Cryopreservation and In Vitro Culture of Isolated Porcine Ovarian Follicles

Cryopreservation of preantral follicles (PAFs) is a promising tool for gene conservation and fertility preservation. However, standardized protocols for the cryopreservation and in vitro culture of isolated follicles-particularly in pigs-are still lacking. This study aimed to analyze the survival and developmental potential of porcine PAFs vitrified using two different methods: open pulled straw (OPS) and cryotube (CT). Ovaries of Hungarian Large White sows were collected from a local slaughterhouse and enzymatically digested to isolate preantral follicles. Morphologically normal follicles were assigned to three groups: fresh control, OPS-vitrified, and CT-vitrified. All follicles were cultured for 10 days in FSH-supplemented medium, with growth, survival, and estradiol (E2) production monitored. Survival rate was lower in the CT group (83.3%) than that of the control and OPS (97.4% and 94.4%, respectively). The follicular area was consistently larger in control than in CT and OPS, with no difference between vitrified groups. E2 production varied among treatments: OPS follicles showed lower E2 levels on Day 2, no differences were detected on Day 7, and CT follicles produced less E2 on Day 10. These results indicate that OPS is the more suitable vitrification method for porcine PAFs and that the culture system supports hormone production; however, it may require refinement to provide long-term follicle maintenance.