Simple SummaryKeel fracture is an important health and welfare problem in laying hens in all production systems. Previous studies have shown that keel fracture can influence hens’ behavior, reduce production performances, and cause pain in laying hens. Additionally, keel fracture also affects their feed intake. However, it is not clear whether the keel fracture induces stress, inflammation, or influences the orexin systems in laying hens. Orexin, also called hypocretin, is associated with the regulation of feed intake, energy homeostasis, and metabolism in poultry animals. Therefore, this study aimed to investigate the effects of keel fracture on stress and inflammatory responses and the activity of the orexin system of laying hens. Our results indicate that keel fracture not only induced stress and inflammatory responses, but inhibited the activity of the orexin system in laying hens. This study provides insights into the adverse effects of keel fracture on laying hens.AbstractKeel fracture has negative effects on the health and welfare of laying hens. We investigated effects of keel fracture on stress, inflammation, and the orexin system in laying hens. Ninety 17-week-old Lohmann white laying hens were palpated and euthanatized at 42 weeks old, and marked as normal keel (NK)/fractured keel (FK) from absence/presence of keel fracture. Serum, brain, liver, and abdominal-muscle samples were collected from 10 NK and 10 FK hens to determine the stress and inflammatory responses and the activity of orexin systems by corticosterone content, expression of heat shock proteins (TNF-α 60, 70, 90), and inflammatory factors (tumor necrosis factor (TNF)-α, nuclear factor-kappa Bp65 (NF-κBp65), inducible nitric oxide synthase (iNOS), prostaglandin E synthases (PTGEs), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β)), orexin (ORX), and orexin-receptor 1/2 (ORXR1/ORXR2). The FK hens had higher serum corticosterone content, Hsps, and inflammatory factor mRNA expression levels than NK hens, although levels of iNOS in the liver and TNF-α in the muscle were similar. Protein levels of Hsp70 and Hsp90 in the brain and liver, iNOS and COX-2 in the liver, NF-κBp65, iNOS, and COX-2 in the brain of FK hens were increased compared with NK hens. Furthermore, FK hens had lower mRNA expression of ORX, ORXR1, and ORXR2 than NK hens. Therefore, keel fracture causes stress and inflammation, and inhibits the expression of the orexin system in laying hens.