## Behavioural and physiological responses of two ovine breeds to human exposure

Mariana Almeida¹, Rita Payan-Carreira¹,², Celso Santos¹ and Severiano R. Silva¹,²

¹Animal and Veterinary Research Centre (CECAV), Universidade de Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal, ²Departamento de Zootecnia, Universidade de Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal; mdantas@utad.pt

The knowledge of temperament enhances management and animal selection, which translates to increased welfare and productivity. This study's intent was to verify the existence of differences in temperament between two sheep breeds – Churra da Terra Quente (CTQ) and Ile-de-France (IF) – by assessing the putative differences in behaviour applying an arena test with a human operator. After an initial one-week habituation period to housing and groups, ten adult female sheep, five CTQ and five IF, were tested during a three-week period. All animals were kept under the same conditions. For practical restrictions, only four animals per group were individually tested to ensure that individual behaviours were not being concealed by group behaviour. The arena was a 6x1.5 m open space, divided into equal parts of 1 m (zone zero to five) and the operator was static for ten minutes inside zone zero. Animal behaviour was filmed, and the footage was analysed while recording a total of 13 behaviours, including proximity and interactions with the operator. Blood samples were collected from 3 tested animal in each group, to screen cortisol serum levels (Immulite 1000, Siemens), immediately before and after the test (<2 minutes) by an experienced handler. These samples were collected into glass test tubes, centrifuged and plasma was transferred into Eppendorf tubes which were then stored at -70 °C. Data analysis consisted of factorial analysis and Fisher-PLSD test (Fisher's Protected Least Significant Difference) to compare means. Statistical analysis revealed significant differences (P<0.05) in locomotor activity, the IF being more active (2.86 vs 2.31 m/min). IF sheep also showed a higher number of loud bleats and a lower latency in bleating for the first time (48 vs 18 and 0.642 vs 4.9 minutes, respectively for IF and CTQ; P<0.05). Both groups displayed a reduction in locomotor activity (P<0.05) from week 1 to week 2 (3.76 vs 1.99 m/min) which might be explained by familiarisation with the operator. Results from cortisol analyses were consistent with existing studies, the IF sheep showing higher values (2.603 vs 2.096 µg/dl; P<0.05). Expectedly, a rise in cortisol levels was observed after the test (1.016 vs 3.684 µg/dl; P<0.05). No significant differences were found between breeds previously to test, but after the test the IF group presented higher cortisol values (4.128 vs 3.239 µg/dl; P<0.05), showing how the two breeds have different physiological reactions when exposed to humans. However, since the number of animals is reduced it would be necessary to conduct further experiments to determine differences between breeds, nevertheless the overall results seem to indicate that IF sheep are more reactive to the operator than CTQ, an autochthonous breed, demanding greater attention in its management.
**Monitoring changes in heart rate and behavioral observations in donkeys during onotherapy sessions: a preliminary study**

Emanuela Tropia, Daniela Alberghina, Maria Rizzo, Gemma Alesci and Michele Panzera
University of Messina, Veterinary Sciences, Polo Universitario dell'Annunziata, 98168, Italy;
emanuela.tropia@hotmail.it

Donkeys are used for assisted therapies (AT) in adults with mental health disorders where they serve as suitable and effective alternative to equestrian therapy. Studies on animal assisted therapy programs have shown that AT could benefit patients treated for schizophrenic disorders since animals could act as social facilitators which are able to increase motivation, mobility and interpersonal contact. Research on characteristics for selection of individual animals or to assess animal welfare during AT are limited. The aim of this study was to evaluate heart rate (HR) and behavioral characteristics of animals involved in donkey therapy (onotherapy) sessions. Six clinically healthy female donkeys (mean age 17.8±5.7 years) were recruited in this study. Behavioral observations were made from 8 am to 8 pm for two consecutive days in order to compile an ethogram of diurnal activity. Subjects were monitored during two work sessions of 30 minutes with a control group (Group C) consisting of six healthy male adults without any previous riding experience and with a therapeutic group (Group T) of six male adults affected by schizophrenic disorders (code ICD-9 diagnosis code 295.8). During session work with both groups the same therapist modulated human-animal interaction. Each session was videotaped in order to score animal responses. Tactile and olfactory interactions between animal and patient, postural behaviors and intraspecific social interaction were evaluated and scored on a 5-point arbitrary scale (0 = very low to 4= very high). Heart rate was recorded during sessions using a Polar heart rate monitor (Polar Equine S-610i, Polar Electro Ltd., Warwick, UK) and transformed using the Polar Equine 4.0 Software. One of the donkeys scored poorly on the behavioral scale and was removed from the study and considered not suitable for AT (results from evaluation of behavioral observation were very low). Mann Whitney Test was used to analyse mean heart rate values and sum of the behavioral scores (Statistica 8 software). There were no statistical differences in behavioral indicators between groups, while mean HR during work sessions with Group T (70.40±7.60) was significantly higher (P<0.05) than with Group C (58.60±2.0). These preliminary results could be indicative of a different emotional state of donkeys probably due to a higher activity of patients (Group T) during riding. These preliminary results suggest that individual aptitude for AT in donkeys should be tested by validated methods and that HR monitoring, in particular it's variability (not considered in this preliminary study), could be useful to evaluate the emotional state of individual animals used in onotherapy. Measure of physiological indicators coupled with behavioral observation may help assess the animal's suitability reactions. Further research is necessary to assess the welfare of donkeys involved in AT.
**Longitudinal stretching while riding (LSR) as a preventive training method of riding horses - will it improve welfare?**

Linn Therese Olafsen¹, Inger Lise Andersen¹, Lars Roepstorff², Juan Carlos Rey² and Sylvia Burton¹

¹University of Life Sciences, Animal-and Aquacultural sciences, P.O. Box 5003, 1432 Ås, Norway,
²SLU, Dep. of Anatomy, Physiology and Biochemistry, P.O. Box 7011, 750 05 Uppsala, Sweden;
inger-lise.andersen@nmbu.no

The aim of the present preliminary project was to assess the immediate effects of a standardized
method of longitudinal stretching of the top line with long reins during riding (LSR) on
behavioral scores, mechanical nociceptive threshold (MNT; may quantify clinical neck and
musculoskeletal sensitivity leading to dysfunction), pressure response score, and stride length,
of 8 riding horses (aged 4 to 11, mixed breeds) subjected to a 20 minute treatment. MNT was
measured at the 6 following locations, left and right of the mid line: muscle Brachiocephalicus
at the basis of the neck, 1 and 2; mid thoracic Longissimus, 10 cm lateral of the dorsal midline,
3 and 4; between Tuber sacral and Tuber coxae on the mid Gluteus muscle, 5 and 6, with a
pressure algometer. Behavioral expression of ears (from score 1: flat orientated backwards to
6: hanging in a relaxed state directed towards each side, eyes (from 1: eyes wide open showing
much eye white to 6: no eye white, no tension around the eyes) mouth (from 1: large opening
between upper and lower jaw, a lot of mouth activity with tongue or lips, rich in foam, lips very
tensed, to 6: no mouth activity, closed, silent, relaxed mouth, very little or no foam), head-and
neck position (from 1: head held high, tensed, nose above the vertical to 6: head and neck
stretched downwards and forward with the mule below front knees, relaxed), collaboration with
the rider (from 1: not willing to move forward, bucking, rearing, or kicking to 6: no negative
behaviours towards the rider), working ability (from 1: low working ability to 6: high working
ability), were scored by two pretrained observers. Four consecutive phases were evaluated: (1)
first 5 minutes lunging; (2) first 10 minutes LSR; (3) last 10 minutes LSR; and (4) last 5 minutes
lunging. Stride length was calculated using the ETB-Pegasus Digital Gait Analysis System.
The last 10 minute LSR resulted in highest scores for all behaviours recorded compared to the
other phases of the treatment, as for the total sum of scores (Genmod procedure: X²-verdi=9.8,
P-value<0.001). Horses with a high score for collaboration with the rider also had a lower,
voluntary head position during riding (Polychoric corr. R=0.62; P<0.0001), a higher score for
eye expression (Polychoric corr. R=0.43; P=0.016), a higher score for ears (Polychoric corr.
R=0.57; P<0.0001), and a higher total sum of behavioural scores (Polychoric corr. R=0.82;
P<0.0001), which may indicate a more positive mental state during riding. After the 20 minutes
with LSR, MNT in five out of 6 locations along the top line were significantly different from
before treatment (Genmod procedure: χ²=588.1; P<0.0001). Pressure response score (0, 1 or
2) was significantly lower after LSR than before irrespective of location (Genmod procedure:
χ²=4.3; P=0.038). Stride length was significantly longer after LSR than before (Glm procedure:
F<sub>3,51</sub>=3.55; P=0.0208). In conclusion, these promising results suggest that we should further
validate this method as a preventive riding method.
# The influence of arena flooring type on behaviour in a test of calves' relationship towards humans

Stephanie Lürzel¹, Andreas Futschik² and Susanne Waiblinger¹

¹Institute of Animal Husbandry and Animal Welfare, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210 Vienna, Austria, ²Department of Applied Statistics, JK University Linz, Altenberger Str. 69, 4040 Linz, Austria; stephanie.luerzel@vetmeduni.ac.at

The behaviour of animals towards humans is often observed in a novel environment. Different degrees of novelty and resulting neophobia can be major confounders and the design of the arena may be essential for obtaining valid results. We investigated whether the presence of bedding has an influence on calf behaviour in a test of the animal-human relationship conducted in a novel arena. We re-evaluated behavioural data recorded from 82 calves that had or had not experienced 42 min of gentle interactions with humans during their first two weeks of life. The test arena measured 1.5×5.8 m² and had a concrete floor, including metal covers of the manure system. For half of the animals, the floor was covered with bedding (wood shavings) and not covered for the other half. The behaviour of the animals was recorded by two cameras placed above the arena. The test consisted of three phases of 3 min each: isolation, presence of an experimenter, isolation. They allow testing for a calming effect of human presence and for negative emotions induced by separation from the human. If the calf approached the experimenter, she stroked it for 30 s. For analysis, the arena was subdivided in five areas, weighted from 1 (at entrance) to 5 (farthest from entrance, in phase 2 with human). An area score was calculated as the sum of the products of the time (s) a calf was in a particular area with the weight of the respective area. The score ranged from 180 to 900, with lowest values indicating that the calf did not move far into the arena. There was a main effect of bedding on area score (mean±SD with bedding 527±166, without bedding 450±229; LMM, P<0.001) and frequency of entering area (10.9±8.3, 5.5±5.5; GLMM, P<0.001), tail flicking (7.6±13.1, 3.3±11.9: P=0.001), running/jumping (1.3±2.5, 0.3±1; P=0.006) and avoidance towards the experimenter (0.3, 0.7; P=0.04). Some of these effects were modified by a trend towards an interaction with phase or towards an interaction with treatment, e.g. frequency of running/jumping, both P≤0.08. For running/jumping, we subsequently ran a model for each bedding condition. No significant main effect of treatment but a trend towards an interaction of phase and treatment (P=0.06) existed in the model 'with bedding', with higher levels of running/jumping occurring in the treatment group than in the control group in phases 2 and 3. In contrast, there was a significant main effect of treatment (P=0.04) in the model 'without bedding', with more running/jumping occurring in the control group. The data show that the properties of the flooring in a novel test arena influence the behaviour of calves. The trends towards interactions with phase and treatment suggest that the results of an arena test may depend on the type of flooring used, and the example of running/jumping shows that the two flooring conditions can lead to partly opposite results regarding the effects of treatment.
**Characterising personality traits and influences on these traits in German Shepherd Dogs**

Juliane Friedrich<fnref n="1" />, Pamela Weiner<fnref n="1" />, Zita Polgár<fnref n="1" /> and Marie J. Haskell<fnref n="2" />

Understanding the expression of personality traits such as playfulness and fear in dogs, and what factors influence these traits, is important for the owners of pet and working dogs. In this study we assessed the effects of housing environment, level and type of training, the type of exercise regime and exposure to social and environmental stimuli on the expression of personality traits in German Shepherd Dogs (GSDs). Owners of GSDs were contacted by the UK Kennel Club and sent two questionnaires: a modified version of the C-BARQ canine personality questionnaire, consisting of 116 questions on behavioural characteristics such as aggression, anxiety, trainability and playfulness, and a ‘management/lifestyle’ questionnaire on care, exercise, training and housing of the dogs. There were 333 respondents to both questionnaires. Principal component analysis was used to derive personality traits from the C-BARQ responses. Linear models using backward selection of variables were used to assess relationships between dog characteristics, management factors and personality traits. The results suggested that 15 factors accounted for most of the variance in the behaviour questionnaire responses, including ‘stranger-directed aggression’, ‘separation anxiety’, ‘stranger-directed fear’ and ‘playfulness’. Regression analysis showed that male dogs exhibited greater separation anxiety than females (t=-3.78, df=331, P<0.001), which has also been shown in Labrador Retrievers. Dogs trained to respond to a higher number of verbal commands had higher playfulness scores (t=6.97, df=330, P<0.001). This suggests that some aspects of the training experience may promote a positive emotional state in the dog, allowing playfulness to be expressed.

<footnote n="1">¹The Roslin Institute, University of Edinburgh, Easter Bush, Roslin EH25 9RG, United Kingdom,</footnote>

<footnote n="2">²SRUC, West Mains Road, Edinburgh EH9 3JG, United Kingdom; marie.haskell@sruc.ac.uk</footnote>
**Social support from companion animals as an important factor influencing therapy and education effectiveness**

Agnieszka Potocka

University of Warsaw, Faculty of Artes Liberales, Nowy Świat 69, 00-001 Warszawa, Poland;
agnieszka.potocka@dogoterapia.net

This paper is a review of research and concerns about the role of companion animals working in Animal Assisted Therapy (AAT) and Animal Assisted Education (AAE). In this paper, based on a review of the research of social effects on humans of interaction with companion animals, I shall present the way to engage dogs, cats or other companion animals in AAT or AAE. Therapeutic interaction with animals could possibly increase effectiveness in therapy or education and without having negative impact on animal welfare. Students, patients or therapy clients who do not have sufficiently strong social support from family members, friends, and relatives may have weaker results in education in school or during any kind of their therapy than persons who have social support. Reason for this could be the feeling of isolation, ostracism, or lowered self-esteem. Dogs, cats and other companion animals can play similar roles as friends or family members and be an additional source of support. Cobb defined social support 'as information leading the subject to believe that he is cared for and loved, esteemed, and a member of a network of mutual obligations'. Social support is needed especially during crisis and difficult life situations, during education or therapy as a difficult relationship with therapist or teacher, fear associated with a new environment, new people, stress during exams or therapy procedures. Anthrozoology research strongly suggests that dogs' and other companion animals' company and trusting relationship with them could reduce anxiety, loneliness, stress and improve social contacts. The theoretical framework explaining the positive effect of human-animal relationship cover physiological mechanism of oxytocin release, behavioral system of attachment and social support. Recognition of significance of these mechanisms as components of therapy efficiency allows proper animal engagement. Satisfying social needs in relationships with others, gives benefits such as a sense of security, belongingness to a group, availability of help and, as a result, increased opportunities for survival, good health and a better quality of life. Dogs, cats, or horses should not be considered as merely tools in the therapeutic process, to be used in any way, without regard for their individual welfare. They should play the role of sensitive, valuable and sentient beings, with whom students, patients or therapy clients can establish a meaningful bond and receive social support from them. This approach facilitates better effectiveness of therapy or education and allows to maintain good welfare of animals.
The welfare of working dogs in cars

Lena M. Skånberg, Yezica Norling, Oskar Gauffin and Linda J. Keeling

Swedish University of Agricultural Sciences, Department of Animal Environment and Health,
Box 7068, 750 07 Uppsala, Sweden; lena.skanberg@slu.se

Working dogs, such as police dogs, spend a large amount of their time in cars. It has been hypothesized that a smaller cage could be safer for the dog during driving, especially at high speed, but that a larger cage could allow better comfort (e.g. for resting) when the car is parked. This presents a conflict. Our study investigated how dogs in a car are affected by cage size. Working dogs of the breeds German Shephard (n=8) and Springer Spaniel (n=8) from the Swedish Police and Customs were tested in four different cage sizes in four different car treatments; parked, slow cruising, normal driving and fast emergency driving. The study was conducted at a test track. Each dog was tested in all combinations of cage and driving style over a two-day period. Each combination involved 30 minutes in the car, except for the fastest driving style that was shortened to 5 minutes. The behaviour of the dogs was recorded by two video cameras in the car and the dog's heart rate and variability were recorded using a monitor (Polar V800). Statistical analyses used a mixed model or a Friedman test followed by a Wilcoxon Signed Rank test. Driving style had the largest effect on the dogs. The faster the driving, the higher the heart rate (P<0.001) and the lower the RMSSD values (P<0.001). Faster driving also resulted in a higher prevalence (P<0.01) of behavioural stress indicators (e.g. lip-licking, panting, shaking). No cage size seemed to be better regarding safety, such as decreasing the prevalence of slipping, losing balance or bumping against the walls of the cage. These behaviours occurred frequently, around 50% of the time, in the fastest driving style. Regarding comfort, the cage that was shorter than the length of the dog (although not the smallest in area), resulted in least time lying down during parking and slow driving (P<0.05) (on average 59% of the time) compared to the other cage sizes (on average 78% of the time lying down). Dogs also had a tendency to stretch more (3.75 times more), after spending 30 minutes in this cage (P=0.058). The largest cage, proportional to the size of the dogs, resulted in 2.6 times more changes in body postures (P<0.05). In conclusion, faster driving, including turns and sudden brakes, appears to be physically demanding and experienced negatively by dogs, irrespective of cage size. However, cage dimension still seems to matter regarding comfort. Transporting a dog in a cage shorter than its length, as measured from nose tip to point of buttock (currently allowed in the Swedish legislation for working dogs) could, due to worse comfort, negatively affect the performance of the dog when taken out of the car.
Do cull dairy cows become lame from being transported?

Kirstin Dahl-Pedersen¹, Mette S. Herskin¹, Hans Houe² and Peter Thorup Thomsen¹

¹Aarhus University, Department of Animal Science, Blichers Allé 20, 8830 Tjele, Denmark.

²University of Copenhagen, Department of Large Animal Sciences, Grønnegårdsvej 8, 1870 Frederiksberg C, Denmark; kirstin.dahpedersen@anis.au.dk

Approximately 200,000 cull dairy cows are transported to slaughter in Denmark each year. Dairy cows are culled for a number of reasons and may be more vulnerable to transport than younger livestock. However, rules and recommendations on animal transport do not mention cull cows specifically and very limited research has focused on this particular group of animals. The aim of this study was to investigate whether transport of cull dairy cows by road for up to 8 hours affect their lameness score. The observational study involved 411 cull dairy cows, transported to slaughter during a 12 month period. The study included one slaughterhouse, trucks from four hauliers and cows from 20 dairy herds. Cows to be included were selected by the owners. An ethical permit allowed inclusion of varying clinical conditions, except for cows defined unfit for transport according to the EU regulation on animal transport (EC 1/2005). All management followed the normal, commercial procedures. The cows were examined clinically on-farm before loading, and then transported to Danish Crown's slaughterhouse in Holsted, Denmark. At the slaughterhouse, the cows went through a second clinical examination. Data were analysed in SAS and the McNemar test was used to compare the proportion of lame cows before and after transport. Logistic regression was used to evaluate the association between risk factors e.g. distance, body condition score and days after calving and the aggravation of locomotion score. Data were collected from 49 journeys. The mean distance was 129 km (range 20-339) and mean duration was 187 min (range 32-510). The mean number of pauses per journey was 2.0 (range 0-6) with a mean total duration of 48 min (range 0-155). The cows were culled after a mean of 2.9 lactations (range 1-10) and a mean of 270 days after calving (range 15-871). The majority of the cows (63%) were in normal body condition, 16% were thin (body condition score≤2.5) and 21% were fat (body condition score ≥4.0). Only 9 cows arrived at the slaughterhouse in a clinical condition, which would have been considered unfit for transport according to current Danish practice. These were all severely lame (locomotion score 5). Before transport the prevalence of locomotion score ≥3 (5-point scale) was 31% and this was increased to 41% after arrival at the slaughterhouse (P<0.0001, df=1). Cows that were lame on-farm (locomotion score ≥3), had an incidence risk of 27% for becoming more lame, whereas cows that were not lame on-farm had an incidence risk of 16% for becoming lame. The maximum change in locomotion score was 3 points, but for 66% of the cows becoming lame or more lame during transport, the locomotion score increased with only one point. For 3% of the cows the locomotion score decreased with one point. Results show that cull cows can become lame during short road transport to slaughter. Further studies are needed to define thresholds for the risk factors and to examine possibilities for improvement e.g. management procedures in relation to culling and transports with special provisions in order to solve this challenge to animal welfare.
**The effect of overstocking different resources within a freestall pen on the behaviour of lactating Holstein cows**

Clay Kesterson, Randi Black, Nicole Eberhart, Erika Edwards and Peter Krawczel

The University of Tennessee, Knoxville, Animal Science, 2506 River Drive, Knoxville, TN 37996, USA; ckesters@vols.utk.edu

Decreasing the availability of resting and/or feeding space has detrimental effects on lying behaviour. However, the interaction of overstocking pressure to varying degrees at these resources has yet to be evaluated to determine how cows may alter behaviour based on priority of use. The objective was to determine the effect of overstocking the freestalls, the headlock, or both at 160% on lying and social behaviour. Holstein cows (n=32) were housed in a naturally ventilated, freestall barn, fed a TMR at 07:00 and 15:30 h, and milked at 08:30 and 18:30 h daily. Groups (n=4) were balanced by parity (1.8±1.1) and DIM (224.3±58.5 d). Treatments were: 100% stocking density at freestalls (FS) and headlocks (HL), 100% stocking density at FS and 160% at HL, 160% stocking density at FS and 100% at HL, and 160% stocking density at FS and HL. Treatments were assigned using a 4×4 Latin square design with 14 d periods followed by a 3-d washout. Accelerometers were affixed to a rear leg of each cow to collect lying behaviours. Displacements 2 hours after each feeding were collected via video data. A mixed model (SAS 9.4) was used to evaluate the effects of treatment on total, left, and right side lying time, bout duration, and number of bouts, and aggression at the headlocks. Relative to 100% at FS and HL, overstocking the FS or overstocking FS and HL at 160% decreased lying time (13.2±0.4 vs 11.4±0.4 or 11.7±0.4 h/d, P≤0.01). Decreased lying time was also evident when FS or both FS and HL were overstocked compared to only overstocking the HL (11.4±0.4 or 11.7 vs 0.4 vs 12.7±0.4 h/d, P≤0.04). No differences in lying time was evident between stocking densities of 100% FS and HL, and 100% FS and 160% HL (P=0.19), or in the two treatments which included overstocking at the freestalls (P=0.47). No other lying behaviours differed (P≥0.15). Surprisingly, no differences in social aggression at the feed bunk were evident (16.6±4.8 n/d at FS stocked at 100% and HL at 160%; 12.8 n/d at FS stocked at 100% and HL at 100%; 8.3 n/d at FS stocked at 160% and HL at 100%; 6.9±4.8 n/d at FS stocked at 160% and HL at 160%; P=0.22). Cows were able to maintain lying time when overstocked at the HL alone, but lost lying time when overstocking was imposed at the freestalls. The lack of treatment effects on social aggression may have been driven by a greater priority given to accessing resting resources or from altering feeding patterns. This indicates that cows may be able to modify their feeding behaviour, but not their lying behaviour to mitigate reduced access to the associated resources. Management of freestall facilities may need to focus on maximizing access to resting space. However, cows enrolled on this study were more than 200 d into lactation, which may have reduced the importance of access to feed. Future studies should evaluate if prioritization of access to resting and feeding resources is shared by different stages of lactation.
**Effect of water temperature on drinking behavior and stress response of sheep**

Shin-ichiro Ogura, Iori Kajiwara and Shusuke Sato

Tohoku University, Graduate School of Agricultural Science, 232-3, Yomogita, Naruko-onsen, Osaki, 989-6711, Japan; shin-ichiro.ogura.e1@tohoku.ac.jp

Appropriate water supply is essential to certify animal welfare of livestock. Water temperature can affect the amount of water intake of animals; for example, cold water in winter may give stress to animals, and in contrast in summer, it may alleviate heat stress of them. Therefore in this study, the effect of water temperature on drinking behavior and stress response was examined under indoor feeding. The experiment was conducted on December (Cool season) and September (Warm season). The mean daily air temperature of each season was 2.6±1.9 °C and 20.2±2.7 °C, respectively. Eight lambs (mean live weight was 38.1-55.8 kg) were fed individually in *ad libitum* of alfalfa hay cube and water. Six animals among the eight ones were used for both seasons. In Cool season, two experiment was done; i.e. water temperature during habituation (5 days) was 25 °C (trial 1) and 5 °C (trial 2). In Warm season, the temperature during habituation (5 days) was 18-23 °C (trial 3). In the experiment period (4 days), water with different temperature (7, 15, 25 and 35 °C) was offered 09:30-13:30, then changed to the same temperature as in the habituation period. The treatment of each animal was changed every day by Latin square design. During the experiment period, water intake and dry matter intake was measured, and drinking behavior (number of drinking and its duration) was recorded by continuous observation. In addition in Warm season (trial 3), heart rate and cortisol concentration in saliva was measured every day in the experiment period, when the treatment of water temperature started. Actual water temperature offered to the animals in the experiment period was 5.5 (5.4-5.6), 18.8 (17.4-19.0), 26.0 (25.8-26.2) and 31.3 (30.9-31.9) °C in Cool season and 7.8 (7.0-8.5), 15.1 (14.7-15.5), 26.1 (23.0-29.1) and 33.5 (29.3-40.7) °C in Warm season. Water intake per live weight was 35 °C > 7 °C (ANOVA, P<0.05) in Cool season (trial 1 and 2), but there was no significant difference in water temperature in Warm season (trial 3). In both seasons, there was positive relationship between water intake and dry matter intake (Pearson's correlation analysis, Cool season: r=0.472, P<0.001; Warm season: r=0.648, P<0.001). In the treatment of 7 °C, water intake per drinking behavior was smallest, thus drinking rate was lowest (ANOVA, P<0.01). Although heart rate was not affected by water temperature, cortisol concentration in saliva was significantly high in 7 °C (1.12 ng/ml) than other temperatures (0.58-0.63 ng/ml) (ANOVA, P<0.05). These results showed that high water temperature (35 °C) increase water intake of animals in cool season, indicating that it increase dry matter intake. This study also suggested that water with low temperature (7 °C) gives stress to animals even in warm seasons.
# Effects of genetic background and selection for egg production on food motivation in laying hens

Anissa Dudde¹, Lars Schrader¹, Mohammad Bashawat² and E. Tobias Krause¹

¹Friedrich-Loeffler-Institute, Institute of Animal Welfare and Animal Husbandry, Dörnbergstr. 25/27, 29223 Celle, Germany, ²Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany; anissa.dudde@fli.de

The motivation to obtain food is a fundamental motivational state. Previous studies have shown that food motivation in chickens increases with food deprivation. However, it is also possible, that this motivation was modified during selection for egg laying productivity. We aimed to find out whether and to which extend hens of different layer strains have increased food motivation with respect to their annual egg productivity and whether they are eager to walk longer distances to reach food. We expect higher egg production to be linked with higher food motivation and, thus, faster locomotion over distances. We used 72 hens from a 2x2 factorial design, (2 genetic backgrounds, indicated by egg colour (white/brown) and 2 productivity level (low 200 vs high 300 eggs/y), thus incorporating four different strains. In their home pens, all hens had *ad libitum* access to food and water and, in addition, free access to grains presented in coloured bowls, which were used as food reward in the tests. For the test, hens were placed individually in a start box, leading to a corridor at which end the food reward was provided at increasing distances (50, 100, 200, 400 cm) on four subsequent days. At each distance, we measured the latency to reach the food reward and the velocity (cm/s). Data were analysed using Linear Mixed Effect Models (LME), with the factors body mass, genetic background, productivity, distances in the test (50, 100, 200, 400 cm), and their two-way interactions. Individual ID was a random factor. The latency to eat was significantly affected by productivity level, genetic background and distance (LME: productivity: $F_{1,65}=6.59$, P=0.01; genetic background $F_{1,67}=8.84$, P=0.004; distance: $F_{1,207}=8.48$, P=0.004; body mass: $F_{1,65}=0.32$, P=0.57). The velocity of hens differed significantly between the productivity levels and the genetic background (LME: productivity: $F_{1,65}=6.08$, P=0.01; genetic background $F_{1,65}=9.06$, P=0.004; distance: $F_{1,207}=102.88$, P<0.0001; body mass: $F_{1,65}=0.45$, P=0.5). All other not mentioned factors were not significant. Body weight was measured at the first test day and differed between white and brown layers (mean ± SD, brown: 1,878±204 g vs white: 1,634±179 g). High productive hens needed less time to feed and had a higher velocity compared to moderate productive hens, even when distance to food reward increased. These differences suggest a higher food motivation in high productive hens resulting from intense selection for egg productivity and their higher nutritional demand, rather than differences in locomotor abilities. Brown layers were faster than white layers. Although body mass was higher in brown layers, it did not affect velocity. Thus, differences in basal metabolism or in walking ability due to higher weights are unlikely be responsible for the higher velocity in brown layers. Instead, they can be explained by phylogenetic difference, which also has been demonstrated for other traits.
**On-farm techniques used to minimize aggression in pigs by US pork producers**

Sarah H. Ison, Ronald O. Bates, Juan P. Steibel, Catherine W. Ernst and Janice M. Siegford
Michigan State University, Animal Science, Room 1290, 474 S. Shaw Lane, East Lansing, Michigan,
48824, USA; shison@msu.edu

Despite decades of research, pig aggression remains a welfare issue. Although various management techniques have proved useful in minimizing aggression; it is not known to what level these techniques are implemented on-farm. One objective of an online survey to US pork producers was to find out what techniques are used, how useful they are perceived to be, and what are the potential barriers to their use. Respondents were given a list of techniques, were asked to select which ones they have used, and indicate how useful they perceived them to be (from 'very useful' to 'not useful at all'). They were also asked about the housing and management of pigs, and demographic information. Chi-square tests of homogeneity compared the frequencies with which different techniques were perceived as useful, and a step-wise binomial logistic regression analyzed the association between other factors and technique implementation (yes or no). Respondents (n=298), had nursery (11,490±4,013 pigs, range: 0-950,000), and/or finisher stage pigs (18,929±5,779, range: 0-1,500,000), and of these, some had both nursery and finisher pigs (n=181), and some had just nursery (n=24), or finisher (n=89) pigs. When asked if they regularly mix or re-group unfamiliar pigs, 36% of all respondents said yes (33% at nursery, 25% at finisher). Of all the respondents, 53% used techniques to minimize mixing aggression. These included pre-weaning piglet socialization (n=19), which was perceived to be more useful than all other techniques (P<0.05). Evening mixing (n=50), was perceived as more useful than odor masking agents (P=0.006, n=38), pre-exposure in a neighboring pen (P=0.03, n=45), and enrichment devices (P=0.008, n=41), but equally as useful as mixing all pigs to a new pen (n=108), barriers (n=25), or using specific mixing pens (n=21). Mixing pigs to a new pen was considered more useful than enrichment (P=0.04). Using mixed weight pens (n=9) was infrequent, but considered equally as useful as other techniques (P>0.05) by those who used it. The best regression model for predicting the odds of using a technique included the factors pig stage (nursery, finisher, or both), regular mixing (none, finisher, nursery, both stages) as significant predictors, and substrate/bedding use (yes, no, sometimes) as a tendency. Respondents with farms specializing in either nursery (log odds=4.08, P<0.001) or finisher pigs (log odds=4.42, P<0.001) only, increased the log odds of using a technique, whereas those that did not mix regularly at any stage (log odds=-5.03, P<0.001), reduced the odds. Those who selected yes to using bedding (log odds=0.88, P=0.077) tended to increase the odds of implementing techniques. These results indicate that, overall, producers avoid mixing. However, when mixing is required, techniques are used to minimize aggression, with pre-weaning socialization indicated as most useful.
If you give the pig a choice: suckling piglets eat more from a diverse diet

Anouschka Middelkoop¹, Raka Choudhury², Walter J.J. Gerrits³, Bas Kemp¹, Michiel Kleerebezem² and J. Elizabeth Bolhuis¹

¹Wageningen University, Adaptation Physiology Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ²Wageningen University, Host-Microbe Interactomics Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ³Wageningen University, Animal Nutrition Group, De Elst 1, 6708 WD Wageningen, the Netherlands; anouschka.middelkoop@wur.nl

Stimulating solid feed intake in suckling pigs is important to ensure a successful weaning transition, exemplified by the correlation between pre- and post-weaning feed intake. In nature, piglets begin to sample food items in a playful manner when only a few days old. In pig husbandry, contrarily, suckling pigs are not encouraged to forage, whereas we hypothesize that this is crucial to increase piglet robustness via improved intestinal and gut microbiota development. One approach to familiarize pigs with solid feed at an early age might be by providing feed in a variety of forms, using diversity or novelty to stimulate the pigs' foraging behaviour. We studied the effect of dietary diversity (i.e. offering two diverse feeds simultaneously) vs novelty (i.e. regularly changing the flavour of one feed) on the foraging behaviour and feed intake of suckling pigs. We also hypothesized that piglets, rather than sampling from one feed only, would prefer a diverse diet. Piglets received *ad libitum* feed from 2 days of age in two feeders per pen. In treatment 1 (T1, n=10 pens) pigs were given feed A and feed B which differed in size, flavour, composition, smell, texture and colour. In treatment 2 (T2, n=9) pigs received feed A plus feed A to which additional novel flavours (4 different ones) were added from day 6 in a daily sequential order. Feeding behaviour was studied by weighing feed remains (d6, 12, 16, 22) and by live observations (4-min scan sampling, 6 h/d; d10, 15, 22; n=6 pens per treatment). Observations were also used to determine 'eaters' (i.e. pigs scored eating at least once). Data were analyzed using mixed models. Piglets did not prefer feed A (d2-22: 196±16 g/pig) or B (152±13) within T1 and did not have an overall preference for feed A with (d6-22: 78±4 g/pig) or without flavour novelty (66±6) within T2. In accordance, just a few piglets (T1: 1.5% and T2: 3.2% out of all eaters per treatment) were observed eating only one of the feeds throughout lactation. Interestingly, T1-pigs (d2-22: 327±28 g/pig) ate much more than T2-pigs (147±9; P<0.0001) and explored the (feed in the) feeders 2.6 times more at d15 (P=0.001). This also implies that feed A, the common feed provided in T1 and T2, was more consumed in T1 (d6-22: 152±13) compared to T2 (68±5; P<0.0001). The percentage of eaters within a litter did not differ over time between T1 (d10: 26%, d15: 78%, d22: 94%) and T2 (20, 71 and 97%). In conclusion, our results suggest that piglets like to eat a varied diet instead of preferring one feed over the other. Dietary diversity by providing two feeds at the same time different in flavour, size, composition, smell, texture and colour stimulated the feed intake and feed-related exploratory behaviour of suckling pigs more than dietary diversity via novel flavours only, but did not elicit pigs to start eating earlier. Further research is needed to explore the most effective dietary diversity to stimulate early feeding.
# Impact of straw particle size and reticulorumen health on the feed sorting behaviour of early lactation dairy cows

Rachael Coon<fnref n="1" />, Todd Duffield<fnref n="2" /> and Trevor Devries<fnref n="1" />

The objective of this study was to determine if feed sorting behaviour in early lactation dairy cows varies with straw particle size in a total mixed ration (TMR) and with reticulorumen health. Forty multiparous Holstein cows were enrolled in a trial ~2 wk before calving. Upon calving, cows were fed either 1 of 2 TMR that included 9% wheat straw (dry matter basis) chopped to: (1) 2.54 cm (Short) (n=20); or (2) 5.08 cm (Long) (n=20). Cows remained on their respective treatments for 4 wk. Fresh feed samples were collected 3x/week to determine particle size distribution. Orts samples were collected every 3 d post-calving to determine feed sorting. A particle separator was used to separate feed samples into 4 fractions: long (>19 mm), medium (<19 mm, >8 mm), short (<8 mm, >4 mm), and fine (<4 mm) particles. Sorting was calculated as: actual intake of each particle fraction expressed as a % of its predicted intake. Wireless telemetry boluses were used to measure reticulorumen pH every 15 min, 24 h/d. Cows were classified as having low reticulorumen pH (LPH) if reticulorumen pH fell below 5.8 for an average of >1 h/d throughout the trial (n=15; mean=280 min/d below pH 5.8). If reticulorumen pH fell below pH 5.8 for an average of <1 h/d cows were classified as having normal reticulorumen pH (NPH) (n=25; mean=16 min/d below pH 5.8). Data were summarized by week and analysed in a repeated measures mixed-effect linear regression model. Significance was declared at P≤0.05, and tendencies were reported if 0.05<P≤0.10. Amongst LPH cows, sorting of long particles differed by treatment (P=0.02); LPH cows on the Long treatment (n=9) sorted against those particles (93±3%; P=0.01), while LPH cows on the Short treatment (n=6) tended to sort for those particles (106±3%; P=0.1). NPH cows on both treatments tended (P≤0.1) to sort against the longest particles (96±2%). Sorting of medium particles tended (P=0.06) to differ by treatment for LPH cows; LPH cows on the Long treatment tended to sort against these particles (98±1%; P=0.1), while LPH cows on the Short treatment sorted in favour of them (103±2%; P=0.05). LPH cows sorted short particles differently by treatment (P=0.05); LPH cows on the Long treatment did not sort for or against short particles (101±1%; P=0.5), while LPH cows on the Short treatment sorted in favour of short particles (104±1%; P<0.001). Depending on treatment, LPH cows sorted the fine particles differently (P=0.01); LPH cows on the Long treatment did not sort for or against those particles (102±2%; P=0.2), while LPH cows on the Short treatment sorted against those particles (93±2%; P=0.002). Sorting was consistent across all weeks. These results suggest that those cows who experienced low reticulorumen pH, and were fed a diet with shorter straw particles, sorted that diet to maximize intake of physically-effective fibre to ameliorate the effects of their low reticulorumen pH.

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

<footnote n="2">²University of Guelph, Population Medicine, 50 Stone Rd East, Guelph, ON, N1G 2W1, Canada;
rcoon@uoguelph.ca</footnote>
## Lowered tail can predict a tail biting outbreak in grower pigs

Helle Pelant Lahrmann¹, Christian Fink Hansen¹, Rick D'Eath², Marie Erika Busch¹ and Björn Forkman³

¹SEGES, Danish Pig Research Centre, Axeltorv 3, 1609 Copenhagen, Denmark, ²Scotland's Rural College, Animal & Veterinary Sciences, West Mains Road, EH9 3JG Edinburgh, United Kingdom, ³University of Copenhagen, Veterinary and Animal Sciences, Grønnegårdsvej 2, 1870 Frederiksberg C, Denmark; hla@seges.dk

Detecting a tail biting outbreak early is essential in order to stop the unwanted behaviour and thereby the risk of pigs getting a tail injury. Previous studies suggest that tail posture and increasing activity level could be used to predict an upcoming outbreak. Therefore, the aim of the present study was to investigate if differences in tail posture and activity could be detected between upcoming tail biting pens (T-pens) and control pens (C-pens) (>7 days away from an outbreak) using video recordings. The video study included 1,745 pigs in 56 pens (mean ± s.d., 31.2±1.5 pigs per pen). Tails were scored three times weekly between 07:00-14:00 h (for wound freshness, severity and tail length) from weaning until a tail biting outbreak. An outbreak (day 0) was defined as 4 or more pigs having tail damage, regardless of wound freshness. On average 9±4.9 pigs had tail injuries in T-pens (50 pens) on day 0. To eliminate the effect of age and housing, an unaffected pen was identified randomly from the same batch and room for use as a control (C-pen, 25 different pens). A C-pen could therefore be paired up with more than one T-pen. Tail posture and activity was recorded in T-pens and in matched C-pens using scan sampling every 30 min. between 08:00-11:00 h and 17:00-20:00 h on day -3, -2 and -1 prior to the tail biting outbreak in T-pens. At each scan sampling the number of active (standing rather than sitting or lying) pigs and their tail posture (curly or hanging) was recorded. Differences in tail posture and number of active pigs between T-pens and C-pens were analyzed using GLM (PROC MIXED, SAS) including fixed effect of group, day, scan period and age-at-outbreak. Differences on day level were estimated using the Least Square Means statement. Tail posture was not affected by scan period (morning/afternoon) or pigs age-at-outbreak, so these were excluded from the model. No difference in activity was observed between T- and C-pens, but more tails were hanging in T-pens than in C-pens on every recording day (Day -3: 23.0% [CI; 20.9-25.2] vs C-pens: 16.7% [CI; 14.6-18.8], t=4.16, P<0.001; Day -2: 24.8% [CI; 22.7-25.2] vs 15.1% [CI; 13.0-17.3], t=6.3, P<0.001 and Day -1: 33.2% [CI; 31.0-35.3] vs 17.4% [CI; 15.2-19.5], t=10.4, P<0.001). No difference in pigs with the tail hanging was observed between day -3 and -2 in T-pens, but more tails were hanging in T-pens on day -1 compared to day -2 (t=-7.58; P<0.001) and compared to day -3 (t=-9.09; P<0.001). In conclusion, although activity did not increase prior to a tail biting outbreak, tail posture might predict a future tail biting outbreak on pen level: Pens which were about to have a tail biting outbreak within the next 3 days had more hanging tails than control pens, and this difference was greater closer to the outbreak.
**Using hen-mounted light sensors to monitor outdoor range use**

Stephanie Buijs, Christine Nicol, Francesca Booth, Gemma Richards and John Tarlton
University of Bristol, School of veterinary sciences, Langford house, BS40 5DU Langford, United
Kingdom; stephanie.buijs@bristol.ac.uk

Studying the ranging behaviour of individual hens is very difficult without the use of an accurate automated monitoring system. To assess the accuracy of a novel light-based monitoring system we compared outputs to direct observations of hen location. Fourteen hens from a commercial flock (British Blacktails, 45 weeks old, total flock size 2,000) were equipped with a device that measured and stored light levels each minute. The device was wrapped in brown tape and fitted to the hen's back using elastic loops around the wing base. Attempting to equip hens that varied in their range use, 7 hens were caught on the range and 7 inside the house. Data were collected 2, 3 and 7 days after equipping. Hen location (in/out) was determined by comparing the hen's sensor to four ambient light sensors placed in the brightest areas of the shed. When the reading of a hen-mounted sensor exceeded that of all inside ambient sensors, the hen was considered to be outside. To avoid collecting data when inside and outside light levels were too similar, two additional ambient light sensors were placed outside, and data were discarded when min(out) <1.1×max(in). This resulted in daily data collection starting between 07.11 and 07.45 and ending between 16.20 and 16.35, corresponding with sunrise and sunset for that time of year (November). In addition, 0-30 minutes of data per day when the sun shone through the popholes (around 08:00 or 14:30) had to be discarded. The accuracy of the system was then evaluated by direct observation of the equipped hens (206 minutes in total). One continuous 5 minute observation was conducted per hen/day, between 10.00 and 15.00 on 3 observation days, balancing individuals over different times within those days. Monitoring and observations were in agreement 92% of the time. The monitoring system indicated that hens originally caught outside spent a much greater percentage of the monitored time on the range than those caught inside (median (Q1-Q3): 42% (40-51) vs 6% (3-17), P<0.001, Wilcoxon rank-sum test). We conclude that the system shows great promise as a tool to monitor range use.
## Housing laboratory mice from the mouse's point of view

Elin M. Weber, Jerome Geronimo, Jamie Ahloy-Dallaire and Joseph P. Garner

Stanford University, Department of Comparative Medicine, Veterinary Service Center, 287

Campus Drive, Stanford, CA 94305, USA; elweber@stanford.edu

Laboratory mice are housed in small, transparent shoebox cages with bedding and small amounts of nesting material. This housing is fundamentally unnatural; it lacks the complexity of a wild environment, preventing many species-typical behaviours. Present cage designs limit what can be added to a cage, so that current enrichment strategies cannot yield anything close to a naturalistic environment. Therefore, instead of refining present housing systems, our goal was to recreate a mouse habitat. We designed a cage from the mouse's point of view, taking into account that mice are mainly nocturnal, thigmotactic, and excellent climbers. Aggression is a serious welfare problem in mouse husbandry and structural enrichment can increase aggression. However, wild mice use structural complexity to escape from aggressive conspecifics. We therefore provided escape routes throughout the cage to stimulate natural behavioural responses, and ran a pilot study to investigate whether housing mice in the complex cage would induce aggression. For 3Rs reduction, we reused mice from a C57BL/6 background that would otherwise be euthanized. Eight groups of four mice were kept in standard cages (SC) for one week before being transferred to the complex cage (CC). Mice were marked, video recorded for three weeks, and checked for wounds every other day in both SC and CC to detect signs of aggression. Data were analysed in JMP using Repeated measures GLMs. Wounds were found in four of the eight groups. No unwounded group started fighting in CC. However, there was a small increase in wounding ($F_{1,233}=45.92; P<0.0001$) that peaked at 6.5 days in CC ($F_{1,233}=50.25; P<0.0001$) and subsequently healed. We observed fighting for 1 h after wound check for four days/housing using continuous recording. No significant differences were found in mean time spent fighting/hour (SC 2.15 min; CC 1.36 min), or mean fight duration (SC 43.9 sec; CC 34.4 sec). The increase in wounds is consistent with that seen after any cage change, but since animals healed, CC does not appear to present a welfare concern in terms of aggression. Location in CC was noted hourly for the first two days; during this period mice used all parts of the cage, but were mainly observed in the two designated resting areas (72% of observations). As soon as 24 hours after being placed in CC, vertical climbing and jumping were observed, and all groups removed food from the feeder and stored it elsewhere. Further behavioural analysis will reveal information on individual behaviour patterns, to investigate if all mice access all parts of the cage or if some mice monopolize certain areas. This caging system is the first to radically redesign mouse housing taking mouse ecology, sensory biology, and behaviour into consideration. The cage also facilitates visual inspection and cage cleaning without handling the animals. These promising early results potentially provide a completely new way of housing laboratory mice in the future.
**Welfare relevance of floor area for single-housed female mink (Neovison vison)**

María Díez-León¹, Samuel Decker², Nora Escribano³, David Galicia³, Rupert Palme⁴ and Georgia Mason¹

¹University of Guelph, 50 Stone Rd E, N1G 2W1 Guelph, Canada, ²Michigan State University, 474 Shaw Ln, 48824 East Lansing, USA, ³University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain, ⁴University of Veterinary Medicine, Vienna, Veterinärpl. 1, 1210 Vienna, Austria; mdiez@uoguelph.ca

Cage size regulations assume larger floor areas improve welfare, as individuals get more space to perform a diversity of behaviours. Canadian regulations for farmed mink have recently increased minimum floor areas, yet these remain smaller than those mandated in Europe. For single-housed adult females – the subpopulation kept on-farm the longest and thus most at risk of experiencing chronic housing effects – the floor sizes of Canadian cages (CC) are almost half those of European cages (EC); a size difference raising concerns that even these new cages underperform in terms of welfare compared to EC. We tested this hypothesis housing 64 pairs of Black females in either EC (2,613 cm²) or CC (1,516 cm²). Each cage housed 2 unrelated females and was provided with an identical nestbox, shelf and 2 enrichments; however, cage size was unavoidably confounded with distance from the aisle. Pairs were split after 3 months (mink being naturally solitary), the remaining 64 females spending 3 more months individually housed in their EC or CC. For 2 weeks at the end of this period we collected data on stereotypic behaviour (SB; % of total activity & % of observations), fearful responses (to handling & in the ‘stick test’), and collected samples for faecal glucocorticoid metabolite (FCM) analyses. Half of our subjects were then humanely killed. *Post-mortem*, we estimated degree of tail chewing, extracted and weighed the thymus, spleen, and adrenal glands, and processed their mandibles for fluctuating asymmetry (FA) analyses to assess housing effects on stress physiology, immune function and developmental stress. The remaining females were then given free access to the cage they had not been raised in. After 2-week habituation, their preference for either cage was assessed (as % of time budget, with chance being a distribution the same as the relative sizes of the floor areas). Results indicate no cage effects on SB or tail chewing, but CC-raised females tended to be more fearful (handling: X²<sub>(dfs.1)</sub>=3.2, P=0.07; stick test: F<sub>1,40.7</sub>=3.1, P=0.08). Stress physiology and immune function were unaffected by cage type (but analyses of FCM & FA are on-going). When a choice of cage was offered, females allocated their overall time equally between EC and CC, but their *active* time was preferentially allocated to the EC (t<sub>31</sub>=9.7; P<0.001), while the reverse was true for their *inactive* time (t<sub>31</sub>=6.1; P<0.001). Our results are consistent with mink preferring larger floor areas for active behaviours; yet their choice to rest more often in CC suggests that relative seclusion and ability to monitor their environment (respectively more likely in CC and EC) may have also played a role in mink’ preferences. Our data cautiously suggest that females housed for 6 months in EC might experience better welfare as they seem to prefer these larger floor areas, particularly when active, and seem less fearful there. This has implications for future cage size regulations.
**Differential effects of enrichment on the subtypes of stereotypic behaviour in mink**

*Andrea Polanco, María Díez-León and Georgia Mason*

University of Guelph, Dept. of Animal Biosciences, 50 Stone Rd East, N1G 2W1, Canada;
apolanco@uoguelph.ca

Stereotypic behaviours (SBs) are common in farmed mink and other captive animals. In carnivores, SBs typically involve whole-body movements (e.g. route-tracing and whole-body bobbing) or head-only movements (e.g. head-twirling). Farmed mink may also repeatedly scratch at the cage walls ('scrabbling'), often in response to the presence of neighbours. Environmental enrichment is commonly used to treat SBs, but typically does not fully eliminate them. We therefore explored whether different subtypes vary in the degree to which they are reduced by enrichment, by assessing enrichment effectiveness on the different SB subtypes of 31 farmed male mink who were individually-housed in standard non-enriched cages from 7 to 12 months of age and then transferred to larger, enriched cages at 14 months old. The amount of change in time spent stereotyping in each subtype was calculated with using two indices: absolute reduction (SB pre-enrichment minus SB post-enrichment) and relative (1 minus SB post-enrichment/SB pre-enrichment) change. Wilcoxon-Signed Rank Tests comparing these metrics for whole-body and scrabbling SB (head-only SB being omitted due to a small *n*) revealed a significant difference in relative change, whole body SBs being reduced more (whole-body SB: Mdn=1, IQR=0; scrabbling: Mdn=0.93, IQR=0.46; z=-2.43, P<0.05), but not for absolute change. Mink were also scored as to whether or not each subtype was 'cured', with 'yes' indicating that the SB was no longer performed post-enrichment, and 'no' indicating continued performance (even if at reduced levels). A logistic regression, with 'cured' as the dependent variable, and SB subtype and mink ID (a random factor) as the independent variables, revealed a trending effect of SB subtype (*x*²=5.32, P=0.07). Specifically, scrabbling was the least likely to be cured by enrichment (P<0.05). Together, the results indicate that scrabbling is less likely to be reduced by enrichment than are other SBs. Thus not all types of mink SB are equally reduced by environmental enrichment, with scrabbling being more resistant to change.
Effects of space allowance and simulated sea transport motion on the behavior of
sheep

Ramazan Col¹,², Grisel Navarro¹ and Clive Phillips¹

¹School of Veterinary Sciences, University of Queensland, Centre for Animal Welfare and Ethics, Centre for Animal Welfare and Ethics, School of Veterinary Sciences, University of Queensland, 4343, Gatton QLD, Australia, ²Faculty of Veterinary Medicine, Selcuk University, Department of Physiology, Department of Physiology, Faculty of Veterinary Medicine, Selcuk University, Campus, 42035 Konya, Turkey; rcol@selcuk.edu.tr

Transporting livestock by ship, especially over long distances, presents significant risks to their welfare. The objective of this study was to assess the effects of space allowance and motion in simulated ship transport on the welfare of sheep. Six sheep (mean weight 25±2 kg) were restrained in pairs in a crate on a programmable platform that generated roll and pitch motion typical of that experienced on board ship for 1 hour/per treatment. Sheep were subjected to movement (regular, irregular motion and control) at Low and High space allowances (SA, 0.26 and 0.52 m²/sheep) in a replicated Latin square factorial design. Irregular movement was programmed as 30 random pitch and roll movements which varied in amplitude (angle of motion) and period (duration of motion). These were selected by software controlling the movement. Regular movement was the mean of these values, which represented approximately 33% of the recommended maximum tolerance for livestock carriers. Behaviour was continuously video recorded by six cameras around the crate. Low space allowance increased the frequency of pushing, but only in the motion treatments (Low regular 38.7, irregular 30.3, control 14.4 events/h, High regular 4.5, irregular 11.7, control 0 events/h, SED 9.30, P=0.04), and increased time spent being aggressive (Low 4.2, High 0.3 s/h, SED 1.18, P=0.04), affiliative behaviour between the sheep, i.e. one head above the other (Low 43.8, High 6.5, SED 32.89, P=0.02) and standing supported by the crate (Low 449, High 122 s/h, SED 181.4, P<0.0001). Sheep in the Low Irregular treatment spent least time with their head up (124 Vs mean 584 s/h for other treatments, SED 269.5, P=0.008). Irregular movement reduced rumination (864 vs Control 1,776 and Regular 1,208 seconds/hour, Standard Error of Difference 432.7, P=0.02) and increased pawing (20 Vs Control 4 and Irregular 10, SED 13.4, P=0.05) and sniffing (31 Vs Control 3 and Irregular 9 events/h, SED 12.8, P=0.003). Thus there was evidence that low space allowance increased interaction between sheep and that it increased agonistic behaviour more during simulated sea motion than in a static, control situation.
# Cooling cows with soakers: effects of flow rate and spray frequency on behavior and physiology

Grazyne Tresoldi¹, Karin E. Schütz² and Cassandra B. Tucker¹

¹Center for Animal Welfare, Department of Animal Science, University of California, Davis, One Shields Avenue, Davis CA, 95616, USA, ²AgResearch, Ruakura Research Centre, 10 Bisley Road, Hamilton, 3240, New Zealand; gtresoldi@ucdavis.edu

Soakers reduce heat load in cattle, however, spray strategies (e.g. flow rate and spray frequency) may affect how cows use this resource and cooling efficiency. Our objective was to evaluate the combined effects of spray frequency (3 min on, 6 min off or 1.5 min on, 3 min off) and flow rates (3.3 or 4.9 l/min) on behavioral and physiological responses to heat load in dairy cows managed in a free-stall barn. In a 2×2 Latin square design, 12 pairs of Holstein cows averaging (±SD) 36±5 kg/d of milk were tested 3 d/treatment when air temperature and relative humidity averaged 23±2 °C and 49±7%, respectively. Water was sprayed at the feedline from 08:15 to 23:30 h (average air temperature ≥19 °C). The overall quantity of water sprayed was not affected by spray frequency; it varied as a function of flow rate (lower: 1,040 vs higher: 1,544 l of water/d). Cows' posture and location within the pen were measured continuously while body temperature was recorded every 3 min 24 h/d. Respiration rates were recorded every 45 min daily from 09:00 to 20:00 h. Data were analyzed with mixed models (SAS) using 24 h averages. Preliminary results indicate that time spent lying down and at the feed bunk were affected by flow rate (P≤0.04), but not by spray frequency (P≥0.35). Under the higher flow rate, cows spent 42 min longer lying down (mean ± SE: 12.1 vs 12.8±0.5 h/d), and used the feed bunk area for 30 min less (5.7 vs 6.2±0.4 h/d). Respiration rate averages were not different among treatments (57 to 59±3 breaths/min, P≥0.22). Although body temperature tended to be reduced when spraying water at higher flow rate (P=0.08) it was within a narrow range, regardless of treatment (38.6 vs 38.7±0.1 °C). Cows adjusted their behavior in response to spray strategies provided. They used the lower flow spray for longer to maintain similar levels of cooling, comparable to those provided by higher flow rates. Spray frequency (when controlling for water volume) did not affect physiological responses or behavior in cattle. Although physiological responses to heat load were not affected, our preliminary behavioral results indicate that other aspects of cow comfort (e.g. less time standing on concrete in front of the feed bunk) are influenced by the cooling strategy used.
## Nesting behaviour of broiler breeders

Anne Van Den Oever¹,²,³, Bas Rodenburg¹,², Liesbeth Bolhuis², Lotte Van De Ven³ and Bas Kemp²

¹Wageningen University, Behavioural Ecology Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ²Wageningen University, Adaptation Physiology Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ³Vencomatic Group, Meerheide 200, 5521 DW Eersel, the Netherlands; anna.vandenoever@wur.nl

Broilers have been selected for growth related characteristics, which are negatively correlated to reproductive traits. This genetic background creates challenges in broiler breeders, as the hens do not make optimal use of the nests provided leading to dirty and cracked eggs. Despite the economic importance of this problem, little research has been done on nesting behaviour in broiler breeders. This project aims to investigate which factors are involved in nest site selection of broiler breeder hens and to create practical improvements in housing design. The first experiment focuses on nest design preferences, where three alternatives to a standard nest will be offered to groups of hens. This preference will then be related to the personality traits sociability and fearfulness, measured with density related permanence and tonic immobility tests. The second experiment will compare two genetic strains on leg health and social interactions. The effect of introducing perches at an age of 20 weeks on leg health, measured by scoring for footpad dermatitis and gait, will be investigated. Leg health will furthermore be related to mobility, fertilisation and production. Social interactions amongst females and between females and males are expected to influence nest site selection as well. Dominant females might attract submissive females to certain nests, but can also prohibit submissive females access to the nests. Feral males are known to escort females to suitable nest sites, although it is unknown whether this holds true for domestic chickens. By scoring social interactions and nest site, these possible relations can be studied. A final field experiment will be conducted at a commercial farm, fitted with nests designed according to the preferences of the first experiment and with or without perches as proven optimal according to the second experiment. Productivity, fertilisation, leg health and behaviour will be recorded to measure the performance of this improved system.
Laying hens' behavioural response to wearing automated monitoring equipment

Francesca Booth, Stephanie Buijs, Christine Nicol, Gemma Richards and John Tarlton
University of Bristol, School of veterinary sciences, Langford house, BS40 5DU Langford, United
Kingdom; francesca.booth@bristol.ac.uk

Laying hens often respond aggressively to flock members with even minor alterations in appearance, causing altered hens to flee, fight or hide. This can be problematic when equipment for automated behavioural monitoring is attached to hens, as the data supplied will no longer be representative of these individuals' normal time budget. We evaluated the effect of 50 g 'backpacks' on behaviour using direct observation. These 'backpacks' contained two monitoring devices (a light sensor and an accelerometer) and a location device. The equipment was wrapped in brown tape and fitted to the hen's back using elastic loops around the wing base. Fourteen hens (British Blacktails, 45 weeks old) were equipped. These hens were housed on a commercial free-range farm in a flock of 2,000 hens. Behaviour of equipped and non-equipped hens was observed 2, 3 and 7 days after equipping (5 min/bird/day, alternating between equipped and non-equipped hens) and compared using Wilcoxon rank-sum tests. None of the equipped hens were ever observed to flee or fight. Although equipment tended to increase the frequency of pecks received on day 2 and 3 (P=0.08 and 0.05, respectively) compared to non-equipped hens, median values were 0 for both groups and differences in interquartile range were small (0-0 vs 0-0.3 and 0-0 vs 0-0.6 pecks/minute, respectively). Equipped hens generally showed no reaction to these pecks (which included all types of pecks except severe feather pecks). Equipped and non-equipped hens were not found to differ significantly in the proportion of time spent eating/drinking, foraging, perching, preening, sitting, standing, walking or in the nestbox, or in their frequency of body-shaking and pecking other hens (all P-values >0.05 for each day). Although further evaluation in other flocks is required, this system seems to have only a very minor impact on behaviour, confirming its suitability for collecting automated behavioural data.
**Effects of stocking density and exercise on incidence of Tibial dyschondroplasia in broilers**

Elmira Kabiri¹ and Ahmad Tatar²

¹Behin Toyour Golestan Aviculture Co., Farhang St. Balajade village, Kordkooy, Golestan province, 4883917893, Iran, ²Ramin Agriculture and Natural Resources University of Khuzestan, Animal Science, Khuzestan, Mollasani, 6341773637, Iran; tatar@ramin.ac.ir

Tibial dyschondroplasia (TD) is the most prevalent skeletal abnormalities associated with rapid growth avian species. It results in deformed bones and lameness. The economic cost of TD as manifested in the rates of mortality, morbidity and condemnation at the processing plant, is enormous as these rates can reach up to 30% of a flock. The exact etiology of TD is unknown. TD is common in fast growing birds, especially in male broilers in which incidence can be high as 30%. Although, rapid growth and other factors like genetic background are contributing factors in the development of TD, they are not the primary factors. Therefore, TD may relate to genetic, nutritional and environmental factors. Placing barriers or ramps within the pen caused an increase in the mass dimensions of broiler breast and leg muscle, indicating that exercise could be encouraged by altering pen design and that such addition to the pen can improve the overall physical condition of the birds. On the other hand, increasing stocking density has been shown to decrease body weight, increase feed conversion and reduce activity level. Reduced activity has also been implicated as a factor involved in the development of leg problems in broiler chickens. In addition to providing birds with exercise, perches in the environment of broiler chickens may improve welfare by providing them with an opportunity to perform a natural behavior and allowing them to exert some control over their environment.
**Effects of enriched environments with perches and dust baths on behavioural patterns of slow-growing broiler chickens**

Hanh Han Quang, Xuan Nguyen Thi and Ton Vu Dinh

Vietnam National University of Agriculture, Faculty of Animal Science, Trau Quy town, Gia Lam district, Hanoi, Vietnam, 10000, Hanoi, Viet Nam; hanquanghanh1304@gmail.com

The present study was conducted to determine the effects of environmental enrichment with perch and dust bath provision on behavioural patterns of slow-growing broiler chickens. A total of ninety-two local crossbred chickens (Ho × Luong Phuong) were distributed equally into two housing systems, a conventional, totally confined housing system, and an enriched, semi-confined housing system with perch and dust bath availability, with two groups (n=23) per housing system. Chickens' behavioural patterns were observed directly by one assessor by instantaneous scan sampling and the number of birds performing different behaviours were recorded in the morning and afternoon or evening from 6 to 13 weeks of age. The Chi-square analysis with Fisher's test was applied to compare the percentage of birds' behaviours between two treatments by Minitab® 16.0. Chickens at the age of 10 to 13 weeks preferred perching at the resting time during the night with a significant higher percentage of birds perching in the evening than that at daytime (P<0.05). Chickens also showed a low percentage of dust bathing behaviour both in the morning and the afternoon (less than 10%) and it were not significantly affected by age (P>0.05). The percentage of birds that showed the play fighting behaviour was low and not different between two housing systems. However, chickens that had outdoor access were more fearful of human (with a significant higher avoidance distance of birds to human, P<0.05) than those in a totally confinement house. Provision of perches and dust baths helps stimulates the expression of several natural behaviours of broiler chickens.
**Farmers' perception of pig aggression and factors limiting welfare improvements**

Rachel S.E. Peden¹, Simon P. Turner¹, Irene Camerlink¹ and Laura Boyle²

¹Scotland's Rural College, Animal Behaviour and Welfare, SRUC, Roslin Institute Building, Easter Bush, Roslin, EH25 9RG, United Kingdom, ²Teagasc, Pig Development Department, Animal & Grassland Research and Innovation Centre, Moorepark, Cork. P61 C997, Ireland; rachel.peden@sruc.ac.uk

Mixing of unfamiliar pigs is common practice in pig husbandry, creating high levels of aggression related to the formation of dominance relationships. Many strategies to reduce aggression have been identified by research but few have been adopted in practice and only by a minority of farmers. As a consequence, aggression remains undiminished in commercial farming. In order to improve welfare by reducing aggression, farmers would have to alter their behaviour and practice regarding pig management. Farmer willingness to change current practice relies strongly on their perception of the situation. Our objective is to approach the welfare problem of pig aggression from the perspective of the industry, focussing on the farmer. UK pig farmers were asked about their management and perception regarding aggression through a postal survey. Of the 167 respondents 73% did not consider aggression at weaning a welfare problem, and 57% did not consider it a problem for grower-finishers. Farmers who did consider aggression a problem for grower-finishers were on average younger than respondents who did not consider aggression a problem (P=0.02). When farmers were asked about pre-weaning co-mingling of adjacent litters, the most researched and one of the most promising methods to reduce aggression, 48% had concerns about the practical management of this technique. If solutions to reduce aggression between pigs were available, 20% of the respondents would consider adopting these methods to manage aggression at weaning, and 37% would do so at the grower/finisher stage. In presenting the results of this research along with data collected in early 2017, we will highlight the contribution of various factors that may limit farmer willingness to change current practice, such as desensitisation, prioritisation of issues, and financial barriers. Knowledge of farmers' perception of aggression, and their perception of proposed strategies to reduce aggression, may help align research questions in this area and find ways to effectively address the issue in a manner that can be adopted by practice.
**Behaviour and welfare of cull sows on the day of slaughter – current knowledge and possible ways forward**

Mette S. Herskin, Katrine Kop Fogsgaard and Karen Thodberg
Aarhus University, Department of Animal Science, P.O. Box 50, 8830 Tjele, Denmark;
mettes.herskin@anis.au.dk

In recent years, international pig production has been characterised by increasing herd sizes and changes in the slaughter industry towards fewer and larger units. Consequently, the needed transport distances from farm to slaughter are increasing. Across animal groups, this development may have welfare consequences. Especially one group of pigs to be transported – cull animals – may be vulnerable towards transport stress and thus pay a price for the current development in the pig industry. However, despite the large proportion of sows being culled from modern production systems each year, the behaviour and welfare of these animals have received almost no scientific attention. For sows in modern production systems, the typical pre-slaughter logistic chain consists of a series of consecutive potential stressors experienced by the animals on the day of slaughter. Sows may be culled as part of a planned strategy, but for the majority of the animals the decision to cull is a consequence of reduced reproduction, injuries or other health issues. Results from our recent study showed that 40% of cull sows were culled directly at weaning. In Denmark, the cull sows may be loaded directly from their home pen or be mixed with other sows and kept in so-called pick-up pens until they are picked up by the truck. In any case, the sows may be mixed with other sows in a transfer vehicle for biosecurity reasons, and either via very short lasting transit, or a stay for up to two hours, pass this vehicle before being picked up by the commercial truck. Danish law specifies that sows can only be transported for up to 8 hours, whereas sows from other parts of the world may be transported considerably longer, and may pass buying stations or similar before they arrive at the slaughterhouse. Results from our recent study showed that the clinical condition of cull sows, such as number of scratches and measures of dehydration, were worsened by commercial transport for up to 8 h. Among the most important risk factors for the clinical worsening were transport duration and temperature. In this presentation, we review the following phases of the pre-slaughter logistic chain for sows: (1) pre-transport in home pen; (2) on-farm pick-up pens; (3) transfer vehicles; (4) transport; and (5) lairage. Across phases we provide an overview of the available knowledge about behaviour and welfare of sows on the day of slaughter, and discuss possible needs for changes in order to secure the welfare of sows while they pass the pre-slaughter logistic chain.
**Clinical consequences of keeping cull sows in on-farm pick-up pens before transport to slaughter**

Cathrine Holm, Karen Thodberg and Mette S. Herskin

Aarhus University, Animal Science, P.O. Box 50, 8830, Denmark; holmen_lojt@hotmail.com

In modern pig production up to 50% of the sows are slaughtered each year. Typically, the period from the decision to cull and until a sow is slaughtered includes a stay in an on-farm pick-up pen (hours to weeks), transport (potentially passing buying stations or similar) and lairage. However, even though cull sows are often mentioned as vulnerable towards challenges such as transport, the behaviour and welfare of these animals have received almost no scientific attention. The aim of this study was to examine clinical consequences of keeping cull sows in on-farm pick-up pens before transport to slaughter. The observational study was conducted in 2016 in a private Danish sow herd with loose-housed gestating and lactating crossbred LYD-sows. As part of the farm management, the farmer decided which sows to cull. The study included all cull sows, except if they were recently medicated, severely lame or showed clinical signs of illness. The dataset consisted of 103 sows with a mean parity of 5.2 (range 1-9) and a BCS of 2.9 (range 2-3). At weaning (after 28 days of lactation), all sows to be included in the study were examined clinically, and then walked to the pick-up pens in another part of the same building. Each pick-up pen holding three sows was 4.15×3.25 m with 50% slatted floor and 50% concrete floor. A chained wooden log was provided as manipulable material. The sows had free access to water from a watercup and were fed twice a day on the floor with a total 2.5 FU per pen. The mean temperature was 21.0 °C (range 18-25.6). The sows were kept in the pens until they were picked up by a commercial truck and transported to slaughter. After 24 h in the pick-up pens the clinical condition of the sows showed signs of worsening (examined by Signed Rank Test), as judged by an increased number of scratches (counted on 6 body parts with a maximum number of 15 per part): median 0 (range: 0-22) vs 9 (0-75); P<0.001) and increased gait score (0-4): median 0 (range: 0-1) vs 0 (0-2); P<0.001) as well as a tendency to an increase in the number of wounds (P=0.0625). No relation between the changes in the clinical condition and the temperature during the stay in the pens was found. These results suggest that a 24 h stay in a pick-up pen may be a challenge to the welfare of cull sows and hence potentially affect their fitness for transport. Further studies are needed in order to understand effects of a stay in a pick-up pen as well as other phases of the pre-slaughter logistic chain on welfare during the day of slaughter and to understand possible links with fitness for transport.
**Development of an indoor positioning system for automatic monitoring of locomotor behaviour of group-housed sows**

Shaojie Zhuang<sup>1,2</sup>, Olga Szczodry<sup>2,3</sup>, Jarissa Maselyne<sup>2</sup>, Johan Criel<sup>4</sup>, Frank Tuyttens<sup>2,3</sup> and Annelies Van Nuffel<sup>2</sup>

<sup>1</sup>Ghent Univeristy, Faculty of Bioscience Engineering, Coupure links 653, 9000 Gent, Belgium,
<sup>2</sup>Institute for Agricultural, Fisheries and Food Research (ILVO), Burg. Van Gansberghlaan 92, 9820 Merelbeke, Belgium, <sup>3</sup>Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium, <sup>4</sup>Sensolus NV, Kortrijksesteenweg 930, 9000 Ghent, Belgium;
olga.szczodry@ilvo.vlaanderen.be

At times when the numbers of animals per farm are rising, it becomes acknowledged that precision livestock farming (PLF) offers promising tools for improving the welfare of the animals by automatically monitoring, among other variables, their behaviour. Several PLF techniques involve sensors that are capable of continuously collecting data at individual level, thereby offering possibilities for ‘management-by-exception’. Our aim was to develop a positioning system that is customizable and accurately tracks animals indoor. For the latter purpose we performed (1) static tests, where position data were collected at 64 predefined locations (approx. 120 s per location, sampling rate 1 Hz, tag height of 1.00 m) and (2) dynamic tests where an investigator walked on a predefined path at a speed of 0~1.50 m/s (1 Hz, 1.00 m tag height). Coordinates of tags were estimated based on tag-to-receiver distances and their accuracy was quantified by their root mean squared distance to the true position. Our preliminary results show that, in static tests, the overall root mean square error (RMSE) of estimated positions is 0.33 m, where 92.2% of test locations have error <0.50 m. The data loss is 3.1%. For the dynamic tests, the RMSE is 0.35 m and 92.6% of the estimates have an error <0.50 m. The data loss is 7.0%. The present results show potential for automatically and continuously collecting position data on animals housed indoor. This offers a wide variety of applications, as for instance, automatic monitoring of locomotor behaviour of sows *via* the estimation of different variables, such as distances covered, walking speed and diversity of areas visited in the pen. Since it has been shown that the latter variables are associated with lameness, the present system has the potential to be used for automatic surveillance of, *inter alia*, lameness status in group-housed gestating sows. This system is currently under development for such an application.
**Exploratory behavior in commercial and heritage breed swine**

Kristina M. Horback¹,² and Thomas D. Parsons²

¹University of California Davis, Department of Animal Science, 1 Shields Avenue, 95616 Davis, California, USA, ²University of Pennsylvania School of Veterinary Medicine, Swine Teaching and Research Center, 382 West Street Road, Kennett Square, PA 19348, USA; thd@vet.upenn.edu

Sows on most commercial farms are the product of intensive breeding programs focused on an ensemble of productivity traits; rarely with any explicit consideration of behavioral responses. This study was designed specifically to see how juvenile behavioral responses to stress-inducing contexts differed between commercial genetics and less intensively selected heritage breed swine. Four neonatal Tamworth litters were fostered onto resident commercial sows (PIC 1050: Landrace-Yorkshire cross) at birth to minimize maternal influences of breed during lactation. Data collection of 14 Tamworth gilts (TAM) and 33 commercial breed gilts (CBG) occurred at processing (1 day old) and 3 weeks old. Measurements recorded include weight (kg), response to restraint via human handling and open field tests. Data were analyzed using SPSS 17.0 with a significance level set of P<0.05. Given that the data was not normally-distributed, differences between breeds were calculated using Mann-Whitney U tests and data is presented as median [IQR]. TAM piglets weighed more (P<0.01) at birth (1.7[0.3] kg) and at 3 weeks old (6.8[1.3] kg) than the CBG piglets (birth: 1.4[0.3] kg, week 3: 5.1[2.5] kg). TAM piglets received higher scores (P<0.05) for the restraint test at 3 weeks old, indicating that this breed showed more resistance to being handled (thrashing and squealing) than CBG. At 3 weeks old, each piglet was given a 5 min open field test (3.4 m² arena), beginning with a 30 s rest in a closed off start box. TAM piglets were quicker (P<0.05; TAM: 13.4[22.2]s, CBG:25.9[43.4]s) to leave the start box, and rested for a longer duration (P<0.001) while in the open field (TAM: 127.2[55.7] s, CBG: 71.1[82.7] s). In contrast, the CGB spent a greater duration nosing the experimental field (P<0.05; CBG: 176.0[96.5] s, TAM: 144.5[65.5] s) and produced longer durations (P<0.05) of high-frequency squeals while in the open field (CBG: 200.6[69.8] s, TAM: 150.6[89.9] s). While TAM were quicker to leave the start box, they moved and squealed less while in the open field. In comparison, the CBG spent more time nosing the ground and walls of the unfamiliar arena and squealed more. These findings demonstrate that TAM and CBG piglets can exhibit different behavioral responses to stress-induced contexts. Exactly how or if these juvenile behavioral differences translate to different coping strategies in adult sows will require additional study. However these apparent differences could contribute to variation in the ability of individual animals to cope with the potential stressors such as the social complexity found on loose housed sow farms and thus have welfare implications for these animals.
**Effect of space allowance and flooring on pregnant ewes' behavior**

Stine Grønmo Vik, Olav Øyrehagen and Knut Egil Bøe

Norwegian University of Life Sciences, Department of Animal and Aquacultural Sciences, P.O.

Box 5003, 1432 Ås, Norway; stine.vik@nmbu.no

Space allowance recommendations for pregnant ewes vary considerably. In Norway there is no regulations for space allowance and the majority of ewes are housed on expanded metal flooring with 0.75 to 0.90 m²/ewe. To investigate the effect of space allowance and type of flooring on the behaviour of ewes, a 3×2 factorial experiment was conducted with 0.75, 1.50 and 2.25 m²/ewe and straw bedding and expanded metal flooring. Six groups with 8 ewes in each group were exposed to each treatment for 7 days. The ewes were video recorded for 24 hours at the end of each treatment period and general activity, lying position in the pen and social lying position were scored every 15 min. Displacements and aggressive interactions were scored continuously from 10:30 h to 14.30 h. Mean values per group were used as statistical units. Increasing space allowance from 0.75 to 1.50 m²/ewe resulted in increased lying time, more simultaneous lying and less aggressive behaviour. Sitting was only observed in the 0.75 m²/ewe treatment and is most likely an effect of difficulties to lay down in such a restricted area. Increasing space allowance further to 2.25 m²/ewe had no significant effect except from increased distance between ewes when lying. Type of flooring had no significant effect on general activity, but ewes in the straw bedding treatment spent more time lying in the middle of the pen than ewes on expanded metal. In conclusion, increasing space allowance from 0.75 to 1.50 m²/ewe had positive effects on activity and behavior in pregnant ewes, but increasing space allowance further to 2.25 m²/ewe had limited effects as well as type of flooring. Hence, recommended space allowance to pregnant ewes should be as close to 1.50 m²/ewe as possible.
# Motivation of dairy cows to enter a milking robot: effects of concentrate type and amount

Julie Cherono Schmidt Henriksen, Martin Riis Weisbjerg and Lene Munksgaard
Aarhus University, Animal Science, Blichers allé 20, 8830 Tjele, Denmark;
[juliec.henriksen@anis.au.dk](mailto:juliec.henriksen@anis.au.dk)

Milk yield is affected by milking frequency and is thus dependent on cows' motivation to go to the robot voluntarily. If this motivation changes then both yield but also the time budget of the cow may be affected. The aim was to investigate the effects of the type and amount of concentrate on milk yield and behaviour in a 2x2 crossover factorial design. Concentrate S was pelleted including wheat, and the other type, O, was a mix of pelleted concentrate and steamrolled, acidified barley. The amount of concentrate was changed from 3 kg to 6 kg, or opposite. The experimental period was divided into two periods with half of the cows receiving type O and the other half receiving type S during the first period. All cows shifted to the other type of concentrate during the second period. Within period, half of the cows were first offered 3 kg, and the other half 6 kg of concentrate, then after one week, the concentrate offer for each cow was changed to 3 kg or 6 kg over 6 days with 0.5 kg per day. The data were recorded during the following week of constant concentrate offered. The 83 cows (42 Danish Jersey, 41 Danish Holstein) included in the experiment were balanced between treatments according to breed, parity and lactation stage. Data were analysed using a proc mixed model in SAS with treatment, parity, breed and interactions as fixed factors, cow as random effect and days in milk as covariate. Day within cow and week was regarded as repeated observations. Contrasts were made where interactions were significant. The frequency of both number of daily milking (3.0 vs 2.8, P<0.001) and visits in the robot (3.4 vs 3.2, P<0.001) was higher when offered type O vs type S, while there was no effect of changed amount of concentrate on daily milking or visits (P=0.25; P=0.78). In both breeds, energy-corrected milk yield was higher when offered type O (36.7 vs 35.8 kg/day, P=0.001) and when offered 6 kg of concentrate (36.6 vs 35.9 kg/day, P=0.034). For the *ad libitum* offered mixed ration on the feed alley, Danish Jersey had a shorter eating time when offered type O vs type S (155 vs 161 min/day, P=0.026) while Danish Holstein ate for longer time when offered type O (200 vs 194 min/day, P=0.05) in the robot. For both breeds, the eating time of the mixed ration decreased when the cows were offered 6 kg compared to 3 kg of concentrate (182 vs 173 min/day, P<0.001). However, this difference may have minor biological significance. The daily lying time for the Danish Jersey cows did not differ between type of concentrate (O vs S) (10.2 vs 10.2 h/day, P=0.089), but the Danish Holstein spent less time lying when offered type O vs type S (11.1 vs 11.8 h/day, P<0.001). In conclusion, both the type and amount of concentrate affected the daily time budget, but only the type of concentrate affected the motivation to visit the milking robot in this study.
**Relationship between daily lying time and weight during lactation in Holstein and Jersey cows**

Lene Munksgaard¹, Julie Cherono Schmidt Henriksen¹, Martin Riis Weisbjerg¹ and Peter Løvendahl²

¹Aarhus University, Animals Science, Blichers Allé 20, Postbox 50, 8830 Tjele, Denmark, ²Aarhus University, Molecular Biology and Genetics, Blichers Allé 20, Postbox 50, 8830 Tjele, Denmark; lene.munksgaard@anis.au.dk

Sufficient lying time has high priority in dairy cows, and devices for automatic recording of lying behaviour on commercial farms is now available. New results suggest that daily lying time changes dramatically during lactation. However, to take advantage of information about lying behaviour we need to know more about how breed, stage of lactation and parity affects lying behaviour. We hypothesized that the relatively large changes in weight during early lactation relate to lying behaviour. Therefore we investigated the relation between daily lying time and weight changes during lactation in Holstein (69 first parity, 87 with average parity 2.77±0.95) and Jersey cows (52 first parity, 86 with average parity 3.05±1.09) kept in a loose housing system with cubicles and automatic milking. Live weight was recorded whenever the cows were entering the robot and an average daily weight was calculated. Lying time was recorded with a leg based sensor (AfiTagII, AfiMilk, Israel). Data from day 4 to day 250 in lactation were included in the analysis. A Wilmink function was fitted to data based on each breed and parity, and slopes of the individual animals were included as random effects. Correlations between random solutions for lying time and weight were estimated. Throughout lactation Jersey cows were spending less time on lying than Holstein (638 vs 707 minutes / day, P<0.0001). In both breeds the profile of lying time during lactation was substantially different between first and later parity cows. In first parity lying time increased until day 100 in Jersey and day 125 in Holstein cows, and stayed afterwards reasonably constant until day 250. In older cows the lying time decreased reaching a nadir approximately 30 days in milk. In contrast to first parity, lying time for older cows increased steadily after nadir until day 250. In both first parity and older cows the weight decreased in early lactation and then increased. Thus the weight change and lying time followed the same pattern over lactation in older cows, but not in first parity cow. Pearsons correlations between weight change and change in lying time estimated by the Wilmink function were weak (numerical <0.25) and non-significant in both first parity and older cows and in both parts of the lactation. In conclusion, lying time in older cows follow the same pattern as the energy balance with a drop during the first month of lactation, however we found only weak correlation between weight change and change in lying time at the individual cow level.
# Effects of stocking density on bedding quality and indicators of cow welfare in hybrid off-paddock facilities

Cheryl O'Connor, Suzanne Dowling and Jim Webster

AgResearch Ltd, Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New Zealand;
cheryl.oconnor@agresearch.co.nz

The dairy industry in New Zealand is pasture-based, but hybrid off-paddock facilities where cows spend part, or all, of the day are becoming more commonplace. It is important to define good practice and to understand the welfare risks associated with these hybrid off-paddock systems. One of the key knowledge gaps is the effect of stocking density on bedding quality and lying time within an off-paddock environment. One hundred and thirty-six non-lactating dairy cows were divided into replicated groups of two commonly-used stocking densities of 28 (7.85 m²/cow) and 39 (5.6 m²/cow) cows. Cows were kept on a pad of post-peelings for 18 h/day, with 6 h/day on pasture, for 30 days during winter. Lying times were recorded continuously for all cows using Onset Pendant G data loggers and analysed using a mixed model smoother. Gait, dirtiness and body condition scores were recorded once a week. Bedding quality measures, moisture content, a visual description of the ratio of post-peelings to muck and a farmer-friendly 'Gumboot score' (the depth that a person standing in boots sinks into the muck, on a scale of 0-3), were taken weekly from four 25x40 cm quadrat samples across each pad. Bedding quality and cow measurement data were analysed using a repeated measures mixed model in Genstat. Daily lying time declined during the trial from 11.7 h/day to 4.7 h/day (SED=0.19; P<0.001). There was heavy rainfall in the last week of the trial, and concurrent troughs in daily lying time. On rainy days, in particular, most of the herd lay for less than the industry minimum recommendation of 8 hours/day. The lying pattern of the herd also changed with more cows lying down sooner, and for longer on pasture. There were no significant effects of stocking density on any of the measures except the mean cow dirtiness score at week 3, and lower rear limb dirtiness overall, which were higher at the higher stocking density. The declines in lying time and bedding quality tended to be earlier and greater at the higher stocking density. Over the 30 days of the trial, bedding moisture content increased from 64 to 86% (P<0.001). The 'gumboot score' and visual percentage muck score also increased significantly over the trial period (P<0.001). The moisture content was over 80% for all groups when daily lying times were less than the recommended 8 hours/day. A combination of visual and gumboot scores provided a good indication of moisture level. When cows are kept in hybrid off-paddock facilities, we recommend that farmers use bedding moisture content indicators and lying behaviour patterns at pasture as convenient comfort indicators for cow welfare.
The effect different tie-rail placements have on ruminating and feeding behavior of
dairy cows housed in tie-stalls

Jessica St John¹, Elsa Vasseur¹ and Jeff Rushen²

¹McGill, Animal Science, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, H9X3V9, Canada, ²University of British Columbia, Dairy Education and Research Centre, 6947 #7 Lougheed Highway, Agassiz, BC, V0M1A0, Canada; jessica.st-john@mail.mcgill.ca

Although the majority of dairy farms in Canada utilize tie-stall housing there is little information on cow comfort based on experimental studies for cows housed in tie-stall barns. From epidemiological studies, potential inadequacies for the current recommendation were observed for tie-rail placement which may lead to reduced cow welfare. With the implementation of animal care assessments through certification programs there is also a need to test and potentially develop new recommendations to help farmers improve dairy cow welfare. The objective of this study was to develop new recommendations for tie-rail placement combining both vertical and horizontal positions of the tie-rail. Four treatments were tested: two new tie-rail positions that follow the neck line of the cows (Neckline1 and Neckline2), current recommendation as a control (Current Recommendation), and the tie-rail position most commonly found on farm (Common on Farm). Forty-eight cows (12 cows per treatment) were divided into two separate start dates and were blocked by number and stage of lactation then randomly allocated to a treatment over a 10-week period. Ear-mounted data loggers were used to continuously recorded rumination and feeding time and the loggers reported the data as percentage of time spent ruminating/feeding per hour. Differences between/within treatments overtime were analyzed using a mixed model where treatment, week, start date, and block were fixed effects and cow was a random effect. Multiple-comparisons with a Scheffé adjustment was performed to analyze differences among the main effects of treatment and week. On average cows housed in the Neckline1 treatment were found to spend 8.5% less time per hour feeding than cows in the Current Recommendation treatment (P<0.01), and up to 4.2% less time per hour feeding than cows housed in the Common on Farm and Neckline2 treatments (P<0.01). Cows in the Current Recommendation treatment spent up to 5.2% more time per hour feeding than cows in the Common on Farm and Neckline2 treatments (P<0.01). The average percentage of time per hour spent ruminating for week 9 (40.1±0.86%/h) was significantly different from week 5 (44.4±0.86%/h; P<0.01), week 6 (43.5±0.86%/h; P<0.05) and week 7 (43.0±0.86%/h; P<0.05). A difference in average time spent ruminating was also found between week 8 (41.2±0.86%/h) and week 5 (44.4±0.86%/h; P<0.05). Results suggest that tie-rail placement may have an effect on time spent feeding but not time spent ruminating.
**Characterizing changes in feeding behaviour of lactating dairy cows during the peri-oestrus period**

Hawar M. Zebari, S. Mark Rutter and Emma C.L. Bleach

Harper Adams University, Department of Animal Production Welfare and Veterinary Services, Edgmond, TF10 8NB Shropshire, United Kingdom; hzebari@harper-adams.ac.uk

The normal time budgets of dairy cows are influenced by oestrus, with cows spending less time resting and eating but more time walking. Cows have been shown to spend approximately 21% less time feeding where the day of oestrus is assumed to be the day of successful artificial insemination. The objective of the present study was to determine whether dry matter intake, feeding duration and the number of visits to feed were affected by behavioural and silent oestrus in lactating dairy cows. Thirty Holstein Friesian cows were used for the study (initial bodyweight 637.2±59.9 kg (mean ± SEM) and 29.6±6.2 days postpartum) at the Harper Adams University dairy unit. Cows were housed in a free-stall barn with 34 cubicles (2.7×1.2 m) and were observed for signs of oestrus during three 30 minute periods (07:30, 12:30 and 19:30) daily. Milk samples (40 ml) were collected on Monday, Wednesday and Friday afternoon and stored at 4 °C until analysis for progesterone concentration by enzyme immunoassay. A cow was considered in behavioural oestrus when points scored at three consecutive observations was >50 and progesterone concentrations were <2 ng/ml, followed by an increase to >15 ng/ml. A cow was considered in silent oestrus when the score was <50 points, during oestrus as defined by milk progesterone profile. Daily dry matter intakes, feeding duration and the number of visits to feed by cows were recorded by a Roughage Intake Control system (Insentec B.V., Marknesse, the Netherlands). The data were analysed by repeated measures ANOVA (GenStat 17th edition). The Animals Experimental Committee of Harper Adams University gave ethical approval for the study. On the day of oestrus, dry matter intake (19.8±0.41 kg/d), feeding duration (2.6±0.09 h/d) and the number of visits to feed (25.3±1.26 visits/d) were significantly lower (P<0.001) compared to 3 days before (22.4±0.41 kg/d, 3.4±0.17 h/d, 28±1.8 visits/d) and 3 days after (22.6±0.5 kg/d, 3.2±0.12 h/d, 27.9±1.23 visits/d). During behavioural oestrus, dry matter intakes, feeding duration and the number of visits to feed were 19.8±0.41 kg/day, 2.6±0.09 h/d and 25.3±1.26 visits/d, respectively compared to silent oestrus, 20.0±0.61 kg/day, 2.9±0.15 h/d and 26.1±1.5 visits per day, respectively. On the day of silent oestrus, only duration of feeding was significantly reduced (P<0.03) compared to one day before (3.4±0.2 h/d) and one day after (3.5±0.2 h/d) oestrus. There was no significant interaction between oestrus expression and time with regard to dry matter intake, feeding duration and number of visits to feed. In conclusion, although dry matter intake, feeding duration and the number of visits to feed were reduced by behavioural oestrus, only feeding duration was significantly lower during silent oestrus.
**Inclusion of feeding behaviour to improve the accuracy of dry matter intake prediction in dairy cows**

Guilherme Amorim Franchi, Natascha Selje-Aßmann and Uta Dickhoefer

University of Hohenheim, Institute of Agricultural Sciences in the Tropics, Section of Animal Nutrition and Rangeland Management (490i), Fruwirthstraße 31, 70599 Stuttgart, Germany; inst490i@uni-hohenheim.de

The inclusion of feeding behaviour parameters into models to predict dry matter intake (DMI) may enhance their accuracy. In this regard, this study aimed at evaluating the accuracy of 16 models published in the literature to predict DMI of dairy cows and at developing a model that additionally incorporates feeding behaviour parameters. Lactating Holstein-Friesian cows, with a mean initial live weight and milk yield of 708.3 kg (standard deviation [S.D.] 81.3) and 27.9 kg/d (S.D. 10.9), respectively, were fed total mixed rations mainly composed by grass silage, maize silage, and a concentrate mixture (forage:concentrate ratios of 67:33 or 70:30). The animals were equipped with noseband pressure sensors to monitor jaw movements and quantify eating and ruminating activities during seven consecutive days per cow, in average (S.D. 1.6). The DMI of individual cows (mean 22.1 kg/d, S.D. 4.4) was recorded by automatic weighing troughs. In summary, a dataset of 336 individual cow observations was obtained and posteriorly divided into two subsets, $n_1=279$ used to calibrate the new model and $n_2=57$ used for model validation. Estimates of DMI from the 16 models were compared to the actual DMI through paired *t* test. Using a stepwise selection procedure (PROC GLMSELECT and PROC REG, SAS 9.4) with $\alpha=0.05$, a new model including parameters of feeding behaviour as well as those used in published prediction equations was created. A linear model regression and a paired *t* test were performed to compare the DMI estimated by the new model to the actual DMI of cows of dataset $n_2$. Finally, the role of the included feeding behaviour parameters in enhancing the DMI prediction accuracy was assessed by removing these parameters from the new model, regressing the new estimates of DMI against the actual DMI, and contrasting the resultant $R^2$ with the one from the first regression analysis. Five models successfully predicted DMI (P>0.05), and their biases ranged from -0.38 to 0.16 kg/d with mean-square prediction errors varying from 6.57 to 17.8 kg$^2$/d. The new model was DMI [kg/d] = 6.65 + (0.001 × Age [d]) - (0.00001 × Lactation length$^2$ [d]) + (3.17 × Lactation length$^{0.178}$ [weeks]) - (0.598 × Mouth Width [cm]) + (12.04 × Milk Protein Yield [kg/d]) + (0.000018 × Liveweight$^2$ [kg]) + (0.012 × Eating Time [min/d]) + (0.178 × Ruminating Chews [n/min]) ($R^2$=0.81, bias = 0.1 kg/d). Estimates derived from this model were highly correlated with ($R^2$=0.75) and did not differ from (P>0.05, bias = -0.03 kg/d) actual DMI of cows in dataset $n_2$. The addition of feeding behaviour parameters and of mouth width as a proxy of bite size improved the accuracy of DMI prediction by 11%, indicating that it may considerably improve the accuracy of DMI predictions in dairy cows. Nevertheless, the proposed model should be validated in trials with more animals and for different dairy farming systems and diets.
# Validation of the ability of a 3D pedometer to accurately determine number of steps in dairy cows when housed in tie-stall

Elise Shepley¹, Marianne Berthelot² and Elsa Vasseur¹

¹McGill University, Animal Science, 21111 Lakeshore Rd, Ste-Anne-de-Bellevue, QC, H9X 3V9, Canada, ²Agrocampus Ouest, 65 Rue de Saint-Brieuc, 35000 Rennes, France; elise.shepley@mail.mcgill.ca

Increasing importance is being placed on the ability of dairy cows to have access to exercise. Automated methods for monitoring activity, such as 3D pedometers, provide the means for researchers and producers to quantify the amount of exercise a cow's environment provides. However, such technologies are not often validated for use in all dairy housing systems with the most frequent gaps being in tie-stall barns – the predominant housing system in Canada. The objectives of the current study were: (1) to determine the ability of the IceTag™ 3D pedometer to accurately measure step data for cows in tie-stall; and (2) to determine whether the leg on which the pedometer is mounted impacts step data. Twenty cows were randomly selected and recorded for six hours each during three separate 2-hour periods. Hours of recording were selected based on when cows in the barn were most active to maximize the number of steps compared. Prior to recording, cows were equipped with a pedometer on each rear leg. Exact start time of the video recordings were documented and lying time was used to confirm that video and pedometer minutes were the same. An ethogram defining step activity was developed by comparing second by second data on the video recording to the pedometer readouts. Two observers were trained to observe for step activity with inter-observer reliability calculated as Kw=0.82. Intra-observer repeatability was calculated as Kw=0.88 and 0.86 for observer 1 and 2, respectively. Video recordings for the 20 cows were then viewed and the total number of steps per minute were recorded for each of the six hours of recordings. Hourly averages for right and left leg data were analyzed separately using a multivariate mixed model with number of steps as a dependent variable and technology and time as fixed effects. This allowed for computation of the correlation between pedometer and video step data. The same model was used to determine correlation between left and right leg step data with number of steps as the dependent variable and leg and time as the fixed effects. The analysis of the video vs pedometer data yielded a high overall correlation for both the left (r=0.93) and right (r=0.95) leg. Furthermore, total number of steps between the left and right leg were found to also be well correlated (r=0.80). These results indicate that the IceTag™ 3D pedometers were reliable for calculating step activity in tie-stall housed dairy cows and can be mounted on either leg with similar step results. The accuracy of this pedometer technology to measure step activity in tied dairy cows provides us with a useful automated tool to determine how to better address exercise needs of dairy cows in tie-stalls.
**Lameness in grazing dairy herds: cow level risk factors**

José A. Bran¹, Rolnei R. Daros², Marina Von Keyserlingk², Stephen Leblanc³ and Maria José Hötzel¹

¹Universidade Federal de Santa Catarina, Departamento de Zootecnia e Des. Rural, Rodovia Admar Gonzaga, 1346, 88034000, Florianópolis, Brazil, ²University of British Columbia, Animal Welfare Program, Faculty of Land and Food Systems, 2357 Main Mall, BC V6T 1Z4 Vancouver, Canada, ³University of Guelph, Population Medicine, Ontario Veterinary College, 50 Stone Road E., N1G 2W1 Guelph, Canada; maria.j.hotzel@ufsc.br

Lameness reduces feed intake, increases reproduction failure and risk of culling in dairy cows. Little information is available on lameness in grazing cows. This study aimed to assess the occurrence and cow level associated factors to lameness in smallholder grazing dairy herds. Farms (n=41) in the south of Brazil were visited twice, and all lactating cows (n=1,110) were gait scored (1-5 points; lame: ≥3). The effects of breed (b), parity (P), presence of superficial hoof abnormalities (ha: scissor claw, inter-digital skin hyperplasia, digital dermatitis, horn fissures) and body condition score (bcs) were assessed. Mixed models (logistic regression using herd as random effect) were fitted for incident ('I' = new case), chronic ('C' = lame on two visits) and recovered ('R' = after the first visit) cases of lameness. Lameness incidence was 28.6%. The ratio of new-cases:recovered-cases was 2, showing progression of the problem, despite the recovery of some cows. The ratio healthy-cases:new-cases was 2.5 and the ratio healthy-cases:chronic-cases was 3. Probability of I cases increased with Holstein breed (OR 3.3), higher parity (OR P 2-3: 2.4; OR P>3: 6.4), bcs≤3 on first visit (OR 2.1), any ha (OR 2.4). Probability of C cases increased with Holstein (OR 7) or crossbred cow (OR 11), old cows (OR p 2-3 8; OR P>3 54), any ha (OR 2.6). Being a Jersey or crossbreed (OR 3.2) or a young (OR p 1-2 3.6) cow increased the probability of recover. The relationship between breed and lameness might not be causal, as breed may be confounded with other factors (e.g. milk yield, feeding practices). The risk factors associated with lameness on smallholder dairy farms seems similar to that previously reported in different dairy cattle housing systems. We also encourage future work to disentangle the interaction between breed and grazing systems.
**Grouping, synchrony and comfort related behaviors of dairy cows are affected by the presence of trees in the paddocks**

José A. Bran, Thomas L. Ferreira, Luã Veiga, Sérgio A.F. Quadros and Luiz C.P. Machado-Filho
Universidade Federal de Santa Catarina, Departamento de Zootecnia e Des. Rural, Rodovia
Admar Gonzaga, 1346, 88034000, Florianópolis, SC, Brazil; pinheiro.machado@ufsc.br

The aim of this study was to assess the effect of tree density on the behaviors of a single herd of 16 lactating dairy cows. The herd was observed over 6 days (8 h/d) in 6 outdoor paddocks: 3 with high (H: 200-300 m² of shadow) and 3 with low (L: 20-60 m² of shadow) tree density. The lying behavior, location (shade or sun), and a synchrony index (SI) of behaviors (grazing, rumination) were recorded via live observation; these behaviors were sampled every 10 min. Grooming and browsing in the trees were sampled continuously. Lying, grooming and browsing were used as indicators of cow comfort behaviors. Weather information was collected. Respiratory rate, rectal temperature and milk yield were used as indicators of each cow's thermoregulatory state. Chi-square test was used to compare the frequency of behaviors and Kruskal-Wallis to compare clustering behavior. A high proportion of cows were grouped in the shadow in the H paddocks (median: 0.63, 0.60, 0.47; L paddocks median: 0.26, 0.06, 0.06, P<0.05). The daily temperature and humidity index (THI) remained within the thermal neutral zone, and we found no effect on the physiological indicators of thermoregulation. The SI was negatively correlated with the THI in all the paddocks (L: -0.92; H: -0.68) indicating that the herd became less synchronous at higher THI. The frequency of grooming with trees (158 vs 55) and browsing (75 vs 11) was higher in H than in L paddocks (P<0.05). The number of lying bouts and rumination while lying 7.4 and 8 times higher in H than in L paddocks (P<0.05). Increasing the number of trees in paddocks may modify the grouping and synchrony of the herd, as well as may influence cow comfort behaviors by occupational, physical and sensitive environmental enrichment
**The accuracy of accelerometer-based, leg-mounted sensors for measuring dairy cow locomotion and lying behaviour at pasture**

Gemma L. Charlton, Carrie Gauld, Emma C.L. Bleach and S. Mark Rutter

Harper Adams University, Animal Production, Welfare and Veterinary Sciences, Shropshire, TF10
8NB, United Kingdom; gcharlton@harper-adams.ac.uk

Lying time is an important measure of cow comfort, and when used in conjunction with other measures it can be used to detect health and welfare problems. Accelerometers can be used to automatically record lying behaviour, and this approach has been previously validated in housed cattle. As previous research shows that accelerometers may overestimate the number of lying bouts in cows at pasture, the objectives of this study were firstly to validate the use of IceQube® accelerometers (IceRobotics Ltd., Edinburgh, UK) for recording lying time, number of steps and transitions between lying and standing and vice versa of dairy cows at pasture. The second objective was to determine whether there are any differences in behaviour, as recorded by accelerometers on the front vs back leg. As part of a larger study at Harper Adams University, Holstein Friesian cows, at various stages of lactation were fitted with IceQube accelerometers; one on the back left leg (BL) and one on the front left leg (FL) for 24 months. The sensors record lying and standing duration, frequency and duration of lying and standing bouts and step count. During the summer of 2015, 48 cows were manually observed for 2 h at pasture. Posture was recorded (lying, standing, walking), as well as all leg movements of the front and back left leg. Movements included number of steps, leg lifts and kicks. During the manual observations the cows spent, on average, 42.5 mins (±3.08; range: 0-120 mins) lying down, they took 332.9 steps (±19.42; range: 0-792 steps) and transitioned 1.2 times (±0.11; range 0-5 transitions) between lying and standing and vice versa. Linear regressions (Genstat, 17th edition, VSN International Ltd., UK) revealed strong, positive correlations between the BL IceQube and manual observations for total lying time (r²=99.5; P<0.001), total number of steps (r²=85.6; P<0.001) and the total number of transitions (r²=76.1; P<0.001). There were positive correlations between FL IceQube and manual observation for total lying time (r²=89.4; P<0.001), total number of steps (r²=68.2; P<0.001) and the total number of transitions (r²=44.6; P<0.001), however these were not as strongly correlated as BL. These findings suggest that IceQube accelerometers on the back leg give an accurate record of lying, stepping and transition behaviour of dairy cows at pasture. The IceQube was designed and calibrated for hind leg use, and this experiment verifies that front leg use would reduce performance.
**Motivation of dairy cows to access a mechanical brush**

Emilie A. McConnachie¹, Alexander J. Thompson¹, Anne-Marieke C. Smid¹, Marek A. Gaworski², Daniel M. Weary¹ and Marina A.G. Von Keyserlingk¹

¹University of British Columbia, 2357 Main Mall, V6T 1Z4, Canada, ²Warsaw University of Life Sciences, Nowoursynowska 164, 02-787, Poland; marina.vonkeyserlingk@ubc.ca

Mechanical brushes are increasingly installed in dairy barns, but little is known about how highly cows value brush use. Motivation testing can be used to assess the importance of different resources for animals. One way to assess motivation is to train animals to push open a weighted-gate for access to the resource, then increasing the weight required to open the gate over time. The more weight the animal is willing to push to access the resource, the more important we infer that resource to be. Our aim was to assess motivation of dairy cows to access a mechanical brush. Over two weeks, 10 Holstein cows were trained to push open a weighted-gate. Motivation to access the mechanical brush was compared with that to access fresh feed (positive control) and an empty pen (negative control), and finally motivation to access the mechanical brush was retested at the end of the experiment. The maximum weight that cows were willing to push was analyzed using Kaplan-Meier survival plots. This analysis showed no difference in motivation to access the mechanical brush and fresh feed (P=0.46; P=0.52; mean max weight ± SEM: brush = 48±7.4 kg and TMR = 56±8.8 kg), but cows were less motivated to access the empty pen (mean max weight = 8±4.4 kg) than the they were to access the either the brush or the feed (P<0.001). We conclude that dairy cows are highly motivated to access a mechanical brush.
**Association of cow- and farm-level factors with lying behaviour and risk of elevated somatic cell count**

Ivelisse Robles¹, David Kelton², Herman Barkema³, Gregory Keefe⁴, Jean-Philippe Roy⁵, Marina Von Keyserlingk⁶ and Trevor Devries¹

¹University of Guelph, Animal Biosciences, Guelph, ON N1G 2W1, Canada, ²University of Guelph, Population Medicine, Guelph, ON N1G 2W1, Canada, ³University of Calgary, Faculty of Veterinary Medicine, Calgary, AB T2N 1N4, Canada, ⁴University of Prince Edward Island, Atlantic Veterinary College, Charlottetown, PE C1A 4P3, Canada, ⁵Université de Montréal, Faculté de Médecine Vétérinaire, Montréal, QC H3T 1J4, Canada, ⁶University of British Columbia, Animal Welfare Program, Vancouver, BC V6T 1Z4, Canada; irobles@uoguelph.ca

The objective of this study was to associate cow- and farm-level factors with the lying behaviour of lactating dairy cows and their risk for elevated somatic cell count (SCC). Cows from 18 commercial free-stall dairy herds in Ontario, Canada were enrolled in a longitudinal study. Four hundred cows in total were selected for the study based on days in milk (<120 d), absence of mastitis treatment in the last 3 mo, and SCC (<100,000 cells/ml). Data on SCC were collected through regular milk testing (~5-wk intervals). The study began within 7 d after a milk test and continued until 3 tests were completed (~105 d), for a total of 3-observation periods/cow. Elevated SCC (eSCC) was used as an indicator of subclinical mastitis. An incident of eSCC was defined as a cow having a SCC>200,000 cells/ml at the end of a period when SCC was <100,000 cells/ml at the beginning of that period. Lying behaviour was recorded for 6 d after each milk sampling, using electronic data loggers. At the end of each recording period, cow body condition score (BCS) was recorded using a 5-point numerical rating scale (1=thin to 5=fat). Details of barn design, stocking density, and herd management were collected. Stall cleanliness was assessed with a 1-m2 metal grid, containing 88 squares, centered between stall partitions of every 10th stall in each farm, and then counting the squares containing visible urine and/or fecal matter. Factors associated with cow lying behaviour were analyzed using multivariable mixed-effect linear regression models. Cows averaged 656±3.3 min/d lying time. On average, under-conditioned cows (BCS≤2.5) spent 37 min/d less (P=0.03) time lying down than over-conditioned cows (BCS≥4.0). On average, cows tended to spend 35.8 min/d more time (P=0.06) lying down in deep-bedded vs mattress-based stalls. Mean proportion of soiled squares/stall was 20.1±0.50%. As the proportion of soiled squares/stall increased across farms, cow lying time decreased (P=0.003); the model predicted a difference in daily lying time of ~80 min/d between the farms with the cleanest stalls as compared to those with the dirtiest stalls. Factors associated with risk of an eSCC were analyzed using a multivariable mixed-effect logistic regression model. Over the study period, 50 eSCC were detected, resulting in an incidence rate of 0.45 eSCC/cow-year at risk. No factors were associated with risk of new eSCC; this is likely due to low frequency of new cases of eSCC across the study period. Overall, these results confirm our knowledge, that on commercial farms, cows prefer to lie down in cleaner and more comfortable environments.
Who's stressed: nonlinear measures of heart rate variability may provide new clues for evaluating the swine stress response

Christopher J. Byrd¹, Jay S. Johnson² and Donald C. Lay, Jr.²

¹Purdue University, Department of Animal Sciences, 125 S. Russell St., West Lafayette, IN 47907, USA, ²USDA-ARS, Livestock Behavior Research Unit, 125 S. Russell St. Rm. 216, West Lafayette, IN 47907, USA; byrd17@purdue.edu

Heart rate variability (HRV) is a well-known proxy for assessing autonomic nervous system function in swine. Several traditional linear HRV indices such as inter-beat interval length (R-R), and standard deviation of inter-beat interval length (long term, SDNN; short term, RMSSD) provide valuable data for understanding an animal's response to common on-farm stressors. Nonlinear HRV indices provide new information by quantifying the complexity of heart rate fluctuations but have not been used in swine welfare studies. The study objective was to evaluate whether nonlinear indices could be used to detect differences in HRV that may go undetected by linear indices following an acute heat stress episode, similar to what swine can experience during summer in the U.S. Methods were approved by the Animal Care and Use Committee (#1604001402). Eight pigs, 13 to 15 weeks old (6 gilts, 2 barrows; 49±1.5 kg) were individually housed in thermoneutral (TN) conditions (22.4±0.1 °C). Each pig was orally administered an ingestible temperature sensor 16 h prior to testing. Heart rate data were collected under TN conditions in their home pens for 1 h (Phase 1). Following Phase 1, pigs were moved to a separate room and exposed to acute HS (Phase 2; 40.3±0.05 °C). When pig body temperature reached 40.5 °C, heart rate was collected for 1 h, or until body temperature reached 41.5 °C. Immediately prior to- and following Phase 2, blood was collected for analysis of cardiac troponin I (cTnI), a metabolite indicative of myocardial stress. Heart rate data were analyzed and corrected using previously published error-correction guidelines. Behavioral data (active/inactive and posture) were collected continuously from video. Datasets of 1000 beats with less than 5% error during inactivity were used for calculation of HRV indices. Data were transformed when needed to meet assumptions of normality and homogeneity of variance. The effect of body weight, breed, sex, and phase (1 or 2) on individual linear (heart rate, R-R, SDNN, and RMSSD) and nonlinear (sample entropy, SD1/SD2, shannon entropy, recurrence rate) HRV indices, cTnI levels, and time spent active were tested using repeated measures mixed model ANOVAs. Comparisons were adjusted using the Tukey test. Sample entropy was higher during Phase 1, indicating greater HRV complexity compared to phase 2 (1.3±0.1 vs 0.8±0.1; P=0.03). Phase was not associated with any of the remaining HRV indices. Levels of cTnI were lower in Phase 1 compared to Phase 2 (0.05±0.01 vs 0.10±0.02; P=0.05). Activity levels in Phase 1 (0.26±0.05) were not different from Phase 2 (0.15±0.05; P>0.05). Breed, sex, and weight were not associated with HRV indices, cTnI concentration, or activity levels. In summary, none of the linear measures of HRV indicated pigs were distressed. In contrast, the non-linear measure, sample entropy, did show the pigs were distressed and may add value to HRV studies focused on improving swine welfare.
**Effects of space allowance and simulated sea transport motion on the behavior and heart rate of sheep**

Grisel Navarro

University of Queensland, Centre for Animal Welfare and Ethics, School of Veterinary Science Building 8134, Gatton Campus University of Queensland, 4343, Australia; grisel.navarrootarola@uq.net.au

We have previously demonstrated that a low space allowance increased interactions between sheep in simulated ship transport, both negative, i.e. pushing, and positive, i.e. affiliative behaviour, where one sheep puts its head beneath the other's head. The net impact on stress to the animals is unclear, therefore we additionally monitored heart rate and its variability. Nine sheep were exposed to three stocking densities (High H, representing the Australian shipping standard for these sheep, Medium M and Low L, 0.78, 0.92 and 1.04 m2/sheep, respectively) in triplets in a replicated Latin square factorial design. Sheep were placed in a crate on a programmable platform that generated roll and pitch motion typical of that experienced on board ship for 1 hour/per treatment. Motions were either irregular (I), programmed as 30 randomly selected amplitude and period values of pitch and roll movements, or regular, the mean of these values, and represented approximately 33% of the recommended maximum tolerance for livestock carriers. Pushing (P=0.006) and stepping behaviours (P<0.05) were increased when the stocking density was high but only when the sheep experienced regular motion, the latter particularly at the beginning of the treatment (P≤0.001). The high stocking density and regular motion also decreased the RMSSD (√mean sums of squares of successive interbeat intervals, P<0.0001), indicating increased stress, and decreased time spent lying (P≤0.001). Regular motion increased head lowered behaviour (P=0.03). Aggression (head to body attack) was increased by the high stocking density (P=0.005) and by irregular movement (P=0.003), the latter particularly at the end of the treatment (P=0.05). The ratio of low to high frequency beat intervals (LF/HF ratio) was reduced in the high stocking density, irregular movement treatment ((P=0.008). Both motion treatments decreased rumination (P=0.03) and tended to increase affiliative behaviour (P=0.06) compared with control. Results suggest that there are two stresses caused by simulated ship motion. In the early stages of exposure sheep push each other in competition for space, especially at high stocking densities and when motion is regular, i.e. they can predict the position of others. In the later stages of exposure frustration builds in irregular movement and high stocking density and aggression results. It is concluded that a high stocking density increases both competition for space and aggression in sheep.
Effects of acute lying and sleep deprivation on behaviour and milk production of
lactating Holstein dairy cows

Jessie A. Kull¹, Gina M. Pighetti¹, Katy L. Proudfoot², Jeffrey M. Bewley³, Bruce F. O'Hara³, Kevin D. Donohue³ and Peter D. Krawczel¹

¹The University of Tennessee, 2506 River Drive, Knoxville, TN 37996, USA, ²The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA, ³Unviesity of Kentucky, 900 W.P. Garrigus Building, Lexington, KY 40546, USA; jkull@vols.utk.edu

Sufficient lying time is important for dairy cows, as cows are highly motivated to lie down after
a period of deprivation. Lying deprivation can result in sleep loss, as cows acquire most of their
sleep while lying down. Evidence from human and rodent research shows that sleep deprivation
can impact immunity, metabolism, and performance, but little research has assessed the
impact of sleep in dairy cows. The objective of this proof-of-concept study was to determine
the impact of sleep deprivation with and without lying deprivation on milk production and
lying behaviour of dairy cows. To ensure cows were not experiencing undo distress, they were
screened every 30 minutes for a set of removal criteria established before the experiment. All
procedures were approved by the University of Tennessee Institutional Animal Care and Use
Committee. Data were collected from 12 mid-lactation Holstein cows (DIM=199±44 (mean
± SD)). Using a cross-over design, cows experienced 24 h of: (1) sleep deprivation without
lying deprivation achieved by noise or physical contact when the cow's posture suggested
the onset of sleep, and (2) lying deprivation created using a wooden grid on the pen floor.
EEGs were used to ensure cows experienced sleep loss. Treatments were separated by a 12-d
washout period. Cows were housed in individual box stalls (mattress base with no bedding)
for habituation (d -3 and -2), baseline (d -1), and treatment (d 0) days. After treatment, cows
returned to a sand-bedded freestall pen for 7 d (d 1 to 7). Lying time and lying bout duration
were recorded from d -1 to 7 using accelerometers. Milk production was recorded twice daily
on the day after treatment (d 1). Data were analyzed using a mixed model in SAS including
fixed effects of treatment (sleep or lying deprivation), day (-1 to 7) and a random effect of cow.
When there was a significant treatment by day interaction (P<0.05), pair-wise comparisons
were made using the PDIFF statement. There was no effect of treatment on lying time or lying
bout duration, but there was an interaction between treatment and day for both variables
(P<0.001). As expected, lying time was lower when the cows were deprived of lying compared
to sleep on d 0 (1.9 vs 8.4±0.7 h/d; P<0.001), but this difference was reversed on d 1 (16.8 vs
13.6±0.7 h/d; P=0.002). Similarly, lying bout duration was lower during lying deprivation
than sleep deprivation on d 0 (15.3 vs 72.9±7.5 min/bout; P<0.001), and was reversed on d 1
(110 vs 89.9±6.8 min/bout; P=0.01). On d 1, milk yield was lower when cows were deprived
of lying compared to sleep (31.8±vs 35.3±2.4 kg/d; P=0.002). The results indicate that 24 h of
lying deprivation resulted in a rebound effect of lying time the day after deprivation. Lying
deprivation also reduced milk yield compared to sleep deprivation, but more research is needed
to determine if sleep deprivation alone has other negative impacts on the cow such as poor
metabolic health and immunity.
Can faecal cortisol metabolites be used as an Iceberg indicator in the on-farm welfare assessment system WelFur-Mink?

Anna Feldberg Marsbøll<fnref n="1" />, Steen Henrik Møller<fnref n="1" />, Tine Rousing<fnref n="1" />, Torben Larsen<fnref n="1" />, Rupert Palme<fnref n="2" /> and Jens Malmkvist<fnref n="1" />

Faecal cortisol metabolites (FCM) are a non-invasive measure of HPA-axis activity. FCM have been found to relate to several aspects of welfare in mink, e.g. FCM levels was reduced by an enriched housing environment, and stereotypic mink had higher FCM levels that non-stereotypic mink. An on-farm welfare assessment system for mink named WelFur-Mink has been developed based on the concept of Welfare Quality*. In this system, a variety of animal- and resource-based measurements are taken on a sample of mink and aggregated into an overall welfare assessment at farm level. As FCM level is linked to stress responses in mink, it is expected to sum up several measurements in the system. We, therefore, aim to investigate if FCM can be used as an 'Iceberg Indicator', i.e. a welfare indicator that summarises many measurements and provides an overall assessment of welfare, with the potential to be used as a more cost-beneficial measurement in WelFur-Mink. In February 2016, the study was initiated with data collection on two Danish mink farms. 150 brown 1st years female mink were included on each farm. Faecal samples were collected 0-5 hours after feeding and the welfare of the individual mink was assessed according to the WelFur-Mink protocol. This includes assessment of stereotypic behaviour, response in a voluntary approach-avoidance test, fur chewing, body condition, and access to bedding material and enrichments. The behavioural measurements were taken first to avoid that the response was affected by the assessors walking behind the cages when collecting the faecal samples. The remaining measurements were taken after collection of faecal samples. Faecal samples were collected from 146 mink on farm 1 and 143 mink on farm 2. FCM levels (ng/g), indicative of circulating cortisol as validated for mink, were analysed using linear models, and differed between the two farms (median [2.5%; 97.5%]: farm 1: 277 [193; 398]; farm 2: 427 [238; 769], F=14, DF=1, P<0.001, ANOVA). The two farms also differed in the prevalence of fur chewing (Farm 1: 32%; Farm 2: 15%, P<0.001, Fisher's exact test), nest boxes with reduced thermal protection (Farm 1: 0%; Farm 2: 48%, P<0.001, Fisher's exact test), and stereotypic behaviour (Farm 1: 44%; Farm 2: 9%, P<0.001, Fisher's exact test). However, it was not possible to standardise the assessment of stereotypic behaviour according to feeding time, thus these prevalences are not comparable. The initial results indicate that FCM may be used to distinguish between farms. In February 2017, data collection on additional five farms is carried out based on the practical experiences from 2016. It will be investigated if mink with a reduced welfare according to WelFur-Mink have higher FCM levels, and if FCM can be used to identify farms with a reduced animal welfare according to WelFur-Mink. This will be investigated in relation to both individual and groups of welfare measurements, as well as the aggregated welfare assessment at criteria, principal, and overall assessment level.

<footnote n="1">¹Aarhus University, Department of Animal Science, Blichers Allé 20, 8830 Tjele, Denmark,</footnote>

<footnote n="2">²University of Veterinary Medicine, Department for Biomedical Sciences, Veterinärplatz 1, 1210 Vienna, Austria; anna.marsboll@anis.au.dk</footnote>
**Key-note:**

**The effect of a low dose of lipopolysaccharide on physiology and behaviour of individually housed pigs**

Janicke Nordgreen¹, Camilla Munsterhjelm², Frida Aae¹, Anastasija Popova¹, Preben Boysen³, Birgit Ranheim¹, Mari Heinonen², Joanna Raszplewicz⁴, Petteri Piepponen⁵, Andreas Lervik⁶, Anna Valros² and Andrew M. Janczak¹

¹Faculty of Veterinary Medicine/Norwegian University of Life Sciences, 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 Sciences, Food Safety and Infection Biology, Ullevålsveien 72, 0033 Oslo, Norway, ⁴Small Animal Teaching Hospital/University of Liverpool, Chester High Road, Neston CH64 7TE, United Kingdom, ⁵Faculty of Pharmacy/University of Helsinki, Division of Pharmacology and Pharmacotherapy, P.O. Box 56, 00014 Helsinki, Finland, ⁶Faculty of Veterinary Medicine/Norwegian University of Life Sciences, Companion Animal Clinical Science, Ullevålsveien 72, 0033 Oslo, Norway; janicke.nordgreen@nmbu.no

Sickness may bring about changes in behaviour that disrupt social interactions. Production animals cannot withdraw socially while sickness lasts. Bad health is a risk factor for tail biting in fattening pigs. One possible mechanism is through cytokine effects on social behaviour, such as during underlying infection when bacterial products like lipopolysaccharide (LPS) may reach the circulation. Before this hypothesis can be tested, detailed knowledge of the changes in behaviour, mood and physiology after immune activation is needed. We describe the changes over three days in time budget, anticipation, cytokines, C-reactive protein, cortisol and immune cells in blood after injection with a low dose of LPS in gilts with permanent central venous catheters in the internal jugular vein. Brain levels of cytokines and monoamines were measured after euthanasia at 72 hours after injection. Pigs were injected IV with an LPS (O111:B4) dose of 1.5 µg/kg (n=7) or a similar volume of saline (n=6). An increase in cortisol (F₁₀, 110=14.05; P<0.0001), TNFα (F₁₀, 110=50.89; P<0.0001), IL-1ra (F₁₀, 110=62.26; P<0.0001), IL-6 (P=0.0034) and IL-8 (F₁₀, 110=27.44; P<0.0001) was seen within the first 6 hours after injection. During that period, activity decreased and sleeping increased in LPS pigs (P<0.05). Pigs were housed in pairs with visual contact (one saline, one control). The level of synchronization did not change significantly during the experiment. No effect on anticipatory behaviour was found. CRP was elevated at 12 and 24 hours after injection (P<0.001 for both) and food intake was lower for the first 24 hours (F 3,33=4,03; P=0.01). Absolute count of peripheral blood mononuclear cells was increased at 48 and 72 h p.i. Three days after the injection the pigs were euthanized by administration of an anaesthetic mixture followed by pentobarbital through the jugular catheter. LPS pigs had lower levels of noradrenaline in their hypothalamus (P<0.062), hippocampus (P<0.042) and frontal cortex (P<0.0006) compared to saline pigs. Thus, a low dose of LPS can induce changes in neurotransmitter levels that persist after inflammatory and stress markers in the periphery have returned to baseline levels. Future field studies on the effects of sickness on behaviour would benefit from following the animals until the clinical symptoms have resolved.
**Effects of embryonic norepinephrine on juvenile and mature quail behaviors**

*Jasmine Mengers and Rachel Dennis*

*University of Maryland, Animal and Avian Science, 8127 Regents Drive, College Park, MD 20742, USA; jmengers@gmail.com*

Poultry breeding flocks experience stress from numerous sources including feed restrictions, confinement, social aggression, changing environments, transport, and stockperson turnover. Maternal diet and stress can increase catecholamine levels, including norepinephrine (NE), and alter tyrosine metabolism. Elevated NE levels impact the embryo and lead to altered survival related behaviors in the developed offspring, such as eating, drinking, and vigilance. In order to determine the effects of NE on feeding and social behaviors, activity level, and fear response, Japanese quail (*Coturnix japonica*) embryos were injected with 10 µl of 0.01 M or 0.05 M of NE or saline at ED1 (n=130) and incubated with intact controls (n=80). Weekly behavioral scan samples (am and pm) were taken from wk 4 (juvenile) to wk 11 (sexually mature) with rehoming between wk 6 and 7 samplings. Body weights were taken every other wk and organ weights were taken at wk 11. Tonic Immobility (TI) tests were conducted at 2, 5, and 9 wks. Data were analyzed in SPSS using ANOVAs with Bonferroni post hoc testing between treatments. Results showed a greater incidence of eating behavior in birds that received 0.01 M and 0.05 M of NE compared to control birds, with eating frequency increasing over time in older NE-treated birds (P<0.05 wks 5, 10, 11). Younger birds that received 0.01 M of NE spent more time drinking compared to birds that received 0.05 M of NE or controls (P<0.05 wks 5, 6). Birds that received 0.05 M of NE foraged more frequently compared to birds that received 0.01 M of NE (P<0.05 wks 6, 8, 10, 11). Following rehoming, NE-treated birds exhibited an increase in inactivity compared to control birds (P=0.007, wk 7). Birds that received 0.01 M of NE weighed significantly less than saline birds (P≤0.01 wks 3, 5, 7, 9). Relative heart, liver, and spleen weights did not significantly differ between treatments. We observed no difference in TI inductions or duration. Our data show that NE injections during early embryonic development have altered feeding and drinking behaviors, activity levels, and weight as well as response to rehoming. These results suggest that increased adrenal hormone *in ovo* can impact both production and behavior, carrying implications for poultry management and indicating a need for further research into maternal stress in birds.
The influence of social stress on three selected lines of laying hens

Patrick Birkl¹, Joergen Kjaer², Peter McBride¹, Paul Forsythe³ and Alexandra Harlander-Matauschek¹

¹University of Guelph, 50 Stone Rd E, ON N1G 2W1 Guelph, Canada, ²Friedrich-Löffler Institut, Dörnbergstraße 25, 29223 Celle, Germany, ³Brain Body Institute, 1280 Main St W, ON L8S 4L8 Hamilton, Canada; pbirkl@uoguelph.ca

When a social animal encounters a group of unfamiliar conspecifics, it faces the challenge of earning itself a spot in their well-established social web. For animals, and even humans, this can involve a range of interindividual interactions which can cause social stress. The aim of this study was to investigate the effects of social stress on three lines of laying hens (HFP, LFP and control). Feather pecking presents an abnormal behavior in laying hens. If this abnormal behavior was indicative of a compromised stress-response, then, we predicted that the HFP genotype would respond more sensitively to social stress in terms of weight-gain variability, changes in amino acid ratios that indicate a stress-response (phenylalanine: tyrosine, PHE: TYR), and increased occurrence of abnormal behavior (feather pecking). For this study, we used 160 laying hens selected for high (HFP) or low (LFP) feather-pecking activity and an unselected control line (C). Birds were housed in floor pens in groups of 16 hens per pen (HFP; n=4, LFP; n=3, C; n=9, 10 pens). At 16 weeks of age, we disrupted the groups in 5 pens by mixing individuals with unfamiliar birds to simulate social stress (this procedure was repeated after two days, to intensify social stress). Procedures were approved by the local animal care committee. Body weight was measured before mixing and again three weeks after the second mixing, to assess weight-gain. Blood plasma was collected one day prior to mixing and two days after the second round of mixing, to determine amino acid concentrations for calculating PHE/TYR ratios. Aggressive pecking and feather pecking were recorded on an all occurrence basis, prior to mixing (baseline), two minutes after mixing, one hour after mixing, and 24 hours after mixing, during 10-minute observation periods per pen and time-point. Data were analysed using a GLIMMIX procedure in SAS. We found that weight-gain showed increased variability for mixed groups, independent of genotype, compared to control groups (mixed: 15.1±0.9 vs unmixed: 4.9±0.9%, P<0.001). PHE/TYR ratios differed between all three lines (HFP: 0.75±0.01 vs LFP: 0.62±0.01 vs C: 0.65±0.01) and in HFP birds exclusively, PHE/TYR ratios were affected by social stress. Socially stressed HFP birds showed significantly lower PHE/TYR ratios than non-stressed birds (HFP unmixed: 0.75±0.02 vs HFP mixed: 0.70±0.02, P<0.02). Occurrence of aggressive pecking was increased two minutes after mixing (mixed: 4.5±0.9 vs unmixed: 1.0±0.5, P<0.05) but was not different from baseline levels one hour post-mixing and 24 hours post-mixing. Social stress had no short-term effect on the occurrence of feather pecking; yet ongoing analysis will investigate potential long term effects. The high variability of weight gain in mixed groups demonstrates physiological consequences of social stress beyond the increased occurrence of aggressive pecking. Our results also show that HFP birds differ in PHE/TYR ratios compared to other genotypes and that social stress reduces this ratio exclusively in HFP birds.
**Feather pecking: is it in the way hens cope with stress?**

Jerine A.J. Van Der Eijk¹,², Aart Lammers¹ and T. Bas Rodenburg¹,²

¹Wageningen University, Adaptation Physiology Group, De Elst 1, 6708 WD Wageningen, the Netherlands, ²Wageningen University, Behavioural Ecology Group, De Elst 1, 6708 WD Wageningen, the Netherlands; jerine.vandereijk@wur.nl

Feather pecking is a serious welfare and economic issue in the laying hen industry. It involves hens pecking and pulling at feathers or tissue of conspecifics, negatively affecting welfare. Excessive damaging behaviours, such as severe feather pecking, are indicative of an animal's inability to cope with the restrictive husbandry environment. The hypothalamic-pituitary-adrenal axis and the serotonergic system play an important role in how animals cope with stress and fear. Therefore, we here investigated whether lines divergently selected on feather pecking behaviour differ in their coping style, stress response and serotonergic system. We used genetic lines selected for high (HFP) and low (LFP) feather pecking and an unselected control line (CON). Lines were housed separately in groups of 19 birds per pen, with 8 pens per line. Group size was reduced by 2-3 birds at 0, 5, 10 and 20 weeks of age. There were two batches that differed 2 weeks in age. At 24 weeks of age birds were subjected to a 5 min. manual restraint test. We recorded latency to vocalize and struggle as well as number of vocalizations and struggles. No distinction was made between types of vocalizations. Blood was collected 10 min. after the test was finished for analysis of plasma-corticosterone levels and whole-blood serotonin levels. Corticosterone concentration was determined by a radio-immunoassay kit. Serotonin concentrations were assessed by a fluorescence assay. Data were analysed using mixed models, with selection line, age, batch, experimenter and test time as fixed factors and pen as random factor. HFP birds had a shorter latency to vocalize compared to CON and LFP birds (HFP=124 s, LFP=173 s and CON=185 s, F<sub>2,180</sub>=5.77, P=0.0037). However, lines did not differ in their latency to struggle or in corticosterone levels after manual restraint. HFP birds did have lower serotonin levels compared to LFP and CON birds (HFP=31.98 nM/ml, LFP=35.84 nM/ml and CON=38.14 nM/ml, F<sub>2,169</sub>=8.63, P=0.0003). These results indicate that HFP birds have a more pro-active coping style than CON and LFP birds. Previous behavioural tests at younger ages also indicated clear differences between the lines in coping style, with HFP birds having a more pro-active coping style in several behavioural tests compared to CON and LFP birds. In conclusion, our results suggest that selection for feather pecking might have an effect on coping style and this is accompanied by changes in the serotonergic system. However, selection did not have an effect on the stress response in this study. These results show that feather pecking seems to be related to coping style which might indicate that birds with a certain coping style are predisposed to develop feather pecking.
Do feather peckers, victims and control hens differ in symmetry of bilateral traits,
body weight and comb size?

Fernanda M. Tahamtani¹, Lena K. Hinrichsen¹, Björn Forkman² and Anja B. Riber¹

¹Aarhus University, Department of Animal Science, Blichers Allé 20, Tjele, 8830, Denmark,

²University of Copenhagen, Department of Veterinary and Animal Sciences, Grønnegårdsvej 8,
1870, Denmark; fernandatahamtani@anis.au.dk

Feather pecking is one of the major welfare issues facing the egg farming industry worldwide. Previous research has found a relationship between cannibalistic behaviour, fluctuating asymmetry of bilateral traits (FA) and body weight in laying hens. As cannibalism is linked to severe feather pecking, it could be suggested that a relationship between feather pecking, FA and body weight also exists. The purpose of this study was to analyse the association between feather pecking behaviour and (1) FA, (2) body weight and (3) comb size in laying hens. Sixty-four laying hens were categorised as feather peckers, victims or control hens based on weekly performance of severe feather pecking behaviour from age 0-23 weeks and their plumage condition at age 23 weeks. Hens with a high number of pecking bouts and low plumage damage score were classified as feather peckers. Hens with a low number of pecking bouts and high plumage damage score were classified as victims. Control hens had few pecking bouts and low plumage damage scores. After culling at 23 weeks of age, the lengths of ulna, length and width of tarsus, lengths of middle toe and widths of hock were measured twice in each side. Each trait was tested for repeatability, directional asymmetry and antisymmetry. Only traits that did not display directional asymmetry and/or antisymmetry were considered appropriate for analysis of FA. Composite FA was calculated using REML for the lengths of ulna, tarsus and middle toe. Control hens displayed less composite FA (0.43±0.08 mm, P=0.0005) and less FA of ulna (0.10±0.12 mm, P=0.0001) than feather peckers (composite: 0.83±0.08 mm, ulna: 0.61±0.11 mm) and victims (composite: 0.84±0.08 mm, ulna: 0.69±0.10 mm). Tarsus length asymmetry differed between all categories, with victims displaying most (0.82±0.08 mm), control hens least (0.15±0.09 mm) and feather peckers intermediate levels of asymmetry (0.40±0.08 mm, P<0.0001). In addition, victims were also lighter in body weight (1,728±43 g) compared to control hens (1,869±46 g) and feather peckers (1,859±45 g, P=0.043). No difference was found in the size of the comb between the three categories (P=0.1). The results suggest that feather peckers and victims were exposed to similar levels of negative experiences, causing developmental instability, whereas control hens were less negatively affected during early life than both feather peckers and victims.
**Investigation of response to the novel object test in pigs in relation to tail biting
phenotypes**

Jen-Yun Chou<sup>1,2,3</sup>, Rick B. D'Eath<sup>2</sup>, Dale Sandercock<sup>2</sup> and Keelin O'Driscoll<sup>3</sup>

<sup>1</sup>University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush, EH25 9RG, Midlothian, United Kingdom, <sup>2</sup>SRUC, Animal & Veterinary Sciences Research Group, Roslin Building, Easter Bush, EH15 9RG, Midlothian, United Kingdom, <sup>3</sup>Teagasc, Pig Development Department, Moorepark, Fermoy, Co. Cork, Ireland; jenyun.chou@ed.ac.uk

Different physiological and behavioural traits of pigs engaged in tail biting have been widely studied, but most research has focused on identifying traits to predict tail biting phenotypes (biters, victims, neutrals). After pigs' involvement in tail biting, the short and long term characterisation of these traits is less understood. This study investigated if there was variation in stress and fear responses to a novel object (NO) test between pigs which were either performers or receivers of tail biting, or not involved in biting events, and whether these responses were still evident later in life. We hypothesised that 'biters' would exhibit a greater response than 'victims' or 'neutrals'. As part of a study on how different enrichment types (rubber floor toy or wooden post) and fibre content (high or low) in the diet affect the occurrence of tail biting, 72 undocked pigs (36 male/female) were selected for the NO test. The pigs were categorised as either a biter (n=24), a victim (n=24), or a neutral (n=24). The test was conducted twice for each pig: one week after they were moved into the finisher house (T1) when traces of biting were evident, and six weeks later (T2) when biting has already settled down. Pigs were placed individually in a test arena, and after a minute of habituation, a bright orange sweeping brush was introduced by dropping down from above. Direct continuous behaviour observation was conducted during the five-minute test. A saliva sample was taken immediately before (baseline) and after the test to determine salivary cortisol level. All data were analysed using mixed models (fixed effects: sex, category, treatment, T1/T2, before vs after; random effect: pen). There was no clear difference in the response to the NO test between the tail biting-associated phenotypes of biter, victim and neutral pig. Overall, cortisol concentration was higher after (0.234±0.017 µg/dl) than before the test (0.152±0.017 µg/dl; P<0.001), and the difference was much greater in T1 (P<0.001) than in T2 (P=0.058). Evidence of habituation to the test was also seen in behaviour: at T2 pigs had a lower frequency of vocalisation (P<0.001), longer latency to vocalise (P=0.05), longer duration of explorative behaviours (P<0.001), and approached the NO quicker (P<0.001) than at T1. Enrichment at the weaner stage had a significant effect on the latency to approach the NO (P<0.05); pigs given floor toys approached the NO faster than ones given wooden post (15.45±5.53 vs 30.04±5.46 s; P<0.05), and spent a longer time interacting with it (P<0.05), possibly because the NO was more similar to the floor toys and therefore was less novel and fearful for them. The habituation and enrichment effect both suggested the NO test was sensitive to detect different levels of stress and fear response, however, the preliminary evidence suggested there was no significant change in pigs' response after their involvement in tail biting events either in the short or long term.
Effects of elevated platforms on fearfulness in fast-growing broilers

Ida J. Pedersen¹, Fernanda M. Tahamtani² and Anja B. Riber²

¹University of Copenhagen, Department of Veterinary and Animal Sciences, Grønnegårdsvej 8, 1870 Frederiksberg, Denmark, ²Aarhus University, Department of Animal Science, Blichers Allé 20, 8830 Tjele, Denmark; idajp@sund.ku.dk

Fear is a general concern in the poultry industry. It may cause smothering, resulting in skin
injuries and, in worst case, mortality. Thus, fear may result in detrimental animal welfare and
economic loss to the farmer. Environmental complexity in the home environment of animals
has been found to reduce fearfulness in several species, including mice, pigs and chickens. The
aim of the present experiment was to investigate whether provision of elevated platforms with
access ramps has an effect on fearfulness of fast-growing broilers, measured as their response
to the tonic immobility test. Six pens (9.5×3 m) with approximately 500 broilers in each (Ross
308, mixed sex), corresponding to a stocking density of 40 kg/m2 were used. Commercial
feed and water were provided *ad libitum*, and wood shavings were used as litter material. The
birds were slaughtered at 35 days of age. The 3 treatment pens included an elevated platform
(540×60×30 cm, length × width × height) made of perforated plastic slats with two access
ramps. The area under the platforms was fenced off and was therefore not included in the net
area used for calculation of stocking density. The 3 control pens did not include platforms,
but otherwise the conditions were similar. At 31 and 32 days of age, 30 birds from each pen
were randomly selected and induced into tonic immobility. The number of inductions needed
for tonic immobility to be achieved (max. 3), the latency to first head movements (max. 10
min) and total duration of tonic immobility (max. 10 min) were registered for each bird. The
statistical model was an ANOVA (GLM) with pen nested in block and treatment as a fixed
factor. Birds with access to platforms had significantly shorter latencies to first head movement
(149.5±135.2 s vs 230.0±191.7 s; F₁,₁₇₅=9.84; P=0.002) and shorter total duration of tonic
immobility (231.6±175.0 s vs 306.9±209.8 s; F₁,₁₇₅=5.73; P=0.017) compared to control birds.
No difference was found in the number of trials needed to induce TI between the control
and treatment groups (1.15±0.42 vs 1.16±0.43; P=1.00). The results suggest that providing
environmental enrichment in the form of elevated platforms can reduce fearfulness, and thus
improve the welfare of fast-growing broilers.
Meloxicam and temperament effects on pain sensitivity and inflammatory response
in surgical or rubber ring castrated calves

Désirée Soares¹, Sonia Marti¹, Daniela Melendez¹, Diego Moya², Eugene Janzen¹, Ed Pajor¹ and Karen Schwartzkopf-Genswein³

¹University of Calgary, 3330 University Dr. NW, T2N 1N4 Calgary, Canada, ²Aberystwyth University, Penglais Campus, SY23 3DD Aberystwyth, United Kingdom, ³Agriculture and Agri-Food Canada, 5403 1 Ave S, T1J 4B1 Lethbridge, Canada; soares.desiree@hotmail.com

The majority of beef operations in Canada use surgical and rubber ring castration before 3 mo. of age and 90% do not use pain mitigation. Meloxicam is a nonsteroidal anti-inflammatory drug which has a label claim to control pain for up to 48 h. The role of animal temperament modulating acute and chronic pain caused by surgical and rubber ring castration is not known. The objectives were to determine the effect of meloxicam and temperament on pain sensitivity and inflammatory response in surgical and rubber ring castrated beef calves. Seventy-two Angus calves (76±2 d of age and 134.5±20.30 kg BW) were randomly assigned to treatments according to a 3×2 factorial design assessing castration technique (CAST): surgical (S), rubber ring (R) or sham castration as control (C) and drug administration (DRUG): single s.c. injection of meloxicam (M) at the time of castration (0.5 mg Metacam®/kg BW) or single s.c. injection of saline solution as control (N) to yield SM, SN, RM, RN, CM, and CN treatments (n=12/treatment). Calves were managed in two groups (GROUP) of 36 to be castrated on two separate days. They were housed on pasture with their mothers and with *ad libitum* access to water. Blood samples were collected via jugular venipuncture to assess plasma haptoglobin concentration (HP, g/l) as an indicator of inflammation. Pain sensitivity of the wound and surrounding skin was measured using a Von Frey anesthesiometer (VA, g). Temperament was assessed by using flight speed (FS, m/s). The variables HP, VA and FS were collected on d -1 and d 0 (immediately prior to castration) as baseline measures; and on d 6, 13, 20, 34, 48 and 62 post castration (SAMPLING DAY). Calves were blocked by the average FS and BW obtained on d -6 and d -1 (prior to castration day). Data was analyzed using a mixed-effects model including CAST, DRUG, SAMPLING DAY, GROUP and their interactions as fixed effects. The average baseline measurements of BW and each dependent variable as well as the average of all FS measurements were used as a covariate. A post-hoc (Tukey) test was used to compare the adjusted means. Our results showed that CAST, DRUG and FS had no effect on HP. Pain sensitivity was lower (P<0.05; greater VA values) in C compared to S and R calves. DRUG had no effect on VA. However, FS was related to pain sensitivity indicating that for each 1 m/s in additional FS the VA increased by 22.1 g (P<0.05). We conclude that a single s.c. injection of meloxicam at the time of castration had no effect on reducing indicators of pain or inflammatory response while more excitable animals showed less pain sensitivity over the 62 d post-castration. Further research is needed to improve the knowledge about pain mitigation post-castration in support of developing new strategies in beef calves and the relationship between temperament and pain sensitivity.
Cat responses to handling: assessment of scruffing, clips, and full body restraint

Carly Moody¹, Georgia Mason², Cate Dewey¹ and Lee Niel¹

¹University of Guelph, Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Road E., Guelph, Ontario, N1G 2W1, Canada, ²University of Guelph, Animal Biosciences, Ontario Agricultural College, University of Guelph, 50 Stone Road E., Guelph, Ontario, N1G 2W1, Canada; cmoody@uoguelph.ca

Cats require regular examinations to ensure optimal health; however, inadequate restraint can increase fear and aggression, resulting in inadequate physical examinations, and poor diagnosis and treatment. There is a lack of science-based evidence to inform best practice for restraint, so the current study evaluated the responses of companion cats to three common restraint techniques: (1) full body (n=19; known negative control); (2) scruff (n=17); and (3) clips applied to the neck (n=16). Cats in each group were also passively handled for use as a baseline comparison (known neutral control). Cats were blocked for sex and age, then randomly allocated to treatment groups. Order of restraint was also counterbalanced within groups. During each 1-min restraint, a mock veterinary examination was performed to assess previously validated measures of negative cat responses to handling. Behavioural (vocalizations- meow, yowl, growl; side and back ear position) and physiological (respiratory rate, pupil dilation) responses were assessed. Cats were initially assessed as either friendly or unfriendly during interactions with a stranger, and this was included as a covariate in analyses. Treatments were compared using linear mixed models that included the effects of sex and age, and cat as a random effect. Data are presented as averages with 95% confidence intervals. Overall, cats showed elevated responses to both full body and clip restraint. In comparison to passive restraint, cats showed larger pupil dilation, a greater number of vocalizations per minute, and a greater odds of having a negative ear position when handled with full body (pupils: 0.45[0.42,0.49] vs 0.54[0.49,0.59], P=0.003; vocalizations: 0.115[0.052,0.253] vs 0.388[0.159,0.947], P=0.009; ear: 0.336[0.170,0.662]; P=0.002), and clip restraint (pupils: 0.45[0.42,0.49] vs 0.53[0.48,0.58], P=0.006; vocalizations: 0.115[0.052,0.253] vs 0.510[0.189,1.374], P=0.007; ear: 0.441[0.220,0.8840], P=0.02). Respiratory rate was also 1.17× faster during full body vs passive restraint (CI: 1.049,1.309; P=0.006), but no effect was seen with the other treatments. In contrast, during scruff restraint cats had reduced pupil dilation and number of vocalizations when compared to full body (pupils: 0.41[0.36-0.47] vs 0.54[0.49,0.59], P=0.002; vocalizations: 0.057[0.011-0.299] vs 0.388[0.159,0.947], P=0.04) and clip restraint (pupils: 0.41[0.36,0.47] vs 0.53[0.48,0.58], P=0.003; vocalization: 0.057[0.011,0.299] vs 0.510[0.189,1.374], P=0.02). However, the odds of having a negative ear position was greater when handled with the scruff restraint in comparison to passive handling (0.346[0.161,0.743]; P=0.008). The current results indicate that responses to clip restraint are comparable to full body restraint, suggesting that the use of clips is negative for cats. While cats showed some negative responses to scruffing in comparison to passive restraint, it was less than that observed for full body or clip restraint.
Evaluation of saliva, milk and hair cortisol as an indicator for increased HPA
axis activity in lactating sows

Winfried Otten¹, Susen Heimbürge¹, Margret Tuchscherer¹, Armin Tuchscherer² and Ellen Kanitz¹

¹Leibniz Institute for Farm Animal Biology (FBN), Institute of Behavioural Physiology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ²Leibniz Institute for Farm Animal Biology (FBN), Institute of Genetics and Biometry, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; otten@fbn-dummerstorf.de

Minimally invasive evaluation of stress over short- and long-term periods is of increasing
interest in biomedical and animal welfare research. Thus, the aim of this study was to
investigate saliva, milk and hair cortisol concentrations after repeated administrations of
adrenocorticotropic hormone (ACTH) as acute or long-term indicators for increased activity
of the hypothalamic-pituitary-adrenocortical (HPA) axis in lactating sows. Fifteen multiparous
German Landrace sows received either i.m. injections of ACTH (Synacthen Depot, 100 IU
per animal) on day 1 until day 13 post-partum once a day (n=6), twice a day (n=5) or were
left untreated (n=4). This procedure has been previously shown to increase plasma cortisol
concentrations for several hours after each administration. During the 2-week treatment
period, saliva and milk samples were taken on seven days and maternal behavior was observed.
On day 1 and 14 post-partum, respectively, two square sections (15×15 cm) were shaved close
to the skin in the caudodorsal region. Hair regrowth in these sections was collected on day 28
and 42, and additional samples of origin hair were taken. All experimental procedures were
ethically approved by the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei
Mecklenburg-Vorpommern (LALLF, 7221.3-1-028/15). Cortisol concentrations in saliva, milk
and hair were analyzed using a commercially available enzyme immunoassay either directly of
after extraction procedures. Data were analyzed using pairwise multiple comparisons of the
least square means by Tukey Kramer tests. Administrations of ACTH did not affect maternal
behavior (e.g. pre-laying behavior, nursing frequency, crushing), but increased the cortisol
concentrations in saliva (P<0.01) and milk (P<0.001) compared to control animals during
the treatment period. On day 14, basal levels were reached 12 or 24 h after the last ACTH
administration. Twice daily administration of ACTH caused higher cortisol concentrations
in saliva compared to ACTH once a day (13.0±1.2 vs 7.9±1.1 ng/ml; P<0.05), while in milk no
significant differences in cortisol levels between the ACTH treatments were found. Cortisol
concentrations were increased in origin hair samples taken on day 42 compared to day 28
(P<0.05) independent of treatment, indicating a higher HPA activity after weaning at day 28
compared to preceding periods. Administration of ACTH generally enhanced hair cortisol
concentrations in origin hair (P<0.05) and in hair samples with regrowth after day 14 (P<0.05).
Our results indicate that an enhanced HPA axis activity in lactating sows is not only reflected in
saliva, but also in milk cortisol concentrations. Hair cortisol can be a suitable and retrospective
indicator for elevated glucocorticoids over longer periods in pigs and may be used for stress
monitoring and severity assessment.
Effect on hair cortisol by the dominance relationship in the cow group

Hideaki Hayashi¹, Mami Matsumoto¹ and Shigeru Morita²

¹School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido, 069-8501, Japan, ²College of Agriculture, Food and Environment Sciences, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido, 069-8501, Japan; hhayashi@rakuno.ac.jp

Dairy cattle experience stress as a consequence of management and production and it is desirable to reduce this as much as possible. Cortisol is a key hormone in the Hypothalamic-Pituitary-Adrenal axis (HPA) reaction, and blood and salivary cortisol are usually used for an index of the stress. However, blood and salivary cortisol exhibit circadian rhythm, and animals experience stress during sampling. Hair cortisol is not affected by the circadian rhythm, and reflects blood cortisol over the long term such as several weeks. However, it is unknown how the physiologic stress evaluation using hair and the behavioral evaluation are related. Therefore in this study we aimed to investigate effect of the dominance relationship in the cow group on hair cortisol. Thirteen Holstein cows a free-stall barn with an automatic milking system were used in this study. Using the dominance relationships for each cow, a dominance value (DV) was calculated: DVi = (number of cows subordinate to cow i) / (number of known dominance relationships of cow i). Cows were divided equally based on DV to three groups of dominant, moderate and subordinate. Activity and rumination time were measured by attaching a 3 dimensional motion sensors tag to the neck of the cow. For each plasma sample, whole blood was collected via jugular venipuncture. White and black hair samples of approximately 1 g each were collected from the shoulder and the hip by severing the hair roots using scissors. Cortisol in collected hair samples was extracted with methanol after washing with isopropanol and then assayed using an enzyme immunoassay kit. The Student's t-test were employed for comparison between groups after ANOVA as the F test. Values were considered to be statistically significant if their P value was <0.05. The plasma cortisol concentration between dominant group and subordinate group was not significantly different. Shoulder white hair (1.12±0.09) and hip black hair (1.62±0.15) cortisol concentration of subordinate group were significantly higher than that (0.57±0.10, 0.97±0.15) of dominant group (P<0.05). Significant positive correlation was shown between plasma and shoulder black hair, plasma and shoulder white hair (0.82, 0.72, P<0.05). However, a significant correlation was not shown between hair and rumination time, activity, milk yield. These results suggest that subordinate cows suffer chronic stress from dominant cows and this stress can be evaluated using hair cortisol.
**Behavioural effects at colostrum feeding when using an oesophageal tube or a nipple bottle in dairy calves**

Carlos E. Hernandez¹, Bengt-Ove Rustas¹, Charlotte Berg², Helena Röcklinsberg², Stefan Alenius³, Kerstin Svennersten-Sjaunja¹ and Lena Lidfors²

¹Swedish University of Agricultural Sciences, Department of Animal Nutrition and Management, P.O. Box 7024, 75007 Uppsala, Sweden, ²Swedish University of Agricultural Sciences, Department of Animal Environment and Health, P.O. Box 234, 53223 Skara, Sweden, ³Swedish University of Agricultural Sciences, Department of Clinical Sciences, P.O. Box 7054, 75007 Uppsala, Sweden; carlos.hernandez@slu.se

New born calves have insufficient immunity to fight against disease and must therefore acquire maternal antibodies by ingesting colostrum soon after birth (transfer of passive immunity (TPI)). One of the main challenges in achieving an adequate TPI in calves fed artificially is the amount of colostrum consumed by the calves and time taken to bottle feed them. Calves that do not consume enough colostrum soon after birth are at increased risk of disease and mortality. For these reasons, some dairy farms force feed colostrum, using an oesophageal tube (OT), to all new born calves. While OT feeding seems ideal for TPI, it is not without risks. OT feeding is an invasive procedure that requires trained personnel to restrain and intubate the calves, thus increasing the risk of stress and discomfort. For these reasons, the aim of this study is to compare the effects of bottle vs OT feeding on the behavioural response of new born dairy calves. At birth, calves were randomly allocated to receive their first colostrum meal via an OT (n=15) or a nipple-bottle (n=30). Calves were fed colostrum 4 h after birth (an amount equal to 8.5% of the calf's body weight). Number of hind leg movements, vocalisations, struggles (i.e. push, slip or laying down) during feeding were analysed from videos. Differences for time feeding were compared using t-test, colostrum intake (express as % of colostrum allowance) with Chi-squared test, leg movements and struggles with Wilcoxon test. Vocalisations could not be analysed due to low frequency of the behaviour. Data are mean ± SEM. All procedures approved by ethics committee. Preliminary data from n=6 OT and n=8 bottle fed calves (analysis of remaining videos is in progress) shows that feeding calves using an OT is faster than with a bottle (370.8±121.8 vs 778.0±397.5 s, P<0.05, respectively). Colostrum intake was similar in both treatments. However, 2 bottle fed calves voluntarily consumed less than 60% of their colostrum allowance (remaining calves consumed >93% of their allowance) and 1 OT calf could only be fed 90% of its allowance due to excessive struggling (all other OT calves were fed >98% of their allowance). More calves vocalised during feeding using an OT than a bottle (3 vs 1 calves, respectively). No difference in the number of leg movements or struggles were found. Using an OT saves time during the first feeding and ensures all calves receive enough colostrum. However, from the welfare point of view, using an OT to feed colostrum to all new born calves appears to be unjustified given that only 25% of calves fail to drink enough colostrum and that 50% of the calves fed with an OT show some sign of distress (i.e. vocalisations). Therefore, preliminary data suggests that the use of an oesophageal tube feeder should be restricted to calves that fail to drink enough colostrum from a bottle.
**Motor lateralisation as welfare risk measure in dogs entering a rescue centre**

Shanis Barnard, Deborah L. Wells and Peter G. Hepper

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

Previous studies have largely reported that shelter dogs may suffer poor welfare especially in
the first few days following entry to a kennel environment. Commonly used welfare indicators
are often resource- and time-consuming. The identification of quick and easy-to-assess
measures to identify those animals that may be at welfare risk when kennelled, would enable
a prompt intervention aimed at improving well-being. It has been suggested that motor bias
(the preferred use of one limb over the other), has the potential to be used as an indicator of
emotional functioning and welfare risk. Our aim was to investigate if motor laterality, in this
case paw preference, could be used as a predictive indicator of stress (measured using cortisol
levels and behavioural observation) in a sample of 35 dogs entering a rescue shelter. Based
on previous literature, we hypothesised that higher stress levels would be associated to a left
paw directional bias or a weak lateralisation. Early morning urine samples were collected on
the animals' first day after kennel admission to assess cortisol levels. We recorded the animals
while undisturbed in their kennel during three bouts of 30 min. observations and analysed
the behaviour (e.g. locomotion, posture, position in kennel, vocalisations) as duration and
frequency of occurrence. Finally, we scored the preferred paw used by the dog during a food-
retrieval task (Kong™ test). Analysis carried out on 12 dogs to date showed a positive correlation
between strength of laterality and barking behaviour (i.e. dogs spent more of their time barking
with increased strength of paw preference; Spearman's rho: R=0.62, P=0.03, tendency after
Bonferroni correction). No significant correlations emerged between the animals' cortisol levels
and laterality (direction and strength of paw bias). Finally, we found significant correlations
between cortisol level and behaviour: dogs with higher cortisol levels spent less time moving
(Spearman's rho: R=-0.67, P=0.017) and barking (R=-0.58, P=0.047) and more time laying
down (R=0.66, P=0.018) than animals with lower cortisol levels. Quieter dogs seamed to cope
less in the new environment and tended to be weakly lateralised. These results only partially
support our hypothesis. Our preliminary analysis suggest that there could be a relationship
between motor laterality and stress levels in dogs entering a kennel; however, a larger sample
is needed to confirm our results.
**Effect of isolation stress on feeding behaviour of caged laying hens using a high-speed digital camera**

Shuichi Ito¹, Mari Iwahara¹, Yumi Nozaki¹, Yuko Suenaga¹, Ken-ichi Yayou² and Chinobu Okamoto¹

¹Tokai University, Department of Agriculture, Kawayo, Minamiaso, Aso-gun, Kumamoto-ken, 869-1404, Japan, ²National Agriculture and Food Research Organization, Animal Waste Management and Environment Research Division Institute of Livestock and Grassland Scien, 2, Ikenodai, Tsukuba, 305-0901, Japan; shuichi_ito@agri.u-tokai.ac.jp

Psychological stress affects feed intake and foraging behaviour in laying hens. Foraging behaviour of caged laying hens can be classified as true feeding, in which pecking is accompanied by food intake, and food tampering, in which pecking is not accompanied by food intake. It is unknown whether psychological stress affects true feeding and food tampering. In this study, we investigated the effect of stressors on laying hens and classified the foraging patterns using a high-speed camera. We randomly selected five commercial laying hens (Boris Brown) that were placed in individual conventional cages as the experimental hens. The hens were housed individually and fed a custom-made pellet diet *ad libitum*. During the control period, these birds were housed in the same room, whereas for social isolation, each test bird was isolated in its own cage by removing peers. The quantity of feed consumed was measured every hour on observation days using electrodes. We categorised feeding behaviours in detail by analysing images captured by conventional high-speed cameras. The number of pecks during each feeding behaviour was divided by the total number of times pecking. Statistical analysis was carried out using a Paired t-test for each feeding behaviour. The feeding behaviours classified by high-speed camera video analysis included hens showing ‘true-feeding’, ‘feeding-failure (dropped the feed)’ and ‘food-tampering’. No differences were observed in the proportions of true-feeding (t=0.6247, df=4, P=0.566) or food-tampering (t=2.093, df=4, P=0.104) between the isolation and control conditions. However, the proportion of hens that feeding-failure was significantly (t=2.979, df=4, P=0.040) greater under the isolated condition (27.45±8.45%) than under the control condition (20.36±6.80%). The quantity of feed consumed was not different between the conditions. We observed an increase in the feeding failure rate in hens held under isolation stress; however, feed intake rate was not affected. Classifying feeding behaviour of caged hens using a high-speed digital camera may be useful to evaluate their behavioural response to stress.
# Effects of overstocking and restricting feed access on the behaviour of lactating Holstein dairy cows

Mac A. Campbell¹,², Peter D. Krawczel³, Heather M. Dann² and Rick J. Grant²

¹The University of Vermont, Department of Animal Science, 102 Terrill, 570 Main Street, Burlington, VT 05405, USA, ²William H. Miner Agricultural Research Institute, 1034 Miner Farm Road, Chazy, NY 12921, USA, ³The University of Tennessee, Department of Animal Science, 2506 River Drive, 258 Brehm Animal Science, Knoxville, TN 37996, USA; krawczel@utk.edu

The cumulative negative effects of overstocked dairy housing and feed restriction, two common stressors, may be greater than either imposed individually. The objective was to determine the interaction between stocking density (100 vs 142% occupancy of feeding and resting space) and restricted feed availability (0 h vs 5 h of feed restriction) on lying, feeding, and rumination behaviours. Multiparous (n=48) and primiparous (n=20) Holstein cows were assigned to 1 of 4 pens (n=17 cows/pen) balanced for parity (2.3±1.1; mean ± SD), days in milk (121±38), and milk yield (47±8 kg/d). Treatments were assigned to pens in a 4x4 Latin square with 14-d periods. A 2x2 factorial arrangement was used to combine two levels of stocking density and feed restriction into 4 total treatments. Total mixed ration was delivered 1×/d (approximately 06:00) and restriction began 19 h later. Time spent lying, feeding, and ruminating were measured using 10-min scan sampling for 72-h from d 8-10 of each period. Feeding and ruminating were summarized as h/d and total min from 0-8 h post-feeding and total min from 17-24 h post-feeding. Pen intake was measured on d 8-14. Data were analyzed using a mixed model in JMP with pen as the experiment unit. Contrary to our hypothesis, there was no interaction of stocking density and feed restriction on behavioural responses, which suggests that there was not a cumulative effect of these potential stressors. Overstocking decreased daily lying time (13.1±0.2 (mean ± SE) vs 12.6±0.2 h/d; P=0.02) and total rumination in a freestall (84.7±1.4 vs 79.1±1.4%; P<0.01). Restricting feed access decreased total feeding time (3.8±0.1 vs 3.7±0.1 h/d; P<0.01). Feeding time in the 8 h following feed delivery was increased by restricting access (96.5 vs 85±3 min), but decreased in the last hours before feed delivery (40±2 vs 60.5±2 min). Intake (25.8±0.3 kg/cow/d) was not affected by this behavioural change (P>0.10). Total rumination time (518±9 min/d) was not affected by feed restriction (P=0.43), but was decreased following feed delivery (153.5±3 vs 162.5±3 min/8-h period) and increased during the later period (199.5±5 vs 187±5 min/ 8-h period). While behavioral changes indicative of a cumulative effect of treatments were not evident, a key behavioural need (lying time) was reduced and diurnal patterns (feeding and ruminating) were altered to maintain total chewing activity. This suggests that both factors result in detrimental effects when presented independently of one another. The lack of a cumulative effect may indicate that cows use different strategies to mitigate these factors. Future research should focus on factors such as overstocking and freestall management, or restricted feed availability and diet composition, which might result in compounding detrimental effects on the behaviour of lactating dairy cows.
**The behaviour and heart rate variability of wool-biting sheep**

Akane Sato, Ken-ichi Takeda and Chen-Yu Huang

Faculty of Agriculture, 8304 Minami-Minowa-mura, Kamiina-gun, Nagano-ken, 399-4598, Japan; chenyo0601@icloud.com

Sheep subjected to wool-biting lose wool and their skin is injured. However, we previously found that some sheep did not try to escape when their wool was being bitten, in which the biters simultaneously ingested quantities of wool that could cause serious health problems. In this study, we attempted to identify (1) the bitten sheep's responses and the relationship between their posture and their hierarchical position and (2) the sheep's heart rate variability (HRV) stress response while having their wool bitten. This study examined 15 Friesland ewes. Each ewe was fed 1 kg of roughage daily at 08:30 and 15:00 and 350 g of concentrate at 15:20. Their behaviour was observed from 16:00 to 18:00, and the posture (standing/lying) and response (tolerate/escape) of the bitten sheep were recorded. The hierarchy of ewes was determined using the feed-scrambling method. We also used a heart rate sensor to record the R-R interval and HRV of four ewes while resting (standing/lying), and being subjected to wool-biting, brushing, wool-pulling and poking with a pencil. The heart rates and the ratio of low frequency to high frequency (LF/HF) in each situation were compared. Over 20 hours, 155 wool-biting events were recorded and 83.9% of the ewes escaped this behaviour rather than tolerated it (P<0.01). The ewes preferred to target higher-ranked individuals while they were lying down. Furthermore, ewes that were lying down tended not to escape when subjected to wool-biting. However, the heart rate and LF/HF were also higher in the lying resting ewes that did not escape. Our results suggest that sheep were under stress despite tolerating their wool being bitten by other individuals.
Does behaviour in novelty tests predict tail biting in pigs?

Sabine Dippel¹, Martina Gerken², Christina Veit¹ and Marie Albers¹,²

¹Friedrich-Loeffler-Institut, Dörnbergstr. 25/27, 29223 Celle, Germany, ²Georg-August-Universität Göttingen, Albrecht Taer Weg 3, 37075 Göttingen, Germany; sabine.dippel@fli.de

Stress can cause tail biting in pigs, which in some cases might be due to detrimental effects of stress on the affective state of the pigs. As affective state can influence how animals react to novel stimuli, we investigated whether behaviour of pigs in tests can predict the onset of tail biting under practical conditions. Tests procedures used were simple in order to allow potential daily use by farmers. A total of 17 groups of 35 to 40 pigs were tested on up to 12 test days in their home pens. The first test day was five days before, the last 24 days after groups were moved from rearing to fattening housing (no mixing). Testing stopped after severe tail lesions occurred. In each group, a voluntary human approach test (VHAT), a novel object test (NOT) and a modified novel object test where the object was thrown into the pen (mNOT) were performed on the same day with alternating objects. Parameters recorded during tests were seconds until first CONTACT and percent of group within one pig length of person/object at 30 seconds after start (INTEREST). During mNOT, ‘percent of group STARTLED when object hits ground’ was additionally recorded. After tests, pigs were scored for tail lesions. All tasks were performed by the same person. Tail biting outbreaks were defined as ≥20% of animals with tail lesions, and for each test, associations between test parameters and the independent factors ‘DAYS in relation to outbreak’ and ‘consecutive test number’ (Tno) were analysed in linear mixed models with batch (n=3) and group as random factors. No significant (P≤0.05) association between DAYS and any test parameter was found. Tail biting outbreaks occurred in all groups at median 6 [Q25=6|Q75=10] days after behaviour tests started, and many pigs already had ear lesions at that point. Tno was significantly associated with CONTACT (-0.83 s, SE=0.18) during VHAT, as well as with CONTACT (-0.82 s, SE=0.24), INTEREST (1.57%, SE=0.60), and STARTLE (-6.91%, SE=1.78) during mNOT. Median Tno per group was 10 [9|12]. Median CONTACT times were 0 s [0|1], 1 s [1|2] and 2 s [1|3] for VHAT, NOT and mNOT, respectively. Median INTEREST was 33% [29|36] during VHAT, 28% [22|33] during NOT and 26% [20|33] during mNOT. Median STARTLE was 40% [20|70]. The almost simultaneous outbreak of tail biting in relation to start of behaviour tests on this farm probably influenced the results due to confounding with habituation. Furthermore, the initial presence of ear lesions might have had an influence. However, although no association between behaviour of pigs in novelty tests and tail biting outbreaks could be established, the variation of behaviour between groups and over time warrant further investigation.
**The occurrence of tail and ear lesions in relation to enrichment and manipulative behavior in weaner piglets**

Christina Veit¹, Angelika Grümpel¹,², Joachim Krieter² and Sabine Dippel¹

¹Institute of Animal Welfare and Animal Husbandry, Friedrich-Loeffler-Institut, Dörnbergstraße 25/27, 29223 Celle, Germany, ²Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Hermann-Rodewald-Str. 6, 24118 Kiel, Germany; christina.veit@fli.de

Tail biting is closely linked with foraging and explorative behavior. While manipulative behavior directed to other pigs has been shown to increase during tail biting outbreaks in experiments, directing manipulative behavior away from pigs contributes to tail biting prevention. We wanted to find out, whether enrichment characteristics could be linked to manipulative behavior and tail and ear lesions of weaned piglets during on-farm assessment. During the application of a tail biting intervention tool in 132 pens on 27 farms, one observer assessed the proportion of active animals per pen which manipulated enrichment (ENRICH%), pen surroundings (PEN%), or other pigs (PIGS%) using scan sampling. Additionally, prevalence of tail and ear lesions and type of enrichment (solid objects only vs loose enrichment with/ without objects; n=42 vs 90 pens) were assessed, and a subjective estimate of 'percent of pigs which can simultaneously access enrichment' made. Data were analyzed using Wilcoxon rank sum tests. Due to skewed distribution, lesion prevalences were collapsed into binary variables using a threshold of 25%. The proportion of pigs manipulating enrichment was higher in pens with loose enrichment (all organic material, e.g. straw; median [Q25|Q75] = 9.5 [2.0|16.0]) compared to solid objects (e.g. objects on a chain; 3.0 [0.0|7.0]; P<0.001). This coincided with higher access for loose enrichment than for objects (median = 40 vs 20%, respectively). The prevalence of tail lesions was 20.5% [10.0 | 37.5] and ear lesions 19.0% [10.0 | 30.0]. Pigs in pens with ≥25% tail lesions (n=59) manipulated enrichment significantly more often (8.0 [3.0 | 13.0]) than pigs in pens with <25% tail lesions (n=73; 1.0 [0.0 | 7.0]; P<0.001). Regarding PIGS% or PEN%, no significant differences were found between pens with <25% tail lesions (median PIGS% and PEN%=0.0) and ≥25% tail lesions (PIGS% and PEN%=3.0). There were no significant differences for ENRICH%, PIGS% or PEN% between pens with <25% ear lesions (0, 0 and 4, respectively) and ≥25% ear lesions (0, 3 and 5, respectively). It can be concluded that provision of loose (organic) enrichment occupies more pigs in a group. Therefore, this combination of attractive material with better simultaneous access might be more effective in tail biting prevention on farm. The higher proportions of pigs using enrichment found in pens with ≥25% tail lesions might be an indicator for increased stress due to biting activities.
**Intramuscular injections induce conditioned place aversion in calves**

Thomas Ede, Marina Von Keyserlingk and Daniel Weary

University of British Columbia, Animal Welfare Program, 2329 West Mall, Vancouver, BC V6T 1Z4, Canada; thomas.ede92@gmail.com

Injections by needle are widely used in the administration of medicines, vaccination and anaesthesia. Injections cause somatic pain in humans, but little is known about how aversive these and alternative administration methods are for animals. We assessed aversion to injections using a conditioned place aversion protocol. Holstein calves (n=15) were randomly assigned to one of four routes of administration for 0.5 ml of saline: intramuscular (0.9×40 mm needle, rump), intranasal (MAD 300, right nostril), subcutaneous (0.9×20 mm, rump skin) and a null control (no treatment). The apparatus consisted of two distinctly colored connected pens with a milk reward available on one side. Approximately 12 h after their last milk meal, calves were allowed to enter the apparatus and received the treatment while consuming 1 l of milk from a bottle (for the first 3 sessions; this reward was reduced to 500 ml, and then to 250 ml, and finally to 0 ml for the last 3 sessions). Over all sessions, we observed a difference between treatments in the latency to enter the pen containing the milk (mixed model with repeated measures, P=0.005). Specified contrasts showed no difference in latency for the subcutaneous (mean ± SE: 15.3±5.0 s) and intranasal (9.6±6.2 s) treatments, with both being similar to the null control (4.4±0.9 s), and both shorter than for intramuscular injections (52.0±11.4 s). Latency increased with lower milk rewards (mixed model with repeated measures, P<0.0001), but there was no interaction with treatment. These results indicate that intramuscular injections are aversive, and that subcutaneous and intranasal injections are a refinement.
**Pain face intensity for quantification of pain in *Bos indicus* bull calves**

Karina Bech Gleerup¹, Teresa Collins², Gabrielle C. Musk², Timothy H. Hyndman², Heidi S. Lehmann², Craig B. Johnson³ and Michael Laurence²

¹University of Copenhagen, Department of Veterinary Clinical Sciences, Højbakkegård Alle 5, 2630 Tåstrup, Denmark, ²Murdoch University, College of Veterinary Medicine, 90 South Street, Western Australia, 6150, Australia, ³Massey University, Institute of Veterinary, Animal and Biomedical Sciences, Private Bag 11 222, 4442, Palmerston North, New Zealand; kbg@sund.ku.dk

*Bos indicus* bulls are most often castrated without the use of analgesia. To determine the most efficacious analgesic protocol, feasible pain evaluation methods are needed. The aim of this study was to investigate if pain face evaluations, using the bovine (*Bos taurus*) pain face, was a feasible method. Forty-one *B. indicus* bulls underwent halothane-anaesthesia allocated to four groups: one non-surgical group (n=5), and three castration treatment groups: CL, intra-testicular and subcutaneous lidocaine (n=12), CM, subcutaneous meloxicam (n=12) and C, no analgesia (n=12). The animals were observed before anaesthesia (t0) and after anaesthesia (t1, t2, t3 and t24) by the same observer (blinded to treatment), trained to evaluate the bovine pain face (*B. taurus*). The face was evaluated as a whole, as described for *B. taurus* but an intensity score was added to the pain face evaluation, using a simple descriptive pain face intensity scale, where 0=no pain face, 1 = mild intensity, 2 = moderate intensity, 3 = severe intensity. All ratings were performed by visual inspection from the distance through binoculars. Thirteen missing ratings were evaluated (blinded) from videos retrospectively. All animals scored 0 (no pain face) before anaesthesia. The non-surgical group scored 0 at every time point. For the castrated animals, the risk of receiving a pain face intensity score 2 or 3 during the first 24 hours following surgery was, 77% for group C, 80% for group CL and 53% for group CM. The results of this study indicate that, a direct pain face evaluation with an intensity score, may be useful for quantifying pain in *B. indicus* cattle. In this study, the animals treated with pre-operative meloxicam (CM), had a lower risk of receiving a moderate-severe intensity pain face score during the first 24 hours, compared to the animals in the C and CL groups.
# ‘Sounding the slaughterhouse’: testing the ‘soundness’ of cow vocalisations as an indicator of cow welfare assessment

Eimear McLoughlin

University of Exeter, Sociology, Philosophy and Anthropology, Amory Building, Rennes Drive, Streatham Campus, EX4 4RJ, United Kingdom; e.mcloughlin@exeter.ac.uk

In an auditory ethnography of the slaughterhouse, I will study how sound is managed, understood and mediated by lairage staff. I will explore how sound, from the biological, like cow vocalisations to the technological, like machinery, impact on and influence the lairage workers. In their daily routines, various sounds and noises are listened to and attended to, whilst others are heard and ignored. Lairage workers, drawing on a breadth of experience, tacit knowledge and certified animal welfare training, define what is important and what is unimportant throughout the day. In this manner, sound can be understood as a mode of surveillance in the lairage that is managed and negotiated by workers. In a sounding of the slaughterhouse, I contend that the lairage workers employ specialised knowledge in daily practices which enables rapid and informed welfare assessments. Lairage work is characterised by efficient and timely processing of animals that are in an unfamiliar environment and are likely to experience heightened levels of stress thus, I will interrogate how the brevity of contact from the human's perspective and the unfamiliarity of the setting from the animals' perspective can be negotiated whilst achieving an accurate welfare assessment. Drawing on the innovative approach of Wemelsfelder's Qualitative Behaviour Approach (QBA) to animal welfare which emphasises the 'wholeness' of the individual animal by integrating developments in philosophy of mind with biological animal welfare scholarship, I will explore how lairage workers assess animals for overall health and wellbeing, with a view to illustrating how assessments are a combination of objective indicators of the animal body and subjective interpretations of lairage workers. In order to validate the tacit knowledge of the lairage staff, I will focus on how cow vocalisations are perceived by workers, as these are typically used as quantitative stress indicators in welfare assessments. The complexity and contextuality of cattle vocalisations is experienced by lairage workers. Therefore, unlike previous animal welfare studies where only the frequency of cow vocalisations is of importance, this study will reproduce and explore the context of the calls, as experienced by lairage staff thereby contributing to a more holistic understanding of the wellbeing of the animal. This research will be conducted in May and will contribute to the growing literature into how qualitative behavioural assessments are both scientifically rigorous but adequately represent the complexity of animal sentience, thereby improving animal welfare standards. This research will unearth the intersection between sound, worker knowledge and welfare assessments thereby contributing to a deeper understanding of the relationship between lairage workers and cattle, as well as furthering slaughterhouse research on worker wellbeing.