Evaluation of mechanical isolation techniques for efficient harvesting of bovine preantral follicles

Preantral follicles constitute a majority of ovarian follicles and are closely associated with a mammalian female's reproductive lifespan and fertility. Culture of preantral follicles to produce competent oocytes is becoming possible with modern technologies. As such, repeatable methods/techniques for harvesting large numbers of viable, high-quality preantral follicles are crucial. Therefore, this study aimed to optimize mechanical techniques to isolate high numbers of viable preantral follicles at various developmental stages (primordial, primary, and secondary) from bovine ovaries. In total, three experiments were conducted: (i) evaluation of 20 μm filtration, neutral red staining, and centrifugation as putative enhancements to an established protocol; (ii) development of a novel mechanical isolation technique [Southern Illinois University (SIU)] and comparison with existing techniques; and (iii) validation of the SIU technique through short-term (7 days) in vitro culture of isolated follicles. Overall, 28,621 preantral follicles were isolated from 22 young cows. The results demonstrate that high numbers of bovine preantral follicles can be reliably isolated using techniques that combine tissue fragmentation, homogenization, serial filtration, neutral red vital staining, and centrifugation. Furthermore, the SIU technique is the first reliable and repeatable method, used in any species, described to isolate a large number of viable primordial follicles. Moreover, follicles of all stages isolated using the SIU technique were able to survive, grow, and develop during a 7-day in vitro culture period. These findings support the concept that preantral follicle isolation cannot be considered a "one-size-fits-all" methodology. The methods used to isolate preantral follicles must be carefully considered depending upon the targeted developmental stages, follicle quality (viability and morphology), and the downstream analyses to be performed.