Experimental Methodology

Due to the logistics of repeated blood sampling birds at regular intervals it was necessary to separate the trial in to two separate study weeks, with a different group of birds in each week. In experimental week one (17-23 January 2011) 16 layer hens were obtained from a poultry egg-laying farm. These chickens were briefly put under a general anaesthetic at Massey University in order to place a catheter in the medial metatarsal vein and in one case the brachial vein. After catheter placement these birds were moved 5 minutes by road to the Massey Poultry Research Unit. Half of the birds became control birds and half became treatment birds. In week two (24-31 January 2011) 18 layer hens were obtained from the poultry farm; 10 of these were put in the control group and 8 were put in the treatment group. These birds were transported directly to the Massey Poultry Research Unit and did not undergo anaesthetic or catheter placement.

The birds used were HyLine Brown hens sourced from Turks Poultry farm (108 Purcell Street, Foxton, New Zealand) and were taken at 47 weeks of age and selected by the staff to be in good physical condition. The birds were selected and removed from the cage by a member of staff and carried upside down with between one to three birds in each hand. The birds were then given a quick physical examination to see if there were any obvious broken bones or lameness. One bird was rejected and replaced as she was considered significantly lower in body condition then the other birds. The birds were placed in chicken crates for transport with either eight or nine birds in a crate. The chickens were transported in the crates for approximately 40 minutes by road to the Poultry Research and Feed Processing Unit. At the poultry unit each bird was given a band so that it could be individually identified and randomly placed in one of two separate but identical rooms. The birds in one of these rooms

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became treatment birds while the birds in the other became control birds. The trial commenced the following day.

Sampling started each day around 8am and alternated between treatment groups where possible. When it was time for sampling, the handler would enter the treatment or control room, identify and retrieve the specific bird by cornering it and picking it up by hand. A blood sample was performed using heparinised 25 gauge needles and 1ml syringes and between 0.5 ml and 1 ml of blood was taken from either a placed catheter, the medial metatarsal vein, the brachial vein or occasionally from the jugular vein. If a sample was taken from a catheter then the catheter was first flushed with 0.5ml of heparinised saline solution and then the first 0.6 ml of fluid withdrawn from the catheter was discarded. All birds with catheters had them flushed at the end of each day also with 0.5 ml of heparinised saline solution.

Control birds were handled and blood sampled at 0, 30, 60, 120 minutes and 24, 48, 72 hours after capture. Between samples at 0, 30, 60, 120 minutes, the control chickens were kept individually in a ventilated box near the sampling area. At 120 minutes they were returned to their rooms and for the 24, 48, 72 hour samples the chickens were picked up from their room and taken to the sampling area, blood sampled and then returned to their room.

All birds in treatment and control groups were handled by necessity for blood sampling at each time point. During this process the birds were carefully removed from either the box (30, 60, 120 minute samples) or from the housing room (0 minute, 24, 48, 72 hour samples) and held either on their backs with their bodies supported with one wing extended or on their side with their body supported and one leg outstretched. Following the blood sample, the bird was held for a short period to ensure haemostasis of the needle site had occurred and then returned to the box or room.

Treatment birds were also handled and sampled at 0, 30, 60, 120 minutes and 24, 48, 72 hours. However in order to simulate the chase that can often happen in wild kiwi capture, treatment birds were first taken from the pens and blood sampled (time = -10 minutes) and then given a simulated chase event where the chickens were walked around an open room by

the handler who kept them moving. The chicken was ‘chased’ in this manner until the sample

at time 0 needed to be taken. Once the time 0 sample had been taken the chicken was given a

‘handling event.’ This involved additional handling of the chicken in accordance with the

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the bird had its legs taped together at the tarsus just below the hock joint, using insulation tape. The bird was then weighed by suspending it from the tape used to bind its legs. All treatment birds were then held by a single handler in the same manner. As prescribed by the kiwi best practice guide, the bird was then held supported on its back with the handler using

one hand firmly holding the bound legs and the other hand (or person’s knees) to support the

birds back. The insulation tape binding the legs was removed immediately prior to the blood sample at 30 minutes. Between samples at 30, 60, 120 minutes, the treatment chickens were kept individually in a ventilated box near the sampling area. At 120 minutes they were returned to their rooms and for the 24, 48, 72 hour samples the chickens were picked up from their room and taken to the sampling area, blood sampled and then returned to their room.