Sex impacts pain behaviour but not emotional reactivity of lambs following ring tail docking.

Studies in humans have shown sex differences in response to painful events, however, little is known in relation to sex differences in sheep. Understanding sex differences would enable improved experimental design and interpretation of studies of painful procedures in sheep. To examine sex differences in response to pain, 80 lambs were tested across five cohorts of 16. The lambs were penned in groups containing two male and two female lambs with their respective mothers. Lambs were randomly allocated from within each block to one of four treatment groups; FRing-Female lamb, ring tail docked without analgesia, MRing-Male lamb, ring tail docked without analgesia, FSham-Female lamb, tail manipulated and MSham-Male lamb, tail manipulated. Following treatment, lambs were returned to their pen and were video recorded for 45 mins for behavioural observations of acute pain and posture. An hour after treatment, lambs then underwent an emotional reactivity test that consisted of three phases: Isolation, Novelty and Startle. Following treatment, Ring lambs displayed more abnormal postures (mean = 2.5 ± 0.5) compared to Sham lambs (mean = 0.05 ± 0.4, P = 0.0001). There was an effect of sex on the display of acute pain-related behaviours in lambs that were tail docked (P < 0.001), with female lambs displaying more acute behaviours (mean count = +2.2). This difference in behaviour between sexes was not observed in Sham lambs. There was no effect of sex on display of postures related to pain (P = 0.99). During the Novelty and Startle phase of the emotional reactivity test, Ring lambs tended to (P = 0.084) or did (P = 0.018) show more fear related behaviours, respectively. However, no effect of sex was observed. The results of this study indicate that a pain state may alter the emotional response of lambs to novel objects and potential fearful situations. It was also demonstrated that female lambs display increased sensitivity to the acute pain caused by tail docking compared to males.

Automated Virtual Fencing Can Effectively Contain Sheep: Field Trials and Prospects.

Virtual fencing technology uses on-animal devices to communicate boundaries via a warning audio tone and electrical pulse signals. There is currently limited validation work on sheep. This study used modified cattle eShepherd® virtual fencing neckbands on reduced-wool sheep with clipped necks to enable automated trials with small groups across both day and night. The first 5-day trial with six Dorper crossbred sheep was conducted in an experimental paddock setting, with a second 5-day trial conducted with 10 Ultra White sheep on a commercial farm. The animals across both trials were contained in the inclusion zone for 99.8% and 92.2% of the trial period, with a mean percentage (±SD) of total audio cues as audio only (i.e., not followed by an electrical pulse) being 74.9% ± 4.6 in the first trial, and 83.3% ± 20.6 for the second trial. In the second trial, sheep crossed over into the exclusion zone on the third night and remained there until they were walked out for their daily yard check in the morning. These preliminary trial results are promising for the use of automated technology on sheep, but suitable devices and algorithms still need to be designed specifically for sheep in the long term.

Impacts of Rearing Enrichments on Pullets' and Free-Range Hens' Positive Behaviors across the Flock Cycle.

Enrichment during the indoor rearing of pullets destined for free-range systems may optimize pullet development including increasing motivated natural behaviors (termed 'positive behaviors') including foraging, dust bathing and chick play. Hy-Line Brown® chicks (n = 1700) were floor-reared indoors across 16 weeks with three enrichment treatments (n = 3 pens/treatment): (1) standard control, (2) weekly novel objects-'novelty', (3) perching/navigation structures-'structural'. At 16 weeks, pullets (n = 1386) were transferred to nine identical pens within rearing treatments with outdoor range access from 25 to 65 weeks. Video cameras recorded the pullet pens, adult indoor pens, and outside range. During rearing, observations of play behavior (running, frolicking, wing-flapping, sparring) in chicks at 2, 4 and 6 weeks (total of 432 thirty-second scans: 16 observations × 3 days × 9 pens) showed no overall effect of rearing treatment (p = 0.16). At 11 and 14 weeks only the 'novelty' hens were observed to increase their foraging across age (p = 0.009; dust bathing: p = 0.40) (total of 612 thirty-second scans per behavior: 17 observations × 2 days × 2 age points × 9 pens). Observations of adult hens at 26, 31, 41, 50, 60 and 64 weeks showed that the structural hens exhibited overall more dust bathing and foraging than the control hens (both p < 0.04) but both novelty and/or structural hens showed small increases depending on the behavior and location (total of 4104 scans per behavior: 17 observations × 2 days × 6 age points × 9 pens × 2 locations = 3672 + an additional 432 observations following daylight saving). Across age, adult hens differed in the degree of dust bathing performed inside or outside (both p ≤ 0.001) and foraging outside (p < 0.001) but not inside (p = 0.15). For litter-reared pullets, additional enrichments may result in some long-term increases in positive behaviors.

Behavior and welfare impacts of water provision via misting in commercial Pekin ducks.

Ducks will access water to maintain feather condition and exhibit natural water-related behaviors such as wet preening. Providing water to ducks commercially is challenging as it may reduce litter and air quality leading to higher duck mortality or illness. This research aimed to measure the behavioral and welfare impacts of water provision via a misting system for commercial Pekin grower ducks in Victoria, Australia. Seven grower flocks were observed (four misted and three nonmisted in open-sided sheds) during May and November 2021. From 26 until 33 d of age, treatment ducks were provided 1 h of misting with shed curtains closed in both treatment and control sheds. At the start and end of the misting application period, external health and welfare measures were taken directly on the ducks via transect walks throughout each shed and catch-and-inspect observations on a sample of 150 ducks from each shed. Video recordings were also made of the misted and nonmisted ducks for 3 h representing time periods prior to, during, and after the 1-h misting across all sheds for all 8 d of the treatment period. Observations were made of all behavior that ducks exhibited at 10-min scan sample intervals across four cameras per shed, totaling 4,198 scans across the seven sheds. General linear mixed models showed the misting application predominantly had impacts on the patterns of behavioral change across the treatment time periods between the misted and nonmisted ducks rather than increasing or decreasing the overall expression of specific behaviors (interaction terms all P ≤ 0.003). The misted ducks increased drinking, tail wagging, and walking, and reduced preening, rooting litter, sitting, and stretching during misting relative to what they showed prior. The nonmisted ducks showed less sitting and more panting during misting relative to prior. Pearson's Chi-square tests showed some differences between the treatment groups in feather cleanliness on the back and wings (both P < 0.0001), likely resulting from pre-existing differences between sheds in blood from pin feathers. Most welfare indicators showed no positive or negative effect of the misting treatment. These results indicate overhead misting does affect duck behavior to some degree without compromising their welfare, but further research with larger water droplet sizes resulting in greater accumulation of surface water or extended durations of misting may lead to greater effects.

Ducks use bathing water for wet preening and feather maintenance. Commercially, it is challenging to provide clean bathing water without compromising litter quality and duck health. Overhead misting may be a mode of water delivery that will wet the ducks' bodies with continuously clean water. This study compared seven grower flocks of Pekin ducks (four misted and three nonmisted treatment flocks) in open-sided sheds during May and November 2021 in Australia. From 26 until 33 d of age, treatment ducks were provided 1 h of misting with shed curtains closed in both treatment and control sheds. External welfare measures were taken directly on the ducks at 26 and 33 d of age. Daily video recordings were made to observe if behaviors differed before, during, or after the 1 h of misting in both treatment and control sheds. Results showed the misting application predominantly affected the way behaviors changed across time between the misted and nonmisted ducks rather than increasing or decreasing the overall expression of specific behaviors. The differences may have in part been related to the curtain closure. Most welfare indicators showed no positive or negative effect of the misting treatment. Larger water droplet sizes may have greater effects on duck behavior.

Social influence on the effectiveness of virtual fencing in sheep.

Early virtual fencing trials have effectively contained small groups of sheep within set areas of a paddock when all animals were wearing manual electronic collars. With sheep farming commonly involving large flocks, a potential cost-effective application of virtual fencing would involve applying equipment to only a proportion of the flock. In this study, we tested the ability of virtual fencing to control a small flock of sheep with differing proportions of the group exposed to the virtual fence (VF). Thirty-six Merino sheep were identified as leaders, middle or followers by moving them through a laneway. The sheep were then allocated to groups balanced for order of movement. The groups (n = 9 per group) included applying the VF to the following proportions of animals within each group: (1) 100% (n = 9 VF) (2) 66% (n = 6 VF; n = 3 no VF) (3) 33% (n = 3 VF; n = 6 no VF) (4) 0% (no VF; free to roam the paddock). The groups were given access to their own paddock (80 × 20 m) for two consecutive days, six hours per day, with the VF groups prevented from entering an exclusion zone that covered 50% of the north side of the paddock. During these hours, VF interactions, behavioural time budgets, and body temperature were recorded as measures of stress, and location was tracked with GPS. Group 100% VF and Control were tested on the first two days and groups 33% VF and 66% VF were tested on the following two days. During VF implementation the 100% VF and 66% VF group were successfully prevented from entering the exclusion zone. Having only 33% of the flock exposed to the virtual fence was not successful, with the sheep pushing forward through the VF to join flock mates in the exclusion zone. For learning to respond to the audio cue, sheep in the 33% group received more electrical stimuli with a 0.51 proportion for the ratio of electrical stimuli to audio cue, compared to 0.22 and 0.28 for the 100% and 66% groups, respectively. There were small differences in behavioural patterns of standing and lying on both days of testing, with the 100% VF and 66% VF groups spending more time lying. Although stress-induced hyperthermia did not occur in any of the VF groups, body temperature differed in the 33% VF group. There were no differences in temperature measures between the control and 100% VF animals. This study demonstrates that for a short period, controlling two-thirds of the flock was equally as effective as virtually fencing all animals, while controlling one-third of a flock with a virtual fence was not effective. For the short term, it appears that implementing the VF to a proportion of the flock can be an effective method of containment. Due to the limitations of this study, these results warrant further testing with larger flocks and for longer periods.

The Influence of Predictability and Controllability on Stress Responses to the Aversive Component of a Virtual Fence.

To ensure animal welfare is not compromised, virtual fencing must be predictable and controllable, and this is achieved through associative learning. To assess the influence of predictability and controllability on physiological and behavioral responses to the aversive component of a virtual fence, two methods of training animals were compared. In the first method, positive punishment training involved sheep learning that after an audio stimulus, an electrical stimulus would follow only when they did not respond by stopping or turning at the virtual fence (predictable controllability). In the second method, classical conditioning was used to associate an audio stimulus with an electrical stimulus on all occasions (predictable uncontrollability). Eighty Merino ewes received one of the following treatments: control (no training and no stimuli in testing); positive punishment training with an audio stimulus in testing (PP); classical conditioning training with only an audio stimulus in testing (CC1); and classical conditioning training with an audio stimulus followed by electrical stimulus in testing (CC2). The stimuli were applied manually with an electronic collar. Training occurred on 4 consecutive days with one session per sheep per day. Sheep were then assessed for stress responses to the cues by measuring plasma cortisol, body temperature and behaviors. Predictable controllability (PP) sheep showed no differences in behavioral and physiological responses compared with the control treatment (P < 0.05). Predictable uncontrollability of receiving the aversive stimulus (CC2) induced a higher cortisol and body temperature response compared to the control but was not different to CC1 and PP treatments. CC2 treatment sheep showed a higher number of turning behaviors (P < 0.001), and more time spent running (P < 0.001) than the control and PP treatment groups, indicating that predictability without controllability was stressful. The behavior results also indicate that predicting the event without receiving it (CC1) was less stressful than predicting the event then receiving it (CC2), suggesting that there is a cost to confirmation of uncontrollability. These results demonstrate that a situation of predictability and controllability such as experienced when an animal successfully learns to avoid the aversive component of a virtual fence, induces a comparatively minimal stress response and does not compromise animal welfare.

Pharmacologically-induced stress has minimal impact on judgement and attention biases in sheep.

The emotional impact of exposure to stressors has not been well quantified in animals. We hypothesised that exogenous induction of stress in sheep would induce a pessimistic judgement bias and increased attention towards a threatening stimulus, suggestive of a negative emotional state. Stress was induced pharmacologically by administering synthetic adrenocorticotropic hormone. Judgement bias was assessed using a spatial go/no-go task after exposure to acute stress (one injection), chronic stress (21 daily injections) and acute-on-chronic stress (2 min isolation after 28 daily injections). Attention bias was assessed during chronic stress only (22 daily injections). In contrast with our hypotheses, there was no strong evidence that Synacthen administration altered judgement bias or attention bias at any stage of the experiment. Stressed sheep were more likely to approach ambiguous locations than saline Control animals, however, statistical evidence for models fitting treatment group was very weak. Overall, our findings suggest that elevated levels of cortisol may not fully explain changes to judgement bias observed in previous studies after environmentally-induced stress. Further studies are required to better understand which aspects of environmentally-induced stress alter judgement bias and to further validate cognitive methods of assessing affect in sheep.

The influence of pharmacologically-induced affective states on attention bias in sheep.

When an individual attends to certain types of information more than others, the behavior is termed an attention bias. The occurrence of attention biases in humans and animals can depend on their affective states. Based on evidence from the human literature and prior studies in sheep, we hypothesized that an attention bias test could discriminate between pharmacologically-induced positive and negative affective states in sheep. The test measured allocation of attention between a threat and a positive stimulus using key measures of looking time and vigilance. Eighty 7-year-old Merino ewes were allocated to one of four treatment groups; Anxious (m-chlorophenylpiperazine), Calm (diazepam), Happy (morphine) and Control (saline). Drugs were administered 30 min prior to attention bias testing. The test was conducted in a 4 × 4.2 m arena with high opaque walls. An approximately life-size photograph of a sheep was positioned on one wall of the arena (positive stimulus). A small window with a retractable opaque cover was positioned on the opposite wall, behind which a dog was standing quietly (threat). The dog was visible for 3 s after a single sheep entered the arena, then the window was covered and the dog was removed. Sheep then remained in the arena for 3 min while behaviors were recorded. Key behaviors included time looking toward the dog wall or photo wall, duration of vigilance behavior and latency to become non-vigilant. In contrast with our hypothesis, no significant differences were found between treatment groups for duration of vigilance or looking behaviors, although Anxious sheep tended to be more vigilant than Control animals (P < 0.1) and had a longer latency to become non-vigilant (P < 0.001). A total of 24 of 80 animals were vigilant for the entire test duration. This censoring of data may explain why no differences were detected between groups for vigilance duration. Overall, a lack of difference between groups may suggest the test cannot discriminate positive and negative states in sheep. We suggest that the censoring of vigilance duration data, the use of insufficient drug doses, the potential influence of background noise and the age of the sheep may explain a lack of difference between groups. Due to these potential effects, it remains unclear whether the attention bias test can detect positive states in sheep.

An attention bias test to assess anxiety states in laying hens.

Fear is a response to a known threat, anxiety is a response to a perceived threat. Both of these affective states can be detrimental to animal welfare in modern housing environments. In comparison to the well-validated tests for assessing fear in laying hens, tests for measuring anxiety are less developed. Perception of a threat can result in an attention bias that may indicate anxious affective states in individual hens following playback of an alarm call. In Experiment 1, an attention bias test was applied to hens that differed in their range access to show that hens that never ranged were more vigilant (stretching of the neck and looking around: P < 0.001) and slower to feed following the second alarm call playback (P = 0.01) compared with hens that ranged daily. All hens showed a reduction in comb temperature following the first alarm call (P < 0.001). In Experiment 2, an open field test was used to determine an effective dose of 2 mg/kg for the anxiogenic drug meta-Chlorophenylpiperazine (m-CPP) in adult laying hens. Hens dosed with 2 mg/kg showed reduced locomotion compared with a saline solution (P < 0.05). In Experiment 3, 2 mg/kg m-CPP or saline was administered to adult hens previously habituated to the open field arena to pharmacologically validate an attention bias test as a measure of anxiety. Hens dosed with m-CPP were slower to feed (P = 0.02) and faster to vocalize following a second alarm call playback (P = 0.03) but these hens did not exhibit the same vigilance behavior as documented in Experiment 1. The m-CPP hens also spent more time stepping and vocalizing (both P < 0.001) than the saline hens. An attention bias test could be used to assess anxiety. However, behavioral responses of hens may vary depending on their age or test environment familiarity, thus further refinement of the test is required. In these tests, 2 mg/kg of m-CPP resulted in motionless behavior when the environment was novel, but more movement and vocalizing when the environment was familiar. The extreme behavioral phenotypes exhibited by individually-tested birds may both be indicators of negative states.

Controlling Within-Field Sheep Movement Using Virtual Fencing.

Virtual fencing has the potential to greatly improve livestock movement, grazing efficiency, and land management by farmers; however, relatively little work has been done to test the potential of virtual fencing with sheep. Commercial dog training equipment, comprising of a collar and GPS hand-held unit were used to implement a virtual fence in a commercial setting. Six, 5-6 year-old Merino wethers, which were naïve to virtual fencing were GPS tracked for their use of a paddock (80 × 20 m) throughout the experiment. The virtual fence was effective at preventing a small group of sheep from entering the exclusion zone. The probability of a sheep receiving an electrical stimulus following an audio cue was low (19%), and declined over the testing period. It took an average of eight interactions with the fence for an association to be made between the audio and stimulus cue, with all of the animals responding to the audio alone by the third day. Following the removal of the virtual fence, sheep were willing to cross the previous location of the virtual fence after 30 min of being in the paddock. This is an important aspect in the implementation of virtual fencing as a grazing management tool and further enforces that the sheep in this study were able to associate the audio with the virtual fence and not the physical location itself.

Attention Bias Test Differentiates Anxiety and Depression in Sheep.

Negative affective states such as anxiety and depression pose a risk to animal welfare, however, practical tests for assessing these states in animals are limited. In humans, anxious individuals are shown to pay more attention toward threatening information than non-anxious individuals, known as an attention bias. Previously, an attention bias test was developed and validated as a measure of anxious states in sheep, where more anxious sheep showed increased attention toward a threat (dog) and were more vigilant than Control animals. Studies in humans suggest that attention biases also occur in depressed individuals, with observations of attention biases toward threats, as well as biases away from positive stimuli. Given these findings, we hypothesized that an attention bias test for sheep could also be used to assess states of depression. We predicted that Merino ewes in pharmacologically induced Depressed (para-chlorophenylalanine) and Anxious (m-chlorophenylpiperazine) states would show greater attention toward a threat than Control animals (saline), but that the Depressed sheep would show relatively less interest in a positive stimulus (photograph of a conspecific). During testing, Depressed sheep paid more attention toward the threat and less toward the photograph than Control animals as predicted (Analyses of Variance, P < 0.05, n = 16 per treatment). Interestingly, Anxious sheep showed an attention bias in the opposite direction, paying more attention toward the photograph and less toward the threat than Control animals (P < 0.05). Both Anxious and Depressed sheep were more vigilant than Control animals (P = 0.002). These results suggest the attention bias test can be used to measure and differentiate states of depression and anxiety in livestock. The bidirectional nature of the attention bias identified between treatments highlights the importance of measuring multiple behaviors in the test and considering the context in which the test is applied. This will enable a clearer characterization of the affective state of an animal, as an aspect of its welfare.

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.

Developing an Ethically Acceptable Virtual Fencing System for Sheep.

To ensure animal welfare isn't compromised when using virtual fencing, animals must be able to associate a benign conditioned stimulus with an aversive stimulus. This study used an associative learning test to train 30, four-year-old, Merino x Suffolk ewes, to associate an audio cue with an electric stimulus. Collars manually controlled by a GPS hand-held unit were used to deliver the audio and electric stimuli cues. For the associative learning, when sheep approached an attractant at a distance of three m from the trough, an audio cue was applied for one s. If the sheep stopped or changed direction, the audio cue ceased immediately and no electrical stimulus was applied. If the sheep did not respond to the audio cue it was followed by a low-level electrical stimulus. Approaches to the attractant significantly decreased from day one to day two. It took a mean of three pairings of the audio cue and electrical stimulus for a change in behaviour to occur, after which sheep that approached the attractant had a 52% probability of avoiding the electrical stimulus and responding to the audio cue alone. Further research is required to determine whether sheep can be trained to associate an audio cue with a negative stimulus for use in group grazing situations.