# Effect of long or short pre-slaughter handling on meat quality and physiological response of broiler chickens

A.F. Soleimani, A.O. Bello and Z. Idrus

Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Selangor, Malaysia; abdoreza@upm.edu.my

Finishing broiler chickens are commonly subjected to a range of stressors including catching, handling, crating, and transportation. These changes in a chicken's microenvironment activate stress response pathways in a range between mild to severe and affect health and welfare and meat quality. This study aimed to test the physiological effects of pre-slaughter handling in broiler chicken and conformed to the ISAE Ethical Guidelines. 96 day-old male broiler chicks were assigned in groups of 4 to 24 battery cages. On d 42, handling treatment was carried out over a short or long distance (25 or 100 meters). Handling involved catching, carrying the bird along the empty house, and ended with blood sampling. One bird per cage was caught at random with both hands, held in an inverted position and carried for a short or long distance (two birds per hands at once) through the house until reaching a separate room. The birds were then placed on a table and blood samples were drawn from wing vein. The average times between initial capture and end of blood sampling were 1:20 min for short handling group and 3:30 min for long handling group. Blood sampling procedure less than 3 min is considered to have no influence on corticosterone response. The same treatment procedure was carried out with another 24 birds, without blood sampling. These birds were crated (12 birds/crate) and loaded onto an open truck and transported for 2 h between 8:00 to 10:00 am with average speed of 80 km/h. Immediately upon arrival, blood and breast samples were collected for determination of drip loss, cooking loss, color and shear force at 24 h postmortem and serum levels of serotonin (5-HT), corticosterone (CORT) and heat shock protein 70 (HSP70). Data were analyzed as one-way ANOVA using SAS software package. The results revealed that the meat from short handling birds had greater drip loss and shear force compared to long handling group (5.49±0.26% and 103.29±4.82 N vs 4.28±0.33% and 91.61±2.86 N, respectively). Meat color was not affected by handling distance (P>0.05). The short handling birds also consistently showed higher HSP70 levels than their long handling counterparts (before transportation: 2.22±0.19 vs 1.73±0.09 ng/ml; after transportation: 2.17±0.18 vs 1.54±0.14 ng/ml). The 5-HT concentration increased in long handling birds compare to short handling groups only after transportation (36.64±3.57 vs 29.88±2.42 ng/ml). Although long handling resulted in higher levels of CORT than short handling before transportation (1.64±0.25 vs 1.00±0.14 ng/ml), it showed an opposite effect after transportation (0.98±0.08 vs 1.21±0.13). The present study showed that long handling, as practiced here, did not compromise welfare and meat quality of chicken. Long handling may decrease the incidence of PSE (pale, soft, exudative) meat in birds under transportation stress.
**Key-note:**

**From Pavlov to Bekhterev – and the pros and cons of positive and negative reinforcement**

Janne Winther Christensen¹, Andrew McLean² and Jan Ladewig³

¹Aarhus University, Animal Science, Blichers Allé 20, 8830 Tjele, Denmark, ²Australian Equine Behaviour Centre, 730 Clonbinane Rd, Clonbinane, 3658 Victoria, Australia, ³University of Copenhagen, Veterinary and Animal Sciences, Grønnegårdsvej 8, 1870 Frederiksberg, Denmark; jwc@anis.au.dk

Pavlov and Bekhterev were rivals in their study of conditioned reflexes. Instead of using food to produce saliva, Bekhterev used mild electrical stimulation to study motor reflexes. According to Bekhterev, one of Pavlov's major research flaws included using the saliva method. If the animal is not hungry, food may not elicit the desired response, acting as evidence of the method's unreliability. Similarly, food motivation may influence an animal's performance in food-rewarded learning tasks. Nevertheless, applied animal ethologists often use food as a positive reinforcer in learning tasks to investigate the cognitive abilities of farm animals, and to test effects of various treatments, such as environmental enrichment, on animal cognition. One should be mindful, however, that any treatment that affects food motivation may also influence performance in food-rewarded learning tasks. Negative reinforcement (NR) is used less frequently in learning tests for farm animals and has been regarded as an inevitably negative (i.e. 'bad') training method, inducing poor welfare. However, recent research showed that horses trained through NR were more optimistic in a judgement bias test, compared to horses trained using positive reinforcement. Learning tasks based on NR may help circumvent some of the challenges with food motivation in food-rewarded learning tasks. Moreover, learning performance in positively and negatively reinforced tasks have been found to show poor correlations suggesting that interpretation of results based only on a food-rewarded test may not reflect the animal's learning performance in other reinforcement regimes. In addition, NR learning may be easily overlooked in experiments involving unpleasant stimulation. For example, studies on nociceptive thresholds (pain sensitivity) frequently use repeated stimulation. Typically the stimulation is removed when the animal shows an avoidance response, e.g. a foot-lift response, which is therefore negatively reinforced through removal of the stimulation. When stimulated again the animal is likely to show the same response at a lower threshold. Results may be interpreted as increased pain sensitivity whereas this sensitivity may in fact interact with NR learning. An increased focus on NR learning, and the interaction between animal motivation and the different learning modalities is likely to enhance the interpretative value of applied animal behaviour research.
**Development of an ethical virtual fencing system for sheep**

Danila Marini¹, Dennis Muleman², Sue Belson³, Bas Rodenburg² and Caroline Lee³

¹University of New England, Armidale, 2350, Australia, ²Wageningen University, De Elst 1, 6700 Wageningen, the Netherlands, ³CSIRO, New England Highway, 2350, Armidale, Australia; dmarini2@une.edu.au

Virtual fencing (VF) has potential application in livestock management where the use of conventional fencing may be impractical. To ensure that animal welfare is not compromised using a VF system, the animal must be able to associate an audio cue with the negative stimulus (a mild electric shock), then learn that altering its behaviour in response to the audio cue will allow it to avoid receiving the negative stimulus. There has been limited work conducted on training sheep to VF. The aims of this study were to (1) find an appropriate stimulus level that would prevent sheep from reaching an attractant and (2) test if sheep can learn to associate an audio warning cue with an electric stimulus by responding to the audio cue alone. This study used 60, 4-year-old, Merino × Suffolk crossbred ewes with an average body weight of 66 kg. Electric dog collars which were manually controlled by a GPS monitor (Garmin, Australia) and had a stimulus range from 1 (low) to 18 (high) were used to deliver the audio and shock cues to the sheep. To determine an appropriate electric shock level, 30 sheep were trained to approach a feed trough and eat a feed reward (pellets). Once trained, sheep received a stimulus as they approached the trough, beginning at level 1 and increasing until all sheep displayed the desired response (to avoid the feed trough). Level 1 and 2 did not elicit a response in the sheep. When set to level 3, 1 out of 11 sheep reached the feed trough after receiving 5 stimulus applications. On level 4 no sheep reached the trough, this was determined to be an appropriate stimulus level. For the associative learning test, a further 30 sheep were trained to approach a feed trough. During the test sheep were fitted with the Garmin collar and allowed to approach the trough, at a distance of 4 m from the trough, an audio cue was applied for 1 s. If the sheep stopped or changed direction, the audio cue ceased immediately and no electric stimulus was applied. If the sheep did not respond to the 1 s audio cue, it was immediately followed by the electric stimuli (level 4) for 1 s. Testing ended after 5 min or if an individual sheep received a maximum of 5 shocks. The test was repeated once per day for 5 days. All 30 sheep approached the trough on day 1 and this was reduced to 8 on day 2, 9 on day 3, 11 on day 4 and 7 on day 5. There was a decrease in the number of shocks that were given across the days, however, this was not correlated to audio warnings. It is apparent that sheep readily learnt not to approach a trough after receiving a negative stimulus, however, the audio cues were ineffective in preventing sheep from receiving a shock. Further research is required to determine whether sheep can be trained to associate audio cues with a negative stimulus for use in virtual fencing.
**Motor self-regulation by goats (*Capra aegagrus hircus*) in a detour-reaching task**

Jan Langbein

Leibniz Institute for Farm Animal Biology (FBN), Institute of Behavioural Physiology, Wilhelm-Stahl-Allee 2, 18196, Germany; langbein@fbn-dummerstorf.de

Motor self-regulation, the ability to inhibit a prepotent motor response, is an important aspect of a cluster of processes generating effective inhibition of behaviour. In primates, it has been suggested that high level of sociability correlates with distinct inhibitory skills and as a consequence with higher behavioural flexibility. To broaden the range of species for a comprehensive discussion of this phenomenon, I studied motor self-regulation in a highly social animal, the goat. I investigated the behaviour of female dwarf goats (n=20; age 15-22 month) in a detour-reaching task, the cylinder task. Goats were trained to approach an opaque cylinder (length 20 cm × diameter 17 cm) from which they could retrieve a reward. After six correct trials in a row, subjects advanced to the test. In the test, an identical transparent cylinder was substituted for the opaque cylinder. Baiting of the cylinder by a human in full view of the animals was counterbalanced for side (left/right). I measured the goats' approach behaviour over a total of 10 trials. A trial was coded as 'accurate' if the goat retrieved the reward directly and 'inaccurate' if the goat tried to reach the visible reward directly by bumping into the cylinder. Additionally, I recorded duration of contact with the exterior of the cylinder, the side (left/right) the goat detoured the cylinder and latency to retrieve the reward. I used Generalised Linear Mixed Models to analyse the impact of trial on approach behaviour and of side of human baiting on the side the goats detoured the cylinder, while Mixed Linear Models were used to analyse the impact of trial on duration of contact and on latency to retrieve the reward. All statistical analyses were done using SAS. The overall average accuracy during testing was 62.5% (±SE: 10.86%). Trial had no impact on accuracy (F<sub>9,171</sub>=0.85; n.s.). Latency to retrieve the reward decreased during testing (Max=35 s, Min=5 s; F<sub>9,134</sub>=7.85; P<0.001), while duration of contact did not change (Mean=1.28 s; F<sub>9,171</sub>=0.93; n.s.). The side of baiting the cylinder by a human had no impact on the side goats detoured it (F<sub>1,151</sub>=0.22; n.s). For goats, living in complex social groups with a high level of fission-fusion dynamics, we found a level of behavioural inhibition equal or even higher compared to various bird and primate species. However, only a minority of subjects improved motor self-regulation during testing. This was in concordance with the constant duration of contact with the exterior of the cylinder across trials. Taken together, goats got faster in expressing the correct motor pattern to retrieve the reward but did not reliable improve inhibition of the prepotent motor response on the group level. Results show the importance to consider individual reaction patterns when analysing motor self-regulation. Effective behavioural inhibition might play an important role for group-housed farm animals living under restricted spatial conditions.
**Cylinder size affects cat performance in the motor self-regulation task**

Katarzyna Bobrowicz<fnref n="1" />,<fnref n="2" /> and Mathias Osvath<fnref n="2" />

katarzyna.bobrowicz@lucs.lu.se

We tested domestic cats in the so-called cylinder task, and found that they perform better if the cylinder is larger. We also found that their highest performance parallels that of great apes and corvids, which are known as the best performing animals on this task. The cylinder task is used to test animals' motor self-regulation: the inhibition of unproductive, but prepotent, movements in favour of productive movements that require a slight detour. Recently a large-scale study tested 36 species on this task and found that absolute brain size correlate with the performance; with great apes as top performers. Another study showed that corvids perform as good as great apes despite having smaller absolute brain size. We questioned whether average brained animals has as poor motor self-regulation as suggested, as it appears highly maladaptive; instead the results could be a reflection of the sensorimotor set-up of different species in relation to the materials used. No cats has yet been tested on the task. As ambush and sneak hunters, cats would arguably have high levels of motor self-regulation, but on the other hand their brain size and neuronal numbers are not above average in mammals. Eight adult domestic cats were tested in four versions of the task. We manipulated the size and materials to test whether that influenced performance: two large cylinders (16 cm diameter) out of glass and plastic respectively, and two small cylinders (9 cm diameter) of the same two materials. Each of the four conditions had two phases with a 24-hour delay in between. Each phase consisted of 10 consecutive trials. On the first day, a subject learned to retrieve a reward from an opaque cylinder. Next day, the cat was tested on a transparent cylinder. A retrieval of the reward without touching the cylinder's front counted as a successful trial. The success rate differed between conditions, and reached 98.75% in the 'big glass' condition, and 97.5% in the 'big plastic' condition, and 83.75% in the 'small glass', and finally 73.75% in the 'small plastic' condition. Two-Factor ANOVA for two within variables revealed a significant main effect of the cylinder size on the success rate [F(1,7)=64.06, P<0.001]. Neither a main effect of the material nor an interaction effect of size and material was statistically significant. The size effect was seen in all subjects. Failure rates did not decrease over time in any condition, so no learning curve was detected. Our results show that cats parallel great apes and corvids in the cylinder task as long as it is 16 cm in diameter and made of glass, despite their average mammalian neural characteristics. There are several possible explanations such as that a bigger size allows for more options of retrieval (e.g. mouth or paw), and/or requires less precise retrieval; it could also be that the distance to the reward is perceived as different. This calls into question whether the large-scale study took into account the sensorimotor architecture of each species, and more importantly, whether the task always measures motor self-regulation.

<footnote n="1">¹University of Warsaw, Faculty of Psychology, Stawki 5/7, 00-183 Warszawa, Poland,</footnote>

<footnote n="2">²Lund University, Department of Philosophy, Helgonavägen 3, 22100 Lund, Sweden;</footnote>
**Factors affecting dairy calves' abilities to learn to use colour cues to locate a source of milk**

Alison Vaughan, Jeffrey Rushen and Anne Marie De Passille

University of British Columbia, Main Mall, Vancouver, BC, Canada; jeffrushen@gmail.com

With the rise in automation in dairy farms it is important to understand the way cattle learn to interact with automated equipment. Dairy calves can have difficulties learning to use automated feeders or in learning to use a new food source, for example during weaning. Using colour cues to signpost resources may be an effective way of helping calves learn to use these food sources. We examined some factors affecting calves' ability to learn to associate colour cues with a milk source by placing unweaned dairy calves (2-6 weeks of age) in a Y maze. In one of the arms, a teat supplied milk while the teat in the other arm was dry. Colour cues (solid red or yellow boards) identified the arms with and without the milk. In exp. 1, calves (n=21) learned to locate the milk reward faster when the colour cues and the milk was always in the same arm of the Y-maze (fixed location) than when the colour cue and milk changed arms (changing location) (mean ±SD number of sessions: fixed side = 2.27±0.47 and changing side = 9.83±3.91; P<0.001). However, when calves initially trained with a fixed location were subsequently tested with changing location, they did no better (P>0.10) than those that initially trained with the changing location, suggesting that they were overlooking the colour cues altogether. In exp. 2, calves (n=8) were trained to find milk in one arm of a Y maze (fixed location) and then tested in a reversed Y maze (configuration rotated by 180 degrees). Most (7/8) calves turned to the same side (left or right) rather than choosing the same arm of the maze (P<0.001) suggesting that they had learned a particular response (e.g. 'turn left') rather than the location of the milk in the maze. In exp. 3 we investigated whether prior exposure to the colour cue outside of the Y maze would aid calves to subsequently learn to use colour cues to choose the correct arm. Calves (n=19) were either pre-trained to associate one colour (solid red or yellow) with milk reward or were exposed to colour cues which did not predict presence or absence of reward prior (control calves). When re-tested in the Y-maze with a variable location, most (9/10) pre-trained calves learned but no control calves learned to choose the correct colour cue within the allotted number of sessions. The most common error made by both pre-trained and control calves was to choose the same side as their previous choice. Most (5/7) control but no pre-trained calves displayed a clear side preference. When training calves to associate a milk reward with a colour cue, the colour cue should not be presented in a fixed location as the calves may ignore the colour and learn only to turn in a particular direction. Pre-training calves to use colour cues is effective at improving their performance in a Y maze test and may reduce the development of a side preference. Improving understanding of how cattle learn cues may help us improve training protocols.
Goats prefer happy human faces

Christian Nawroth¹,², Natalia Albuquerque³,⁴, Carine Savalli⁵, Marie-Sophie Single⁶ and Alan G. McElligott²

¹Leibniz Institute for Farm Animal Biology, Institute of Behavioural Physiology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ²Queen Mary University of London, Biological and Experimental Psychology, Mile End Road, London E1 4NS, United Kingdom, ³University of Lincoln, School of Life Sciences, Brayford Pool, Lincoln LN6 7TS, United Kingdom, ⁴University of São Paulo, Department of Experimental Psychology, Butantã, São Paulo 03178-200, Brazil, ⁵Federal University of Sao Paulo, Department of Public Policies and Collective Health, Vila Clementino, São Paulo 04021-001, Brazil, ⁶Technical University of Munich, Physiology Weihenstephan, Alte Akademie 8, 85354 Freising, Germany; nawroth.christian@gmail.com

Facial expressions are highly informative for humans and other animals. However, little is known about whether livestock animals perceive human emotional facial cues. In particular, discriminating emotional expressions in heterospecifics is considered especially challenging because emotions are not necessarily expressed in similar ways across species. We investigated whether goats show preferences for human faces with different emotional valences. To ensure exploration in subsequent test trials, goats first received a brief training session in which they had to approach an experimenter (with a neutral facial expression) at the opposite side of a test arena to receive a food reward. After training, two images of an unfamiliar human face (male or female) with different emotional valences (happy or angry) were presented to the subjects, one on the left and one on the right side of the arena. Goats (n=20) were free to explore the arena and interact with the stimuli; each test trial lasted 30 s. Each goat received a total of four trials, counterbalanced for position of the emotional faces (left vs right) and gender. We analysed first interaction, rate of interaction and time spent interacting with each face, using a Generalized Estimated Equation model for binary data with a logit link function. Goats interacted first, more often and for longer with the images on the right side (first interaction: X²=9.63; P=0.0019; rate: X²=10.47; P=0.0012; duration: X²=7.67; P=0.0056). Moreover, we found an interaction effect of side (left vs right) and preference (happy vs angry): the side of presentation of the positive face affected the preference of goats for all three parameters (first interaction: X²=8.90; P=0.0029; rate: X²=8.01; P=0.0047; duration: X²=8.24; P=0.0041). For happy faces, goats interacted first, more often and for longer when they were positioned on the right side. No preference was found when the happy faces were placed on the left side. Face gender and goat sex had no effect on their preference for any parameter. We show that goats can discriminate human facial expressions with different emotional valences and prefer happy ones.
**Responses in novelty tests are associated with grain intake and unrewarded visits to the milk feeder in dairy calves**

Heather W. Neave, Joao H.C. Costa, Katrina Rosenberger, Daniel M. Weary and Marina A.G. Von Keyserlingk

University of British Columbia, Animal Welfare Program, 2357 Main Mall, Vancouver, BC, V6T 1Z4, Canada; hwneave@gmail.com

The feeding behaviour of dairy calves is known to be highly variable between individuals. We tested the hypothesis that personality features, as revealed by standardized tests, can account for this variation. Our hypothesis was that more interactive and less fearful calves in novel situations would explore their feeding environment more than other individuals, particularly when calves are learning to eat solid feed or when they must find novel food sources when milk is limited. We also expected these calves to be more persistent in attempting to gain milk during weaning. Fifty-six dairy calves were housed in 7 groups, and randomly assigned within group to either 6, 8, 10 or 12 l/d of milk (n=14 each) until d 41 of age, when milk was reduced to 50% of allowance. At d 50, milk was gradually reduced by 20%/d until calves were completely weaned at d 55. Calves had *ad libitum* access to calf grain and hay. We measured DM intake of grain and the number of unrewarded visits to the robotic milk feeder during the 4 d before the 50% reduction of milk allowance (d 38-41), the day before weaning (d 49), the day after weaning (d 50), the 2 weeks after weaning (d 55-69), as well as for the total experimental period (d 7-69). At d 30 and d 70 of age, each calf was subjected to a human approach test (10 min with an unknown stationary human), a novel object test (15 min with a black 110 l bucket) and a food neophobia test (30 min with two identical white 20 l buckets, one empty and the other filled with a novel food, a fresh cow total mixed ration). Seven behaviours (vocalizations, time spent touching, latency to touch human/object, looking at or disengaged with human/object, time spent playing, and intake of novel food) were recorded, and data were averaged across age and then across tests. These behaviours were examined in a Principal Components Analysis where 2 factors explained 66% of the variance (Factor 1 = 49% and Factor 2 = 18%). Factor 1 had high positive loadings on time spent in contact with the human/object and time spent playing, and a high negative loading on time spent disengaged with the human/object. Factor 2 had high positive loadings on the number of vocalizations and time spent disengaged with human/object, and high negative loadings on time spent looking at human/object, latency to touch human/object and intake of novel food. Factor 1 and Factor 2 loadings for each calf were regressed against feeding behaviour variables for each feeding period with milk allowance, birth weight, sex and group included as covariates in the model. Calves with high Factor 2 loadings had greater starter DM intake during the day before weaning (R²=0.33; P<0.01) and a higher number of unrewarded visits to the milk feeder during the total experimental period (R²=0.61; P<0.01). These results indicate that personality differences, as revealed in standardized tests, are predictive of grain intake before weaning and of continued attempts to receive milk when it is unavailable.
**Light during incubation and noise around hatching affect cognitive bias in laying hens**

T. Bas Rodenburg<sup>1,2</sup>, Nathalie J.T. Scholten<sup>2</sup> and Elske N. De Haas<sup>1,2</sup>

<sup>1</sup>Wageningen University & Research, Adaptation Physiology Group, P.O. Box 338, 6700 AH Wageningen, the Netherlands, <sup>2</sup>Wageningen University & Research, Behavioural Ecology Group, P.O. Box 338, 6700 AH Wageningen, the Netherlands; bas.rodenburg@wur.nl

Incubation and hatching conditions can influence development of laying hens. In commercial conditions, chicks are incubated in the dark and hatch in a noisy environment. This may affect their behavioural and cognitive development in a negative way and may have lasting effects on their assessment of ambiguous stimuli in a cognitive bias test. The aim of this study was to investigate the effects of light during incubation and noise around hatching on cognitive bias in adult laying hens. To meet that aim, eggs were incubated and hatched according to four different treatments in a 2×2 cross design: during the first 16 days of incubation, they would either be incubated in the dark or in a 12L:12D light schedule. From 17 to 21 days of age, half of the eggs from each light treatment would either be incubated in a noisy environment (playback commercial incubator noise; 90 dB) or a quiet environment (60-70 dB). Birds were kept in groups of maximum 10 birds in floor pens separated per treatment from 0 to 35 weeks of age (seven groups per treatment). At 35 weeks of age, 32 hens (eight per treatment) were trained for a cognitive bias test, where they had to learn that one side of the test arena was rewarded (mixed grains and mealworms) and the other side was not (smell of feed only). The rewarded side was varied according to a balanced design. The experimenter was blind to birds' treatments. When a bird had learned the task, she was tested with ambiguous cues, where the feeder would be placed in the centre (50%), nearer the unrewarded side (25%) or nearer the rewarded side (75%). This was done by subjecting each hen to 12 trials, where two rewarded (R) trials and one unrewarded (U) trial (order RRU or RUR, balanced design) were followed by one of the ambiguous trials. For all hens, the first ambiguous trial was the 50% trial, followed by the 25% trial and the 75% trial. Latency to approach the feeder was analysed using a mixed model with treatment, trial order and type of cue as fixed factors (including all interactions) and pen nested within treatment as random factor. Over all treatments, birds had a shorter latency to reach the feeder with the rewarded cue compared to all other cues, and with the 50% and 75% compared to the unrewarded and 25% cue as expected (F4,360=211.4; P<0.001). There was a significant interaction between treatment and type of cue (F12,360=2.11; P<0.05). Birds from the light and noise treatment were slower than birds from the other groups in the ambiguous 50% trial (F1,360=3.49; P<0.05), which is interpreted as a more pessimistic response to the ambiguous cue. The light treatment during incubation may have resulted in a faster embryo development. This may in turn have resulted in chicks that were more sensitive to the noise treatment that was applied around hatching. In conclusion, incubation and hatching conditions in laying hens can affect cognitive bias in laying hens. Creating more quiet incubators and could have positive effects on laying hen welfare.
Facial expressions of emotion influence sheep learning in a visual discrimination task

Lucille Bellegarde¹,²,³, Hans W. Erhard², Alexander Weiss¹, Alain Boissy⁴ and Marie J. Haskell³
¹The University of Edinburgh, School of Philosophy, Psychology and Language Sciences, EH89JZ Edinburgh, United Kingdom, ²INRA, UMR Modélisation Systémique Appliquée aux Ruminants, Université Paris-Saclay, 75005 Paris, France, ³SRUC, West Mains Road, EH93JG Edinburgh, United Kingdom, ⁴INRA, UMR Herbivores, Centre de recherche Auvergne-Rhône-Alpes, 63122 Saint-Genes-Champanelle, France; lucille.bellegarde@sruc.ac.uk

Faces are an essential source of information for social species. Sheep are one of the most studied farm species in terms of their ability to process information from faces, but little is known about their face-based emotion recognition abilities. We investigated whether sheep could use images of faces displaying different emotional states as cues in a simultaneous discrimination task. To that end, we took photos of faces of four sheep in three social situations: two with a negative valence (social isolation or aggressive interaction) and a neutral situation (ruminating in the home pen). In a two-armed maze, sheep (n=16) were then presented with pairs of images of the same familiar individual taken in the neutral situation and one of the negative situations. Sheep had to learn to associate one type of image from a pair with a food reward. Once they had reached the learning criterion, sheep then had to generalise the task to new pairs of images of different conspecifics displaying the same emotions as in the previous phase. For every run in the maze, the latency to choose an arm and the outcome of the choice (success or error) were recorded, as well as the total number of runs needed to learn the task (learning speed). Influence of the type of image rewarded and of the side of presentation of the rewarded image were analysed by linear mixed models and learning speed was analysed by Mood's median test. All sheep learned the task with images of faces. Sheep that had to associate a negative image with a reward learned faster that sheep that had to learn the neutral-reward combination (medians: 40 vs 75 runs, χ²=4.00, df=1, P=0.046). With the exception of sheep from the Aggression-rewarded group, sheep could generalise the discrimination task to images of new faces (F3,26.6=3.37, P=0.033). Sheep chose an arm correctly more often (F₁,288.1=5.02, P=0.026) and more quickly (F₁,289.3=23.92, P<0.001; right: 8.4±0.6 sec, left: 9.6±0.8 sec) when the rewarded image was displayed on the right side, suggesting the influence of a right hemisphere/left visual field bias in face-based perception of emotions. Our results strongly suggest that sheep can perceive the emotional valence displayed on faces and that this valence affects learning processes.
**Free-choice exploration in laboratory-housed zebrafish**

Courtney Graham, Marina A.G. Von Keyserlingk and Becca Franks

University of British Columbia, Applied Animal Biology, 2357 Main Mall, Vancouver BC, V6T

1Z4, Canada; courtney3graham@gmail.com

Cognitive stimulation has been shown to be rewarding and capable of eliciting positive
emotions in several species. In contrast to the abundant learning and exploration opportunities
available in nature, captive environments can be under-stimulating – with the potential to
induce anhedonia and reduce welfare. Zebrafish are now a popular scientific model, in part
because of their high cognitive function and sensitivity to environmental manipulations, yet
little is known regarding their response to opportunities to explore. As a first step toward
understanding the role that cognitive enrichment may play in zebrafish welfare, we housed
zebrafish within six large structurally-enriched tanks (10 fish/110 l tank) for nine months and
then removed a divider to expose 10 cm of additional novel tank space. This approach allowed
us to measure free-choice exploratory behaviour (latency and number of inspections) as well
as anxiety (bottom-dwelling) and social behaviour (aggression, cohesion and coordination).
We collected video data on each of four days: the day before (baseline), the day of, the day
after, and two weeks after divider removal. The number of inspections was scored in 30-sec
bins (120 lines of data for each observation) and bottom-dwelling from spatial location was
recorded every two minutes (32 lines) for one hour. Social behaviours were scored every two
minutes from the time immediately after divider removal to tank lights out at 18:00 h (210
lines). Using linear mixed models, we found that after the divider was removed, zebrafish
moved into the novel space on average within 9.7 (±7.6 SD) seconds, and that the number
of inspections increased on each day observed (P<0.003). We found no evidence of bottom-
dwelling (P>0.73), which, as a well-established measure of anxiety in zebrafish, indicates that
the manipulation was not anxiety-provoking. Further, the opportunity to explore altered social
behaviour: it reduced aggression on the day of and day after (P=0.02), and increased cohesion
on the day of (P=0.04) and coordination on the day after (P=0.04) divider removal. These results
contrast with previous research that has shown that zebrafish living in larger tanks tend to have
lower group cohesion. Our finding that group cohesion increased in response to additional
space suggests a different mechanism may be at play (i.e. cognitive rather than physical). In
summary, we found that zebrafish readily engage in free-choice exploration and that such
opportunities do not induce anxiety behaviour. Moreover, these opportunities alter social
behaviour: reducing aggression and increasing cohesion and coordination. Considering that
their natural environment would normally include many such information-gain opportunities,
our findings suggest that high aggression and low cohesion and coordination may be signs of
abnormal behaviour in captive zebrafish. This study provides novel evidence and adds to the
growing body of literature on the role cognitive stimulation may play in zebrafish welfare. It
indicates that zebrafish are good candidates for further cognitive enrichment research and we
encourage future research to elucidate our findings.
**Cognitive bias and paw preference in the domestic dog (Canis familiaris)**

Deborah Wells, Peter Hepper, Adam Milligan and Shanis Barnard

Queen's University Belfast, Animal Behaviour Centre, School of Psychology, Queen's University
Belfast, BT7 1NN, United Kingdom; d.wells@qub.ac.uk

Cognitive bias measure, designed to detect the valance of affective states, have been proposed as a tool for assessing animal emotions and welfare risk. Such measures are not always practical in applied contexts. Limb use, an indicator of hemispheric functioning, may be a useful predictor of cognitive bias. This study explored the association between motor asymmetry and cognitive bias in the domestic dog. Thirty pet dogs had their paw preferences assessed using the Kong ball test, in which the animal had to use its paw/s to stabilise a food-filled ball. Affective state was assessed following training on a cognitive bias test used previously on dogs. Here, latency to approach a bowl placed in one of 3 ambiguous positions (referred to as Near Positive, Middle, Near Negative) between a baited food bowl (Positive) and a non-baited food bowl (Negative) was recorded. Positive bowls were placed, unblinded, on the left-hand side of the room for 50% of the dogs, and on the right side for the remaining subjects. Friedman tests were conducted for left, right and ambilateral animals to determine if their speed to approach the 3 ambiguous bowls differed according to location. Pearson moment correlations examined the relationship between the direction and strength of the dogs' paw preferences and their latency to reach the 3 ambiguous bowls. Kruskal-Wallis tests revealed no significant relationship between the dogs' paw preferences and their latency to approach the bowl located at the Negative (P=0.99) or Positive (P=0.46) position. Right-pawed (Friedman test=7.40, df=2, P=0.02) and ambilateral (Friedman test=22.15, df=2, P<0.001) dogs differed in their latency to approach the 3 ambiguous food bowls, becoming increasingly slower the further the bowl was positioned from the baited food bowl. Left-pawed animals showed no significant difference in their latency scores for the bowls positioned at the 3 ambiguous locations (Friedman test=3.71, df=2, P=0.16). Analysis revealed a positive correlation between the direction of the dogs' paw use scores and their latency to approach the food bowl positioned at Near Positive (r=0.66, n=30, P<0.001). Animas became slower to reach the bowl located in this position with increasing left-pawedness. The strength of the dogs' paw preferences was correlated with their speed to reach the bowl located at Near Positive (r=0.39, n=30, P=0.03). Animas became slower to reach the bowl at this location with increasing lateralisation. The study points to a relationship between cognitive bias and paw preference in the dog, with left-pawed animals being more negative in their cognitive outlook than right-pawed or ambilateral individuals. Limb preference testing might offer a more practical way of identifying individuals at risk from poor welfare by virtue of how they perceive the world than more time-consuming cognitive bias tests.
**Breed and behavioral characteristics of dogs that differ in owner reported tendency to attend to visual stimuli on a TV**

Miki Kakinuma, Izuru Nose, Yumiko Uno, Shuichi Tsuchida, Hitoshi Hatakeyama, Sayaka Ogawa and Miyoko Matoba

Nippon Veterinary and Life Science University, 1-7-1 Kyonancho, Musashino-shi, Tokyo, 180-8642, Japan; kakinuma-miki@nvlu.ac.jp

Dogs are known to understand human forms of communication such as pointing and that they are born with the ability to communicate with humans. On the other hand, some researchers suggest that experience of living close to human is essential in doing so. Our previous study indicated that therapy dogs attend to visual stimuli on a TV screen more than regular pet dogs. We also found breed differences in attending to visual stimuli on a TV screen. Based on the description of infant TV watching behavior, such as attending to the screen, trying to touch the screen, talking to the person on the screen or looking behind the monitor, we asked owners for similar behavior in their pet dogs. We conducted the dog personality tests and collected oral swabs for DNA analysis and compared the results. In a study on dog behavior survey by owners 179 pet dog owners were recruited in Tokyo, Japan. We excluded 17 dogs that had no access to TV at home. Dog owners were asked to fill in a three page survey with dog breed and basic demographic information, reaction to a TV screen and dog personality test. Of the 161 dogs 31 were Toy or Miniature Poodles, 18 mixed breeds, 13 Shiba Inu, 11 Dachshunds and 10 Border Collies. Of those with access to TV, 52 attended to TV screens. Border Collie attended to the TV screens, Dachshund did not ($\chi^2(3)=16.10, P<0.01$). The result of dog personality test showed that TV screen attending dogs were higher in 'trainability' than non-attending dogs ($t(161)=4.44, P<0.01$). TV attending Poodles scored higher in 'trainability' than non-attending Poodles ($t(31)=3.26, P<0.01$) In a study on DNA analyses, oral swabs of 120 pet Toy Poodles were collected. The genomic DNA samples were extracted from oral swabs using DNA extraction kit. We focused on a gene, *FNBP1L*, which was estimated to be one of associated polygenic genes to human intelligence by gene-wide association study. The 14 coding regions in canine *FNBP1L* gene were amplified by PCR and carried out direct sequencing. By comparing the nucleotide sequences of the *FNBP1L* gene, the efficiency of the gene is evaluated in the dog behavior concerning attention to TV screens. The analysis of results is still underway. Since small mall breeds are popular in Japan, our findings may not apply to other societies with larger breeds or outdoor dogs. Our findings suggest that dogs attending to TV may have better relationship with the owners, thus are more adapted to the human society. The DNA analysis may help us understand the meaning of the owner reported personality traits. Nippon Veterinary and Life Science University ethics committee approved the experiments (S27K46). This research was supported by MEXT-KAKENHI Grant Number 15K04086.
Effects of birth weight and gender on spatial learning and memory in pigs

Sanne Roelofs¹,², Ilse Van Bommel²,³, Steffi Melis²,⁴, Franz Josef Van Der Staay¹,² and Rebecca Nordquist¹,²

¹Brain Center Rudolf Magnus, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands, ²Utrecht University, Department of Farm Animal Health, Yalelaan 7, 3584 CL Utrecht, the Netherlands, ³Utrecht University, Graduate School of Life Sciences, Heidelberglaan 8, 3584 CS Utrecht, the Netherlands, ⁴HAS University of Applied Sciences, Onderwijsboulevard 221, 5223 DE 's Hertogenbosch, the Netherlands; s.roelofs@uu.nl

In current pig farming, selection for large litter sizes has led to an increased number of low birth
weight (LBW) piglets being born and surviving to slaughter age. In humans, LBW is a risk factor
for various cognitive deficits. In pigs, the consequences of LBW are less well known. Cognitive
deficits may influence animal welfare if they result in an impaired ability to cope with housing
and rearing conditions. Previous studies have provided contradictory results when comparing
cognitive performance of normal birth weight (NBW) and LBW piglets in a spatial holeboard
task. It is possible that gender acted as a confounding factor on these results, as both males and
females were tested without using gender as a factor in statistical analysis. Therefore, this study
compared cognitive performance of LBW and NBW piglets of both sexes. Forty piglets from 15
available litters were selected (as NBW-LBW sibling pairs of the same gender). This resulted in
twenty NBW piglets (1,446±49 g) and twenty LBW piglets (810±24 g), each group consisting of
ten males and ten females. They were moved to the research facility at approximately 4 weeks of
age and housed in groups of 10 in mixed sex groups, but separated by birth weight. All piglets
were housed in pens enriched with straw. The piglets were trained to search for the locations
of four hidden food rewards (chocolate M&M's) in the spatial holeboard, an open arena
containing a 4×4 matrix of potentially baited holes. The holeboard allows for the simultaneous
assessment of multiple cognitive variables, such as working memory (assessed by how many
revisits a pig makes during a trial) and reference memory (assessed by how well a pig learns
its rewarded configuration). All piglets received 44 acquisition trials. Testing in the holeboard
lasted until the pigs were approximately 3 months of age. Effects of birth weight and gender
were analysed using a mixed model ANOVA. LBW piglets showed an impaired reference
memory performance (F=5.16; df=1,360; P=0.024) compared to NBW piglets throughout the
acquisition phase. No effect of birth weight was found on working memory scores. Birth weight
did not influence the latency to visit the first hole of a trial, indicating comparable motivation
to perform the task for LBW and NBW piglets. However, LBW piglets were on average slower
to find their first reward (F=8.54; df=1,360; P=0.004) than NBW piglets. Gender did not affect
any of these measures. Together, these results confirm our hypothesis that LBW causes cognitive
deficits in pigs. Furthermore, male and female piglets show comparable cognitive performance
in the acquisition of the holeboard task. This suggests the negative cognitive effects of LBW
are not gender-specific.
**Never wrestle with a pig**

Dorte Bratbo Sørensen

University of Copenhagen, Department of Veterinary and Animal Sciences, Grønnegardsvej 15,
1870 Frederiksberg C, Denmark; brat@sund.ku.dk

The use of positive reinforcement training (PRT, ‘clicker training’) has slowly spread from the world of zoos and pets to laboratory animal facilities housing non-human primates, chimpanzees or dogs. Especially in apes and non-human primates, the power of PRT to decrease stress and facilitate veterinary procedures has been demonstrated, but similar studies are lacking in pigs. Compared to primates, the pig is a much more commonly used lab animal, but even though the pig is easy to train, often only simple habitution- and luring techniques (if any) are used to reduce stress and fearfulness in the pigs. According to the EU directive (2010/63/EU Directive on the protection of animals used for scientific purposes; preamble 31), animal welfare should be given the highest priority. Hence, training of laboratory animals should always be implemented to the highest possible extent. This presentation will provide an overview of basic positive reinforcement training principles and pit-falls and examples of individual effects of PRT on laboratory pigs. We have used simple PRT such as target training to rapidly and effectively gain benefits such as improved human-animal contact, less fear of the animal caretaker and a quicker and smoother performance of e.g. weighing of the pigs. Moreover, by implementing PRT in daily management, we simply add control, predictability and positive anticipation to the environment of the pigs; features which are all said to increase animal welfare. This presentation will discuss the choice of training method (PRT, luring, habituation, etc.) in relation to the purpose of training and the importance of competences and engagement of the trainers and the equipment used. Also the time allocated for training and initial socialization to humans needs to be considered, as pigs traditionally are used for short-term studies, where time for training is seldom included. This lack of time for optimization of training, however, seems not to be in accordance with the EU directive and hence there is definitely room for improvement. Videos demonstrating training techniques and training outcomes when using simple luring compared to PRT are presented and discussed as are welfare gains and economic/other benefits of training laboratory pigs.
Exploring the effect of automated milk feeding stall design on dairy calf behavior

Tanya Wilson, Stephen Leblanc, Trevor Devries and Derek Haley
University of Guelph, Population Medicine, 50 Stone Rd E, N1G 2W1, Canada;
tdevries@uoguelph.ca

There is evidence that the health and welfare of young dairy calves may be improved by
increasing milk allowance and by providing milk through a teat. These aspects are easily
incorporated into automatic milk feeding systems, which promote group housing that has
been demonstrated important for social development of calves. Little is known about how
calves interact with automatic milk feeders. We investigated the effect of stall design features
on calves learning to use the automatic feeder. Sixty-six male and 53 female Holstein calves
were enrolled at 4 d of age and introduced to a group pen and trained on an automatic feeder;
they were allowed to suck on a trainers' fingers and guided to the teat. Calves were allocated to
1 of 2 different stall designs: one with gated (open) side walls (n=59), the other with solid side
walls (n=60). Over a 72-h period after training on the automatic feeder, data from the automatic
feeder was collected and calf behaviour was monitored by video. The hypothesis for this study
was that solid stalls would result in a longer latency to approach and feed from the automatic
feeder. Main outcomes measured were latency to first voluntary visit to the feeder, latency to
first feeding, time spent in feeder, amount of milk drank, and exploratory behaviour such as
sniffing and licking of the feeder. Data were analyzed using a mixed-effect linear regression
model. The overall latency for calves to first voluntarily drink from the feeder was 5.3 h greater
(P=0.05; SE=2.7) with a gated side compared to solid. The average time taken to first drink was
23.7 h (min 2.9, max 69.1) with gated sides and 15.1 h (min 3.2, max 54.5) with solid sides.
Male calves also drank 2.0 l more (P=0.05; SE=1.0) milk over the 72-h period than females.
Results from this experiment show that simple features of a stall on an automatic milk feeder
can influence dairy calf behaviour. A shorter latency to first voluntary milk feeding from the
automatic feeder with solid sides compared to gated, suggests simple differences between
automatic feeders can influence calves feeding behaviour, and should therefore be taken into
account by producers and companies manufacturing the equipment.
Poor reversal learning in dairy cows is associated with endometritis risk after calving

Kathryn Proudfoot¹,², Becca Franks², Alexander Thompson² and Marina Von Keyserlingk²

¹The Ohio State University, Veterinary Preventive Medicine, 1920 Coffey Rd, Columbus, OH 43210, USA, ²The University of British Columbia, Animal Welfare Program, 2357 Main Mall, Vancouver, BC, V6T 1Z4, Canada; proudfoot.18@osu.edu

Intensively housed dairy cows face a number of stressors at calving, and are at high risk of disease. How individual cows cope with these stressors may influence their health, but little research has identified characteristics of at-risk cows. Individual differences in neuroticism have been found to be associated with disease risk in other species; neuroticism is characterized by a prolonged negative affective state, vulnerability to stressors, and cognitive deficits including poor ‘reversal learning’ or the inability to change from a previously-rewarded behaviour to a new behaviour. Our aims were to determine if dairy cows that perform poorly on reversal learning tasks before calving have a higher risk of endometritis after calving. Cows were cared for according to a protocol approved by the UBC’s Animal Care Committee. Four weeks before calving, cows were assigned to one of 7 groups (4 cows/groups, n=28) and moved into a free stall pen with 4 electronic feed bins, 8 lying stalls, and 4 non-experimental cows each assigned to a single feed bin throughout the experiment. Feed bins recorded feed intake during each visit and the number of times a cow tried to access a bin that she was not assigned to. On the first day, cows were assigned to have access to one feed bin that they had to share with a non-experimental partner. For the next 4 weeks, cows were assigned to a new bin and a new partner every other day, exposing them to continuous reversal-learning challenges until calving. To determine if cows showed stable individual differences in reversal learning, we collected two variables on days that cows were assigned to a new bin: (1) the number of times a cow tried to visit the bin she had been assigned to the previous day after successfully feeding from her new bin, and (2) the number of times a cow visited any bin after successfully feeding from her new bin. Visits to the previous rewarded bin were divided by total visits (rewarded and unrewarded) to determine the proportion of time a cow persisted at her previous bin. Endometritis (>5% neutrophils) was diagnosed using a uterine cytology smear 3-5 wk post-calving. Cows showed stable individual differences in their persistence in attempting to visit their previous, now unrewarded, bins: the correlation between persistence during the first and last two weeks was 0.55 (P<0.01). There was no correlation between poor reversal learning and feed intake (P>0.6). However, logistic regression revealed that the most persistent cows had an increased probability of developing endometritis (P<0.04). In conclusion, some cows were consistently poor at changing a previously-rewarded behavior, and these cows were more likely to develop endometritis regardless of feed intake. The results provide evidence that one characteristic of neuroticism is associated with endometritis risk in dairy cows; more research is needed to determine the relationship between other characteristics of neuroticism and health in dairy cows.
**Artificial intelligence algorithms as modern tool for behavioural analysis in dairy cows**

Håkan Ardö¹, Oleksiy Guzhva², Mikael Nilsson¹, Anders Herlin², Lena Lidfors³ and Christer Bergsten²

¹Lund University, Centre for Mathematical Sciences, Sölvegatan 18A, Box 118, 221 00 LUND, Sweden, ²Swedish University of Agricultural Sciences, Biosystems and Technology, Sundsvägen 16, Box 103, 230 53 Alnarp, Sweden, ³Swedish University of Agricultural Sciences, Animal Environment and Health; Applied Ethology Unit, Gråbrödragatan 19, Box 234 532 23 Skara, Sweden; oleksiy.guzhva@slu.se

Behaviour is an invaluable tool for monitoring health and welfare of dairy cows. While the behaviour of an individual has a direct correlation with actual health and stress levels, it is also influenced/influences behaviour of other individuals within the group. In dairy cows, group hierarchy and group dynamics are essential indicators of cow's performance. Correct classification of behaviours occurring within the group is of importance as some interactions are positive, and strengthen the bond between cows and others are possibly deleterious and can reflect a struggle for resources. Therefore, gaining knowledge about dyadic relationships within the group could help in creating an animal-friendly environment and reduce stress related to competition. The aim of this study was to take the first step towards an automated system for behavioural analysis. The complete dataset contained 4 months, from two different seasons, of recordings (400 million frames, among which 2,148 were randomly selected and annotated) collected from 3 cameras installed in a holding pen of a conventional dairy barn with 252 Swedish Holstein cows. Then an algorithm removed uninteresting parts (e.g. those, containing behaviours that were not relevant or without animals in the scene) of the recorded video material. The annotated subset contained in total 9,278 cows. A simple 'watchdog' algorithm extracting frames containing two or more cows was implemented, and it was possible to discard 38% of the recordings as uninteresting. To remove more of the uninteresting video, additional features such as the minimum distance (as cows need to be close to interact) between the cows in the scene and duration of contact, were also extracted. Then a short sequence containing interactions consisting of 187 frames evenly sampled over 10 minutes was extracted and manually annotated by an expert to be then used for cross-validation with automatically annotated data. Five different interactions (used in previously developed 7-point shape model) were manually identified as relevant for this study: body pushing, butting, head butting, head pressing and body-sniffing. Frames where any of these interactions were present, were considered interesting and all other frames uninteresting. Most, 97.1%, of the images were perfectly interpreted, i.e. all cows present were detected with no extra detections, with false alarm rate of 2.9%. The proposed algorithm was able to discard 50% of uninteresting video material, while only losing 4% of potentially interesting fragments. This, in combination with features for proximity analysis, allows to conclude that modern computer algorithms could help in assessing group structure in dairy cows as well as save valuable expert time.
**Best friends forever, new approach to automatic registration of changes in social networking in dairy cows**

Per Peetz Nielsen¹, Luis E.C. Rocha², Olle Terenius³, Bruno Meunier⁴ and Isabelle Veissier⁴

¹University of Copenhagen, Department of Veterinary and Animal Sciences, Grønnegårdsvej 8, 1879 Frederiksberg C, Denmark, ²Karolinska Institutet, Department of Public Health Sciences, Solnavägen 1, Solna, 171 77 Stockholm, Sweden, ³Swedish University of Agricultural Sciences, Department of Ecology, P.O. Box 7044, 750 07, Sweden, ⁴UCA, INRA, VetAgro Sup, UMR1213 Herbivores, 63122 Saint-Genes-Champanelle, France; ppn@sund.ku.dk

Until recently, it was not possible to record social network in dairy cows over a long period. However, thanks to the development of systems using ultra-wideband for real time location, it is now possible to follow the exact position of a large group of cows over an unlimited period. The aim of this study was to examine the changes in social contact that occurs when cows are removed from a stable group and at the same time adding new cows into the remaining group. We used a position system using UWB to record x-y position of dairy cows at the INRA research farm (UE1414 Herbipole) in Marcenat, France. The tag was placed on the neck of the cow. This technology enables us to look more deeply into the social network and structure of dairy cows and examine how disturbances affect this structure. Furthermore, it can be used to develop automated systems for the detection of dairy cows at risk of being infected by other cows and for the reduction of the spread of infection in the herd, with the information on which cows that have been exposed to contaminated areas. A social network was defined by a group of nodes, representing the cows, connected by links, representing the social interactions between the cows. Every second two cows were within a radius of 1.25 m of each other, we considered it as a link. Such links were defined only for cows outside the resting/cubicle area. The value of 1.25 m corresponds approximately to twice the distance between the neck and the nose of the cow and thus captures potential face-to-face interactions. We aggregated all links during the day and assigned the total time to the aggregated link between the respective cows. The connectivity (or node degree) of a cow to another cow was defined as the total time exceeding 1,500 seconds of connections during a day. The data consisted of a group of 26 cows where 9 cows were moved and after that, 8 different cows were added into the group. Sociograms (a graphic representation of social links that an individual has) of the links between the cows showed that the first seven days after the new cows are added into the group, there were many social links, but these links changed each day. The number of average connections during the week that exceeded 1,500 seconds per day was smaller in the new constellation and mainly occurred between the original cows in the group. We interpret this as an increased connectivity due to a build-up of a new hierarchy. It is furthermore concluded that cow-tracking systems have a huge potential for examination of the social structure and changes of these structures in dairy cows. Further studies are needed with more groups of cows in order to conclude how the hierarchy develops over time in new constellations of dairy cows.
**Phenolab: ultra-wide band tracking shows feather pecking hens spent less time in close proximity compared to controls**

Elske N. De Haas¹, Jerine A.J. Van Der Eijk¹, Bram Van Mil² and T. Bas Rodenburg¹

¹Wageningen Universit & Research, Behavioural Ecology Group, Adaptation Physiology Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ²Noldus Information Technology B.V., Nieuwe Kanaal 5, 6709 PA Wageningen, the Netherlands; elske.dehaas@wur.nl

Hens which feather peck can cause multiple victims in a group. As a result pen-mates could keep a greater distance from feather peckers. Our aim was to relate time spent in close proximity between birds selected divergently on feather pecking. Automatic ultra-wide band location data was obtained by using an active sending tag (Ubisense®) placed in a backpack on the birds. A group of birds was then placed for 15 minutes in a barren test-room equipped with four receiving beacons. We used 37-week old White Leghorn laying hen lines selected for high (HFP) or low feather pecking (LFP), and an unselected control (CON) line (n=76 in total, 5-7 hens per pen; 4 pens per line). Hens were habituated to the backpacks four weeks prior to observations, and were tested for 5-min to the test-room individually one week prior to group testing. Sample rate of tags was set to twice per second, detecting location of each tag separately and simultaneously. Location data was provided by TrackLab software (Noldus, Wageningen, The Netherlands) and compared between tags by Excel calculations. Percentage of time in close proximity was defined as being less than 25 cm apart from another tag, calculated for each bird and averaged per group. Behavioural sampling of feather pecking of 2x20 min at 28-29 wks of age was included on pen-level and used for identification of peckers (>2 bouts/20 min). Percentage of time in close proximity was correlated to feather pecking (pearson correlation), tested to differ between peckers and average of the group (t-test of groups with peckers) and tested to differ between lines (ANOVA including the number of animals per pen as covariate). Feather pecking on pen-level was not correlated to proximity measures (r=-0.28, P>0.10), but birds spent less time in close proximity of the feather pecker as opposed to time spent on average with other pen-mates (17.6±1% vs 21.6±1.2, t16=-1.18, P=0.04). Hens of the high feather pecking line spent a lower percentage of time in close proximity compared to controls (HFP: 18.9±2.2% vs CON: 39.8±10.1% vs LFP: 27.0±1.6%; F<sub>2,12</sub>=5.4, P=0.05). The number of hens per group did not influence proximity (F<sub>1,12</sub>=0.39, P=0.93). This data shows that selection on feather pecking has affected proximity. Less time in close proximity in HFP vs LFP groups and with feather peckers vs non-peckers could be explained by higher activity of peckers and in HFP groups. As feather damage was pre-existing in HFP birds, proximity differences between HFP and LFP groups could not be used as predictors for feather pecking. Whether this pattern of proximity is linked to the divergent genetic selection or a response to the actual occurrence of feather pecking will be further examined on group level in other home-pen situations. This automatic tracking method seems promising to pick up within group differences in behavioural patterns and might ultimately be used to predict feather pecking in laying hens.
**Floor feeding over multiple drops does not increase feeding opportunities for
submissive sows.**

Natalia Zegarra¹, Paul. H. Hemsworth¹ and Megan Verdon²

¹The Animal Welfare Science Centre, The University of Melbourne, Parkville, Victoria, 3010, Australia, ²Tasmainian Institute of Agriculture, The University of Tasmainia, Burnie, Tasmania, 7320, Australia; megan.verdon@utas.edu.au

Feeding sows from the floor is one of the simplest and cheapest method of feed delivery, but often results in high levels of aggression as sows compete for access to food. This may lead to subordinate sows sacrificing the opportunity to feed in an attempt to avoid receiving aggression. This study examined whether floor feeding sows their daily ration over multiple bouts per day creates more opportunities for subordinate sows to feed. Two-hundred pregnant gilts were mixed into pens of 10 (1.8 m²/gilt) within 7 days of insemination and floor fed four times per day (07:30, 09:00, 11:00, 15:00 h). The identity of gilts that were feeding (head down, rooting the ground/feed, chewing) was recorded, along with her location in the pen [on the cement area where feed was scattered (i.e. feeding area; F), directly under the feeder (i.e. high feed availability; HF), on the slats at the back of the pen (i.e. little or no feed availability; NF)], every 30 s for 20 min after each feed drop on days 2, 9 and 51 post-mixing. Gilts were also classified as dominant (D), subdominant (SD) or submissive (S), as described elsewhere. GLMM in conjunction with LSD tests were used to analyse the effects of gilt classification, day and feeding bout on the time gilts spent feeding in each location. Each model included a repeated effect of day and feeding bout within gilt, and pen nested within replicate as a random effect. Regardless of feeding drop (P>0.05) or day (P>0.05), D spent the most, and S the least, time feeding at the HF location. In feed drops 1, 2 and 4, the time SD spent HF was intermediate to S and D, but in drop 3 the time SD spent HF was comparable to S (classification × drop interaction P=0.01). Irrespective of feed drop (P>0.05), S gilts spent less time in the feeding area (P<0.001), and more time in the NF location (P=0.03), than D and SD, while the latter two did not differ. Feeding gilts over multiple drops does not increase feeding opportunities for subordinate sows. Regardless of feed drop or day, D gilts occupied the area of high feed availability and likely had the highest feed intake. That SD appear to adopt a strategy of feeding between and around the D gilts in areas of reduced feed availability. However, S gilts appear to be at greatest risk of low feed intake, which may result in reduced body condition and increased hunger and frustration. Further research is required to determine how the feed intake of subordinate sows can be increased in floor feeding systems, while also protecting these animals from aggression.
The effect of socialization before weaning on social behaviour and performance in
piglets (Sus scrofa) at a commercial farm

Laura Salazar¹, Irene Camerlink², Heng-Lun Ko³, Chung-Hsuan Yang¹ and Pol Llonch³

¹Edinburgh University, Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, EH25 9RG, Edinburgh, United Kingdom, ²Scotland's Rural College, Animal and Veterinary Sciences Research Group, West Mains Rd., EH9 3JG, Edinburgh, United Kingdom, ³Universitat Autònoma de Barcelona, School of Veterinary Science, Bellaterra, 08193, Cerdanyola del Vallès, Spain; laurasalazarhofmann@gmail.com

Although positive effects of socializing piglets pre-weaning on behaviour and performance have been confirmed, it has not been proven in commercial conditions. The aim of the present study was to investigate behaviour (locomotor play, aggressive and social behaviour), aggressions (skin lesion score) and performance (weight and average daily gain (ADG)) of piglets socialized at different ages during lactation at a large scale commercial farm. We hypothesized that socialization increases social behaviour during lactation, reduces skin lesion score (less aggression) and results in improved weight gain after weaning. After approval from IACUC, piglets were either not socialized (CON; n=12 litters) or socialized at 7 (M7; n=20 litters) or 14 days of age (M14; n=20 litters) by removing the separating barrier between two adjacent pens. At weaning (day 25), piglets were regrouped (~39 pigs/group, 8 groups in total) with unfamiliar piglets of the same treatment. Social, aggressive and locomotor play behaviour was measured using instantaneous scan sampling on day 7, 14, and 21 in all treatment groups. Skin lesions were counted before (-1) and after (+1 and +2) mixing at socialization and weaning. Weight was recorded weekly from one day after farrowing until ten days after weaning (day 35). Data were analysed using mixed models accounting for sow and pen effects. Socialized (M7 and M14) piglets showed three times more play behaviour (1% of observations) compared to CON piglets (0.2% of observations) on day 14 (P=0.03). At the time of socialisation, aggression was more often observed in M14 (1.5%) than in CON (0.6%; P=0.04) and tended to be higher in M7 (1.4%) than in CON (P=0.07). Skin lesion score increased after socialisation in M7 (before: 0.40±0.09; after: 1.53±0.20; P<0.001), but not in M14 (before: 0.82±0.12; after: 0.80±0.13; P=0.68). After weaning, skin lesions increased in CON (before: 0.21±0.06; after: 1.03±0.21; P<0.001), but not in M7 (before: 1.11±0.39; after: 0.74±0.21; P=0.007) neither M14 (before: 0.97±0.33; after: 1.20±0.32; P=0.44). Treatment groups did not significantly differ in birth weight (CON 1.7±0.03; M7 1.5±0.03; M14 1.5±0.03 kg; P=0.41) but from day 7 to 21 the control group was heavier than socialized piglets (P<0.05). Although the decline in ADG after weaning was not significantly different between treatments (CON 0.093±0.016; M7 0.082±0.012; M14 0.093±0.011 kg/d) (P=0.68), the final weight (day 35) was not different among treatments (CON 7.04±0.54; M7 6.07±0.44; M14 6.40±0.44 kg) (P=0.27). In conclusion, in a commercial farm, socialization can increase social behaviour during lactation and reduce aggression when mixing with unfamiliar piglets at weaning without reducing performance, which is in line with research outcomes under controlled experimental facilities.
**Socialisation, play behaviour, and the development of aggression in domestic pigs**
*(Sus scrofa)*

Jennifer Weller¹, Simon Turner², Irene Camerlink², Marianne Farish² and Gareth Arnott¹

¹Queen's University Belfast, School of Biological Sciences, Queen's University Belfast, Belfast, BT7 1NN, United Kingdom, ²Scotland's Rural College (SRUC), Animal Behaviour and Welfare, RSUC Roslin Institute, EH25 9RG, United Kingdom; jweller01@qub.ac.uk

Aggressive behaviour post-weaning in domestic pigs is a major welfare concern that remains to be fully addressed. The role of play behaviour in the development of aggression has received little attention, yet it has been suggested that early life social play facilitates the development of later life social skills, including those used to resolve agonistic encounters. We hypothesise that socialising piglets during early life will increase the opportunity for play experience, and that individuals that have experienced more play will be less aggressive with conspecifics during later life. Additionally, we examine sex differences in play behaviour, making connections to later life aggression and expected social roles. This study was conducted using 196 piglets from 19 litters. Of these, 8 litters were selected to undergo socialisation and were allowed to interact freely with the individuals of a neighbouring pen between days 14-26 after birth. The remaining 11 litters acted as controls and remained isolated within their litter groups until post-wean mixing. In both socialised and control pigs, all occurrences of social play with other piglets (play fighting, nudging and chasing) and sows (naso-naso contact and climbing) were recorded over a 6 hour period for 5 days that spanned the socialisation period. At 7 weeks old, piglets (n=109) from both treatments had their aggressiveness assayed using the previously validated resident-intruder (RI) test, repeated with different intruders on two consecutive days. Latency to attack was recorded, or the interaction terminated if no attack occurred within five minutes. All work conformed to required legislation, receiving ethical approval and adhered to ISAE guidelines. Linear mixed effects models revealed that socialisation did not significantly increase total play fighting behaviour (P=0.83) or play behaviour as a whole (P=0.33). Despite this, socialised piglets were found to have a shorter attack latency than controls (P=0.04, Soc=258.9 s, SE=±23.5 s, Con=384.3 s, SE±27.0 s) suggesting that some other aspect of socialisation influences aggression. Controls were found to interact with the sow significantly more than socialised individuals (P=0.02, Con=13.4, SE±0.9, Soc=6.8, SE±0.5) and there was a positive correlation between sow-aimed play and attack latency (P=0.04, rho=0.19). Regarding sex differences, males engaged in more play fighting than females (P<0.001, Male=27.0, SE±1.7, Female=18.3, SE±1.4) although females showed a shorter attack latency than males (P=0.0003, Female=261.4 s, SE±28.9 s, Male=352.2 s, SE±23.6 s). Thus, early life socialisation resulted in increased aggressiveness in the context of an RI test, possibly reflecting an increased ability to rapidly establish a dominance hierarchy. This assertion will be explored, while also examining sex effects, in due course by considering the effects of socialisation on the assessment strategies used during contests and mixing practices in later life.
**Intensity of aggression in pigs depends on their age and experience at testing**

Irene Camerlink¹, Marianne Farish¹, Gareth Arnott² and Simon P. Turner¹

¹Scotland's Rural College (SRUC), Animal Behaviour & Welfare, Animal Veterinary Sciences Research Group, West Mains Rd., EH9 3JG Edinburgh, United Kingdom, ²Queen's University, School of Biological Sciences, Institute for Global Food Security, University Road, BT7 1NN Belfast, United Kingdom; irene.camerlink@sruc.ac.uk

Type and frequency of behaviour in any species is strongly dependent upon the situation in which it is measured. Our aim is to illustrate, using the example of aggression, how research outcomes can change due to age and experiences of the animals at testing. Data from three experiments on pig aggression (approved by ethical committees) were combined. All trials included dyadic contests with the same test set-up, carried out on the same farm. Within and between tests, pigs varied in age and experience before the contest. Pigs were either tested at 8, 10 or 13 weeks of age and had either never encountered an unfamiliar pig (E0, n=550 pigs of 8 and 10 wk of age), had been co-mingled with another litter pre-weaning for 2 wks (EL, n=184; 8 wk age), had encountered a single unfamiliar pig at 10 wk age (E1, n=136; 13 wk age) or had been subject to group mixing at 11 wks of age (EM, n=168; 13 wk age). Comparisons for age were made between E0-8 and E0-10 whereas the effect of experience was compared between E0-8 vs EL-8 and E1-13 vs EM-13. For the contest, two unfamiliar pigs simultaneously entered an arena (2.9×3.8 m). Contest duration, skin lesions as a result of bites, and agonistic behaviour were recorded. Contests lasted max 30 min and had strict end-points to prevent lasting harm. Data were analysed using mixed models that accounted for trial and pen in the random effects. Contests with an escalated fight were longer (fight 287 s; no fight 158 s; P<0.001) and fight occurrence was thus included as a fixed factor. Contest duration increased with age, with naïve pigs (E0) on average spending 164 s in contest at 8 wk of age and 240 s at 10 wk of age (P<0.001). Experience of co-mingling decreased the contest duration at 8 wk of age from 253 s (E0) to 202 s (EL; P<0.01). In contrast, experience of group mixing at 10 wk of age increased the contest duration at 13 wk of age from 199 s (E1) to 299 s (EM; P<0.001). Skin lesions (overall average of 45 lesions) increased with contest duration (P<0.001). When corrected for contest duration, the number of lesions were unaffected by age when comparing pigs of 8 vs 10 wk age (P=0.52), and were unaffected by experience (E0-8 vs EL-8: P=0.11; E1-13 vs EM-13: P=0.50). Ritualized display behaviour, such as foaming and piloerection, increased with age, but almost exclusively in males. Foaming was performed by 19% of the males when 8 wk of age, by 26% when 10 wk of age, and by 68% when 13 wk of age. In females the percentage was 5, 5, and 15%, respectively. This highlights strong age related sex differences even at a pre-pubertal age. We recommend that the age and experience of test animals are carefully considered prior to conducting behavioural research, in particular for research on aggressive behaviour.
# Effect of overstocking during the dry period on the behaviour and stress level of dairy cows, and on pre-weaning calf growth

Mayumi Fujiwara¹,², Kenny Rutherford², Marie Haskell² and Alastair Macrae¹

¹University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, EH25 9RG, United Kingdom, ²Scotland Rural College, West Mains Road, Edinburgh, EH9 3JG, United Kingdom; mayumi.fujiwara@sruc.ac.uk

On commercial dairy farms, dry cows are usually kept in dynamic social groups. Limited access to resources such as feed and/or lying space is known to increase competition and alter feeding behaviour in dairy cows. Stressful maternal experience can have a negative impact on fetal development, which could affect the offspring throughout its postnatal life. The aim of this study was to investigate the effects of overstocking during the dry period on the behaviour and welfare of dairy cows, and to investigate the effects of maternal social experience on calf bodyweight and growth. Forty-eight Holstein cows (calving over a six month period) were dried off 8-9 weeks before their expected calving date, and kept in cubicle sheds with a yoke feed barrier during the first six weeks (far-off) and then kept in straw yards with a post-and-rail feed barrier until calving (close-up). Cows were allocated to either high (H) or low (L) stocking density groups after dry-off. H and L cows had 0.5 vs 1.0 yokes/cow and 1.0 vs 1.5 cubicles/cow respectively in the far-off period, and 0.3 m vs 0.6 m feed face/cow and 6 m² vs 12 m² lying space/cow respectively in the close-up period. The time spent feeding during the 3 hours after feed delivery was recorded three days per week, and agonistic social interactions were observed two days per week (0-20 min, 40-60 min and 80-100 min after feed delivery). Faecal glucocorticoid metabolites (FGCM) were measured during the dry period at 5 time-points. Bodyweight of calves (n=44) was measured at birth and at weaning (at 49 days old), and daily gain from birth to weaning was calculated. Data were analysed by Residual Maximum Likelihood (cow data) or General Linear Model (calf data) using GenStat following log transformation where necessary. Means and standard errors are presented, and means obtained from the transformed data were back-transformed to the original scale, and 95% confidence intervals (CIs) are reported. H cows spent less time feeding (72.1±4.4 min) than L cows (86.8±4.8 min) during the 3 hours after feed delivery (W=5.5, P=0.023), but engaged in more agonistic interactions (H: 3.45/group, 2.92-4.05; L: 1.93/group, 1.57-2.34). The concentration of FGCM was not affected by treatment, but FGCM during the dry period were significantly higher than that at dry-off (dry-off: 184.1 ng/g, 80.1-423.1; week2: 379.3 ng/g, 161.1-893.1; week5: 323.6 ng/g, 140.8-743.7; week6: 368.1 ng/g, 160.1-846.1; pre-calving: 357.3 ng/g, 150.0-851.2, W=85.6, P<0.001). No effect of maternal treatment was found in the calf bodyweight at birth and weaning, or in the pre-weaning daily gain. Overstocking of dry cows resulted in a shorter feeding time during the 3 hours post feeding and more frequent competition at the feed face. However overstocking did not affect the concentrations of faecal glucocorticoid metabolites. Maternal competitive experience and altered feeding behaviour during the dry period did not have negative consequences on calf bodyweight or pre-weaning growth.
**Relationship between social rank and reproductive, body and antler traits of pampas deer (Ozotoceros bezoarticus) males**

Matías Villagrán¹, Florencia Beracochea¹, Luděk Bartoš² and Rodolfo Ungerfeld¹

¹Facultad de Veterinaria, Universidad de la República, Departamento de Fisiología, Lasplases 1620, 11600, Uruguay, ²Institute of Animal Science, Prague, Department of Ethology, Přátelství 815, 104 00, Czech Republic; matiasv06@gmail.com

The aim of this work was to determine the relationship between social rank and parameters of body size, antler size, testosterone concentration, and semen characteristics of pampas deer males. The study was performed in a semi-captive population of pampas deer allocated in the Estación de Cría de Fauna Autóctona Cerro Pan de Azúcar (Uruguay), in February and March (early to mid-rut). Data was collected from five groups of 4-7 adult males (2-7 y old) in four different seasons. Overall, 17 animals were used, but as 6 of them were allocated in different groups in different years, the total number of data considered for the analysis was 26. The agonistic behavior was recorded during the rut identifying the dominant and the subordinate animal in each interaction. The individual Success Index (SI) was calculated as the number of individuals displaced/(number of individuals displaced + number of individuals that displaced it) (range = 0 to 1). Animals were anesthetized, and blood was collected to measure testosterone concentration. Body weight, neck perimeter, and shoulder height, circumference of the antlers' coronet, length of second and third tines and total antler length were measured. Semen was collected by electrojaculation of the anesthetized animals. Semen volume, semen concentration, and percentages of spermatozoa with progressive motility, normal morphology, membrane integrity, and acrosomal integrity were determined. The total number of spermatozoa of each variable was calculated (percentage of the variable × total number of spermatozoa). Applying a principal component analysis, following components were created: semen vitality (variables: total number of spermatozoa with progressive motility, membrane integrity, and normal morphology), acrosome integrity (percentage of spermatozoa with intact acrosome), antlers (circumference of the coronet, and length of the second and third tine), and body size (neck perimeter, body weight, shoulder height). The relationship between dependent variable SI and age and the components was determined using a generalized linear mixed model designed for repeated measurements. Success Index was basically dependent on age [F(5, 7.7)=10.0; P=0.003] and body size [F(2, 12.7)=5.7; P=0.02]. In 1-4 y old males, males with higher SI had greater *body size*, while in fully mature, 5-7 y old males increasing body size was observed with decreasing SI. Social rank tended to be associated with greater values of antlers component [F(1, 10.3)=3.9; P=0.07] but not on testosterone concentration (P=0.16). The semen vitality component was positively associated with SI [F(1, 2.7)=44.9; P=0.01] while the acrosome integrity was not related to SI at all (P=0.34). In conclusion, while social rank of pampas deer males allocated in small groups in semi-captivity was positively associated to semen vitality and probably to antlers size, in younger individuals SI was positively associated to body size.
**Rearing bucks (*Capra hircus*) isolated from females affects negatively their sexual behavior when adults**

Lorena Lacuesta¹, Julia Giriboni¹, Agustín Orihuela² and Rodolfo Ungerfeld¹

¹Facultad de Veterinaria/Universidad de la República, Departamento de Fisiología, Lasplaces 1620, 11600, Uruguay, ²Facultad de Ciencias Agropecuarias/Universidad Autónoma de Morelos, Cuernavaca, 62209, Mexico; lacuesta16@gmail.com

In some domestic ruminants, contact with females is necessary for normal development and display of sexual behavior. The aims of this experiment were to determine if rearing bucks isolated from females affects negatively their sexual behavior when adults, and if this negative effect is overcome after four short-term contacts with females. Sixteen Saanen male kids were maintained during one year in two groups: kids reared in permanent direct contact with four adult goats (FEM; n=7), and kids that remained isolated from females (ISO; n=9). When bucks were 12 mo-old, females were removed and both groups were joined in the FEM pen. Nine months later all bucks were individually exposed four times to unrestrained estrous females in 20 min tests, each performed 10 days apart, recording courtship (sum of anogenital sniffs, flehmens and lateral approaches) and mounting behaviors (sum of mount attempts, mounts without and with ejaculation) and the latency to the first ejaculation. Data were compared with the glimmix proc assuming a Poisson distribution of data and the combination of the number of bucks that ejaculated and the latency at which they did it was compared with a survival analysis. The number of courtship behaviors was greater in FEM than ISO bucks (P<0.0001), varied with number of test (P<0.0001) and there was an interaction between treatment and number of test (P<0.0001) (test 1: 75.7±16.3 vs 45.5±17.9; 2: 86.4±24.6 vs 43.4±25.7; 3: 44.3±6.7 vs 48.0±21.6; 4: 51.1±15.2 vs 29.1±8.5, FEM and ISO respectively). The interaction was explained by a greater number of courtship events in FEM than in ISO bucks in the first, second and fourth tests (P<0.0001 for all these tests). Bucks reared with females displayed more mounts with ejaculation and total number of mounts than ISO bucks (mounts with ejaculation: 3.2±0.9 vs 1.5±0.5, FEM and ISO respectively; total mounts: 3.6±0.9 vs 1.75±0.6, FEM and ISO respectively, P<0.0001 for both). Mounts with ejaculation and total number of mounts did not vary according to the number of test and there was not interaction between treatment and number of test. The combined effect of number of bucks that ejaculated and the time at which they first ejaculated (survival analysis) favored FEM bucks in the first and second tests (test 1: 7/7 and 46.0±15.3 s vs 5/9 and 159.0±109.6 s; test 2: 7/7 and 43.8±11.5 s vs 5/9 and 196.0±159.0 s, FEM and ISO respectively; P<0.03). We concluded that the lack of contact with females during the rearing period affects negatively adult bucks' sexual performance, effect that cannot be overcame with at least four repeated exposures to estrous does. In bucks, contact with females during the rearing period leads to develop normal male sexual behavior. As goats kids are reared in females groups, this allows bucks to mate females effectively at an earlier age and thus, increase the reproductive success of the specie.
**Social behavior and adrenocortical activity in goats up to four wees after grouping**

Susanne Waiblinger¹, Eva Nordmann¹ and Nina Maria Keil²

¹Institute of Animal Husbandry and Welfare, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Wien, Austria, ²Center for Proper Housing of Ruminants and Pigs, Agroscope Reckenholz-Tänikon, Federal Veterinary Office, Agroscope Reckenholz-Tänikon ART, Research Station ART, Tänikon, 8356 Ettenhausen, Switzerland; susanne.waiblinger@vetmeduni.ac.at

Changing group composition is frequently practiced in farm animals and known to be stressful. Most studies in goats investigated integration of few animals in a larger group for only a short time. This study investigated the course of agonistic social interactions, adrenocortical activity and injuries up to week 4 after merging two groups. Seven groups of 7 and one group of 6 female dairy goats (n=55, 3-8 years old) were grouped together to form 4 groups of 14 or 13 animals. Two groups consisted only of horned goats, the other 2 only of hornless (polled or dehorned) for experimental reasons. The groups were housed in pens with 2.2 m²/goat on deep litter with an elevated feeding place. For adrenocortical activity, faecal cortisol metabolites (FCM) were measured in samples taken rectally on 2 days per period: 2 weeks before grouping (Before), after grouping on day1 and 2 (Week1), 2 weeks after grouping (Week2), 4 weeks after grouping (Week4). Observations of agonistic interactions by continuous behaviour sampling were conducted in Week1 to 4. Initiator and receiver of agonistic interactions were identified individually. Number of agonistic interactions in total (AgoTotal), with body contact (AgoBodyC) and without body contact (AgoNoBodyC) were calculated per animal per 2.25 h. Goats were examined for injuries once per period. Behaviour (log-transformed) and FCM were analysed by mixed models with animal nested in group as random factor; injuries are depicted descriptively. Adrenocortical activity increased from Before (261 ng/gr) to Week1 (312 ng/gr) and then decreased (Week2: 245, Week4: 240 ng/gr; main effect period: P<0.001, F<sub>3,123</sub>=6.59). In contrast AgoTotal and AgoBodyC peaked in Week2 (model estimate: hornless: 0.833, horned: 0.620). In Week4 the number of these interactions was still higher (model estimate: AgoTotal: 0.744; AgoBodyC: 0.517) than in Week1 (0.547; 0.440) for hornless goats, but lower for horned goats that also in general were on a lower level than hornless goats (Week4: 0.481; 0.240; Week1: 0.520; 0.381; interaction period×horn status: P=0.042, F<sub>2,105</sub>=3.26 and P=0.054, F<sub>2,103</sub>=3.01). AgoNoBodyC increased from Week1 to 2 and stayed on this level until Week4 (main effect period: P<0.001, F<sub>2,105</sub>=13,53), independent of horn status. The number of injuries (mostly superficial lesions and scars) increased in the course of the experiment. Total number of injuries in the 28 hornless goats were 28, 30, 45 and 57, respectively, for the four periods and 7, 10, 23 and 39, respectively, in the 27 horned goats. Most injuries in hornless goats occurred at the front region, where the horns would grow. Results show, that regrouping should be avoided in goats. While physiological stress is highest directly after grouping, the development of a stable social hierarchy takes more time as indicated by the elevated levels of agonistic interactions and injuries in Week4, at least in hornless goats. Yet, having horns may facilitate this development.
**Social behaviour in groups of intact organic fattening boars – a cause for concern?**

Jeannette C. Lange and Ute Knierim

University of Kassel, Farm Animal Behaviour and Husbandry Section, Nordbahnhofstr. 1a, 37213

Witzenhausen, Germany; jlange@uni-kassel.de

While it is suggested to avoid castration of male pigs for animal welfare reasons, it should be recognised that other welfare problems may arise due to the boars' social behaviour. Under certain housing conditions, increased frequencies of lameness as well as skin and penis injuries have been reported. The aim of this study was to investigate this issue under commercial organic conditions in Germany. We compared 41 boar and 32 control groups (26× barrows, 3× mixed, 3× gilts) on five farms. At three observation points (around 80 kg live-weight, at slaughter age before and after marketing of first pigs), agonistic and mounting behaviour were observed by continuous behaviour sampling for 15 minutes every alternate light hour from 48 h videos (same number of light hours per farm and batch in boars and controls). Concurrently all 625 boars, 433 barrows and 83 females of these groups were scored for skin lesions and lameness according to the Welfare Quality® protocol. Randomly selected penises of 123 boars and 14 barrows were scored for lesions. Prevalences of pigs being lame at least once in the three scorings were analysed by Chi-square-test. Frequencies of interactions and lesions (group means) were analysed with mixed models in R, with the random factors 'group', nested in 'batch' and within 'farm'. Fixed factors were 'boars vs controls' and, if significant, 'season' and either 'age' or 'observation point'. Further factors (including group size and stocking density) were not significant. More biting and head knocking (2.1 events vs 1.5/pig×hour, P<0.001), more fighting (0.4 events vs 0.1/pig×hour, P<0.001), and more mounting events (0.44 vs 0.05/pig×hour, P<0.001) in the boar groups did not result in significantly more skin lesions (4.8 vs 4.1 on one body-side, P=0.48) or lameness (0.8 vs 2.1%, P=0.06). Apparently, the relatively spacious and structured pens allowed a considerable amount of non-injurious social behaviour, which in part may even have been play behaviour. Most lesions were scratches and wounds <2 cm (98.1%). Wounds of 2-5 cm (1.7%) or >5 cm (0.2%) occurred similarly seldom in boars and controls. Also, the maximum lesion number in boars (34) and controls (29) was rather similar. Following the Welfare Quality®-evaluation only 0.1% of controls, but 0.2% of boars were severely injured (WQ-score 2). In addition, 1 control vs 7 boar groups had conspicuously increased lesion levels (>twice than average), with particular contribution to these cases by two farms. All penises of barrows were uninjured whereas in 10% of dissected boars small penile injuries were found. Their absence on two farms with generous straw litter management was noticeable. Results reflect a certain welfare risk due to the higher social activity of intact boars that, however, under organic husbandry conditions and good management can be kept to acceptable limits. In this regard, boar fattening presents a practicable alternative to castration.
Effect of removing the heaviest pigs from a group of grower pigs on aggression and the dominance hierarchy

Sabine Conte¹, Niamh O'Connell² and Laura Boyle³,⁴

¹Agriculture and Agri-Food Canada, Dairy and Swine R&D Centre, Sherbrooke, J1M 0C8 QC, Canada, ²Queen's University Belfast, Institute for Global Food Security, School of Biological Sciences, Northern Ireland Technology Centre, 97 Lisburn Road, BT9 7BL Belfast, United Kingdom, ³Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Department of Animal Behaviour and Welfare, ul. Postepu 36A, Jastrzębiec, 05-552 Magdalenka, Poland, ⁴Teagasc, Pig Development Department, Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy, Co. Cork, Ireland; laura.boyle@teagasc.ie

Split marketing, whereby the heaviest pigs in a group are sent for slaughter before their penmates, is a common practice. However, there are concerns that every time pigs are removed from a group that there is some re-organisation of the dominance hierarchy (DH) coupled with aggression and that this has negative consequences for pig welfare particularly in entire male production systems. Hence the aim of this study was to investigate the effect of removing the heaviest animals from stable groups of male and female grower pigs on the DH and aggression. Grower rather than slaughter pigs were chosen for this work because slaughter weight pigs can be injured when fighting on fully slatted concrete floors and being heavier, inflict more damage when fighting. At 8 wks of age (17.5±2.0 kg), 64 pigs were assigned to four groups of 8 males and four groups of 8 females. After 5 d all occurrences of aggression and the identity of the pigs involved were recorded over a 25 min period (Pre-test1). Thereafter the troughs were removed from the pen (16:00 h) until 10:00 h the following morning when pigs were subjected to a food competition test (FCT) of 20 min duration. All occurrences of aggressive behaviour in the pen were recorded and a dominance score (DS) was calculated for each pig according to the no. of pigs it displaced and the no. of pigs that displaced it. 12 d after grouping, the 2 heaviest pigs were removed from each pen and the remaining pigs were observed for 25 min (Pre-test2). A FCT was performed the next day as previously described. Data were analysed by ANOVA, Spearmans rank and Pearsons correlation tests and Goodman and Kruskals gamma test in Genstat. There was no difference in the no. of aggressive behaviours performed between the 8 pigs in Pre-test1 and the no. of aggressive behaviours performed by the 6 pigs in Pre-test2 (mean of 4 aggressive behaviours/pig; P>0.05). However, there were fewer aggressive behaviours between the 6 pigs of interest (excluding the aggressive behaviour given and received by the two heaviest pigs) at Pre-test1 than at Pre-test2 (i.e. after removal of the two heaviest pigs) (14.8 vs 39.0, respectively, SEM=3.37; F<sub>(1,6)</sub>=25.96; P<0.01). There were no gender or time effects on aggressive behaviours performed during the FCT (P>0.05). The rank order within the 6 remaining pigs before the removal which was a function of DS measured during the FCT, was not correlated with the rank order after the removal (Gamma=0.19, P>0.05). In conclusion, removal of the two heaviest pigs from a pen led to a two fold increase in aggression and re-organisation of the DH. However, it is not known if this represented a permanent re-establishment or temporary confusion of the DH.
Behavior and growth of pigs with divergent social breeding values

Joon-Ki Hong¹, Ki-Hyun Kim¹, Na-Rae Song¹, Hyun-Su Hwang², Jae-Kang Lee², Tae-Kyung Eom² and Shin-Jae Rhim²

¹National Institute of Animal Science, Rural Development Administration, Swine Science Division, 114 Shinbang-1-gil, Cheonan, 31000, Korea, South, ²Chung-Ang University, School of Bioresource and Bioscience, 4726 Seodongdaero, Ansung, 17546, Korea, South; sjrhim@cau.ac.kr

This study was conducted to characterize the behavior and growth of pigs with divergent social
breeding values (SBV). Positive (+, n=5) and negative (-, n=5) SBV groups of finishing pigs
(n=70, males: 35, females: 35, age: 64.4 days after birth, weight: 30 kg) were housed in 10 test
pens (3.0×3.3 m, 7 pigs/pen). Males and females were evenly distributed between groups. Pigs
were observed with the aid of video technology for 9 consecutive hours on days 1, 15, and 30
after mixing. Observation duration and methods are the same for all pens. Moreover, pigs were
weighed at approximate 90 kg body weight that was then calculated at the number of days to
reach 90 kg to compare the growth between groups. On day 1 after mixing, agonistic behavior
was significantly higher in the -SBV than in the +SBV group (Mann-Whitney U test, Z=2.21,
P=0.027). Feeding and mate feeding (feeding behavior together with pen-mates) behaviors
were significantly higher in the +SBV group on days 1 and 30 after mixing (Z=-5.11~-4.22,
P<0.001). Moreover, the day that pigs reached 90 kg was significantly different between the
+SBV (128 days) and -SBV (135 days) groups (t-test, t=3.20, P=0.002). The divergent SBV of
pigs were affected by responses to social behavior. Such social interactions among pen-mates
might affect their growth rate and feed intake. Selection for SBV might be indirect technique
for improving the growth performance of pigs. The experimental protocols were reviewed and
approved by IACUC, National Institute of Animal Science, Republic of Korea (NIAS 2014-289).
**Influence of breed of sire on the occurrence of tail-biting in pigs**

Friederike Katharina Warns¹, Felix Austermann², Tobias Scholz³ and Ernst Tholen⁴

¹University of Goettingen, Department of Animal Science, Albrecht-Thaer-Weg 3, 37075 Goettingen, Germany, ²Agricultural Chamber of North Rhine-Westphalia, Department of Animal Production, Haus Duesse, 59505 Bad Sassendorf, Germany, ³Agricultural Chamber of North Rhine-Westphalia, Agricultural Test Center VBZL Haus Duesse, Haus Duesse, 59505 Bad Sassendorf, Germany, ⁴University of Bonn, Institute of Animal Science, Endenicher Allee 15, 53115 Bonn, Germany; friederike.warns@agr.uni-goettingen.de

Tail-biting reduces animal welfare in pigs. In this study we focused on genetic effects as a risk factor for tail-biting. 180 non-docked crossbred piglets (Duroc × Topigs20, n=73 [DU]; Piétrain × Topigs20, n=107 [PI]) were weaned at the age of four weeks and divided into two replicates (DG1=28 DU+63 PI; DG2=45 DU+44 PI). According to weight and sire's breed piglets were grouped and raised in four grower pens (16 to 25 pigs per pen with an average of 0,37 m²/pig) in a conventional flatdeck. Water and feed along with chopped alfalfa hay was offered for ad libitum consumption. Each piglet's tail was evaluated once a week to determine possible injuries, partial loss, fresh blood and swelling. At the age of ten weeks piglets were transferred to finishing pens. Per replicate, piglets of same sire breed were kept together in one pen. During the finishing period pig's tails were scored 4 (DG1) to 5 times (DG2). Statistical analysis was done with R software (Version 0.99.486) by a GLMM which comprises individual birthweight (BW, covariable), sex (S) and piglets' pen (Bu) as fixed effects, and individual sire × dam of piglet as random factor. Pigs of different breeds were not mixed within the pens, so breed effects were calculated by means of linear contrasts. Six days after weaning tail-biting was observed in one group of DG1 with severe bloody injuries, in other groups only small lesions like scratches were determined. During second half of the flatdeck period and first week in grower pens tail-biting occurred in every group irrespective of R or DG. If tail biting occurred an iodine solution was sprayed to all pigs of the affected pen. There was a significant difference in blood (P=0.024) between breeds and the two replication groups differ significantly in injuries (P=0.017) and swelling (P<0.001). Furthermore there was a highly significant difference for injuries, partial loss and swelling (P<0.001, respectively) between grower pens. The factors injuries, partial loss and swelling showed a comparatively high heritability between 0.217 and 0.279, which was derived from the variance of the random factor sire × dam. Because the results were strongly affected by replication and pen group it is not possible to identify a clear genetic influence on tested evaluation factors. Nevertheless our study indicates a moderate heritability for tail-biting, which motivates to verify the results in further studies.
**The effect of illness on social behaviour, cytokine levels and feed intake in undocked boars**

Janicke Nordgreen¹, Camilla Munsterhjelm², Frida Aae¹, Mari Heinonen², Kristin Olstad³, Torunn Aasmundstad⁴, Andrew M. Janczak¹ and Anna Valros²

¹Faculty of Veterinary Medicine/Norwegian University of Life Science, Production Animal Clinical Science, Ullevålsveien 72, 0033 Oslo, Norway, ²University of Helsinki, Production Animal Medicine, P.O. Box 3, 00014 Helsinki, Finland, ³Faculty of Veterinary Medicine/Norwegian University of Life Science, Companion Animal Clinical Science, Ullevålsveien 72, 0033 Oslo, Norway, ⁴Topigs Norsvin, Storhamargata 44, 2317 Hamar, Norway; andrew.janczak@nmbu.no

Tail biting is detrimental to animal welfare and has negative economic consequences. Even though several risk factors are known, they do not reliably predict tail biting outbreaks. Bad health is one of the risk factors for tail biting and the first aim of this study was to test for links between health status and behavior related to tail biting at the individual level. The second aim of this study was to test whether variation in cytokines was related to variation in social behavior. Cytokines, proteins produced by the immune system as a response to infection, but also in response to social stress and contaminated feed, may be mediators between the risk factors of bad health and behaviours increasing the likelihood of tail biting. Cytokines can influence behaviour either in the direction of withdrawal or increased aggression, both of which can increase non-functional social behaviour and thereby the risk of a tail biting outbreak. To investigate this, we collected videos, health data, feeding data and blood samples from undocked boars at a test station farm in Norway. We compared groups with three different diagnoses: osteochondrosis diagnosed by computer tomography scanning (OCSAN; n=30), osteochondrosis diagnosed by clinical examination (OCCLIN; n=14) and respiratory tract disease (RESP; n=13), with healthy controls (CTR; n=37). We tested whether the diagnoses were associated with social behavior and cytokine levels and whether there were correlations between cytokine levels and social behavior. Social behavior differed between categories with OCSCAN pigs receiving more social behavior –both aggressive (ear- and tail biting) and non-aggressive (sniffing)– compared to CTR (P<0.05 for both), and RESP pigs tending to perform more ear- and tail-biting than controls (P<0.1). Ear biting was defined as ‘taking the ear of another pig in the mouth followed by an immediate reaction by the receiver’. Tail biting was defined as ‘taking the tail of another pig in the mouth followed by an immediate reaction by the receiver’. There were no differences in absolute levels of cytokines between categories, but in particular IL1-ra and IL-12 showed correlations (calculated as Spearman Rank correlations) with several behaviours that have been shown by others to be associated with current or future tail biting activity; IL1-ra levels and tail biting: 0.34, IL1-ra and ear biting: 0.31, IL1-ra and received tail sniffing: 0.30. IL-12 and received tail sniffing: 0.37, IL-12 and received attacks: 0.33. To our knowledge, this is the first published study showing a difference in social behaviour for pigs with different diagnoses and the first to report correlations between cytokines and social behaviour. The possible link between health and tail biting warrants further investigation.
**Bash item or clash item – examining pigs’ enrichment material priorities and their effects on social behaviour**

Linda Wiesner, Hanna Ifland, Rahel Ambiel, Christiane Schalk, Birgit Pfaffinger and Volker Stefanski

University of Hohenheim, Behavioral Physiology of Livestock, Garbenstr. 17, 70599 Stuttgart, Germany; wiesner.linda@gmail.com

Public opinion on the question of how farm animals should be treated has changed remarkably over the last decades. Enhanced housing to improve animal welfare of livestock is one important demand by many consumers in the EU. The question is, however, how welfare can be determined. In the past, indicators for poor welfare, such as injuries or bad physical condition, were used to assess the welfare status of animals. The evaluation of welfare and positive affective state of animals is still a challenge. One approach to assess ‘what animals want’ is the use of preference tests. Preference testing is a suitable method to draw conclusions regarding the importance of key housing elements. The aim of the present study was to examine pigs’ preferences for different enrichments and the enrichments’ effects on social interaction among the pen mates. The data are the first of a series of experiments in a larger animal welfare project. 12 castrated male pigs (German Landrace × Piétrain) were housed in six pairs of two. They were about 3 months of age and weighed about 40 kg each at the beginning of the experiment. In a successive preference test, animals were offered access to 4 pre-selected enrichments (soil, straw, metal chains and sisal ropes) consecutively. Each enrichment was presented for 4 days, provided that the order of enrichments was determined randomly. The pigs’ behaviour was observed by video recordings on one control day prior to enrichment (empty pen), as well as on the first and third day of enrichment access. Material manipulation and social interactions were analysed by 15 intervals of 6 minutes (1.5 h observation time per day) by continuous recording distributed throughout daylight period (9:00-18:00 h) with *The Observer XT*®. Preliminary analysis indicates that on the first day of enrichment pigs frequented the enrichment materials soil and straw on average 48 times and 42 times, while the enrichment objects sisal rope and metal chain were used on average 18 and 15 times per 1.5 h daily observation time, respectively. So the pigs frequented enrichment materials soil and straw significantly more often than enrichment objects metal chain and sisal rope (P<0.003), whereas there was no significant difference among enrichment materials as well as among enrichment objects (P>0.05). Analysis of social data before and after enrichment is in progress and results will be presented at the meeting. So far, we can conclude that our preference test is a useful tool to examine pigs’ priorities for enrichments. Following experiments will also include the measurement of physiological parameters (e.g. urinary cortisol) to validate the importance of certain enrichments, especially if access is subsequently denied. We are confident that this approach will allow us to decide which enrichments are of particular importance for pigs and therefore could contribute to the improvement of housing conditions.
# Effects of long-term selection for productive traits on sexual behaviour of Taiwan Country chicken

Hsi-Wen Hung

Hualien Animal Propagation Station, Livestock Research Institute, No.38, Sec. 6, Ji'an Rd., Ji'an Township, Hualien County, 973, Taiwan; gingsu@gmail.com

Taiwan Country chickens are vigorous meat-type chickens which is one of the most important chicken breeds in Taiwan. Our hypothesis is that long-term selection of meat and egg production will not only influence the breed on the genetic level but also on the behavioral level. Chickens selected based on their resource allocation of meat and egg production will have different behaviors, and chickens of non-selected strains will retain their own resource allocation percentage and the corresponding behaviors. This study compared the sexual behavior of two long-term selected strains, B for meat production and L2 for egg production, and two randomly-bred local breeds, Quemoy and Shek-ki. Paired mating tests were conducted at 25-26 and 35-36 weeks of age. Each male was paired with a different female of the same strain for four different days. During the test, male and female were separated by a curtain for 20 min to adapt to the environment. After removing the curtain, chickens' behaviors were recorded for 5 minutes. The result shows B strain females performed less crouch in both age period than any other strains (P<0.05). The frequency of crouch response in females was over 47% at 35-36 weeks of age, except B strain. Quemoy males performed more waltz, attempt-to-mount, mount and successful mating in both age period than any other strains. The selected strains had less successful mating than did the non-selected strains. Each strain had higher successful mating at 35-36 than at 25-26 weeks of age. In all strains, over 50% males displayed force mating and no significant difference among four strains. Except for B strain, the frequency of force mating decreased with age. In conclusion, long-term selection for productive traits might influence fowls' mating behavior, and fowls will improve mating behavior by age and experience.
**Key-note:**

**What can facial expression reveal about animal welfare? Supporting evidence and potential pitfalls.**

Kris Descovich¹,²,³, Jennifer Wathan⁴, Matthew Leach⁵, Hannah Buchanan-Smith³, Paul Flecknell⁶, David Farningham⁷ and Sarah-Jane Vick³

¹University of Queensland, Centre for Animal Welfare and Ethics, 'White House' Building 8143, Gatton 4343, Queensland, Australia, ²Unitec Institute of Technology, Environmental and Animal Sciences, Private Bag 92025 Victoria Street West, Auckland 1142, New Zealand, ³University of Stirling, Psychology, Faculty of Natural Sciences, Stirling FK9 4LA, United Kingdom, ⁴University of Sussex, School of Psychology, Room 2A13 Pevensey Building, Falmer BN1 9QH, United Kingdom, ⁵Newcastle University, School of Agriculture, Food and Rural Development, Agriculture Building, Newcastle upon Tyne NE1 7RU, United Kingdom, ⁶Newcastle University, Comparative Biology Centre, Newcastle upon Tyne, NE1 7RU, United Kingdom, ⁷Medical Research Council, Centre for Macaques, Porton Down, Salisbury SP4 0JQ, United Kingdom; kdescovich@unitec.ac.nz

Animal welfare is a key issue for all industries that use or impact upon animals. The assessment and improvement of animal welfare is dependent on measures that are both reliable and valid and these may include behavioural, physiological, and clinical indicators. Animals may respond to internal and external environments both in generalised (e.g. species-specific) and individual ways. Although no single measure can provide a comprehensive picture of animal well-being, attention in animal welfare science is increasingly turning towards the determination of affective states. Affective states are drivers of good or poor welfare, but are challenging to measure in non-human species, because verbal self-report is not possible. Facial expressions are extensively used as measures of emotion in humans, however they are infrequently used in animal studies, despite evidence that facial changes indicate pain states in many mammals. This empirical review of the science aims to assess whether the measurement of facial expressions is valuable within an animal welfare context. Evidence for facial representations of underlying affective states in animals will be discussed, as well as the relevance of facial communicative signalling to welfare. Facial expressions in mammals are widespread and many movements are conserved across species. Evidence suggests that facial expressions have the potential to indicate psychological and emotional experiences in animals, and can provide information on temporal or stimuli specific reactions. Facial expressions also have social, reproductive and health functions and can therefore be more broadly relevant to welfare assessment than exclusively as indicators of affective state. It is proposed that irrespective of the proximate causes of facial expressions, which are primarily communication and affective state, facial muscle movements or changes in the frequency of such, may be reliably linked to underlying welfare states. Validated tools for measuring facial movement are outlined, and the potential of expressions as honest signals are discussed, alongside other challenges and limitations to facial expression measurement within the context of animal welfare. It is concluded that the measurement of facial behaviour in animals is a useful but infrequently used measure that complements existing tools in the assessment of welfare.
# Do dogs smile when happy – an objective and comparative study of dog and human facial actions in response to emotional trigger

Cátia Correia-Caeiro, Kun Guo and Daniel Mills

University of Lincoln, Brayford Pool, Ln58LP, Lincoln, United Kingdom; ccorreiacaeiro@lincoln.ac.uk

One way of investigating emotion cognition processes in animals is by applying comparative psychology methods and look at phylogenetically distant species facing similar present adaptation problems (e.g. domestic dogs-humans). The dog is unique in having shared the same social environment with humans for more than 10,000 years, facing similar challenges and acquiring a set of socio-cognitive skills in the process. Furthermore, the orofacial musculature seems to be relatively well conserved across mammal species, including the domestic dog. Given this commonality in facial musculature, and its known link to emotion expression in humans, it might be expected that similar emotional reactions share communicative correlates in other animals as well. Therefore, this hypothesis was tested. The spontaneous and naturalistic facial behaviour of 50 humans and 100 dogs was analysed and compared, by coding available database videos (e.g. AM-FED, Youtube). These videos were selected based on a list of criteria ranging from quality of image to emotional trigger presence. No concerns regarding animal welfare were raised, since already available videos recorded in day-to-day situations were used. Furthermore, the ethical committee in the School of Psychology, University of Lincoln approved this study. The objective and anatomically-based coding tools, Facial Action Coding System (FACS) and DogFACS, were applied in a range of contexts associated with four classes of emotional response: a) happiness, b) positive anticipation, c) fear, and d) frustration (e.g. thunderstorm-phobic dog, human winning a high stake game). The full range of facial movements in both species was coded, including Action Units (AU), Action Descriptors (ADs) and Ear Action Descriptors (EADs); these are the basic units of independent muscle movement that form facial expressions. Significant differences were found between each emotional response: fearful dogs displayed more AD126 (panting); dogs anticipating a positive event, showed more AD37 (lip wipe), AD137 (nose lick) and EAD102 (ears adductor); and happy dogs, produced more AU27 (mouth stretch); however, frustrated dogs did not display higher rates for any of the facial actions. There were also significant differences between species: when fearful, dogs displayed more AD19 (tongue show) than humans; when frustrated, dogs produced more AU45 (blink); when anticipating a positive event, dogs displayed more AD19 and AD37; and when happy, dogs showed more AD19. This study reports two new important scientific findings: Dogs, like humans, produced differentiated facial movements in response to a particular emotional context; However, the individual facial actions were different between humans and dogs, and, consequently, different facial expressions of emotion were displayed in response to comparable emotional triggers. As such, it challenges some of the arguments relating to the shared mammalian origin of emotion expression, as similar muscle groups are being used to produce different facial expressions of emotion in dogs and humans. These results have important theoretical and practical implications not only for the origin of emotion expression, but also for how humans interact with dogs.
**Facial expression of harbour seal pups (*Phoca vitulina*) in response to painful stimuli**

Amelia Mari Macrae, David Fraser and Joanna Makowska

University of British Columbia, Animal Welfare Program, 2357 Main Mall, Vancouver, V6T 1Z4,
Canada; amarimacrae@gmail.com

Facial expressions have proven to be a repeatable, accurate and valid way to identify pain in
multiple species. Currently, there are no clear species-specific indicators of pain in harbour
seals (*Phoca vitulina*) and facial expression has not been examined in any species of pinniped.
The aim of this study is to investigate if changes in harbour seals' facial expressions change
immediately after a painful procedure. Nineteen pups (healthy, >60 d old) admitted to a
Canadian rehabilitation facility had their facial expressions video-recorded during routine
procedures of tagging (tag placed inter-digitally through axillary webbing of hind flipper)
and micro-chipping (subcutaneous). Analgesia is not standard practice for these procedures.
Pups were filmed for 6 min: 2 min before, 2 min during tagging and chipping and 2 min
after. For each seal, a 90-sec segment of both before and after the procedure was extracted
(within 5 s of noxious stimuli) from the videos. Two observers, blind to treatment, watched
the 38 video clips in randomized order. Orbital tightening, bunching of the nose, whisker
position, mouth position, as well as behaviours such as vocalizing, looking around (inquisitive),
struggling, trembling, open-mouth breathing and biting were scored as present or absent for
each 1-sec interval of each 90-sec clip. Observers also assigned an overall pain score (pain
present or absent, scored subjectively) for the entire clip. Observers were considered to be
'experienced' (>8 yr working with pinnipeds) and 'inexperienced' (no direct experience with
pinnipeds). Paired sample t-tests showed that orbital tightening increased from before to after
the procedure (t=10.80, df=18, P<0.0001), whereas the behaviours of looking around (t=-5.92,
df=18, P<0.0001), struggling (t=-3.43, df=18, P<0.003) and trembling (t=-2.91, df=18, P<0.01)
all decreased from before to after the procedure. Inter-observer reliability ranged from r=0.89
to 0.92 for the four types of behaviour. For both observers, the subjective scoring of whether
pain was present or absent was very accurate (95% for experienced and 89% for inexperienced
respectively). Each animal acted as its own control; however, a separate trial with 5 pups had
shown no change in the behavioural responses to sham tag/chip procedures indicating the
response seen in this trial was due to pain rather than handling. As there are currently no
published pain scales based on facial expression of this species, these results show promise for
facial expression and other behaviour to be used as a tool for acute pain assessment in seals.
The startle response as a measure of fear in dairy calves

Sarah J.J. Adcock and Cassandra B. Tucker

Center for Animal Welfare, Animal Science, University of California, Davis, CA, 95616, USA;

sadcock@ucdavis.edu

There are, at present, no well-validated tests for fear in cattle. A particularly powerful tool that has been used to study fear in humans and rodents is the startle response – a ubiquitous, cross-species defensive reflex to sudden stimulation. Our objective was to assess the validity of the startle response as a measure of fear, defined here as the behavioral and physiological response to a perceived threat, in dairy calves. We predicted that calves exposed to predator odor, a fear-eliciting stimulus, would have a higher startle magnitude, elevated heart rate, and longer latency to feed in response to a sudden noise than calves exposed to the odor of a herbivore or water. Thirty-four calves were assigned to 1 of 3 treatments (coyote urine, deer urine, water) in a between-subjects design. Each calf was fitted with an accelerometer and heart rate monitor and tested individually in 10 min trials in a familiar arena on 3 consecutive days. A bucket feeder containing 500 ml of milk replacer was secured to the side opposite the arena entrance. Immediately before the trial, the odor was dispersed into the arena with fans. The startle stimulus, a 0.4 s, 105±2 dB burst of white-noise, was delivered upon the calf's arrival at the feeder. We assessed treatment (coyote, deer, water) and test day (1, 2, 3) effects on startle magnitude and heart rate with linear mixed models using the restricted maximum-likelihood method and calf identity fitted as a random effect. The effect of treatment and test day on latency was modeled using a parametric survival model. Correlations between variables were made using Spearman's rho. The magnitude of the behavioral startle response was higher in the coyote treatment than in both controls on the first day of testing (mean total acceleration on day 1 ± SE: Coyote: 199±15 g; Deer: 166±3 g; Water: 172±8 g; Treatment×Day: F<sub>4,50</sub>=2.56, P=0.050). Calves exposed to coyote odor tended to take longer to return to the feeder following the sudden noise compared to control animals (mean ± SE: Coyote: 11±5 s; Deer: 4±2 s; Water: 4±1 s; Treatment: X<sub>2</sub><sup>2</sup>=8.32; P=0.016; post-hoc tests: P≤0.086). The cardiac startle response did not differ between treatments (mean heart rate ± SE: Coyote: 164±6 bpm; Deer: 167±6 bpm; Water: 177±6 bpm; F<sub>2,30</sub>=1.29, P=0.290) nor days (F<sub>2,52</sub>=0.02, P=0.979). Startle magnitude and latency to return to the feeder were positively correlated (rho=0.60, P<0.0001). Unexpectedly, heart rate correlated negatively with both startle magnitude (rho=-0.26, P=0.019) and latency to return to the feeder (rho=-0.46, P<0.0001), which suggests fear brachycardia – a defense mechanism characterized by a decrease in heart rate – may accompany the startle response. In conclusion, the results provide support for using the behavioral startle response as a measure of fear in dairy calves.
Pre-weaning environment affects pigs' emotional reactivity

Oceane Schmitt¹,², Laura Boyle², Emma M. Baxter¹ and Keelin O'Driscoll²

¹SRUC, Animal Behaviour and Welfare, Animal and Veterinary Science Research Group, West Mains Road, Edinburgh EH9 3JG, United Kingdom, ²Teagasc, Pig Development Department, Moorepark, Fermoy, Co. Cork, Ireland; oceane.schmitt@teagasc.ie

Artificial rearing systems such as Rescue Decks (RD) are of growing interest to facilitate the rearing of super-numerous piglets born in large litters, where survival is compromised. However, as piglets are separated early from their dam (i.e. 7 days-old), and have less space allowance in the RD than in a farrowing pen, rearing in a RD might affect pigs' emotional reactivity. This study compared the post-weaning reactions of sow-reared (SOW) piglets and artificially reared (RD) piglets (i.e. milk replacer in RD from 7 days-old until weaning) to a variety of novel situations, using four behaviour tests. Tests were carried out on groups in the home pen at approximately 1 week post-weaning (W1; 33±3 days-old, n=26 pens), at transfer to the second stage weaner accommodation (W2; 69±1 days-old, n=18 pens) and at transfer to the finishers accommodation (F; 100±1 days-old, n=14 pens). The tests were: (1) Startle Test (ST): the group's reaction to the opening of a red umbrella in front of the pen (1: >60% froze/ startled; 0: no reaction) and the latency to recover from startling (i.e. return to normal activity for 50% of startled group) were recorded; (2) Novel Object Test (NOT): the latency of the first pig to touch a novel object (yellow Frisbee, pink paddle or blue bucket) was recorded; (3) Human-Animal Relationship Test (HART): the reaction of pigs to a familiar human's contact was scored (1: avoidance of stationary human or human contact, 0: acceptance of contact); and (4) Open Door Test (ODT): the latency of the first pig to exit the pen, and the max proportion of pigs outside the pen during 5 min after the pen door was opened were recorded. Latencies were analysed using survival curve analysis. Prevalence of startling reaction and percentage of fearful pigs during HART were analysed using Generalised Linear Mixed Models with the total number of pigs in the pen being a random factor. There was no interaction between time and pre-weaning environment, thus only pre-weaning environment effects are reported. There was no effect of pre-weaning environment on the ST reaction, but SOW pigs took longer to recover compared to RD pigs (16.64±2.16 vs 11.65±1.20 sec; P<0.05). Pre-weaning environment did not affect latency to touch the novel object. However, more SOW pigs showed fear of human contact than RD pigs (0.58±0.05 vs 0.32±0.05; P<0.001). Latency to exit the pen was not affected by pre-weaning environment (P>0.05), but the maximum proportion of pigs observed outside the pen was higher in SOW than in RD (0.83±0.03 vs 0.72±0.03; P<0.05). Overall, RD pigs seemed calmer than SOW pigs in the presence of a human, as indicated by a faster return to normal activity after startling, and more willingness to accept human contact. This could be a result of increased exposure to human contact prior to weaning. However, fewer of these pigs spontaneously left the home pen, indicating greater reluctance to explore a novel environment.
Lying around with nothing to do: boredom in farmed mink

Rebecca K. Meagher<fnref n="1" />,<fnref n="2" />, Dana L.M. Campbell<fnref n="1" /> and Georgia J. Mason<fnref n="1" />

Boredom, a negative emotional state resulting from too little or monotonous stimulation, has received little empirical study in non-human animals, but is commonly believed to be prevalent in captive environments. If true, this would mean that millions of animals worldwide are experiencing an aversive state that might lead to other negative consequences such as depression and poor health. Boredom should, by definition, result in motivation to obtain novel stimulation. In a previous experiment, we demonstrated that, compared to mink in elaborately enriched cages previously shown to improve welfare, mink in non-enriched farm cages showed exaggerated interest in a wide range of stimuli, suggesting that they do indeed experience boredom-like states. According to some measures, this also correlated with time spent lying awake when undisturbed. We conducted a second study to attempt to replicate these findings. We used 20 adult male mink, half of which were housed in standard non-enriched cages, while half had been housed in enriched cages since 3 months of age. Spontaneous behaviour (locomotor stereotypies and time spent lying awake but inactive) was assessed by 340 scan samples over 20 days in the winter. Tests of interest in stimulation were conducted in the spring. Mink were exposed to 11 stimuli, categorized as aversive, rewarding or ambiguous based on biological relevance and prior observation, for five minutes each. Interest was measured via the latency to touch, time in contact with, and time oriented to the stimuli, as well as consumption of food treats in three separate tests. MANOVAs showed that time oriented to and in contact with stimuli were higher in non-enriched mink, although the magnitude of these effects differed across stimuli (interaction terms: $F_{9,51}=2.62, P=0.016$ and $F_{7,33}=2.38, P=0.047$, respectively). When analysed separately by stimulus type, for aversive stimuli only, these measures were higher in non-enriched than enriched mink (oriented to: $F_{1,8}=4.12, P=0.038$; in contact: $F_{1,8}=6.97, P=0.015$). Latency to contact and food treat consumption did not differ between treatments in this study. Time lying awake again correlated with average orientation time (general linear model: $F_{1,8}=9.49, P=0.015$) within the enriched treatment. Overall, this study broadly confirms our previous findings that mink in non-enriched cages express a core symptom of boredom.

<footnote n="1">¹University of Guelph, Animal Biosciences, 50 Stone Road East, Guelph, N1G 2W1, Canada,</footnote>

<footnote n="2">²University of Reading, School of Agriculture, Policy and Development, Whiteknights, P.O. Box 237, Reading RG6 6AR, United Kingdom; rkmeagher@gmail.com</footnote>
Can minor, easily applied alterations of routines during the rearing period reduce fearfulness in adult laying hens?

Margrethe Brantsæter¹, Fernanda Machado Tahamtani², Janicke Nordgreen¹, Ellen Sandberg³, Tone Beate Hansen⁴, Bas Rodenburg⁵, Randi Oppermann Moe¹ and Andrew Michael Janczak¹

¹Norwegian University of Life Sciences, Department of Production Animal Clinical Science, Ullevaalsveien 72, 0033 Oslo, Norway, ²Aarhus University, Department of Animal Science, Blichers Allé 20, DK-8830, Tjele, Denmark, ³Norwegian University of Life Sciences, Department of Chemistry, Biotechnology and Food Science, Arboretveien 40, 1432 Ås, Norway, ⁴Animalia, Norwegian Meat and Poultry Research Centre, Lørenveien 38, 0513, Oslo, Norway, ⁵Wageningen University & Research, Behavioural Ecology Group, Adaptation Physiology Group, Droevendaalsesteeg 4, 6708, Wageningen, the Netherlands; margrethe.brantsaeter@nmbu.no

Exaggerated fear-reactions are associated with injuries, smothering, feather pecking and other
events that compromise laying hen welfare. Provision of litter during the rearing period may
reduce fearfulness. The aim of this study was to test the hypothesis that chicks with access to
litter during the first five weeks of life would be less fearful as adults compared to birds reared
without access to litter. The hypothesis was tested in 23 commercial avian layer flocks in
Norway. Five rearing farmers divided the pullets into two groups within their rearing houses.
During the first five weeks of life, paper substrate, on which food and other particles could
accumulate, covered the wire mesh floor in the treatment group, whereas the control group
was reared on bare wire mesh. The egg producers were instructed to follow their normal
management procedures. At 30 weeks of age, 23 layer flocks (11 control flocks and 12 paper
reared flocks) were visited. A stationary person test and a novel object test were conducted to
test fearfulness of the adult hens. In addition, data on provision of environmental enrichment
was collected as a binary yes/no variable. Provision of environmental enrichment to adult birds
tended to reduce the latency to approach within 2 m of the stationary person (P=0.08). For
birds without environmental enrichment as adults, access to litter during rearing increased the
number of birds that approached the novel object compared with birds reared without paper
(P=0.04). For birds with access to environmental enrichment during production, the access
to litter during rearing had no effect on the number of birds that approached the novel object
as adult (P=0.99). These results indicate that both providing chicks with paper substrate from
the first day of life and providing them with environmental enrichment as adults, are practical
and simple alterations of management that reduce fearfulness in laying hens.
Assessing anxious states in sheep: a more practical attention bias test

Jessica Monk¹,², Rebecca E. Doyle³, Sue Belson¹, Greg Cronin⁴ and Caroline Lee¹

¹CSIRO, Agriculture and Food, FD McMaster Laboratory Chiswick, Armidale NSW 2350, Australia, ²University of New England, Natural Resources Building, Armidale NSW 2350, Australia, ³Animal Welfare Science Centre, University of Melbourne, Alice Hoy Building, Parkville VIC 3010, Australia, ⁴University of Sydney, JL Shute Building, Camden NSW 2570, Australia; jessica.monk@csiro.au

Animals experiencing increased states of anxiety show greater attention towards threatening stimuli than calmer individuals, a response termed attention bias. Tests for attention bias potentially offer more rapid assessment of affective state in animals than existing cognitive methods. We have previously developed and pharmacologically validated an attention bias test as a measure of anxious states for sheep. Experiment 1 in the current study aimed to confirm that the responses being measured in the test were directed towards the dog and not just other aspects of the test such window cover movement. Experiment 2 aimed to refine the test, shortening the duration from 3 min per sheep and removing the need to train sheep to feed from a bucket. In each experiment, the attention bias test consisted of an enclosed arena (4×4.2 m) with Lucerne hay in the centre and a small window on one wall. When a sheep entered the test, a dog sitting quietly was visible through the window for 3 s, then the window was covered. The sheep remained in the test for 3 min while behaviours were recorded. Duration of vigilance (head above shoulder height) was analysed using Kruskal-Wallis one-way ANOVA and feeding behaviour was analysed using survival analysis. For experiment 1, sheep either underwent the described procedure or were exposed to an empty window at the beginning of the test instead of a dog (n=20 per group). Sheep exposed to the dog spent more time displaying vigilant behaviour (mean ± s.e. 165±2.8 s vs 147±5.3 s; P=0.004), were less likely to feed (log rank test P<0.001) and were less likely to sniff the window (Fisher's exact test P=0.008) than sheep exposed to an empty window. These findings indicate the behaviours recorded in the test were at least partially a response to the dog. In experiment 2, 60 sheep were divided into 3 groups (n=20) and given either an anxiolytic drug (diazepam, 0.1 mg/kg i.v.), an anxiogenic drug (1-(m-chlorophenyl)piperazine (mCPP), 2 mg/kg i.m.) or a control treatment (saline i.m.) before testing. Behaviours during the first 45 s of testing were also assessed to determine whether the test duration could be shortened. During the first 45 s, mCPP treated sheep spent more time displaying vigilance (35±0.6 s) than control (29±1.6 s) and diazepam (24±2.2 s) groups (P<0.001) and were less likely to feed (log rank test P<0.001). This indicates differences in anxious state may be detected using a shorter test. Only 4 animals in the control and diazepam groups failed to eat the novel feed within 180 s, showing it is not necessary to train sheep prior to testing. Overall the current study refined the attention bias test by removing the need for prior training and shows the test duration can be shortened. It also confirms that the measured responses were towards the dog and provides further pharmacological validation that the attention bias test may be useful for assessing anxious states in sheep.
Development of behavioural tests for WelFur on-farm welfare assessment of foxes

Jaakko Mononen<fnref n="1" />, Eeva Ojala<fnref n="2" />, Hannu T. Korhonen<fnref n="1" /> and Tarja Koistinen<fnref n="1" />

The WelFur on-farm welfare assessment protocol for foxes includes two behavioural tests. The Feeding Test (FT) is a well validated test that measures human-animal relationship (HAR). It is the only measure in the ‘Good HAR’ criterion of the WelFur fox protocol. In the test the assessor delivers feed to a hungry fox and records whether the animal eats or not within 30 seconds while the assessor stands near the animal’s cage. The Stick Test (ST), in turn, is supposed to measure explorative behaviour, and forms a part of the ‘Positive Emotional State’ criterion in the protocol. In the test the assessor inserts 20-30 cm of a 150 cm stick into the cage of a fox, and records whether or not the fox touches the stick in an explorative way within 10 seconds. ST is performed as the very first part of collecting data at the cage level, and the assessor tries to stay as far as possible from the cage in order to minimize human effect. ST has not been, however, properly validated, and since exploration may be affected by the presence of human we studied whether the results of FT and ST overlap. In addition we studied whether a Subjective Evaluation of HAR (SE) during the 1.5-3.0 min period when other animal and resource-based data are collected from a cage could substitute for the more laborious FT in the WelFur protocol. In SE the tendency of a fox to approach (low score) or withdraw from (high score) the assessor was scored on a scale from one to five. All adult blue fox (n=354) and silver fox (n=94) vixens on a private Finnish farm were tested with FT, ST and SE during two days in October. The results from FT and ST were compared with Logistic Regression to study the overlap hypothesis. Species was in the model as a blocking factor. SE scores (ordinal scale) between the animals eating and not eating in FT were compared with Mann-Whitney U-test separately for the two species to study the substitution hypothesis. Logistic Regression revealed that FT and ST were not independent of each other (P<0.01), but species had no effect (P=0.65). The 93% precision (251/271 foxes; species pooled) of ST in predicting FT indicates that ST measures to a great extent the same character as FT, most probably HAR. On the other hand, sensitivity of ST in predicting FT was only 69% (251/362 foxes; species pooled), i.e. ST failed to recognize almost one-third of the foxes eating in FT. The SE score was lower (P<0.01, Mann-Whitney) in foxes eating than not eating in FT in both species: blue foxes, 2.5-3.0-3.5 (quartiles Q1-Q2-Q3, n=282) vs 3-3-3 (n=72); silver foxes, 1.0-2.0-2.5 (n=81) vs 2.5-3.0-3.5 (n=13). However, the distributions of the scores overlapped markedly between the two groups of foxes in both species. We conclude that ST is rather a HAR test than a test of explorative behaviour, and ST could probably substitute for FT in the WelFur fox protocol. Instead, SE would require further development aiming at increasing its discriminative power before it could replace FT.

<footnote n="1">¹Natural Resources Institute Finland, Green Technology, Halolantie 31A, 71750 Maaninka, Finland,</footnote>

<footnote n="2">²Kannus Research Farm Luova Ltd., Turkistie 6, 69100 Kannus, Finland; jaakko.mononen@luke.fi</footnote>
Dietary tryptophan supplementation and affective state in pigs

Jenny Stracke¹,², Winfried Otten², Armin Tuchscherer³, Maren Witthahn²,⁴, Cornelia C. Metges⁵,
Birger Puppe²,⁴ and Sandra Düpjan²

¹University of Veterinary Medicine Hannover, Institute for Animal Hygiene, Animal Welfare and Farm Animal Behaviour, Bischofsholer Damm 15, 30173 Hannover, Germany, ²Leibniz Institute for Farm Animal Biology (FBN), Institute of Behavioural Physiology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ³Leibniz Institute for Farm Animal Biology (FBN), Institute of Genetics and Biometry, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ⁴Faculty of Agricultural and Environmental Sciences, University of Rostock, Behavioural Sciences, Justus-von-Liebig-Weg 6, 18059 Rostock, Germany, ⁵Leibniz Institute for Farm Animal Biology (FBN), Institute of Nutritional Physiology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; jenny.stracke@tiho-hannover.de

The assessment and provision of welfare in farm animals has become a major issue in animal
science. Affective states in general, and positive affective states in this particular study, are
a key element for providing good welfare. As the serotonergic system plays a central role
in regulating affective behaviour, an increase in centrally available serotonin (5-HT) via
dietary supplementation of its precursor tryptophan (TRP) might be an approach to induce
positive affective states. Therefore, the aim of our study was to investigate the effects of dietary
TRP supplementation on brain TRP metabolism and 5-HT levels, but also on affective state
(cognitive bias test) and behavioural reactivity (combined open field/novel object test) in
pigs. The study was conducted on 52 female, juvenile pigs in total (20 for part 1; 32 for part
2). All experimental procedures were ethically approved (LALLF M-V/AZ:7221.3-1-066/13).
Animals were fed a standard diet until eight weeks of age, then feed was changed for all
animals, with half the animals receiving a diet with a standard TRP content (2.5 g/kg; control),
while the other half received a TRP enriched diet (10.2 g/kg; TRP+). In part 1 of our study,
we investigated the effects of the dietary TRP supplementation on TRP metabolism in brain
areas related to affective and cognitive processing (data analysed blind to treatment). We found
significantly increased concentrations of TRP (all F>82.7; all DF=1,15; all P<0.001) and its
metabolites in nearly all analysed brain tissues. In part 2 of our study, we analysed the effects
of these alterations on the affective state as measured in a cognitive bias test, namely the spatial
judgement task (SJT) which we had validated for pigs in a previous study (data not shown).
Here, animals had to learn to discriminate between a positively reinforced and a negatively
reinforced location, and were subsequently confronted with ambiguous, intermediate locations.
Additionally, a combined open field/novel object test (OFNO) was conducted to measure
behavioural reactivity (activity/exploratory behaviour). The TRP enrichment revealed no
significant behavioural changes in the OFNO tests (all P>0.05). In the SJT, the TRP+-group
showed more pessimistic behaviour after dietary change than before. Therefore, our results
do not support the suggestion that TRP supplementation induces positive affective states and
thus improves animal welfare in pigs.
# Playing or exploratory behaviour in pigs: neurohormonal patterns in mini-pigs and suggestions for welfare management

Míriam Marcet Rius, Alessandro Cozzi, Cécile Bienboire-Frosini, Eva Teruel, Camille Chabaud, Philippe Monneret, Julien Leclercq, Céline Lafont-Lecuelle and Patrick Pageat
Research Institute in Semiochemistry and Applied Ethology, Quartier Salignan, Apt 84400, France; m.marcet@group-irsea.com

The European Directive 2008/120/EC laying down minimum standards for the protection of pigs establishes that pigs must have permanent access to a sufficient quantity of material, such as straw, to enable proper investigation and manipulation activities. The aim of this study was to investigate benefits that different types of enrichment, triggering different behaviours, like exploratory with straw or playing with dog toys, could provide in pigs. Peripheral oxytocin and its evolution over time was measured. Play behaviour is a commonly observed and characteristic behaviour of young mammals, and exploratory behaviour, a high priority behaviour in pigs, i.e. one of the behavioural needs. Housing and experimental procedure were carried out according to European legislation and in the respect of ethical principles. It was approved by IRSEA's Ethics Committee (125) and the French Ministry of Research (AFCE_201602_01). 18 minipigs were divided into 2 halls and housed in pens of 2 individuals. 'Enriched group' received dog toys during play sessions, every day during a total of 30-40 minutes, for 3 weeks, and after it, straw was provided continuously in the pens for 3 more weeks, whereas other group, 'Control group' did not have any material during all this period. Animals of 'Enriched group' were subjected to 10 minutes' video recording per day, in their pens, to analyse the behaviour during the interaction with each material. One blood sample had been drawn before play session and before the provision of straw (T₀), and another one 10 minutes after playing or exploring during 10 minutes (T₁). This procedure was the same for each animal during the 6 weeks, including the animals of the 'Control group' that did not explore nor play. The mostly found behaviours during the interaction with toys were: object play, described as biting, sniffing, pushing, kicking, licking, and chewing the toy, as well as tail movement behaviour, considered as a possible indicator of positive emotions. The mostly found behaviours during the interaction with straw were exploratory behaviour, especially rooting, as well as tail movement behaviour. For play behaviour, oxytocin was significantly higher at T1 than at T0 in the 'Control group' (T0: 5.00±2.65 vs T1: 9.22±11.56; P=0.00); in 'Enriched group' there was no significant difference between T0 and T1 (T0: 4.98±2.97 vs T1: 5.37±3.47; P=0.87). Regarding exploratory behaviour, oxytocin was significantly higher at the 3rd week than the 2nd week (2ndw: 3.90±2.42 vs 3rd w: 6.16±2.72; P=0.02). Results suggest both types of material produce positive emotions and allow the development of natural behaviours. As the provision of toys could be an easier measure for the actual system than the provision of straw, we recommend it as a measure to improve welfare.
**Low rate of stereotypies in pregnant sows is associated to fear related behaviours in their offspring**

Patricia Tatemoto, Thiago Bernardino, Luana Alves, Marisol Parada Sarmiento, Anna Cristina
De Oliveira Souza and Adroaldo José Zanella

Center for Comparative Studies in Sustainability, Health and Welfare, Department of Veterinary
Medicine and Animal Health, USP - VPS, Avenida Duque de Caxias Norte, 225, Pirassununga,
SP, 13635-900, Brazil; patricia.tatemoto@usp.br

The commercial sow housing systems during gestation are frequently environments considered
barren by their low complexity. The reduction of environmental stimuli has been considered a
stressor and cause of stereotypies. The effects of prenatal stress in the brain can generate changes
in the emotionality in the offspring. We studied the effects of the expression of stereotypies
performed by the mother on the welfare indicators in piglets. We evaluated sows kept in
group housing pens (n=11; low rate abnormal behavior = 4, high rate abnormal behavior =
7), recording the behaviors on gestation days 88, 89, 91, 92, 106 and 107, for two consecutive
minutes, with three replicates, totalizing six minutes per female in each day. In the first time,
we observed 35 sows maintained in barren pens or pens with straw (hay). To this part of
analysis, we considered just the females kept in the barren pen (control group) with expression
of abnormal behaviors which was consistent throughout the days. Thus, sows with low rate
abnormal behavior were those that never presented the behavior and females characterized
by a high rate of abnormal behavior were those that presented stereotypies for at least 50% of
the days of observation. To access emotionality, we subjected the piglets (one couple per sow)
individually to an open field test and to a novel object test (totalizing 10 minutes), we recorded
behaviors such as latency for activity, activity duration, exploratory behaviors, freezing and
vocalization. In the open field test, we showed that piglets from females with low rate abnormal
behavior rate presented higher latency for activity (Mann-Whitney, P=0.04) and less activity
duration (walking through the pen; P=0.01). To measure aggressiveness, we counted the
number of skin lesions in each piglet using photographs, on 28, 29, 35, 36, 42 and 43 days after
weaning. There was only difference throughout time (Anova, repeated measures; P=0.0001),
where the lesions were reduced as the hierarchy stabilized within the group. We conclude that
the rate of abnormal behavior expressed by the mother is related to more fearful offspring. These
changes in the phenotype of offspring indicate changes in the limbic system associated with fear.
# Influence of novelty and housing environment on play and tail wagging behavior in pigs

Lisa Christine McKenna and Martina Gerken

University og Goettingen, Animal Sciences, Albrecht-Thaer Weg 3, 37075 Göttingen, Germany;
lisa.mckenna@agr.uni-goettingen.de

To improve the welfare of pigs in intensive husbandry systems, positive emotions should be stimulated. Indicators of positive emotions in pigs are e.g. tail wagging and play behaviour. The present study was conducted to investigate whether female pigs kept in housing environments with different enrichment levels show differences in the occurrence of play and tail wagging behaviour when confronted with novel objects. A total of 36 female animals were tested over a period of 2 weeks when the animals were between 5 and 7 weeks of age. The experimental animals were kept either in a non-enriched (NE) stable (barren, except for a thin layer of sawdust), an enriched (E) stable (sawdust and straw bedding, 2 m²/animal) or in a super-enriched (SE) stable (sawdust and straw bedding, 2 m²/animal plus additional enrichment which was changed weekly). In every environment, there was one test group (n=6) and one control group (n=6). For behavioural testing, the animals were led into the test room in groups of three to avoid social isolation stress. There, three novel objects (the same for each animal) were laid out for the animals of the test groups to explore. For the control animals, the test room remained empty. The novel objects consisted of: a rubber duck, a rubber dog toy filled with grapes, a wallow and potting soil. Test objects were chosen based on their potential to trigger positive emotions in the animals. The animals were videotaped during the testing period of 7 minutes. Play and tail wagging behaviour were recorded continuously. Each animal was tested 4 times (twice per object). The frequency of occurrence of these behaviours was added up and averaged for the test and control groups. This calculation resulted in a Positive Emotion Score (PES) which was compared between test and control groups and the different housing environments. Residuals of this variable were not normally distributed and were therefore analysed with the Mann Whitney U test. We have found that the test groups showed significantly higher PES than the control groups (P=0.001). We also found a tendency for higher PES in the test animals of the NE compared to the E and SE environment (P=0.74, P=0.22, respectively) (PES NE: 4.27 and 0.46, E: 2.25 and 0.10, SE: 1.73 and 0.79 for test and control animals, respectively). Previous studies showed rebound effects in pigs housed in barren environments. The similar tendency found in NE animals was unexpected, as these animals were provided with sawdust. We suggest that novel objects potentially cause positive emotions in pigs as measured by play and tail wagging behaviour.
# The effect of different stimuli on the ‘stick test’ should be taken into account when measuring the mink’s temperament

Britt I.F. Henriksen, Jens Malmkvist and Steen H. Møller

Department of Animal Science, Blichers Allé 20, 8830 Tjele, Denmark; britt.henriksen@anis.au.dk

The temperament of mink can be measured in a voluntary approach-avoidance test called the ‘stick test’. The test is measuring the mink’s reaction when a wooden tongue spatula is put into the front of their cage through the wire netting. Their reaction is categorised as ‘fearful’, ‘exploratory’, ‘aggressive’ or ‘undecided’. We aimed to test the effect of different stimuli on the temperament score obtained in a standardised ‘stick test’ of mink. We tested the influence of (1) additional cage shelf in the front part of the cage (vs none), (2) distance from test person to mink, (3) familiar vs unfamiliar dressed test person, and (4) two types of wooden test spatula (small vs large). We hypothesise that (1) mink may use the cage shelf as a safe place, thus increasing their exploratory behaviour in the test, (2) fear increases when the test person is bending over the cage, (3) unfamiliarity in colour and smell of test person’s coverall results in decreased exploratory behaviour and increased distance to stimuli and that (4) the type of wooden spatula does not affect the score. We tested 600 pair-housed (male/female) brown juvenile mink in October/November 2015 and 500 brown female mink (first and second parity) in January/February 2017. In 2015 the mink were divided into two groups of 150 cages where one group was tested with or without the human tester bending over the cage, and the other group was tested with or without the animals having access to an extra shelf in the front part of the cage. In 2017 the mink were divided into five groups of 100 cages per group testing combinations of the human tester having white or dark blue disposable coverall or a dark blue coverall from the farm, tongue spatula (150×17mm) or a coffee stick (140×5mm). Each animal was tested twice with a different combination. Statistical analysis (prevalence of scores) was done in logistic binomial mixed models taking repeated measures and two mink per cage into account. We did not find any significant effect of the human position on the scores (P>0.05). However, of the non-exploratory animals, the human tester bending over the cage resulted in fewer animals in the first half of the cage close to the test stimuli (χ²=4.0, P<0.05). The prevalence of exploratory animals was significantly higher (χ²=29.1, P<0.0001) and the prevalence of fearful and undecided animals significantly lower (fearful: χ²=28.8, P<0.0001 and undecided: χ²=7.3, P<0.001) when the animals had access to an extra shelf. Thus, mink lying on the shelf during testing were more often scored explorative (χ²=41.6, P<0.0001). This is in line with other studies showing that a shelf increase exploration/decrease flight in the stick test of mink. Data from the trials in 2017 is upcoming. We conclude that the position of the test person has minor influence on exploration/fear in mink, not affecting the score, whereas a cage shelf close to the test area significantly increases mink scored explorative.
**Stress in male Japanese macaques living in vegetated and non-vegetated enclosures**

Josue S. Alejandro, Michael A. Huffman and Fred Bercovitch

Primate Research Institute, Kyoto University, Ecology and Social Behavior, 41-2, Kanrin, Inuyama
City, Aichi Prefecture, 484-8506 Japan, Japan; josue.pastrana.64s@st.kyoto-u.ac.jp

Improving captive environments for primates has been an important tool to enhance animal welfare. To investigate the benefits in which living in naturalistic environments decreases stress and promotes general animal well-being we observed two outdoor housed groups of Japanese macaques (*Macaca fuscata*) in the Primate Research Institute (PRI). The two groups are located in the same area (similar proximity to road, human contact) and were selected for the study based on similar age-sex class composition (5 mature males, 15-20 mature females, and immatures for both conditions), group size (42-45 individuals), rearing histories, and year of establishment. The vegetated enclosure (3,900 m²) contains trees, a pond, natural ground cover as well as artificial perching. The enclosure is divided in two sections and animals are moved once a year to allow for re-growth of the vegetation. The non-vegetated enclosure (960 m²) is a concrete outdoor enclosure with perching, swings, climbing structures, artificial river, and platforms with shelters to protect animals from the weather. 10 individuals (5 mature and 5 immature) from both conditions were observed and their behaviors recorded using 10 minute continuous focal sampling across seasons. All observations were conducted inside the enclosure and the observer habituated to both groups for a period of 3 months. Their activity budgets, rates of agonistic, affiliative (groom, play) and abnormal behaviors, as well as coat conditions were recorded using a 0 to 1 scale. We found that animals in the naturalistic enclosure had activity budgets more similar to their wild counterparts; they spent significantly less time moving (U=17, n₁=10, n₂=10, P=0.013), less time in agonistic interactions during the mating season (T=4.624, df=19, P=0.001), and immatures spent more time in social play than animals in the non-vegetated enclosure (U=2, n₁=10, n₂=10, P=0.012). We found no differences in social grooming or self-grooming, but we did note significantly better coat conditions in mature individuals in the vegetated enclosure (U=0, n₁=5, n₂=5, P=0.008) and a higher incidence of abnormal behaviors in the non-vegetated enclosure. Animals in highly enriched, vegetated enclosures allocated their time across activity patterns in a proportion more similar to that of their wild counterparts. Behaviors associated with optimal living standards, such as playing and foraging have an important role in reducing stress and they seem to be more prevalent in highly enriched enclosures. Perhaps because living in an enriching environment is less stressful, animals spend less time in aggression and stereotypic behaviors than animals living in conventional non-naturalistic environments. One limiting factor in our study was the difference in size of the enclosures, which might have influenced on rates of aggression. Nonetheless, monkeys living in enclosures that promote species-specific behavior seem to prevent or at least ameliorate welfare concerns. Therefore, to the extent possible, primates in non-vegetated environments should have their condition improved in order to provide a healthier living area.
**Algorithm for real-time classification of mental responses of horses during physical activity**

Vasileios Exadaktylos¹, Anne Marleen Van Aggelen², Tomas Norton² and Daniel Berckmans²

¹BioRICS, Technologielaan 3, 3001 Leuven, Belgium, ²KU Leuven, Biosystems Engineering, Kasteelpark Arenberg 30, 3001 Leuven, Belgium; marleen.vanaggelen@kuleuven.be

A horse is a living organism and consequently each horse has an individual response to a
specific situation. The physical and mental status of a horse plays an important role in the
animal's behaviour. In order to understand the behaviour of horses better, it's important to
gain insight in mental status beside the physical status of the animal. Numerous ways exist
to quantify the physical status of a horse (e.g. medical examination, exercises tests, etc.) by
audio-visual scoring in a manual way. Although this methodology exists, the quality of the
mental status in terms of positive, neutral or negative status of a horse in full action remains a
challenge. This work aims to classify the mental status of a physically active horse as positive,
neutral or negative in real-time by using wearable technology. Ten horses participated in an
experiment where each horse was lunged at walking speed for 5 minutes and then at trotting
speed for 10 minutes. The total of 15 minutes was then immediately repeated a second time.
Halfway through each of the two trotting segments, a mental response was induced to the horse,
either positive or negative, and in a random order to know when a certain mental status could
be expected and reduce order effects. To induce a positive mental response, the experimenter
shaked with a bowl of food (concentrate and carrots) at the edge of the lunging circle. The
negative mental response was induced by means of opening and closing an umbrella abruptly,
to evoke fear. Transitions to walk or trot were gently asked by voice to avoid additional stress
in the horse. Heart rate and activity were continuously recorded during the experiment and
each experiment was completely captured on film. Twenty-seven experiments in total were
done and mental events were automatically identified using the Cumulative Sum (CUSUM)
control chart algorithm. Each event was labelled as positive, neutral or negative based upon
audio-visual scoring of the behaviour of the horse on the recorded videos. The classification
performance of a linear and quadratic discriminant classifier, and a neural network were
investigated by stratified 10-fold cross-validation. The neural network performed best on a
dataset of 123 labelled mental events in terms of accuracy, with a value of 0.7182. The results
indicate that it must be possible to classify mental events as positive, neutral or negative based
on measurements of heart rate and activity.
**The use of selective serotonin reuptake inhibitors to try to develop a pharmacological model of positive affect in sheep**

Andrew Fisher¹, Leigh Atkinson¹, Emily Houghton², Caroline Lee³ and Rebecca Doyle¹

¹The University of Melbourne, Animal Welfare Science Centre, Faculty of Veterinary and Agricultural Sciences, Victoria 3010, Australia, ²Cardiff University, School of Biosciences, Cardiff, CF10 3AT, United Kingdom, ³CSIRO, Animal Behaviour & Welfare, Locked Bag 1, Armidale NSW 2350, Australia; adfisher@unimelb.edu.au

There is growing interest in animal welfare research in understanding and measuring positive emotional states in animals. To enhance future methodological research on improving measurement of positive affect in animals, the aim of this study was to develop a pharmacological model of positive affect in sheep. The hypothesis was that sheep treated with fluoxetine or citalopram would have a more positive affective state and thus be more likely to approach ambiguous bucket positions than control sheep. Following approval by the University Animal Ethics Committee, 32 female Merino sheep (9-18 months of age and initially naïve to the testing facility) were trained and evaluated to have successfully and individually learned a cognitive bias testing procedure in which approach to a positive feed bucket location was rewarded by feed present in the bucket, whereas a negative bucket location had no feed present and a loud buzzer sounded if approached. Animals were then randomly allocated to treatments: (1) Control saline injection subcutaneously 30 min before testing; (2) Fluoxetine 40 mg administered orally on day -2 and day -1 and saline injection 30 min before testing; (3) Citalopram 0.5 mg/kg bodyweight injected 30 min before testing; (4) Citalopram 0.2 mg/kg bodyweight injected 30 min before testing. Each sheep was exposed to three of the four treatments (i.e. 3 testing days), with a 1-week period between treatments. Cognitive bias testing measured sheep go/no-go behaviour and latency to approach the two training bucket positions (negative and positive) and three intermediate ambiguous positions. Latency data were analysed using survival analysis with a Cox's proportional hazards mixed effects model. Results showed that bucket position significantly affected latency to approach (P<0.001), with approach behaviour declining incrementally as bucket positions became close to negative. Day of testing effects revealed that sheep were more likely to approach ambiguous bucket positions on Day 1 (66%) than on Days 2 and 3 (53% and 51%, respectively; P<0.007). There were no effects of treatment on approach responses (P=0.71), although a treatment × day effect was present (P=0.013), whereby citalopram at 0.5 mg/kg caused sheep to approach all bucket locations more on Day 2 than Day 1, whereas sheep on other treatments showed a decline in approach from Day 1 to Day 2. Approach to the positive bucket was consistent, suggesting pharmacological changes in appetite were not mediating the effects recorded. In conclusion, treatment with fluoxetine and citalopram at the dosages used in this study did not appear to induce a positive judgement bias in Merino sheep. Further work on pharmacological agents and doses, or development of alternative, behaviourally-based models may need to be undertaken in order to provide validated models of positive affect in sheep.
**Video footage captured in a walk-over-weigh (WoW) system can be used to assess welfare state in sheep**

*Emily Grant, Amy Brown, Sarah Wickham, Fiona Anderson, Anne Barnes, Patricia Fleming and David Miller*

Murdoch University, School of Veterinary and Life Sciences, 90 South Street, Murdoch, 6150, Australia; e.grant@murdoch.edu.au

Extensive sheep farmers often don't have the time or resources to regularly monitor the health and wellbeing of their animals. Recent technological advancements in automated data capture have made this task easier, though to date this technology has not included behavioural monitoring. We tested whether quantitative and qualitative behavioural assessment (QBA) methods could be used to assess sheep in different welfare states as they traversed a walk-over-weigh (WoW) system. Video footage was remotely collected from thirty-six Merino sheep within four treatment groups; control (n=12), habituated (n=8), lame (n=8) and inappetant (n=8) as they traversed the WoW system. The habituated sheep were exposed to a low-stress handling regime for six consecutive days prior to filming. At the same time, the feeding behaviour of 870 electronic ID tagged sheep at an automated feeder was recorded and those animals that had an average intake more than two standard deviation units below the population mean over the six days were designated as inappetant. A lameness scoring system was also employed to identify lame individuals, and the control animals were selected ensuring that they were not lame, inappetant or habituated. The footage from these 36 sheep was presented at random to 18 observers, blind to the treatments, for QBA analysis. Using a free choice profiling approach, observers used their own descriptive terms to score the behavioural expression of each animal along a visual analogue scale. Footage was also used to measure quantitative behavioural traits. There was a high level of agreement between the observers (P<0.001), with the generalised Procrustes analysis (GPA) consensus profile explaining 58.2% of the variation between observer scores. Two main dimensions of behavioural expression were identified from this consensus, explaining 73% and 9% of the variation between sheep scores, respectively. Treatment differences were identified on this first dimension (P<0.05); habituated and lame sheep consistently scored higher (more focused/collected/assured) compared to the control sheep (more reluctant/tense/wary). There was no difference between the inappetant sheep compared to the controls. No treatment differences were observed on GPA 2. There was agreement between the qualitative and quantitative behavioural measures. Those sheep that baulked more frequently at the entrance to the WoW system ($R_s=-0.70; P<0.001$) or had a higher number of circling incidences ($R_s=-0.68; P<0.001$) were described as more reluctant/ tense/wary, and those that recorded higher walking speeds ($R_s=0.65; P<0.001$) or spent less time standing stationary ($R_s=-0.48; P<0.01$) were more focused/collected/assured. In conclusion, differences in welfare states (lame and habituated vs control) can be identified from video footage as sheep traversed the WoW system. These findings suggest that behavioural measures could be collected remotely to provide meaningful assessments of sheep welfare.
How does a stationary cow brush affect cattle physiologically and behaviourally?

Daisuke Kohari, Kenji Shimizu and Tsuyoshi Michikawa

Ibaraki University, College of Agriculture, Ami 4668-1, Inashiki-gun, Ibaraki, 300-0331, Japan;

daisuke.kohari.abw@vc.ibaraki.ac.jp

Cow brushes are used as an enrichment device to facilitate self-grooming of cattle. The degree to which they are used cattle and the brushing areas of body parts have been well reported from many studies. However, few reports of the relevant literature describe studies of brush use effects on cattle. To clarify brush effects on cattle, we investigated their heart rate variability and their behaviour when they used it. Experiments were conducted at experimental pens (3×4 m) at the Field Science Center of Ibaraki University. Five beef steers observed for this study were housed alone in the experimental pen during test days. A stationary cow brush (B2; DeLaval Inc.) was set at the side fence of the pen, adjusted to the height of their withers. Before observation, a heart rate monitor transmitter (RS300CX; Polar) was attached to each animal. Behavioural observations were conducted for 2-6 h per day for each animal. We observed them for 10.6-24.4 h during 22 days. Brush grooming durations were recorded by continuous sampling. The average heart rate and root mean square successive difference (rMSSD) between adjacent R waves of heart rate variables were calculated 30 s before, during, and 30 s after each bout of brush grooming. Furthermore, maintenance behaviour (standing or lying resting, grazing, self-grooming, brush grooming, defecation, others) and social behaviour (agonistic interaction or social grooming with cattle in neighbouring pens) of each animal were observed every 1 min using instantaneous sampling. Differences of the average heart rate and rMSSD before, during, and after brush grooming were analysed using repeated ANOVA for each animal. Also, the brush grooming duration was compared when the average heart rate or rMSSD was increased and decreased from before to after brush grooming using t-tests. Furthermore, data of maintenance and social behaviours immediately before and after brush grooming were extracted from all behavioural data and their percentages were calculated. We observed 98 bouts constituting 1,015 s of brush grooming during 81.9 h observation. Neither the average heart rate nor rMSSD of any steer was significantly different before, during, or after brush grooming. The brush grooming duration was not different with the average heart rate transitions from before to after brush grooming. However, the brush grooming duration was significantly longer in rMSSD increased from before to after brush grooming (41.4±26.3 s) than in rMSSD decreased (24.7±20.3 s) (t=2.17, P<0.05). Before brush grooming, the most frequently observed behaviour was standing resting (61.5%) followed in rank by self-grooming (17.9%). Self-grooming was also the most frequently observed behaviour before standing resting: 31.8%. The most frequently observed behaviour after brush grooming was standing resting (57.9%), followed in rank by grazing and lying resting (13.2%). These results suggest that the brush grooming duration affects calming levels and that cattle might indicate satisfaction behaviour by resting after brushing.
**Key-note:**

**The affective dyad: the role of maternal behaviour in positive affective states of mother and young**

Cathy M. Dwyer

SRUC, Animal and Veterinary Sciences, Roslin Institute Building, Easter Bush Campus, Edinburgh,
EH25 9RG, United Kingdom; cathy.dwyer@sruc.ac.uk

Maternal care provides offspring with nutrition, warmth, protection, comfort and opportunities for social learning, which serve to promote the survival of the offspring. In farmed mammalian livestock, the first two benefits of maternal care are well recognised, even in early weaning systems. However, the other attributes are less recognised and provision for these less likely to be considered in management systems. Further, any benefits of providing maternal care for the welfare of the mother are rarely considered. The onset of maternal care in mammals is associated with a complex interplay of foetal signals, steroidal hormones and the release of oxytocin at parturition. Oxytocin is also stimulated in the mother with suckling interactions and mother-offspring contact. Oxytocin has been shown to be associated with positive emotional states in humans and repeated oxytocin treatment decreases anxiety in rats. In addition, ventral contact between mother and pups has also been shown to reduce maternal anxiety, with anxiety increasing the longer mothers are away from their pups. In sheep and rats, where these processes have been well studied, parturition is also associated with alterations in dopamine signalling which may reflect rewarding properties of interacting with offspring. It seems plausible, therefore, that the expression of maternal care in mammals is associated with positive emotional states and could be considered as a component of positive welfare. For the offspring, mother-rearing shapes behavioural development, influencing social and environmental preferences, and reduces expression of fear, stress and pain responses. Mothers have also been shown to respond to pain expression in their offspring, for example increased maternal attentiveness when lambs were castrated or tail docked. The amount of maternal care expressed correlates with active pain behaviours of the lamb, although whether this contributes to decreasing pain experience of the young is not known. Thus, it is well known that maternal care contributes to the positive welfare of the offspring. However, whether these benefits can only be achieved by the biological mother, or if similar effects can be elicited by the presence of other conspecifics, or a human care giver, still needs further investigation. With the increase in interest in the importance of positive emotional states in animals, management systems that can provide prolonged periods of mother offspring contact may be important sources of positive welfare for both mother and young.
Dairy cows and heifers prefer to calve in a bedded pack barn or natural forage compared to open pasture

Erika M. Edwards¹, Katy L. Proudfoot², Heather M. Dann³ and Peter D. Krawczel¹

¹The University of Tennessee, 2506 River Drive, Knoxville, Tennessee 37996, USA, ²The Ohio State University, 1920 Coffey Road, Columbus, Ohio 43210, USA, ³William H. Miner Agricultural Research Institute, 1034 Miner Farm Road, Chazy, New York 12921, USA; eedwar24@vols.utk.edu

Despite years of research, the incidence of disease after calving in dairy cattle remains stagnant. Designing a calving environment that accommodates cow preferences and natural calving behaviours may allow cows to better cope, thereby improving calving outcomes. Cows will seek a secluded area to calve when housed alone, but this behaviour has not been established in group-housed cows. The objectives were to determine dairy cows' preference for calving location when grouped and provided access to a natural environment, and to evaluate the relationship between cows' activity during the last 21 d of gestation and preference for calving location. Multiparous Holstein dairy cows (n=33) and nulliparous Holstein heifers (n=34) were enrolled together as a dynamic group with size ranging from 21 to 1, as cows were added once weekly and removed after calving. Cows were housed in a bedded pack barn with free access to 2.06 hectares of pasture beginning 21 d before expected calving date. The bedded pack barn was 13.94 m² and included 22 headlocks where cows were provided a TMR daily. From -21 d until after calving, cows had access to the bedded pack (section 1), open pasture subdivided into areas of approximately equal size (~0.23 hectares; sections 2 through 8), and an area of natural forage at the far end of the pasture (section 9). Man-made hides (n=6) were created to provide an alternative option for seclusion in the pasture; hides were rotated among sections 3 to 8 every week. Video data were collected to determine calving location. Dataloggers were attached to each cows' rear fetlock 21 d prior to their expected calving date to record steps (n/d). To determine preference for a calving location, data were analyzed using a chi-square test in SAS. To determine the relationship between steps and calving location, linear regression was used. Twenty-three calvings (34%) occurred on the bedded pack, and 25 (37%) calvings occurred in the natural forage area. The remaining 19 calvings (28%) occurred in one of the 7 sections of open pasture, and of these, 3 cows calved within close proximity to the hides. There was a preference for the bedded pack barn (P=0.005) and natural forage area (P=0.002) for calving over the open pasture. Within sections, heifers calved in the area of natural forage more often than cows (P=0.03), and cows tended to calve in the barn more often than heifers (P=0.06). There was no relationship between the mean steps taken daily during the 21 d prior to calving and calving location (3,512.7±44 n/d (overall mean ± SE); P=0.60; R²=0.0045). The results provide evidence that both heifers and cows seek seclusion in a group setting when provided access to a covered barn and an area of natural forage. Cows preferred the covered barn to calve, whereas heifers preferred natural forage. This difference may be driven by previous experience; cows had greater exposure to indoor housing, whereas heifers had only been housed on pasture.
**Factors affecting maternal protective behavior in Nellore cows**

Franciely Costa¹,², Tiago Valente¹, Marcia Del Campo³ and Mateus Paranhos Da Costa¹,⁴

¹ETCO Group, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, Sao Paulo, Brazil, ²Postgraduate Program in Animal Science, FCAV, State University of Sao Paulo (UNESP), Department of Animal Science, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, Sao Paulo, Brazil, ³National Program of Meat and Wool, INIA, Ruta 5, Km 386, Tacuarembo, Uruguay, ⁴FCAV, State University of Sao Paulo (UNESP), Department of Animal Science, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, Sao Paulo, Brazil; mdelcampo@tb.inia.org.uy

The aim of this study was to evaluate the effect of different factors on maternal protective behavior (MPB) of Nellore cows. A total of 3629 cows were evaluated around 24 h after calving, while their newborns were handled for navel care, ear tattoo and weighing. MPB was assessed by observing each cow reactions when its calf was caught in a corral pen. MPB score consisted of 5 levels, going from 1 (the cow paid no attention to the calf and remained indifferent to the procedure) to 5 (the cow showed aggressive behaviors toward the handler, trying to attack). The cow body condition was registered using the scale from 1 to 5. Information regarding cow age at calving was available from the farm and six classes were defined (1: one to two years old, 2: two to three years old, 3: three to four years old, 4: four to five years old, 5: five to six years old, and 6: six to eight years old at calving). A generalized linear mixed model was fitted to evaluate the effect of cow body condition, cow age at calving, calf sex and calf birth weight on MPB. The cow body condition score, its age at calving and the calf sex were considered as fixed effects. Additionally, calf birth weight was used as a covariate and the cow was included as a random effect in the model. Cows with the greatest body conditions were more protective with their calves (F=9.79, P<0.0001). Younger cows were less protective to their offspring (F=4.66, P=0.0003). Adjusted means ± SE of MPB for each cow age class were: 2.41±0.06 (1), 2.56±0.04 (2), 2.62±0.03 (3), 2.67±0.04 (4), 2.73±0.06 (5) and 2.86±0.11 (6). The calf sex had a significant effect on MPB (F=6.25, P=0.0126) being higher for mothers of females calves (2.68±0.03) compared to male calves (2.60±0.03). Results from this study also showed that cows were more protective towards heavier calves at birth (F=19.05, P<0.0001). In conclusion, older Nellore cows, with a better body condition at calving, with females and heavier offsprings showed more intensive maternal protective behavior toward their newborns.
Preference for mother does not last long after weaning in lambs

Rodolfo Ungerfeld¹, Aline Freitas-De-Melo¹, Raymond Nowak² and Frédéric Lévy²

¹Facultad de Veterinaria, Universidad de la República, Lasplaces 1620, Montevideo 11600, Uruguay, ²UMR INRA, CNRS, Université F. Rabelais, IFCE, 37380 Nouzilly, 37380 Nouzilly, France; rungerfeld@gmail.com

Our hypothesis was that although lambs prefer their mother after weaning, this preference does not last long. The aim of this experiment was to study the effects of various durations of mother-young separation after weaning on the maintenance of a preference for the mother. Eighty-two single Corriedale lambs stayed with their mother until weaning at 3 months of age. Preference for the mother was tested using a two-choice test between their own and another mother from the same flock, matched for age and physiological status. Lambs were tested in a 10×10×7 m triangular testing enclosure with one ewe placed in each of the two holding pens. A plastic fence line prevented lambs from approaching the ewes at less than 1 m. A proximity zone near the ewes was marked in the floor (1×4 m). The location of the mother and the other ewe in the holding pens was alternated between tests. The total time spent and the number of vocalizations in the proximity zone near each ewe was recorded for 3 min. Lambs were habituated to this enclosure before testing. All the lambs were tested 3 days before weaning, and 30 (n=25), 40 (n=15) or 60 days (n=42) after weaning. Before weaning, lambs vocalized more frequently (16.2±1.2 vs 3.4±0.5, respectively; P<0.0001) and stayed in the contact zone for longer (104.1±25.3 s vs 20.3±4.2 s respectively; P<0.0001) when near their mother than near the other ewe. Thirty days after weaning, lambs still vocalized more and stayed in the proximity zone of their mother for longer than in that of the alien ewe (vocalizations: 5.9±1.5 vs 1.5±0.6; P=0.005; time in the zone: 36.6±9.5 s vs 13.4±4.8 s; P=0.016). By contrast, when tested 40 or 60 days after weaning there were no differences in any of these variables. The decrease in the number of vocalizations emitted by the lambs in the contact zone of their mother was more pronounced in lambs tested 40 and 60 than in those tested 30 days after weaning (16.9±2.9, 15.2±1.8, and 9.1±2.4, respectively P=0.04). There was no difference in the number of vocalizations emitted in the proximity zone of the other ewe between the three testing periods after weaning (2.1±0.9, 2.0±1.9, and 1.7±1.0). Before weaning, when lambs were almost 3 months old, they had a clear preference for their mother than for an alien ewe. However, that preference decreased 30 days after weaning and disappeared completely 10 days later. Although it may be expected that according to their age, lambs tested 40 and 60 days after lambing can easily recognize their mother' face, they failed to prefer her toward another known ewe coming from the same flock. Considering that though it has not been studied in lambs, ewes can remember faces from other individual ewes during at least 2 years, this implies that although lambs can recognize her, they lost the motivation to maintain the preference for their mothers shortly after weaning. Overall, the preference of lambs for their mother fades rapidly after 30 days of mother-young separation suggesting that attachment to the mother does not last long after weaning.
**Behavioural responses of dairy calves to separation: the effect of nutritional dependency on the dam**

Julie Føske Johnsen¹, Cecilie Mejdell¹, Annabelle Beaver², Anne Marie De Passille², Jeffrey Rushen² and Dan Weary²

¹Norwegian Veterinary Institute, Department of health surveillance, P. O. Box 750, 0106 Oslo, Norway, ²University of British Columbia, Faculty of Land and Food systems, 2357 Main Mall, Vancouver, BC V6T 1Z4, Canada; julie.johnsen@vetinst.no

Rearing a calf with the cow has health and welfare benefits, but separation and weaning are challenges. We studied how the calf's nutritional dependency on the cow affects calf behavioral response to separation. Dependent calves (D, n=10) and semi-dependent (SD, n=10) calves could suckle from their dam at night, but SD calves also had continuous *ad libitum* access to an automated milk feeder (AMF). Independent (I, n=10) calves had *ad libitum* access to an AMF and their dams wore an udder net that prevented suckling. Separation took place after six weeks. Calf responses were recorded using live observations for 4 days of partial separation (with fence-line contact with the dam) and then for 3 more days after removal of the dam from the barn (total separation). Vocalizations were classed as high-pitched (open mouth) or low-pitched (closed mouth). From d 1 of partial separation, all calves had *ad libitum* access to the AMF. We hypothesized that the calves accustomed to use the feeder before separation (i.e. SD and I calves) would continue to use it, and that calves would show fewer responses to separation if they were well established on the AMF. Differences were analyzed using non-parametric statistics and results presented as, median (25th percentile, 75th percentile). During partial and total separation, I calves produced fewer high-pitched vocalizations/d than D and SD calves combined (0.00 (0.00,0.00) vs 7.2 (0.75,23.51), Mann-Whitney U=12.2, z=-3.21, P=0.001 and 0.00 (0.00,0.00) vs 0.00 (0.00,1.00), U=40.5, z=-2.25, P=0.024), and also tended to produce fewer low-pitched vocalizations during partial separation (0.00 (0.00,0.05) vs 1.17 (0.00,2.54), U=29.5, z=-1.90, P=0.057). During the separation phases, 23 calves (four, nine and ten calves of D, SD and I treatments, respectively), consumed at least 1.5 l/d from the AMF; these calves produced fewer high-pitched vocalizations/d than calves not using the AMF (0.00 (0.00,7.83) vs 8.33 (5.13,24.83), U=16.0, z=-2.219, P=0.027) and tended to produce less low pitched vocalizations (0.00 (0.00,1.67) vs 0.8 (0.00,2.91), U=21.5, z=-1.731, P=0.083). During partial separation, calves' high pitched vocalizations were strongly negatively correlated to daily milk intake, (Spearman's rho=-0.770, P<0.001) and digestible energy intake (r=-0.544, P<0.011). During total separation, calves using the AMF spent more time playing (s/d) compared to calves not using it implicating that they were separated and weaned at the same time (3.67 (0.00,29.00) vs 0.00 (0.00,0.17), U=28.0, z=-1.990, P=0.047). The results indicate that nutritional independency from the dam can reduce the behavioural responses to separation from the dam. The results also show that disentangling separation from the dam and weaning from milk can improve calf welfare at separation.
Does farrowing duration affect maternal behavior of outdoor kept sows?

Cecilie Kobek Thorsen¹, Sarah-Lina Aagaard Schild¹, Lena Rangstrup-Christensen¹, Trine Bilde² and Lene Juul Pedersen¹

¹Aarhus University, Animal Science, Blichers alle 20, 8830 Tjele, Denmark, ²Aarhus University, Bioscience, Ny Munkegade 116, 8000 Aarhus C, Denmark; cecilie.thorsen@anis.au.dk

Piglet mortality is high the first three days after farrowing in Danish outdoor production. The use of high prolific sows is associated with long farrowing duration due to the greater number of piglets born. Prolonged farrowing may exhaust the sow and cause pain, which may affect maternal behavior and cause increased piglet mortality. The behavior of thirty-eight outdoor kept sows were video recorded from inside their individual hut during farrowing and the following three days. Farrowing duration, nursing pattern, sow posture changes, carefulness when lying down and latency to leave the hut after birth of the last piglet were recorded. The effect of farrowing duration on sow behavior was analyzed as a continuous variable using Proc Mixed in SAS. A mean farrowing duration of 7.5 h (range 2-29 h) to last live born piglet was recorded with a mean of 17 total born piglets (range 11-25). The only effect found of farrowing duration was that the latency to leave the hut after birth of the last piglet increased by one hour for every hour increase in farrowing duration (F1,110=7.37, P=0.008, mean=15.7±7 h). Farrowing duration had no effect on nursing frequency, duration sows presented the udder, number of posture changes nor the risk of 'flopping' when lying down. The results, however, did show normal patterns of increased activity as the days passed. Thus, prolonged farrowing did not seem to cause exhaustion to an extent where maternal behaviour was affected.
**Does dietary tryptophan around farrowing affect sow behavior and piglet mortality?**

Jeremy N. Marchant-Forde¹, Brian T. Richert², Jacob A. Richert² and Donald C. Lay, Jr.¹

¹USDA-ARS, LBRU, West Lafayette, IN 47907, USA, ²Purdue University, Department of Animal Sciences, West Lafayette, IN 47907, USA; jeremy.marchant-forde@ars.usda.gov

Piglet mortality remains a serious welfare and economic problem. Much of the early mortality is due to crushing by the sow. Tryptophan has been shown to reduce aggression and have a calming effect on behavior, which may reduce the number and type of posture changes, thereby altering risk of crushing. The aims of this experiment were to determine if feeding dietary tryptophan around farrowing would affect quantitative measures of posture-changing behavior and piglet mortality. Twenty-four multiparous sows (parity 2, 3 and 4) were moved to the farrowing house on d 110 of pregnancy and randomly assigned to one of two treatments: (1) standard lactation diet (CTL); or (2) experimental lactation diet with same energy content but containing four times the amount of tryptophan contained in the standard diet (TRYP). Treatments were applied from entry to the farrowing house until 3 d post-farrowing, with all sows receiving standard lactation diet thereafter. Sow behaviour was video-recorded continuously from entry until 7 d post-farrowing and all occurrences sampling was used to determine number and type of posture changes from 3 d before until 2 d after birth of first piglet. Posture changes recorded were all transitions between standing, sitting, kneeling, lying sternally and lying laterally. Production data recorded included sow weight on entry to farrowing crate, sow weight at weaning, sow feed intake, number of piglets born alive, dead and mummified, birth weight, 24 h weight, mortality and cause (physical inspection in conjunction with video data), number weaned, and weaning weight. Data were analyzed using the MIXED procedure accounting for repeated measures when appropriate. Dietary treatment had no effect on total litter size or number of piglets born alive (P=0.30), but TRYP sows tended to give birth to fewer dead piglets than CTL sows (1.0±0.4 vs 2.5±0.7, P=0.07). Total feed intake over the immediate pre- and post-farrowing period and sow lactation weight loss was not different between treatments, but on the day of farrowing, TRYP sows tended to eat more than CTL sows (3.0±0.4 vs 1.8±0.4 kg, P=0.09). Piglet birthweight, weaning weight and growth rates did not differ and total piglet mortality (born dead + liveborn mortality) was similar between treatments (TRYP 17.0±2.8 vs CTL 24.3±5.4%, P=0.29). The number and type of posture changes varied over time, but the only difference between treatments during the critical immediate 2 d post-farrowing period, was that CTL sows transitioned less between sitting and lying than TRYP sows (TRYP 34.9±5.6 vs CTL 21.2±2.0, P=0.03). Overall, feeding a high tryptophan diet around the time of farrowing had little effect on sow posture-changing behavior and no effect on liveborn piglet mortality. There may be a beneficial effect on stillbirth incidence and farrowing day feed intake, which could affect early lactation milk production, but this requires further investigation.
Relationships between sow conformation, accelerometer data and crushing events
in commercial piglet production

Stephanie M. Matheson¹, Rob Thompson², Thomas Ploetz², Ilias Kyriazakis¹, Grant A. Walling³
and Sandra A. Edwards¹

¹Newcastle University, Agriculture, Food and Rural Development, Newcastle upon Tyne, NE1 7RU, United Kingdom, ²Newcastle University, Computing Science, Newcastle upon Tyne, NE1 7RU, United Kingdom, ³JSR Genetics Limited, Southburn, East Yorkshire, YO25 9ED, United Kingdom; stephanie.matheson@newcastle.ac.uk

Pig selection for meat production maximises growth rate and length of back with a resulting change of body shape and difficulty in control of posture change in sows. As a consequence, many piglets are crushed, either as the sow lies down or rolls from side to side. This study investigated how characteristics of posture change movements in the sow (in conjunction with leg conformation) affect the likelihood of piglet crushing. Piglet births (n=11,752) were recorded for 21 weeks in a population of approx. 750 Landrace sows crossed with either White Duroc or Large White sires. All sows had leg conformation data collected (using previously published criterion), with a subset having rump-mounted accelerometer data (n=315). Data extracted from the accelerometers included the mean rate of change in movement around the X-axis (ROLL-CHANGE) and the mean rate of change of movement around the Y-axis (PITCH-CHANGE). The farrowing floors were either concrete/plastic, concrete/metal, fully metal or fully plastic. Piglet data gathered at processing (18-24 hours after birth) were piglet weight, sex, IUGR-status determined by head morphology (normal, light-IUGR and severe-IUGR) and reason for death. All piglets remained in their birth litters until processing, but were fostered thereafter. Females were individually identified but males were unidentifiable after processing. There were 349 piglets (both males and females) crushed between birth and processing (CPROC); 146 females were crushed between processing and weaning (males were unidentifiable after processing) giving a total of 495 crushed piglets (CTOT). Non-significant effects (GLMM at piglet level with sow-week as a random factor) on both CTOT and CPROC included sow parity, piglet sire breed, gender and the direct effects of sow conformation. Significant effects on CPROC were an interaction between piglet weight and IUGR-status (P=0.004), the type of flooring in the farrowing house (P=0.038), ROLL-CHANGE (P=0.043), PITCH-CHANGE (P=0.008), the interactions between PITCH-CHANGE and the hind pastern angle (P=0.039) and PITCH-CHANGE and placement of the hind feet (P=0.035). There was also an interaction between ROLL-CHANGE and the shape of the hind leg (P=0.077). For effects on CTOT, the piglet weight/IUGR-status interaction retained significance (P=0.002), as did PITCH-CHANGE (P=0.002) and the interactions between PITCH-CHANGE and the hind pastern angle (P=0.032) and PITCH-CHANGE and hind feet placement (P=0.018). The heritabilities for the accelerometer traits were: PITCH-CHANGE, h²=0.32±0.171 and ROLL-CHANGE, h²=0.03±0.110. In conclusion, accelerometer-derived measurements of sow movement have an effect on early piglet crushing. Sow limb conformation influences crushing events through interactions with sow movements. The interactive effects of sow conformation and accelerometer-derived data merit more detailed investigation. This research was funded by the EU FP7 Prohealth project (no. 613574).
**Effects of litter size on maternal investment and neonatal competition in pigs with different genetic selection pressures**

Marko Ocepek, Ruth C. Newberry and Inger Lise Andersen

Norwegian University of Life Sciences, Department of Animal and Aquacultural Sciences, P.O.
Box 5003, 1432, Norway; marko.ocepek@nmbu.no

Artificial selection of the domestic pig (*Sus scrofa domesticus*) offers a useful model for investigating changes in behaviour associated with reproductive trade-offs between litter size and fitness of offspring. The aim of this study was to evaluate effects of litter size on teat stimulation, sibling competition and pre-weaning survival and growth in three populations of domestic pigs subjected to different selection pressures (a maternal line emphasizing high weaned pig production (Norsvin Landrace), a paternal line emphasizing meat traits (Norsvin Duroc), and a crossbred line (Norsvin Landrace and Yorkshire)). We predicted that, with increasing litter size, piglets would spend more time in teat massage, be less likely to gain access to a teat during milk letdown and, if surviving to weaning, have lower, more variable body weights. We also predicted that maternal line sows would wean more piglets of higher weight, despite larger litter sizes, than paternal line sows. Sows (maternal line, n=12, paternal line, n=12, crossbred line, n=14) were loose-housed with their litters in individual farrowing pens. We collected data on piglet behaviour during nursing at 1 day of age, when sibling competition was expected to be most intense. Piglets were weaned at 35 days of age, when they were weighed and cumulative mortality was calculated. Effects of 1-day litter size, genetic line and their interaction on pre- (mean ± SE, 153±9 s) and post- (552±39 s) udder massage duration, piglets missing milk letdown (10.7±1.4%), nursing interval (54±1.9 min), nursing success (92.5±1.6%), piglet mortality to weaning (10.9±0.5%), mortality due to starvation (3.2±1.3%) and crushing (4.5±1.3%), piglet weight at weaning (11.1±0.3 kg) and variation in weight at weaning (14.9±1.1%) were analysed using a general, or generalised, linear model (depending on distribution of residuals). With increasing litter size, piglets spent more time in pre- (P=0.050) and post- (P<0.001) letdown massage and had shorter nursing intervals (P=0.018). Increasing litter size resulted in larger litters at weaning (P=0.002), but at a cost of more frequent termination of nursing prior to letdown (P<0.001), more piglets without a functional teat at letdown (P<0.001), an increased risk of mortality due to starvation (P<0.001) and crushing (P=0.002), and lower (P=0.039), more variable (P=0.002) body weights at weaning. Despite more post-letdown massage in the maternal line (litter size × breed: P<0.001), nursing intervals were longer (P<0.001) and mortality due to crushing was higher (P<0.001) with increasing litter size in this line, without increments in number, weight or weight uniformity of weaned piglets with increasing litter size (litter size × breed: P>0.1). Our results suggest that continued selection pressure to increase sow reproductive capacity is socially unsustainable due to the increasing animal welfare costs resulting from increased piglet competition.