In vitro technology is one of the most dynamically developing and more and more stable biotechnological methods today accelerating selection, intensifying reproductive and genetic potential of breeding animals, allowing to increase breeding young animals production by one champion cow up to 5–10 calves per year, reduce generation interval and significantly accelerate process of updating and qualitative improvement of livestock. However, obtaining oocytes competent for in vitro development is one of the critical factors determining success of the method and depending on a number of biological and technical factors. This paper presents results of studies on effect of biological factors of direct and indirect impact on efficiency of obtaining oocytes in the system of transvaginal aspiration for the first time conducted in the Republic of Belarus. Yield of excellent and good quality oocytes increased during aspiration during the luteal phase of estrous cycle and remained almost unchanged during aspiration into the follicular phase. Presence of follicles with diameter over 8 mm in the ovaries during aspiration reduced yield of excellent and good quality oocytes averagely by 9.4 percentage points. Removing the dominant follicle 72 hours prior to aspiration allowed increasing the number of aspirated follicles by 41 %, and yield of oocytes – by 22.9 %. Microstimulation of ovaries prior to aspiration by follicle-stimulating hormones FSG-super and Plusset increased efficiency of aspiration in terms of the main indicators by 19.2–45.9 %. Follicular cyst or persistent corpus luteum in one of the ovaries reduced both quantitative and qualitative indicators of aspiration. The data obtained are of practical importance for development of technology for in vitro embryo production in the system of transvaginal aspiration of oocytes which will help to accelerate breeding process and increase efficiency of breeding work in livestock production in general.Acknowledgments. The research was conducted within the two state research programs: “Biotechnology”, subprogram “Development of biological science, biological education and biological industry for 2007–2011 and for the period up to 2020”, “High technologies and equipment for 2016-2020”, subprogram 1 “Innovative biotechnologies–2020”.