Cows were presynchronized beginning at 32 ± 6 DIM using 2 injections of 25 mg (i.m.) of PGF 2α (dinoprost tromethamine; Lutalyse, Zoetis, SP, Brazil) given 14 d apart. Cows were then synchronized based on the protocol described by Pereira et al. (2015) (link): 11 d following the second PGF 2α injection of the presynchronization, cows initiated the following synchronization protocol: at d -11: intravaginal progesterone implant (Eazi-Breed controlled internal drug-releasing implant containing 1.9 mg of progesterone, Zoetis) + 2.0 mg (i.m.) of estradiol benzoate (Gonadiol, Zoetis) + 100 µg (i.m.) of gonadotropin-releasing hormone (gonadorelin diacetate; Cystorelin, Merial, SP, Brazil); at d -4: 25 mg (i.m.) of PGF 2α (dinoprost tromethamine; Lutalyse, Zoetis); at d -2: removal of intravaginal progesterone insert + 1.0 mg (i.m.) of estradiol cypionate (ECP, E.C.P., Zoetis) + 25 mg (i.m.) of PGF 2α ; and at d 0: TAI performed using commercial semen from 10 randomly selected bulls.
Positive pregnancy diagnosis was made when an amniotic vesicle with viable embryo was detected via rectal palpation with ultrasonography (Honda HS 101V with a 5.0-MHz linear probe, Honda, Tokyo, Japan) 32 and 60 d after TAI.