Name: a standardized protocol for preference testing to assess fish welfare
Uploaded: 2020-02-22T13:30:00.000+00:00
Duration: 7 min 29 s
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 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>

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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) 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><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&#039;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 ...

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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

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Environment

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Neuroscience

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Medicine

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPI&#160;assay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

Here we describe a protocol that utilizes commercially available automated systems to pharmacologically validate the prepulse inhibition (PPI) assay in larval zebrafish.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, ...

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.