Volume 76 (2026) Issue: 2026 No#2

Equine whole saliva: daily reproducibility of selected biochemical parameters and protein electrophoretic patterns (pilot study)

Author(s): Stefan Đoković, Ana Mandić, Aleksandra Mitrović,Stefan Radosavljević, Anja Ilić Božović, Milena Radaković, Milica Kovačević Filipović

Keywords:horse, salivary analytes, SDS-PAGE, variability

Salivary analytes are known to exhibit variability associated with time of day and seasonal influences, reflecting circadian rhythmicity. Whole saliva provides a more comprehensive representation of salivary composition compared to commonly studied parotid saliva. Objective of the study was to investigate reproducibility of selected equine whole-saliva constituents when measured at different times of day across four consecutive days, and to qualitatively access stability of salivary protein fractions using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Six clinically healthy Warmblood horses were enrolled in the study. Saliva samples were taken twice a day, in the morning and in the evening. Data were assessed descriptively and expressed as median (range), with P<0.05 considered significant. Linear mixed-effects models revealed that time of day had no significant effect on most salivary parameters in horses, with the exception of adenosine deaminase (P=0.020). The sampling day had a significant effect on uric acid (P=0.003), total proteins (P=0.007), adenosine deaminase (P=0.011), alkaline phosphatase (P<0.001), γ-glutamyl transferase (P=0.013) and calcium (P=0.005). Interclass correlation coefficients (ICC) were low for most parameters (ICC≈0.00–0.16), indicating that within-horse variability exceeded between-horse variability. The relative proportions of salivary electrophoretic fractions were highly stable and not significantly affected by sampling day, time of day, or their interaction. Despite the identified variability, the overall stability of the salivary electrophoretic profile supports the potential use of salivary protein fractions as qualitative biomarkers.