Handling, welfare and stress

Stress is built into the water maze: it's what makes the animal want to escape. Too much, though, and the animal stops learning, or stops trying. Good handling is the difference.

Questions people ask

How should I handle and habituate rats?

You should handle rats gently for several days before testing, so they are calm with you. Tickling (about 15 seconds a day for 3 days) can help them become relaxed with you. Stop if the rat avoids your hand. Your local ethics committee, licence conditions and vet always take precedence.

More detail

About 15 s per rat daily for 3 days; stop on avoidance, defensive posture or 22-kHz calls [NC3Rs]. Tickling induces positive affect and reduces fear of humans [Cloutier 2018].

Going deeper

Accustoming animals to handling reduces stress [Home Office].

Sources: nc3rs.org.uk/3rs-resources/rat-tickling; doi:10.3791/57190; gov.uk/government/publications/care-and-accommodation-of-animals-in-science-advisory-standards

How does stress affect maze results?

Stress can make animals learn worse, hug the wall or swim slower, which looks like a memory problem when it is not. Keep handling, temperature, light, noise and timing the same for every animal. Your local ethics committee, licence conditions and vet always take precedence.

More detail

Water maze training raised corticosterone more than the Barnes maze, and learning fell as corticosterone rose [Harrison 2009]. Stressed animals perform worse, so keep temperature, light and noise constant [Bromley-Brits 2011]. Thigmotaxis reflects anxiety [Prut & Belzung 2003].

Going deeper

If stress could differ between groups (for example a drug that changes anxiety), consider a land maze as well [Harrison 2009; Patil 2009].

Sources: Harrison et al. (2009). Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behavioural Brain Research 198(1):247-251. doi:10.1016/j.bbr.2008.10.015; Bromley-Brits et al. (2011). Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice. Journal of Visualized Experiments (53). doi:10.3791/2920; Prut & Belzung (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European Journal of Pharmacology 463(1-3):3-33. doi:10.1016/s0014-2999(03)01272-x; Patil et al. (2009). Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze. Behavioural Brain Research 198(1):58-68. doi:10.1016/j.bbr.2008.10.029

Can the experimenter affect the results?

Yes. Who handles the animals, and how, can change behaviour. Use the same person (or balance people across groups), record who ran each animal, and keep them blind to group where you can. Your local ethics committee, licence conditions and vet always take precedence.

More detail

Rodents showed a stress response to male experimenters and their odour [Sorge 2014]. Experimenter effects are an important water maze factor [Vorhees & Williams 2024]. The lab environment affects behaviour [Chesler 2002; Crabbe 1999].

Going deeper

Assisted escape is avoided partly because no two experimenters guide animals the same way [Vorhees & Williams 2024].

Sources: doi:10.1038/nmeth.2935; doi:10.1016/j.crtox.2024.100151; doi:10.1038/nn1102-1101; doi:10.1126/science.284.5420.1670

How can I reduce stress in my animals?

You reduce stress mainly through gentle handling and a calm, unchanging routine. Handle gently (tunnel or cup for mice; tickling for rats), habituate animals, keep the room calm and constant, keep water at your approved temperature, and dry and warm animals after swims. Your local ethics committee, licence conditions and vet always take precedence.

More detail

Stress is reduced by gentle, non-aversive handling (a tunnel or cupped hands for mice, tickling for rats), habituation, constant room conditions and keeping animals warm and dry after swims. Evidence: non-aversive handling [Gouveia & Hurst 2017; NC3Rs]; constant conditions [Bromley-Brits 2011; Home Office]; temperature and drying [Iivonen 2003; Vorhees & Williams 2024].

Going deeper

Refinement and good science align: reduced variability can reduce animal numbers [Home Office].

Sources: doi:10.1038/srep44999; nc3rs.org.uk/3rs-resource-library/mouse-handling; Bromley-Brits et al. (2011). Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice. Journal of Visualized Experiments (53). doi:10.3791/2920; gov.uk/government/publications/care-and-accommodation-of-animals-in-science-advisory-standards; doi:10.1016/s0166-4328(02)00369-8; doi:10.1016/j.crtox.2024.100151

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