In vitro culture of sheep early-antral follicles: Milestones, challenges and future perspectives

Early antral follicles (EAFs) represent the transitional stage between pre-antral and antral follicles, containing oocytes that have completed most of their growth phase. Therefore, they offer an easily exploitable reserve for producing mature oocytes and preserving genetic resources, given their higher abundance compared to antral follicles (AFs) and shorter culture period than other pre-antral follicles (PAFs). Despite these advantages, the culture of EAFs remains challenging, and the success rates of in vitro embryo production (IVEP) from EAFderived oocytes are still far below the standard achieved with fully grown oocytes in ruminant species.The difficulty is related to developing suitable in vitro culture systems tailored with nutrients, growth factors, and other signaling molecules to support oocyte growth. In this review, we focus on the in vitro development of sheep EAFs to provide an informative reference to current research progress. We also summarize the basic aspect of folliculogenesis in sheep and the main achievements and limitations of the current methods for EAF isolation, in vitro culture systems, and medium supplementation. Finally, we highlight future perspectives and challenges for improving EAF culture outcomes.