Synchronization of follicle wave emergence among heifers before ovarian superstimulation and ovum pick-up (OPU) affects in vitro embryo production (IVP) in non-pregnant donors. Utilization of IVP has steadily increased, therefore, there is need for optimization of pregnant animal treatments for oocyte donors. The objective, therefore, was to evaluate effects of synchronization of follicle wave emergence among heifers, before ovarian superstimulation and OPU on embryo production in pregnant heifers. Pregnant (47-69 days of gestation) Holstein heifers (n = 64) 19.0±0.3 months of age were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence by mechanical ablation of all follicles 6 mm (FA) or untreated control (no-FA). Superstimulatory treatments consisting of 160 mg of porcine follicle stimulating hormone (p-FSH), administered in four injections 12 h apart, were initiated 36 h after FA or at random stages of the follicular wave in the no-FA group. Ovum pick-up (recovery of oocytes) was performed in all heifers 40 h after the last p-FSH administration. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Recovered oocytes were processed in groups by treatment, and IVP was performed. Differences between treatment groups were evaluated using generalized linear mixed models. Total number of follicles was not different between treatment groups (P=0.61), however, heifers in the FA group had a greater (P=0.01) proportion of medium sized (6-10 mm) follicles than those in the no-FA group (61.2±2.8% and 49.9±3.4%). Conversely, heifers in the no-FA group had a greater (P=0.01) proportion of small sized (6mm) follicles than those in the FA group (41.1±3.7% and 29.6±2.4%). Cleavage rate (85.5% and 72.4%; P=0.04) and blastocyst rate (48.4% and 33.1%; P=0.04) were both greater in heifers of the FA than no-FA group which led to larger (P=0.01) numbers of embryos produced per heifer in the FA (8.9±1.2) than no-FA (5.9±1.0) group. In summary, synchronizing timing of follicle wave emergence among pregnant heifers, prior to ovarian superstimulation and OPU, results in a greater proportion of medium size follicles and improved developmental competence of the recovered oocytes which leads to increased embryo production.