Simple SummaryChronic inflammatory lameness is a common painful condition in dairy cows that affects animal welfare and produces significant yield losses. Bovine chronic lameness can lead to a chronic pain condition due to the participation of pro- and anti-nociceptive mediators and reactive oxygen species. The results of this study revealed increased concentrations of substance P and β-endorphin in lame dairy cows, indicating chronic pain development and sustained endogenous analgesics release. Moreover, the results revealed decreased disulfide levels and α-tocopherol concentrations in the lame group, suggesting an overproduction of reactive oxygen species, which may indicate their involvement in the development of chronic pain in bovine lameness.AbstractInitial lameness inflammation leads to chronic lameness and development of chronic pain due to the release of pro-inflammatory mediators such as reactive oxygen species (ROS), which are implicated in the transition from acute to chronic pain, and free radical scavengers countering thiol, substance P (SP), and β-endorphin (BE). The present study aimed to evaluate the dynamic thiol–disulfide homeostasis, α-tocopherol concentrations and SP and BE concentrations in the spinal cord of chronically lame dairy cows. Ten lame and 10 non-lame cows with a parity range of 2–6 were selected for the study. Lame cows had a history of up to 3 months of lameness. Spinal cord samples were obtained from the L2 to L4 lumbar vertebrae aspect of each animal. A thiol–disulfide homeostasis assay was performed using absorbance, and the α-tocopherol concentration was determined by HPLC. SP and BE concentrations were measured using ELISA kits. The results indicated that SP and BE were significantly higher in the spinal cord of lame cows. In contrast, disulfide levels and α-tocopherol concentrations were significantly lower in the spinal cord of lame cows. In conclusion, disulfide levels and α-tocopherol concentrations indicated a defective antioxidant response in cows with chronic lameness. The results of SP and BE concentrations suggested chronic pain and a defective endogenous analgesic response.