Study On Cryopreservation Of Spermatoza And Methods For Detecting The Sperm Quality Of Atrina Pectinat

Dimethyl sulphoxyde(DMSO), glycerol(Gly), Sucrose were used as antifreezeprotective agent to freeze the sperms of Atrina pectinat. The objective of research is try toestablish basic procedure of freezing Atrina pectinat sperms.Fluorescent dye was used tostain the sperm of Atrina pectinat to its membrane intergrity and mitochondrial activity.Sowe can offer basic data for building its sperm quality detection and evaluation1Study on Cryopreservation of Spermatoza in Atrina pectinatThe spermatozoa were obtained from the Atrina pectinat gonad by dissection.Thedimethyl sulphoxyde (DMSO)、glycerol(Gly)、Soucrose melted by filtrated seawater wasused as cryoprotectant. We added different rypes, different concentration of cryoprotectantto mature sperms.Than the specimens were transferred into LN2by4temperature loweringprocesses. And, we compared the effects of mortality with different concentration ofDMSO、volume、equilibration times and thawing method after freezing-thawing.Theconclusion showed that the concentration of cryoprotectants and the volume of samplesand the ttime of prefreezing and the thawing method were related to the survival rate ofcryopreserved spermatozoa.The effects of cryoprotectants is DMSO better than glycerol,DMSO mixed with soucrose, and the optimum concentration of DMSO is8~10%. Thevolume of samples between1.0to1.5mL is suitable for cryopresering spermatozoa ofAtrina pectinat.The optimal time of prefreezing is15minutes at0℃.The optimum thawingtemperature is38~40℃.2Research on membrane integrity of Atrina pectinat sperms with a combination offluorometric stainingWe investigated the combination of Fluorescein diacetate(FDA) and Propidiumiodide(PI) to study membrane integrity.We found that optimal concentration of FDA and PIwas10μg/mL and the suitable staining time was10min.Fresh sperms were mixed withthose sperms which were killed at60℃(water bath) to obtain nine rations (for dead sperms)concentrations (10%,20%,30%,40%,50%,60%,70%,80%,90%),which were used astheoretical mortality and detected with FDA and PI staining. Results indicated that deadsperms could be discriminated from live ones by FDA and PI. Dead sperms gave out redfluorescence, live ones glowed green fluorescence. Furthurmore, correlation did existedbetween practicalmortality and theoretical of sperms(P<0.01). Also the membrane integritysperm after cryopreservation was detected by FDA-PI staining, the results showed that the mortality remarkably decreased after freezing-thawing(P<0.01) and the membrane wasdamaged.3Research on mitochondrial activity of Atrina pectinat sperms with a combination offluorometric stainingTwo fluorescent dyes, Rhodaminel23(Rh123) and Propidium iodide (PI), were usedto detect the mitochondrial activity of the spermatozoa of Atrina pectinata, and the testingeffect of different staining time and dye concentration were also compared, and themitochondrial activity of sperm after cryopreservation (frozen in liquid nitrogen for24h)was detected. The result showed that the most effective staining appeared when theconcentration of Rhodaminel23was10μg/mL, and the optimum reaction time was10min.The activity of mitochondria sperm glowed green fluorescence, and dead sperm shined redfluorescence, and sperm with damaged plasmalemma and undamaged mitochondria gaveout red-green fluorescence. Therefore, Rh123-PI double staining was feasible. Frozensperm’s mitochondrial activity and plasma membrane integrity was61.8%,56.8%,respectively, and was lower than fresh sperm (95.3%,93.5%). The result indicated that thefreezing-thawing process damaged sperm structure and Rh123-PI double staining coulddetect the quality of the spermatozoa of Atrina pectinata.