**3. Results**

Least square means ± standard errors (LSM ± SE) for normalised mRNA counts in peripheral leukocytes with respect to the 12 genes of interest in control and meloxicam-administered calves, prior to as well as at 4 and 24 h post-disbudding, are presented in Figure 1a,b. Significant di fferences in mRNA counts, between groups as well as between time points, were observed with regard to *IL8*, *IFN*γ, *IL1B*, *NOS1*, and *PGHS2* genes. The *IL8* gene exhibited significantly higher transcription (compared to baseline mRNA counts) at 4 and 24 h post-disbudding in both groups (Figure 1a). The *IL8* mRNA counts were particularly high in the control group calves at 4 h post-disbudding, which significantly di ffered from those in the meloxicam-administered calves. *IL1B* transcription was also significantly higher post-disbudding in both groups, with the mRNA counts at 4 and 24 h in the control group and at 4 h in the meloxicam group being significantly higher compared to the respective baseline values (Figure 1a). Furthermore, the *IL1B* mRNA counts at 4 h post-disbudding in the meloxicam group were significantly higher compared to those in the control group.

A slight but significant increase in *IFN*γ gene transcription, compared to time point 0, was evident in the meloxicam group at 4 and 24 h post-disbudding (Figure 1a). In addition, the mRNA counts for this gene at 24 h post-disbudding in the meloxicam group were significantly higher than those in the control group. The mRNA counts for *NOS1* and *PGHS2* genes were significantly higher in the control group at 24 h post-disbudding compared to their respective baseline values as well as the meloxicam-administered group (Figure 1b).

**Figure 1.** Transcription profile of selected cytokine and neuroactive ligand-receptor genes in peripheral leukocytes from calves, in response to cautery disbudding. *Two groups of calves (aged 31 to 41 days, n* = *8 per group), that were either administered meloxicam or no analgesic (control), were disbudded using an electric debudder. mRNA counts in peripheral leukocytes, as determined by amplification-free nCounter gene expression assay (NanoString, Seattle, WA, USA), were normalised based on the RNA counts of three reference genes included in the assay. An asterisk (\*) over the mean bars indicates a significant (p* < *0.05) di*ff*erence of transcription compared with respective pre-disbudding mean, within treatment, while significant di*ff*erences between the treatment and control group means, within each time point, were denoted with braces and respective p-values on top of the bars. (a) Data pertaining to angiotensin II receptor type 2 (ATGR2), corticotropin-releasing hormone (CRH), interleukin 8 (IL8), interferon gamma (IFN*γ*), interleukin 10 (IL10), and interleukin 1 beta (IL1B) are shown in this graph. (b) Data pertaining to interleukin 6 (IL6), nerve growth factor (NGF), nitric oxide synthase 1 (NOS1), prostaglandin-endoperoxide synthase 2 (PGHS2), tachykinin precursor 1 (TAC1), and tissue necrosis factor* α *(TNF*α*) are shown in this graph.*

A significantly higher transcription, compared to respective group baseline mRNA counts, was observed in the case of *AGTR2* (in meloxicam as well as control groups at both 4 and 24 h post-disbudding), *IL10* (at 4 h in meloxicam group), *IL6* (at 24 h in control group), *NGF* (at 4 h in control group), *TAC1* (at 24 h in control group), and *TNF*α (at 24 h in both control and meloxicam groups) genes (Figure 1a,b). In the case of the *CRH* gene, although the mRNA counts were relatively low, a between-group difference was observed at 4 h post-disbudding (Figure 1a). The mRNA counts for this gene in the control group calves were significantly higher compared to the meloxicam-administered calves.

Post-hoc power analysis results indicated a power of 0.706 for detecting a significant difference in the *IL8* mRNA counts between the meloxicam and control groups at 4 h post-disbudding, and 0.922 in the case of *PGHS2* mRNA counts between the two groups at 24 h post-disbudding. A sample size of 10 and 16 animals per group would increase the power of detection to 0.8 and 0.9, respectively, in case of the *IL8* gene. Similarly, powers of 0.966 and 0.999 were realised in the case of detecting a within-group (control) difference (compared to baseline values) in *IL8* mRNA at 4 h and *PGHS2* mRNA at 24 h post-disbudding, respectively.
