Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival

Acceptable fertility using cryopreserved ram sperm is currently only achieved using laparoscopic intrauterine insemination. Improving the cryosurvival of ram sperm may permit greater fertility rates using more practical techniques. This study was conducted to determine if treating ram sperm with six different cyclodextrins pre-loaded with cholesterol (CLC), prior to cryopreservation increases sperm cryosurvival and if this technology can be used with neat semen. Subsequent experiments evaluated how adding CLC to sperm affected sperm cholesterol content, sperm osmotic tolerance limits, sperm post-thaw survival after incubation and the capacity of sperm to bind to zona pellucidae of cattle and sheep oocytes. Sperm treated with 2-hydroxypropyl-β-cyclodextrin prior to cryopreservation exhibited greater percentages of motile sperm (62%) compared to the control (no CLC treatment) samples (43%, P <0.05), after thawing. In addition, samples treated with methyl-β-cyclodextrin exhibited percentages of motile and viable sperm similar to samples treated with 2-hydroxypropyl-β-cyclodextrin. Other CLC-treated samples were similar to the control. The CLC concentration that optimized sperm cryosurvival was 2mg CLC/120×10⁶ sperm for both methyl-β- and 2-hydroxypropyl-β-cyclodextrin when added to neat semen prior to cryopreservation. Addition of 2mg CLC not only maintained greater percentages of motile sperm compared to the control samples, but maintained greater percentages of motile sperm during a 3h incubation after thawing. In addition, 2-hydroxypropyl-β-cyclodextrin pre-loaded with cholesterol maintained greater percentages of viable sperm (33%), than control sperm (18%; P <0.05). Treating ram sperm with CLC increased the sperm cholesterol content>1.9-fold and although some cholesterol was lost from the sperm during cooling and cryopreservation, the cholesterol content remained greater in CLC-treated sperm after cooling and after thawing than in control sperm (P <0.05). In addition, CLC-treated sperm maintained greater percentages of motile sperm through a wide range of osmotic solutions (150 and 425mOsm) while control sperm lost motility in solutions outside a more narrow range (270 to 370mOsm). Greater numbers of CLC-treated sperm bound to zona pellucida than control sperm (P <0.05), although number of sperm binding cattle and sheep oocytes, was similar (P >0.05). In conclusion, treating ram sperm with CLC increases sperm cryosurvival rates and sperm longevity after thawing. It also increases the cholesterol content, osmotic tolerance, and zona-binding capabilities of sperm. Finally, CLCs can be added to neat semen, making this technology feasible for practical application using current cryopreservation techniques for ram semen.