In the first week of the experiment, hair from the left forelimb was shaved in all ewes. Later, the same surface-hair samples were obtained twice, in Week 5 (W5) , when ewes were in the first half of pregnancy and in Week 10 (W10). Samples were processed following a methanol-based extraction protocol previously described (Davenport et al., 2006, Tallo-Parra et al., 2015). Cortisol concentrations in hair samples were determined by using a competitive cortisol enzyme immunoassay (EIA) kit. Saliva samples were taken in all ewes on Weeks 0, 3, 5, 8 and 10 (W0, W3, W5, W8, W10), and sampling was always performed between 16:00 and 17:30. Samples were obtained from a sponge of Salivette® tubes (Sarstedt, Aktiengesellsschaft & Co.; Nümbrecht, Germany). Samples were subsequently centrifuged at 3000 rpm for 10 minutes and then stored at -200C until analysis. Measurement of cortisol concentrations in saliva samples and the assay validation tests were performed using a commercial Enzyme-Linked ImmunoSorbentAssay (ELISA) kit (Neogen Corporation©; Ayr, UK). The assay was validated for the analysis of cortisol in saliva of Ovis aries following the criteria for an immunological validation (Midgley et al., 1969; Reimers & Lamb, 1991). The dilution test showed an R2 = 98.54 % and a mean recovery rate of 105.90% suggesting high specificity of the test.

...