid metabolites were lower in broilers aged 42 d compared with those aged 35 d (P < 0.03) even though these levels are generally lower in younger broilers (Hochleithner, 1994; Capitelli and Crosta, 2013). However, in a study of seagulls, it is generally lower in adults (Alonso-Alverez, 2005). Birds may also present greater uric acid after ingesting high-protein diets, prolonged fasting, or severe muscle degeneration (Gregory, 2003; Lierz, 2003; Campbell, 2004). No differences (P > 0.05) were found in creatinine levels, suggesting that renal function was similar between the NORM and DCM groups. Creatinine levels across bird species are normally constant (between 0.1 and 0.4 mg/dl) independent of muscle mass (Hochleithner, 1994). However, Rajman et al. (2006) proposed that elevations are related to increased muscle activity and food restrictions in some species. CONCLUSIONS Based on these results, we confirmed that DCM caused alterations in the histomorphometric characteristics of the muscle fibers in the ALD muscle in broilers, with a striking presence of multifocal regenerative myodegeneration and of fibrosis. A decrease in Vvm and an increase in Vvc (P < 0.001) associated with DCM indicates that the hardness found in the ALD muscles in broilers with DCM can be explained by this fibrosis. It is suggested that DCM is signified by a localized muscle lesion because serum levels of CK, AST, ALT, and LDH enzymes were strongly associated with the DCM group. Also, CK, the primary organ-specific serum enzyme linked to muscle lesions, was found at greater levels in the DCM group. This, accompanied by much greater AST levels, suggests not only muscle damage, but also hepatic damage. High serum activities of LDH and ALT highlight that even though they are considered less specific indicators, they are nevertheless good indicators of muscle damage in broilers. Additional studies are needed to enhance our understanding of this myopathy because it is an emerging problem in the poultry industry, and DCM lesions are associated with fast-growing broiler strains with the greatest slaughter weights. ACKNOWLEDGMENTS The authors are appreciative for the support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) throughout this Project. REFERENCES Alonso-Alvarez, C Age-dependent changes in plasma biochemistry of yellow-legged gulls (Larus cachinnans). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 140: Amaral, P. C., L. R. dos Santos, M. L. R. Provin, L. F. Pedrotti, and E. L. Dickel Dorsal cranial myopathy as a cause of sentencing in broiler slaughterhouses. 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