Figure 2. With TRAD conditions, 14 male mice required separation from their home cage as a direct result of fighting incidences. Conversely, NON-TRAD conditions required no separations.
Figure 1. TRAD cage conditions included a red igloo, nesting material contained within its packaging, a cotton nesting square, and an intermediate chewing device (not photographed).
more, we learned that male mice are stressed by the scent of a human male’s sweat.4 From these articles, we implemented some changes in housing and husbandry conditions for our subsequent IACUC approved protocol. We realized we had a unique opportunity as both studies were identical in design and procedures with the only differences being the study drugs (which were all amalgamated in a Western diet base) and study length.
Second Study: NON-TRAD
The second study, which we referred to as “non-traditional” (NON-TRAD), employed 71 male C57BL/6N mice (Envigo), both litter mates and non-litter mates, at age 3-4 wk. Observations were made throughout the study length of 433 d. Regarding the NON-TRAD housing conditions, we removed the igloo and the outer packaging of the nesting material (Figure 4). The nesting material pouches were simply opened and discarded as mice were previously observed using the pouch as a makeshift hut. We hypothesized that both igloo and pouch were being territorialized and should not be included in the NON-TRAD cages. This is contrary to conventional thinking as the act of pouch destruction is
Figure 3. Fighting incidences were recorded on average of every 1.74 days with TRAD conditions. This period between fight incidences increased almost three times longer to 5.15 days with NON-TRAD conditions.
generally considered positive environmental enrichment. Mice for NON-TRAD were still allotted a cotton nesting square and given a wooden gnawing stick. While Van Loo et al. suggested housing 3 mice per cage, their study did not test a density of 4 mice per cage. Since overall costs are a huge concern for investigators, we chose to house 4 per cage to reduce costs and to fill in this gap in knowledge. To further reduce stress, we moved our observations to 6 a.m. during the week to coincide with the institution’s light cycle (on at 6 a.m., off at 6 p.m.). We also performed all cage and water bottle changes ourselves (a dedicated female research team member) to reduce the amount of cage disturbances to once per day, and roughly half of the “dirty” nest was moved to the clean cage and mixed with the new nesting material. The results were quite astonishing. Mirroring the TRAD conditions, fighting began at 6 wk in age. Unlike TRAD conditions, however, we only observed 84 fight incidences versus 107, but no separations were required (Figure 2) and no mice were euthanized due to fight wounds. This resulted in an average of 5.15 d between fight incidences (Figure 3), which is almost three times longer when compared to the TRAD conditions. Peak fighting occurred around the
March 2019 Laboratory Animal Science Professional 39