{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T04:02:50Z","timestamp":1792814570124,"version":"4.5.0"},"reference-count":53,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2025,3,9]],"date-time":"2025-03-09T00:00:00Z","timestamp":1741478400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,5,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>We recently reported successful equine in vitro fertilization using fresh semen pre-incubated for a prolonged period (22\u00a0h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine in vitro fertilization with frozen\u2013thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the sperm separation device method, fertilization rates with sperm pre-incubated for 15\u00a0min, 3, 6, and 9\u00a0h were 7.1, 22.2, 38.5, and 73.3% respectively (9\u00a0h vs. 15\u00a0min or 3\u00a0h, P\u2009&amp;lt;\u20090.05). Fertilization rates differed significantly (45.9% vs. 85.5%) between freezing extenders. Blastocysts were produced using frozen\u2013thawed semen from each of three stallions and transfer of nine vitrified-warmed blastocysts to mares yielded seven embryonic vesicles. Anti-protein tyrosine phosphorylation staining of the entire sperm tail increased over pre-incubation, and sperm both with and without staining in the tail bound to the oocyte cumulus after co-incubation. Using the stain DiSC3(5) and flow cytometric analysis, a population of apparently hyperpolarized sperm was identified at 22\u00a0h in fresh sperm that was not seen at any time in frozen\u2013thawed sperm. We conclude that frozen\u2013thawed equine sperm can successfully fertilize oocytes after a shortened pre-incubation time of 9\u00a0h, suggesting that the freeze\u2013thawing process induces capacitation-related changes. Our findings on evaluation of pre-incubated sperm indicate that the mechanisms by which frozen\u2013thawed sperm become capable of fertilization may differ from those found in fresh sperm.<\/jats:p>","DOI":"10.1093\/biolre\/ioaf043","type":"journal-article","created":{"date-parts":[[2025,3,9]],"date-time":"2025-03-09T20:29:41Z","timestamp":1741552181000},"page":"867-879","source":"Crossref","is-referenced-by-count":19,"title":["Equine in vitro fertilization with frozen\u2013thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes"],"prefix":"10.1093","volume":"112","author":[{"given":"Matheus R","family":"Felix","sequence":"first","affiliation":[{"name":"Department of Clinical Studies\u2014New Bolton Center, University of Pennsylvania School of Veterinary Medicine , Kennett Square, PA 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Kennett Square, PA ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Okada","sequence":"additional","affiliation":[{"name":"Department of Clinical Studies\u2014New Bolton Center, University of Pennsylvania School of Veterinary Medicine , Kennett Square, PA ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebeca","family":"Santos","sequence":"additional","affiliation":[{"name":"Department of Clinical Studies\u2014New Bolton Center, University of Pennsylvania School of Veterinary Medicine , Kennett Square, PA ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrin","family":"Hinrichs","sequence":"additional","affiliation":[{"name":"Department of Clinical Studies\u2014New Bolton Center, University of Pennsylvania School of Veterinary Medicine , Kennett Square, PA 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