Name: a standardized protocol for preference testing to assess fish welfare
Uploaded: 2020-02-22T13:30:00
Description: Watch this Scientific Journal Video about A Standardized Protocol for Preference Testing to Assess Fish Welfare at JoVE.com

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. 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. 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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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Research

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Behavior

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 ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

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 ...