Although fixed-time artificial insemination (FTAI) protocols are available for sheep, estrus resynchronization has not been previously reported. The objectives of this study were to evaluate the effect of estrus resynchronization with exogenous progestogen on endogenous progesterone levels and to compare pregnancy rates after two consecutive estrus synchronizations in ewes. In Experiment 1, ewes (n = 20) received an intravaginal device (IVD) containing 60 mg medroxyprogesterone acetate (MPA) for 10 days. At the IVD withdrawal (D0), ewes received 250 IU eCG and were allocated into two treatments: either no further treatment (Control; n = 10) or estrus resynchronization (Resynch; n = 10) from D12 to D19. Serum progesterone (P4) levels did not differ at D12 and D19 (P > 0.05), but were greater at D15 for the Control compared with the Resynch group (P < 0.05). In experiment 2, ewes (n = 250) were submitted to a first synchronization protocol followed by estrus detection and either artificial insemination (AI) or natural mating (NM). Subsequently, ewes were divided into two groups: Control (n = 104): which received no further treatment and were bred by NM; and Resynch (n = 146): which were submitted to a second synchronization starting on D14 (first IVD withdrawal = D0) and to NM after second IVD withdrawal (D20). Cumulative pregnancy rates did not differ between the Control (67.3%, 70/104) and Resynch (62.3%, 91/146) groups. In a third experiment, ewes (n = 83) were bred by two consecutive FTAI within a 20-day interval. Pregnancy rates after the first (30.1%, 25/83) and the second FTAI (36.2%, 21/58) did not differ (P > 0.05). In conclusion, although exogenous progestogen supplementation reduced circulating levels of P4, pregnancy maintenance was unaffected. Estrus resynchronization in ewes is feasible, resulting in similar fertility after the first and the second services. The use of resynchronization coupled with artificial insemination using semen from genetically superior rams may potentially accelerate genetic improvement in sheep herds by allowing a higher differential selection compared with natural breeding.

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