Implementation of timed artificial insemination (TAI) programs in beef cattle increases beef reproductive and production efficiency, and improves profitability [1,2]. Heifers or cows that become pregnant in the first 42 days of the breeding season produce heavier calves at weaning and have more time to become pregnant in the next breeding season resulting in greater longevity in the herd and more calves produced during productive life [3,4]. In beef cattle, TAI allows ∼50% of cows to become pregnant on the first day of the breeding season [5]. After first TAI, cleanup bulls have been used to increase the number of heifers/cows that are pregnant before 42 days of the breeding season. In one study, however, more than 60% of nonpregnant cows returned to an anovular condition after induction of ovulation during the first TAI protocol [6]. To overcome these negative impacts of anovulation on beef cattle operations, serial use of TAI programs can be used to induce and synchronize ovulation during second and later TAI [[7], [8], [9]]. Use of TAI at first and later AI can increase the number of pregnant cows during the breeding season and increase the percentage of cows that are pregnant early in the breeding season.

In the past decades, multiple resynchronization (Resynch) strategies have been reported with interbreeding intervals of 24–40 days [[10], [11], [12]]. These protocols have used different approaches for pregnancy diagnosis and Resynch treatments, often initiating the protocol before pregnancy diagnosis, i.e. in all cows without knowledge of gestational status of the cow. Two features of Resynch protocols that have limited their widespread use are: (1) animal management for each TAI is on different days of the week with schedules that limit the number of groups of cows that receive first AI and Resynch AI during the breeding season and (2) most Resynch protocols only allow a maximum of two TAIs by day 42 of the breeding season, a critical time for efficiency of beef operations.

The ReBreed21 resynchronization program was developed to improve reproductive efficiency on beef production operations [13] by assuring that all TAIs occur on the same day of the week which optimizes its implementation and applicability. Furthermore, the ReBreed21 protocol allows three TAI by day 42 of the breeding season increasing the number of cows that are pregnant early in the breeding season. In our first report of ReBreed21, heifers had larger ovulatory follicle size at the time of reinsemination compared to another Resynch strategy. However, the follicle dynamics and endocrine profiles of animals submitted to the ReBreed21 protocol were not evaluated.

We developed a series of studies to understand physiological and reproductive outcomes using the ReBreed21 program in large beef production operations. This particular manuscript reports the results of a study designed to assess the follicular dynamics and endocrine profiles of Bos indicus Nelore beef heifers during the ReBreed21 program. In addition, a further modification to the original protocol is tested endeavoring to optimize the endocrine and follicular profile leading to reinsemination. We hypothesized that (1) GnRH treatment at the start of ReBreed21 will increase synchronization of follicular wave emergence (within 1–3 days); (2) ReBreed21 protocol does not alter the timing of structural luteolysis but synchronizes ovulation by maintaining circulating concentrations of P4 (P4) ≥ 1 ng/ml until after removal of P4 device; (3) The ReBreed21 protocol, including eCG treatment on d19, will increase follicle growth rate (d19 to d21) and increase follicle size on d21; and (4) The ReBreed21 protocol, including GnRH treatment on d21 (expected time of AI), will increase synchronized (d22 and d23) and overall ovulation compared to Control non-pregnant heifers.