不同抗冻剂对牙鲆胚胎毒性研究以及玻璃化颗粒冷冻保存方法的应用  

Toxicity of different cryoprotectants to Japanese flounder(Paralichthys olivaceus) embryos and application of vitrified droplets method

作　　者：刘本伟[1] 陈松林[2] 田永胜[2] 孔晓瑜[1] 王春花[2] 

机构地区：[1]中国海洋大学,山东青岛266003 [2]中国水产科学研究院黄海水产研究所农业部海洋渔业资源可持续利用重点开放实验室,山东青岛266071

出　　处：《中国水产科学》2007年第5期733-742,共10页Journal of Fishery Sciences of China

基　　金：国家自然科学基金资助项目(30570259);国家863计划项目(2003AA603510)

摘　　要：以牙鲆(Paralichthys olivaceus)不同发育期胚胎为实验材料,研究了7种渗透性和5种非渗透性抗冻剂对牙鲆尾芽期和心跳期胚胎的毒性,同时对玻璃化液在不同胚胎发育期的毒性作用,以及玻璃化颗粒冷冻过程中,冷冻颗粒降温和解冻的时间进行了研究。结果表明,单一渗透性抗冻剂对牙鲆胚胎的毒性随着抗冻剂浓度的升高、平衡时间的延长而提高,其毒性由大到小依次为乙二醇(EG)、酒精(EtOH)、甘油(Gly)、二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、甲醇(MeOH)、1,2-丙二醇(PG)。非渗透性抗冻剂中聚乙烯吡咯酮(PVP)对牙鲆尾芽期胚胎的毒性最强,其次是蔗糖和D果糖,葡聚糖和葡萄糖毒性最弱。在总体积分数一定的情况下,PG、MeOH与DMSO体积比为9∶6∶5的混合抗冻剂对牙鲆尾芽期胚胎毒性最低;各发育期胚胎经过玻璃化液平衡后,尾芽期以前胚胎的成活率随胚胎发育期逐渐升高,心跳期以后逐渐降低,尾芽期和心跳期成活率最高。含胚胎的玻璃化颗粒冷冻降温时间最短为15.09 s,解冻时间最短为6.22 s;而不含胚胎的玻璃化颗粒冷冻降温和解冻时间分别为(13.83±1.86)s和(7.20±0.90)s。将PG、MeOH与DMSO按体积比9∶6∶5配成总体积分数为35%的混合溶液,再添加5%的蔗糖配制成玻璃化液,采用此玻璃化颗粒冷冻方法对173粒牙鲆尾芽期至心跳期胚胎进行超低温冷冻保存,解冻后共获得4粒成活胚胎。The objectives of this study were to analyze the toxicity of seven permeable and five non-permeable cryoprotectants on flounder embryos and to find a suitable method for cryopreseration of flounder embryos. The proportion of permeable cryoprotectants in vitrification, the toxicity of vitrification on embryos at different stages and the equilibrated effect on embryos of every step in five step method were also studied. The results showed that the toxicity of different cryoprotectants on flounder increased with concentration and exposure time. The toxicity of permeable cryoprotectants was： EG 〉 EtOH 〉 Gly 〉 DMF 〉 DMSO 〉 MeOH〉 PG. In the five non-permeable cryoprotectants, the toxicity of PVP on flounder embryos is the highest, and the toxicity of glucose and dextran is the lowest. When the volume proportion of PG and MeOH is 3： 2 and PM： DMSO is 3 ： 1, the toxicity of combined cryoprotectant on flounder embryos is the lowest. The survival rate of the embryos at tail-bud stage and heart beating stage is higher than other stages after exposure in the selected vitrification solution. The freezing and thawing time of vitrified droplets were tested. And the shortest freezing and thawing time of the droplets with embryos were 15.09 s and 6.22 s, respectively. The flounder embryos from tail-bud stage to heart beating stage were crryopreserved using selected vitrification solution and vitrified droplets method, and 4 survival embryos were obtained. [ Journal of Fishery Sciences of China, 2007,14 （5） ： 733 - 742 ]

关 键 词：牙鲆(paralichthys olivaceus) 胚胎 抗冻剂 玻璃化 颗粒冷冻保存法 

分 类 号：S91[农业科学—水产科学]