Technical Abstract: High fertility using cryopreserved ram sperm is currently only achieved using laparoscopic intrauterine insemination. Improving the cryosurvival of ram sperm may permit higher fertility rates using more practical techniques. This study was conducted to determine if treating ram sperm with different cyclodextrins pre-loaded with cholesterol (CLC), prior to cryopreservation increases sperm cryosurvival and if this technology can be used with neat semen. Prior to cryopreservation, sperm were treated with six different cyclodextrins pre-loaded with cholesterol. Sperm samples treated with 2-hydroxypropyl-beta-cyclodextrin resulted in higher percentages of motile sperm (62%) compared to the control (no CLC treatment) samples (43%, P < 0.05). In addition, samples treated with methyl-beta-cyclodextrin exhibited similar percentages of motile and viable sperm as samples treated with 2-hydroxypropyl-beta-cyclodextrin. Other CLC treated samples were similar to control. The CLC concentration that optimized sperm cryosurvival was 2 mg CLC/120 x 106 sperm for both methyl-beta- and 2-hydroxypropyl-beta-cyclodextrin. Addition of 2 mg cholesterol-loaded methyl-beta- and 2-hydroxypropyl-beta-cyclodextrin/120 x 106 sperm to neat ejaculates prior to cryopreservation resulted in higher percentages of total motile sperm (42 and 50%, respectively), compared to the control samples (32%; P < 0.05) and higher percentages of viable sperm for samples treated with 2-hydroxypropyl-beta-cyclodextrin pre-loaded with cholesterol (33%), compared to the control sperm (18%; P < 0.05). In conclusion, CLC treatment prior to cryopreservation improves the cryosurvival of ram sperm. The CLCs can be added to neat semen, making this technology feasible for practical application using current ovine cryopreservation techniques.