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Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm cryosurvival¹

Department of Biomedical Sciences, Colorado State University, Fort Collins 80523

² Corresponding author: jkgraham{at}colostate.edu

Damage occurring to spermatozoa during cryopreservation results in a loss of motile cells and cells that are functionally normal, compared with fresh sperm samples. Treating bull sperm with cholesterol-loaded cyclodextrins (CLC) before cryopreservation results in increased sperm cryosurvival. However, in previous studies, CLC were always added to sperm samples that had been highly diluted. The aim of this study was to develop a procedure for adding CLC to whole bull ejaculates that would optimize sperm cryosurvival. Adding 2 or 4 mg of CLC/120 x 10⁶ sperm to sperm samples ranging in concentration from 120 to 2,000 x 10⁶ sperm/mL resulted in greater (17 to 28 percentage points; P < 05) numbers of live cells compared with control samples (no CLC treatment), regardless of the sperm concentration, except for samples at 120 x 10⁶ sperm/mL treated with 4 mg of CLC. Incubating sperm with CLC at 23 or 37°C before cryopreservation resulted in similar sperm cryosurvival. The cooling rate used to cryopreserve CLC-treated cells did not affect sperm cryosurvival. Finally, adding CLC to undiluted ejaculates (2 mg of CLC/120 x 10⁶ sperm) resulted in greater percentages of live sperm compared with the control samples (62 vs. 45%; P < 0.05), although the percentages of motile sperm were similar for both CLC-treated and control samples (58%). In conclusion, bull sperm cryo-survival can be improved if spermatozoa are treated with CLC before freezing. In addition, CLC can be added to fresh ejaculates at either 23 or 37°C. This technique is simple, practical, and can be easily integrated into current cryopreservation protocols.

Key Words: bull sperm • cholesterol • cryopreservation • cyclodextrin • freezing rate

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