Because animals are unable to report their pain as humans can [1], the recognition of pain in animals requires the ability to understand the behaviour of the target species, the behavioural changes typically observed in animals in pain and the specific changes that occur in each animal's behaviour in response to pain. In this context, the video records obtained in this study served as an initial survey of items that might be appropriate for use in building and subsequently validating a scale for the assessment of pain in cattle.
The use of video recording for the validation of scales and for behavioural assessment is a common tool [14]-[16],[20] that permits the simultaneous analysis of an animal by multiple evaluators to be performed as often as necessary. In this study, following evaluation of the videos by the researcher, the films were edited according to the behaviour observed at different points in time. After reviewing the films, changes not covered in the initial scale were identified and items deemed irrelevant were excluded. The behaviour of animals in pain (M2) showed a reduction in eating and moving around, and when animals in pain did move around, they did so with restrictions and/or short steps and/or hunched backs. In addition, animals in pain spent more time lying down with their heads on or near the ground. When in the standing position, these animals assumed an abnormal posture, e.g., hunched and rigid and/or with the hind limbs extended caudally. Arched-back movements were also observed more frequently in animals in pain, along with cranial extensions of the neck while lying down, kicking, wagging the tail abruptly and looking at and licking the surgical wound. Given their relationship to pain, these behaviours were incorporated into the scale.
Some of the behaviours observed in this study have been described previously in cattle subjected to orchiectomy. These behaviours include remaining idle for longer periods, assuming an abnormal standing posture [26],[49] and exhibiting gait changes involving shorter, more cautious steps [32],[33]. With respect to time spent lying down, the results reported in the literature vary according to whether xylazine and analgesics were used. It would seem that animals spend more time in the standing position when xylazine is not used [26],[30],[31],[50] and that they spend more time lying down and less time moving around when it is used [33],[51]. In the present study, although xylazine might contribute to lying down after the surgery, the fact that administration of additional analgesia resulted in less rather than more lying behaviour suggests that this behaviour is due to pain rather than to the sedative effect of xylazine.
The use of xylazine in the present study was necessary because the Nellore breed is skittish and difficult to control. No animal showed recumbency after orchiectomy when leaving the restraining chute, once again demonstrating that the sedation was mild. Low doses of xylazine (0.015 to 0.025 mg/kg IV or IM) generally promote sedation without recumbency in ruminants [38]. In conclusion, decreased activity in cattle may be a good indicator of pain.
The reduced time spent eating observed in our study is consistent with other findings in the literature that report reductions in grazing time [29],[50], eating frequency [49],[50],[52] and, in the case of calves, suckling time [26]. The benefit of rescue analgesia with respect to this behaviour was also evident from the fact that the time spent feeding increased after rescue analgesia was performed.
Kicking and abrupt wagging of the tail were observed more frequently at M2, as described previously [26],[27],[29]. These events may occur after the local anaesthetic effect has lost its effectiveness [29] but may also be related to the presence of flies, which might represent one limitation of the study. Although it is impossible to completely eliminate flies from the environment, care was taken to reduce the number of flies present by using fly repellent, and the study was conducted in the winter when there is a low incidence of insects. One possible indication that flies had little effect on the kicking and abrupt tail wagging observed in this study was that kicking the abdomen was not observed at baseline; furthermore, the behaviour of wagging the tail abruptly and repeatedly is very characteristic and differs from the motion the animal makes to ward off flies. Thus, it would appear that these two behaviours are also related to pain in cattle [26].
Although it might be expected that the number of steps taken by the animals would be reduced after orchiectomy, no significant difference was observed in the number of steps recorded before and after orchiectomy. This finding differs from results previously reported in the literature [32]. The difference may be explained by the fact that the animals in the previous study did not receive analgesia [32], in contrast to this study, in which there was only a short (4-hour) span during which analgesia was not provided. The short period of pain experienced by the animals in the present study was most likely insufficient to influence the data obtained during the 24-hour evaluation.
The larger number of lying bouts observed after orchiectomy may be related to the restlessness and discomfort of the animals in the period prior to the application of the rescue analgesic. A similar phenomenon was observed in previous studies in cattle [27],[31]. Lying-down behaviour is also evaluated on the pain scales commonly used for dogs [10],[12],[13] and cats [14],[16], showing that although it is important to develop species-specific tools to assess pain, some pain behaviours are common among species.
Because the pedometer is not expensive and is relatively easy to handle, it can be a useful tool in the assessment of pain in cattle, especially when data analysis is carried out over relatively short periods.
Methods other than pain scales have also been used to investigate pain following castration in cattle. These methods include the assessment of physiological and neuroendocrine changes, such as serum cortisol concentration, and infrared thermography [53]. Facial expression of pain and kinematic and force platform gait analysis have been used in mice and in horses and dogs, respectively [54]-[56]. However, these methods have not yet been validated in cattle, and they either do not provide information in real time or require special equipment that is not currently available and/or is impractical under field conditions.
Validity and reliability are the key attributes of a scale that can be used to identify and quantify pain in animals. Reliability demonstrates the ability of the scale to reproduce the results regardless of the evaluator and at different times by the same evaluator [22]. In this study, the assessment of content validity was performed using the judgment of experts in the field who analysed the representativeness of each item in relation to the scale as a whole [57]. This methodology, which is well accepted [16],[17],[42], refers to the scope and adequacy with which the instrument reflects the phenomenon of interest, in this case, pain [22].
Criterion validity tests the effectiveness of a scale's measurement by comparing results obtained using that scale to results obtained using a previously validated method [12]. Criterion validity can be predictive when evaluating the criterion after testing and concurrent when evaluating the instrument and the criterion simultaneously [57]. In tests of criterion validity, the correlation between the scale and another instrument, ideally the gold standard [16],[22], is evaluated.
Considering that, to our knowledge, no gold standard instrument has been developed to evaluate pain in cattle and that correlation of the total scores obtained using our proposed scale with the scores determined by VAS may be questionable, an alternative method was used to investigate criterion validity in this study. The method involved comparing the agreement between pain scores assigned by blinded evaluators and a "gold standard" evaluator, in this case, the local evaluator. This method has been used with instruments designed for use in cats [16] and in young children [58].
Although the VAS, SDS and NRS may not show inter-observer reliability when tested on animals, they are nonetheless widely used to validate veterinary pain scales [12],[13],[16],[59] because the gold standards of verbal expression and self-assessment evaluation are not available in animals. Although inter-observer reliability may not be adequate when using VAS [59], intra-observer agreement or reliability is consistent over time [37] and may be a good option for measuring and comparing pain assessed by the same trained observer over time, as was done in this study [37].
The same methodology used for criterion validity in studies in cats [14],[15] was used to refine the scale by comparing the pain scores determined by blinded observers with those determined by the local evaluator. Subsequently, criterion validity should be evaluated by correlating the results obtained using the proposed scale and another instrument considered the gold standard (concurrent validity) [13]. Given the absence in the literature of validated scales for pain assessment in cattle, the pain scores on the scale proposed in this study were compared with the scores obtained using three other classical scales used in animals, the VAS, the NRS and the SDS. There was a high correlation between the results obtained using the four scales. Although these scales have not been validated in animals, this approach has been widely used to evaluate pain scales in veterinary medicine [12],[13],[16],[20].
Using factor analysis, it is possible to determine the dimensionality of the scale [45], i.e., the number of factors (dimensions or domains) represented by different variables [42]. Because the scale in question generated only one factor, it was considered unidimensional, in contrast to the scales validated for cats, which were considered multidimensional based on this analysis [16],[42]. Factor analysis is commonly used to develop an instrument and to relate a large number of variables such that the items that define specific parts of the construct are grouped together [60].
Despite the low reliability observed for the items standing posture and head position and the low correlation of the item attention to the surgical wound with the total scale score in this study, it was deemed important to retain a behaviour for each item.
The behaviours rigid hind limbs, hunched back and head below the line of spinal column may not have been clearly visible in the videos, and this may have resulted in the observed poor correlation between the blinded observers and the local observer on the items standing posture and head position. Conversely, the behaviour head below the line of spinal column obtained satisfactory agreement when considering only M2. Regarding the item attention to the surgical wound, the description of the behaviour looking at the surgical wound may not have been wholly appropriate because it produced different results when assessed by the local evaluator and the blinded observers. A description such as moves the snout in the direction of the surgical wound might have clarified observation of this behaviour; the description looking at the surgical wound was subjective because it could denote looking at the abdomen and/or to the side for another reason, a fact that may have confused the observers.
According to the Cronbach's α value, the scale employed in this work has excellent internal consistency [45],[60]. Internal consistency ensures that the scores of the items comprising the scale can be summed to produce a total score related to the overall assessment of pain intensity [16].
The moderate to good inter-observer agreement found in this work demonstrates the consistency of the results obtained by different evaluators and the ability of the instrument to produce consistent results [23]. The lower level of agreement for the item locomotion may be due to the short video analysis time and the animals' way of walking, which may have hindered the definition of the category. Thus, the results of both inter- and intra-observer reliability tests demonstrated good repeatability and stability of the scale.
The analysis grouping of M2 and M4 was important to confirm the reliability of the scale because these points represent the two most challenging times for pain assessment. A similar approach was used to validate a postoperative acute pain scale in cats [15],[16], but in that case, only M2 was considered separately. In our study, M4 was included because it also represents a challenging time, given the reduction in analgesic effect and the manifestation of pain-related behaviour that typically occurs after 24 hours.
Construct validity examines whether a given instrument detects predictable changes in the construct [22]. It can be evaluated by the well-known group method. This method determines whether the instrument detects differences between groups and is based on testing the hypothesis that time and intervention, both surgical and analgesic, should alter the pain scores [16]. The observed differences between pain scores at the time of greatest pain (M2) and the scores at other time points confirm the construct validity used in this work by verifying the reduction in pain scores in response to analgesia and over time [36]. This method has also been used to validate scales in veterinary medicine [14],[16] and attests to the responsiveness of the scale using a similar approach.
ROC curve analysis was used to determine the minimum score required for analgesic intervention [48], as was previously performed for a pain assessment scale in cats [15],[16]. The determination of scores that suggest a need for the use of analgesics assists the professional's clinical decision, affirms the effectiveness of analgesic treatment [15] and helps avoid unnecessary suffering in animals. Based on the balanced sensitivity and specificity criteria observed in this study, an optimum cut-off of > 4 was identified, i.e., additional analgesia is recommended when the pain score is ≥ 5 (0-10 point scale). It should be emphasised that according to clinical evaluation, additional analgesia must be performed if deemed necessary even if the score is lower than the cut-off point.
The high AUC observed (0.963) in this study indicates that the scale has excellent discriminatory ability and high accuracy, i.e., the instrument can correctly classify subjects with or without pain [47],[48]. Similar results were observed in the validation of a pain scale in cats [15],[16].
A possible limitation of this study is the absence of a control or uncastrated group of animals. The inclusion of a control group was considered when the study was designed, and a pilot study was performed to address this point. Subsequently, the authors decided to use only one castrated group and a larger number of animals based on the rationale that the animals' behaviour during the time period immediately prior to surgery could be considered a control because, at this point, the animals had already adapted to the environment and no management changes were performed during the study that could influence the results. This methodology has previously been used in cats [14],[16], dogs [12],[13] and horses [20]. The results observed here, which show significant changes in pain scores before surgery, after surgery and after analgesia, support the validity of the construct as well as the responsiveness of the scale. Additional support for the idea that the pre-surgical time period provided an appropriate control comes from the fact that the observers were blinded to the test moments and the order of the videos was randomised to avoid any bias. Although, in a very few cases, it was possible to observe the region of the testicles in the videos, it was not possible to determine from the video footage whether the animal had already been castrated. Another consideration is that it would be difficult to compare a different, uncastrated control group of animals with a group of castrated animals because the response to pain varies according to each individual.
The results of this study allow us to state that the UNESP-Botucatu unidimensional pain scale for assessing acute postoperative pain is valid and reliable. However, clinical tests with different analgesics and surgical protocols are recommended to assess the scale's clinical applicability.
