Ovum pick-up (OPU) is a repeatable technique that is used for the retrieval of large numbers of immature oocytes from the antral follicles of live animals. The aim of the present study was to determine the effect of repeated OPU application on cumulus-oocyte complex (COC) number, oocyte quality, and blastocyst rates. The animal material for the study consisted of 10 Holstein heifers. Heifers selected as donors were administered OPU a total of 9 times on random days of the cycle at 1-week intervals. All antral follicles with a diameter of 2-8 mm were aspirated during the OPU procedure. In vitro embryo production (IVEP) was performed on the oocytes obtained after quality assessment. Various degrees of adhesion and connective tissue thickening were found in the ovaries of the animals after 9 repeated applications of OPU. It was found that the average number of A-quality oocytes was higher than the number of B, C, and D-quality oocytes. The number of cleavages per OPU was 2.69 (cleaved oocytes/opu session), and the number of blastocysts was 0.72 (number of blastocysts/opu session). It was determined that the general average of cleavage ratios was 73.62%, and blastocyst rates were 18.97%. It was observed that the number of oocytes, cleavage, and blastocysts obtained as a result of repeated OPU applications was not affected by repeated OPU. It was concluded that in order to achieve the target number of oocytes and blastocysts with recurrent OPU, experiments should be carried out with varying frequencies of OPU, and prophylactic measures should be taken to prevent adhesions.