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Ovulatory response to the first GnRH of Ovsynch is the critical determinant for successful synchronization of ovulation in dairy cows. Our objective in this study was to develop a pre-Ovsynch treatment that increased the percentage of cows that ovulated in response to the first GnRH injection of Ovsynch. To accomplish our goal, we evaluated a hormonal strategy that consisted of PGF_2α and GnRH before the first GnRH of Ovsynch. Lactating dairy cows (n = 137) were assigned to receive either no treatment before Ovsynch (control) or 25mg of PGF_2α (PreP) followed 2 d later by 100μg of GnRH (PreG), administered 4 (G4G), 5 (G5G), or 6 (G6G) d before initiating the Ovsynch protocol. Transrectal ultrasonography was performed to assess follicular size and resulting ovulation, and blood samples were collected to measure circulating concentrations of progesterone and estradiol immediately before each hormonal …