Investigating conditioned pain modulation in horses: can the lip-twitch be used as a conditioning stimulus?

Study objective was to evaluate whether the application of a lip twitch could be proposed as conditioning stimulus in the context of a novel Conditioned Pain Modulation (CPM) assessment paradigm for use in horses. The study was a prospective, experimental, randomized trial. Twelve healthy horses were evaluated in two experimental sessions. The lip twitch was used as the conditioning stimulus in both sessions; electrical stimulation was used as the test stimulus in one session, while mechanical and thermal stimulations were used in the other. Differences between thresholds recorded before and during twitching (Δ) as well as their percent (%) change were computed for each stimulation modality as a measure of CPM. Heart rate and respiratory rate were recorded throughout the experiments to monitor physiological reactions, while the general level of stress and aversiveness toward twitching were scored using <i>ad hoc</i> behavioural scales. Based on these scores, interruption criteria were defined. Ten and seven horses completed the electrical and mechanical/thermal experimental sessions respectively. For electrical stimulation, median (IQR) Δ was -2.8 (-3.9, -1.1) mA and% change 87.9 (65.7-118.2)%; for mechanical stimulation, Δ was -18.2 (-6.4, -21.4) N and% change 343.5 (140, 365.3)%; for thermal stimulation, Δ was -3.1 (-9.2, -2.1)°C, while% change was not calculated. Heart rate and respiratory rates varied significantly over time, with higher values recorded during twitching. Median stress and aversion scores did not differ between the two sessions. As lip twitching consistently affected thresholds to all stimulation modalities, it can be proposed as effective conditioning method for CPM assessment in horses. The exclusion of subjects due to severe aversion shows that this paradigm cannot be indistinctively applied to all horses and that stringent interruption criteria are necessary to guarantee adequate welfare during testing.