Biotinidase as a novel biomarker for pain assessment in dairy cattle.

Introduction: Freedom from pain is a key component of animal welfare. However, unlike in humans, objective methods for assessing and managing pain in farm animals remain limited. Pain assessment is particularly challenging in cattle, due to their inherently stoic nature. Objective: We investigated biotinidase, an enzyme with amino-exopeptidase activity on opioid neuropeptides, as a novel, short-term pain-biomarker in dairy cattle, in comparison with cortisol and substance P. Methods: Plasma levels of biotinidase, substance P, and cortisol were quantified by ELISA in Holstein cows subjected to 3 distinct pain stimuli: freeze branding (routine husbandry), topical application of capsaicin cream (nonclinical pain model), and spontaneous clinical mastitis. Results: Biotinidase levels increased significantly within 30 minutes after freeze branding compared to controls (1.36 difference of normalized values; P = 0.021), and after capsaicin application (from 18.5 ± 2.5 to 21.5 ± 2.7 ng/mL; P = 0.002). Neither cortisol nor substance P exhibited significant changes after freeze branding or capsaicin exposure. Biotinidase levels elevated significantly in mastitic cows (28.1 ± 8.2 ng/mL) compared to healthy controls (13.63 ± 5.62 ng/mL; U = 74.0, P = 0.038), whereas substance P and cortisol showed no significant differences. Receiver operating characteristic analysis suggests that biotinidase (AUC: 0.84, 0.52, 0.81) outperformed substance P and cortisol in distinguishing painful from painless states. Conclusion: These findings provide compelling new evidence that plasma biotinidase responds robustly to diverse pain stimuli in nonhuman mammals. Further studies should investigate its diagnostic performance individually, and alongside other biochemical, physiological, and behavioral indicators, to enhance pain assessment accuracy and of noninvasive pain detection protocols for farm animals.