Using Cholesterol-Loaded Cyclodextrin to Enhance Cryo-Survivability and Reduce Cryo-Capacitation of Bovine Semen Frozen over Static Liquid Nitrogen Vapor

This study evaluated the use of cholesterol-loaded cyclodextrin (CLC) to mitigate cryo-damage, aiming to enhance sperm cryo-survivability and reduce cryo-capacitation. Different CLC levels were tested, and post-thaw motility was higher (p < 0.05) at 1.5-2 mg/mL CLC added to extended semen containing 120 million sperm/mL. Subsequent investigations compared untreated and 2 mg/mL CLC-treated bovine sperm (n=5) and assessed membrane and acrosome integrity, mitochondrial potential, and capacitation status using flow cytometry. Moreover, this study is the first to quantify the post-thaw binding ability of CLC-treated bovine sperm and evaluate their capacitation window using an IVF approach. CLC-treated sperm showed no differences in flow cytometric results. CLC treatment significantly (p < 0.0001) increased the number of viable bound sperm cells to oviduct cells (1.65 × 10⁶ µm²) from 118 ± 15 to 267 ± 15 after three hours, and from 70 ± 15 to 127 ± 15 after seven hours (p < 0.05) of coincubation. When equal numbers of motile sperm were incubated with oocytes, CLC-treated sperm showed penetration and pronuclei formation rates comparable to the control, indicating a similar capacitation window. In conclusion, CLC addition significantly improved post-thaw sperm motility and binding ability, while maintaining sperm capacitation and fertilizing ability.