【作者】 于海涛；

【导师】 张秀梅； 李军；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2004， 硕士

【摘要】 本研究对花鲈和红鳍东方鲀的精子以及杂色蛤、太平洋牡蛎的胚胎及幼虫进行了超低温保存实验，确定了其最佳保存条件和方法，并对造成精子超低温冷冻损伤的原因进行了探讨。 对花鲈(Lateolabrax japonicus)精子的超低温冷冻研究发现，采用15％甘油+5％蔗糖+5％葡萄糖的抗冻液配方，冻精复活率可达到86.5％；离体精子活率在前2h内下降缓慢，3h后活率下降为50.0％；甘油的最佳添加浓度为15％；DMSO的最佳添加浓度为10％；甘油对精子的保护效果好于DMSO；快速解冻法好于慢速解冻法。 对超低温冷冻前后的红鳍东方鲀(Takifugu rubripes)精子超微结构的观察发现，受冷冻损伤精子的被膜膨胀、破裂、脱落；线粒体内嵴变形，结构模糊，甚至脱落；鞭毛在基部或主段、末段断裂，或被膜膨胀脱落。结果表明，经超低温保存后，膜结构损伤、线粒体脱落和鞭毛轴丝断裂是导致红鳍东方鲀精子活力下降的主要原因。 对杂色蛤(Ruditapes variegata)胚胎和幼虫的超低温冷冻保存发现，杂色蛤囊胚和面盘幼虫的保存复活率显著高于担轮幼虫和D形幼虫的复活率(P＜0.05)；杂色蛤囊胚的解冻复活率达42.3％(4.0M DMSO)，相同DMSO浓度条件下，高蔗糖浓度组保存效果好于低蔗糖浓度组，但差异不显著(P＞0.05)；在相同蔗糖浓度条件下，抗冻液中DMSO浓度较高组的保存效果好于DMSO浓度较低的组，但差异不显著(P＞0.05)。 对太平洋牡蛎(Crassostrea gigas)的胚胎和幼虫的超低温冷冻保存发现，担轮幼虫的解冻复活率最高，达61.1％(3.0M DMSO)，显著高于其他发育阶段幼虫的保存结果(P＜O.05)；牡蛎担轮幼虫的保存结果显示，在相同DMSO浓度条件下，高蔗糖浓度组保存效果好于低蔗糖浓度组，但差异不显著(P＞0.05)。更多还原

【Abstract】 In the present paper, to develop simple, reliable and efficient methods for Cryopreservation are reported.Survival rate of 86.5% was obtained in Cryopreservation of the spermatozoa of seabass Lateolabrax japonicus. The prescription of the best antifreeze solution are glycerol 15%+ sucrose 5%+ glucose5%. The motility of fresh spermatozoa descends slowly within the former 2 hours and it is 50.0% at the third hour. The better density of glycerol and DMSO are 15% and 10%. The antifreeze effect of glycerol is better than that of DMSO. The effect of quick thawing is significantly better than that of slow thawing.The ultrastructure of Takifugu rubripes spermatozoa before and after Cryopreservation was studied by means of ultrathin-section TEM technique. The results indicate that cryodamage may cause the membrane of sperm head swelling or disintegrating, the abnormality of the head, the transmutation of mitochondria, the loss of mitochondria.After Cryopreservation of embryo and larvae of manina clam Ruditapes variegata, the results indicate the survival rate of blastula embryo and vlegier are significantly better than that of trochophore and D-shaped larvae(P<0.05); the survival rate of blastula embryo is 42.3% (4.0M DMSO) and the effect of high sucrose density is better than that of low sucrose density with the same DMSO dendity.After Cryopreservation of embryo and larvae of pacific oyster Crassostrea gigas, the results indicate the survival rate of trochophore larvae is 61.1% and significantly better than that of other stage embryo or larvae(P<0.05).The effect of high sucrose density is better than that of low sucrose density with the same DMSO density.更多还原