Assessing nest-building performance in mice: A multifactorial study of strain, age, sex, and environmental factors (2026)

Cintra, L., Hernandes, C. F., Alexandre-Ribeiro, S. R. et al.

Abstract

Providing nesting material is one of the main forms of environmental enrichment for laboratory mice. Nest building is an innate behavior, widely observed in wild mice, which construct complex, dome-shaped, multi-layered structures. Although various materials can be used in microisolator cage systems, this behavior remains underexplored under laboratory conditions. Evidence indicates that laboratory mice can build nests comparable to those of their wild counterparts, reinforcing the relevance of this enrichment for animal welfare. The aim of this study was to evaluate nest-building behavior in mice, considering biological and environmental variables, including strain, sex, age, nest location within the cage, seasonal variation, ambient temperature, and the use of cardboard tunnels. A total of 620 observations were analyzed from 106 cages, each containing 2–5 mice of both sexes, distributed across three strains (B6J, Nude, and BTBR). Nests were assessed weekly over the course of one year using a scoring system adapted from previous studies, in which higher scores indicate more elaborate, dome-shaped structures, whereas lower scores correspond to incomplete, flat, or less cohesive constructions. The use of cardboard tunnels provided as enrichment was also recorded (intact, shredded, or soiled with excreta). Other variables evaluated included age (20–109 days), and the position and side of the nest within the cage. Most mice built well-structured nests, with 39% of cages reaching the maximum score of 5. Statistical analysis showed that nest quality was significantly influenced by strain, season, and nest location within the cage. Compared to Nude mice, B6J and BTBR strains showed reduced nest-building performance. Scores were lower in spring compared to winter, while nests located at the cage edges, particularly on the right side and at the front, had slightly higher scores than those in the center. In conclusion, strain is the primary determinant of nest quality, while environmental and positional factors exert additional effects. Nude mice, during winter or colder periods, and nests located at the edges and front of the cage were associated with more complex nest constructions.