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Studies have demonstrated that chronic heat stress can accelerate glycolysis, decrease glycogen content in muscle, and affect muscle quality. However, the consequences of chronic heat stress on glycogen synthesis, miRNA expression in pectoralis major (PM) muscle, and its regulatory functions remain unknown. In this study, high-throughput sequencing and cell experiments were used to explore the effects of chronic heat stress on miRNA expression profiles and the regulatory mechanisms of miRNAs in glycogen synthesis under chronic heat stress. In total, 144 cocks were allocated into 3 groups: the normal control (NC) group, the heat stress (HS) group, and the pair-fed (PF) group. In total, 30 differently expressed (DE) miRNAs were screened after excluding the effect of feed intake, which were mainly related to metabolism, signal transduction, cell growth and death. Furthermore, the gga-miR-212-5p/GYS1axis was predicted to participate in glycogen synthesis through the miRNA-mRNA analysis, and a dual-luciferase reporter test assay confirmed the target relationship. Mechanistically, chronic heat stress up-regulated gga-miR-212-5p, which could inhibit the expression of GYS1in the PM muscle. Knocking down gga-miR-212-5p alleviates the reduction of glycogen content caused by chronic heat stress; overexpression of gga-miR-212-5p can reduce glycogen content. This study provided another important mechanism for the decreased glycogen contents within the PM muscle of broilers under heat stress, which might contribute to impaired meat quality.

This experiment aimed to reveal the dynamic changes of protein post-translational lactylation modifications and their correlations with the glycolytic process in broiler breast muscle within 48 hours of postmortem acidification. The experiment involved 12 male AA broilers, 42 days old, with similar body weights (2.8 ± 0.05 kg). The breast fillets (Pectoralis major) were collected after slaughter, and samples were taken at various time points: 0, 15 min, 30 min, 45 min, 60 min, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, and 48 hours postmortem. The results showed that the rate of glycogen decline in the muscle was highest at 45 minutes postmortem, and glycogen levels tended to stabilize at 8 hours postmortem. The lactate content in the breast reached its highest level at 4 hours postmortem and began to decrease, stabilizing at 24 hours postmortem. Additionally, the glycolytic potential increased gradually in the first 4 hours postmortem, decreased rapidly from 4 to 8 hours. Similarly, lactylation modification levels were highest at 8 hours postmortem, but stabilized at 12 hours postmortem. During this process, the protein expression of the enzymatic lactylation modifier p300 showed no significant difference, while the content of the non-enzymatic lactylation substrate lactoylglutathione significantly decreased at 8 hours and 24 hours postmortem. Correlation analysis found that lactylation levels were negatively correlated with glycogen content, glucose content, glycolytic potential, and pH value, while positively correlated with lactate content. Besides, there was a positive correlation between lactylation levels and the protein expression of hexokinase, phosphoglycerate kinase 2, phosphoglucomutase 1, and triosephosphate isomerase. Additionally, lactylation levels were positively correlated with the activities of lactate dehydrogenase and phosphofructokinase. In summary, our experiment elucidated the dynamic changes in the entire glycolytic pathway in broiler pectoral muscle during acidification. During this process, lactylation modifications may participate in the glycolysis process by regulating the protein expression and activity of glycolytic enzymes.

Heat stress (HS) largely impairs the quality of broiler breast meat through protein oxidative modification. This study aimed to investigate the carbonylation pattern of Ca2+channels and apoptotic proteins in the breast muscle of heat-stressed broilers. A total of 144 28-day-old male Arbor Acres broilers were randomly divided into three treatment groups. The normal control (NC) group was kept at 22°C and provided with unlimited feed. The HS group was exposed to 32°C and provided with unlimited feed. The pair-fed (PF) group was kept at 22°C and given an amount of feed equivalent to that consumed by the HS group on the previous day. Results showed that broilers under HS conditions had a higher respiratory rate than those in NC and PF groups (P< 0.05). HS disrupted the morphology and structure of breast muscle fibers by decreasing the average diameters and average density of myofibers compared to the NC group (P< 0.05). HS increased the mean fluorescence intensity of the positive carbonyl signal in breast muscle compared with the NC group (P< 0.05). Besides, the pectoral Ca2+concentration in the sarcoplasmic reticulum, cytoplasm, and mitochondria was elevated by HS when compared with the NC group (P< 0.05). In comparison to the NC and PF groups, HS increased the apoptosis rate and caspase-3 activity in the breast muscle (P< 0.05). Furthermore, HS elevated the relative protein expressions of plasma membrane Ca2+-ATPase, Na+/Ca2+exchanger 1, and sarco/endoplasmic reticulum calcium transport ATPase 1 compared to the NC group (P< 0.05). Higher relative protein expression of μ-calpain and lower relative protein expression of cytosolic cytochrome complex were found in the HS group than the NC group (P< 0.05). HS decreased the carbonylation levels of transient receptor potential canonical 1 and inositol 1,4,5-trisphosphate receptor compared to the NC group (P< 0.05). Additionally, the carbonylation levels of cleaved caspase-3 and precursor caspase-9 were increased and decreased, respectively, by HS treatment compared to the NC group (P< 0.05). In conclusion, HS damages the myofiber based on Ca2+dyshomeostasis and apoptosis, which are potentially associated with protein carbonylation. These results shed new light on the possible mechanism behind the development of poor meat quality in broilers due to HS.

Approaches for the diagnosis of wooden breast (WB) myopathy in live birds are urgently required before applying intervention strategies to reduce occurrence and severity for the poultry industry. The objective of this study was to characterize the serum metabolic profiles in male broilers affected by WB and to identify biomarkers related to this myopathy. Broilers were categorized into normal (CON) and WB groups based on gross scoring and histological evaluation. Gas chromatography-mass spectrometry-based metabolomics, multivariate analysis and orthogonal partial least squares discriminant analysis revealed a clear separation between CON and WB. A total of 73 significantly different (P< 0.05) metabolites with 17 upregulated and 56 downregulated were identified, which were mainly involved in pathways of alanine, aspartate and glutamate metabolism, carbohydrate metabolism and taurine and hypotaurine metabolism. By using the nested cross-validation function of random forest analysis, 9 significantly altered (P< 0.05) metabolites (cerotinic acid, arabitol, phosphoenolpyruvate, terephthalic acid, cis-gondoic acid, N-acetyl-d-glucosamine, 4-hydroxymandelic acid, caffeine and xanthurenic acid) were identified as biomarkers with an excellent discriminant performance for WB myopathy. Collectively, this study provides new insights for a deeper understanding of the pathogenesis and provides metabolites as biomarkers for diagnostic utilization of WB myopathy.