Professor Jane Hurst (William Prescott Professor of Animal Science at the University of Liverpool) and Mr John Waters (Named Animal Care and Welfare Officer at the University of Liverpool) answer your questions about the refined handling techniques.
- Do the tunnel and cup handling methods work for young mice?
- Do the tunnel and cup handling methods work on all strains of mice?
- Are there differences in adapting to tunnel and cup handling methods between male and female mice?
- Can the tunnel and cup handling methods be used in IVCs?
- What size of tunnel is appropriate for my cage?
- Do I have to take environmental enrichment out of the cage if using a tunnel?
- Can I share a tunnel between cages? How often should I clean the tunnel? How do I clean the tunnel?
- Where can I find a supplier of clear tunnels that are suitable for autoclaving?
- How long does it take to transition to a tunnel or cup handling method?
- Won’t the animals become anxious anyway once they’re restrained for scientific procedures?
Young mice are more active and exploratory, and like the challenge of getting out of the cages when the lid is removed. Tunnel handling of mice at weaning age can be a little more difficult, especially if they have NEVER encountered a tunnel before. However, this can be overcome by placing a handling tunnel in the breeding cage so the weaners are used to it pre-weaning. As they are very fast, a slight adjustment is needed. When guiding weaners into a tunnel, leave both ends open so they are not discouraged from entering, then you can place a hand over one end and slightly tilt the tunnel upwards to keep them in the tunnel. If you need to handle them directly, it is then easy to locate their tails while they are at the bottom of the tunnel. This is also a very easy way to sex them and/or take a genetic sample.
We find we are also able to move weanling mice from transport boxes to cages using a tunnel, even when they are unlikely to have had previous experience of a tunnel at the suppliers. The key is to make slight adjustments to the protocol for each scenario.
Weaner mice are not suited to sitting on the hand without restraint using the cup method, due to their hyperactivity and lack of habituation to the hand. Although they can be moved between cages between closed hands, we would recommend use of a tunnel for about three weeks post-weaning to habituate them to handling. Then they can usually be picked up by cupping on the hand without any problems.
The positive response to tunnel handling appears to be very general. We find this works very well for all strains of laboratory mice that we have kept, and also for wild house mice, Mus spicilegus, wood mice, harvest mice, bank voles, field voles, laboratory rats and fat dormice – indeed, every small rodent that we have tried. Given this generality across small rodents, it seems unlikely that there will be a strain of laboratory mouse that it does not work with. However, local protocols may sometimes impinge on the practicality of tunnel handling.
Strains do differ in how quickly they habituate to sitting on the hand without restraint. We find that B6 mice can be more unsettled on the hand and try to get off as soon as possible, unless they have extensive experience of cup handling, so are better being handled by a tunnel. Other strains such as BALB/c, BALB.k and outbred ICR (CD-1) habituate much more quickly and show settled behaviour with both methods.
In general, there is no noticeable difference according to sex. However, males of some strains (e.g. B6, BALB/c) can take longer to habituate really well to cup handling, although they still show a much better response than those picked up by the tail. Also, some males can be a little more reluctant to enter tunnels and may need a bit more encouragement from the handler.
Yes. Although we do not use IVCs routinely in our unit, our colleagues elsewhere (including in other universities) successfully use the tunnel method with IVC cages and cleaning cabinets without problems.
As a guide, we suggest tunnels 5cm in diameter and between 10cm and 18cm in length, depending on how much room you have available in your cages. Make sure that the length of tunnel is not very similar to the width of the cage, so that animals can never become trapped in the tunnel. In any case, the tunnel is best positioned along the length of the cage for ease of capture of the mice.
No. Once skilled with tunnel capture, you should be able to capture mice when there is enrichment in the cage. Our videos show capture in an empty cage for visibility, so you can easily see the technique and responses of the animals. But we have plenty of enrichment in our mouse cages and use tunnel handling as a routine method. Placing a tunnel in the home cage also provides enrichment, as mice like to climb on the tunnel as well as go inside. Of course, you have to find a balance between how much enrichment you place within the cage and the ability to easily observe the animals. Clear tunnels have a real advantage as animals can be seen inside them.
This should be judged according to local protocols. Ideally, it is beneficial to have a tunnel in each cage. If it is not possible to have a tunnel in each cage, at a minimum you need separate handling tunnels for males and females to discourage aggression that can be stimulated by transfer of odour between the sexes. Whether to use the same handling tunnel between cages will depend on your local biosecurity rules. If you need to avoid any possible contamination between cages (for example, you always use clean gloves for each cage and wipe down surfaces between handling each cage to ensure no contamination), you will need a clean tunnel for each cage. Use of home tunnels within cages is particularly beneficial in this situation. Otherwise have a set of clean tunnels available that can be cleaned and dried between each cage. Fortunately, animals do not normally urinate in tunnels during brief handling.
We prefer not to clean home tunnels at every clean out, so that the animals keep some of their home scents. We judge the need to clean on the degree of soiling. This will depend on numbers of animals in the cage and habits of those particular mice, but typically we find that we only need to clean tunnels at every two to three cage cleans. However, allowing animals to keep some of their own scent on the tunnels is not necessary for tunnels to have a beneficial effect on handling. Animals used to a tunnel will respond happily to a clean tunnel.
How you clean the tunnels will depend on your local biosecurity protocol and you should follow those rules.
Datesand Ltd. supply clear plastic (polycarbonate) tunnels which meet all the guidelines required. These tunnels are available in 10cm and 13cm lengths, are 5cm in diameter and can be autoclaved. Tunnels can also be sourced from Braintree Scientific Inc., LBS and SerLab. We will add other suppliers as they become known to us.
Like any new skill, it does take new users a few days or weeks to get highly skilled and able to apply these new handling methods very efficiently in any situation. Animals will adapt surprisingly quickly. Reports from other users who are phasing the tunnel method into their practices have found that leaving a tunnel in a cage for at least five days has dramatically reduced time taken for capture and release. We highly recommend this practice, as it will reduce having to invest any “extra” time in habituating new animals. Using the tunnel to transfer animals between clean cages is normally sufficient to habituate animals to being picked up, and they will then settle much more quickly on the open hand.
We have found that mice habituated to these non-aversive handling methods are much more tolerant of being restrained by scruffing for further procedures than those picked up by the tail. Mice accustomed to non-aversive handling will accept physical restraint without losing tameness towards the hander.
In our studies, mice that were habituated to non-aversive handling rather surprisingly failed to show signs of anxiety immediately after a minor procedure, such as subcutaneous injection, and they did not attempt to avoid handling after such experience. This was the case even when the procedure was repeated. By contrast, picking up mice by the tail appears to induce background anxiety in animals that then exacerbates the stress of being restrained for scientific procedures.
Although there are many reports that restraint itself is the main cause of stress associated with minor procedures, it has not been recognised that using non-aversive methods for routine handling can substantially reduce the stress of restraint. And of course it is also important to bear in mind that we are duty bound not to induce stress unnecessarily in animals as part of their routine husbandry, when this can easily be avoided.