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Being able to assess pain in nonhuman primates undergoing biomedical procedures is important for preventing and alleviating pain, and for developing better guidelines to minimize the impacts of research on welfare in line with the 3Rs principle of Refinement. Nonhuman primates are routinely used biomedical models; however, it remains challenging to recognize negative states, including pain, in these animals. This study aimed to identify behavioral and facial changes that could be used as pain or general wellness indicators in the rhesus macaque (Macaca mulatta). Thirty-six macaques scheduled for planned neuroscience procedures were opportunistically monitored at four times: Pre-Operative (PreOp), Post-Operative (PostOp) once the effects of anesthesia had dissipated, Pre-Analgesia (PreAn) on the subsequent morning prior to repeating routine analgesic treatment, and Post-Analgesia (PostAn) following administration of analgesia. Pain states were expected to be absent in PreOp, moderate in PreAn, and mild or absent in PostOp and PostAn when analgesia had been administered. Three potential pain indicators were identified: lip tightening and chewing, which were most likely to occur in PreAn, and running, which was least likely in PreAn. Arboreal behavior indicated general wellness, while half-closed eyes, leaning of the head, or body shaking indicated the opposite. Despite considerable individual variation, behavior and facial expressions could offer important indicators of pain and wellness. They should be routinely quantified and appropriate interventions applied to prevent or alleviate pain and promote positive welfare.
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