Constructing a nest is a deliberate behavioral strategy used by mice to improve fitness and survival. Video courtesy of M. Rock/Tufts University.

When it comes to nesting, lab mice are really not all that different than people. They crave the comforts of home, and construct nests that are sophisticated and surprisingly multifunctional.

Mice use nests for rearing and maternal activity, of course. But nests also help filter out harsh light—which mice despise—protect against predators and regulate body temperature. Evidence suggests constructing a nest is a deliberate behavioral strategy used by mice to improve fitness and survival, so there’s no reason why normal, healthy, uninjured mice shouldn’t be motivated to perform nesting behaviors quickly and consistently.

Right now, labs evaluate many of the same behaviors that afflict humans when we are in pain—appetite, fluctuating body weight, posture, mood changes, or activity. All these methods are valid and quantifiable, but they have limitations. Many require handling, sample analysis or concurrent controls, and therefore are not ideal for clinical real-time evaluation.

With this in mind, Charles River Laboratories in collaboration with our colleagues at Tufts University hypothesized that nesting behavior might be a useful indicator of well-being in laboratory mice. We developed an objective, simple and quick diagnostic screening tool—which we dubbed the time-to-integrate-to-nest test or TINT—and evaluated its ability to identify mice with compromised welfare, such as pain.

To conduct the TINT, a nominal amount of nesting material is added to a mouse cage. A positive TINT results when a mouse integrates the new nesting material into the main nest site within 10 minutes; failure to interact with the nesting material during this 10 minutes is defined as a negative TINT. Our results, published this month in the Journal of the American Association for Laboratory Animal Science, suggest that TINT could potentially be a fast and simple cage-side screening technique for flagging mice in distress, though identifying the cause and treatment for the distress may require additional evaluation. 

Nature and Nurture  
In our first experiment, we found that nearly all of the 10 mouse strains tested had positive TINT outcomes. However, the C3H/HeNCrl mice, used widely in cancer research, needed a few more testing sessions before figuring out the TINT procedure. One possible explanation for the early failure rates might have been the fact that C3H/HeNCrl are blind by six weeks of age. However, the FVB/NCrl mouse, who are also blind, only failed once out of the 12 tests. So perhaps the C3H/HeNCrl mouse is just behaviorally different.

By way of comparison, the BALB/cAnNCrl mouse, one of the oldest inbred mice used by researchers, shared the gold medal with the 129S2/SvPasCrl mouse as the top performer. Over 12 trials, it took all strains, on average, less than seven minutes to integrate new nesting materials, supporting our theory that nesting is a innate and motivated behavior in otherwise healthy mice.

Our second experiment looked at whether the TINT outcome was altered after a potentially painful, surgical procedure such as carotid artery injury (CAI) and minipump implantation. The implantation of the minipump did not appear to affect the TINT results but mice were significantly more likely to fail the TINT for two days after CAI surgery, though the scores improved thereafter. Another telling observation: Regardless of surgical treatment, mice living alone were more likely to fail the TINT than mice cohabitating with at least one other mouse.

TINT is not the only method of this kind that has been used to gauge pain in research mice. The Mouse Grimace Scale is a useful and increasingly validated technique to study pain. The tool captures facial grimaces on video and scores them. While our study sets up TINT nicely as a rapid, easy way of detecting abnormal behavior in mice, in real time, more research will be needed in order to truly validate TINT as an indicator of distress in research mice.

And determine more precisely what prevents mice from whistling why they work.

For more about TINT, check out this Journal of Visualized Experiments (JOVE) video.