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Handling Techniques to Reduce Stress in Mice

Published: September 25th, 2021



1Campbell Family Mental Health Research Institute of CAMH, 2Department of Psychiatry, University of Toronto, 3Department of Pharmacology and Toxicology, University of Toronto, 4Departamento de Investigación, Universidad Central de Queretaro, 5Centre National de la Recherche Scientifique, UMR 5287, Institut de Neurosciences Cognitives et Intégratives d'Aquitaine, Université de Bordeaux

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal's capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

Mice and rats are essential assets to preclinical studies1,2 for multiple purposes, including endocrinal, physiological, pharmacological or behavioral studies2. From the increasing number of studies involving animals, it arose that uncontrolled environmental variables including human interaction influence various outcomes in biomedical research3,4,5. This is responsible for significant variability observed across experiments and research laboratories4,

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Procedures involving animal subjects were approved by the CAMH animal care committee and conducted in compliance with the Canadian Council on Animal Care guidelines.

NOTE: The handling method described herein can be used in various mouse strains, including non-transgenic (C57/BL6, BalbC, CD1, SV129, etc.) and transgenic lines. It can also be used with young or old mice, noting that young adult (4-6 weeks old) mice tend to be slightly more active than adult or old mice, especially on day 1.


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Two separate studies were performed with C57BL/6 mice. Study #1 included 6-month-old males and Study #2 included 2.5-month-old females (N=36/study) from Jackson Laboratories (Cat #000664). Mice arrived in the facility at the age of 2 months. While Study #2 females were handled and tested two weeks after arrival, Study #1 males were only handled and tested at the age of 6 months (delay due to global pandemic shutdown). During this time, one mouse from Study #2 died, prior to starting handling experiments. The Study #1 mal.......

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This study and method development are based on the observation that handling techniques in mice are still overlooked by the scientific community, and that some labs are still reluctant to implement habituation or handling techniques to reduce stress and reactivity of their animals prior to experiments. While representing a time commitment, animal handling provides beneficial effects to the animals that may contribute to the success of the experiments to be performed and prevents experiments from having to be performed mu.......

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The authors thank the Animal Care Committee of CAMH for supporting this work, as well as the animal caregivers of CAMH who provided extensive feedback on the usefulness of the procedure, motivating the execution of the described experiments and submission of the detailed protocol for other users. This work was in part funded by CAMH BreakThrough Challenge, awarded to TP, and by internal funds from CAMH.


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Name Company Catalog Number Comments
23 G x 1 in. BD PrecisionGlide general use sterile hypodermic needle. Regular wall type and regular bevel. BD 2546-CABD305145 Needles for Blood collection
BD Vacutainer® Venous Blood Collection EDTA Tubes with Lavender BD Hemogard™ closure, 2.0ml (13x75mm), 100/pk BD 367841 EDTA Coated tubes for blood collection
Bed’o cobs ¼” Corn cob laboratory animal bedding Bed-O-Cobs BEDO1/4 Novel bedding for novelty suppressed feeding
Centrifuge Eppendorf Centrifuge 5424 R For centrifugation of blood.
Corticosterone ELISA Kit Arbor Assays K003-H1W
Digital Camera Panasonic HC-V770 Camera to record EPM/Experimenter interactions
Elevated Plus Maze Home Made n/a Custom Maze made of four black Plexiglas arms (two open arms (29cm long by 7 cm wide) and two enclosed arms (29 cm long x7 cm wide with 16 cm tall walls)) that form a cross shape with the two open arms opposite to each other held 55 cm above the floor
Ethanol Medstore House Brand 39753-P016-EA95 Dilute to 70% with Distilled water, for cleaning
Ethovision XT 15 Noldus n/a Automated animal tracking software
Laboratory Rodent Diet LabDiet Rodent Diet 5001 Standard Rodent diet
Memory Card Kingstone Technology SDA3/64GB For video recording and file transfer
Novelty Suppressed Feeding Chamber Home Made n/a Custom test plexiglass test chamber with clear floors and walls 62cm long, by 31cm wide by 40cm tall .
Parlycarbonate tubes Home Made n/a 13 cm in length and 5cm in diameter
Purina Yesterday’s news recycled newspaper bedding Purina n/a Standard Bedding
Spectrophotometer Biotek Epoch Microplate Reader

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