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The aim of the study was to determine the luteolytic dose of cloprostenol administered directly into the corpus luteum (CL; intra-luteal treatment, ILT) in dairy cattle. Cows of two control groups were treated with 500 μg of cloprostenol (Estrumate®) intramuscularly (IM-500) or via ILT with 0.2 mL of physiological solution (ILT-0). Cows of four experimental groups were treated by ILT with cloprostenol in doses 5, 25, 50 and 100 μg (ILT-5, -25, -50 and -100 groups). Progesterone concentrations (P4) and size of CL were evaluated to assess luteolysis at 0, 0.5, 1, 2, 4, 8, 24 and 48 h or at 0, 24 and 48 h after ILT, respectively. Cows in the ILT-0 and -5 groups were unaffected by ILT. The P4 concentrations were less in cows of the IM-500, as well as ILT-25, -50 and -100 groups at 48 h subsequent to ILT. The size of the CL was less in cows of IM-500, as well as ILT-25, -50 and -100 groups at 48 h after ILT. There were P4 concentrations of about 1 ng/mL 48 h after ILT in cows of the IM-500, as well as ILT-50 and -100 groups. In conclusion, the cloprostenol dose of 50 μg administered intra-luteally is a luteolytic dose in cows., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2020 Elsevier B.V. All rights reserved.)