星斑川鲽胚胎的玻璃化冷冻保存  

Cryopreservation of Starry flounder embryos by vitrification

作　　者：李振通 田永胜[1,2] 唐江[1,3] 成美玲 孙宗哲[5] LI Zhentong;TIAN Yongsheng;TANG Jiang;CHENG Meiling;SUN Zongzhe(Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071;Laboratory for Marine Fisheries,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266273;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023;Penglai Zongzhe Aquaculture Company,Penglai 265617)

机构地区：[1]中国水产科学研究院黄海水产研究所,农业农村部海洋渔业可持续发展重点实验室,青岛266071 [2]青岛海洋科学与技术试点国家实验室,海洋渔业科学与食物产出过程功能实验室,青岛266273 [3]上海海洋大学水产与生命学院,上海201306 [4]大连海洋大学水产与生命学院,大连116023 [5]蓬莱市宗哲水产养殖公司,蓬莱265617

出　　处：《安徽农业大学学报》2020年第3期373-379,共7页Journal of Anhui Agricultural University

基　　金：山东省良种工程(2019LZGC020,2016LZGC009);山东省重点研发计划(2019GHY112063);烟台市重点研发计划(2016JH021);烟台市高端人才引进“双百计划”项目;黄海水产研究所基本科研业务费(20603022019002,20603022018019)共同资助

摘　　要：为研究星斑川鲽胚胎玻璃化冷冻保存,首先,于室温条件下采用玻璃化液五步平衡法处理星斑川鲽的尾芽期胚胎30 min,比较分析分别含有葡萄糖、果糖、蔗糖和海藻糖的4种不同玻璃化液(PMG3G、PMG3F、PMG3S、PMG3T)的毒性,发现含有海藻糖的玻璃化液(PMG3T)对尾芽期胚胎的毒性最小,胚胎的成活率与孵化率相对较高,分别为75.00%±5.43%和50.00%±4.53%(P<0.05)。其次,分析玻璃化液PMG3T对星斑川鲽囊胚期至出膜前期7个时期的胚胎的毒性,发现尾芽期胚胎和心跳期胚胎经处理后成活率较高,分别为69.33%±6.43%和72.67%±3.94%(P<0.05),但心跳期胚胎的孵化率较高,为65.33%±3.91%(P<0.05),尾芽期胚胎次之,说明心跳期胚胎对PMG3T的耐受能力相对于尾芽期胚胎更好,更适宜进行胚胎的玻璃化冷冻保存实验。最后,采用玻璃化液PMG3T对心跳期胚胎进行–196℃冷冻实验,发现所处理的60粒胚胎中有7粒成活,可漂浮水面,继续培养后可见进一步发育,但未孵化出膜。另外,将100 mL未受精卵(未经玻璃化液处理)置于–20℃,分别在冷冻5~70 min时取出10 mL复温并进行授精,结果显示,冷冻5 min的卵的受精率为89.83%±6.51%(P>0.05),随着冷冻时间的延长,受精率越来越低,冷冻70 min时受精率为3.71%±1.27%,畸形率越来越高,在冷冻70 min时达到100%。本研究筛选出比较适宜的玻璃化液、低温耐受性较高的胚胎发育时期,为星斑川鲽胚胎低温冷冻保存技术的建立和种质的长期保存和应用提供了实验数据。In this study,the vitrification cryopreservation of Starry flounder(Platichthys stellatus)embryos was studied.Firstly,the tail bud stage embryos was treated 30 min with vitrification solution by five-step equilibrium method at indoor temperature,The toxicity of 4 different vitrification solution(PMG3G,PMG3F,PMG3S,PMG3T)containing glucose,fructose,sucrose and trehalose were compared and analyzed.It was found that trehalose-containing vitrification solution(PMG3T)had the least toxicity to the tail bud stage embryos,and the embryo survival rate and hatching rate were relatively high,75.00%±5.43%and 50.00%±4.53%,respectively(P<0.05).Secondly,the toxicity of PMG3T to seven stage embryos from blastula stage to pre-hatching stage was analyzed,the result reveals that the survival rate of treated tail bud stage embryos and heartbeat stage embryos were higher,69.33%±6.43%and 72.67%±3.94%(P<0.05),however,the hatching rate of heartbeat stage embryos was relatively higher,65.33%±12.48%(P<0.05),followed by tail bud embryos,which suggests that heartbeat stage embryos was of higher endurance capacity to PMG3T than tail bud stage embryos,and the heartbeat stage embryos was unfit for cryopreservation experiment.Finally,the heartbeat embryos treated with PMG3T were used to perform a-196℃freezing experiment,it was found that 7 of the 60 embryos survived and floated on the water.After further cultivation,further development was observed,but no embryos hatched in the end.In addition,100 mL ovum were placed in-20℃and taken out 10 mL ovum when refrigerating for 5 to 70 minutes,rewarm and inseminate,the results show that fertilization rate of ovum frozen for 5 min was 89.83%±6.51%,and with the extension of the freezing time,the fertilization rate was getting lower and lower,and the deformity rate was getting higher and higher,the fertilization rate and deformity rate of ovum after freezing for 70 min were 3.71%±1.27%and 100%.In this study,We have selected suitable vitrification solution and embryonic development stages with hi

关 键 词：星斑川鲽 胚胎 玻璃化 冷冻保存 

分 类 号：S984.1[农业科学—捕捞与储运] Q492.6[农业科学—水产科学]