Sperm cryopreservation has provided an effective way for human sterility cure, livestock breeding and biodiversity preservation, since Polge et al (1949) found the cryoprotective effect of glycerol and set up the milestone of cryobiology. However, due to the lack of basic knowledge about reproductive biology and cryobiology of most species, the study of sperm cryopreservation is empirically depended. Even with the most modern freezing protocol and equipments, only about 50% spermatozoa can survive after freezing and thawing. By now, only one offspring of rhesus monkey has been obtained using cryopreserved spermatozoa. The difficulty of cryopreserving nonhuman primate spermatozoa may be due to their fragility. In order to improve the fertility of cryopreserved sperm, factors affecting sperm cryo-survival were studied with rhesus monkey as experimental animal. Sperm motility, plasma membrane integrity, acrosome integrity and in-vitro fertilization were used as functional assessment for frozen-thawed spermatozoa. The present study included: 1) Suitable freezing medium and freezing protocol for rhesus monkey spermatozoa cryopreservation; 2) Effects of different glycerol and dimethyl suifoxide levels on the sperm cryopreservation; 3) Effects of four penetrating cryoprotectants: glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol on the sperm cryo-survival and function; 4) Cryoprotective roles of monosaccharide, disaccharide and trisaccharide. The Results showed that: 1) rhesus monkey spermatozoa could be successful cryopreservation with TTE; 2) five percent of glycerol is suitable for rhesus monkey sperm freezing; 3) the cryoprotective effects of various penetrating cryoprotectants are different. Dimethyl sulfoxide and propylene glycol are not suitable for rhesus monkey sperm freezing, especially propylene glycol. Ethylene glycol with similar cryoprotective properties to glycerol could be successfully used in the cryopreservation of rheusus monkey spermatozoa; and 4) the cryoprotective effects of various sugar types are different, and combination using of various types of sugar at proper concentration is better than that using one type of sugar alone. The results will be beneficial for spermatozoa cryopreservation of non-human primate and improving the cryo-survival of cryopreserved sperm.
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