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.

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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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...

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 laboratories4,5, posing a major caveat in animal research.
Various approaches have been implemented with the goal of limiting the impact of environmental stressors and reducing reactivity to human interaction. For example, to limit the impact of environmental stressors, standardization of housing conditions and automated housing systems6,7 have been implemented across laboratories. Regarding interaction with human beings, commonly used approaches for handling and transporting animals had little regard for animal discomfort and stress. For instance, picking up animals by their tail or using forceps8 increases baseline anxiety9,10,11, reduces exploration9,12 and contributes greatly to inter-individual variability within and across studies13,14. As a result, other approaches were developed, such as the cup handling technique, which is applicable to mice and rats. In this approach, the animals are &#34;cupped&#34; out of their cage, and held by the experimenters with their hands forming a cup9,10,11. Another useful alternative to tail handling involves the use of a polycarbonate tunnel to transfer mice9,10,15. This approach eliminates direct interaction between the mouse and the experimenter. Both the cup and tunnel approaches showed efficacy in reducing anxiety-like behaviors and fear of the experimenter that can be exaggerated by aversive handling techniques, such as tail pick up/tail handling9,10.
Therefore, increasing evidence demonstrates the usefulness of proper mouse handling for reducing variability between individuals9,11, and improving animal welfare10. However, the techniques mentioned above are still faced with limitations. The cup handling technique has been implemented with schedules ranging from 10 days (10 sessions over 2 weeks16) up to 15 weeks17, which is a considerable amount of time for facility staff and experimenters. Additionally, the effectiveness of cup handling varies by strain9 and conventional cup handling in open hands may lead to na&#239;ve mice or particularly jumpy strains to jump from the hand9,18. Tunnel handling results in more consistent and generally quicker results in gentling19. Tunnels are also used as home cage enrichment. They help animals habituate to handling quickly and provide the added benefits of enrichment. Tunnel handling, however, has limitations when transferring animals between apparatuses. Interestingly, Hurst and West9, and Henderson et al.20 demonstrated that using gentle and brief manual handling to transfer animals from the tunnel to the apparatus does not affect their phenotype.
To provide an alternative to existing methods, with achievable habituation in a short period of time, this article describes a novel technique that expands on the cup handling technique, therefore requiring no particular equipment. This approach uses milestones to gauge the level of comfort mice have with the handling process. It shows efficacy at decreasing mouse reactivity and stress (at the behavioral and hormonal levels), facilitates routine handling and contributes to reducing variability between animals. Details of this technique are provided here, and its efficacy at reducing anxiety-like behaviors, improving interaction with experimenters, and limiting peripheral stress-hormone (corticosterone) release are demonstrated in two separate studies (male and female mice), in comparison with tunnel handling (positive control) and tail handling techniques (negative control).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>23 G x 1 in. BD PrecisionGlide general use sterile hypodermic needle. Regular wall type and regular bevel.</td>
			<td>BD</td>
			<td>2546-CABD305145</td>
			<td>Needles for Blood collection</td>
		</tr>
		<tr>
			<td>BD Vacutainer&#174; Venous Blood Collection EDTA Tubes with Lavender BD Hemogard&#8482; closure, 2.0ml (13x75mm), 100/pk</td>
			<td>BD</td>
			<td>367841</td>
			<td>EDTA Coated tubes for blood collection</td>
		</tr>
		<tr>
			<td>Bed&#8217;o cobs &#188;&#8221; Corn cob laboratory animal bedding</td>
			<td>Bed-O-Cobs</td>
			<td>BEDO1/4</td>
			<td>Novel bedding for novelty suppressed feeding</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5424 R</td>
			<td>For centrifugation of blood.</td>
		</tr>
		<tr>
			<td>Corticosterone ELISA Kit</td>
			<td>Arbor Assays</td>
			<td>K003-H1W</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Panasonic</td>
			<td>HC-V770</td>
			<td>Camera to record EPM/Experimenter interactions</td>
		</tr>
		<tr>
			<td>Elevated Plus Maze</td>
			<td>Home Made</td>
			<td>n/a</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Medstore House Brand</td>
			<td>39753-P016-EA95</td>
			<td>Dilute to 70% with Distilled water, for cleaning</td>
		</tr>
		<tr>
			<td>Ethovision XT 15</td>
			<td>Noldus</td>
			<td>n/a</td>
			<td>Automated animal tracking software</td>
		</tr>
		<tr>
			<td>Laboratory Rodent Diet</td>
			<td>LabDiet</td>
			<td>Rodent Diet 5001</td>
			<td>Standard Rodent diet</td>
		</tr>
		<tr>
			<td>Memory Card</td>
			<td>Kingstone Technology</td>
			<td>SDA3/64GB</td>
			<td>For video recording and file transfer</td>
		</tr>
		<tr>
			<td>Novelty Suppressed Feeding Chamber</td>
			<td>Home Made</td>
			<td>n/a</td>
			<td>Custom test plexiglass test chamber with clear floors and walls 62cm long, by 31cm wide by 40cm tall .</td>
		</tr>
		<tr>
			<td>Parlycarbonate tubes</td>
			<td>Home Made</td>
			<td>n/a</td>
			<td>13 cm in length and 5cm in diameter</td>
		</tr>
		<tr>
			<td>Purina Yesterday&#8217;s news recycled newspaper bedding</td>
			<td>Purina</td>
			<td>n/a</td>
			<td>Standard Bedding</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biotek</td>
			<td>Epoch Microplate Reader</td>
			<td></td>
		</tr>
	</tbody>
</table>

handling techniques to reduce stress in mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;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&#39;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.

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...

Watch this Scientific Journal Video about Handling Techniques to Reduce Stress in Mice at JoVE.com

Handling Techniques to Reduce Stress in Mice

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 ...

My name is Dr.Thomas Prevot. I am a scientist in the Center for Addiction and Mental Health, or CAMH, and an assistant professor at the University of Toronto in the Department of Psychiatry. My group has a particular interest at reducing baseline anxiety and reactivity in mice in order to reduce data variability and improve animal welfare.Mice have proven to be invaluable assets in basic science and preclinical research, however, very few labs around the world control for baseline anxiety and the reactivity of their laboratory animals, therefore, complicating replicability of data between labs. A major contributing factor to baseline anxiety is uncontrolled environmental variables, such as housing conditions, environmental enrichment, and exposure to stress. While standardization of housing conditions and enrichment has helped improve this issue, exposure to stress during interaction with experimenters is still largely unaddressed in mice.Tail handling, the process of lifting the mouse by the tail, is still the mouse widely used mouse handling technique, despite it being particularly stressful. Though alternatives to tail handling have emerged including tunnel handling and cup handling. Adoption of these techniques has been slow.Here we describe a novel technique, similar to cup handling, which can be implemented with as little as three days of habituation, or 3D handling. By gradually increasing the degree and complexity of interaction and progressing in response to the reactivity of the mouse to certain handling milestones, we can reduce stress and facilitate routine handling. We assess the efficacy of 3D handling in mice in behavioral tests, assessing anxiety-like behaviors, interaction with experimenters and peripheral stress hormone, corticosterone, levels, and provide a comparison with tunnel handling and tail handling techniques.Gently open the cage and place the lid on the side. Introduce your gloved hand to the home cage before attempting to pick up the mouse. Allow the mouse habituate to the presence of the hand for about 30 seconds.Then attempt to pick up the mouse in cupped hands. If the mouse can't easily be picked up, cup both hands together and use them to guide the mouse to the corner of the cage. Then use both hands to pick up the mouse.Once the mouse is removed from the cage, keep your hands as flat and open as possible. Allow the mouse to freely explore. This provides a flat platform for the mouse to step onto and limits the risk of biting.Hold the hand open and flat, with the palm up. Place your other hand adjacent to the mouse and allow the mouse to move freely from hand to hand, without any restraint. After one minute of handling with flat hands, relax the palm of your hand, and slightly cup the mouse in your head, prior to gently rolling the mouse between hands.Continue for four minutes, alternating between gentle rolling between hands and free exploration of your open hands. During the last two minutes, perform a shelter test. Let the mouse move to edge of your hand and bring the two hands together.Very slowly, cup your hands so the mouse fits inside a shelter formed by the hands. Make sure to leave an opening, so the mouse can escape if needed. Try to keep the mouse in the shelter for five to 10 seconds without any restraint.Repeat the shelter test three to four times over the last two minutes. Do not rush the process. If the mouse appears stressed by being confined inside the hand, continue with rolling between hands and free exploration for 30 seconds, and then retry the shelter test.Allow free exploration in hands for 30 seconds, then gently replace the mouse in its cage. Clean the bench top of any feces and urine with 70%ethanol or an appropriate cleaning solution. Clean the gloves of feces and urine as necessary, and when changing to a new cage, rinse gloves with 70%ethanol or change gloves.By day two, it should already be feasible to remove the mouse from the cage in cupped hands. If the mouse seems reactive, perform the flat hand and free exploration of open heads for 30 seconds to a minute. Otherwise, proceed to the roll between hands and shelter test.Perform the role between hands and shelter steps from day one for two to three minutes. Perform each step several times. If the mouse seems at ease in the shelter test, attempt to pet the mouse on the back and head.If the mouse does not attempt to flee contact, proceed to the nose poke. Gently touch the mouse on the tip of the snout. If it does not bite or attempt to turn its head, this is a good sign of habituation.For the petting and nose poke test, do not rush the process. If the mouse appears stressed by being confined inside the hands, or does not want to be touched, continue with the rolling between hands for a few minutes and then retry. Stop this session after about three minutes of handling, if the animal reacts well to the shelter, head petting and nose poke.And if the animal appears to be willing to explore your hands without any signs of stress, such as attempts to escape, jumping from the hands or avoiding contact with the hands. If the mouse continues to exhibit signs of stress or is not reacting well to the shelter test or nose poke test, continue the session until reaching five minutes, as in day one. Replace the mouse in its cage and clean the bench top and gloves.On the third day, proceed through the same steps that day two. Pick up the mouse in the palm of the hand. Transfer the mouse between hands.Gently confine the mouse in cupped hands. If the mouse responds well to these steps, try petting the mouse and attempting the nose poke test. On the third day, the mouse should be relaxed enough to sit in the palm of their hand without moving off, and you should be able to proceed through these steps over approximately two to three minutes.Replace the mouse in its cage, and clean the bench top and gloves. On the third day. if the animal will be restrained for experimental purposes, such as oral gavage or intra peritoneal injection, the mice can be subjected to the neck pinch test.Grasp the mouse by the nape of the neck, between your thumb and forefinger and lift the mouse five centimeters above your hand for two to three seconds. This is normally an uncomfortable position for adult mice, and if the mice remain near immobile, they are well habituated to handling and will be easy to restrain for injections. Place the mouse back on your flat hand and allow it to freely explore for one minute.Animals were tested for their willingness to voluntarily interact with the experimenter and the ease of handling in an experimental context. In study one, mice handled by either tube handling or 3D handling were less likely to flee when an experimenter attempts to pick up a mouse. Additionally, study one mice handled using the 3D technique spent more time in the same quadrant as the experimenter's hand, indicating an increased willingness to interact with the experimenter.In study two, mice handled by either tube handling or 3D handling were less likely to flee when an experimenter attempts to pick up the mouse compared to tail handled mice. However, in study two, handling technique did not significantly influence the time spent in the same quadrant as the experimenter's hand. The effect of 3D and tunnel handling were compared to tail handling in two tests of anxiety-like behaviors, the novelty suppressed feeding test and the elevated plus maze.In study one, in the novelty suppressed feeding test, there was a trend to reduced anxiety in tunnel handled and 3D handled mice. In the elevated plus maze, there was no overall effect of handling. When summarized as a Z-score, there was a significant reduction in overall anxiety in tunnel handled and 3D handled mice compared to tail handled mice.In study two, there was no overall effect of handling on anxiety measures in the novelty suppressed feeding test or the elevated plus maze. When summarized as a Z-score, handling technique had no overall effect on anxiety. As a physiological measure of stress, we collected serum from tail handled, 3D handled and tunnel handled mice 15 minutes after a brief handling session and tested corticosterone levels.There was no overall effect of handling in study one. In study two, 3D handling significantly reduced corticosterone levels in comparison to tail handling. Based on observations that handling techniques in mice are not widely recognized by the scientific community, we described the use of a novel handling technique.In both studies presented, tunnel and 3D handling reduced attempts of mice to flee when being picked up, potentially facilitating routine handling. Additionally, in older male mice, this technique reduced anxiety-like behaviors and increased interaction with the experimenter. In young females.this technique decreased corticosterone levels observed in response to handling. Implementing this technique has the potential to reduce data variability, improve animal welfare and facilitate routine handling.

handling-techniques-to-reduce-stress-in-mice

Research

JoVE Journal

Behavior

34.1K vistas.  Campbell Family Mental Health Research Institute of CAMH. Mi nombre es Dr.Thomas Prevot. Soy científico en el Centro de Adicciones y Salud Mental, o CAMH, y profesor asistente en la Universidad de Toronto en el Departamento de Psiquiatría. Mi grupo tiene un interés particular en reducir la ansiedad y la reactividad basales en ratones para reducir la variabilidad de los datos y mejorar el bienestar animal.Los ratones han demostrado ser activos invaluables en la ciencia básica y la investigación preclínica, sin embargo, muy pocos laborato...