The Cryopreservation Investigation On Mouse Microamount Round Spermatids From In Vitro Culture

Part ⅠThe cryopreservation investigation on mousemicroamount round spermatids from in vitro cultureObjective：To explore the optimal cryopreservation protocol, freezing condition ofmicroamount round spermatids from spermatogenic cells in vitro culture Methods:Spermatids motility by vitrification and standard slow freezing with different glycerolconcentrations (5%,7%and9%) and equilibrium times (0,15,30,45and60min) wereevaluated respectively after thawing. Results:(1) The optimal spermatids motilitycould be obtained with both vitrification and standard slow freezing under thecondition of7%glycerol concentration and equilibrium time of30min.(2) Spermatidsmotility of vitrification were significantly higher than that of standard slow freezing（P＜0.05）. Conclusion: The optimal frozen-thawed result of microamount roundspermatids could be obtained with vitrification under the condition of7%glycerolconcentration and equilibrium time of30min.PartⅡ The application value of frozen-thawed mouse roundspermatids from in vitro cultureObjective：To investigate the application value of frozen-thawed mouse round spermatids from in vitro culture on in vitro fertilization. Methods: The frozen-thawedround spermatids were stained with DAPI，then round spermatid injections（ROSI）were performed. The procedures of fertilization and embryonic development wereobserved.Results: DAPI labeling rate of frozen-thawed round spermatids was100%.After ROSI, DAPI fluorescence signals were observed in80.0%male pronucleus and77.3%two cell embryo respectively,and the rates of fertilization, two cell embryo,four-eight cell embryo, the morula and blastocyst were51.9%、51.0%、41.8%and12.2%respectively..Conclusion: The frozen-thawed round spermatids possessfertilization capacity and participate in the development of embryo.