One animal-based method to evaluate welfare is the presence of stress. In dairy ruminants, the responses to stressors include the activation of both neuroendocrine and autonomous nervous system that can be evaluated through an endocrine assessment. The present study aimed to validate a radioimmunoassay method for cortisol in buffalo milk. Three formulations (whole and skimmed milk and whey) and three solvents (methanol diethyl ether and dichloromethane) were tested: methanol was characterised by the best extraction efficiency (69.88%), whey cortisol concentrations
showed a significant correlation with whole extracted milk and were not affected by
fat content variation during the milking session. The RIA used in the present study showed good precision, sensitivity and specificity: the dilutions test indicated the high reproducibility of the results, overlapping of the dilution curve and standard curve highlighted high specificity and the lack of interfering factors by buffalo whey matrix. It is concluded that the present assay suits the cortisol measurement in buffalo milk and the ranges described could be employed in the calibration of a biosensing technologies directly integrated in milking parlour systems.
