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On D 0, in both periods, plasma concentrations of glycerol, glucose, insulin, NEFA, urea, plasma osmolality and, in whole blood, RBC indices were determined from samples drawn from jugular vein before morning feeding at 08:00. On D 3, plasma concentrations of glycerol, glucose, insulin, NEFA, urea, and plasma osmolality were also determined from eight consecutive samples, while RBC indices from two samples (at 08:00 and at 10:00). Repeated sampling on D 3 were collected after jugular cannulation. At each sampling, from each ewe, two blood samples were collected: one using 2-mL vacuum collection tubes with glycolytic inhibitor (5.0 mg sodium fluoride, 4.0 mg pot. ox. - Vacutainer Systems Europe; Becton Dickinson, MeylanCedex, France) for glucose assay; one using 2.0 mL vacuum collection whole blood tube with spray-coated K₂EDTA (Vacutainer Systems Europe; Becton Dickinson, MeylanCedex, France) for other metabolites, insulin and RBC indices determination. Immediately after recovery, samples were cooled to 4 °C. RBC indices were determined within 2 h of blood collection. The other blood samples were centrifuged at 1500 g for 15 min at 4 °C degrees. Individual plasma was removed and stored in vial at − 20 °C until assayed.
Plasma osmolality (Osm/kg) was measured using a freezing point osmometer (Osmomat 030, Gonotec, Berlin, Germany).

The saliva was obtained as previously reported [3 (link),22 (link)]. The sponges (Esponja Marina, La Griega E. Koronis, Madrid, Spain) soaked with saliva were placed in a commercially available device immediately after sampling (Salivette, Sarstedt, Aktiengesellschaft & Co, Nümbrecht, Germany). The cases were only included if guaranteed that horses did not receive any feed for at least 12 h and if the dirtiness degree of the saliva was 0 or 1 according to the colour scale previously reported (0–4 score) [23 (link)]. After saliva sampling, blood was obtained by jugular venepuncture and transferred into tubes containing a clot activator (Becton Dickinson Vacutainer Systems Europe). Tubes with saliva and blood were centrifuged at 3000× g for 10 min at 4 °C within 30 min of sampling. Saliva and serum were then transferred into Eppendorf tubes and stored at −80 °C until analysis. Horses that yielded serum samples with visual gross haemolysis or blood-contaminated saliva samples were excluded from the study.
The samplings in the EGUS and healthy population were performed before intravenous sedation and gastroscopy but immediately after the horses were placed in the examination stand. In the CIE group, the samples were obtained after the arrival at the hospital, immediately after horses were placed in the examination room.

After each ovarian ultrasound examination, blood samples were collected via jugular venipuncture in vacutainer tubes containing heparin (Vacutainer ® Systems Europe, Becton Dickinson, Meylan Cedex, France). Centrifugation of blood samples was performed at 4100 g for 10 mins, and the plasma was stored at −80°C until further analysis. Plasma concentrations of P⁴ were measured by using a commercially available double antibody radioimmunoassay kit (Immunotech, Prague, Czech Republic) with a 125I-labelled tracer. The interassay and intraassay coefficients of variation for P⁴ assay were 6.2% and 3.5%, respectively, with a minimum detection limit of 0.3 ng/ml.