A Standardized Protocol for Preference Testing to Assess Fish Welfare

Summary

A fundamental aspect of assessing the welfare of animals in captivity is to ask whether the animals have what they want. Here, we present a protocol to determine housing preference in the zebrafish (Danio rerio) with respect to the presence/absence of environmental enrichment and access to flowing of water.

Abstract

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the addition of physical objects or conspecifics in the housing environment) is often a way to give captive animals the opportunity to choose who or what they interact with and how they spend their time. A fundamental component of the aquatic environment that is often overlooked in captivity, however, is the ability for the animal to choose to engage in physical exercise. For many animals, including fish, exercise is an important aspect of their life history, and is known to have many health benefits, including positive changes in the brain and behavior. Here we present a method for assessing habitat preferences in captive animals. The protocol could easily be adapted to look at a variety of environmental factors (e.g., gravel versus sand as a substrate, plastic plants versus live plants, low flow versus high flow of water) in different aquatic species, or for use with terrestrial species. Statistical assessment of preference is carried out using Jacob's preference index, which ranks the habitats from -1 (avoidance) to +1 (most preferred). With this information, it can be determined what the animal wants from a welfare perspective, including their preferred location.

Introduction

The regulations governing how laboratory animals should be housed in captivity are explicit and well-defined. The Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International oversees and manages all organizations and institutions that work with research animals and has specific guidelines for species-appropriate husbandry and housing. For example, The AAALAC's Guidance on the Housing and Care of Zebrafish, Danio Rerio¹ "strongly encourages" the use of enrichment (the addition of physical objects or conspecifics in the housing environment) when housing zebrafish in captivity. The guide goes on to state, "Providing artificial plants or structures that imitate the zebrafish habitat allow animals a choice within their environment."

Evidence suggests that enrichment can stimulate the growth of new neurons (neurogenesis) in areas of the brain involved in processing spatial information², and it is thought that these neural changes are associated with enhanced learning ability³. The effects of enrichment on neurogenesis and learning have been widely studied across various taxa, including fish^4,5, birds⁶, reptiles⁷, and mammals⁸. Although these types of studies are important to understand the effects of enrichment on the brain and behavior, they do not take into consideration the particular choices or preferences of animals for a particular environment over another.

A fundamental question to ask when assessing the welfare of captive animals is whether or not the animals have what they want⁹. A way to investigate this question that provides tangible evidence is to provide animals with choices that allow us to understand their subjective preferences. For example, two studies have investigated whether zebrafish prefer access to either an enriched or a plain environment, with both studies indicating a preference for areas that contain enrichment^10,11. However, it has also been suggested that zebrafish appear indifferent to environmental enrichment¹², so the answer to the question is obviously not clear-cut. Another application of preference testing associated with animal welfare extends to trying to understand how different aspects of an enriched environment play a part in the choices an individual animal makes. In fish alone, different types of enrichment have differential effects on the brain and behavior, and this relationship is further complicated by individual differences in personality traits¹³. Moreover, preference testing could be useful for comparative studies of environmental enrichment. Even across different fish species, enrichment has been shown to have an effect on many different types of behavior, including aggression¹⁴, boldness¹⁵, locomotion¹⁶, and risk-taking behavior¹⁷.

Jacob's preference index is a statistical test that is used frequently to quantify housing preferences¹⁸. Jacob's preference index assigns a value to each different habitat based on the number of animals present in each habitat type at different time points, where preference ranges from -1 (avoidance) to +1 (most preferred). Here we describe a method for using Jacob's preference index to investigate housing preferences in fish and use the example of assessing two important characteristics of the aquatic environment: 1) the presence or absence of enrichment; and 2) the flow of water¹⁹. However, the protocol could easily be adapted to look at a variety of environmental factors (e.g., gravel versus sand as a substrate, plastic plants versus live plants, low versus high water flow) across different species and landscapes (e.g., aquatic and terrestrial).

Protocol

The current study has approval and complies with all requirements of the animal care and use protocols of the Pennsylvania State University; IACUC no. 46466.

1. Setup of preference apparatus

1. Attain approval from the institute's Animal Care Committee (or equivalent organization) for all experimental and husbandry procedures involving live animals before commencing the experiment.
2. Use an experimental tank made of opaque white plastic. The walls between zones are made from grey acrylic that is fixed in place with silicon sealant.
   NOTE: The size of the experimental tank is dependent on the size of the species of interest and the number of individuals used (e.g., for 8 adult zebrafish, a tank of 76 cm L x 76 cm W x 30 cm H is recommended).
3. Split the experimental tank into four zones that vary in accordance with the specific habitat parameters to be tested. Examples of different types of enrichment to investigate include sandy vs. rocky substrate, artificial plants vs. shelters, or flow of water vs. presence of artificial plants (Figure 1).
   1. If using flow of water as a parameter of interest, use small pumps to supply jets of water (see Table of Materials). Set the pumps at a chosen velocity so that they provide a constant and directed flow of water. Choose the desired velocity based on the species of interest's ecology and life history (e.g., 14 cm/s for zebrafish).
4. In the middle of the experimental tank, have a central arena where food is delivered (Figure 1). Access to the central arena from each zone is through a small opening in the separating walls. The opening is large enough for the species of interest to move between zones unhindered, but small enough to reduce any visual cues the fish might experience from other zones.
5. Place a biofilter and a heater in each corner of the tank, but outside the experimental area so as not to disturb the flow of water and to ensure a constant water temperature across all zones.
6. Set up additional experimental tanks as space dictates. Rotate the different zones in each experimental tank to limit any sequential bias. Ensure that all replicate tanks have uniform conditions (same light levels, water temperature, etc.)
7. Place cameras (see Table of Materials) on tripods directly above each experimental tank, so that all zones are visible. Avoid wide-angle lenses and ensure the memory cards have enough space for recording.
8. Set the room lighting on a gradual (e.g., 1/2 h) 12 L: 12 D cycle to simulate sunrise and sunset. Maintain water temperature at 25 ± 1 °C.

2. Capture, acclimation, and procedure

1. Keep fish in home tanks when they are not being tested. Net all test fish from their home tanks and place in the center arena of the experimental tank (Day 1). Minimize capture times to reduce stress (e.g., less than 30 s).
   NOTE: An alternative procedure for transferring fish from their home tank to the experimental tank that may minimize stress is to transport the fish in a beaker of tank water.
2. Keep the number and gender of the fish in each experimental tank constant across replicate tanks and choose based on the species size and ecology.
3. On days 1–4, fish spend time acclimatizing and exploring the different zones. Do not collect data on these days.
   NOTE: Extend or reduce the number of days for acclimation depending on the particular experimental protocol. However, the acclimation period should be sufficient to minimize the effects of handling as well as to get the fish accustomed to feeding in the apparatus.
4. During the acclimation period, monitor water quality closely by conducting regular water quality tests (e.g., pH, nitrate, or nitrite levels) and replace the water if any problems are detected (see Table of Materials).
5. Feed the fish flake food (see Table of Materials) in the central arena using a floating food ring (see Table of Materials) attached to the wall of the central arena at the water's surface. A food ring ensures food particles stay within the central arena and do not present a bias for zones due to food drifting.
6. Give the fish .5 h to feed ad libitum before removing the leftover food from the experimental tank with a dip net. Feed the fish once in the morning and once in the afternoon.
7. Assess behaviors on days 5–7. Switch cameras on and record fish behaviors for 2 h after each scheduled morning and afternoon feeding. On day 8 remove all fish from the experimental tanks with a dip net and place them back in their home tanks.
8. Depending on how much sump water is available, replace at least a 1/3 of the water in the experimental tank with fresh sump water to reduce any effects of stress hormones on fish in following replicates.
9. Set up the experimental tanks in accordance with the zone rotation schedule for that week. Rotating the zones decreases the chance of any behavioral bias occurring as a result of the placement of any zone relative to each other. Then begin the testing process again with a new batch of fish.

3. Measurements and data analysis

1. Download the videos to a computer at the end of each recording day. This ensures there is space on the memory card before every use.
2. Use video software (see Table of Materials) to quantify zone preference. Manually count the number of fish in each zone at 5 min intervals in each 2 h recording period (include the central arena in these counts). Define the gender of the fish during analysis if differentiation between males and females is possible from the video footage.
3. To analyze habitat preference, calculate the mean number of fish per zone for each replicate tank (i.e., average all data across the 3 days). In order to obtain a preference score for structure use, calculate Jacobs' preference index¹⁵ as
   
   J = (r_x − p)/[(r_x + p) – 2*r_x*p]
   
   where x is the zone of interest, r_x is the ratio of fish in zone x to the total number of fish in all zones, and p is the available proportion of all zones in the experimental tank. The index ranges between +1 for maximum preference, and −1 for maximum avoidance.
4. To determine if there are any changes in the rate at which fish switch between zones during an observation period, calculate the switch rate, r_sr, in the first and last 5 min of every observation period, where r_sr is the number of times a fish enters each zone from the central arena, divided by the total number of fish.
5. Consider a fish to have entered into a zone when the fish's whole body crosses through the opening separating the zones. Calculate a starting and a finishing mean switch rate for each replicate tank. Carry out all behavioral observations by the same experimenter to reduce any experimenter observation bias.
6. Using statistical software (see Table of Materials), conduct relevant statistical analyses. Suggested analyses include a one-way ANOVA, with preference index as the dependent variable and zone as the predictor variable, and a paired t-test on the starting and finishing mean switch rate for each tank.
7. Apply Tukey's multiple comparison post hoc test to further investigate zone comparisons, where each zone is compared to each other. More complex statistical analysis includes mixed models that assess time effects, arena effects, sex effects, or even individual differences in behavior.

Representative Results

We used the preference test to investigate housing preferences in zebrafish given a choice between varying enrichment including 1) plastic plants and sandy substrate; and 2) water flow. These were divided into four zones: (i) Enriched Only; (ii) Flow Only; (iii) Enriched and Flow; (iv) Plain; and a Central arena where food was delivered¹⁹. Zebrafish showed the highest preference for the Enriched and Flow zone, which was significantly different than all other zones (Enriched Only, Flow Only, Plain, and Central Arena; p < 0.01). Fish avoided both the Flow Only and Plain zones, spending more time in the Central Arena¹⁹ (Figure 2A). In addition, zebrafish moved between different habitat zones more often at the start of the observation period than at the end (Figure 2B).

Figure 1: Examples of different experimental designs to test for habitat preferences. (A) Setup of an experimental tank to test the preference of a sandy versus a rocky substrate. (B) Setup of an experimental tank to test the preference of enrichment (plastic plants) versus a shelter. (C) Setup of an experimental tank to test the preference of enrichment (plastic plants) versus a flow of water. In all figure panels, the four corner compartments were not accessible to the fish and only contained heaters and filters. Please click here to view a larger version of this figure.

Figure 2: Representative data showing the results of a habitat preference test on zebrafish. (A) Jacobs' preference index for each zone: (i) Enriched only; (ii) Enriched and Flow; (iii) Flow Only; (iv) Plain; and a neutral Central Arena. Positive and negative values indicate preference and avoidance, respectively. The boxes indicate the 25 ± 75th percentile range and contain the median line; bars represent the 10th and 90th percentile values; open dots represent points outside these values. a = significant difference from all zones (p < 0.05); b = significantly different from Enriched and Flow, Enriched Only, and the Central Arena (p < 0.05); and (B) box plots showing the switch rate at the beginning and the end of the observation period (boxes indicate the 25 ± 75th percentile range and contain the median line; bars represent the 10th and 90th percentile values). Figure 2A has been modified from DePasquale et al.¹⁹. Please click here to view a larger version of this figure.

Discussion

Here we present an experimental design that allows us to investigate the preferences of fish for different types of habitats. Some critical steps that are important in preference testing include: 1) ensuring that uniform conditions are maintained across different replicates (e.g., external noises or movement, experimenter, water chemistry, light levels); 2) ensuring that the zones are rotated between replicates and a significant amount of water is replaced with fresh sump water between tests to reduce biases; (3) ensuring that an appropriate sample size is used to detect significant results, both in terms of number of individuals in each group and number of replicate tanks; and 4) if trials are recorded, optimizing and ensuring proper video recording and file transfer.

Modifications to the current protocol include exposing fish to a variety of other habitat types, such as different enrichment items, different substrates, or even different flow rates. In addition, it may be possible to use animal tracking software to further understand how the fish are using the space in each zone (e.g., do the fish spend time swimming against the flow of water in the flow zones, or do they avoid that part of the habitat altogether). However, the walls of the experimental tank may need to be modified to accommodate this type of tracking software. Finally, the preference test described here could be adapted to any fish species, or potentially any aquatic organism that the experimenter wants to investigate.

A limitation of the current protocol is that preference testing is limited by the resources that are presented to the animals. Therefore, the animal may not be choosing a preferred choice, but the least unpleasant of those presented²⁰. However, it may be that having a choice in the first place is better for welfare than only being given limited options (i.e., access to the most preferred habitat only). Also, it has been suggested that zebrafish find light backgrounds aversive²³, thus an alternative tank color (e.g., black) may be more suitable. Moreover, preference testing is often limited to observations made in a small window of time, where the animal in question may be acting on immediate cues rather than future needs^21,22. In addition, gender, group size, and social context are factors that affect group dynamics and therefore potentially habitat preferences in fish, so it is important to try to keep these factors consistent across replicates.

With our representative results we showed that zebrafish preferentially choose both Enriched and Flow and Enriched Only zones and avoid Flow Only and Plain Zones. In sum, the Enriched and Flow zone was preferred over all other zones. A preference for enriched environments, and in particular the Enriched and Flow Zone, may be the result of an increased need for sensory stimulation (exploration) or it could be the need to find places to hide (reduced competition from conspecifics). Interestingly, there was a slight preference for the Central Arena over the Flow Only and Plain zones, suggesting that the potential of food being delivered was a higher motivational factor than swimming. In terms of movement between the zones, there was more switching between zones in the beginning of the observation period than at the end. The increase in movement at the beginning of the observation period may correspond to the timing of feeding (fish were fed half an hour before recording started), thus they may have been more motivated to move and look for additional food. In summary, the protocol described in the current study is an effective tool for looking at habitat preferences in fish.

Materials

Name	Company	Catalog Number	Comments
Artificial Aquarium Plants	Smarlin	B07PDZQ5M5
Artificial Seaweed Water Plants for Aquarium	MyLifeUNIT	PT16L212
Experimental tanks	United State Plastic Corporation	6106
Floating food ring	SunGrow	B07M6VWH9V
Flow meter	YSI	BA1100
Jager Aquarium Thermostat Heater	Ehiem	3619090
Master Water Quality Test Kit	API	34
SPSS Statistics for Macintosh	IBM	Version 25.0
Submersible Pump, SL-	Songlong	SL-381
TetraMin Tropical Flakes	Tetra	16106
Triple Flow Corner Biofilter	Lee's	13405
Video camera	Coleman	TrekHD CVW16HD
Windows Media Player (video software)	Microsoft	Windows Media Player 12