Materials and methods .1 Ethical considerations .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

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of birds used considered to be the minimum required to obtain a statistically significant difference between treatments.

4.2.2 Experimental design and overview of treatments

This study was conducted in an experimental room in the Comparative Biology Centre (CBC) using a cross-over design (each bird served as a control for itself) having two phases: in Phase 1, on Day 6 Cort-Dex birds were offered mealworms injected with deuterated corticosterone (Cort d8; C21D8H22O4; a stable isotope, 4-pregnen-11β, 21-diol-3, 20-dione; QMX Laboratories, Thaxted, UK) and dexamethasone (Sigma Aldrich, Gillingham, UK) dissolved in a non-polar solvent, namely DMSO (Sigma Aldrich, Gillingham, UK),whereas Control birds were offered mealworms injected with DMSO only. In the literature, it has been reported that an acute dose of corticosterone (4μg) offered to white crown sparrows elevated plasma corticosterone levels for 60 minutes, after which they returned to baseline values (Breuner et al., 1998). In a different study, a dose of 500μg of dexamethasone offered to pigeons required a period of 52 h for corticosterone levels to return to baseline values (Westerhof et al., 1994).

Therefore, the decision was made to adopt a three-day interval before the second phase (Phase 2) of the study began on Day 9. The treatments were swapped over so that control birds now received mealworms injected with corticosterone/dexamethasone, and birds previously on the Cort-Dex treatment now received control mealworms (injected with DMSO only). There were four replicate pens with three birds per pen. The arrangement of the experiment and timing of sampling is presented in Table 4.1. To ease the workload, application of treatment was staggered over two days.

4.2.3 Animals and application of treatments

A total of 12female broiler chickens (Ross 308, 32 days old and approximately 1.1 kg body weight) purchased from Oakland Farms Ltd., York, UK. On Day 0, birds arrived at the laboratory and were allocated to four pens (each 30 cm tall and 90 cm in diameter bedded with wood shavings 5 cm deep; Goodwill’s Wood shavings and Timber Products Ltd, Ponteland, UK) at three birds per pen. Female broilers were chosen because of their rapid excretion of radiolabelled corticosterone in the urine (Hirschenhauser et al., 2012). In each pen, two birds were randomly assigned to the control treatment and one bird to the Cort-Dex treatment. One dosage of Cort d8 equivalent to 4mg/Kg bodyweight(Post et al., 2003) was tested against the control. This

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non-invasive method of administering corticosterone involved on Day 6 offering the birds mealworms which had been previously injected with either Cort d8) and dexamethasone both dissolved in DMSO) or DMSO only. On Days 1 to 3, the birds were offered standard mealworms (nothing injected) once a day in their pen so that they become accustomed to feeding on mealworms before the application of treatment.

4.2.4 Preparation of experimental mealworms Hormone preparation

To prepare the deuterated corticosterone (Cort d8), 25mg of Cort d8 was dissolved in 0.25ml of DMSO and vortexed giving a concentration of 100 mg/ml. Dexamethasone was prepared by dissolving 50 mg of dex. in 1.0 ml of DMSO and vortexed giving a concentration of 50 mg/ml.

Preparation of mealworms

Before injecting mealworms they were made inactive by placing them inside a plastic bag which was then placed on ice for approximately 20 mins. According to treatment, Cort d8, dexamethasone and DMSO or DMSO only were injected into the mealworms using a 100μL Hamilton syringe (Hamilton Bonaduz AG, CH-7402 Bonaduz, Switzerland). To the control birds, a dose equivalent to 40 μl of DMSO was administered per bird by offering the birds four mealworms each injected with 10 μl of DMSO (Figure 4.2c). Cort-Dex birds were offered seven meal worms, the first two mealworms were injected with Cort d8 (20 μl of the corticosterone 100mg/ml preparation was injected into each mealworm to arrive at a dose of 4mg per bird) while the other five mealworms were injected with dexamethasone (20 μl of the dexamethasone 50mg/ml preparation was injected into each mealworm to arrive at a dose of 5mg per bird). As soon as the mealworm was injected, it was offered to the bird as shown in Figure 4.2d.

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4.2.5 Experimental procedure

Commercial-specification poultry feed was provided ad libitum to birds in a feed hopper placed on the floor of the pen (20% CP, 4% oil, 6 % ash and 13.0 MJ/kg ME, Poultry Pro Finisher Pellets, W.E. Jameson & Son Ltd, Masham, UK). Fresh water was provided daily in a bell drinker (Wells Poultry Equipment, UK). Daily checks on the health and welfare of the birds were conducted.

At the start of each Phase, a clean heavy duty waterproof plastic membrane sheet was spread on the floor of the experimental room. A plastic ring similar in size to the home pen (30 cm tall and 90 cm in diameter) was then placed on the plastic membrane sheet to be used as the holding pen. A transparent perspex divider was used to divide this holding pen into three equal sections and one bird was placed in each section (Figure 4.2a). The birds were left in this pen until they produced the first dropping (basal

Figure 4.2a: Birds placed in pen awaiting the production of baseline guano samples

Figure 4.2b: Birds provided with feed and water after baseline drooping and plasma samples were collected

Figure 4.2c: Injecting mealworms with DMSO

Figure 4.2d: A broiler bird being offered a mealworm

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sample), which was within 20 minutes of being placed in the holding pen. Immediately a dropping was produced, the bird was picked up and a blood sample taken within 2 mins to act as a basal sample. Then the bird was placed back in its section and offered the appropriate mealworms according to treatment. Feed and water was then provided to the bird (Figure 4.2b).

At 10 mins and 2 h post treatment, the 2^nd and 3^rd blood samples were taken from each bird, again within 2 mins of the bird being picked up. All droppings produced between 10 mins and 2 h post ingestion of mealworms were collected and labelled according to treatment and time post ingestion of mealworms.