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. 2022 Feb 15;12:2550. doi: 10.1038/s41598-022-06395-1

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© The Author(s) 2022

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Irwin score. As compared to female adult animals, handling-associated vocalization (a) and defecation (c) scores were increased in prepubescent animals. Handling-associated urination (b) in sexually mature female animals exceeded that of female adult animals (a, F(4,95) = 3.387, p = 0.0123; b, F(4,95) = 3.437, p = 0.0114; c, F(4,95) = 3.474, p = 0.0108). Rectal body temperatures (d) in female mice exceeded those in male mice in all age groups except for P25, and body temperatures in pubescent animals of both sexes were higher as compared to those in young adult animals (interaction p < 0.0001, age phase p < 0.0001, sex p < 0.0001). One-way ANOVA, followed by Bonferroni multiple comparison tests for (a–c), and two-way ANOVA, followed by Bonferroni multiple comparison tests for (d); *p < 0.05. Colored dots refer to the respective age groups. Error bars indicate the standard error of the mean (SEM).