| Guppies of Poecilia reticulata are not only known as famous aquarium fish, but also are important behavioral research models. As one species of live-bearing fish, it has special reproduction mode that adapted to its function. Despite the study of sperm cryopreservation in some 200 species of freshwater and marine fishes, sperm cryopreservation has just been initiated in live-bearing fishes as a group, for examples, with Xiphophorus helleri and X. couchianus. However, sperm cryopreservation is essentially unexplored in Poecilia. The goal of this study was to develop practical techniques for guppy sperm cryopreservation, and areas of investigation included: 1)basic parameters for sperm collection and processing; 2)evaluation of optimal conditions for refrigerated storage; 3)optimization of cryopreservation procedures; 4) establishment of method for quick determination of sperm concentration; and 5)development of artificial insemination with cryopreserved sperm.Studies of basic parameters for sperm collection and processing suggested: 1)Testis weight had significant linear relationships with standard length, body wet weight and the number of sperm cells, but not sperm density; 2)Intact testis could be stored at 4℃for＞96 h without significant reduction of sperm motility, and sperm had the highest motility in isotonic solutions(～305 mOsmol/kg)and remained motile in solutions with osmolalities of between 200 and 430mOsmol/kg, however, hypotonic or hypertonic solutions beyond this range lead to sperm immobilization; 3)Sperm collection at either 25℃or 4℃had no effect on the duration of refrigerated storage; 4)Sperm collected by cutting testis had significant higher motility than sperm released by crushing testis; 5)pH had a significant effect on sperm motility during refrigerated storage; 6)Dilution ratios of 1:50 to 1:500(sperm to HBSS)yielded no significant difference on sperm motility; 7)Different storage volumes in 1.5ml tubes were found to affect sperm motility significantly. Based on these findings, the highest motility of refrigerator storage occurred when sperm were suspended in HBSS at 300 mOsmol/kg with pH of 5.0 to 8.0, a dilution ratio of 1:200(sperm to HBSS), and a loading volume of between 100 to 500μl in 1.5ml centrifuge tubes,Major findings of studies of sperm cryopreservation included: 1)The post-thaw motility of samples cryopreserved with DMSO, methanol and DMF varied with cryoprotectant concentration, but stayed constant for glycerol in the tested range of 5 to 20％; 2)Equilibration time of 20 min or less yieIded the highest post-thaw motility; 3)Reasonable post-thaw motility(16±9％)could be obtained with samples cryopreserved with DMSO(5, 10 and 15％), methanol(2, 6 and 10％)and DMF(2, 5 and 8％), but the highest was with glycerol of 5, 12 and 20％; 4)Addition of nonpermeating cryoprotectants such as sucrose did not show any significant improvement on post-thaw motility of guppy sperm; 5)Cooling rates of 15-35℃/min from 4 to -80℃had no significant difference on post-thaw motility; 6)Sample loading volume in 0.25ml straws had no significant difference on post-thaw motility; 7)Dilution ratio of 1:20(sperm to HBSS)before freezing yielded the best post-thaw motility, but final sperm-to-extender ratios of 1:20～1:120 with single(before freezing)and second dilution(after thawing)had no significant difference on post-thaw motility. Overall, the highest post-thaw motility occurred when sperm were suspended in HBSS at 300 mOsm/kg with 14％glycerol with a dilution ratio of 1:20(weight to volume; mg:ml), equilibrated for 10min in 120-μl aliquots in 0.25-ml French straws, cooled at -25℃/min from 4 to -80℃, helding for 5min at -80℃before plunging into liquid nitrogen, and thawed at 40℃in a water bath for 7 s.A spectrophotometric device, namely enzyme-labeled instrument, was used for rapid estimation of sperm concentration. Wavelengths of 405, 450, 490 and 630nm were compared, and 405 and 450nm were found to be the most sensitive. A linear relationship between sperm concentration and photometric absorbance was observed for sperm concentrations between 6×10~5 and 4×10~8 cell/ml, but its reliability needs further validation. Artificial insemination of thawed sperm with virgin females produced live offsprings when sperm were washed and concentrated to 2×10~9 cell/ml with centrifugation at 2000×g for 10min.
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