**5. Conclusions**

Hot iron disbudding induced significant changes in the expression of a complex network of inflammatory cytokine mRNA in the peripheral blood leukocytes of calves. The subcutaneous administration of meloxicam one hour before disbudding significantly attenuated the upregulation of IL6, PGHS2, TAC1, NOS1, and CRH gene transcription post-disbudding. The mRNA expression levels of acute and specific pro-inflammatory cytokines such as IL1β, IFNγ, IL8, and TNFα were significantly increased after disbudding, and meloxicam did not suppress the elevation of these cytokine mRNA compared to the control group. The current study results indicate that the administration of only a systemic NSAID could not completely reduce the acute inflammatory response following disbudding. nCounter gene expression assay (NanoString, Seattle, WA, USA) was used in the current study, which is a promising tool to study the expression of cytokine genes and thus could be used for the pre-clinical evaluation of novel analgesics. However, it would be useful to corroborate these gene transcription results at protein levels in further studies.

**Author Contributions:** K.K., V.S.R.D. and C.B.J. conceived and supervised the study, K.K., V.S.R.D., C.B.J. and J.W. planned and carried out disbudding of calves and sample collection. V.S.R.D., H.M.T., A.H. and A.M. were involved in planning and execution of gene expression analysis. K.K. and V.S.R.D. prepared the paper drafts. All authors contributed to editing the article and approved the final manuscript.

**Funding:** This research received no external funding and was funded by the McGeorge research fund, Massey University, New Zealand.

**Acknowledgments:** We acknowledge the help of Alison Cullum, Veterinarian and Animal Welfare Officer, AgResearch, Ruakura, New Zealand, with disbudding the calves.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
