Rebecca E. Doyle

Measuring judgement bias and emotional reactivity in sheep following long-term exposure to unpredictable and aversive events

Animal welfare research is now starting to measure the cognitive component of affective states in an effort to improve welfare assessments of animals. Twenty-six Romane ewe lambs were trained to a spatial location task previously demonstrated to test for judgement bias in sheep. This required a go/no-go response according to the location of a bucket in a pen, with one location being positively reinforced (with a feed reward) and the other negatively reinforced (with a fan-forced blower). While training in the judgement bias arena continued, half of the sheep (n=13) were subjected to a chronic, intermittent treatment that consisted of stressful events common to production systems. After 3 weeks of treatment, all sheep were tested for biases in judgement by placing the bucket in ambiguous locations between the two learnt reference locations. The emotional reactivity, as characterised by behavioural and physiological responses, of all sheep to events that were unexpected, novel or sudden was then tested. A significant treatment × bucket location interaction was seen on day 3 with Stressed sheep approaching the bucket locations less than Control sheep (p=0.007). This may reflect a bias in judgement, however it is also possibly a treatment-induced difference in learning. Cardiac data did not indicate treatment differences, however the RMSSD of Control sheep in novel and unfamiliar situations was always higher than the Stressed animals. No meaningful treatment differences in emotional reactivity behaviours were evident. This paper provides further evidence that affective states exist and can be measured in animals.

The effect of repeated testing on judgement biases in sheep.

Testing judgement biases of animals may provide insight into their affective states; however important questions about methodologies need to be answered. This experiment investigated the effect of repeated testing using unreinforced, ambiguous cues on the response of sheep to a go/no-go judgement bias test. Fifteen sheep were trained to differentiate between two locations, reinforced respectively with feed (positive) or with the presentation of a dog (negative). The responses to nine ambiguous locations, positioned between the positively and negatively reinforced locations, were tested repeatedly over 3 weeks. Sheep exhibited a symmetrical gradation in response to ambiguous locations between the positive and negative reinforcers. There was a significant decline (P=0.001) in the total number of approaches to the ambiguous positions over time (weeks). This effect of time suggests that sheep learnt that the ambiguous locations were unrewarded. This result supplies evidence of a limitation identified in current judgement bias methodology, due to repeated testing, which has the potential to provide misleading results.

Administration of serotonin inhibitor p-Chlorophenylalanine induces pessimistic-like judgement bias in sheep

Judgement bias has potential as a measure of affective state in animals. The serotonergic system may be one mechanism involved with the formation of negative judgement biases. It was hypothesised that depletion of brain serotonin would induce negative judgement biases in sheep. A dose response trial established that 40 mg/kg of p-Chlorophenylalanine (pCPA) administered to sheep for 3 days did not affect feeding motivation or locomotion required for testing judgement biases. Thirty Merino ewes (10 months old) were trained to an operant task for 3 weeks. Sheep learnt to approach a bucket when it was placed in one corner of the testing facility to receive a feed reward (go response), and not approach it when in the alternate corner (no-go response) to avoid a negative reinforcer (exposure to a dog). Following training, 15 sheep were treated with pCPA (40 mg/kg daily) for an extended duration (5 days). Treated and control sheep were tested for judgement bias following 3 and 5 days of treatment, and again 5 days after cessation of treatment. Testing involved the bucket being presented in ambiguous locations between the two learnt locations, and the response of the sheep (go/no-go) measured their judgement of the bucket locations. Following 5 days of treatment, pCPA-treated sheep approached the most positive ambiguous location significantly less than control sheep, suggesting a pessimistic-like bias (treatment × bucket location interaction F(1,124.6)=49.97, p=0.011). A trend towards a significant interaction was still evident 5 days after the cessation of pCPA treatment (p=0.068), however no significant interaction was seen on day 3 of testing (p=0.867). These results support the suggestion that judgement bias is a cognitive measure of affective state, and that the serotonergic pathway may be involved.

Attention bias to threat indicates anxiety differences in sheep

Humans and animals show increased attention towards threatening stimuli when they are in increased states of anxiety. The few animal studies that have examined this phenomenon, known as attention bias, have applied environmental manipulations to induce anxiety but the effects of drug-induced anxiety levels on attention bias have not been demonstrated. Here, we present an attention bias test to identify high and low anxiety states in sheep using pharmacological manipulation. Increased anxiety was induced using 1-methyl-chlorophenylpiperazine (m-CPP) and decreased anxiety with diazepam, and then we examined the behaviour of sheep in response to the presence of a dog as a threat. Increased attention towards the threat and increased vigilance were shown in sheep that received the m-CPP and reduced in sheep receiving the diazepam. The modulated attention towards a threat displayed by the m-CPP and diazepam animals suggests that attention bias can assess different levels of anxiety in sheep. Measuring attention bias has the potential to improve animal welfare assessment protocols.

Stakeholder Perceptions of Welfare Issues and Indicators for Extensively Managed Sheep in Australia

Simple Summary This survey was designed as the first step in the development of a welfare assessment framework with the aim of identifying potential causes of welfare compromise and useful indicators for sheep in extensive Australian production systems. We asked the general public, sheep producers, service providers and sheep industry related scientists to provide their thoughts on the importance of a range of sheep welfare issues and possible key indicators. All respondents thought sheep welfare was adequate but that improvement was desired. Issues perceived to cause the most risk to sheep related to factors influenced by the environment (i.e., nutrition and food supply), heat stress and lameness while key indicators useful to assess welfare were nutrition and food availability, mortality/management issues, pain and fear related indicators, and numbers of illness/injuries. Women and the general public perceived all issues and indicators to be more important than other groups of respondents. These results highlight the need to consult a wide range of stakeholders in order to develop a broadly acceptable assessment system. Abstract An online survey was designed to form the basis of a framework for the welfare assessment of extensively managed sheep in Australia. The survey focused on welfare compromise and useful welfare indicators. A total of 952 people completed the survey in its entirety, representing four stakeholder groups: Public (53.6%), Producer (27.4%), Scientist (9.9%), and Service provider (9.1%). Animal welfare was considered to be important by all participating groups in this survey (average score of 3.78/4). Respondents felt the welfare of grazing sheep was generally adequate but improvement was desired (2.98/5), with female members of the public rating sheep welfare significantly worse than other respondents (p < 0.05). Environmental issues were considered to pose the greatest risk to welfare (3.87/5), followed by heat stress (3.79), lameness (3.57) and husbandry practices (3.37). Key indicators recognised by all respondents were those associated with pain and fear (3.98/5), nutrition (4.23), mortality/management (4.27), food on offer (4.41) and number of illness/injures in a flock (4.33). There were gender and stakeholder differences in the perceived importance of both welfare issues and indicators with women and the public consistently rating issues (all p < 0.01) and indicators (all p < 0.05) to be of greater significance than other respondents. These results highlight the importance of including all stakeholders and an even balance of genders when developing a welfare framework that can address both practical and societal concerns.

Evaluating pharmacological models of high and low anxiety in sheep.

New tests of animal affect and welfare require validation in subjects experiencing putatively different states. Pharmacological manipulations of affective state are advantageous because they can be administered in a standardised fashion, and the duration of their action can be established and tailored to suit the length of a particular test. To this end, the current study aimed to evaluate a pharmacological model of high and low anxiety in an important agricultural and laboratory species, the sheep. Thirty-five 8-month-old female sheep received either an intramuscular injection of the putatively anxiogenic drug 1-(m-chlorophenyl)piperazine (mCPP; 1 mg/kg; n = 12), an intravenous injection of the putatively anxiolytic drug diazepam (0.1 mg/kg; n = 12), or acted as a control (saline intramuscular injection n = 11). Thirty minutes after the treatments, sheep were individually exposed to a variety of tests assessing their general movement, performance in a ‘runway task’ (moving down a raceway for a food reward), response to startle, and behaviour in isolation. A test to assess feeding motivation was performed 2 days later following administration of the drugs to the same animals in the same manner. The mCPP sheep had poorer performance in the two runway tasks (6.8 and 7.7 × slower respectively than control group; p < 0.001), a greater startle response (1.4 vs. 0.6; p = 0.02), a higher level of movement during isolation (9.1 steps vs. 5.4; p < 0.001), and a lower feeding motivation (1.8 × slower; p < 0.001) than the control group, all of which act as indicators of anxiety. These results show that mCPP is an effective pharmacological model of high anxiety in sheep. Comparatively, the sheep treated with diazepam did not display any differences compared to the control sheep. Thus we suggest that mCPP is an effective treatment to validate future tests aimed at assessing anxiety in sheep, and that future studies should include other subtle indicators of positive affective states, as well as dosage studies, so conclusions on the efficacy of diazepam as a model of low anxiety can be drawn.

Towards a more practical attention bias test to assess affective state in sheep

Tests for attention bias potentially offer more rapid assessment of affective state in animals than existing cognitive methods. An attention bias test has previously been developed for sheep and validated as a measure of anxious states. The 3 minute test assessed behavioural responses of sheep in an enclosed arena after brief exposure to the threat of a dog. Experiment 1 of the current study aimed to refine the previously developed method, removing the need for a habituation period and shortening the test duration. Sheep were given either an anxiolytic drug, an anxiogenic drug or a control treatment prior to testing to induce contrasting affective states. Differences in behaviour were found between the treatment groups within the first 45s of the test, indicating the original test duration could be shortened from 180 s. During testing, 36 of 40 animals in the control and anxiolytic groups ate the novel feed offered in the test, indicating it is not necessary to habituate animals to a feed container. Experiment 2 aimed to confirm the responses measured in the test were primarily towards the dog rather than other aspects of the test environment. Sheep exposed to an empty window at the beginning of the test behaved differently to those which were exposed to a dog, indicating sheep behaviour in the test is at least partially a response to the dog. A third group of sheep were also tested with the dog immediately after having small data loggers attached to their necks. Behaviour of these sheep did not differ from the sheep tested without loggers, indicating data logger attachment did not impact their behaviour in the test. In both experiments, treatments did not appear to modify activity (zones crossed), which we propose indicates the test was primarily detecting valence of the affective state rather than arousal.

Physiological and Behavioral Responses of Horses to Wither Scratching and Patting the Neck When Under Saddle.

Riding is considered to be an arousing activity for horses. It has been suggested that wither scratching may be a more useful tool for relaxation compared with the common practice of neck patting. In the current study, 18 horses were exposed to 3 treatments, including control or no interaction, neck patting, and wither scratching, for 1 min each following a short obstacle course. Heart rate, heart rate variability, and a variety of behaviors were measured in the horses. Wither scratching produced a significantly longer duration of relaxed-type behaviors. Wither scratching could be a useful tool to help a horse relax while under saddle. Additionally, the study identified 2 ear positions that may be useful for future research in horse behavior.

An assessment of African lion Panthera leo sociality via social network analysis: prerelease monitoring for an ex situ reintroduction program

Abstract The wild population of the African lion Panthera leo continues to decline, requiring alternate conservation programs to be considered. One such program is ex situ reintroduction. Prior to release, long-term monitoring and assessment of behavior is required to determine whether prides and coalitions behave naturally and are sufficiently adapted to a wild environment. Social network analysis (SNA) can be used to provide insight into how the pride as a whole and individuals within it, function. Our study was conducted upon 2 captive-origin prides who are part of an ex situ reintroduction program, and 1 wild pride of African lion. Social interactions were collected at all occurrence for each pride and categorized into greet, social grooming, play, and aggression. Betweenness centrality showed that offspring in each pride were central to the play network, whereas degree indicated that adults received (indegree) the greatest number of overall social interactions, and the adult males of each pride were least likely to initiate (outdegree) any interactions. Through the assessment of individual centrality and degree values, a social keystone adult female was identified for each pride. Social network results indicated that the 2 captive-origin prides had formed cohesive social units and possessed relationships and behaviors comparable with the wild pride for the studied behaviors. This study provided the first SNA comparison between captive-bred origin and a wild pride of lions, providing valuable information on individual and pride sociality, critical for determining the success of prides within an ex situ reintroduction program.

Exploring African lion (Panthera leo) behavioural phenotypes: individual differences and correlations between sociality, boldness and behaviour

Increasing our understanding of personality, at an individual and group level, is crucial to the pre-release assessment of social species within ex situ reintroduction programs. We conducted the first exploration into the personality of a captive-origin pride of African lions (Panthera leo), assessing behavioural variations and consistencies in daily activity, social and hunting behaviour, and boldness. Data were collected via direct observations, while a species-specific protocol for testing boldness, using playbacks, was developed. Differences in sex, age and session time for the activity budget were evaluated using Pearson correlations and repeated-measures ANOVA, while social interactions were analysed using social network analysis. Spearman’s correlations were conducted to assess for associations between boldness scores, activity and sociality. The two boldness tests provided a range of scores per lion, indicating that the test was effective. Correlations and variations in individual behaviour indicated that adults and sub-adults have specific roles within pride behaviour. Correlations between boldness and activity and social behaviours provided information on the role of individuals, allowing investigation into the behaviour of a dominant and a social keystone. Our study indicates that evaluating various aspects of behaviour in conjunction with boldness has the potential to assist the pre-release assessment of a pride within an ex situ reintroduction program.