Introduction

In the recent literature, emotion has been considered to include behavioural, physiological, cognitive, and subjective components [1]. The first two components are relatively simple to measure in animals (e.g. vocalizations, cortisol levels), and give an indication of emotional intensity but not valence (i.e. pleasant or unpleasantness). This is problematic, as emotional valence is more informing of an animal's welfare status compared to emotional arousal. Because animals are unable to verbally convey their subjective experiences, researchers have turned to the cognitive component of emotion as a method of assessing emotional valence. Emotional responses in humans are associated with changes in cognitive functioning, including attention, memory, and judgement bias [1]. The effect of emotion on judgement making has been explored in a number of areas, including risk-taking, future expectations, and interpretation of ambiguous stimuli. Depressed or anxious people interpret ambiguous stimuli more negatively [2], while people in positive states have more optimistic interpretations [3], [4].

Recent research has attempted to use changes in cognitive processes as a method of assessing emotions of animals [5]. The first published study on cognitive bias in animals [6] focused on judgements of ambiguous stimuli presented in an operant discrimination task. Rats were housed in predictable and unpredictable housing conditions; the latter condition was intended to induce a negative affective state in the rats. Compared to the control rats, rats in the unpredictable housing condition showed fewer positive responses to the ambiguous stimulus. More recent studies have examined different species exposed to various other affect manipulations [5]–[12] and have found similar biases, illustrating that judgement biases can be useful in identifying emotional states in animals.

Pain is defined by the International Association for the Study of Pain [13] as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage.” Indeed, pain is one of the most highly studied emotions in animals [14], and the rich interplay between pain and cognitive processes is well studied in humans [15], [16].

Research on pain assessment in animals has focused on behavioural and physiological measures, but these measures only provide an understanding of the arousal of the emotional experience. Studies in rodents have also shown cognitive changes in response to pain, including attentional biases [17], conditioned place aversion learning [18] and social modulation [19]. Research on negative judgement bias resulting from pain has been limited to the human literature [20]; we hypothesize that animals in pain exhibit similar judgement biases. Such biases may be functional in that animals experiencing pain are likely more vulnerable to fitness threats and thus may preferentially benefit from interpreting ambiguous stimuli as threatening [21].

Removal of horn buds from dairy calves (i.e. disbudding, also termed dehorning), like other routine surgeries on commercial farms, is commonly performed without use of post-operative analgesics [22]. Post-operative pain from disbudding is associated with behavioural (e.g. head rubbing, head shaking, ear flicking, vocalizations) and physiological changes (e.g. plasma cortisol concentrations) that persist for at least as long as 24 h after the procedure [22]–[24]. The aim of this study was to assess whether the pain-induced changes in emotional state in calves are associated with a judgement bias. We predicted that calves experiencing pain after disbudding would exhibit a pessimistic bias in their responses to ambiguous stimuli.

Materials and Methods

(c) Animals and experimental testing pen

Experiment 1.

We used eight male Holstein calves starting at approximately three days of age. Calves were housed in individual sawdust bedded pens (1.2 m×2.0 m). Management of the calves from birth to enrolment followed standard farm protocol. A separate pen, identical to those the calves were housed in, was used for the cognitive bias task (Fig. 1). A 48-cm computer monitor was fastened at the rear of the pen, approximately 50 cm above the pen floor. Images on the monitor were controlled using a laptop computer. All calves were handled, trained and disbudded by the same person, and calves were trained and tested in the cognitive bias task at the same time each day, twice per day. Training and testing sessions lasted approximately 15 min per calf. Although there was individual variability in learning speed, each step of the training process was tailored to the individual calf (i.e. calves did not proceed to the next stage until the specified criterion was met).

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Figure 1. Experimental apparatus for training and testing in the judgement bias task.

Calves entered through the front gate to begin each session. The computer controlled the display of positive, ambiguous and negative screens.

https://doi.org/10.1371/journal.pone.0080556.g001

Experiment 2.

Nine male Holstein calves were enrolled at approximately five days of age. A 38-cm video monitor was used to present training and test screens. All calves were handled, trained and disbudded by two previously trained individuals.

Results

(b) Response to probes

When tested with ambiguous intermediate screen colours before disbudding, the calves treated the near-positive and near-negative screen colours as being similar to the training screens, approaching these screens in 92% and 23% of trials, respectively; calves were more ambivalent about the halfway red screen, approaching it in 69% of the trials (Fig. 2). After disbudding calves approached the ambiguous screens less frequently compared to before disbudding (p = 0.0004). Numerically, this negative bias was weakest for the near-positive probe (with calves approaching this probe 4% less frequently after disbudding) and more prominent for the halfway and near-negative probes (declining 14 and 11% respectively).

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Figure 2. Mean ± SE approach responses of calves to each screen colour before and after disbudding.

Calves were trained to approach the positive screen colour for a milk reward, and to avoid approaching the negative screen colour, and were tested with unreinforced ambiguous colours. Calves responded to the ambiguous colours less frequently after versus before disbudding (p = 0.0004); as expected, there was no effect of disbudding on the responses to positive and negative training colours.

https://doi.org/10.1371/journal.pone.0080556.g002

Individual calves differed somewhat in their responses to the probes (Table 1), but most calves (13 of 17) responded less frequently to the probe screens after disbudding compared to before. Three of the 17 calves showed no difference and only 1 calf responded more frequently to the probes after disbudding.

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Table 1. Calf approach responses to each screen before and after disbudding (%).

https://doi.org/10.1371/journal.pone.0080556.t001

Discussion

Calves exhibited a negative judgement bias for at least 22 hours after hot-iron disbudding; calves were less likely to approach ambiguous screen colours in two test sessions after the procedure. This ‘pessimistic’ negative bias is indicative of a negative emotional state in calves following hot-iron disbudding.

These results provide the first evidence of judgement bias associated with pain in non-human animals. Previous studies examining depression and anxiety have shown different types of judgement bias. In humans, depression is associated with a decreased expectation of positive events, while anxiety is associated with an increased expectation of negative events [29], [30]. Studies in animals have reported both types of biases, interpreted based upon their proximity to the “positive” or “negative” reference stimuli in the judgement bias task.

The strongest bias observed in this study was at the halfway and near-negative colours, indicating an increased expectation of negative events. A more pronounced bias at the near-negative probes was also described by Burman et al. [31], Matheson et al. [32] and Pomerantz et al. [6] who induced negative emotional states in rats, starlings, and monkeys, respectively. Based on the direction of the bias, both Burman et al. [31] and Pomerantz et al. [6] proposed that the emotion experienced by the animals was negatively valenced and of high intensity, similar to anxiety. Following this reasoning, we suggest that the pain experienced by these calves was associated with an anxiety-like rather than depression-like emotional state.

The post-operative pain associated with hot-iron disbudding is known to persist at least 24 h (reviewed by [24]). Calves exhibit elevated plasma and salivary cortisol levels and heart rate for up to several hours after hot-iron disbudding [33], and have higher frequencies of head shaking, ear flicking, and head rubbing for up to 24 h after hot-iron disbudding [22], [23]. When given a post-operative analgesic such as ketoprofen or meloxicam following hot-iron disbudding, calves have a marked reduction in ear flicks and head shakes [34], [35] and lower plasma cortisol and heart rates [36] compared to calves without post-operative medication.

In our study, we tested calves' judgement biases at 6 h (the peak of pain behaviours and cortisol levels) and 22 h (toward the end of the post-operative pain period) following disbudding. Interestingly, there was no difference in responses to the ambiguous screens between these two sessions following disbudding, suggesting that the impact of disbudding on the emotional state of calves persists for at least 22 h.

Decreased feeding motivation after disbudding cannot explain the reduced response to the probes as there was no difference in the number of responses to the reinforced training screen after disbudding showing that calves continued to be motivated to drink milk. It is possible that the reduced responding to the probe screens after disbudding was due to calves learning to not respond to the unreinforced probes, but we used a low rate of probes and partial reinforcement specifically to prevent this type of learning and we found no evidence of reduced responding to the probes over multiple test sessions before or after disbudding. However, future studies should include test sessions in the days following disbudding to ensure that calves return to baseline. Finally, the negative bias may have resulted from recovery from the sedative (or the local anaesthetic, or some other part of the procedure) rather than from the pain of disbudding per se. Previous work has shown that calves exhibit little or no behavioural responses during recovery from xylazine and local anaesthetic when disbudding does not occur [23] but no research has investigated how calves feel when recovering from these drugs; we encourage further work on this topic. Future studies should include a sham condition where calves are exposed to the xylazine and local block but are not disbudded. We also encourage future research to include a treatment where animals receive post-operative pain control, for example a non-steroidal anti-inflammatory drug such as ketoprofen or meloxicam.

To conclude, this study provides the first evidence of judgement bias in animals following a painful procedure. These results support the use of cognitive bias tasks in the assessment of animal emotions.

Supporting Information

Acknowledgments

The authors would like to thank Mike Mendl for his helpful suggestions in the development of this manuscript, and the staff and students of the UBC Dairy Education and Research Centre for their help during the project. We are grateful to Maria Hötzel, Alisson Martendahl, and Haley Nace for their help in developing the techniques used in the current research.