Non-invasive measurements of glucocorticoid (GC) concentrations, including cortisol and corticosterone, serve as reliable indicators of adrenocortical activities and physiological stress loads in a variety of species. As an alternative to invasive analyses based on plasma, GC concentrations in saliva still represent single-point-of-time measurements, suitable for studying short-term or acute stress responses, whereas fecal GC metabolites (FGMs) reflect overall stress loads and stress responses after a species-specific time frame in the long-term. In our study species, the domestic guinea pig, GC measurements are commonly used to indicate stress responses to different environmental conditions, but the biological relevance of non-invasive measurements is widely unknown. We therefore established an experimental protocol based on the animals’ natural stress responses to different environmental conditions and compared GC levels in plasma, saliva, and fecal samples during non-stressful social isolations and stressful two-hour social confrontations with unfamiliar individuals. Plasma and saliva cortisol concentrations were significantly increased directly after the social confrontations, and plasma and saliva cortisol levels were strongly correlated. This demonstrates a high biological relevance of GC measurements in saliva. FGM levels measured 20 h afterwards, representing the reported mean gut passage time based on physiological validations, revealed that the overall stress load was not affected by the confrontations, but also no relations to plasma cortisol levels were detected. We therefore measured FGMs in two-hour intervals for 24 h after another social confrontation and detected significantly increased levels after four to twelve hours, reaching peak concentrations already after six hours. Our findings confirm that non-invasive GC measurements in guinea pigs are highly biologically relevant in indicating physiological stress responses compared to circulating levels in plasma in the short- and long-term. Our approach also underlines the importance of detailed investigations on how to use and interpret non-invasive measurements, including the determination of appropriate time points for sample collections.
Sex-specific effects of dietary fatty acids on saliva cortisol and social behavior in guinea pigs under different social environmental conditions