The present study aimed to investigate whether acute pain associated with castration and tail docking of male piglets may modulate the expression of salivary microRNAs (miRNAs) and to explore their potential use as biomarkers. Thirty-six healthy 4-d-old piglets (Hermitage × Duroc) were randomly assigned to three groups: the first group (12 piglets) has been pretreated with anesthetic and anti-inflammatory drugs (ANA) and then castrated and tail docked; the second one (12 piglets) has been castrated and tail docked without any drugs (CONV); the third one (12 piglets) has been only handled (SHAM). Saliva was collected 10 min before (control group) and 30 to 45 min after the procedures. Salivary cortisol has been quantified. The expression concentrations of seven miRNAs, namely miR-19b, miR-27b-3p, miR-215, miR-22-3p, miR-155-5p, hsa-miR-365-5p, and hsa-miR-204, were measured and assessed as potential biomarkers of pain by quantitative Polimerase Chain Reaction using TaqMan probes. The area under the receiver operating curve (AUC) was used to evaluate the diagnostic performance of miRNAs. The concentration of salivary cortisol increased after treatment in CONV and ANA, while no significant variation was observed in the SHAM group. The comparative analysis demonstrated that the concentrations of salivary miR-19b (P = 0.001), miR-27b (P = 0.042), and miR-365 (P < 0.0001) were significantly greater in CONV as compared with pretreatment. The AUC of pretreatment vs. CONV and CONV vs. ANA were excellent for miR-19b and miR-365 and fair for miR-27b. Combining two miRNAs, namely miR-19b and miR-365, in a panel increased the efficiency of distinguishing between pre- and post-treatment groups. No differences have been identified between SHAM and ANA groups. mRNA potential targets of differentially expressed-miRNA were investigated, and genes related to pain and inflammation were identified: miR-19b potentially modulates TGF-beta and focal adhesion pathways, miR-365 regulates cytokines expression (i.e., IL-1, Tumor Necross Factor-alpha, and IL-8 cytokine), and miR-27b regulates macrophage inflammatory protein pathways (i.e., MIP1-beta). In conclusion, we demonstrated that the abundance of miR-19b, miR-27b, and miR-365 increases in the saliva of piglets castrated and tail docked without the administration of pain-relieving drugs. Further studies are needed to assess their potential during routine husbandry procedures and to extend their assessment in other stressful events, such as weaning or chronic pain.

中文翻译：

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唾液微RNA是潜在的生物标记物，可用于精确和精确地鉴定仔猪的尾巴对接和去势后的炎症反应。

本研究旨在调查与雄性仔猪去势和尾巴对接相关的急性疼痛是否可以调节唾液微RNA（miRNA）的表达，并探讨其作为生物标记物的潜在用途。将36只健康的4日龄仔猪（Hermitage×Duroc）随机分为三组：第一组（12头小猪）已用麻醉和抗炎药（ANA）进行了预处理，然后rated割并尾巴对接；第二只（12头）被been割，尾巴停药而没有任何药物（CONV）；第三只（12头）只被处理过（SHAM）。手术前10分钟（对照组）和手术后30至45分钟收集唾液。唾液皮质醇已被定量。七个miRNA的表达浓度，即miR-19b，miR-27b-3p，miR-215，miR-22-3p，miR-155-5p，hsa-miR-365-5p，使用TaqMan探针通过定量Polimerase链反应测量和评估hsa-miR-204和hsa-miR-204作为潜在的疼痛生物标志物。接收器工作曲线（AUC）下的面积用于评估miRNA的诊断性能。CONV和ANA治疗后唾液皮质醇浓度增加，而SHAM组未观察到显着变化。对比分析表明，与预处理相比，CONV中唾液中miR-19b（P = 0.001），miR-27b（P = 0.042）和miR-365（P <0.0001）的浓度明显更高。对于miR-19b和miR-365，预处理相对于CONV和CONV相对于ANA的AUC极好，对于miR-27b则相当。将两个miRNA（即miR-19b和miR-365）组合在一起可以提高区分治疗前和治疗后组的效率。在SHAM和ANA组之间没有发现差异。研究了差异表达的miRNA的mRNA潜在靶标，并鉴定了与疼痛和炎症相关的基因：miR-19b可能调节TGF-beta和粘着斑途径，miR-365调节细胞因子的表达（即IL-1，肿瘤交叉因子） -α和IL-8细胞因子）和miR-27b调节巨噬细胞炎性蛋白途径（即MIP1-β）。总之，我们证明了在不使用缓解疼痛药物的情况下，cast割和尾巴停泊的仔猪唾液中miR-19b，miR-27b和miR-365的含量增加。需要进行进一步的研究以评估其在常规饲养过程中的潜力，并将其评估扩展到其他压力事件中，例如断奶或慢性疼痛。