In the present study, we present a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with enzymes, proposing an optimized protocol useful for all further applications related to follicle in vitro growth and reproductive tissue engineering. The porcine follicles were isolated using mechanical technique alone (hand blender and scalpels) or in combination with collagenase or Liberase Dispase High (DH) at different doses applying different protocols. Finally, the number, morphology, and stage of isolated follicles were compared between the protocols. Moreover, the follicle viability (live/dead assay) and morphology (rhodamine phalloidin and 4′,6-diamidino-2-phenylindole staining and scanning electron microscopy analysis) were evaluated after 10 days of culture. We found an optimum protocol for intact follicle isolation using the mechanical technique in combination with enzymes at a concentration of 0.5 mg/mL. However, the number of total isolated follicles and primordial follicles was significantly higher when collagenase was used compared to Liberase DH (p < 0.05), while Liberase DH could isolate a significantly higher percentage of preantral follicles. After 10 days of culture, the morphology and health status of follicles were statistically higher when Liberase DH was used in comparison with collagenase. Moreover, on the follicles extracted with Liberase DH, it was possible to observe theca cells covering part of the follicle surface. In conclusion, we demonstrated that the intact primary or secondary follicles could not be obtained using only mechanical methods, which led to the isolation of denuded oocytes and dramatically damaged follicles. We concluded that the collagenase-based follicle isolation could negatively affect the morphology and developmental potential of the follicles. Moreover, the incubation of ovarian cortex tissues with Liberase DH solution is an optimized protocol for porcine ovarian follicle isolation with developmental competence.

中文翻译：

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优化猪卵巢卵泡分离方法以获得更好的发育潜力。

在本研究中，我们提出了单独使用机械技术或与酶结合使用的猪卵泡分离产物之间的比较分析，提出了可用于与卵泡体外相关的所有其他应用的优化方案生长和生殖组织工程。单独使用机械技术（手动搅拌器和解剖刀）或结合胶原酶或Liberase Dispase High（DH）以不同剂量应用不同方案分离猪卵泡。最后，比较了方案之间分离的卵泡的数量，形态和阶段。此外，在培养10天后评价卵泡的生存力（活/死分析）和形态（若丹明鬼笔环肽和4'，6-二mid基-2-苯基吲哚染色和扫描电子显微镜分析）。我们发现使用机械技术与浓度为0.5 mg / mL的酶结合的完整卵泡分离的最佳方案。然而，p <0.05），而Liberase DH可以分离出更高百分比的窦前卵泡。培养10天后，与胶原酶相比，使用Liberase DH时卵泡的形态和健康状况在统计学上更高。此外，在用Liberase DH提取的卵泡上，可以观察到覆盖卵泡表面一部分的卵泡膜细胞。总之，我们证明了仅使用机械方法无法获得完整的初级或次级卵泡，这导致了裸露的卵母细胞的分离并严重破坏了卵泡。我们得出的结论是，基于胶原酶的卵泡分离可能会对卵泡的形态和发育潜能产生负面影响。此外，