Nine adolescent female Danish Landrace pigs acquired from a commercial farm were included (23–40 kg). The animals were housed in pairs in iron enclosures with a 13:11 h light-dark cycle. Commercial food was provided twice daily, and nipple drinkers allowed the animals unlimited access to water. The room was maintained at ≈24°C. The study was approved by the Danish Veterinary and Food Administration under the Ministry of Environment and Food of Denmark (protocol number: 2020-15-0201-00514).

The pigs were habituated and trained daily at roughly the same time (08:00 to 10:00 AM) to decrease stress levels and increase the method's reliability. The pigs were habituated to the stable, the caretakers, the researchers, the equipment and separation from the mate for one week after arrival at the facility. The pigs then underwent clicker training individually for one week to train them to stand still and accept the mounting of the boots. When both the active and dummy boot were mounted, the animal received a food bowl with their regular commercial food to allow them to relax and stand still. At the conclusion of the training period, no retraction of the limbs was observed during the mounting boot attachment to the leg. In the third week, measurements of the MNT were conducted while pigs were eating calmly by their food bowl with both boots mounted.

The pressure algometer system consisted of an active actuator mounted in a placement boot on the animal's forelimb and connected to a strap and buckle kit to fixate the probe (ProdPro, Topcat Metrology Ltd, United Kingdom). A blunt-ended pin (2 mm diameter) protruded from the placement boot to allow the experimenter to apply pressure on the animal's limb. The blunt-ended pin was positioned approximately two cm below the middle carpal joint, roughly 45° from the sagittal plane. The pressure was manually induced via air injection from a syringe. The injection rate was controlled with the assistance of green and red lights that indicated if the pressure rate should be increased or decreased to maintain a constant slew rate of 2 N/s. The pressure algometer kit ensured a perpendicular angle between the pressure point and the skin surface. Finally, both limbs were tested, and while the mounting boot and actuator were placed on one limb, a dummy actuator and mounting boot were placed on the contralateral limb (see Figure 1).

The pressure was increased gradually until a foot lift was visually observed at which the respective force level was annotated. Stimulation was also immediately stopped when the cut off force of 25 N was reached. Three measurements were obtained on each limb at each experimental session, with a minimum rest interval of approximately 15 s. Finally, the animals were measured for three days, with a one-day interval between each measurement day. In total, 162 measurements were obtained, representing nine animals measured three times per session, on three sessions, at both limbs. The experimental procedure is described in Figure 2.

The force measurements were converted to pressure by dividing the force by the probe area. The statistical analysis was split into two parts: the first to assess internal consistency (within-session) reliability and the second to assess stability (between-session) reliability (19). Within and between-session systematic errors were tested with the one-way repeated measures ANOVA.

The relative reliability was determined by calculating the intraclass correlation coefficient 2-way mixed-effects model type absolute agreement (ICC_2,k) where k indicates the average of three repetitions. The average form, rather than the single measurement of ICC, was selected because a few repetitions are often performed in MNT. ICC values were interpreted based on a previously proposed category, according to which an ICC between than 0.81 and 1.00 is considered almost perfect, from 0.61 to 0.80 it is considered substantial, values between 0.41 and 0.60 are considered moderate, and below 0.40 it is considered unacceptable (20).

The absolute reliability was evaluated by the coefficient variation (CV) and standard error of measurements (SEM). CV was computed by the within-subject standard deviation as a proportion of the within-subject mean, indicating the stability of a measure across repeated trials (21). The SEM was defined as SEM=SD1−ICC and reflects the precision of individual scores on a test, meaning that it can be considered an estimation of expected random variation when no real change has occurred.

Finally, results are presented as mean and SD unless otherwise specified. The adopted significance level was 0.05, and the assumptions of normality and homoscedasticity were verified through residual analysis (Q–Q plots and histograms). Statistical analysis was performed with R software (22).

Six relative reliability measures were obtained with one almost perfect ICC (>0.81), two substantial ICCs (0.61–0.80), two moderate ICCs (0.41–0.60), and one unacceptable ICC (<0.40). The average ICC for the within-session analysis was substantial (i.e., 0.71), while the average ICC for the between-session analysis was moderate (i.e., 0.45), confirming that a pressure algometer is a valuable tool in assessing the nociception in the forelimb of pigs. We have also found that the within-session reliability was higher than the between-session reliability, indicating that the most considerable variability occurs between different days, even though measurements were obtained at roughly the same time every day to control for circadian patterns. This result could be explained by subtle changes in the position of the blunt-ended pin. Contrary to the hand-held algometer, where the exact position can be seen, the placement boot masks a clear view of the pin location.

Earlier studies were carried out to quantify the reliability of these methods in other species or at different body locations of the pig, such as the tail (16), where the authors used a hand-held pressure algometer in three distinct tail regions for animals with different ages and obtained on average, ICCs ranging from 0.33 to 0.46, depending on the tail region. A study investigating mechanical thresholds at the back of the metacarpus in piglets’ legs found across different days ICC values in the range of 0.29 to 0.65 (18). The results obtained in this study indicate that in terms of relative reliability, our method is consistent with previous reports and substantiates the use of MNT in large animals. It must be stated that human studies tend to find higher reliability coefficients; for instance, in a study investigating interrater reliability of pressure pain threshold, the authors found high ICCs (>0.92) for several body sites such as wrist, leg, neck and back (27). It is expected that human studies display higher reliability since instructions can be given such that the participants specifically respond to a painful stimulus. In animals, however, one cannot be sure whether the animal is responding to a noxious sensation or other sensations. Another likely reason for higher ICCs in humans is that humans offer a more heterogeneous sample and therefore larger individual differences. As relative reliability is dependent on the between-subjects variability, this, in turn, can result in a higher ICC.

Regarding absolute reliability, we obtained a grand-average CV of 19.1%, lower than previous studies that found an MNT CV of 25.5% (17) and 35% (14) for the limbs of pigs and dogs, respectively. A CV of 19.1% can be considered low and is comparable to values obtained in human studies (14.6% for the leg and 17.7% for the arm) (28). Only a few studies reported the SEM, making the comparison with existing literature challenging. Still, the SEM is an important parameter to be compared with future studies as it indicates the precision of individual scores on the test (29). The grand-average SEM of 249.5 kPA obtained in this study is considerably higher than the 93 kPA obtained in the leg of humans (27), which can be explained by the fact that SEM tends to increase at higher scale values (20), and pigs have a higher MNT than humans. Therefore, to allow for comparison across different species, we also calculated the SEM in terms of percentages of the mean, which resulted in an average value of 13.7% and 19.5% for the within- and between-session, respectively. These values are comparable to human studies (20).

No systematic differences in MNT were observed between the sessions, suggesting no effect of habituation (i.e., increased thresholds) or sensitisation (i.e., decreased thresholds). A systematic difference was observed only within session 2, where MNT increased in trials 2 and 3, suggesting some adaptation has occurred. Still, the lack of systematic changes between sessions highlights the importance of adequate training of the animals for the task prior to the experiment so that measurements are not obtained in a period where the animal's familiarisation curve is changing. A prior study investigating MNT in dairy cows reported that pre-test habituation decreased the variability and increased the reliability of MNT (30). In sows, a study assessing anatomical and methodological factors influencing MNT reported increased MNTs over measurement days up to a stabilisation in the fourth and fifth day, indicating habituation to the stimulus (17). We did not observe a difference in the average MNT between the right and left limbs; however, contradictory evidence exists in the literature, with studies also reporting no left-to-right differences in MNT (31) and others reporting different values on the left vs. right side of the body, which might be a result of left- or right-side dominance (32).

An advantage of the “remotely-controlled” actuator used in this study is that the animal has no visual cue when the stimulus is given, which can generate anticipation of the stimulus (17). Another factor concerning the device that may influence the measurements’ reliability is the pressure application rate; the device uses a light system (green and red diodes) to indicate if the pressure is at the selected level. We observed that prior training with the device was enough for the researcher to keep the pressure rate stable during the experiment. Still, further improvements could include computer-controlled algometers with fixed pressure rates.

The limbs are particularly important in neuropathic pain models, where the disease is induced by some form of peripheral nerve injury (33). In animals, it is challenging to make the subjects stay still long enough to obtain accurate measures, especially in large animals. For instance, rodents can be immobilised with the hand to obtain the measurements (34). A “forced” immobilisation in large animals would be practically impossible and can generate stress-induced analgesia, affecting the measurements by increasing thresholds (35). Still, recent studies have shown the feasibility of assessing the nociceptive system in the limbs of pigs using von Frey filaments (36), laser stimulation (37), and mechanical stimulation (38). The latter used a perforated test platform to which the animals were acclimatised. In our study, the animals were tested in their pen, and a food bowl was sufficient to keep them standing still for the duration of the task.

It must be noted that several factors can influence the MNT; therefore, caution must be taken when translating the results from this experiment to other studies. Previous studies demonstrated that mechanical threshold increases with larger tip diameters (39) and time of the day, where thresholds are higher in the morning than in the afternoon (17). The range of MNT values obtained in this study is similar to other studies in pigs at the same weight range (38), but it is far smaller than MNTs in the limbs of heavier animals. In pigs weighing an average of 267 kg, thresholds of 16,500 kPA were observed (31). Therefore, the direct comparison of MNT between studies should also consider the effect of animal weight, as mechanical thresholds are positively correlated with body weight (18).

This study was conducted only on female pigs for two reasons. First, female subjects are underrepresented in preclinical pain research (40), despite the fact that the majority of chronic pain sufferers are female (41). This trend resulted in a male-based literature (42). The second reason relates to swine housing. Due to the fact that the animals in this experiment were housed in pairs, it is known that entire male pigs tend to exhibit more aggressive behaviour and fighting activity, particularly during puberty (43), which could impede the continuation of the study. Additionally, housing mixed-sex groups also result in more aggressive behaviour than housing only females (44). Sex differences in MNT have been investigated in dogs (39) and piglets (18), and none of the studies reported significant differences between males and females.

The effect of different examiners on reliability was not investigated in this study and should be considered in future experiments, as different examiners can display significantly different reliability levels that may be related to the examiner's experience or timing in detecting avoidance reactions (15).