Backgraund: The first step in the in vitro embryo production (IVEP) procedure is to obtain high-quality and viable oocytes. Therefore, the ovum pick-up (OPU) method was developed in cattle. Ovum pick up in cattle is usually performed once or twice a week. However, when a fixed time follicular aspiration procedure is planned, the OPU application is conducted on different days of the donor's estrous cycle. Furthermore, there is no recommendation as to the optimum day within the estrous cycle for OPU. Therefore, the main objective of the present study was to determine the effect of OPU application on oocyte yield, quality and blastocyst rates on different days of the estrous cycle. Materials, Methods & Results: In the study, 18 Holstein heifers were administered OPU on days 2-3, 4-7, 8-12 and 13-16 of the estrous cycle. The OPU application was performed once in one oestrus cycle for all donors. The following OPU application waited at least one estrous cycle. Grade A quality oocytes were obtained in greater numbers in D 2-3 and D 4-7 groups than in D 13-16 group (P < 0.05). There was no difference in other oocyte quality and total number of oocytes between the groups. The number of viable oocytes was similar between D 2-3 and D 4-7 groups, but greater than D 13-16 group (P < 0.05). Concurrently, the number of viable oocytes was greater in D 4-7 group than in D 8-12 group (P < 0.05). There was no difference between D 2-3 and D 8-12 groups in terms of viable oocyte number. The number of blastocysts in D 2-3 and D 4-7 groups was greater than D 8-12 and similar to D 13-16 (P < 0.05). Discussion: A follicle may be in 1 of 4 phases during the follicular wave: growth, dominance, static, or regression. In present study, the greater number of oocytes with greater quality and viability rate in D 2-3 and D 4-7 groups is thought to be due to the absence of follicles in the dominant stage in these two groups. In OPU/IVEP method, a greater number of oocytes per donor increases the rate of blastocysts and viable embryos and accordingly positively affects the post-transfer pregnancy rate. In current study, the number of Grade A oocytes and viable oocytes were greater in D 2-3 and D 4-7 groups of the cycle compared to D 13-16 group of the cycle. The reason for this is that when OPU is used on days 13-16 of the cycle, different follicular groups can be found on the ovary. In addition, the present study demonstrated that the number of viable oocytes and blastocysts was greater in the D 4-7 group compared to the D 8-12 group. This may be due to the greater number of atresia follicles (subordinal follicles) between days 8-12 of the cycle (compared to days 4-7) and the lower developmental competence of oocytes in the subordinal follicle. The most significant factors influencing the success of an in vitro embryo production process are the quality and viability of the oocyte. In the present study, the number of Grade A oocytes was greater in the D 2-3 and D 4-7 groups than in the D 13-16 group, but there was no difference in the number of blastocysts. However, in parallel with the number of viable oocytes, the number of blastocysts obtained in groups D 2-3 and D 4-7 was greater than in group D 8-12. In conclusion, the determination of the appropriate day of the cycle for OPU will rule out synchronisation or stimulation of the follicular wave and ablation of the dominant follicle. Therefore, the oocyte quality, number and most importantly the number of blastocysts can be increased by performing OPU between 2-3 or 4-7 days of the estrous cycle.