**Di** ff**erential Transcription of Selected Cytokine and Neuroactive Ligand-receptor Genes in Peripheral Leukocytes from Calves in Response to Cautery Disbudding**

**Kavitha Kongara 1,\*, Venkata Sayoji Rao Dukkipati 1,2, Hui Min Tai 3, Axel Heiser 1,4, Alan Murray 1, James Webster 5 and Craig Brian Johnson 1**



Received: 13 May 2020; Accepted: 6 July 2020; Published: 14 July 2020

**Simple Summary:** Calf disbudding is a painful husbandry practice on dairy and beef cattle farms. Continuing e fforts to enhance the accuracy of pain assessment can aid in the application of e ffective anti-nociceptive (analgesic) agents in non-verbal animals. The aim of this study was to evaluate the changes in the expression of genes involved in inflammation and pain sensitisation in response to removal of horn buds in calves, using hot-iron cauterization. The e fficacy of an analgesic, meloxicam, was also tested in attenuating the changes in expression of the studied genes post-disbudding. It was revealed that cautery disbudding induces significant changes in the expression of genes involved in inflammation. Meloxicam was able to blunt the increased expression of some of the genes at 4 h and 24 h after disbudding, while it could not attenuate the increased expression of a few other genes associated with inflammation.

**Abstract:** Calf disbudding is a painful husbandry practice on dairy and beef cattle farms. An objective measurement of pain is useful to reliably evaluate the pain intensity and anti-nociceptive (analgesic) efficacy of therapeutic agents. The aim of this study was to investigate the changes in peripheral leucocyte inflammatory cytokine gene expression in calves after disbudding, and to assess whether the changes in cytokine gene expression could be an indicator of the e fficacy of analgesic drugs. In a randomised controlled study, 16 calves (aged 31 to 41 days and weighing 58 to 73 kg), undergoing routine disbudding, were randomly allocated into two groups (*n* = 8 in each group). Calves in the control group received no analgesic, while those in the treatment group received 0.5 mg kg−<sup>1</sup> meloxicam subcutaneously prior to disbudding. Disbudding was performed using an electric debudder. Blood (10 mL) was sampled from the jugular vein just before and 4 and 24 h post-disbudding, RNA was extracted from leukocytes, and the transcription of 12 genes of interest was assessed using nCounter gene expression assay. The results showed significantly higher transcription (compared to baseline values) of the studied genes (except *CRH*, *IFN*γ, and *IL10*) in the control group calves at either 4 or 24 h post-disbudding. The administration of meloxicam one hour before disbudding significantly attenuated the upregulation of *IL6*, *PGHS2*, *TAC1*, *NOS1*, and *CRH* gene transcription post-disbudding, while it did not suppress the elevated transcription of acute and pro-inflammatory cytokines such as *IL1*β, *IFN*γ, *IL8*, and *TNF*α genes. In conclusion, nCounter gene expression assay seems to be a promising tool to study the expression of cytokine genes and thus could be used for the pre-clinical evaluation of novel analgesics.

**Keywords:** calves; disbudding; gene transcription; cytokines; pain; meloxicam; nCounter
