Proteins as indicators of stress and pain in lambs after castration by a rubber ring with and without pain relief

The objective assessment of pain in animals is challenging because indicators that are used are complex to interpret, inconsistent in presentation, and unable to distinguish between pain that varies in intensity and severity. The present study evaluated substance P, thiol oxidation, and plasma proteins as candidate indicators of pain in lambs. Merino ram lambs at 10-12 weeks of age were assigned, based on live weight, to one of four treatments: ring castration (n = 13), ring castration with pain relief (Meloxicam and Bupivacaine, n = 14), sham castration (n = 14), or sham castration with pain relief (n = 13). Samples of whole blood and plasma were collected pre-treatment and periodically after the treatment. The plasma concentration of cortisol was increased in the lambs 60 min after castration by a rubber ring without pain relief, while there was no change in the other treatment groups. There was no meaningful difference or change over time in substance P or thiol oxidation among the treatment groups. Proteomic analysis revealed no difference in the plasma proteins that were quantified between the lambs that were castrated by a rubber ring with and without pain relief. However, there was a significant increase in the number of unique proteins present 30 min and 2 h after castration in the lambs that were not provided with pain relief. Proteomics remains a promising avenue and targeted proteomic analysis is warranted to validate the proteins that were identified as indicators of stress or pain in lambs.