Cattle strongly mask signs of pain and do not show obvious pain behavior (1), which makes pain recognition difficult (2, 3) and has often lead to the assumption that cattle are insensitive to pain (1). Therefore, pain as well as pain management in cattle remain a major welfare problem in veterinary medicine (4, 5).

One parameter used to evaluate nociception in cattle and to differentiate between distress caused by nociception and stress is substance P (SP) (6, 7). SP is a neurotransmitter involved in processing noxious information to the brain (8), and a neuromodulator of pain (9). It is synthesized in ribosomes as a prepropeptide and transported via axons to the nerve ends. Following a noxious stimulation, SP is released from the neurons of the spinal ganglion and can be found in afferent neurons of the dorsal horn of the spinal cord, in cells of the dorsal ganglion, and in the dorsal roots of spinal nerves (10). SP is released slowly, with a delayed onset of excitation of the dorsal horn of 20 to 40 s, and a slow response which lasts 30 to 90 s (11–13).

In 2008, Coetzee et al. found that SP concentrations in surgically and sham castrated calves differed significantly, contrary to cortisol concentrations (6). Since then, SP concentrations have been described in cattle undergoing different painful procedures and conditions. However, studies provide conflicting results about the suitability of SP as a biomarker for nociception. SP concentrations were significantly lower in meloxicam-treated compared with placebo-treated calves following castration (14) and scoop dehorning (15). Other studies found no effect of flunixine meglumine on SP following castration (16) or cautery dehorning (17).

The major limitation of using SP as a biomarker for nociception is that research in experimental animals showed that SP controls the migration of inflammatory cells to an inflammatory site (10) and is an inflammatory marker (18). Local injury and tissue damage result in plasma extravasation due to increased plasma permeability, resulting in a release of SP from sensory nerve endings as an inflammatory mediator (19). Recent research found a positive and significant correlation between SP and leucocyte count in calves (20). In animal models, SP is also released in the course of a stress response, and SP and Neurokinin (NK)-1 receptors are located in those neurons of the neuroaxis which are part of the integration of pain, stress, and anxiety (8). The majority of studies in bovine medicine assessed SP concentrations following a painful stimulation resulting in tissue damage, and therefore, local inflammation (6) or during conditions involving the immune system (21, 22). Basic scientific research about the influence of nociception without tissue damage, stress, or inflammation without nociception on SP concentrations in cattle has not been done so far. This study is part of a project with the purpose to evaluate the influence of these stimuli on SP concentrations in cattle. A companion study assessing the influence of stress on PSPC was recently published (23).

The subject of this pilot study was to investigate the effect of nociception without tissue damage on SP. The objectives of the present study were to (1) evaluate the plasma substance P concentrations (PSPC) in calves experiencing a repeated electrical stimulation compared to a control group, (2) describe plasma cortisol concentrations (PCC), and (3) assess behavioral changes during and after the stimulation.

The primary motivation for the present study was to describe PSPC following a nociceptive stimulus not resulting in tissue damage, to distinguish between nociception associated with tissue damage, as described for castration (6) or inflammation such as metritis (21), and nociception occurring in the absence of tissue damage. The hypothesis of the present study was that a noninvasive acute peripheral electrical stimulation results in an increase of substance P, but not cortisol concentrations. This experiment is important, as until now, no biomarker for the assessment of only nociception has been described in cattle and basic research work about SP, which has been used as a biomarker for mostly nociception in cattle, is rare. The results of this study could help other researchers when planning studies including the assessment of SP.

All experimental procedures in the present study were approved by the ethics committee of the government of Upper Bavaria (reference number 55.2-2532-Vet_02_20–176). The present study is a pilot study with 12 animals included per group (24–26). Sample collection was done from May 2023 to May 2024.

According to our findings, PSPC decreased following an electrical stimulus, with no significant changes to PCC, and only mild behavioral reactions. Therefore, our hypothesis that an electrical stimulus increases PSPC has to be rejected. To the best of our knowledge, this study is the first to present changes to PSPC during and following an electrical stimulation, which is the major strength of this study.

We used an electrical impulse for an electrical nociceptive stimulation as was done before in cattle (27), to reduce the risk of tissue damage or inflammatory processes influencing the PSPC, as would have been possible following a thermal, chemical, or mechanical stimulus. As electrical stimulation is noninvasive (31), it was the best option for the purpose of this study.

Electrical stimulation is not a natural type of stimulus. It excites all peripheral fibers, including large diameter fibers which are not directly involved in nociception. The electrical thresholds of these individual fibers are related to their diameters. This results in a stimulation of first Aβ-, followed by Aδ- and C-fibers with increased intensity of the stimulus. Therefore, electrical stimulation of a sensory nerve results in pain due to nonselective activation of all types of peripheral fibers (31). Contrary to electrical stimulation, thermal stimulation excites thermosensitive and nociceptive fibers. Mechanical stimulation activates low-threshold mechanoreceptors and nociceptors, and result in tissue damage if they are truly nociceptive (31).

Electrical stimulation was done every 5 min over a duration of 25 min (5 times), to imitate the continuous nociceptive stimulation of a painful surgery (28). Evasive behavior during stimulation could have been provoked by nociception, which is the physiologic process leading to pain perception (19), or by pain, which is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage (19). However, the withdrawal response or reflex, an automatic response of the spinal cord to protect the body from harmful stimulation from noxious stimuli (32) could also have resulted in the evasive behavior (shaking of hind limb) shown by calves. Electrical stimulation was terminated when calves showed obvious signs of pain (shaking of the leg). After removal of the electrodes, following adspection and palpation, no tissue damage or alterations were recorded macroscopically, similar to adult cattle where only a hyperemia was recorded (27). As no biopsies were taken for histological examination, tissue damage cannot be ruled out microscopically, which is a limitation.

Even though calves in PAIN showed altered behavior indicative of pain, such as shaking and withdrawal of the leg (27) as well as half closed lids and head held below the dorsal line (3), PSPC did not differ between groups. PSPC decreased during the painful stimulus with the lowest concentrations at 25 min (last stimulus), and an increase after that stimulus. These findings are not in accordance with previous literature about PSPC in cattle exposed to nociception, where an increase in PSPC was found (6, 22), but similar to a recently published study, which found a decrease of PSPC during a stressful stimulation (23). Also, our findings are somewhat comparable to a previous study in bulls, which described that PSPC following electroejaculation (77.2 ± 17.2 pg./mL) did not differ from probed (79.1 ± 17.2 pg./mL) and control (93.4 ± 17.2 pg./mL) bulls. The authors concluded that electroejaculation did not result in nociception (33). Other studies only assessed PSPC in calves submitted to long lasting nociception in combination with the destruction of tissue (6, 7). Electrical stimulation is not a natural stimulus, but excites peripheral fibers, which are not directly involved in nociception (31), contrary to mechanical, chemical, or thermal stimuli which induce action potentials via nociceptors (19). SP is reported to be released following electrical stimulation in rats (34). However, transcutaneous electrical stimulation in rats supposedly reduces the production of SP in the dorsal root ganglion, resulting in analgesic effects by suppression of nociception via the C-fibers in the peripheral nerves (35). Research in cats showed that a single electrical stimulation of C-fibers was not mediated by NK-1 receptors, indicating that SP release is only mediated by specific patterns of firing nociceptors. This was further confirmed by the stimulation not being blocked by a NK-1 antagonist, suggesting that the response to the stimulus is not mediated by SP (36). Transcutaneous electrical nerve stimulation was found to inhibit the up-regulation of SP following skin/muscle incision and retraction in rats (37, 38). These findings could explain the absence of an increase of SP in our study population following electrical stimulation. However, a differentiation between procedures involving tissue damage, such as castration (6), inflammation such as due to lameness (22), or no tissue damage during electroejaculation (33) or a stressful stimulation (23) should be made regarding the evaluation of PSPC. It is possible that tissue damage, as well as inflammation is needed to raise PSPC. Therefore, PSPC might not be a good biomarker for nociception in the model used in the present study.

It is recommended to simultaneously determine PSPC and PCC to differentiate between acute stress due to handling and distress due to nociception (6, 7). It was found that the peak volume of PCC was reached 10 min after cattle being stressed by social separation (39). In the present study, there were no significant differences between groups, indicating that exposure to stress was the same for both groups. All animals had at least seven (8 to 15) days after purchase for acclimatization at the clinic and were trained to stand in the crush for 5 to 7 days. The acclimatization rate that it took heifers to move through a novel funnel structure in a yard was found to be decreased on days 1 and 2, and then consistent and increased on days 3 and 4 of training, respectively (40). Therefore, acclimatization and training period should have been sufficient for the present study. Different factors, such as restraint or presence of humans (7), as well as management and external environmental factors (41) can influence PCC. Significant differences within groups can be explained by calves being used to the procedure of the trial and of blood sampling at the end of the trial, resulting in less stress.

There was a negative and significant correlation between PSPC and PCC overall and in PAIN, which is not in accordance with previous findings in cattle which found a positive correlation between PSPC and PCC (23, 42). As a recently published study also found a significant decrease of PSPC in stressed calves but a positive correlation between PSPC and PPC (23), our findings could indicate that nociception and stress result in different interactions of cortisol and SP.

The observed behavioral responses are hard to compare with other studies, as previous research describing pain scales for cattle were working with a score system and defined thresholds or cut-off values associated with clinically relevant pain (3, 43). A grimace scale for pain and stress developed for calves also refers to a scoring system from scores zero to two (44). Therefore, an indication as to whether the observed behavioral responses reached thresholds associated with clinically relevant pain in calves cannot be made. As the stimulation calves were submitted to was in the context of a controlled trial and was limited until the moment calves showed continuous evasive behavior, it is possible that behavioral changes are more consistent with avoidance behavior than with nociception of sustained pain.

During stimulation, shaking of the stimulated hind leg was done significantly more often by PAIN calves and was the most frequent sign for discomfort and distress resulting in termination of the electrical stimulus. Seconds until termination decreased with significant differences from stimulus 1 to stimulus 4 and 5. Therefore, sensitization, which means that nociceptors are activated or become hypersensitive to subsequent stimulation (19, 45), cannot be ruled out. Shaking of the leg was not shown immediately following the start of the electrical stimulus as would have been expected in case of a withdrawal reflex, and calves often expressed stronger reactions to the subsequent stimuli and in some cases stereotypical behavior such as licking their halter, indicating that calves showed a conscious avoidance reaction. Frequency of occurrence of other parameters did not differ significantly, which can be explained by these behavioral patterns not being as specific for nociception as “shaking of the hind legs” in our study setup and is in accordance with a previous study investigating electric shocks in steers for vocalization, movements of the tail, and moving forwards and backwards in the crush (46). “Kicking with hind limbs” was mostly seen after the last electrical stimulus when the fixomull and the electrodes were removed from the skin and was therefore also recorded both in PAIN and CON. Contrary to previous findings describing more frequent head tossing in steers submitted to an electric shock compared to a control group (46), there was no difference in movements of the head between PAIN and CON in our study. This difference can be explained by the electrodes being placed behind the poll in those steers (46), and above the coronary band in the calves included in the present study. Behavioral reactions to the electrical stimulation were mild, contrary to an experiment in cattle which was terminated due to the severity of the behavioral responses of the animals (47). However, we stopped the electrical stimulation as soon as animals showed a continuous evasive behavior. It is possible that reactions would have been stronger and more severe if the electrical stimulation had not been stopped but continued until the end after 60 s. In cattle, the severity of behavioral responses was found to increase with the intensity of the electric shock (47). A study investigating the effect of an electrical prod on behavior in weaned beef cattle stated that beef cattle which had been touched, buzzed, and shocked with the electrical prod showed more escape reactions (such as running, kicking, or pushing through a block) compared with cattle that had only been touched or touched and buzzed (48). As the voltage in our study was continuously increased until a maximum of 60 s as opposed to a shock with a duration of 1 s (48), a comparison is hard to make. It should also be noted that the electrical stimulation used for the present study was not intended to model commercial handling procedures, but to serve as a controlled experimental tool. To limit the influence of investigators on behavior (49), the same number of people was always present during the period of the electrical or sham stimulation. Due to the setup of the study, the observer was not blinded to the grouping of the animal as recommended (49), which is a limitation. Also, interobserver reliability was not investigated, as behavioral assessment was either always done by TT (videos), or TT supervising HK.

Behavioral parameters assessed prior to every blood sampling did not differ between groups before, during, and after stimulation, except for the position of the head during, and expression of the eyes during and after the stimulation, with a higher occurrence of “lids half closed” in PAIN calves during and after the stimulation. The facial expression of animals changes when submitted to painful stimulation (50) and the facial grimace scale has been used for pain assessment in previous studies in cattle (4). The higher number of animals expressing “lids half closed” could be indicative of calves in PAIN experiencing nociception or pain during the electrical stimulation, even if the other parameters (ears, facial muscles, nuzzle) (3) did not differ between groups.

HR was significantly higher at 35 min compared with baseline in both groups, but did not increase following the electrical stimulation, as was also found for ten minutes following an electric shock in steers (46). Contrary to our findings, the heart rate of cattle increased monotonically immediately following an electrical shock in another study (47). The assessment of RR is an indirect measure to evaluate pain, even if not a satisfactory one (51). A previous study found no differences in RR between treatment groups in cattle submitted to surgery for left displaced abomasum with or without ketoprofen treatment (52). Significant differences in HR and RR between baseline and at 0 min compared with 35 min in both groups can be explained by walking the calves from the trial room to their igloos. Other factors, such as temperature humidity index, as well as posture of animals, seem to have an influence on RR (53), limiting the suitability of RR as an indicator for pain (52).

As electrical stimulation in PAIN was done above the coronary band of the right hind limb, changes to activity and steps taken due to pain should have been recorded. Pedometers directly record the locomotion and are therefore valuable for the assessment of lameness (54) and musculoskeletal pain (55). Similar to a previous study in calves undergoing tail docking (29), downs did not differ between CON and PAIN. Number of steps in our calves was similar with the baseline values of calves prior to tail docking housed under similar conditions. Contrary to our findings, number of steps was found to decrease within 1 h following tail docking (29), as well as following experimentally induced lameness in calves (54). The fact that there was no lameness in any calf or any differences between groups indicates that the electrical stimulation was not strong enough to result in tissue damage or long-lasting pain. However, limited room for movement restricts the evaluation of the “walking time.”

Calves weight was between 68 and 88 kg on the day of purchase. Body weight was not assessed on the day of either electrical or sham stimulation. A study showed that women’s tolerance to endure electrical muscle stimulation positively correlated with body weight, body fat mass, visceral fat area, and hip circumference (56). As applicators were placed around women’s abdomen (56), and the electrodes in the present study were placed above the coronary band of the right hind leg, where no large muscle or fat tissue can be found, an influence of body weight on our results seems unlikely. As cortisol and body weight are not correlated (57), body weight should not have had an effect on the PCC in the present study population.

To the best of our knowledge, this study is the first to present changes to PSPC during and following a noninvasive electrical stimulation, which is the major strength of this study, and to evaluate these changes in German Simmental calves.

One limitation of the present study is the low number of animals. The present study was conducted as a pilot study to assess the influence of an electrical stimulation on PSPC. A follow up study with a larger number of animals and comparison of different nociceptive stimuli, such as mechanical, thermal, or chemical should follow this study to further assess the suitability for SP as a biomarker for either nociception itself, or rather nociception as well as inflammation due to tissue damage.

Another limitation is the fact that calves needed to be clinically healthy on the day before and of the trial. Calves were submitted to a clinical examination prior to buying, but as calves were bought at a market, exposure to diseases was increased (58). Diarrhea and respiratory disease are the most common infectious diseases in calves (59), which was also the case in the present study population. As previous research showed that leucocyte count is significantly and positively related to PSPC in calves (20), calves were only included in the present study if leucocyte count on the day prior to the trial was within the clinic’s reference ranges to reduce the influence of inflammatory processes on PSPC. Calves were also excluded from the study if they had been treated either with an antibiotic or an inflammatory agent within 48 h prior to the trial, to limit the influence of diseases on the study results, and as the administration of analgesics results in a decrease of PSPC (14, 15).

The present study shows that a repeated short electrical stimulation does not increase, but continuously decrease PSPC in calves during the stimulation, even if animals show behavioral expressions of discomfort. It should be considered that, even if calves were clinically healthy and leucocyte count was within the reference ranges on the day before the trial, and animals had been trained to get used to the conditions of the trial, influence of pre-existing health conditions and stress on our study results, as well as of environmental factors, cannot be completely ruled out. As our findings are in contrast with other studies reporting an increase in PSPC following painful procedures in cattle, the authors could not assess if the evaluation of PSPC is a suitable method for assessment of noninvasive nociception in cattle which can be used individually and without relying on other parameters. Evaluation of PSPC may be a good marker for nociception in combination with tissue damage or inflammation but does not seem to be a reliable biomarker for nociception without tissue damage or inflammation. Studies to assess the influence of different nociceptive stimuli, such as mechanical, thermal, and chemical, with a larger number of animals will follow this study to establish basic research work regarding PSPC in the bovine.