Multiple exposures to familiar conspecific withdrawal is a novel robust stress paradigm in ewes.

Paradigms used so far to study the effects of social isolation in sheep confound the effects of social isolation with those of other stressors (e.g. new environment) and showed contradictory effects after multiple social isolations. We propose here to characterize and examine the repeatable effects of social isolation induced by the familiar conspecific withdrawal (FCW). This latter test consists of socially isolating the ewe by the removal of group mates from the room test for 3 h. Behavioral and endocrine responses of adult ovariectomized-estradiol implanted ewes were compared 90 min before and 90 min after FCW, which was applied three times every fourteen days. We observed that each FCW induced significant increases in plasma cortisol level, in the number of vocalizations, foot pawing, circling attempts and a significant decrease in time spent lying down. An increase in plasma cortisol levels and decrease in duration of maintenance behaviors were significantly lower after the third FCW than after the first one. These differences could be explained by higher plasma cortisol levels and lower duration of maintenance behaviors before the third FCW than before the first FCW suggesting an anticipation of the social isolation period. These data indicate that social isolation is sufficient to induce distress with stable stressful responses after multiple exposures to familiar conspecific withdrawal.