马氏珠母贝精子质量检测方法的的研究

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1、同的冷冻保存体积对马氏珠母贝精子存活率的影响，结果显示，体积过大或过小都会影响精子的存活率，冷冻时最佳的保存体积为1．2一1．6mL。3、吖啶橙(AcridineOrange，AO)染色法检测马氏珠母贝精子DNA的完整性本文用AO染色来检测马氏珠母贝精子DNA的完整性。结果显示，精液经AO染色后，在荧光显微镜下可观察到发出绿色荧光、红色荧光或橘黄色荧光的精子，其中发绿色荧光的精子DNA双链结构完整，发红色或橘黄色荧光的精子DNA双链已受到不同程度的损伤，DNA双链结构已断裂为单链。通过不同染色浓度和时间的比较

2、发现，最适的AO染色浓度为200ug／mL，染色时间为5min。在此基础上，用AO染色法检测评价超低温冷冻前后精子DNA的完整性。结果显示，冷冻对精子的DNA双链结构会有不同程度的损伤。关键词：马氏珠母贝，精子，存活率，质膜完整性，DNA完整性nStudyontheMethodsforDetectingtheSpermQuality磺PinctadamartensiiAbstractInthispaper,thesemenofPinctadamartensiiwasusedasthemodelforstudy

3、ingitsviabilityrate，membraneandDNAintegritywithtyballblueandfluorescentstainingthroughamicroscope．TheseCanofferbasicdataforbuildingitsspermqualitydetectionandevaluation．1．StudyonspermsviabilityrateofPmartensiiwithtypanbluestainingThespermsofPmartensiiwerest

4、ainedwithtypanblue，whichappearedobviousdifferencebetweenthedeadandlivesperms．Thelivespermswerenotstained，whilethedeadoneswerestainedbytypanblueandthesizeofdeadoneswasbiggerthanthelives．Thenwecomparedifferentconditiondyeconcentrationanddyingtime，thebestcondi

5、tionwasthat0。2％trypanblueunderstainingl5min．Then，thespermswerekilledat60。C(waterbath)weremixedthefleshonestoobtaindifferentrations(fordeadsperms)concentrations(10％、20％、30％、40％、50％、60％、70％、80％、90％)，whichwereidentifiedastheoreticalmortalityanddetectedwith0。2％

6、typanblueand0．04％esionrTheresultsshowedthatthereWassignificantcorrelationbetweentheoreticalmortalityandpracticalmortality(P<0．01)，andtherewasapositivecorrelationbetweentwostainingmethods(尸≮0．01)．Finally，Wemadeatrialaboutthetoxicityofdyeandthenaturalmortalit

7、yatroomtemperature(28℃)．Theresultindicatedthatthetoxicityofthesedyewillaffecttheviabilityrateofspermswhentheyreachedacertainlevel．But，thereWasnoremarkabletoxiceffectonviabilityrateofspermswithin45min．Inconclusion，theoptimumdyeconcentrationwas0．2％andstaining

8、timewas15min．TybanbluestainingWasavaluabletooltoassessingthespermsofPmartensii．2．ReseacrhonspermsviabilityrateandmembraneintegrityofPmartensiiwithacombinationoffluorometricstainingWeinvestigatedthecomb