animals, treating human infertility, and further developing reproductive techniques, such as artificial insemination(AI), in vitro fertilization(IVF) and embryo transplantation(ET). The first attempt to cryopreserve spermatozoa of nonhuman primate (NHP) was made more than 30 years ago, and up to now sperm of 15 primate species out of more than 200 NHP in the world have been frozen and only five species (i.e. gorillar, chimpanzee, cynomolgus monkey, marmoset and rhesus monkey) have been documented to produce offspring when frozen-thawed spermatozoa were artificially inseminated so far. As few knowledge has been obtained on sperm cryobiology of NHP and the mechanisms of sperm cryo-injury as well as cryoprotection by some compounds are lacking, post-thaw sperm survival and fertility decrease dramatically, which will in turn cause a low rate of artificial insemination in frozen-thawed spermatozoa. In order to probe into the mechanisms of sperm cryo-injury and improve the fertility of cryopreserved sperm, factors influencing the efficiency of sperm cryopreservation were studied using cynomolgus and rhesus monkey as experimental models. Sperm motility, plasma membrane and acrosomal integrity were examined to evaluate the cryopreserved sperm function. The present study included:!) Effect of amino acids on cryopreservation of cynomolgus monkey sperm; 2) Cryopreservation of cynomolgus monkey spermatozoa in a chemically defined extender; 3) Effects of various extenders and permeating cryoprotectants on cryopreservation of cynomolgus monkey spermatozoa; 4) Comparative studies with six extenders for sperm cryopreservation in cynomolgus monkey and rhesus monkey. The results showed that: 1) Adding a limited amount of amino acids to the extenders is beneficial for freezing cynomolgus monkey sperm but higher concentration of amino acid had negative effects on sperm freezing; 2) cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender. 5% glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity; 3) TTE, DM, mDM, LG-DM, G-DM and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation. The mechanism of action of permeating cryoprotectants are not affected by extender composition. Ethylene glycol has a similar cryoprotective efficacy to glycerol; 4) TTE, DM, mDM, LG-DM, G-DM and TCG are equally suitable for cryopreservation of spermatozoa from cynomolgus and rhesus monkeys. These results will be beneficial for increasing the efficiency of sperm cryopreservation in NHP by improving sperm freezing procedures. Additionally, the results will also be helpful for researchers to understand and study the mechanisms of sperm cryo-injury as well as cryoprotection given by some compounds. This work will contribute some information to the area of cryobiology and bio-diversity preservation.
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