栉江珧和近江牡蛎的精子超低温冷冻保存  

Cryopreservation of Spermatozoa in Pen Shell(Atrina pectinata) and Suminoe Oyster(Crassostrea rivularis)

作　　者：于非非[1] 余祥勇[1] 郑娇[1] 曹静[1] 王梅芳[1] 

机构地区：[1]广东海洋大学水产学院,湛江524088

出　　处：《农业生物技术学报》2016年第2期305-312,共8页Journal of Agricultural Biotechnology

基　　金：广东海洋大学优秀青年骨干教师培训项目(No.2014004);广东省海洋渔业科技推广专项项目(No.A201308A11)

摘　　要：栉江珧（Atrina pectinata）和近江牡蛎（Crassostrea rivularis）是我国重要的海产经济贝类,建立其精子冷冻保存技术及精子冷冻库,对种质资源的保护和遗传育种具有重要意义。本研究对其精子冷冻保存过程中冷冻剂种类和浓度的选择、冷冻程序、平衡时间、保存体积、精液稀释方式、保存时间等多个影响因素进行了比较筛选,并利用显微镜直接检测和曙红Y（eosin Y）染色检测结合的方式检验精子存活率。结果表明,二甲亚砜（dimethyl sulfoxide,DMSO）作为抗冻剂的保存效果要明显优于甘油（glycerol,Gly）,终浓度为8%~10%的二甲亚砜保存效果最好,其栉江珧和近江牡蛎精子活率均可达60%以上,而甘油保护下精子活率小于40%。精液的稀释方式采用将抗冻液等分为3份,依次与精液混合,每次间隔8 min,整个过程约30 min;此种精液稀释方式与一次性混合方式相比,精子活率可提高约30%。稀释后精液需要在4℃下平衡15~25 min,平衡样品与直接冷冻样品相比,精子活率提高约50%。精液降温程序为在样品投入液氮前,于液氮面以上15 cm处停留5 min,5 cm处停留10 min,精子活率可达60%以上;而未经此降温程序直接投入液氮中的样品精子活率接近0。与此降温程序相对应的样品冷冻体积在1.0~1.4 m L范围内为宜。冷冻180 d内复苏的精子活率由90%左右（鲜精）下降至60%左右,180 d后精子活率下降至56%左右。研究结果为栉江珧和近江牡蛎精子超低温冷冻保存、双壳类种质保护和遗传育种提供了理论和技术依据。Atrina pectinata and Crassostrea rivularis are important marine economic shellfish,but their output is declining sharply due to overfishing,disorderly use and environmental deterioration. The establishment of sperm cryobank and cryopreservation technology was significant to their germplasm resources preservation and genetic breeding. In this study,in order to establish a technology of sperm cryopreservation suitable for Atrina pectinata and Crassostrea rivularis,the series of selecting experiments for type and concentration of cryoprotectant,freezing procedure,balance time,sample volume,sperm dilution method and freezing time were designed and compared. The sperm survival rate was detected by both direct microscopic examination and eosin Y staining detection. The data indicated that the protection of dimethyl sulfoxide（DMSO） to sperm was obviously superior to glycerol（Gly）. The sperm survival rates of both Atrina pectinata and Crassostrea rivularis were up to 60% with the protection of DMSO,while only 40% or so with the protection of Gly. The terminal concentration of DMSO was 8%,which was suitable for Atrina pectinata,and 10% suitable for Crassostrea rivularis. The cryoprotectant was divided into 3 parts and added to sperm by 3 times,interval 8 min every time. Using this dilution method,the sperm survival rate was enhanced about 30% than that using oncemix method. The precooling of samples for 15~25 min at 4 ℃ was necessary and could increase sperm survival rate by about 50% compared with direct freezing method. The optimum freezing procedure was firstly to prefreeze for 5 min at 15 cm above liquid nitrogen level,then to prefreeze for 10 min at 5 cm above liquid nitrogen level,and to freeze in liquid nitrogen at last. The sperm survival rate was more than 60% with this freezing procedure,while near to zero without any freezing procedure. Under this freezing procedure,the volume of sample between 1.0 to 1.4 m L could make the sperm survival rate of 60% or so,and less than 1.0m L or more than 1.8 m L wou

关 键 词：栉江珧 近江牡蛎 精子 冷冻保存 

分 类 号：Q492[生物学—生理学]