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Before (study planning)
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| • Regulations |
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Prepare a study protocol according to legal, ethical, and institutional regulations applied to the project.
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Submit the protocol to the ethical committee before study onset.
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Usually, all procedures involving laboratory animals, from transportation to the arrival in the laboratory until the humanitarian endpoints, should be included in the protocol.
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Consult your institution about regulations applied to your project.
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Perform experiments and other procedures involving laboratory animals after institutional approval of the protocols to avoid ethical or legal issues.
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| • Experimental design |
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Define the experimental groups, allocation ratio (if sampling will be balanced between groups or not), minimal biological effect size, statistical model, statistical power and alpha to calculate the sample size a priori.
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Consider to use randomized block experimental designs is useful for controlling confounding factors-related variability
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Define how allocation to the groups will be performed.
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Define how blinding of experimenters will be performed.
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Define inclusion and exclusion criteria beforehand, explain how sample size will be kept.
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Register online the protocol and the experimental design you are going to execute
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PREPARE guidelines and electronic assistant EDA may help you to make a complete experimental and analytical plan.
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A priori sample size calculation will help you to avoid p-hacking and data dredging.
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Sequence generation and allocation concealment help you to avoid selection bias.
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Randomized block designs are more powerful than completely randomized designs being in accordance with the “reduce” principle.
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Blinding (or masking) of experimenter to animals‘ treatment and outcome assessments will help you to avoid performance and detection bias, respectively.
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Predefined exclusion criteria help you to avoid cherry picking and attrition bias.
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Make available (online) your planning will help you to avoid HARKing behavior (“Hypothesizing After the Results are Known”)
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| • Personnel training |
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Training (any procedures with animals) under supervision.
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Training on the routine procedures in the animal facility.
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Training on the routine procedures in the experimental rooms.
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Novices should be instructed on how to dress in the animal facility or experimental rooms (use a dedicated lab coat; avoid circulating outside the animal facility with the dedicated lab coat; avoid use perfume or creams, shampoos, other products with fragrance; avoid lab coat washing with laundry softener or perfumed soap).
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Novices should be instructed on how to behave in the animal facility or experimental rooms (avoid speak loudly or make sudden noises; avoid using headphones to listen to music or speak on
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the mobile phone).
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Activities planned to the experiment (transportation from a box/room/apparatus to another; injections, animal handling and restraining methods, blood samplings, surgeries; behavioral testing; etc.) should be rehearsed as much as necessary to be learned and make experimenters confident about all the steps trained.
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Complete personal training before real experiments onset.
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| • Environment settings |
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Check devices controlling animal room settings (light, temperature, humidity, light cycle)
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Check availability of the resources to the home cage (bedding, enrichment, food/water availability)
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Check devices controlling experimental room settings (light, temperature)
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Advertise to all personnel and keep visible posters with the rules, procedures, and routines in the animal house and experimental rooms.
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Animal room settings, home cage conditions and experimental room settings should be decided according to species-specific homeostatic needs and experimental requirements as specified in the protocol approved by the ethical
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committee. Restrict the access to the animal and experimental rooms to authorized, trained personnel.
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Consider using environmental enrichment as the standard condition (see below), as recommended by the legislation.
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| • Animals and environmental enrichment |
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Observe animals’ appearance (fur, color of skin, eyes, body weight) and specifications (species, strain, sex, age, number of animals, batch number) upon arrival in the laboratory.
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Be aware of the characteristics of the species (and strain when applicable) that will be used in the study (rat, mouse, fish, worm, fly, etc.).
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Keep animals in suitable (enriched) environments to improve animal welfare.
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Type (species, strain, sex, age) and number of animals should be as prespecified in the protocol approved by the ethical committee.
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Perform periodical assessment and record of animals’ appearance (fur, color of skin, eyes, body weight, secretions, feces), behavior (general activity, food, and water intake) and home cage conditions (bedding, enrichment, food/water availability) during their stay in the animal facility.
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Notify unexpected events to the staff responsible for the animal experimentation in the laboratory.
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| • Animal models and behavioral tests |
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Choose an animal model suitable for testing the study’s hypothesis.
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Be familiarized with the key features of your animal model, the particular behavioral and physiological characteristics and/or responses to interventions
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Choose an appropriate behavioral assay to test your animal model or treatments/interventions administered to your model. Define the behavioral and physiological variables to be measured in your test.
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Understand the validity and limitations of the model in question. i.e., What aspects (if any) of a human pathology is modeled by your animal? Is it a model or a behavioral assay (e.g., tests design for drug screening).
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Check the original source for the model or test development and validation process. Avoid citing or relying on subsequent work that may have misinterpreted the or modified the model or test parameters.
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Does the test allows for the animal to express the expected behavioral/physiological output to be measured (e.g., in the case of models that present some kind of motor dysfunction, a test that relies heavily on motor function should be avoided).
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During experimentation
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| • Experimenters |
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Prepare experimental settings.
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Inform other personnel that behavioral experiments are in progress.
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Make experimenter blind to animals’ treatment (or experimental group).
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Follow the prespecified protocol.
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Use a dedicated lab coat (different of that used in the other sectors of the laboratory).
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Use fragrance-free products in the body, hair, and clothes (including lab coat).
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Record unexpected events and deviations of the protocol in the lab book (or equivalent)
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| • Experimental room |
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Illumination, temperature, and level of noise in the experimental room should be set right before behavioral testing.
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Position the video camera and video recording settings should be right before behavioral testing.
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Organize an adjacent room with appropriated settings to receive experimental animals after behavioral testing.
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Time-to-time check the settings of experimental room, adjacent room and video recording devices during every experimental session (include this in your time schedule).
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Only bring the experimental animal to the experimental room when the environment is prepared to the behavioral testing.
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| • Experimental procedures and data collection |
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Identify animals according to the rule prespecified in the step of allocation to the groups.
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Bring animals to the experimental room following the prespecified order (preferentially randomly selected)
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Consider using automated methods (such as hardware and software) to collecting data.
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Blinding of animals’ treatments will help you to avoid performance bias.
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Avoid bringing animals directly from the home cage to the experimental procedures, include an interval in the experimental room before behavioral testing.
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Avoid returning animals directly from experimental procedures to the animal house or home cage where are untested animals.
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Perform behavioral testing, or invasive procedures, in a laboratory animal away of the conspecifics (many species communicate using ultra-sonic vocalizations and scent).
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Automatization of data collection helps to minimize observer bias and also between laboratory variations. It also mimics blinding procedure during data collection.
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| • Animals and environmental enrichment |
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Observe animals’ appearance (fur, color of skin, eyes, body weight) and specifications (species, strain, sex, age, number of animals, batch number) throughout the experiment.
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Keep animals in suitable (enriched) environments to improve animal welfare.
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Social animals must be kept in groups (except for procedures that require isolation). When keeping animals in groups, be careful not to compromise population density.
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When using territorial animals (such as mice), avoid complete exchange of objects between home cage hygienization, as this can increase aggressiveness.
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After
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| • Animal models and behavioral tests |
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For example, the administration of a certain drug, might reverse the immobility of mice subjected to chronic unpredictable stress (a model for depression) in the tail suspension test. This does not necessarily equals the reversal of depression. Since the measurement is immobility time, the drug might increase the overall activity of mice without having a true antidepressant effect.
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| • Analysis |
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Follow the prespecified analytical plan.
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Make blind assessment of the outcome when statistically analyzing the data.
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Be careful about outlier exclusion
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Make sure that the data met the assumptions for the chosen statistical method.
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Deviations of analytical plan and post hoc analysis should be acknowledged in the publication.
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Blinding of outcome assessment will help you to avoid detection bias.
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Do not include data obtained from animals that for some reason have experienced methodological problems (e.g., power outages during behavioral testing). They should not be treated (or tested) as outlier once deviation from normality is probably a consequence of problems during data acquisition.
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When considering the exclusion of outlier, do it only after blinding the researcher to the groups.
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Consider to use alternative statistical approach when the data does not met the assumptions for running the chosen statistical method. For example, using GLM as an alternative for ANOVA.
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| • Reporting |
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Report hypothesis, methods and results transparently.
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Make your data (including video recordings) available to the community.
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Choose an adequate descriptive statistic to represent the data.
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Report all the data collected (data points)
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Report the effect sizes with confidence intervals
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ARRIVE guidelines may help you to make complete report of the study.
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A complete report helps you to avoid reporting bias.
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There are several data repositories where you can share your data with your peers. This is in accordance with the 3R principles once other researchers can use (explore) your data instead of performing a whole independent experiment again.
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Consider to represent the data as median and range (instead of mean ± standard deviation) when data are not normally distributed. Alternatively, represent the data with confidence intervals (e.g., 95% CI). Avoid using standard error of the mean to represent data variability.
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The confidence interval for the effect indicates how precisely the effect has been estimated
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The effect size is a quantitative measure that estimates the magnitude of differences between groups, or strength of relationships between variables.
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