Myodegeneration With Fibrosis and Regeneration in the Pectoralis Major Muscle of Broilers

This study investigated the effects of acute stress on breast meat quality, redox status, and mitochondrial function in pectoralis major (PM) muscle of broilers. A total of 168 broiler chickens (42-d-old, Ross 308) were randomly divided into control (CON) and preslaughter transport (T) treatments. A broiler was an experimental unit. Each treatment consisted of 84 broilers, and they were put in 12 crates with 7 broilers each. Broilers in the T group were transported according to a designed protocol, and the CON broilers were kept in crates under normal living conditions before slaughtering. Based on the meat quality traits assessed at postmortem 24 h, all PM muscles of the transported broilers were further classified into normal (T-NOR) and pale, soft, and exudative (PSE)-like (T-PSE) groups for the determination of redox status in PM muscle and isolated mitochondria, energy metabolites, mitochondrial electron transport chain complexes activities, as well as mitochondrial function-modulating genes expression. Compared with CON, the extent of lipid peroxidation as well as protein oxidation were significantly increased in both PM muscles and mitochondria in T-PSE (P < 0.05), whereas not in T-NOR. Higher activities of glutathione peroxidase, total superoxide dismutase, and Cu-Zn superoxide dismutase were observed in PM muscle of T-NOR broilers when compared with CON (P < 0.05). Preslaughter transport increased the generation of reactive oxygen species, as well as enhanced antioxidant capacity in PM mitochondria of broilers (P < 0.05). Compared with CON, the ATP content, activities of complexes I and III, as well as relative mitochondrial membrane potential and swelling were significantly decreased in T-PSE (P < 0.05), whereas no significant changes in either ATP content or complex I activity were observed in T-NOR. Preslaughter transport enhanced the mRNA expression of regulators involved in the glutathione system, thioredoxin 2 system, and mitochondrial biosynthesis in PM muscle of broilers (P < 0.05). Moreover, we noticed a more evident enhancement effect in T-NOR than in T-PSE (P < 0.05). Overall, this work indicates that acute stress-induced redox imbalance and mitochondrial dysfunction have significant implications for the development of PSE-like meat.

Tonic immobility (TI), which can be divided into short (STI) or long (LTI) duration, is a trait related to fear and stress response. In a previous study, we found that in broilers that LTI phenotype and chronic corticosterone (CORT) administration caused retarded growth and lower muscle weight compared with their control counterparts. The aim of this study is to determine whether the mitochondrial DNA (mtDNA) copy number and mitochondrial oxidative phosphorylation (OXPHOS), the vital factors involved in regulating energy homeostasis, have been changed by LTI or CORT treatment. The results showed that STI broilers had higher mtDNA copy number and cytochrome c oxidase (COX) enzyme activity compared with LTI broilers. Analysis of mtDNA-encoded OXPHOS genes revealed that the mRNA expression of the COX subunit 1, 2, NADH dehydrogenase (ND) subunits 1, 3 and 6, were also increased in STI broilers compared with LTI broilers. Regarding the transcriptional regulation of mtDNA-encoded OXPHOS genes, no difference was found in the methylation of the mitochondria control region between the TI phenotypes or the CORT treatments. The PGC-1a protein level was higher in STI broilers, but the av uncoupling proteins, did not show significant difference at the protein level between TI phenotypes. These results suggest that the mitochondrial function in pectoralis major muscle of STI broilers is better than that of LTI counterparts. However, chronic CORT administration did not affect the mitochondrial metabolism, indicating the mitochondrial insensitivity to CORT treatment in pectoralis major muscle.

Research Note: Expression levels of collagen-related genes in PSE conditions and white striping features of broiler pectoralis major muscle

Shotgun proteomics reveals changes in the pectoralis major muscle of broilers supplemented with passion fruit seed oil under cyclic heat stress conditions

Pale, soft, and exudative (PSE) meat is a severe quality problem in chicken production. In this study, HSP70-interacting proteins in normal and PSE-like chicken pectoralis major (PM) muscles were identified using Nano-LC-ESI-MS/MS analysis. The results showed that HSP70-interacting proteins were mostly enriched in pathways of glycolysis/gluconeogenesis, biosynthesis of amino acids, and the calcium signaling pathway (FDR <0.001). Immunoprecipitation, immunofluorescence, and molecular docking confirmed the specific interaction between HSP70 and SERCA1 in the PM muscle of broilers. Enzyme activity assays and in vitro experiments confirmed that HSP70 alleviates the heat-induced decrease in SERCA activity (P < 0.05). Overall, our study reveals that the HSP70-SERCA1 interaction in the PM muscle of broilers alleviates the decrease in SERCA activity in the sarcoplasmic reticulum (SR) of broiler skeletal muscle caused by acute stress, which may provide a further understanding of the mechanism of meat quality changes under acute stress.

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

Two experiments were performed to study the interaction between the standardized ileal digestible (SID) leucine (Leu) and valine (Val) levels on the mRNA expression of genes involved in the mechanistic target of rapamycin (mTOR) pathway (experiment I) and determine the requirement of these amino acids in low-protein diets, and their effects on performance, serum parameters and muscle fiber diameters of broilers (experiment II) from day 1 to day 21 post hatch. Broiler chickens were distributed in a completely randomized design in a 3 × 3 and 5 × 5 factorial arrangement for a total of 9 and 25 treatments in experiments I and II, respectively. There was no (P &gt; 0.05) interaction between the SID Leu and Val levels on mRNA expression of mTOR, S6 kinase 1 (S6K1), 4E-binding protein-1 (4EBP1), eukaryotic elongation factor 2 (eEF2), and insulin-like growth factor-1 (IGF-1) genes in pectoralis major muscle. Leucine supplementation increased (P &lt; 0.05) mRNA expression of mTOR and S6K1 genes in muscle tissue, whereas Val supplementation did not affect (P &gt; 0.05) mRNA expression of the genes investigated. Interaction was observed (P &lt; 0.05) between dietary Leu and Val levels on feed intake and gain:feed. Leucine supplementation may stimulate mRNA expression of mTOR and S6K1 genes in pectoralis major muscle of broilers from day 1 to day 21 post hatch. The SID Leu and Val levels required for the optimization of feed intake, weight gain, and gain:feed in low-crude protein diets for broiler chickens from day 1 to 21 post hatch were estimated at 1.29% and 0.96%, 1.28% and 0.92%, and 1.27% and 0.91%, respectively; however, these requirements may be greater to maximize muscle fiber growth.

Research Note: Evaluation of the incidence of white striping and underlying myopathic abnormalities affected by fast weight gain in commercially fed broiler chickens

This study was planned to determine the effects of feed restriction applied at different times and levels on the occurrence of white striping and wooden breast myopathies, performance, carcass characteristics and some blood parameters in broilers. In total, 600 male broilers were randomly placed to 5 groups with eight replicates. Control group was fed ad libitum throughout experimental period. T1 and T2 groups received feed as 80% and 70% of control group from 11 to 24 days of age, respectively. T3 and T4 groups received feed as 80% and 70% of control group from 25 to 39 days of age, respectively. T2, T3 and T4 groups had the lower mean values for feed intake and body weight gain from 0 to 49 days of age, final body weight, carcass weight, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, compared to control group. T3 and T4 groups had lower dressing percentage, breast meat yield, serum aspartate aminotransferase (AST) and creatine kinase (CK) levels than control. T2, T3 and T4 groups had lower incidence for white striping and wooden breast myopathies than control. T3 and T4 groups had also the lower incidence for severe degree white striping and wooden breast myopathies than control. In conclusion, feed restriction to 70% of ad libitum intake between 11 and 24 days of age and feed restriction to 80% and 70% of ad libitum intake between 25 and 39 days of age may reduce the incidence and severeness of white striping and wooden breast myopathies, although there is some loss in performance traits. Feed restrictions to 80% and 70% levels of ad libitum intake between 25 and 39 days of age may be more effective in reducing these myopathies on pectoralis major muscle of broilers.

Chronic heat stress inhibits glycogen synthesis through gga-miR-212-5p/GYS1 axis in the breast muscle of broilers

Functional and transcriptional responses of chicken macrophages to hypoxia

Introduction: We have previously shown that angiotensin II (Ang II) type 2 receptor (AT2R) positively regulates skeletal muscle stem cell (MuSC) differentiation. Systemic angiotensin II (Ang II) increase or congestive heart failure (CHF) suppress AT2R expression, likely contributing to the development of skeletal muscle atrophy and fibrosis associated with these conditions. Hypothesis: We hypothesized that AT2R overexpression in MuSCs could prevent skeletal muscle atrophy and fibrosis associated with CHF. Approach: Two mouse models, systemic Ang II infusion and left anterior descending coronary artery (LAD) ligation were used in this study. MuSC differentiation, muscle atrophy and muscle fibrosis were assessed by analyzing gene expressions (e.g. myogenin and atrogin-1), myofiber cross sectional area, and the collagen deposition (Collagen I immunohistochemical staining and picrosirius red staining). Primary MuSCs were isolated from mouse hindlimb muscles. MuSC-specific AT2R-overexpressing transgenic mice were generated by crossing Pax7CreER/+ mice (tamoxifen-inducible cre recombinase is expressed under Pax7 promoter) and CAG-stopflox-AT2R mice (AT2R is expressed under CMV early enhancer element, chicken β-actin gene promoter upon cre-mediated excision of floxed stop codon). Results: AT2R expression was robustly increased during MuSC differentiation both in vitro and in vivo. In both Ang II infusion and LAD ligation models, AT2R induction in MuSCs was significantly suppressed, associated with reduced muscle regeneration and increased atrophy and fibrosis. Consistently, AT2R knockdown significantly suppressed MuSC differentiation in vitro and skeletal muscle regeneration in vivo, leading to increased skeletal muscle fibrosis. Restoring AT2R levels via MuSC-specific AT2R overexpression in vivo increased muscle regenerative capacity (increased regenerative marker expressions), reduced muscle atrophy (reduced atrophy markers and increased myofiber cross sectional area) and fibrosis (decreased collagen) in CHF models. Conclusions: AT2R induction in MuSC is suppressed in CHF, possibly via increased Ang II. Decreased AT2R expression and its downstream signaling in MuSC lead to lowered muscle regenerative capacity and increased muscle atrophy and fibrosis. AT2R overexpression in MuSCs restores their regenerative potential, and prevent muscle atrophy and fibrosis in CHF condition.

Simple SummaryWooden breast myopathy is a muscle abnormality resulting from the genetic improvement practiced by the poultry industry and which is widespread throughout the planet. Recent research has tried to unravel this myopathy for years and how the quality of the meat is affected, in search of its etiology and the solution to this problem. Unfortunately, the problems related to the myopathies of the affected birds will only be solved when the cause of the abnormality is in fact unveiled and controlled; in the meantime, it is up to us researchers to contribute scientifically to research on this type of meat that is fit for consumption but that, for often presenting non-standard appearance for sale in the form of whole fillets, is discarded.This study aimed to characterize the effects of wooden breast myopathy (WBM) on quality of broiler chicken breast meat. Normal samples (absence of myopathy), moderate-degree samples (hardness only in one area of the breast fillet) and severe-degree samples (hardness throughout the breast fillet) were classified. In macroscopic analysis, the pectoral muscle affected by the WBM showed, in general, pale color with stiff, irregular and reddish regions (suffusions and petechiae), with the presence of white striations. In microscopic analysis, the myopathy was characterized by loss of the polygonal aspect of the muscle fibers. Samples with moderate degree of the myopathy showed greater (p = 0.0266) water retention capacity. There was an increase (p = 0.004) in total collagen concentration in samples from the severe-degree group 0.29% in normal samples to 0.43% and 0.48% in samples from moderate- and severe-degree groups, respectively. Samples of chicken breast affected by the severe-degree WBM showed lower (p < 0.0001) myofibrillar fragmentation index (64.51) and lower (p = 0.0002) fat concentration (2.17%) than normal chicken samples (80.45 and 3.79%, respectively). Samples affected by WBM are larger and heavier and present poorer physical quality when compared to normal chicken meat. Histologically it is possible to observe loss of the polygonal aspect of muscle fibers.

High leucine levels affecting valine and isoleucine recommendations in low-protein diets for broiler chickens

Effect of dietary lysine on performance and expression of electron transport chain genes in the pectoralis major muscle of broilers