This protocol gives us a better understanding of what fish want, by looking at the choices that they make regarding habitat preferences, which is fundamental to their welfare. This protocol can easily be adapted to look at a variety of different environmental factors, including gravel versus sandy substrate, live versus plastic plants, or even across different aquatic species. To set up a preference apparatus, use an experimental tank made of opaque white plastic, split into four zones with walls made from acrylic, fixed in place with silicone sealant.
Place the appropriate enrichment materials into each zone in accordance with the specific habitat parameters to be tested. For example, include sandy versus rocky substrates, artificial plants versus shelters, or flow of water versus artificial plants. If flow of water is to be used as a parameter of interest, place small pumps to supply jets of water into the tank and set the pumps at a specific velocity, so that they provide a constant and directed flow of water based on the species of interest's ecology and life history.
In the middle of the experimental tank, have a Central Arena where the food will be delivered. Access to the Central Arena from each zone should be through a small opening in the separating walls that is large enough, for the species of interest, to move between the zones unhindered, but small enough to reduce any visual cues the fish might experience from other zones. Place a biofilter and a heater in each corner of the tank outside of the experimental area, so as not to disturb the flow of water and to ensure a constant water temperature across all of the zones.
Set up any additional experimental tanks as space dictates, ensuring that all the replicate tanks have uniform conditions. Place cameras on tripods, directly above each experimental tank, so that all of the zones are visible. After confirming that the memory cards have enough space for recording, set the room lighting on a gradual 12-hour, light/dark, cycle to simulate sunrise and sunset.
And set the water temperature to 25, plus or minus one degree Celsius. Keep the fish in home tanks when they are not being tested. On the day of the experiment, use two nets to quickly capture all of the test fish from their home tanks, and place them into the Central Arena of the experimental tank within 30 seconds of capture.
On days one through four, the fish will spend time acclimatizing and exploring the different zones. Do not collect data on these days. During the acclimation, conduct regular water quality tests, replacing the water if any problems are detected.
To feed the fish, use a floating food ring, attached to to the wall of the Central Arena at the water surface, to deliver flake fish food to the fish. After 30 minutes of ad libitum access, use a dip net to remove the leftover food from the tank. On days five through seven, switch on the cameras, and record fish behaviors for two hours after each scheduled morning and afternoon feeding.
On day eight, use the dip net to transfer all of the fish back into their home tanks. Depending on how much sump water is available, replace at least one-third of the water, in the experimental tank, with fresh sump water to reduce any effects of stress hormones on the fish and subsequent replicates. Then, set up the experimental tanks in accordance with the zone rotation schedule for that week, and begin the testing process with a new batch of fish.
At the end of each recording day, download the videos to a computer. And use video software to manually count the number of fish in each zone, at five minute intervals, in each two hour recording period. To analyze habitat preferences, calculate the mean number of fish per zone for each replicate tank.
To obtain a preference score for structure use, calculate Jacobs'preference index using the formula as indicated. Consider a fish to have entered into a zone, when the fish's whole body crosses through the opening between the zones. To determine if there are any changes in the rate at which the fish switch between zones, during an observation period, calculate the number of times a fish enters each zone from the Central Arena, divided by the total number of fish in the first and last five minutes of every observation period.
And calculate a starting and a finishing mean switch rate for each replicate tank. Using statistical software, conduct relevant statistical analysis, such as a one-way ANOVA, with a preference index as the dependent variable and the zone as the predictor variable, and a paired t-test on the starting and finishing mean switch rate for each tank. To compare each zone to each other zone, apply Tukey's multiple comparison post-hoc test.
In this representative experiment, the zebrafish demonstrated the highest preference for the Enriched and Flow zone, and avoided the Flow Only and Plain zones, spending more time in the Central Arena. In addition, zebrafish moved between the different habitat zones more often at the start of the observation period than at the end. It is critical that uniform conditions, such as external noise, or movement, water chemistry, or light levels are maintained across replicate tanks.
Preference testing extends to trying to understand how different aspects of the environmental enrichment influence an individual's decisions. But also is useful for comparative environmental enrichment studies.
