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Methods

Behavioural assays

Each bird was subjected to four behavioural assays. Each assay was carried out on different days to reduce the affect the tests may have on each other. The assays were carried out when the birds were 4, 6, 8, 16, 34 and 40 weeks old and it took approximately 12 days to test all 36 birds in all 4 tests.   Tests were carried out between 7am and 11am, and 2pm and 5pm (local times), coinciding with the peaks of activity of the birds. A different handler tested the birds when they were 4, 6 and 8 weeks, compared to the other tests where all observations were done by the author. At the research station, the behavioural assays were filmed using video cameras.

Prior to testing, lights were dimmed and birds were caught by using nets. This was done with the aim to standardise the stress that each bird was exposed to prior to each test, and to reduce the stress of being caught. Birds were randomly caught according to a pre-prepared list of the test order of birds. Birds were tagged with metal wing tags for final identification, but were also fitted with coloured leg rings to facilitate identification without needing to catch the birds.

Novel arena

Exposure to a novel arena has been used to estimate a range of behavioural responses in various animals. Initially, the test measures the general fear of an individual to being in a novel place, and subsequently when the individual has familiarised itself some to the arena, the test can be used to measure variation in exploration and activity.

I used this test to estimate variation in fearfulness, boldness and activity.

Weeks 4 to 8

Young birds (aged 4, 6 and 8 weeks) were tested in novel arenas on the university campus, 2 minutes away from the hatchery where they were housed. Chicks were transported to the test room in a cardboard box and kept in an enclosure similar to their home pen. The birds were allowed 60 minutes to acclimatise to the new home pen before being re-caught and introduced into the novel arena.

Different sized arenas were used for each age to accommodate for the growth of the birds (figure 1 A-D). Obstacles (empty food and water containers) were used to block the bird’s eyesight, so to fully explore the area, the bird would have to walk around them. To ensure novelty and prevent the birds from habituating to the arena between test occasions, the floor covering and the placement of obstacles were changed for each time the individual was tested (Figure 1 A-C). Behaviours (Table 1) were recorded with a 10 second interval for 15 minutes. Vocalisations (every time the chick peeped) were recorded continuously for 15 minutes. A wire net covering was used to stop the birds from escaping. 

Weeks 16 to 40

After each bird was caught in the home pen, it was placed in a dark transportation box for 5 minutes with the aim to standardise the stress each bird was exposed to before testing. After 5 minutes the bird was transferred to the novel arena. Behaviours (Table 1) were recorded with a 10 second interval for 15 minutes. Vocalisations were recorded when they occurred (Table 1). To generate novelty, the ground substrate and placement of obstacles (empty food and water containers) in the novel arena were changed between test occasions of individuals (figure 1 D-F).

Figure 1. Novel arenas used to score behavioural responses of red junglefowl of various ages. Figures show size and distribution of empty food and water containers (grey dots), and floor covering used at A) 4 weeks, B) 6 weeks, C) 8 weeks, D) 16 weeks, E) 34 weeks and F) 40 weeks in the novel arena. The dashed lines represent imaginary boundaries of sub areas used as a measure of activity (Table 1).

 

Table 1. Behaviours of red junglefowl of various ages, observed in the novel arena, and novel object and startle tests.

Behaviour
Definition
Latency to walk

The initial time it took for the bird to take a step after being introduced into the arena. N/A to the startle test.

Vigilance

Alert body stance, either moving or stationary, and eyes are fully open.

Foraging

Looking at the ground and/or using its beak to explore or peck at the floor.

Preen
An individual grooming itself with its beak.

Alarm calls

“Screaming”, high intensity calls to both aerial and ground predators.  

Vocalisations

Clucking, crowing or peeping (not including alarm calls).

Activity

The number of times the bird crossed the imaginary boundary of sub-areas in the arena. N/a to startle test.

Exploration

Latency it took for the bird to visit all of the imaginary sub areas of the arena. N/a to the startle test.

Novel object test

Exposure to a novel object is used to test neophobia and general fearfulness of individuals.

Directly after the novel object test and in the same arena, the novel object test was performed. At the start of the novel object test, the object was placed in the arena .  The reason for using the same arena was to be able to disentangle responses due to being in an unfamiliar place, and responses due to fear of novel objects. The same behaviours as in the novel arena test, as well as some additional ones, were recorded at each 10 second for 10 minutes (Table 1). The additional behaviours recorded were if the bird went in the same ‘imaginary square’ as the object and if the bird touched it.

Weeks 4 to 8

The novel object was placed in the opposite corner relative to the position of the bird. Different novel objects were used during each test period to prevent habituation: a 19 cm grey toy mouse at 4 weeks, a 21cm white and beige toy rabbit at 6 weeks, and a 30 cm white toy dog with black spots at 8 weeks.

Weeks 16 to 40

The novel object was placed on the floor in the centre of the arena. Different novel objects were used during each test period: a 10 cm green toy worm at 16 weeks, a 10 cm bright orange toy moose at 34 weeks, and a 19 cm white toy mouse at 40 weeks.

Tonic immobility test

A tonic immobility test tests how long an individual remains in a state of tonic immobility when this state has been induced. The tonic immobility response is used in applied ethology as a measure of fear, as it is meant to replicate a predator attack.

To induce tonic immobility, the individual was placed on its back in a v-shaped wooden stand. The observer applied a slight pressure to the bird’s chest with a hand for 10 seconds after which the hand was slowly removed. If the bird righted itself within 3 seconds, the process was repeated. If the bird did not go into a state of tonic immobility after three attempts, the duration of the tonic immobility was recorded as 0 seconds. If the bird lay on its back for over 600 seconds, the test was interrupted. The time it took from the removal of the hand until the first head movement and for the bird to stand were recorded.

Startle test

Startle tests typically measures the reactions of an individual when subjected to a strong startle, and individual’s strategies with how they deal with such a stressful situation such as an anti-predatory attack.

When the birds were 16, 34, 40 weeks old (thus not when younger), they were subjected to a simulated aerial predator test. Each bird was placed in a 150 x 50 x 50 cm test arena covered with a netting to prevent the birds from escaping. The birds were then allowed a five minute period to acclimatise to the enclosure. After this, the bird’s behaviours were recorded every 10 seconds for 5 minutes (Table 2). A wooden hawk model (length 31 cm, width 62 cm) slid along a rope that was hung roughly 120 cm above the top of the enclosure, using pulleys, controlled by the observer. The instantaneous reaction was noted (Table 2), and the bird was observed for another 5 minutes post-startle. The model hawk was hidden whilst not in use.

Table 2. Instantaneous reaction of red junglefowl to a hawk model in a simulated aerial predator test (sensu Håkansson & Jensen 2005 Behavioural and morphological variation between captive populations of red junglefowl (Gallus gallus) - possible implications for conservation).

Immediate Response

Definition
0

No visible change in bird’s behaviour.

1

The bird reacts by quickly lifting its head once and reverting back to normal behaviour.

2

The bird reacts as for score 1, but does not revert back to normal behaviour for > 3 seconds. The bird may also freeze for 3 - 5 seconds.

3

The bird reacts as for score 2 but also attempts to escape or run. The bird freezes > 5 seconds.


Responsible for this page: Agneta Johansson
Last updated: 05/02/13