Enzymatic isolation of porcine preantral follicles impairs oocyte viability and long-term <i>in vitro</i> growth

The enzymatic isolation of preantral follicles (PAFs) is considered the most efficient method for retrieving a large number of intact follicles, offering significant advantages in terms of yield and processing time. However, the low success rate of enzymatically isolated follicles in long-term culture raises concerns regarding their impact on oocyte quality and developmental potential. This study addresses a critical gap in understanding how enzymatic retrieval of PAFs affects the oocyte-granulosa cell connection and its relationship with high mortality and culture failure observed during in vitro growth (IVG). By systematically comparing crude collagenases (IA and IV) and purified collagenases (Liberase TM and DH) with a mechanical isolation protocol, we identified the optimal enzyme concentrations that maximize follicle yield while minimizing cellular damage. Our results reveal that the enzymatic retrieval of PAFs corresponds to the loss of transzonal projections (TZPs) post-isolation, as well as premature oocyte extrusion and follicle deformities during IVG. Our findings also highlight the differential apoptotic responses in oocytes and granulosa cells. Although these enzymes sustain follicle cell integrity, they compromise oocyte viability during isolation. Notably, crude collagenases impair oocyte growth during prolonged culture, whereas purified collagenases preserve the developmental potential of oocytes. This study also provides the first evidence that enzymatic isolation of PAFs adversely affects TZPs. Overall, our study highlights the importance of selecting an appropriate method, enzyme type, and concentration for preserving the integrity of oocytes, follicles, and their connections, thereby supporting successful in vitro culture. Additionally, our results suggest that mechanical protocols and high-purity enzymes are preferred for maintaining oocyte competence.