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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\u2009\u03bcm 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\u2009days) 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 \u201cone-size-fits-all\u201d 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.<\/jats:p>","DOI":"10.1093\/reprod\/xaag020","type":"journal-article","created":{"date-parts":[[2026,2,4]],"date-time":"2026-02-04T12:46:05Z","timestamp":1770209165000},"source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of mechanical isolation techniques for efficient harvesting of bovine preantral follicles"],"prefix":"10.1093","volume":"171","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8304-4644","authenticated-orcid":false,"given":"Ana N P","family":"Morais","sequence":"first","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]},{"name":"Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary Medicine, State University of Cear\u00e1 , Fortaleza, Cear\u00e1,","place":["Brazil"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1573-8000","authenticated-orcid":false,"given":"Kendall A","family":"Hyde","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5251-9598","authenticated-orcid":false,"given":"Andr\u00e9 L","family":"Concei\u00e7\u00e3o-Santos","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]},{"name":"Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary Medicine, State University of Cear\u00e1 , Fortaleza, Cear\u00e1,","place":["Brazil"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-0170-4206","authenticated-orcid":false,"given":"L\u00e9a","family":"Engelman","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0043-0137","authenticated-orcid":false,"given":"Melba O","family":"Gastal","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9349-4369","authenticated-orcid":false,"given":"Jones B","family":"Graceli","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United States"]},{"name":"Laboratory of Cellular Toxicology and Endocrinology, Department of Morphology, Federal University of Esp\u00edrito Santo , Vitoria, Esp\u00edrito Santo,","place":["Brazil"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2528-4874","authenticated-orcid":false,"given":"Anna C","family":"Denicol","sequence":"additional","affiliation":[{"name":"Department of Animal Science, University of California Davis , Davis, CA,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8855-6980","authenticated-orcid":false,"given":"Eduardo L","family":"Gastal","sequence":"additional","affiliation":[{"name":"Animal Science, School of Agricultural Sciences, Southern Illinois University , Carbondale, IL,","place":["United 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