1. Institut de Recherche en Sémiochimie et Ethologie Appliquée Department of Clinical Sciences / Department of Agronomy and Aquaculture THE POSSIBLE ROLE OF OXYTOCIN AS STRESS INDICATOR IN PUPPIES DURING THE FIRST SOCIAL ISOLATION. Patrick Pageat*, C. Bienboire Frosini, C. Chabaud, C. Lecuelle Lafont, F. Noel, A. Cozzi Dvm, MSc, PhD, BDFVS, Dipl ECVBM-CA Professor of Applied Ethology and Animal Welfare EI Purpan – INP Toulouse Senior Researcher in the Department of Agronomy and Aquaculture of the IRSEA Chairman of the Scientific Advisory Board of the IRSEA p.pageat@irsea.info

2. Why looking at oxytocin? Oxytocin is a nonapeptidic hormone, involved in numerous behaviors, including maternal, social and non-social, with emotional components Oxytocin is known to be strongly involved in parental behavior in rodents ( Landgraf et al 1991 ), in rabbits ( Caba et al 1996 ) or in ewes ( Levy et al 1995 ) More recently oxytocin has been shown to play a role in intraspecific social relationships in different species and in interspecific relationships between dogs and humans ( Handlin et al 1991, Oliva et al 2012 ).

3. A simple cycle of life illustrates numerous points at which Oxytocin may affect behaviors and physiology to facilitate the propagation of the species. (H.-J. Lee et al. / Progress in Neurobiology 88 (2009) 127–151)

4. Isolation Test 4 Methods Where Bio2M – CEDS Dog Breeding Facility Beagles - Domaine des Souches 89130 MEZILLES France Animals Physiological and behavioural parameters were evaluated, on 5 Beagle puppies aged 7/8 weeks, 3 males and 2 females, during a standardized test: the first social isolation after puppy litter separation Puppy with littermates Separation and isolation 15 min. End of test MATERIAL AND METHODS

5. Physiological parameters considered were: Plasma concentration of oxytocin Plasma concentration of ACTH Plasma concentration of Cortisol Methods Physiological parameters 5 T0: before the separation T1: after 15 minutes of separation and social isolation

6. Methods for hormones dosification Cortisol and ACTH: chemiluminescent immunoassay with Immulite 2000, automated immunoassay analyzer Oxytocin: Enzyme ImmunoAssay (EIA); ( Oxytocin EIA kit, ADI-900-153, Enzo Life Sciences )

7. Results

8. Cortisol Distribution is normal  possibility to use Student t test for paired samples The increase in cortisol plasma level, between T0 and T1, is not statistically significant (p= 0.065)

9. ACTH Distribution is not normal  Wilcoxon test for paired samples Significant increase in ACTH plasma level, between T0 and T1 (p= 0.043)

10. Oxytocin Distribution is normal  possibility to use Student t test for paired samples The decrease in oxytocin plasma level, between T0 and T1, is stastistically significant (p= 0.031)

11. Discussion

12. Criticisms There is currently no test kit validated for the measurement of oxytocin in canine plasma: we have first managed a validation process that will be soon submitted for publication. Small population: decision related to ethical concerns Short duration of the separation and no study of recovery: oxytocin mediated reaction was assumed to be quite immediate, further research to be done with oxytocin during other standardized tests (Ainsworth strange situation test) Influence of puppies age? Maybe a way to assess the most favorable age for separation from the litter

13. Other comments Separation from mother and litter is proved to be a serious stress Could it influence further development and interspecific socialization? Some puppies seem to display no or very mild decrease in oxytocin: is it related to the quality of primary attachment (secure or unsecure?)

14. Conclusion Oxytocin plasma level may be a good indicator for stress and assessment of attachment quality Further research is required to validate clinical correlations It may be a way to assess the role of attachment in separation-related disorders

15. Thank you for your attention