Nestlet pressed cotton square

Two weeks before the pathological examinations, the mice were individually housed for 5 h before the nesting test. A Nestlet pressed-cotton square (Ancare, Bellmore, NY, USA) was placed in each cage 1 h before the beginning of the dark cycle. Photographs of the Nestlet were taken at specific time points, and the test lasted 52 h. Nest construction was graded on a five-point scale by two persons who were blinded to the treatments, as described in a previous study [53 (link)]. A score of 1 indicated that the Nestlet was >90% intact, whereas a score of 5 indicated that it was <10% intact and a nest with an obvious crater had been constructed.

Mice were individually housed for 5 h, then a nestlet pressed-cotton square (Ancare, Canterbury, Kent, UK) was put into each cage 1 h before the dark cycle started. Nest construction was scored using a 5-point scaling system 16 h later [34 (link)]. Nest score of 1 to 5 indicates a >90%, >60%, >30%, >10% and <10% intact nestlet, respectively, and the nest reaching score 5 has an obvious crater. The duration required for constructing a nest reaching scored 5 was recorded with a recording interval for 2 h.

After a 24 h rest period, mice were housed individually and tested for nest building (adapted from Deacon, 2006 (link); Wesson and Wilson, 2011 (link)). Two hours prior to the onset of the dark phase of the lighting cycle, individual cages were supplied a commercially available Nestlet pressed cotton square (Ancare, UK agent, Lillico). The next morning (~16 h later) cages were inspected for nest construction. Pictures were taken prior to evaluation for documentation. Nestlet nest construction was scored blind to genotype using the system of Deacon (please see Deacon, 2006 (link), for detailed scoring standard). Briefly, in this 5 point scale, 1 indicates a >90% intact nestlet, whereas a 5 indicates a nestlet torn >90% and a clear nest crater.