Multi-oocyte follicles in the bovine ovary: occurrence, activation, and growth during early in vitro folliculogenesis in a dynamic culture bioreactor

Purpose: Multi-oocyte follicles (MOFs) have been identified in various mammals, including humans, yet their origin and function remain controversial. This study aimed to investigate the occurrence and early developmental dynamics of MOFs in bovine ovaries, comparing them to single-oocyte follicles (SOFs). Methods: Ovarian tissues from Bos taurus taurus individuals, including one case with an unusually high MOF incidence, were cultured in vitro under dynamic conditions. MOF frequency and their progression through early folliculogenesis stages were assessed histologically and via confocal microscopy. Results: MOFs were observed at varying frequencies, with one individual showing a notably high incidence (15.8%). In vitro culture confirmed that MOFs can activate and progress to secondary stages, similar to SOFs. However, differences in activation rates and oocyte number dynamics were noted between MOFs and SOFs, and between fresh and cultured tissues. No de novo formation of MOFs was detected in vitro. Conclusions: MOFs retain the capacity for early folliculogenesis comparable to SOFs, without increased atresia. Their stable frequency postculture supports a prenatal origin. These findings offer new insights into MOF biology and suggest a possible physiological relevance in mammalian reproductive systems.