Task validation – manipulation of affective state in broiler chickens

To validate a cognitive bias task, it was necessary to show that the animals’ judgment of ambiguous stimuli is sensitive to their affective state. This required us to manipulate affective state. In most previous tests authors have elected to manipulate state indirectly via changing the animals’ environment (enrichment, light intensity), but in this study we decided to manipulate state more directly by feeding the stress hormone corticosterone

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to mimic a state of chronic stress. Changes in environmental conditions such as unpredictable housing conditions (Harding et al., 2004), predatory attack (Bateson et al., 2011), barren environment (Douglas et al., 2012) have been associated with pessimistic judgement biases and hence by assumption with negative affective states.

Recently, Pomerantz et al. (2012) measured both cognitive bias and faecal corticosterone level in capuchins exhibiting stereotypic behaviour. They found that capuchins which showed higher level of stereotypic behaviour and had higher faecal corticosterone levels were in a negative affective state. Also, honey bees subjected to predatory attack also showed physiological changes related to stress (Bateson et al., 2011). Together, these results suggest that stress has a link with the induction of negative affective state (Paul et al., 2005). The secretion of corticosterone during short-term (acute) stress is beneficial because it prepares the animal to cope with the situation.

However, long-term (chronic) stress response is detrimental to the birds (Dickens et al., 2010) thereby compromising welfare. Some of the negative consequences of chronic stress are breakdown of muscle proteins and fat and suppression of immune function (Hill et al., 2008) changes in behaviour, disruption of the HPA (Dickens et al., 2010) and impaired cognitive functions (Lupein et al., 2007).

Normally, the negative feedback system regulates the concentration of corticosterone in circulation, but at times the negative feedback system is inhibited and this consequently leads to a continuous increase in the concentration of corticosterone (Pariante and Lightman, 2008), indicative of chronic stress (Wolf, 2008). In the wild, birds are faced with series of stressors such as predators, harsh weather and lack of food which cause their levels of corticosterone to increase (Pravosudov, 2005). Broilers could similarly experience multiple stressors including high temperature, high RH, lighting condition, wet litter, increased ammonia level and noise (Rosales, 1994).

Chronic stress can be mimicked by implanting animals with stress hormones (Puvaldopirod and Thaxton, 2000; Olanrewaju et al., 2006) or feeding hormones through drinking water (Post et al., 2003; Shini et al., 2008) to achieve a continuous elevation of corticosterone level for several days (7-10 days). Puvaldopirod and Thaxton (2000) undertook a comprehensive study to investigate changes in broiler chickens associated with chronic stress. Their study showed that as early as 4 days post implant of an ACTH mini-osmotic pump (delivering 8IU/kg BW/day), the performance (depression in body weight), immune system (reduction of relative spleen weight) and metabolism (increased blood glucose and relative liver weight) were affected. Chronic

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stress also induces changes in acid-base balance in broilers (Olanrewaju et al., 2006).

Changes in internal organs and acid-base status could serve as indicators of chronic stress in replacement of levels of plasma corticosterone which is known to be easily affected by procedures involved in blood sampling such as catching and handling, especially if blood is not sampled within 2-3 minutes of catching the bird (Mormede et al., 2007). A regulated and non-invasive way of administration of exogenous corticosterone in white crown sparrows was reported by Breuner et al. (1998) where birds were offered mealworms injected with either 4μg of corticosterone dissolved in dimethyl sulfoxide (DMSO) or DMSO only. Breuner et al. (1998) reported a significant peak of in the levels of corticosterone in the plasma seven minutes after ingestion and this stimulated the activity level of the birds in terms of an increase in perch hopping behaviour.

To obtain further confirmation that we had manipulated the affective state of the birds, we chose to use the novel object test as a behavioural assay of fear. As mentioned earlier, increased standing alert behaviour in mother hens subjected to an aversive stimulus, namely an air puff, was an indication of fear (Edgar et al., 2011). Fear is generated when an animal is faced with threatening conditions such that behavioural and physiological responses are triggered (Cockrem, 2007) in preparation for danger (Forkman et al., 2007). Fear is considered to be a type of emotion (Forman et al., 2007), obviously a negative one because animals cannot be fearful in the absence of a negative stimulus. Fear can be measured through the use of a novel object test (Wichman et al., 2012), such that an animal that avoids a novel object is considered fearful. Other tests of measuring fearfulness in animals are open field test and tonic immobility (Cockrem, 2007). The use of an open field test does not however indicate the true level of fear because it is masked by the effect of isolation (Forkman et al., 2007). Interestingly, Wichman et al. (2012) found a positive correlation (P<0.05) between the tonic immobility and novel objects tests of fearfulness. Hence, a novel object test seems to have promise in measuring fear because it is based on how threatening the bird appraises the novel object. Destrez et al. (2013) found that sheep which had been chronically stressed sheep for 6 weeks were more fearful in a novel object test as indicated by displaying emotional responses such as increased vocalisation and making less contact with the novel object. In another study of Destrez et al. (2013), chronically stressed sheep (6 weeks) judged ambiguous cues (empty buckets placed in three ambiguous positions in between the two trained positions) in a cognitive bias test

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pessimistically. In amphibians, a frog exposed to a toad had increased level of corticosterone metabolites in its urine indicative of stress and subsequently displayed a longer duration of tonic immobility which implies a condition of fear (Narayan et al., 2013). Hence it seems that animals subjected to stressful conditions tend to be more fearful and this subsequently reduces productivity of the birds in terms of body weight, egg weight and even mortality (de Haas et al., 2013).

In summary therefore, the aim of this study was to develop an improved cognitive bias task for broiler chickens similar to that of Wichman et al. (2012) but with some modifications. We elected the use of a ‘punisher’, in this case an air puff, in the unrewarded position instead of an empty food bowl. We hypothesised that the averseness of the air puff would reduce the time required by birds to learn the spatial discrimination necessary for the task. Additionally, birds will be presented with 12 trials per day of training (after Burman et al., 2008 and 2009). We hypothesised that by giving the birds more trials per day, we would further reduce the number of days necessary to learn the discrimination. This new task will be validated on broilers whose internal state has been manipulated to mimic chronic stress by direct administration of corticosterone.

We hypothesised that birds whose internal state had been manipulated to mimic chronic stress would be more likely to show a pessimistic interpretation of ambiguous positions.

We also predicted that the manipulation would cause changes in internal organs and blood parameters indicative of chronic stress. Finally, we predicted that broilers under a condition of mimicked chronic stress would be more fearful to a novel object and exhibit behaviours different from the control birds.

5.2 Materials and methods 5.2.1 Ethical considerations

This experiment was conducted under Project Licence number PPL 60/4270 of the Animals (Scientific Procedures) Act 1986. Approval for the project was given at both national (Home Office) and local (Ethical Revenue Committee) level, with the number of birds used considered to be the minimum required to obtain a statistically significant difference between treatments.

130 5.2.2 Experimental design and location

The experiment was a between-subjects design with a single factor which was the treatment at two levels namely: i) Control birds fed mealworms (Tenebrio larvae) injected with DMSO solvent and ii) Corticosterone birds fed mealworms injected with corticosterone dissolved in DMSO (a non-polar solvent) for quick delivery of corticosterone into the bloodstream (Breuner et al., 1998). Birds were trained in a spatial task to differentiate between a rewarded and unrewarded position after which they were randomly allocated to treatment, either Control or Corticosterone. On the third day of treatment, birds were subjected to a cognitive bias test were three ambiguous locations between the rewarded and unrewarded locations were introduced.

The cognitive bias test was undertaken for three consecutive days (Days 3 to 5 of treatment). On Day 6, birds undertook a novel object test in the test arena. On the last day of the study, Day 7, the behaviour of the birds in their home pen was recorded after which they were weighed, blood sampled, euthanized and the internal organs recovered during post mortem.

The study was conducted in the controlled environment rooms in the Ridley Building of Newcastle University in two batches, i.e. first and second replicate. The second replicate commenced one week after the end of the first replicate; details of the replicates are as follows:

1^st replicate: 15 Ross 308 broiler chickens, 17 days of age (average weight of 1.3 kg) were purchased from a poultry farm (Oakland Farms, Moor Monkton, York, UK). The medical history showed that the birds had been given repeat vaccinations of an infectious bronchitis (IB) vaccine.

2^nd replicate: 27 Ross 308 broiler chickens, 13 days of age (average weight 950 g) were purchased from a poultry farm (Oakland Farms, Moor Monkton, York, UK). The medical history showed that the birds had received treatment for Chronic Respiratory Disease (CRD) for 3 days when they were 1-3 days old, and were given an IB vaccine when they were 10 days old.

On arrival at the laboratory in Newcastle, the birds were weighed individually and placed in holding pen, 5 birds were randomly allocated to each plastic pen (diameter of 90 cm and height of 30 cm) containing wood shavings (Goodwill’s Wood shavings and Timber Products Ltd, Ponteland, UK) approximately 5cm deep. Commercial specification broiler feed (20%CP, 13.0MJ/kg ME, 4% oil and 6% ash; W.E Jameson &

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Son Ltd, Masham, UK) and tap water was provided ad libitum. Lighting conditions were 14L:10D whilst temperature and relative humidity were maintained at 20^oC and 50% RH respectively. The birds were allowed to settle for three days to adjust to the new environment and to become familiar with the feeding and drinking apparatus. The birds were offered standard mealworms once a day (approximately 6 mealworms per bird) to allow them to become accustomed to feeding on mealworms. On Day 1 of the experiment (three days after arrival), the birds commenced the training (fully described in Section 5.2.5). Learning was confirmed when the birds showed significant difference between the rewarded and unrewarded positions.

After the learning was established (4 days), birds were randomly allocated to one of two treatment groups as described previously, namely Control (fed mealworms injected with DMSO only) or Corticosterone (fed mealworms in which corticosterone dissolved in DMSO solvent had been injected) as described in Section 5.2.3. Because of the diurnal nature of basal corticosterone levels (Hess, 2006), corticosterone was fed indirectly via mealworms twice a day i.e. 10 am and 5 pm. We mimicked chronic stress by feeding corticosterone for 7 days according to Puvaldopirod and Thaxtion (2000). The first two days was referred to as the ‘corticosterone build up stage’ and on the 3^rd day of treatment, the cognitive bias test commenced. Previous study has shown that the use of 4 mg/kg in drinking water elevated plasma levels of corticosterone from the day 1 to 8 of treatment (Post et al., 2003), so this level was chosen as an appropriate dose of corticosterone. The initial weights of the birds were similar between treatment groups.