**1. Introduction**

Calf disbudding is a routine managemen<sup>t</sup> practice on dairy and beef cattle farms. The veterinary medical associations in several countries recommend this procedure to be performed at an early age [1]. Since late 2019, the disbudding of calves in New Zealand requires the administration of a local anaesthetic [2]. However, it is still performed without the provision of analgesia in most countries. Pain is a subjective state, and it can only be measured indirectly in non-verbal animals [3]. Previous studies have used a variety of pain assessment methods following calf disbudding. These include changes in behaviour [4] and/or physiological variables such as plasma cortisol [4–7], substance P levels [8,9], heart rate, eye temperature [7,8,10], nociceptive thresholds [7,8], and performance measures such as activity and weight gain [11].

During the recent decade, several studies have focussed on evaluating the effect of local anaesthetics and nonsteroidal anti-inflammatory drugs (NSAID) in mitigating the acute pain and distress following the disbudding/dehorning of calves [3,9,11]. The findings of those studies varied regarding the effects of local anaesthetics and/or analgesics on measures of pain and welfare following disbudding in calves. While some studies demonstrated that pre-procedural administration of local anaesthetics and/or analgesics resulted in benefits such as acute pain relief, increased weight gains, and growth rates [3,11], a few other studies did not find any significant reduction of physiological and behavioural changes [8,9,12]. None of the pain assessment methods have been described as the gold standard for measuring the nociceptive blocking ability of analgesics.

The molecular mechanisms behind nociception and the resultant peripheral and central sensitisation are gradually being unravelled. Cytokines are small proteins secreted by different cell types at the site of injury and by systemic immune cells in response to injury [13]. They appear rapidly following injury due to active gene transcription and translation by the injured cells. They initiate the acute phase response and induce the production of other pro-inflammatory cytokines resulting in amplified response to injury. Increased expression of inflammatory cytokine genes has been demonstrated post-surgery in humans and rodents [14], while perioperative analgesia attenuated the rise in inflammatory cytokine levels and postoperative pain scores in humans [14]. In addition, a significant increase in the expression of inflammatory cytokine mRNA was evident in tissue samples from the testes, epididymis, and scrotum after the castration of calves [15]. Important pro-inflammatory cytokines include interleukin 1 beta (*IL1B*), interleukin 6 (*IL6*), interleukin 8 (*IL8*), tissue necrosis factor α (*TNF*α) and interferon gamma (*IFN*γ) [13], while interleukin 10 (*IL10*) is an anti-inflammatory cytokine, and its induction has been shown to attenuate systemic inflammatory response [16]. Apart from pro-inflammatory cytokines, several molecules involved neuroactive ligand–receptor interaction, such as the nitric oxide synthase 1 (*NOS1*), prostaglandin-endoperoxide synthase 2 (*PGHS2*), angiotensin II receptor type 2 (*AGTR2*), corticotropin-releasing hormone (*CRH*), nerve growth factor (*NGF*), and tachykinin precursor 1 (*TAC1*), have an important role in nociception pathways [17]. Very few studies looked at the expression of inflammatory cytokine genes in response to calf disbudding [18,19]. A precise understanding of the cytokine response to injury can be exploited therapeutically to improve animal welfare.

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is routinely used to investigate gene expression. However, the enzymatic reactions (reverse transcription and polymerization) used in this method could contribute to variability. nCounter gene expression assay enables the amplification-free multiplex detection of nucleic acids by the molecular barcoding of target molecules using a colour-coded probe pair [20].

The aim of this study was to investigate the changes in peripheral leucocyte inflammatory cytokine gene expression, using nCounter assay, 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. It was hypothesised that the cautery disbudding of calves would induce significant changes in systemic inflammatory cytokine gene transcription, and that meloxicam (an NSAID analgesic) administered before disbudding would significantly attenuate the changes in cytokine gene expression.

### **2. Materials and Methods**

### *2.1. Animals, Groups and Ethics Approval*

This trial was undertaken on Holstein–Friesian Jersey crossbred calves born at the AgResearch farm, Tokanui, Hamilton, New Zealand, during the July to September calving season. The study protocol (#15/61) was approved by the Massey University Animal Ethics Committee, Palmerston North, New Zealand. Sixteen 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). Randomisation was performed by an online software QuickCalcs (GraphPad Software, San Diego, CA, USA). Calves in the control group received no analgesic before disbudding, and those in the treatment group received meloxicam prior to disbudding. During the study period, the calves were housed together indoors in a calf-rearing unit floored with wood shavings and maintained under normal farm practice.

### *2.2. Disbudding and Blood Sampling*

Calves in the treatment group received 0.5 mg kg−<sup>1</sup> meloxicam (Metacam 20 mg/mL for injection, Boehringer Ingelheim, NZ Ltd., Manukau, New Zealand) subcutaneously (SC) 1 h prior to disbudding. No analgesic was administered to the control group calves. Calves in both groups were sedated with 0.1 mg kg−<sup>1</sup> Xylazine (Xylazine 2% injection, Phoenix Pharm Distributors, Auckland, New Zealand) intravenously 10 min before disbudding. Disbudding was performed by a single veterinarian in all calves by cautery, using an electric debudder (Shoof International Ltd., Cambridge, New Zealand). The contact time between the cautery iron and each horn bud was maintained for 12–15 s [11]. Blood (10 mL) was sampled from the jugular vein into heparinised vacutainers just before and 4 and 24 h post-disbudding and transported to the laboratory at ambient temperature for processing. The 4 and 24 h sampling time points post-disbudding were chosen with a view to study the kinetics of the early and late transcription of cytokine and neuroactive ligand-receptor genes. Since calves in the control group received no analgesic either prior to disbudding, meloxicam (0.5 mg kg−<sup>1</sup> SC) was administered soon after the final sampling at 24 h post-disbudding to alleviate pain.

### *2.3. RNA Extraction and Purification*

Two ml of heparinised blood contained in a 50 mL conical tube was mixed with 20 mL pre-warmed tris-bu ffered ammonium chloride (TAC) bu ffer (46 mM Tris-Cl, pH 8.1, and 1 mM CaCl2), incubated at 37 ◦C for 10 min, and centrifuged at 350× *g* for 7 min at room temperature. The supernatant was discarded, and the pellet was re-suspended in one mL of RNeasy Lysis Bu ffer (Qiagen GmbH, Hilden, Germany). RNA in the lysates was purified using a QIAamp RNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany), and its quantity and quality were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA concentrations in the samples were adjusted to ≥25 ng/μ<sup>L</sup> and stored at −80 ◦C.

### *2.4. Enumeration of Gene-Specific RNA*

The detection and enumeration of RNA specific to 15 genes (12 of interest and 3 reference) was performed using nCounter gene expression assay [20]. Forty-eight RNA samples pertaining to the 16 animals, over three time points, were analysed using a custom-designed probe panel for 15 genes. Details of the genes, their Genebank accession numbers, and the position of target sequences are shown in Table 1. The simultaneous detection of mRNA of the 15 genes (Table 1) was undertaken using this assay as per the protocol outlined in the nCounter ™ Gene Expression Assay Manual, v.20090807 (NanoString, Seattle, WA, USA). In brief, the gene-specific capture and reporter probes were hybridised to complementary target mRNA (around 100 ng) in solution by overnight incubation at 65 ◦C in a thermocycler, followed by washing o ff the excess probes and non-target transcripts in the solution by a two-step magnetic bead-based purification on the robotic nCounter ™ Prep Station (NanoString, Seattle, WA, USA). Subsequently, the tripartite molecules were eluted and immobilised on cartridge for the enumeration of di fferent colour-coded probe-mRNA hybrids, using a nCounter ™ Digital Analyzer (NanoString, Seattle, WA, USA) with maximal sensitivity (555 fields of view, FOV).


**Table 1.** Details of genes analysed for transcription in peripheral leukocytes.

### *2.5. Processing mRNA Expression Data*

Tabulated data in comma-separated value (CSV) format, obtained from the nCounter ™ Digital Analyzer as reporter code count (RCC) file, was input into nSolver ™ Analysis Software, version 4.0 (https://www.nanostring.com/products/analysis-software/nsolver) and analysed as per the "nCounter Gene Expression Data Analysis Guide", MAN-C0011-04 (NanoString, Seattle, USA). The reporter library file containing the CodeSet information specific for the genes in this study was used to undertake the quality control routine. Default quality control (QC) settings were used: an Imaging QC (a measure of the percentage of requested fields of view successfully scanned in each cartridge lane) of <75; a Binding Density QC (a measure of reporter probe density in each cartridge lane) range of 0.1 to 2.25; a Positive Control Linearity QC (a measure of correlation between the counts observed for the positive control probes and the concentrations of the spike-in synthetic target nucleic acids) of <0.95; and a Positive Control Limit of Detection QC (indicates whether the counts for the positive control E probe and target sequence spiked in at 0.5 fM, assumed to be the system's limit of detection, are significantly above the counts of the negative control probes) of ≤2 SD above the mean of the negative controls. All samples (except a 24 h sample for an animal in the meloxicam group) passed the quality control. A background minimisation of counts was undertaken by subtracting the number of counts for the highest negative control +2 SD from all the mRNA counts. Subsequently, a positive control normalisation of RNA counts was then performed using the geometric mean of the 6 positive controls included in the nCounter assay. Finally, a biological normalisation of gene-specific RNA counts was undertaken based on the RNA counts of the chosen three mRNA reference genes (glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*), glucuronidase beta (*GUSB*), and 14-3-3 protein zeta/delta (*YWHAZ*), and the final counts were exported into an Excel workbook.
