Assisted reproductive technologies are being increasingly utilised by the agricultural industry to meet the growing demand for animal protein (1). Commercial in vitro production of bovine embryos uses oocytes from the antral follicles of donor cow ovaries collected via ovum pick-up (2-3). However, only ~25 antral follicles are present per ovary, resulting in low oocyte yields per cow (4). The number of follicles from earlier stages is greater with ~4,000 secondary and ~250 tertiary follicles per ovary (5-6), and thus are a promising way to increase oocyte yield. This study aims to isolate viable secondary and tertiary follicles from fresh bovine ovarian tissue and develop a culture system that allows for successful follicular growth and oocyte maturation in vitro. Follicles were successfully cultured from the secondary to preovulatory stage, with the addition of insulin-transferrin-selenium to culture medium being beneficial for secondary follicle growth from Day 1 to 21 and for tertiary follicle growth during early culture. To further improve this growth, Factor A and Factor B were added in addition to insulin-transferrin-selenium. The addition of Factor A to culture medium was beneficial for secondary follicle growth during late culture and for tertiary follicle growth from Day 1 to 21. The addition of Factor B to culture medium had no effect on secondary follicle growth but was beneficial for tertiary follicle growth from Day 1 to 21. In conclusion, isolation and culture of secondary and tertiary follicles to produce mature oocytes offers a favourable approach for the in vitro production of bovine embryos to produce large numbers of high-quality livestock.