大黄鱼胚胎在不同稀释液及抗冻剂中的成活及超低温冷冻保存试验  

Screening of optimal diluents and cryoprotectants for cryopreservation of Larimichthys crocea embryos and evaluation of preservation effect

作　　者：杨海燕 高心明 杜晨 程顺 沈伟良[1,3] 竺俊全[1] YANG Haiyan;GAO Xinming;DU Chen;CHENG Shun;SHEN Weiliang;ZHU Junquan(Key Laboratory of Applied Marine Biotechnology of the Ministry of Education,Ningbo University,Ningbo 315832,China;Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,China;Ningbo Marine and Fisheries Research Institute,Ningbo 315012,China)

机构地区：[1]宁波大学教育部应用海洋生物技术重点实验室,浙江宁波315832 [2]浙江省淡水水产研究所,浙江湖州313001 [3]宁波市海洋与渔业研究院,浙江宁波315012

出　　处：《宁波大学学报（理工版）》2020年第3期7-12,共6页Journal of Ningbo University：Natural Science and Engineering Edition

基　　金：浙江省科技计划重大项目(2016C02055-7);宁波市农业重大专项(2015C110005);浙江省农业(水产)新品种选育重大科技专项(2016C02055-7-2);国家海水鱼产业技术体系(CARS-47-Z08).

摘　　要：为了解大黄鱼胚胎在不同稀释液与抗冻剂中的成活及超低温冷冻保存的效果,开展了大黄鱼胚胎在不同稀释液及抗冻剂中的成活试验.结果表明,大黄鱼原肠期胚胎在BS2稀释液中培育30 min后的成活率与对照组无显著差异,说明BS2可能适合作胚胎冷冻保存用稀释液;单一抗冻剂对大黄鱼胚胎的毒性依次为丙二醇(PG)<甲醇(Me OH)<二甲基亚砜(DMSO)<二甲基甲酰胺(DMF)<乙二醇(EG),抗冻剂使部分胚胎的卵黄囊皱缩,胚体发生损伤,影响胚胎的成活率及孵化率;混合抗冻剂PMD(PG:Me OH:DMSO=9:6:5)对大黄鱼心跳期胚胎的毒性较小,不影响胚胎的成活率及孵化率.以BS2为稀释液,40%PMD为抗冻液,采用两步降温法进行超低温冷冻保存大黄鱼胚胎试验,解冻后成功复活卵裂期及尾芽期冻胚各1粒,原肠期冻胚2粒,心跳期冻胚3粒.To determine the optimal diluents and cryoprotectants for cryopreservation of Larimichthys crocea embryos,the survival rate of embryos under different diluents and cryoprotectants were analyzed.The results showed that there were no significant differences between control and experimental group treated by BS2 for 30 min in the survival rate of gastrula stage embryos.The results indicated that BS2 may be an optimal diluent for L.crocea embryos.The crumpled yolk sac and damaged embryonic body were observed in given embryos treated with five most commonly used cryoprotectants:1,2-propylene glycol(PG),methanol(MeOH),dimethyl sulfoxide(DMSO),dimethylformamide(DMF)and ethylene glycol(EG),causing reduction of the survival rate and hatching rate of embryos.The toxic effect of five cryoprotectants on L.crocea embryos was in the following sequence:PG<MeOH<DMSO<DMF<EG.The toxic effect of mixed cryoprotectants on the heart beating of L.crocea embryos was tested.The PMD(PG:MeOH:DMSO=9:6:5)was identified as suitable cryoprotectant for cryopreservation of L.crocea embryos because it did not significantly decrease the survival rate and hatching rate of embryos.The cryopreservation of embryos was finally implemented,using 40%PMD by two-step cooling method.A signfiicnat protion of frozen embryos were successful resurrected,including one frozen embryo in cleavage stage,two frozen embryos in gastrula stage,one frozen embryo in tail-bud stage and three frozen embryos in heart beating stage.

关 键 词：大黄鱼 胚胎 稀释液 抗冻剂 冷冻保存 

分 类 号：S917.4[农业科学—水产科学]