The Hen Y-Maze is an adaptation of the conventional Y-Maze apparatus and is used in the investigation of choice behaviors of fowls. Understanding preferences is crucial in the development of welfare programs, especially for domesticated fowls raised for meat and eggs. The Hen Y-Maze consists of a start area leading to a choice area that branches into two choice arms. Each section of the maze is separated from each other using gates. The choice arms have provisions for placement of rewards or other cues. In a simple preference testing paradigm, each choice arm is equipped with a resource, and the subject is observed for its choice over a session.

The Hen Y-Maze can also be applied to observe behaviors such as spontaneous alternation behavior, which can provide insights into the decision-making process of fowls. Learning behaviors based on associative learning paradigms can be effectively applied in the Hen Y-Maze by the introduction of different types of cues such as visual and olfactory in the choice arms. Additionally, the guillotine gates dividing each section can be taken advantage of in application of delay protocols to allow assessment of retention memory as well as the introduction of novelty in a task. Popular learning protocols such as discrimination testing and delayed nonmatch to sample protocols, thus, can be easily replicated for hen using the apparatus. Additionally, the simplicity of the design also makes it highly modifiable and adaptable for investigations other than those concerned with animal welfare. The Hen Y-Maze can be modified to evaluate social preferences and behaviors similar to the Rodent Social Y-Maze.

Other Y-Mazes include the Rodent Y-Maze, the Zebrafish Y-Maze, the Octopus Y-Maze, the Drosophila Y-Maze, and the Porcine Y-Maze.

The Hen Y-Maze is constructed using solid galvanized steel walls and has an overall length of 2.4 m. The start area measures 0.6 × 0.6 m and is separated from the decision area by a mesh gate. The decision area, together with the start area, has an overall length of 1.4 m and bifurcates into choice arms, each having a solid guillotine door at their entries. The choice arms have provisions for holding resources. All doors in the maze can be operated from a single point. The maze is covered with a removable mesh roofing and is raised 49 cm off the ground. Modifications such as placement of cues on the back walls of the choice arm are possible.

Depending on the type of investigation, the cleaning of the apparatus between trials may be needed to remove lingering cues. The subjects may need to be food-restricted or maybe put under other restrictions required as a pre-requisite for the investigatory aim. Prior to Hen Y-Maze training, birds need to be habituated to handling by the experimenters to avoid the influence of stress on the performances.

An automated tracking and recording system such as the Noldus EthoVison XT may be used to assist with the observations.

The following are sample protocols for Hen Y-Maze tasks.

Preference Testing Protocol

Habituation and Pre-training

Open the gates of the choice arms and do not place any cues or resources in them. With the start-box gate closed, place the subject in the start area. Open the start-box gate after 20 seconds and allow the subject 30 seconds to exit the start area; use gentle touch to encourage movement if the subject doesn’t exit the area. Close the start-box gate once the subject is in the decision area and allow it to enter one of the choice arms in 30 seconds. If the subject shows no movement, use gentle touch to encourage movement. Once the subject chooses an arm, close the opposite arm entry for 2 minutes. Remove the subject after the 2 minutes have elapsed, and place it back in the start box. Repeat the trial with the arm previously visited closed and alternating the closed arm for the next trial. Perform the free-choice and the two-forced choice trials consecutively per day for 3 days.

Hen Y-Maze Preference Training

Restrict access to the resources or stimuli at least 30 minutes prior to the training. Place the resources or stimuli to be tested in the corresponding arms. With the start gate closed, place the subject in the start area. Close the entry to one of the choice arms and begin the trial. Perform two trials per choice arm, with the other arm closed during the trials. Perform a total of 4 consecutive trials per subject per day for 2 days.

Hen Y-Maze Preference Testing

Restrict access to the resources or stimuli the night before testing. Place the resources in their corresponding arm, ensuring that the pairing is the same as that used during training. Place the subject in the start-box with the gate closed. Begin trials with both the entries to the choice arms open and observe the preference behavior. Once the subject enters a choice arm, block the access to the opposite arm completely. Perform two consecutive trials per test day.

Visual Discrimination Protocol

For discrimination trials instead of solid guillotine doors, colored curtains serving as the visual cue can be used, or visual cues can be placed next to the entries of the maze. The following protocol is based on curtains as the visual cues.

Habituation and Pre-training

Introduce the subject to the maze with all gates open and without the presence of any cues or rewards. Allow the subject to explore the maze for 5 minutes. Repeat trial for 3 consecutive days.

Hen Y-Maze Visual Discrimination Training

Place the discrimination cues in the entrance of the corresponding arms and bait the arm with the cue associated with the reward. Place the subject in the start-box with the doors closed. Allow it 20 seconds to observe the maze through the mesh gate. Lift the gate and begin trials. Allow the subject to consume the reward on the correct choice. If the subject makes an incorrect choice or does not make a choice in the allocated time, end the trial and begin the next trial after a predetermined inter-trial interval. Alternate the cues during the training sessions to mitigate side-bias. Perform at a maximum of 10 trials per day. Repeat sessions until the subjects meet the learning criterion.

Hen Y-Maze Visual Discrimination Testing

Following a retention period, perform visual discrimination testing. Repeat trials as in the training sessions.

Investigation of cognitive differences in two strains of hens

Von Waldburg-Zeil, van Staaveren, and Harlander-Matauschek (2018) investigated the differences in the performances of LSL and UCD-003 strains of laying hens in a visual discrimination task. Hens of both strains aged 71 weeks were trained in a visual discrimination task using red and green visual cues (arm-entry curtains paired with matching arm-back wall cue) performed in a Hen Y-Maze. The birds were trained to associate food rewards (corn kernels) with one of the visual cues. In odor to balance odor cues, the reward container in the non-rewarded arm was covered with a perforated plastic top to prevent access to the food. Following a 3-day habituation period, birds performed 10 trials per day for 7 days until they met the learning criteria of 5 out of 6 consecutive correct runs. During the training, the visual cues were systematically alternated to control for side-bias. The memory tests were then performed 21 days after the initial habituation trial. Both strains displayed similar levels of activity during the entirety of the Hen Y-Maze task. During the training, the LSL strain required fewer trials (26.0 ± 3.01 runs) than the UCD-003 strain (41.8 ± 4.76 runs). However, memory performance between the two strains did not significantly differ.

Investigation of the effects of altered motivational state on preferences in laying hens

Arnold and Hemsworth (2013) investigated the choice behaviors of HyLine Brown strain laying hens in a Hen Y-Maze. Three resources, feed, sawdust (dust bath substrate), and social contact, were used in pairwise comparisons following different restriction combinations of the resources prior to evaluation. Groups of 48 birds were tested for each pairing. In the experiment, birds were deprived of either only one of the resources, both the resources or neither resources of the pair tested using a 2 × 2 factorial design. Feed restrictions were applied 3 hours prior to the trials, while dust bath restrictions were applied by removal of the substrate the night before the experiment until the end of the experiment. Social interactions were restricted by placement of solid rubber partitions the day before beginning trials up until the end of the experiment. Social restriction was mainly imposed as a visual social restriction. In order to standardize resource contact prior to testing, the feed and dust bath were removed 30 minutes prior to testing for the birds in the corresponding non-restricted groups. Each bird was evaluated in two consecutive tests per day for 12 days. For the first pair, feed vs. dust, overall preference for feed (78% of the trials) was observed. On analysis of preferences using only the first trial of the days, birds that were feed restricted showed an overwhelming preference for feed in comparison to their non-restricted counterparts. Additionally, feed restricted birds also displayed a decrease in the latency to exit the start box as trials progressed in comparison to birds that were not feed-restricted. When dust vs. social contact was evaluated, no significant overall preference could be observed. Birds that were restricted access to dust-baths showed a higher preference for the dust arm than those that were not restricted when only first trials with no encouraging push were considered. Social-contact restricted birds required fewer pushes and were observed to move faster in the maze in comparison to unrestricted social contact. Dust restricted birds were also observed to require fewer pushes in comparison to the unrestricted access to dust bath birds. Preference assessment in feed vs. social contact set-up revealed an overall significant preference for feed (88% of the trials) by the groups. Based on the observations made in the three experimental set-ups, the feed was concluded to be the higher valued resource for the birds, while dust bath preference over social contact was only observable when birds were dust restricted.

Depending on the investigation needs, the following behaviors and parameters can be recorded in the Hen Y-Maze task

• Latency to initiate the trial
• First choice
• Number of trials required to meet the learning criteria
• Rate of acquisition
• Number of incorrect choices
• Number of correct choices
• Time spent in a choice arm
• Time spent exploring the resource/stimulus
• Success rate
• Trial duration
• Resource or stimuli preference (preference testing)
• Head direction
• Number of encouragement pushes required

Strengths

Hen Y-Maze uses a simple 2-choice design to allow assessment of learning, memory, and choice behaviors. The maze comes equipped with guillotine doors that divide the apparatus into different sections. The guillotine doors are useful in experiments requiring forced-choices or creating novel arms. The mesh guillotine door allows the visual contact with the remainder of the maze, which is useful in reducing pre-trial stress and familiarization with the apparatus. The apparatus can be easily modified, such as by extending the start area or replacing the guillotine doors with one-way or curtain entries to goal arms to allow the application of different learning and memory protocols. Additionally, social behaviors can also be accessed in the maze by the inclusion of goal boxes that hold social stimuli at the end of the choice arms.

Limitations

Pre-trial handling and maintenance can have a significant impact on the performances of the birds in the task. In investigations of preferences, the value of the resource or stimuli used may result in different motivations. For experiments that involve food rewards, changes in the quantity or quality of the reward between trials may greatly impact performances. Pushes to encourage exploration may have an unintentional impact on the choice of the birds. Factors such as strain, age, and sex may also affect performances.

• The Hen Y-Maze can be used in the evaluation of preference behaviors and cognitive functions.
• The apparatus consists of guillotine doors that separate each section of the maze and can be used to introduce task delays or novel areas.
• The gate separating the start-box from the decision area of the maze is made of mesh, which allows the subject visual access to the maze prior to trials.
• The apparatus can be adapted to different investigation needs, such as for social preference testing or visual discrimination task, by introducing simple modifications.
• Handling and post-trial environment factors can impact task performances.

1. Arnold, N. A., & Hemsworth, P. H. (2013). Examining the usefulness of a Y-maze choice method to measure the preferences of laying hens. Animal Production Science. doi:10.1071/an12390
2. Von Waldburg-Zeil, C. G., van Staaveren, N., & Harlander-Matauschek, A. (2018). Do laying hens eat and forage in excreta from other hens? Animal, 1–7. doi:10.1017/s1751731118001143