三角帆蚌和褶纹冠蚌的精子冷冻保存方法建立及其应用  

DEVELOPMENT AND APPLICATION OF CRYOPRESERVATION METHODS FOR SPERM OF HYRIOPSIS CUMINGII AND CRISTARIA PLICATA

作　　者：李长志 吴玉扬 王贺 白志毅[1,2,3,4] LI Chang-Zhi;WU Yu-Yang;WANG He;BAI Zhi-Yi(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;Zhejiang Wuyi Freshwater Pearl Science and Technology Yard,Shanghai Ocean University,Jinhua 321200,China;Shanghai Aquaculture Engineering Research Center,Shanghai Ocean University,Shanghai 201306,China;Shanghai Collaborative Innovation Center of Aquatic Animal Breeding and Green Aquaculture,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学、农业农村部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学浙江武义淡水珍珠科技小院,浙江金华321200 [3]上海海洋大学、上海市水产养殖工程技术研究中心,上海201306 [4]上海海洋大学、上海市水产动物良种创制与绿色养殖协同创新中心,上海201306

出　　处：《海洋与湖沼》2025年第2期413-422,共10页Oceanologia Et Limnologia Sinica

基　　金：国家重点研发计划,2022YFD2400105号;国家现代农业产业技术体系,CARS-49号。

摘　　要：三角帆蚌和褶纹冠蚌均为我国主要淡水育珠蚌,其所产珍珠占世界珍珠总产量的90%以上,建立两种蚌精子冷冻保存技术,对种质资源保护和遗传育种具有重要意义。比较优化冷冻保护剂、平衡时间、精子包装、精子稀释剂及降温程序等关键技术参数,以精子活力和存活率作为保存效果评价指标,建立了三角帆蚌与褶纹冠蚌精子最优冷冻保存技术方案。结果表明,二甲基亚砜(DMSO)在5%和8%浓度下分别对两种蚌精子具有最佳保护效果,添加0.05 mol/L海藻糖保护效果显著提升;0.5 mL麦管为两种蚌精子的最适容器,优于0.25 mL冷冻麦管及0.5 mL的冻存管;平衡50 min为三角帆蚌精子在5%DMSO浓度下最佳平衡时间,而褶纹冠蚌则在8%DMSO平衡10~30 min表现最佳。D溶液(稀释10倍的HBSS溶液)作为两种蚌精液稀释剂的效果均优于生理盐水和任氏液,并发现过滤后的养殖池塘水对褶纹冠蚌精子稀释效果与D溶液相当。通过对比6种降温程序,确定三角帆蚌精子保存最优降温程序为:4℃平衡30 min,-7℃/min降至-30℃,再平衡2 min,-12℃/min降至-80℃,最后平衡2 min后迅速浸入液氮中;褶纹冠蚌精子保存最优降温程序为:4℃平衡30 min,-7℃/min降至-80℃,平衡2 min后迅速丢入液氮。两种蚌冷冻精子在40℃水浴中解冻复苏8 s均获得最佳活力和存活率,三角帆蚌复苏精子最佳活力和存活率分别为28.57%±2.02%和71.64%±3.04%,褶纹冠蚌复苏精子最佳活力和存活率分别为33.38%±3.80%和64.04%±2.40%。利用冻存后的三角帆蚌精子进行人工受精实验,受精率达43.93%±3.40%。该研究创新了蚌精子冷冻保存方法,助力杂交育种及现代生物技术发展。Hyriopsis cumingii and Cristaria plicata are the primary freshwater pearl mussels in China accounting for over 90%of the global pearl production.Developing sperm cryopreservation techniques for these is essential for germplasm conservation and genetic breeding.This study optimized cryopreservation techniques for both species by comparing key parameters such as cryoprotectants,equilibration times,packaging,extenders,and cooling procedures,using sperm motility and survival rates as evaluation indicators.Optimal conditions included 5%and 8%DMSO for H.cumingii and C.plicata sperm,respectively,supplemented with 0.05 mol/L trehalose.The 0.5 mL straw was identified as the optimal container for both species,outperforming 0.25 mL straws and 0.5 mL cryovials.For H.cumingii,the ideal equilibration time was 50 minutes with 5%DMSO,whereas C.plicata showed the best performance with 8%DMSO and an equilibration time of 10 to 30 minutes.Solution D(10-fold dilution of HBSS)outperformed physiological saline and Ringer's solution as a sperm extender,with filtered pond water showing comparable results for C.plicata.Comparison of six cooling protocols identified the optimal cryopreservation procedure for H.cumingii sperm as follows:equilibration at 4℃for 30 minutes,cooling at-7℃/min to-30℃,holding for 2 minutes,followed by cooling at-12℃/min to-80℃,holding for another 2 minutes,and finally plunging into liquid nitrogen.For C.plicata sperm,the best protocol involved equilibration at 4℃for 30 minutes,cooling at-7℃/min to-80℃,holding for 2 minutes,and plunging into liquid nitrogen.Both species achieved the highest motility and survival rates when thawed in a 40℃water bath for 8 seconds.Post-thaw motility and survival rates were 28.57%±2.02%and 71.64%±3.04%for H.cumingii,and 33.38%±3.80%and 64.04%±2.40%for C.plicata,respectively.Artificial fertilization experiments using cryopreserved H.cumingii sperm resulted in a fertilization rate of 43.93%±3.40%.These findings represent a significant advancement in sperm cryopre

关 键 词：三角帆蚌 褶纹冠蚌 精子冷冻保存 人工授精 

分 类 号：S917.4[农业科学—水产科学] Q492[生物学—生理学] S966.2