Abstract <p>Assisted reproductive technologies to produce embryos from live donors are commonly recognized tools used to preserve the genetic characteristics of local cattle breeds. The ongoing research is the first work focused on evaluating the potential for in vitro embryo production (IVP) with the oocytes isolated from the Istoben cattle with the ovum-pick-up (OPU) technique. The OPU procedure was conducted in sexually mature heifers (<i>n</i> = 8) in two schemes: initially twice a week (2W) and then once a week (1W). Overall, nine consecutive OPU system sessions each two scheme variants were conducted. With respect to the procedure conducted once a week (1W), the number of aspirated follicles per OPU session was lower than that for scheme variant 2W (6.99 vs. 8.75, <i>p</i> &lt; 0.001) because of a gradual decrease of this parameter to the ninth session, while the oocyte retrieval rate per punctured follicle was, on the contrary, significantly higher (57.3 ± 3.7 vs. 43.2 ± 3.1, <i>p</i> &lt; 0.01). In addition, the number of collected oocytes (4.1 ± 0.3 oocytes) and the proportion of oocytes selected for in vitro culturing (63.5 ± 3.9%) were comparable with that in scheme variant 2W (3.9 ± 0.4 oocytes and 59.4 ± 4.4%, respectively). Moreover, a similar pattern was observed for the rates of maturation, cleavage, and development of the selected oocytes reaching the blastocyst stage with in vitro culturing for both scheme variants 1W and 2W, comprising 85.6 ± 3.1 and 88.3 ± 2.9, 67.6 ± 4.1 and 68.0 ± 4.7, and 23.2 ± 3.8 and 20.7 ± 3.9%, respectively. The conclusion about both comparability of the study-based data with the datasets produced for the cattle of the other local breeds and the IVP embryo production efficiency similar in the 1W and 2W OPU scheme variants has been drawn. Therefore, the OPU/IVP embryo production technology described in the article may be used in the reproduction programs for the Istoben cattle breed and the programs on preserving its unique gene pool.</p>