Curcumin, the major active compound of turmeric, has shown potential benefits for poultry health and production in various studies. However, its specific role in enhancing the egg quality and liver health of laying hens, as well as its underlying mechanisms, have yet to be determined. Here, a total of 600 Su Qin No.1 Laying hens, aged 55 weeks and with similar laying rates, were randomly placed into five groups, with 10 replicates of 12 hens each. Curcumin doses of 0, 100, 200, 400, and 800 mg/kg were added to the basal diet to form the experimental groups. After an 8-week feeding period, no significant changes were observed in the production performance of laying hens due to curcumin supplementation. However, additional tests revealed that a 200 mg/kg curcumin supplementation improved albumen height, yolk color, Haugh unit, and eggshell thickness, while reducing the thin albumen’s weight and proportion. This was accompanied by a significant down-regulation of the mRNA expression level of the Prolactin Receptor (Prlr) in the oviduct magnum. Furthermore, the number of hepatic lipid droplets and the hepatic triglyceride (TG) content, as well as malondialdehyde (MDA) levels were significantly reduced, indicating improved hepatic lipid metabolism and oxidative status. This was accompanied by a significant reduction in the expressions of sterol regulatory element binding protein-1 gene (Srebp-1), fatty acid synthase gene (Fasn), as well as fatty acid synthase (FASN), which are closely related to fatty acid synthesis in the liver. Overall, these findings suggest that curcumin supplementation at a dosage of 200 mg/kg could lead to significant improvements in egg quality and hepatic lipid metabolism.

Ferroptosis participates in the occurrence and development of neurological disorders. Modulating ferroptosis may have therapeutic potential in nervous system diseases. Therefore, TMTbased proteomic analysis in HT-22 cells was performed to identify erastin–induced differentially expressed proteins. The calcium-transporting ATP2B3 (ATP2B3) was screened as a target protein. ATP2B3 knockdown markedly alleviated the erastin–induced decrease in cell viability and elevated ROS (p < 0.01) and reversed the up-regulation of oxidative stress-related proteins polyubiquitin-binding protein p62 (P62), nuclear factor erythroid 2-related factor2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase-1 (NQO1) protein expression (p < 0.05 or p < 0.01) and the down−regulation of Kelch-like ECH-associated protein 1(KEAP1) protein expression (p < 0.01). Moreover, NRF2 knockdown, P62 inhibition, or KEAP1 overexpression rescued the erastin–induced decrease in cell viability (p < 0.05) and increase in ROS production (p < 0.01) in HT-22 cells, while simultaneous overexpression of NRF2 and P62 and knockdown of KEAP1 partially offset the relief effect of ATP2B3 inhibition. In addition, knockdown of ATP2B3, NRF2, and P62 and overexpression of KEAP1 significantly down-regulated erastin–induced high expression of the HO-1 protein, while HO-1 overexpression reversed the alleviating effects of ATP2B3 inhibition on the erastin–induced decrease in cell viability (p < 0.01) and increase in ROS production (p < 0.01) in HT-22 cells. Taken together, ATP2B3 inhibition mediates the alleviation of erastin–induced ferroptosis in HT-22 cells through the P62–KEAP1–NRF2–HO-1 pathway.

Tonic immobility (TI) serves as an indicator of innate stress response recovery in poultry. Broilers with different TI phenotypes exhibit varying levels of aggressive behavior, which can significantly impact their welfare. However, the influences of TI phenotypes on broiler aggression remain largely unexplored. In this study, broiler chickens were stratified into two distinct phenotypic groups based on the TI duration: short TI (STI) and long TI (LTI). The impacts of TI phenotypes on broiler aggression were investigated by analyzing cecal intestinal morphology, cecal bacteria, plasma metabolites, and corticosterone levels. Compared to LTI broilers, STI broilers showed significantly reduced plasma corticosterone (CORT) levels (p < 0.05) and a decreased frequency of aggressive behaviors, including dominant and subdominant types (p < 0.01). Histological analysis revealed that STI broilers have an increased duodenal villus height and villus-height-to-crypt-depth ratio (p < 0.01), a decreased jejunal crypt depth with an increased villus-height-to-crypt-depth ratio (p < 0.01), and a reduced ileal crypt depth and villus height (p < 0.01) compared to LTI broilers. 16S rDNA sequencing and Linear discriminant analysis effect size (LefSe) identified differential cecal bacterial abundance, notably in the genus cc115 belonging to Firmicutes. Specific microbiota in LTI broilers exhibited significant positive correlations with aggressive behavior and plasma corticosterone, while those in STI broilers showed significant negative correlations. Untargeted plasma metabolomics revealed 21 downregulated and 17 upregulated metabolites between TI phenotypes. Correlation analysis showed that the genus cc115 and 10 plasma metabolites were positively correlated with aggressive behavior, whereas 8 metabolites were negatively correlated. LTI broilers have higher plasma corticosterone content and more intense aggressive behavior than STI broilers. The distinct behavioral and physiological profiles observed in broilers with different TI phenotypes are strongly correlated with their specific gut microbiota and differential plasma metabolite profiles. The identified gut microbial signatures serve as key biomarkers for regulating aggressive behavior in broilers, while the differential plasma metabolites represent potential early indicators for detecting stress and behavioral issues in poultry farming.

Heat stress leads to excessive hepatic lipid deposition and oxidative imbalance in laying hens, especially during peak laying period. Chlorogenic acid (CGA), a dietary polyphenol with antioxidant and lipid-modulating properties, may improve hepatic lipid homeostasis, yet its effects under heat-stress conditions remain unclear. In this study, 240 Hy-Line Brown laying hens at 36 weeks of age were randomly assigned to one of two treatments (120 hens per treatment, with six replicates of 20 hens each): a basal diet or a basal diet supplemented with 300 mg/kg CGA and subjected to heat-stress conditions for 8 weeks. CGA supplementation significantly reduced liver weight (25.3%), liver index (14.4%), hepatic triglyceride content (29.1%), and serum triglyceride level (61.7%) (p < 0.05). Histological assessment revealed lower steatosis and inflammation scores, alongside increased hepatic SOD activity (13.6%) and decreased MDA content (58.7%) (p < 0.05). RNA-seq analysis identified 420 differentially expressed genes that were significantly enriched in PPAR signaling and fatty acid β-oxidation pathways. CGA upregulated fatty acid oxidation-related genes (ACSL1, CPT1A, ACOX1, ACAA1) and downregulated lipogenic markers (FASN, ACACA). Serum metabolomics revealed coordinated changes in lipid and carbon metabolism. These results indicate that dietary CGA alleviates hepatic lipid accumulation and oxidative stress in heat-stressed peak-laying hens, potentially via PPARα-mediated enhancement of fatty acid oxidation and inhibition of de novo lipogenesis.

Heat stress (HS) has emerged as a major environmental stressor, inducing oxidative stress, hepatic steatosis and impairing production performance and health in laying hens, with limited evidence-based nutritional interventions available. This study investigated the hepatoprotective effects of dietary silymarin (SIL) against chronic HS. In a 10-week trial, 252 43-week-old Hy-Line Brown hens were exposed to daily HS (32 ± 1℃, temperature-humidity index [THI] > 73) and fed either a basal diet or one supplemented with 100 mg/kg SIL. SIL significantly increased laying rate (P < 0.05) and improved albumen height, Haugh units, and shell strength by week 8 (P < 0.05). Histological analysis showed a 48% reduction in non-alcoholic fatty liver disease (NAFLD) activity score, with significantly decreased hepatic triglyceride content (P < 0.05); Oil Red O staining confirmed reduced lipid droplet accumulation. SIL restored redox balance by increasing plasma and hepatic total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) (P < 0.05), increasing hepatic catalase (CAT) and glutathione (GSH) levels while decreasing malondialdehyde (MDA) (P < 0.05). Untargeted plasma metabolomics identified 11 key metabolites related to 2-oxoglutarate and purine metabolism, while hepatic transcriptomics revealed 835 differentially expressed genes primarily in the PPAR signaling and fatty acid biosynthesis pathways. SIL suppressed de novo lipogenesis via downregulation of ACACA and FASN, and enhanced β-oxidation through upregulation of CPT1A and ACSL1 (P < 0.05). Molecular docking and Western blotting confirmed strong SIL binding to these targets and corresponding protein changes. Correlation networks associated ACSL1 and CPT1A with improved performance and antioxidant indices, while FASN, ACACA, and xanthosine showed inverse relationships. These findings emphasize the potential of SIL as a sustainable animal nutrition antioxidant additive, which can alleviate HS induced lipid disorders in the liver of laying hens and provide insights for livestock applications.

Heat stress (HS) has emerged as a major environmental stressor, inducing oxidative stress and hepatic steatosis and impairing production performance and health in laying hens, with limited evidence-based nutritional interventions available. This study investigated the hepatoprotective effects of dietary silibinin (SIL) against chronic HS. In a 10-week trial, 252 43-week-old Hy-Line Brown hens were exposed to daily HS (32 ± 1 °C, temperature–humidity index [THI] > 73) and fed either a basal diet or one supplemented with 100 mg/kg SIL. SIL significantly increased laying rate (p < 0.05) and improved albumen height, Haugh units, and shell strength by week 8 (p < 0.05). Histological analysis showed a 48% reduction in non-alcoholic fatty liver disease (NAFLD) activity score, with significantly decreased hepatic triglyceride content (p < 0.05); Oil Red O staining confirmed reduced lipid droplet accumulation. SIL restored redox balance by increasing plasma, hepatic total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) (p < 0.05), increasing hepatic catalase (CAT) and glutathione (GSH) levels while decreasing malondialdehyde (MDA) (p < 0.05). Untargeted plasma metabolomics identified 11 key metabolites related to 2-oxoglutarate and purine metabolism, while hepatic transcriptomics revealed 835 differentially expressed genes primarily in the PPAR signaling and fatty acid biosynthesis pathways. SIL suppressed de novo lipogenesis via downregulation of ACACA and FASN, and enhanced β-oxidation through upregulation of CPT1A and ACSL1 (p < 0.05). Molecular docking indicated favorable binding affinities between SIL and these targets, which was further supported by corresponding changes in protein expression via Western blotting. Correlation analysis revealed a consistent alignment between the upregulation of ACSL1/CPT1A and improvement in performance and antioxidant status, suggesting a coordinated metabolic shift. These findings emphasize the potential of SIL as a sustainable animal nutrition antioxidant additive, which can alleviate HS-induced lipid disorders in the liver of laying hens. Importantly, these hepatoprotective effects were demonstrated exclusively under chronic heat stress conditions; further studies incorporating a normothermic baseline are required to distinguish stress-specific mitigation from general metabolic stimulation.