All procedures were approved by the Ethics Committee on Animal Experimentation of the University of Lleida (license numbers CEEA.09–01/12 and CEEA.09–01/13).
During the weekly reproductive visit, open cows with more of 50 days in milk and with no reproductive disorders such as ovarian cysts and endometritis detected by ultrasound were randomly assigned to one of the following groups: 2PGG, 2PGGe, 2PGe and PGe (Fig. 1

Treatment protocols used to synchronize estrus for fixed-time AI (FTAI) in high-producing dairy cows. All cows (n=232) were fitted with a progesterone releasing intravaginal device (PRID-DELTA, containing 1.55 g of progesterone; CEVA Salud Animal, Barcelona, Spain) for 5 days (PRID-5 days).

). Cows in the 2PGG group were treated with a progesterone-releasing intravaginal device (PRID) (PRID-DELTA, containing 1.55 g of progesterone; CEVA Salud Animal, Barcelona, Spain) plus GnRH (100 μg i.m.; Cystoreline, CEVA Santé Animale, Libourne, France) at PRID insertion. The PRID was left for 5 days, and these animals were also given PGF2α (25 mg dinoprost i.m.; Enzaprost, CEVA Santé Animale, Libourne, France) at PRID removal. Twenty-four hours later, the cows received a second PGF2α dose, and they were inseminated and received a second GnRH dose 36 hours after receiving the second PGF2α dose. The remaining groups were treated with the same P4-based protocol but with the following differences: cows in the 2PGGe group received 500 IU of eCG i.m. (Syncostim, CEVA Santé Animale, Libourne, France) at PRID removal; cows in the 2PGe received eCG at PRID removal and no GnRH was given at PRID insertion; and cows in the PGe group received eCG at PRID removal
and no GnRH at PRID insertion nor the second dose of PGF2α. In this latter group, cows were fixed-time inseminated 60 h after PRID removal. Only healthy cows with no signs of mastitis, lameness or digestive disorders were included in the study. Two experiments were performed to investigate effects of treatments on follicular/luteal dynamics (Experiment I) and fertility (Experiment II).
Cows diagnosed as not pregnant received no further treatment related to the study. This meant that a cow receiving a five-day P4-based protocol was included only once in both experiments. All gynecological exams and pregnancy diagnoses were performed by the second author.