Reference intervals for serum macro- and microminerals in clinically healthy horses in Northwestern Spain: Influence of age, sex, breed and diet

• Serum reference intervals (RI) were established for 14 minerals in healthy horses. • Age, sex and breed had minimal impacts on mineral concentrations. • Diet influenced selenium; pasture-fed horses near lower RI limit. • The RI are valuable for clinical, nutritional, and equine mineral research. Serum mineral concentrations are key indicators of nutritional status, metabolic function and health in horses; however, contemporary reference intervals (RI) derived from large, well-characterised populations remain scarce. To establish serum RI for macro- and microminerals in clinically healthy horses in Northwestern Spain and to evaluate the influence of sex, age, breed and diet on mineral status. Blood samples were collected from clinically healthy horses (n= 211), and serum concentrations of essential macrominerals (calcium [Ca]; phosphorous [P]; magnesium [Mg]; sodium [Na], potassium [K] and sulphur [S]) and microminerals (cobalt [Co]; copper [Cu]; iron [Fe]; iodine [I]; manganese [Mn]; molybdenum [Mo]; selenium [Se] and zinc [Zn]) were determined using precise, accurate multielement techniques (Inductively Coupled Plasma: ICP-OES and ICP-MS). Reference intervals were established for all minerals in accordance with the clinical and laboratory standards institute (CLSI) C28-A3 guidelines. Biological factors (age, sex, breed) contributed minimally to variability in concentrations, with extensive overlap between groups, indicating partitioning was unnecessary. Diet significantly influenced the concentrations of several elements, particularly Se; pasture-fed horses had lower levels of this element, often close to the lower limit of the RI, than horses receiving commercial concentrate diets. This study provides robust serum RI for macro- and microminerals in horses, providing valuable data for clinical assessment, nutritional monitoring and research on mineral metabolism.