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. 2019 Dec 30;9:20305. doi: 10.1038/s41598-019-56860-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Response of mice familiarised with tunnel or tail handling to subcutaneous injection. Mice were familiarised with brief (2 s) pick up by tunnel or tail in 10 daily sessions. They were then either picked up, restrained by scruff and given a subcutaneous injection (i) or just picked up briefly without restraint or injection (c). (A) Duration of voluntary interaction with the handling device (hand for tail and cup, or hand holding tunnel) assessed immediately after 10^th handling familiarisation, 1^st injection or control pick up, and after 5^th injection or control pick up (mean ± sem). Pre-inject: for the 10^th handling familiarisation, mice assigned to the injection treatment are compared to those in the control pick up treatment, although neither group had experienced injection at this point. (B) Percentage of mice reluctant to be handled when approached by the handler. (C) Exploratory (positive score) versus anxious behaviour (negative score) in a modified open field test conducted after 5^th injection or control pick up (mean ± sem). P values from Mann-Whitney (A, Table 7), repeated measures ANOVA (C, Table 8), or contingency chi-squared tests (B). N = 20 females/10 cages per treatment group.