This work was supported by a Research Collaboration Fellowship and the Huck Institute at The Pennsylvania State University, as well as USDA AES 4558. The research complied with all requirements of the animal care and use protocols of the Pennsylvania State University; IACUC no. 46466.

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
<ol>
	<li>Attain approval from the institute&#39;s Animal Care Committee (or equivalent organization) for all experimental and husbandry procedures involving live animals before commencing the experiment.</li>
	<li>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).</li>
	<li>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).
	<ol>
		<li>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&#39;s ecology and life history (e.g., 14 cm/s for zebrafish).</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.)</li>
	<li>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.</li>
	<li>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 &#177; 1 &#176;C.</li>
</ol>
2. Capture, acclimation, and procedure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>On days 1&#8211;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.</li>
	<li>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).</li>
	<li>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&#39;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.</li>
	<li>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.</li>
	<li>Assess behaviors on days 5&#8211;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Measurements and data analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39; preference index15 as 
	 
	J = (rx &#8722; p)/[(rx + p) &#8211; 2*rx*p] 
	 
	where x is the zone of interest, rx 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 &#8722;1 for maximum avoidance.</li>
	<li>To determine if there are any changes in the rate at which fish switch between zones during an observation period, calculate the switch rate, rsr, in the first and last 5 min of every observation period, where rsr is the number of times a fish enters each zone from the central arena, divided by the total number of fish.</li>
	<li>Consider a fish to have entered into a zone when the fish&#39;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.</li>
	<li>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.</li>
	<li>Apply Tukey&#39;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.</li>
</ol>

<ol>
	<li>Reed, B., Jennings, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+on+the+housing+and+care+of+zebrafish,+Danio+rerio.">Guidance on the housing and care of zebrafish, Danio rerio.</a> AAALAC International. , 36 (2010).</li><li>van Praag, H., Kempermann, G., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+consequences+of+environmental+enrichment.">Neural consequences of environmental enrichment.</a> Nature Reviews Neuroscience. 1, 191-198 (2000).</li><li>Oomen, C. A., Berkinschtein, P., Kent, B. A., Sakisda, L. M., Bussey, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+hippocampal+neurogenesis+and+its+role+in+cognition.">Adult hippocampal neurogenesis and its role in cognition.</a> Wiley Interdisciplinary Reviews - Cognitive Science. 5 (5), 573-587 (2014).</li><li>DePasquale, C., Neuberger, T., Hirrlinger, A. M., Braithwaite, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+complex+and+threatening+environments+in+early+life+on+brain+size+and+behaviour.">The influence of complex and threatening environments in early life on brain size and behaviour.</a> Proceeedings of the Royal Society B: Biological Sciences. 283 (1823), 1-8 (2016).</li><li>Salvanes, A. G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+promotes+neural+plasticity+and+cognitive+ability+in+fish.">Environmental enrichment promotes neural plasticity and cognitive ability in fish.</a> Proceedings of the Royal Society B: Biological Sciences. 280, 1-7 (2013).</li><li>Barnea, A., Pravosudov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birds+as+a+model+to+study+adult+neurogenesis:+bridging+evolutionary,+comparative+and+neuroethological+approaches.">Birds as a model to study adult neurogenesis: bridging evolutionary, comparative and neuroethological approaches.</a> European Journal of Neuroscience. 34 (6), 884-907 (2011).</li><li>LaDage, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+territoriality,+spatial+environment,+and+hippocampal+neurogenesis+in+male+side-blotched+lizards.">Interaction between territoriality, spatial environment, and hippocampal neurogenesis in male side-blotched lizards.</a> Behavioral Neuroscience. 127 (4), 555-565 (2013).</li><li>Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+New+Neurons?+Possible+Functions+for+Adult+Hippocampal+Neurogenesis.">Why New Neurons? Possible Functions for Adult Hippocampal Neurogenesis.</a> Journal of Neuroscience. 22 (3), 635-638 (2002).</li><li>Dawkins, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+behaviour+to+assess+animal+welfare.">Using behaviour to assess animal welfare.</a> Animal Welfare. 13, 3-7 (2004).</li><li>Kistler, C., Hegglin, D., W&#252;rbel, H., K&#246;nig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+structured+environment+in+zebrafish+(Danio+rerio)+and+checker+barbs+(Puntius+oligolepis).">Preference for structured environment in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis).</a> Applied Animal Behaviour Science. 135, 318-327 (2011).</li><li>Schroeder, P., Jones, S., Young, I. S., Sneddon, L. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+zebrafish+want?+Impact+of+social+grouping,+dominance+and+gender+on+preference+for+enrichment.">What do zebrafish want? Impact of social grouping, dominance and gender on preference for enrichment.</a> Laboratory Animals. 48 (4), 328-337 (2014).</li><li>Matthews, M., Trevarrow, B., Matthews, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+virtual+guide+for+zebrafish+users.">A virtual guide for zebrafish users.</a> Lab Animal. 31 (3), 34-40 (2002).</li><li>N&#228;slund, J., Johnsson, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+for+fish+in+captive+environments:+Effects+of+physical+structures+and+substrates.">Environmental enrichment for fish in captive environments: Effects of physical structures and substrates.</a> Fish and Fisheries. 17 (1), 1-30 (2016).</li><li>Oliveira, K. V., Barreto, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+reduces+aggression+of+pearl+cichlid,+Geophagus+brasiliensis,+during+resident-intruder+interactions.">Environmental enrichment reduces aggression of pearl cichlid, Geophagus brasiliensis, during resident-intruder interactions.</a> Neotropical Ichthyology. 8 (2), 329-332 (2010).</li><li>Brydges, N. M., Braithwaite, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+environmental+enrichment+affect+the+behaviour+of+fish+commonly+used+in+laboratory+work.">Does environmental enrichment affect the behaviour of fish commonly used in laboratory work.</a> Animal Behaviour Science. 118, 137-143 (2009).</li><li>Ahlbeck Bergendahl, I., Miller, S., Depasquale, C., Giralico, L., Braithwaite, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Becoming+a+better+swimmer:+structural+complexity+enhances+agility+in+a+captive-reared+fish.">Becoming a better swimmer: structural complexity enhances agility in a captive-reared fish.</a> Journal of Fish Biology. 90 (3), 1112-1117 (2017).</li><li>Roberts, L. J., Taylor, J., de Leaniz, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+reduces+maladaptive+risk-taking+behavior+in+salmon+reared+for+conservation.">Environmental enrichment reduces maladaptive risk-taking behavior in salmon reared for conservation.</a> Biological Conservation. 144 (7), 1972-1979 (2011).</li><li>Jacobs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+food+selection.">Quantitative measurement of food selection.</a> Oecologia. 14, 413-417 (1974).</li><li>DePasquale, C., Fettrow, S., Sturgill, J., Braithwaite, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+flow+and+physical+enrichment+on+preferences+in+zebrafish.">The impact of flow and physical enrichment on preferences in zebrafish.</a> Applied Animal Behaviour Science. 215, 77-81 (2019).</li><li>Bekoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Animal Rights and Animal Welfare, 2nd edition. , 53 (2009).</li><li>Fraser, D., Nicol, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+and+motivation+research.">Preference and motivation research.</a> Animal Welfare. , 183-199 (2011).</li><li>Franks, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+animals+want.">What do animals want.</a> Animal Welfare. 28, 1-10 (2019).</li><li>Blaser, R. E., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+anxiety+in+zebrafish+(Danio+rerio):+dissociation+of+black/white+preference+and+novel+tank+test.">Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test.</a> PLoS One. 7 (5), 1-8 (2012).</li></ol>

The authors have nothing to disclose.

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 needs21,22. 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.

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&#39;s Guidance on the Housing and Care of Zebrafish, Danio Rerio1 &#34;strongly encourages&#34; 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, &#34;Providing artificial plants or structures that imitate the zebrafish habitat allow animals a choice within their environment.&#34;
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 fish4,5, 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 enrichment10,11. 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&#39;s preference index is a statistical test that is used frequently to quantify housing preferences18. Jacob&#39;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&#39;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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artificial Aquarium Plants</td>
			<td>Smarlin</td>
			<td>B07PDZQ5M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Seaweed Water Plants for Aquarium</td>
			<td>MyLifeUNIT</td>
			<td>PT16L212</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental tanks</td>
			<td>United State Plastic Corporation</td>
			<td>6106</td>
			<td></td>
		</tr>
		<tr>
			<td>Floating food ring</td>
			<td>SunGrow</td>
			<td>B07M6VWH9V</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow meter</td>
			<td>YSI</td>
			<td>BA1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Jager Aquarium Thermostat Heater</td>
			<td>Ehiem</td>
			<td>3619090</td>
			<td></td>
		</tr>
		<tr>
			<td>Master Water Quality Test Kit</td>
			<td>API</td>
			<td>34</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics for Macintosh</td>
			<td>IBM</td>
			<td>Version 25.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Submersible Pump, SL-</td>
			<td>Songlong</td>
			<td>SL-381</td>
			<td></td>
		</tr>
		<tr>
			<td>TetraMin Tropical Flakes</td>
			<td>Tetra</td>
			<td>16106</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Flow Corner Biofilter</td>
			<td>Lee&#39;s</td>
			<td>13405</td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Coleman</td>
			<td>TrekHD CVW16HD</td>
			<td></td>
		</tr>
		<tr>
			<td>Windows Media Player (video software)</td>
			<td>Microsoft</td>
			<td>Windows Media Player 12</td>
			<td></td>
		</tr>
	</tbody>
</table>

a standardized protocol for preference testing to assess fish welfare

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&#39;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.

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 &#60; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60674/60674fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60674/60674fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60674/60674fig2.jpg" /> 
Figure 2: Representative data showing the results of a habitat preference test on zebrafish. (A) Jacobs&#39; 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 &#177; 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 &#60; 0.05); b = significantly different from Enriched and Flow, Enriched Only, and the Central Arena (p &#60; 0.05); and (B) box plots showing the switch rate at the beginning and the end of the observation period (boxes indicate the 25 &#177; 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. <a href="https://www.jove.com/files/ftp_upload/60674/60674fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Standardized Protocol for Preference Testing to Assess Fish Welfare at JoVE.com

A Standardized Protocol for Preference Testing to Assess Fish Welfare

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.

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the ...

a-standardized-protocol-for-preference-testing-to-assess-fish-welfare

Nanyang Technological University

Research

JoVE Journal

Behavior

6.8K Views.  Pennsylvania State University - Altoona. 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.

Video: A Standardized Protocol for Preference Testing to Assess Fish Welfare

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

A Within-subjects Experimental Protocol to Assess the Effects of Social Input on Infant EEG

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants socially interact. Electroencephalography (EEG) power is an advantageous technique to assess infant functional neural activation. However, many studies record infant EEG only during one baseline condition. This protocol describes a paradigm that is designed to comprehensively assess infant EEG activity in both social and nonsocial contexts as well as tease apart how different types of social inputs differentially relate to infant EEG. The within-subjects paradigm includes four controlled conditions. In the nonsocial condition, infants view objects on computer screens. The joint attention condition involves an experimenter directing the infant's attention to pictures. The joint attention condition includes three types of social input: language, face-to-face interaction, and the presence of joint attention. Differences in infant EEG between the nonsocial and joint attention conditions could be due to any of these three types of input. Therefore, two additional conditions (one with language input while the experimenter is hidden behind a screen and one with face-to-face interaction) were included to assess the driving contextual factors in patterns of infant neural activation. Representative results demonstrate that infant EEG power varied by condition, both overall and differentially by brain region, supporting the functional nature of infant EEG power. This technique is advantageous in that it includes conditions that are clearly social or nonsocial and allows for examination of how specific types of social input relate to EEG power. This paradigm can be used to assess how individual differences in age, affect, socioeconomic status, and parent-infant interaction quality relate to the development of the social brain. Based on the demonstrated functional nature of infant EEG power, future studies should consider the role of EEG recording context and design conditions that are clearly social or nonsocial.

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...