Changes of milk fatty acid composition in four lipid classes as biomarkers for the diagnosis of bovine ketosis using bioanalytical Thin Layer Chromatography and Gas Chromatographic techniques (TLC-GC)

The aim of this study was to extend the limited research available on the association between the concentration of milk fatty acids and the elevated plasmatic value of β-hydroxybutyrate (BHB) in early lactation of dairy cows. Fifty-four Holstein Friesian dairy cows were enrolled in the study. All animals were classified on the basis of their blood BHB concentration: BHB ≥ 1.0 mmol/L (BHB-1, sick group) and BHB ≤ 0.99 mmol/L (BHB-0, healthy group). Using Thin Layer Chromatography (TLC), four lipid classes (cholesterol esters -CE-, phospholipids -PL-, free fatty acids -FFA- and triacylglycerols -TAG-) were separated, and then the fatty acid (FA) composition was determined by High Resolution Gas Chromatography coupled with Flame Ionization Detector/Mass Spectrometer (HRGC-FID/MS). The FA profiles were used to investigate the diagnostic potential value of milk fatty acids for the correct classification of cows with BHB concentration above the established threshold (BHB < 1.0 mmol/L). Boruta Test and Receiver Operating Characteristic curves (ROC) were used to identify which FA and their thresholds of concentration could be used when animals presented hyperketonemia. The research has identified fourteen FA, belonging to CE, FFA, and TAG classes, useful for an association with BHB-1. These compounds, with predictive value for the development of hyperketonemia, could be considered valuable biomarkers. Further studies on a wider sampling, based on clinical and therapeutic approach, will be necessary to confirm, by bioanalytical chromatographic approaches, if these predictive FA will change between healthy and sick animals. New approaches in relation on the administration of different diets or supplements, and administration of drugs might improve the prevention of hyperketonemia.

Keywords: Biomarkers; Cholesterol esters; Free fatty acid; Ketosis; Phospholipids; Triglycerides.