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In cattle, blood lactate was measured in various conditions such as parturition and dystocia. To our knowledge, to date, no handheld device has been validated for the use in cows and only one handheld device was validated for the use in calves. When determining plasma lactate concentrations blood samples have to be processed carefully. Sodium fluoride was recommended to inhibit glycolysis and to stabilize plasma lactate concentrations during transport. However, its effect on measurements conducted with electrochemical meters has not been studied. The objectives of 3 experiments were to study factors influencing measures of L-lactate in dairy cows (e.g., different anticoagulants, different methods) and to validate a handheld device (Lactate Scout, SensLab GmbH, Leipzig, Germany) to determine L-lactate concentration in dairy cows and calves. In a first approach, blood samples from 49 cows were analyzed by 2 different laboratories. Measures of L-lactate concentration were correlated between the different laboratories in both lithium heparin plasma (r=0.98) and sodium fluoride plasma (r=0.99). In a second approach, these samples were analyzed using 3 methods [Lactate Scout, Biosen C_line (EKF Diagnostics GmbH, Barleben, Germany), and commercial laboratory]. Concentrations of L-lactate measured in lithium heparin did not differ when analyzed with the Lactate Scout (0.99±0.35 mmol/L), the Biosen C_line (0.81±0.26 mmol/L), or the laboratory (1.0±0.36 mmol/L). Concentrations of L-lactate measured in sodium fluoride, however, were higher when analyzed with the Lactate Scout (1.85±0.66 mmol/L) compared with those measured with the Biosen C_line (0.92±0.37 mmol/L) and by the commercial laboratory (0.72±0.45 mmol/L). In the second and third experiments, blood samples from 173 cows and 106 calves were analyzed using the 3 methods (Lactate Scout, Biosen C_line, and commercial laboratory). L-Lactate concentrations measured with the 3 methods were correlated (cows: Lactate Scout vs. Biosen C_line: r=0.97, Lactate Scout vs. laboratory 1: r=0.98, Biosen C_line vs. laboratory 1: r=0.99; calves: Lactate Scout vs. Biosen C_line: r=0.97, Lactate Scout vs. laboratory 1: r=0.98, Biosen C_line vs. laboratory 1: r=0.99). In conclusion, Lactate Scout and Biosen C_line measure blood L-lactate concentrations reliably compared with a commercial laboratory as the reference method in dairy cows and calves. However, attention needs to be paid to the choice of anticoagulant used in sample collection.