稀有鮈鲫精子超低温冷冻保存  

SPERM CRYOPRESERVATION OF GOBIOCYPRIS RARUS

作　　者：李鑫 叶欢[1] 何勇凤[3] 余乐 程佩琳 杜浩[1] 吴金明[1] LI Xin;YE Huan;HE Yong-Feng;YU Le;CHENG Pei-Lin;DU Hao;WU Jin-Ming(Key Laboratory of Freshwater Biodiversity Conservation,Ministry of Agriculture and Rural Affairs,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;lnstitute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;School of Animal Science and Nutritional Engineering,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]中国水产科学研究院长江水产研究所,农业农村部淡水生物多样性保护重点实验室,武汉430223 [2]上海海洋大学水产与生命学院,上海201306 [3]中国科学院水生生物研究所,武汉430072 [4]武汉轻工大学动物科学与营养工程学院,武汉430023

出　　处：《水生生物学报》2024年第10期1745-1757,共13页Acta Hydrobiologica Sinica

基　　金：国家重点研发计划子课题“川陕哲罗鲑抢救性保种与繁育技术攻关”(2021YFD1200304)资助。

摘　　要：为建立稀有鮈鲫(Gobiocypris rarus)精子超低温冷冻保存技术,研究了稀有鮈鲫精子在不同种类和浓度稀释液与抗冻剂、降温程序中的冷冻保存效果,并开展授精实验,以期建立稀有鮈鲫精子超低温冷冻保存方法。首先,比较了5种精子稀释液对稀有鮈鲫精子活力的影响,发现在BSMIS(Buffered Sperm Motility-inhibiting Solution)稀释液中,精子活力最高(95.55±2.35)%,精子曲线运动速度(VCL)、直线运动速度(VSL)和平均路径速度(VAP)分别为(103.00±29.82)、(84.00±26.21)和(126.67±27.57)μm/s,寿命(37.67±0.58)s,与鲜精无显著差异(P>0.05);其次,筛选了不同浓度的6种渗透性抗冻剂,发现当BSMIS稀释液含有10%甲醇时,精子活力最高(22.33±2.08)%;再次,筛选和优化了不同浓度的非渗透性抗冻剂,发现当BSMIS稀释液含有8%甲醇和90 mmol/L葡萄糖时,精子活力最高(46.00±4.00)%,且VCL、VSL和VAP分别为(125.02±7.42)、(107.10±4.92)和(116.99±6.34)μm/s,寿命(32.67±2.52)s,其中VSL、VAP与鲜精相比无显著差异(P>0.05)。最后,比较了4种液氮蒸气中平衡高度和3个液氮蒸气中平衡时间对稀有鮈鲫精子冷冻保存效果的影响,发现液氮蒸气中平衡高度为3 cm且平衡时间为10min时,精子活力显著高于其他实验组。采用流式细胞仪检测冻精质膜完整性、线粒体活性和DNA完整性,分别为(55.78±2.13)%、(56.18±0.64)%和(55.07±2.00)%。进一步通过授精实验评估冻精质量,冻精受精率和孵化率分别为(87.31±1.38)%和(98.84±1.02)%,与鲜精无显著性差异(P>0.05)。上述结果表明,稀有鮈鲫精液以BSMIS稀释液、8%甲醇和90 mmol/L葡萄糖为抗冻保护液,液氮蒸气中3 cm处平衡10min后置于液氮的超低温冷冻保存效果最好。研究初步建立稀有鮈鲫精子超低温冷冻保存技术,为其种质资源的长期保存提供技术支撑。Gobiocypris rarus is listed as a national second-class protected animal and serves as an important aquatic experimental organism.This study aims to establish ultra-low temperature cryopreservation technology for Gobiocypris rarus sperm by investigating the effects of different types and concentrations of extenders,cryoprotectants,and cooling procedures on sperm cryopreservation.Insemination experiments were conducted to assess the quality of cryopreserved sperm.Firstly,the effects of five types of sperm extenders on sperm motility were compared,revealing that sperm in the BSMIS exhibited the highest motility(95.55±2.35)%.The sperm curvilinear velocity(VCL),straight line velocity(VSL),average path velocity(VAP),and sperm longevity were(103.00±29.82),(84.00±26.21),(126.67±27.57)μm/s,and(37.67±0.58)s,respectively,with no significant difference observed compared to fresh sperm(P>0.05).Secondly,six permeable cryoprotectants at different concentrations were screened,and the highest sperm viability(22.33±2.08)%was achieved when the BSMIS dilution contained 10%methanol.Subsequently,non-permeable cryoprotectants were screened and optimized,with the highest sperm viability(46.00±4.00)%observed when the BSMIS dilution contained 8%methanol and 90 mmol/L glucose.The VCL,VSL,VAP,and longevity were(125.02±7.42),(107.10±4.92),(116.99±6.34)μm/s,and(32.67±2.52)s,respectively,where VSL,VAP were not significantly different(P>0.05)compared to fresh sperm.Finally,the effects of four equilibrium heights and three equilibrium times in liquid nitrogen vapors on the cryopreservation of Gobiocypris rarus were compared.The highest sperm viability was observed when the equilibrium height and time were 3 cm and 10min,respectively.The plasma membrane integrity,mitochondrial activity and DNA integrity of frozen sperm detected by flow cytometry were(55.78±2.13)%,(56.18±0.64)%,and(55.07±2.00)%,respectively.The quality of frozen sperm was further assessed by insemination experiments,and the fertilization and hatching rate of frozen

关 键 词：精子 冷冻保存 精子质量 流式细胞仪 稀有鮈鲫 

分 类 号：S965.1[农业科学—水产养殖]