Improved cognitive bias task for broilers i) Training i) Training

This section discusses whether the introduction of an air puff improved acquisition of the cognitive bias task. Ad hoc observations suggested that birds found the air puff aversive. Birds jumped, stepped back or froze for some time after receiving an air puff on their face. Freezing is a response to threatening situation (Wang et al., 2013). In fact, in an experiment using rats, a single air puff for one second deterred the animals from entering the part of the arena where they had received an air puff the previous day (Moriarty et al., 2012). Thus the introduction of an air puff in the current study could have contributed to the rapid discrimination between the rewarded and unrewarded positions.

The results of the current study showed an improvement over that of Wichman et al.

(2012) who reported that none of the laying birds learnt a spatial discrimination task

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after six training sessions. Indeed the laying birds used by Wichman et al. (2012) required an average of 12 training sessions before they could discriminate between the rewarded (corn) and unrewarded positions (empty bowl). However, in our study birds discriminated between rewarded (mealworm) and unrewarded (air puff) positions after only 6 training sessions. Similar findings was reported in rats that initially required 6 days to attain the learning criterion in a spatial task involving a visible but inaccessible food in the unrewarded position (Burman et al., 2008). Furthermore, the introduction of an aversive stimulus (feed treated with quinine) reduced to training period to 2 days (Burman et al., 2009).

Regarding issues relating to the relatively small proportion of birds that learnt the task, although it was our intention to train as many birds as possible for the cognitive bias task, only 60% and 22% of the birds from Replicates 1 and 2 respectively were able to attain learning criterion. A lower number of the younger birds in Replicate 2 learnt the task. In contrast, some other studies have reported success in training 4 day old birds (Salmeto et al., 2011; Hymel and Sufka, 2012), albeit using a different task. Therefore the difference may not be related to age per se, but perhaps a lower level of motivation by the younger birds to participate in the training or the possibility that younger birds were more fearful in the test arena. In the study of Salmeto et al. (2011), the latency to reach a goal box was longer in the presence of a chick stimulus cue (still picture of a chick) than a mirror cue where the birds could see a moving image. Our observation was that most of the birds that were excluded during the training could be categorised into one of three main groups namely, i) birds that failed to feed on the mealworms during the acclimatisation or pre training period, ii) birds that fed only in the presence of a companion but failed to feed when tested singly in the test arena, and iii) birds that failed to flip open the cones to access the mealworms hidden underneath it. Perhaps if we had persisted with training for a longer time, a higher number of birds would have learnt the task. Previous studies have demonstrated the need to extend the training period to avail animals the opportunity to learn a task. Douglas et al. (2012) reported that training of pigs had to continue for ten days to allow all the pigs to reach learning criterion. In another study on rate, after three days of training only 65.2% of animals learnt a discrimination task so an extra training period (4-5 days) was provided for rats that were yet to learn the task (Ritcher et al., 2012).There is a possibility that the few birds that learnt the task in our study were the fast learners; hence conclusions from the

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cognitive bias task might be restricted to the subset of animals that successfully learnt the cognitive bias task in three days.

The impact of stress on the performance of birds in a cognitive bias task was not evident in the current study despite the report that imposing stress on animals after learning a task could affect their memory in subsequent tests (Mendl, 1999). After 20 minutes of restraint (the stress), zebra finches failed to continue in a visual task but not in a spatial task (Hodgson et al., 2007). In the current study, birds which had successfully learnt the task and which were subsequently offered corticosterone-injected mealworms for two days before the cognitive bias test still demonstrated significant discrimination between the two trained positions, except for a single bird which failed to approach the cone in any of the five positions. It seems that the effect of stress on the performance of animals in a cognitive bias task depends on the particular task.

ii) Cognitive bias task

This is the first study to report the direct effect of elevated corticosterone on cognitive bias, and hence by assumption, the affective state of birds. The current study found that corticosterone-treated birds took longer to approach ambiguous positions compared to the control birds, a reaction typical of a pessimistic judgement suggesting therefore that the corticosterone birds may well be in a negative affective state.The overall latency to approach the cones in the five positions was longer in corticosterone birds which could indicate less motivation of corticosterone birds to perform the task. Hence, we decided to adjust the latencies of individual birds to approach the ambiguous cones according to their latencies to approach the rewarded and unrewarded positions. Despite this adjustment, corticosterone birds still took longer to open cones in the ambiguous positions. A similar finding was reported by Enkel et al. (2010), where rats subjected to a pharmacological treatment to increase stress (in this case a noradrenergic-glucocorticoid treatment) were pessimistic in their judgement of ambiguous cues.

5.6.2 Validation of the non-invasive method of mimicking chronic stress in broilers In this section, the effect of elevated corticosterone on physiological changes, level of fear and behaviour of birds will be discussed.

151 i) Physiological indicators

Chronic stress causes several anatomical changes within the body system of birds. The current study therefore considered the use of other blood parameters apart from corticosterone and changes in internal organs as indicators of chronic stress. The method adopted in this study involved feeding corticosterone-injected mealworms to birds and was first reported by Breuner et al. (1998) in white crowned sparrows. The corticosterone dosage (4 mg/kg body weight) was adopted from Post et al. (2003) and Shini et al. (2008) who offered similar levels of corticosterone to birds through drinking water.

The current study found physiological changes indicative of chronic stress similar to that reported in previous studies such as suppression of the relative spleen weight, increase in relative liver weight and (albeit a tendency for) increase in blood glucose levels (Puvaldopirod and Thaxton, 2000; Post et al., 2003; Shini et al., 2008; Wang et al., 2013). In a different study, significant changes in the internal organs (relative spleen and liver) of broilers were reported as early as 4 days post treatment with ACTH (Puvaldopirod and Thaxton, 2000). The suppression of the relative spleen weight suggests an impaired immune system (Post et al., 2003) or an immunosuppressive effect of corticosterone (Wiepkema and Koolhaas, 1993). Furthermore, the suppression of the relative spleen weight in broilers was accompanied by an inhibition of antibody production in response to sheep red blood cell vaccine (Post et al., 2003). Since the immune system is responsible for safeguarding the body from infections and foreign material (Hill et al., 2008), an impairment of the immune system could easily predispose animals to disease infections which consequently result in mortality.

The increased relative liver weight is caused by the accumulation of lipid in the liver during the process of fat breakdown for the production of glucose (Puvaldopirod and Thaxton, 2000; Wang et al., 2013). Gluconeogenesis is the process whereby non-carbohydrate substrates like proteins and fats are converted into glucose to avail birds with the required energy to survive during stress (Ognik and Sembratowicz, 2012) because of the suppression of the digestive processes (Øverli et al., 2002). In the current study, there was a tendency for corticosterone birds to have a greater level of blood glucose, however this is in contrast with Olanrewaju et al. (2006), Vahdatpour et al.

(2009) and Lin et al. (2004) who all found that corticosterone-treated birds had increased blood glucose levels.

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One of the detrimental effects of elevated corticosterone is a depressed body weight. In our study, the body weight gain of the corticosterone birds was 14.4g less than the control birds (P>0.05). It could be that if the study had persisted for longer or if a higher dosage of corticosterone had been used then this reduction in weight gain might have been significant. Previous studies undertaken for a longer duration (either 10 or 49 days) found significant reduction in body weight of chronically stressed birds (Post et al., 2003 and Vahdatpour et al., 2009 respectively). Decline in body weight was reported in birds treated with a higher dosage of ACTH (8IU/kg BW/day), Puvaldopirod and Thaxton (2000). The reduction in body weight arising from elevated corticosterone could be explained by the inhibitory effect of corticosterone on growth hormone (Hill et al., 2008) or the catabolic effect of corticosterone on muscle tissues.

Apart from changes in internal organs and body weight, elevated levels of corticosterone act on the kidney thus impairing the blood acid-base balance. Although we found no effect of corticosterone supplementation on levels of blood pCO2 or pH, Olanrewaju et al. (2006) reported that chronic stress increased the level of arterial pCO2

but had no effect on pH. The kidney regulates blood pH controlling the levels of bicarbonates (Powell, 2000). In addition, the reduced level of Na⁺ in the blood of corticosterone birds could be attributed to increased urinary excretion (Ewing et al., 1999). Stress has been reported to increase the water content of the excreta of birds by as much as 187% to that of control birds (Puvadolpirod and Thaxton, 2000). The loss of sodium could be detrimental because of its role in the regulation of blood pressure (Goldstein and Skadhauge, 2000). Probably increased level of corticosterone restricts the production of aldosterone, which is a steroid hormone secreted by the zona glomerulosa of the adrenal cortex that regulates the epithelial cells of the late distal tubule and early collecting duct of the nephron to reabsorb Na⁺ into the interstitial fluid and plasma for the conservation of Na⁺ (Hill et al., 2008).

ii) Response to a novel object test

This section will discuss whether chronic stress increases behavioural indicators of the level of fear in birds. The novel object test was conducted in the same test arena used for the training and cognitive bias task because it was believed that birds were accustomed to being tested alone in the arena, so it was considered an appropriate venue to estimate the level of fear in the birds. Previous studies suing the chronic mild stress paradigm on rats, quails and sheep have shown that simulated chronic stress increases the level of fear (Yang et al., 2006; Laurence et al., 2012; Destrez et al., 2013).

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Therefore we expected that in the current study the corticosterone-treated birds would be fearful, however there was no difference in the response of the corticosterone and control birds to the novel object. Corticosterone birds displayed fewer escape attempts during the novel object test than control birds; however this difference did not reach statistical significance perhaps because of the low sample size used so that it might be worth repeating this test in further investigations. During the training and cognitive bias test, the birds were fast in approaching the cone placed in the rewarded position (<15 seconds). With the novel object placed behind the cone in the rewarded position, all the birds refused to open the cone during the two-minute test period. One corticosterone bird opened the cone and accessed the mealworm within 3 seconds of stepping into the test arena, after which the bird raised its head up and discovered the novel object and then it hurriedly moved away suggesting that the bird was afraid of the novel object (personal observation).

iii) Behaviour of birds in their home pen

It is known that there is a bidirectional relationship between hormones and behaviour such that the secretion of certain hormones could trigger the display of specific behaviour or the other way round (Nelson, 2005). In the current study, the levels of preening, feeding, drinking and foraging were similar both treatment groups. Wang et al. (2013) found that the percentage of broilers walking increased under chronic stress but stress had no effect on other behaviours such as feeding, drinking, sitting, standing, foraging and dust-bathing. Our study also found an increase in walking behaviour in corticosterone birds (P>0.05) during the novel object test. Probably corticosterone enhances motion/walking and could be a signal of restlessness or anxiety in broilers. In fact, increased locomotion is one of the behavioural responses of the mother hen when they perceive that their chicks were subjected to aversive conditions (Edgar et al., 2013). The opposite trend was observed in rainbow trout where locomotor activity was greater in rainbow trout subjected to a single day cortisol treatment than those given a three-day cortisol treatment.

In the current study, behavioural recording started 7 minutes after the birds had ingested mealworms injected with either corticosterone or DMSO with the aim of finding a significant change in behaviour at the point when the plasma levels of corticosterone is at its peak (Breuner et al., 1998). After an acute dose of corticosterone, Breuner et al.

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(1998) reported an increase in perch hopping behaviour which coincided with the period when the plasma corticosterone level was at its peak (7 mins); however, this did not persist throughout the 60 minute period during which plasma corticosterone levels were elevated before returning to baseline values (Breuner et al., 1998). Breuner et al. (1998) suggested that the initial increase in perch hopping behaviour was needed to rapidly adjust to the stressful conditions. Hence, behavioural changes with respect to increased level of corticosterone may be more of an acute than chronic effect. Such a rapid adjustment in behaviour was not found in this study as behaviour recording was undertaken on the seventh day of treatment so the results cannot be compared to that of a single dose.

A cumulative effect of corticosterone (after the second and fourth dose) on feeding behaviour was reported in red-eyed vireos Vireo (Lõhmus et al., 2006) with an increase in the number of visits to the food bowl compared to control birds. The current study did not find a difference in preening behaviour between birds which were in a positive (control birds) or a negative (corticosterone birds) affective state although a behavioural study by Zimmerman et al. (2011) reported increased preening in birds expecting a positive stimuli (mealworm) compared to those treated to an unconditioned negative stimuli (water spray).

5.7 Conclusions

The improved spatial task developed by employing the use of an air puff as a punisher in the unrewarded position enhanced the learning process compared to previous reports in laying hens. To validate this task, physiologically stressed birds whose internal state was manipulated through a non-invasive administration of corticosterone (indirectly through mealworms), took longer to open cones placed in the ambiguous positions which is interpreted as a pessimistic judgement indicating a negative affective state. In addition, physiological stress in broilers was associated with reduced relative spleen weight, reduced sodium ion levels but increased plasma glucose levels and relative liver weight. Physiologically-stressed broilers had a similar level of fear to that of control birds. Body weight gain, feeding, preening, drinking and foraging behaviour were not different from that of the control birds. In conclusion, welfare markers of physiological stress in broilers include suppression of relative spleen weight and levels of sodium ion, increase in relative liver weight, blood glucose and the presence of negative affective state. Out of all these physiological indicators of chronic stress, only the relative liver

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weight was positively correlated with the latency to open cones in the ambiguous positions.

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Chapter 6: Validation of techniques to measure the core body