Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 3;10(1):14562.
doi: 10.1038/s41598-020-71476-y.

Benefits of tunnel handling persist after repeated restraint, injection and anaesthesia

Lindsay J Henderson  1   2   3 , Bridgette Dani  4 , Esme M N Serrano  4 , Tom V Smulders  5   4 , Johnny V Roughan  4
Affiliations

Benefits of tunnel handling persist after repeated restraint, injection and anaesthesia

Lindsay J Henderson et al. Sci Rep. .

Abstract

Millions of mice are used every year for scientific research, representing the majority of scientific procedures conducted on animals. The standard method used to pick up laboratory mice for general husbandry and experimental procedures is known as tail handling and involves the capture, elevation and restraint of mice via their tails. There is growing evidence that, compared to non-aversive handling methods (i.e. tunnel and cup), tail handling increases behavioural signs of anxiety and induces anhedonia. Hence tail handling has a negative impact on mouse welfare. Here, we investigated whether repeated scruff restraint, intraperitoneal (IP) injections and anaesthesia negated the reduction in anxiety-related behaviour in tunnel compared with tail handled BALB/c mice. We found that mice which experienced repeated restraint spent less time interacting with a handler compared to mice that were handled only. However, after repeated restraint, tunnel handled mice showed increased willingness to interact with a handler, and reduced anxiety in standard behavioural tests compared with tail handled mice. The type of procedure experienced (IP injection or anaesthesia), and the duration after which behaviour was measured after a procedure affected the willingness of mice to interact with a handler. Despite this, compared with tail handling, tunnel handling reduced anxiety in standard behavioural tests and increased willingness to interact with a handler within hours after procedures. This suggests that the welfare benefits of tunnel handling are widely applicable and not diminished by the use of other putatively more invasive procedures that are frequently used in the laboratory. Therefore, the simple refinement of replacing tail with tunnel handling for routine husbandry and procedures will deliver a substantial improvement for mouse welfare and has the potential for improving scientific outcomes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Study designs and timelines for experiments (BALB/c mice, N = 48 mice). (A) Experiment 1: Mice were equally split between tail or tunnel handling methods, and then divided into three experimental groups (N = 8 mice per handling method and restraint group). Handling only; mice experienced their designated handling method daily for 9 days. Pinch Restraint; handling only day 1–5, then restraint on days 6–9. Head Support Restraint; handling only day 1–5, then restraint on days 6–9. Voluntary interaction tests were conducted on day 1, 5 and 9. All mice were then tested in an elevated plus maze (EPM) and an open field test (OFT). (B) Experiments 2 and 3: Tail and tunnel handled mice were split into two experiments; IP injection (N = 20 mice, N = 10 mice per handling method) or anaesthesia (N = 24 mice, N = 12 mice per handling method). For Experiments 2 and 3 all mice experienced handling only for the first 5 days followed by an OFT on day 6. Mice were then anaesthetised or received an IP injection on three occasions, three days apart (day 7, 11 and 15). Voluntary interaction tests were conducted on day 1 and 5, and immediately after each procedure, and on the following day immediately after handling. This was followed by a final EPM test.
Figure 2
Figure 2
Effect of handling method and restraint upon time spent interacting with the handler (N = 24 cages, mean ± 1SEM). (A) Voluntary interaction (VI) tests were conducted on two days; day 1 the first day of daily handling and day 5 after five days of handling. VI tests were conducted both before (pre-) and after (post-) the animals were handled via either tail or tunnel handling. (B) Time spent interacting with the handling between treatment groups immediately after restraint or handling only, on day 5 before daily restraint began and on day 9. Mice were either handled only using tail or tunnel methods (handling only) on day 1–9 or were handling from day 1–5, then restrained daily using two methods of restraint (Pinch or Head Support) from day 6–9. ** denotes significant effects where P < 0.01.
Figure 3
Figure 3
Influence of handling methods upon behaviour in the elevated plus maze (EPM) and open field test (OFT) (N = 48 mice, mean ± 1SEM). (A) number of entries onto the open arms of the EPM, (B) proportion of time spent in the open arms of the EPM, and (C) number of entries into the centre of the OFT, by tail and tunnel handled mice, that experienced three treatments; Handling only, Pinch Restraint or Head Support Restraint. ** denotes significant effects where P < 0.01.
Figure 4
Figure 4
Influence of handling method upon voluntary interaction with a handler and behaviour in the open field test (OFT). (A) Time spent voluntarily interacting with the handler after a 4–8 week break between experiments on two days; day 1, the first day of daily handling and day 5, after five days of handling. Voluntary Interaction tests were conducted after the animals were handled via either tail or tunnel method (N = 22 cages). (B) Number of entries into the centre of the OFT by tail and tunnel handled mice (N = 44 mice). Graphs show mean ± 1SEM. * denotes significant effects where P < 0.05, ** denotes P < 0.01.
Figure 5
Figure 5
Effect of handling method and procedures (IP injection or anaesthesia) upon voluntary interaction with a handler and behaviour on the elevated plus maze (EPM). (A) time spent voluntarily interacting with the handler over three tests conducted immediately post-procedure and the day after IP injections or anaesthesia (N = 22 cages). (B) Number of entries onto the open arm and proportion of time spent on the open and closed arms of the EPM by tail and tunnel handled mice, that experienced either IP injections or anaesthesia (N = 44 mice). Graphs show mean ± 1SEM. ** denotes significant effects where P < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Holson RR, Scallet AC, Ali SF, Turner BB. “Isolation stress” revisited: isolation-rearing effects depend on animal care methods. Physiol. Behav. 1991;49:1107–1118. doi: 10.1016/0031-9384(91)90338-O. - DOI - PubMed
    1. Rodgers RJ, Dalvi A. Anxiety, defence and the elevated plus-maze. Neurosci. Biobehav. Rev. 1997;21:801–810. doi: 10.1016/S0149-7634(96)00058-9. - DOI - PubMed
    1. Balcombe JP, Barnard ND, Sandusky C. Laboratory routines cause animal stress. J. Am. Assoc. Lab. Anim. Sci. 2004;43:42–51. - PubMed
    1. Cao L, et al. Environmental and genetic activation of a brain-adipocyte BDNF/leptin axis causes cancer remission and inhibition. Cell. 2010;142:52–64. doi: 10.1016/j.cell.2010.05.029. - DOI - PMC - PubMed
    1. Wahlsten D, et al. Different data from different labs: lessons from studies of gene-environment interaction. J. Neurobiol. 2003;54:283–311. doi: 10.1002/neu.10173. - DOI - PubMed

Publication types