**8. Conclusions**

Sensitive, specific, and well-validated methods of assessing pain provide the cornerstone for developing e ffective analgesic medications. Unfortunately, there are few such methods available for assessing pain associated with castration in neonatal piglets. This is confounded by the neonatal piglet's physiological response to restraint, handling, and surgical stress due to tissue trauma, and the seemingly subtle, and short-lived expression of pain in the post-operative period. An understanding of the strengths and weaknesses of currently available methods for pain assessment is critical to identifying and developing e ffective pain mitigation strategies in neonatal piglets. Employing methodologies that lack specificity or reliability risks underestimating both piglet pain, and the e fficacy of pain-relieving medications, and creates welfare concerns associated with unproductive or counter-productive research. In the absence of a validated "gold standard" method of assessment, use of a number of di fferent methods are required and, indeed, this is a foundational requirement for any treatment method seeking regulatory approval. This review has discussed the potential strengths and weaknesses of a range of currently available methods of pain assessment in the context of examining the e fficacy of di fferent anaesthetic and/or analgesic treatment options in field trial settings.

Based on the detailed review of di fferent methods for assessing perioperative pain associated with surgical castration of piglets, this review concludes that:


assessment period as closely as possible to the time of acute pain generation. This is considered particularly important if studies are required in the field situation as opposed to acoustically separated environments.


It is hoped that this review may assist the future development of more standardized methods of assessing pain mitigation in neonatal piglets, assist investigators to optimise (reduce and refine) future analgesic e fficacy trials in this field, and support the development and evaluation of innovative effective and practical approaches to improve piglet welfare where surgical castration is still utilised in commercial pig facilities worldwide.

**Author Contributions:** Conceptualization, M.S.; methodology, M.S.; data collection, M.S. and A.P.; writing—original draft preparation, A.P.; writing—review and editing, M.S. and A.P.; supervision, M.S.; project administration M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding for this review was provided by Animal Ethics Pty Ltd. Yarra Glen, Victoria, NSW Australia.

**Conflicts of Interest:** Meredith Sheil is founding Director of Animal Ethics Pty Ltd., and inventor of Tri-Solfen ®, topical anaesthetic formulation. Adam Polkinghorne was funded by Animal Ethics Pty Ltd. for this project.
