Refined Mouse Handling Publications

There have been an increasing number of peer-reviewed publications investigating refined mouse handling. At least 19 peer-reviewed publications support the use of refined mouse handling. They find that refined mouse handling improves mouse welfare and scientific quality.

Key paper findings include that refined mouse handling:

Reduces anxiety as measured by elevated plus maze, social novelty test, open field test, and light-dark box test ^{3, 5, 6, 8, 11, 12, 13, 18, 21, & 22}
Reduces depressive-like behavior as measured by sucrose reward, resilience to negative events, forced swim test and burrowing test ^{5, 7, 12}
Reduces chronic stress as measured by adrenal gland size ⁵
Increases test reliability ¹³
Improves physiological parameters such as improving glucose tolerance & reducing blood glucose & corticosterone ^{17, 18}
Increases voluntary interaction with the handler ^{4, 5, 6, 7, 10, 13, 14, 21}
Improving breeding as measured by larger pups, more pups born & weaned, and a longer breeding productive lifespan ¹

You can find a list of the publications cited here and a brief description of their findings below. For a more extensive summary of each paper’s methods and findings, see NC3Rs mouse handling research summary.

Brief publication descriptions:

1. Hull et al. 2022. Effects of non-aversive versus tail-lift handling on breeding productivity in a C57BL/6J mouse colony. PLoS ONE. DOI:10.1371/journal.pone.0263192

Tunnel handling increased breeding productivity by 5% (i.e., one extra pup was born and weaned over a 6-month period for each breeding pair)
Tunnel handling increased the number of breeding pairs that were able to successfully wean all litters that were produced
Tunnel handling decreased the risk of recurrent litter loss by 20%

NOTE: the results from this study and the projected cost savings as a result of increased breeding productivity were significant enough to persuade a change in the standard of care across the institution (University of Florida)

2. Miller et al. 2022. Welfare assessment, end-point refinement and the effects of non-aversive handling in C57BL/6 mice with Lewis lung cancer. Animals. DOI: 10.3390/ani12010023

Non-aversive handling (cupping and tunneling) did not have a significant effect on the data quality or variability in Lew lung carcinoma tumor-bearing mice

3. Redaelli et al. 2021. Neuroinflammation, body temperature and behavioral changes in CD1 male mice undergoing acute restraint stress: An exploratory study. PLoS ONE. DOI: 10.1371/journal.pone.0259938

Tunnel handling and mechanoreceptive handling protected mice from a lack of weight gain seen in tail-handled mice following exposure to restraint and bright light (15min)

Mechanoreceptive handling increased the time mice spent in the open arm of an elevated plus maze following exposure to restrain and bright light (15min)

4. Sandgren et al. 2021. Using cage ladders as a handling device reduces aversion and anxiety in laboratory mice, similar to tunnel handling. Journal of Laboratory Animal Science. DOI: 10.23675/sjlas.v47i0.1083

Tunnel and ladder handling increased the number of voluntary interactions with handlers

Tunnel and ladder handling reduced anxiety, determined by exploration of the elevated plus maze

5. Clarkson et al. 2020. Negative mood affects the expression of negative but not positive emotions in mice. Proc Biol Sci. DOI:10.1098/rspb.2020.1636

Tunnel handling reduced anxiety, as evidenced by increase in the number of voluntary interactions with handlers and time spent in open arms of elevated plus maze

Tunnel handling reduced adrenal gland size

Tunnel handling increased resilience to negative events

Tunnel handling reduced anhedonic behavior and signs of depression, as evidenced by increased sucrose consumption and lick cluster size

6. Henderson et al. 2020. Benefits of tunnel handling persist after repeated restraint, injection and anaesthesia. Scientific Reports. DOI: 10.1038/s41598-020-71476-y

Tunnel handling increases the time spent interacting with handlers and reduces anxiety, as evidenced by increased time spent in the open arms of the elevated plus maze

Repeated stressful procedures (scruff restraint, i.p. injections, or anesthesia) do not reverse the benefits of tunnel handling

7. Sensini et al. 2020. The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific. Sci Rep. DOI:10.1038/s41598-020-74279-3

Tunnel handling decreased despair-like behavior in males, but not females, as evidenced by immobilization during forced-swim tests and reduced wellbeing-associated burrowing

Tunnel handling increased positive interactions with handlers’ hands (e.g., sniffing, touching, climbing) and reduced negative interactions (defensive burrowing)

8. Ueno et al. 2020. Effects of repetitive gentle handling of male C57BL/6NCrl mice on comparative behavioural test results. Scientific Reports. DOI: 10.1038/s41598-020-60530-4

Repeated daily tail handling led to increased body weight compared to mice handled only during cage changes

Repeated daily tail handling reduced anxiety, as evidenced by increased time spent in the open arms of the elevated plus maze

Repeated daily tail handling improved spatial cognitive function, as evidenced by increased alterations in the Y-maze test

9. Doerning et al. 2019. Assessment of mouse handling techniques during cage changing. JAALAS. DOI:10.30802/AALAS-JAALAS-19-000015

Tunnel handling did not increase contamination during cage changes (the surface of the tunnel vs the gloves used to tail handle)

Home tunnels did not impair the ability of caretakers to perform cage-side health checks

10. Gouveia et al. 2019. Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. Scientific Reports. DOI: 10.1038/s41598-019-56860-7

Handling for only 2 seconds during cage changes is sufficient to familiarize mice with tunnel handling

Brief (10 seconds) but more frequent handling was required to familiarize mice with cupping

Tail handling resulted in strong aversion to the handler compared to tunnel handling or cupping

Exposure to repeated scruff restraint and subcutaneous injections

11. Mertens et al. 2018. Effect of three different forms of handling on the variation of aggression-associated parameters in individually and group-housed male C57BL/6NCrl mice. PLoS ONE. DOI: 10.1371/journal.pone.0215367

Picking up male mice by the tail with forceps, rather than fingers, resulted in stress-induced hyperthermia to a greater degree than tail handling or tunnel handling

Forceps-handled mice exhibited increased aggression after cage cleaning compared to tail- or tunnel-handled mice

Compared to tail and forceps handling, tunnel handling resulted in reduced anxiety, as evidenced by decreased exit latency and fighting

12. Clarkston et al. 2018. Handling method alters the hedonic value of reward in laboratory mice. Scientific Reports. DOI:10.1038/s41598-018-20716-3

Tunnel handling reduced depressive-like behaviors, as evidenced by increased sucrose consumption and lick cluster sizes

13. Nakamura & Suzuki. 2018. Tunnel use facilitates handling of ICR mice and decreases experimental variation. Journal of Veterinary Medical Science. DOI: 10.1292/jvms.18-0044

After 1 week of handling and 1 week of oral gavage, tunnel-handled mice exhibited increased voluntary interactions with the experimenters compared to tail-handled mice

Tunnel-handled mice were easier to handle and urinated and defecated less during handling, as measured by a “rating scale for wildness”

Tunnel handling increased exploration in the open field test and the elevated plus maze compared to tail handling, but did not increase %time spent in the open arms of the elevated plus maze

Tunnel handling reduced the variation for the data collected behavioral tests (open field test and elevated plus maze) after IP administration of saline or diazepam

14. Roughan & Sevenoaks. 2018. Welfare and scientific considerations of tattooing and ear-tagging for mouse identification. Journal of the American Association for Laboratory Animal Science. DOI: 10.30802/AALAS-JAALAS-18-000057

Tunnel handling increased voluntary interaction with handlers compared to tail handling, even after restraint, tattooing, or ear-tagging

Tunnel handling resulted in reduced Mouse Grimace Scale scores after acclimation to handling and following restraint, ear-tagging, or tattooing

15. Gouveia & Hurst. 2017. Optimising reliability of mouse performance in behavioral testing: the major role of non-aversive handling. Scientific Reports. DOI: 10.1038/srep44999

Tunnel-handled female mice showed increased willingness to explore and investigate test stimuli (urine samples collected from male mice), regardless of familiarization or stimulus location, compared to tail-handled mice

Tunnel- and cup-handled mice overall exhibited less caution and increased exploratory behavior compared to tail-handled mice during behavioral testing (habituation-dishabituation paradigm)

16. Wilde et al. 2017. Tail-cuff technique and its influence on central blood pressure in the mouse. Journal of the American Heart Association. DOI: 10.1161/JAHA.116.005204

Handling method (tail, tail-cup, or tunnel) did not elicit differences in blood pressure, heart rate, or body temperature before, during, or after restraint for tail-cuff plethysmography

The authors concluded that restraint induces sustained, high levels of stress in mice and that the findings of this study should be considered in designing studies that involve the tail-cuff technique

17. Ono et al. 2016. Does the routine handling affect the phenotype of disease model mice? Japanese Journal of Veterinary Research. DOI: 10.14943/jjvr.64.4.265

Tail handling resulted in increased severity of glomerulus lesions compared to control (non-handled) mice in a model of ICR-derived glomerulonephritis

Tail handling resulted in increased plasma corticosterone levels in C57BL/6 mice (but not BALB/c mice) compared to controls;

Tunnel handling resulted in increased plasma corticosterone levels in BALB/c mice (but not C57BL/6 mice) compared to controls

The authors noted that it took much longer for BALB/c mice to enter the tube voluntarily (5min) compared to C57BL/6 mice (10-15sec), indicating potential strain differences in handling-induced stress

18. Ghosal et al. 2015. Mouse handling limits the impact of stress on metabolic endpoints. Physiology & Behavior. DOI:10.1016/j.physbeh.2015.06.021

Cupping reduced anxiety-like behaviors and blood glucose levels

Cupping resulted in improved glucose tolerance in mice fed a high fat diet

Cupping + massage resulted in lower glucose levels following an overnight fast

Cupping + massage reduced anxiety-like behaviors and decreased plasma corticosterone levels

19. Miller & Leach. 2015. The effect of handling method on the mouse grimace scale in two strains of laboratory mice. Laboratory Animals. DOI: 10.1177/0023677215622144

Handling method alone did not result in significantly different Mouse Grimace Scale scores (tail vs tunnel handling), demonstrating that handling method is not a confounding factor for determining a baseline score

20. Novak et al. 2015. An exploration based cognitive bias test for mice: effects of handling method and stereotypic behaviour. PLoS ONE. DOI: 10.1371/journal.pone.0130718

No difference was found between handling methods (tail handling vs cupping) in the level of exploration of mice in the radial arm maze (arms were positively or negatively cued); a notable caveat: mice were housed two to a cage such that one mouse was tail handled while the other was handled by cupping, potentially confounding the results if stress is communicated amongst cage-mates (I.e. the mouse handled by cupping may have experienced stress unrelated to the handling method as a result of being housed with a stressed, tail-handled cage-mate)

21. Gouveia & Hurst. 2013. Reducing mouse anxiety during handling: Effect of experience with handling tunnels. PLoS ONE. DOI: 10.1371/journal.pone.0066401

Tunnel handling reduced anxiety in both C57BL/6 and CD-1 mice with both home cage tunnels and shared tunnels, regardless of prior familiarity, as evidenced by increased willingness to approach the handler and behavior in the elevated plus maze)

Home cage tunnels may be more beneficial for anxious strains, as C57BL/6 mice showed faster habituation to handling with a home cage tunnel compared to shared tunnel (evidenced by increased voluntary interaction with the tunnel)

22. Hust & West 2010. Taming anxiety in laboratory mice. Nat Methods. DOI:10.1038/nmeth.1500

Tunnel handling & cupping were found to reduce stress/anxiety, as evidenced by approach behavior with handlers, urination/defecation during handling, and the elevated plus maze, for multiple strains & during both light and dark phases.

Tunnel handling & cupping are compatible with scruff restraint & tail lifting.

Tunnel handling and cupping were effective regardless of handler