Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation of cholesterol efflux from the cell membrane, was examined for its ability to increase the cryosurvival of spermatozoa from Göttingen miniature pigs. The intactness of the acrosome and various motion parameters of spermatozoa after freezing and thawing were used to monitor cryosurvival. Spermatozoa were exposed to MBCD or a combination of MBCD and cholesterol-3-sulfate over a period of 3 hours while being cooled slowly from 25 degrees C to 5 degrees C, and were subsequently cryopreserved by the pellet method. After freezing and thawing, the acrosomal status was monitored by fluorescein isothiocyanate-labeled peanut agglutinin, and the motion parameters were assessed with a computer-assisted sperm motility analysis system. In post-thaw spermatozoa the values of the intact acrosome, motility, progressive motility, progressive velocity, straightness, and linearity of the cell path increased greatly with the concentration of MBCD (P < .05). In contrast, the lateral head displacement amplitude and the beat cross-frequency of post-thaw spermatozoa were not different among all treatments. On the contrary, the addition of cholesterol-3-sulfate to the diluent containing MBCD abolished the protective effect against freeze-thaw injury that MBCD provides to spermatozoa. These results indicate that cryosurvival of boar spermatozoa is enhanced by exposure to MBCD before freezing. This protective effect of MBCD may be due to active depletion of sperm membrane cholesterol.