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 fr...

<ol>
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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. 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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. 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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) ensurin...

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,...

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 ...

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.

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6.7K visualizações.  Pennsylvania State University - Altoona. Este protocolo nos dá uma melhor compreensão do que os peixes querem, olhando para as escolhas que eles fazem em relação às preferências de habitat, o que é fundamental para o seu bem-estar. Este protocolo pode ser facilmente adaptado para olhar para uma variedade de diferentes fatores ambientais, incluindo substrato de cascalho versus areia, plantas vivas versus plásticas, ou mesmo em diferentes espécies aquáticas. Para configurar um aparelho de preferência, utilize um tanque expe...