In order to uncover the role of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K/AKT) signaling pathway in metabolic regulation in Tan sheep meat during chilled storage, the transcriptome changes of lamb meat injected with the PI3K inhibitor LY294002 after 0, 4 and 8 days of chilled storage were studied by high-throughput sequencing. Altogether, 474, 96, and 13 differentially expressed genes (DEGs) between the control and LY294002 treatment groups were identified on days 0, 4, and 8, respectively. Functional enrichment analysis showed that the DEGs were mainly related to metabolic homeostasis, such as amino acid biosynthesis, glycolysis/gluconeogenesis, and glyceride metabolism. In addition, tissue section analysis showed that the gap between muscle fibers in the LY294002 group increased and the diameter of muscle fibers decreased significantly relative to the control group, indicating muscle atrophy. These results indicated that the PI3K/AKT signaling pathway played a crucial role in maintaining metabolic balance during the storage of Tan mutton, which in turn ensured the normal growth of muscle. This study provides a new direction for further elucidating the molecular mechanism by which the PI3K/AKT signaling regulates metabolic homeostasis in Tan mutton during chilled storage.

This study was performed in order to uncover the molecular mechanism by which postmortem glycolysis affects meat quality traits for the purpose of promoting the research and development of prediction technologies for the quality change of chilled Tan lamb and promoting the improvement of its processing, storage and transportation technologies. High-throughput sequencing technology was used for transcriptome sequencing and analysis of the hind leg meat from Yanchi Tan lambs after 0, 4 and 8 days of chilling. The results showed that the differential expression genes (DEGs) were mainly enriched in cellular amide metabolism, ATP metabolism, skeletal muscle contraction and ribosomes and involved in glycolysis, ribosomes and the p53 signaling pathway, and these DEGs constituted a key part of muscle cell function. The phosphofructokinase (PFKM), glyceraldehyde-3-phosphate dehydrogenase (GADPH), phosphoglycerate mutase 2 (PGAM2), enolase 3 (ENO3) and pyruvate kinase (PKM) genes were the core genes involved in the glycolytic metabolism of Tan lambs, which changed meat quality mainly by affecting the structure of muscle fibers, the degradation degree of structural proteins, the activity of glycolytic enzymes, the production of ATP and the activity of complexes.

In order to clarify the relationship between the degree of glycolysis and water-holding capacity during the maturation of Tan sheep meat, the M. longissimus dorsi of six-month-old Tan sheep was aged at 4 ℃ postmortem and evaluated for drip loss, purge loss, calpain-1 activity, myofibril fragmentation index (MFI), ATP content, glucose content, glycogen content, lactic acid content, lactate dehydrogenase (LDH) activity, pyruvate kinase (PK) activity and pH after 0, 1, 2, 3, 4 and 8 days. The results showed that as maturation time increased, drip loss, purge loss and centrifugal loss increased first and then decreased (P < 0.05), reaching their maximum values on day 4. ATP and glycogen contents continued to decrease (P < 0.05). The contents of glucose and lactic acid increased first and then decreased (P < 0.05), and so did LDH activity (P < 0.05), reaching its maximum level on day 4. PK activity showed a constant decreasing trend (P < 0.05). The correlation analysis showed that glycogen content was negatively correlated with drip loss (P < 0.05) as well as purge loss and centrifugal loss (P < 0.01), and positively correlated with nuclear magnetic resonance (NMR) transverse relaxation times (T₂₁, T₂₂ and T₂₃) (P < 0.05 or P < 0.01). LA content was significantly positively correlated with drip loss (P < 0.001) as well as purge loss and centrifugal loss (P < 0.01), but had no correlation with T₂₁, T₂₂ or T₂₃. Therefore, the glycolysis process of Tan sheep meat was activated rapidly after slaughter, which caused change in the activity of glycolysis-related enzymes and the contents of metabolites, thereby resulting in changes in the water adsorption capacity of muscle proteins, and ultimately affecting the water-holding capacity of Tan sheep meat after slaughter.

In order to elucidate the relationship between the oxidative phosphorylation and the color stability of Tan sheep meat during postmortem storage, the changes in the color stability and oxidative phosphorylation of the Longissimus dorsi of 6-month-old Tan sheep were determined after 0, 1, 2, 3, 4, 6 and 8 days of postmortem storage at 4 ℃. The results showed that L*, a* and b* values increased first and then decreased during 0–8 days of storage (P < 0.05), and so did R₆₃₀/R₅₈₀ ratio (P < 0.05). The relative content of metmyoglobin (MetMb) showed a decreasing and then increasing trend (P < 0.05), whereas the relative content of oxymyoglobin (OxyMb) exhibited the opposite trend (P < 0.05), and the relative content of deoxymyoglobin (DeoMb) showed a decreasing trend (P < 0.05). Mitochondrial membrane permeability significantly increased (P < 0.05), and mitochondrial membrane potential significantly decreased (P < 0.05). The activity of mitochondrial complex Ⅰ, Ⅱ, Ⅲ and Ⅳ significantly decreased (P < 0.05), while the activity of complex Ⅴ significantly increased (P < 0.05). Reactive oxygen species (ROS) levels significantly increased (P < 0.05). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) significantly decreased (P < 0.05). Catalase (CAT) activity first increased and then decreased (P < 0.05). The contents of ATP, ADP and AMP significantly decreased (P < 0.05). The relative contents of MetMb, OxyMb and DeoMb and meat color parameters were significantly correlated with oxidative phosphorylation. The above results indicate that the failure of endogenous antioxidant enzymes in Tan sheep meat during postmortem storage led to excessive ROS production in myocytes, which damaged the integrity of mitochondria, and then affected the activities of enzymes related to mitochondrial oxidative phosphorylation and consequently the activity of MetMb reductase in myocytes, which resulted in the failure of MetMb reduction, thereby leading to deterioration of the color stability of Tan sheep meat.

The hindleg meat of Tan sheep was injected postmortem with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 at a concentration of 10 μmol/L and stored at 4 ℃. After 0, 2, 4, 6 and 8 days, the protein expression of PI3K and protein kinase B (AKT)was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot so as to verify whether the PI3K/AKT signaling pathway was effectively inhibited. Meanwhile, we analyzed changes in energy factors, oxidative stress levels, the degree of mitochondrial damage and cysteine aspastic acid-specific protease 3 (caspase-3) activity in order to explore the mechanism by which the PI3K/AKT signaling pathway induces the pathways of cell apoptosis during chilled storage of Tan sheep meat. The results showed that during storage, the protein expression levels of PI3K and AKT showed a decreasing trend, indicating that LY294002 inactivated the PI3K/AKT signaling pathway. Compared with the control group, LY294002 treatment significantly reduced mitochondrial ATP, glycogen content, succinate dehydrogenase (SDH) and citrate synthase (CS) activity (P < 0.05), gradually increased mitochondrial reactive oxygen species (ROS) levels, elevated the levels of oxidative stress and the opening of mitochondrial permeability transition pore (MPTP), significantly decreased membrane potential (P < 0.05), and significantly augmented the activity of caspase-3 (P < 0.05), indicating that the PI3K/AKT signaling pathway mediates the activation of downstream caspase-3 by promoting the production of ROS to interact with PI3K, damaging the structure and function of mitochondria, leading to the opening of MPTP and decreased membrane potential, and ultimately inducing cell apoptosis.

In order to clarify the mechanism underlying the regulatory effect of oxidative phosphorylation (OXPHOS) on the color stability of Tan sheep meat during postmortem storage, the M. longissimus dorsi of 6-month-old Tan sheep was stored at 4 ℃ after slaughter and evaluated for the activity of endogenous antioxidant enzymes and the content of reactive oxygen species (ROS) after 0, 4 and 8 days. Proteomics based on isobaric tags for relative and absolute quantitation (iTRAQ) was used to study protein expression in Tan sheep meat at different storage times. The results showed that during 0–8 days of storage, the activity of endogenous antioxidant enzymes showed a decreasing trend (P < 0.05), and the ROS content significantly increased with storage time (P < 0.05). Eight differentially expressed proteins were identified as the core proteins affecting the color stability of Tan sheep meat, namely ATP synthase F1 subunit alpha (ATP5A1), ATP synthase peripheral stalk subunit OSCP (ATP5O), ATP synthase peripheral stalk subunit D (ATP5H), cytochrome c oxidase subunit Ⅱ (COX2), cytochrome c oxidase subunit 5A (COX5A), succinate dehydrogenase complex iron sulfur subunit B (SDHB), ADP/ATP translocase 1 (SLC25A4), and cytochrome c (CYCS). These proteins affected the color stability of Tan sheep meat mainly through the energy metabolism and redox processes. These results indicated that the loss of the activity of antioxidant enzymes in Tan sheep meat during postmortem storage caused the accumulation of ROS, which led to the destruction of the mitochondrial integrity of muscle cells and aberrant gene expression of complex subunits such as ATP5A1, ATP5O, ATP5H, COX2, COX5A, SDHB, SLC25A4 and CYCS in the oxidative phosphorylation pathway, thus contributing to changes in the structure and function of mitochondrial complexes and blockage of the oxidative phosphorylation process, and ultimately affecting the color stability of Tan sheep meat.