Pups: weaning, monitoring and isolation housing

Litters were weaned according to their treatment allocation and pups were housed in same-sex groups of four animals per cage. Pups were considered for inclusion at weaning if their weight was within one standard deviation of the mean litter weight—experimental subjects were randomly selected from this pool of animals within litter. At weaning, pups were assigned a permanent intradermal ear-tattoo for identification and were given food pellets within the cage twice daily. Additionally, in experimental week 6, all animals were assigned a unique fur mark using a non-toxic animal paint marker (Stoelting Co, Illinois, USA) for individual identification during video recording (see Fig. 1). During routine weekly husbandry, one randomly selected focal animal, between ages 49–52, was removed from each cage of four animals and singly housed for the remainder of the experiment (Fig. 1). The age of single housing was selected because it is typically associated with dispersal from the natal nest and sexual maturity^47–49.

Experimental timeline until isolation housing of focal animal.