利用显微注射技术冷冻保存牙鲆胚胎  

Cryopreservation of flounder (Paralichthys olivaceus) embryos using microinjection

作　　者：丁浩[1,2] 田永胜[1] 武鹏飞[1,3] 刘洋[1] 陈松林[1] 

机构地区：[1]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 [2]上海海洋大学,上海200090 [3]中国海洋大学,山东青岛266003

出　　处：《中国水产科学》2008年第5期866-872,共7页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(30570259);国家高技术研究发展计划(863计划)项目(2003AA603510);农业科技成果转化资金项目(2007GB2C600174)

摘　　要：在鱼类胚胎的冷冻保存中,需要抗冻剂有效地渗入胚胎,才能对胚胎起到保护作用。本研究采用显微注射技术将抗冻剂直接注射到牙鲆（Paralichthys olivaceus）胚胎内,以实现牙鲆胚胎的玻璃化低温冷冻保存。研究中,首先对抗冻剂种类进行了选择,将抗冻剂的毒性由大到小依次排列为：乙二醇（EG）、甘油（Gly）、二甲基亚砜（DMSO）、甲醇（MeOH）、1,2丙二醇（PG）、PM21（PG∶MeOH=2∶1）;对胚胎发育时期和注射剂量进行了筛选。实验结果显示,注射600pL（1pL=10-6mL）抗冻剂PM21后,牙鲆的心跳期胚胎成活率显著高于尾芽期胚胎（P〈0.05）,成活率为（64.04±2.05）%;对卵黄囊、卵膜与卵黄膜间隙作为注射部位进行了选择,发现采用卵黄囊内注射的胚胎成活率高于＂五步平衡法＂,并显著高于通过卵黄膜间隙注射（P〈0.05）,其成活率为（44.24±7.88）%。结果表明,注射600pL35%PM21至牙鲆心跳期胚胎卵黄囊内,平衡10min,然后进行玻璃化冷冻保存,取得了68.2%~79.4%透明胚胎,能够对牙鲆胚胎提供很好的保护。说明显微注射方法可以成功地将抗冻剂注射入牙鲆胚胎,并在牙鲆胚胎的冷冻及胚胎的完整性方面发挥有效的保护作用。Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the permeation of cryoprotectants have failed to protect all embryo compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to make cryoprotectants penetrate into the yolk sac of flounder （Paralichthys olivaceus） embryos. Several factors relating to cryopreservation of flounder embryos with microinjection were investigated which were cryoprotectants, stages of embryo developement, injection volumes of cryoprotectants and injection sites. The toxicity sequence of cryoprotectants were got from high to low as follows： Ethylene glycol （EG）, Gly, DMSO, methanol （MeOH）, 1,3-propanediol （PG）, PM21 （PG： MeOH=2 ： 1 ）. To test the effects of developmental stage of embryos and volumes of cryoprotectant injection, embryos at tail bud and heart beating stage were injected with different volumes of cryoprotectants. After microinjecting with 600 pL 35% PM21 to embryos, survival rate of embryos at heart beating stage is （64.04± 2.05） %, obviously higher than that of embryos at tail bud stage （P〈0.05）. Through the choice of injection sites which are intermembranous space and yolk sac, the results indicate that the optimal injection site is yolk sac, with significant higher survival rate of（44.24± 7.88）% compared with intermembranous space injection group （P〈0.05）.The results show that this method can provide a sufficient protection for the embryos by injecting 600 pL 35% PM21 into the yolk sac of flounder embryos at heart beating stage before vitrifying. With this method 68.2%-79.4% transparent embryos were got. Although after thaw the embryos didn＇t survive because of limited time and embryo quality, it is still testified that microinjection allowed delivery of high concentrations of cryoprotectants into yolk sac without deleterious e

关 键 词：牙鲆 胚胎 显微注射 玻璃化 冷冻保存 

分 类 号：S91[农业科学—水产科学]