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1 黄海大头鳕胚胎发育过程于道德 1, 刘名 1, 刘洪军 1 2,, 姜云荣 3 1, 官曙光 (1., ; 2., ; 3., ) 摘要 : 驯化野生大头鳕 (Gadus macrocephalus Tilesius) 作为亲鱼, 通过自然受精和人工授精两种方法获得受精卵, 自然受精卵通过虹吸法收集受精卵孵化条件为水温 6~8, 盐度 32~33, ph7.8~8.2, 微充气在奥林巴斯 SZ61 解剖镜下对大头鳕的胚胎发育进行了观察, 并使用 CCD 图像传感器拍照, 描述了各发育时期的发育时序和形态特征大头鳕受精卵为圆球形端黄卵, 弱黏性, 无油球, 卵膜上具龟裂结构, 卵子直径 0.95~1.12mm, n=30, 卵黄均匀透明类似于大部分硬骨鱼类, 其胚胎发育可分为胚盘形成期卵裂期桑椹期囊胚期原肠期神经胚期器官发生期尾芽期肌肉效应期在水温 6~8, 盐度 32~33 的条件下, 受精卵历时约 336h 完成胚胎发育过程, 孵化出膜在大头鳕胚胎发育过程中, 具有 4 个不同于其他硬骨鱼类的地方 :(1) 发现早期分裂球具有不规则现象 ; (2) 很多胚胎发育过程不出现柯氏囊, 有柯氏囊的胚胎仅占 10% 左右, 且出现晚, 消失快 ; (3) 胚胎孵化为仔鱼与发育进程关联性不大, 孵化的仔鱼有 3 种主要形态 ; (4) 胚胎各个时期都有较高死亡率, 以原肠期最为严重关键词 : 大头鳕 (Gadus macrocephalus Tilesius); 胚胎发育 ; 柯氏囊中图分类号 : S917.4; Q959.4 文献标识码 : A 文章编号 : (2014) doi: /hykx (Gadus macrocephalus Tilesius), (Pacific cod), (Perciformes) (Gadidae)(Acanthopagrus), (G. morhua)(g. ogac) 3,,,, ;,, [1],, [2], (, ~10 /kg),,,,, [3] [4], [5], [1],, [6],,,,, 1 材料与方法 1.1 亲鱼收集和驯化 , 54 ( 40~60cm), 6m 6m 1.2m,, 25W, , 2, 1/3 2.4, 自然产卵与人工授精 ,, : ; : : (2013GHY11509) : (1978-),,,,, : , @163.com;,,,, : / 2014 / 38 / 3

2, 微充气在奥林巴斯 SZ61 解剖镜下对大头鳕的胚胎发育进行了观察, 并使用 CCD 图像传感器拍照, 描述了各发育时期的发育时序和形态特征大头鳕受精卵为圆球形端黄卵, 弱黏性, 无油球, 卵膜上具龟裂结构, 卵子直径 0.95~1.

2 3~4 ; ,,,,,,, 2~3kg, 100~280mL, 2~3kg 700~1200mL 1.3 受精卵及孵化条件,,,,, 6 ±0.5, 32~33, ph 7.8~8.2,, 2.4m 1m 0.8m 1.4 观察方法,, 3 h,, 3 SZ61, ; CCD SONY H-50 ; DN-2,, 3, 30 50% 2 结果,,,,,,, 0.95~1.12mm 6 0.5, 33, 2h, ; 7 h, 24h 35h,,,, 70h, 90%,, KV(Kupffer s vesicle, KV),,, 110h,,,, KV 10%, 192 h,, 210 h ;,, 278h,, 278~336h, 讨论 3.1 低温发育,,,, ( 2), 48h,,, [7],,,,,,,,, 3.2 黄海大头鳕卵子的物理特性, [3],,,, [8-9] (32~34), [7], [10] 3 7,,,,, 1,,,,, ( ),,,, , 1-30, 1-31,, Marine Sciences / Vol. 38, No. 3 /

5, 33, 2h, ; 7 h, 24h 35h,,,, 70h, 90%,, KV(Kupffer s vesicle, KV),,, 110h,,,, KV 10%, 192 h,, 210 h ;,, 278h,, 278~336h, 1 1 3 讨论 3.

3 表 1 大头鳕的发育时序及发育特征 Tab.1 Developmental duration and characteristics of Gadus macrocephalus Tilesius embryo (h) ~1.12mm 1-1 2,, , 2 1-3, , , 8,, , 16, ,, ,,, ,, , ,,, %,, %,, 1-14, %, 70 90%,, KV,,,, ,,,, KV 10%, 4~ ,, 8~12,,, KV 3/4,, 黒, KV, KV, 12~16,,,, 黒,,,,, 18~20 1/5,, 20 /min, 1/3 1/4,,, 40 /min,, 10 /min,, 1/2 1/3,,,,, 1-26, 1-29,,,, 1-27, 1-30,1-34,,,,,, 1-31,,,, 1-32, ,, 3.3 成熟卵子与黏性,, 3 82 / 2014 / 38 / 3

1-14, 1-15 54 50 %, 70 90%,, KV,,,, 1-17 110,,,, KV 10%, 4~6 1-18 144 164 178 192 210 240 258 278 298 336 336,, 8~12,,, KV 3/4,, 黒, KV, KV, 12~16,,,, 黒,,,,, 18~20

4 Fig.1 图 1 大头鳕的胚胎发育 Embryonic Development of Gadus macrocephalus 1-1. 未受精卵; 1-2. 胚盘形成; 细胞侧面观; 细胞背面观; 细胞; 细胞; 细胞侧面观; 细胞; 细胞期; 桑椹期; 高囊胚期; 低囊胚期; 原肠早期; 原肠中期侧面观; 1-15.原肠中期背面观, 可见胚环; 原肠晚期; 原口关闭前期; 原口关闭期; 视囊期; 色素形成期; 晶体形成; 尾芽期; 心跳期; 肌肉效应期; 孵化前Ⅰ期; 孵化前Ⅱ期; 孵化前Ⅲ期; 孵化前Ⅳ期; 1-29.Ⅱ期孵化仔鱼; Ⅲ期孵化仔鱼; 初孵仔鱼; 示孵化仔鱼卵黄囊形态; 示孵化仔鱼卵黄囊形态; 示Ⅲ期孵化仔鱼, 眼球色素沉淀尚不完全 1-1. unfertilized eggs; 1-2. blastodisc forming stage; cell stage, lateral view; cell stage, abdomen view; cell stage; cell stage; cell stage; cell stage; cell stage; morula stage; high blastula stage; low blastula stage; early gastrula stage; middle gastrula stage, lateral view; middle gastrula stage, abdomen view, showing embryo ring; late gastrula stage; blastopore closing stage; blastopore closed stage; optic vesicle forming stage; pigment forming stage; lens forming stage; tai bud formed stage; heart beating stage; muscle effect stage; pre-hatching Ⅰperiod; pre-hatchingⅡperiod; pre-hatching Ⅲperiod; pre-hatching Ⅳperiod; newly- hatched larva during Ⅱ period; newlyhatched larva during Ⅲperiod; newly-hatched larva; newly-hatched larva, showing yolk sac; newly-hatched larva, showing yolk sac; newly-hatched larva during Ⅲ period, showing incomplete pigment sediment eye 表 2 大头鳕与条斑星鲽胚胎发育比较 Tab.2 Comparison of embryonic development between Gadus macrocephalus Tilesius and Verasper moseri 项目发育温度大头鳕条斑星鲽悬浮性 1~12 6~11 完全沉性(个别浮性) 低盐度(26)沉性油球无无胚盘形成 2h 2.5h 第一次卵裂 7h 不同步卵裂现象第二次卵裂 4h 第三次卵裂裂球均一程度差早差稍晚仔鱼孵化温度敏感期 10 d(6 ±0.5 ) 整个胚胎发育过程 9 d(8~10 ) 原肠期之前空泡胚胎有(正常) 有(病态) 精的过程中发现: 成熟度最好的一次(3 月 7 日, 受精率高达 92%), 轻压腹部, 卵子自动流出, 在烧杯中呈现流动状态, 与混合其中的卵巢液可轻易倒入塑料盆中, 仅在烧杯壁黏有个别卵子, 其卵子表现为弱黏性而在 2 月 28 日进行的人工授精过程, 卵子黏性很大, 烧杯壁黏满卵子, 流水冲洗也不易剥落, 其卵子相互挤压, 为不规则的圆形, 受精率仅 30, 发育至原肠期, 几乎全部停止发育死亡其究竟为未成熟或者是过熟还不得而知在 3 月 10 日, 进行人工授精与 3 月 7 日为同一条雌性亲鱼, 发现卵子仍为弱黏性, 但是受精率已经降低至 1%以下, 卵子表现局部发白, 应该为过熟的卵子(图 2) 通过受精率来判断, 第 1 次人工授精中受精率 Marine Sciences / Vol. 38, No. 3 /

孵化前Ⅱ期; 1-27. 孵化前Ⅲ期; 1-28. 孵化前Ⅳ期; 1-29.Ⅱ期孵化仔鱼; 1-30. Ⅲ期孵化仔鱼; 1-31. 初孵仔鱼; 1-32. 示孵化仔鱼卵黄囊形态; 1-33. 示孵化仔鱼卵黄囊形态; 1-34. 示Ⅲ期孵化仔鱼, 眼球色素沉淀尚不完全 1-1. unfertilized eggs; 1-2. blastodisc forming stage; 1-3.

Fig.2 2 3 Comparison of three times artificial insemination of Gadus macrocephalus 2-1. ( 1 ); 2-2. ( 2 ); 2-3., ( 3 ); 2-4. ( 1 ); 2-5.8 ( 1 ); 2-6., ( 1 ) 2-1.

5 Fig Comparison of three times artificial insemination of Gadus macrocephalus 2-1. ( 1 ); 2-2. ( 2 ); 2-3., ( 3 ); 2-4. ( 1 ); ( 1 ); 2-6., ( 1 ) 2-1.showing fertilizing eggs(first artificial insemination); 2-2. showing fertilizing eggs(second artificial insemination); 2-3.morphology of mature egg, unfertilized(third artificial insemination); cell stage(first artificial insemination); cell stage(first artificial insemination); 2-6.early gastrula stage, showing the chap on egg membrance(first artificial insemination) 30%,, 3 (),, 3, 3.4 柯氏囊 Kupffer 1868, (Kupffer s vesicle) [11] (monociliated cells), [12-13] KV,,,, (Oplegnathus fasciatus Temmine) KV, [14] (Acanthopagrus schlegelii(bleeker, 1854)) KV,,, 10% KV, KV,, KV,,, ( , 1-23, 1-24) KV 3.5 孵化时期,,,, 6h,,, 60h,, ,,,,,,,,,, 3.6 胚胎高死亡率,, 6,, 92%,, 54%, 84 / 2014 / 38 / 3

2-cell stage(first artificial insemination); 2-5. 8-cell stage(first artificial insemination); 2-6.

6 , 20%, 15.8%,, 5%,,, [6] 4~6,, 3 11 ( ), 8, : [1]. [D]. :, [2],,,. (Gadus macrocephalus)[j]., 2012, 43(5): [3] Sakurai Y, Hattori T. Reproductive behavior of Pacific cod in captivity[j]. Fisheries science, 1996, 62(2): [4] Hanna S K, Haukenes A H, Foy R J, et al. Temperature effects on metabolic rate, swimming performance and condition of Pacific cod Gadus macrocephalus Tilesius[J]. Journal of Fish Biology, 2008, 72(4): [5],,,. Gadus macrocephalus Tilesius [J]., 2003, 4: [6]. [D]. :, [7],,,. [J]., 2008, 32(2): [8] Ahlstrom E H, Moser H G. Characters useful in identification of pelagic marine fish eggs[j]. Reports of California Cooperative Oceanic Fisheries Investigations, 1980, 21: [9] Riis-vestergaard J. Energy density of marine pelagic fish eggs[j]. Journal of Fish Biology, 2002, 60: [10] Selman K, Wallace R A, Cerda J. Bafilomycin A1 inhibits proteolytic cleavage and hydration but not yolk crystal disassembly or meiosis during maturation of sea bass oocytes[j]. Journal of Experimental Zoology, 2001, 290: [11] Kupffer C. Beobachtungea uber die Entwicklung der Knochenfische[J]. Arch. Mikrob. Anat, 1868, 4: [12] Kreiling J A P, Williams G, Creton R. Analysis of Kupffer s vesicle in zebrafish embryos using a cave automated virtual environment[j]. Developmental Dynamics, 2007, 236(7): [13] Essner J J, Amack J D, Nyholm M K, et al. Kupffer s vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut[j]. Development, 2005, 132(6): [14],,,. [J]., 2008, 32(3): Marine Sciences / Vol. 38, No. 3 /

Temperature effects on metabolic rate, swimming performance and condition of Pacific cod Gadus macrocephalus Tilesius[J]. Journal of Fish Biology, 2008, 72(4): 1068-1078. [5],,,.

7 The Embryonic Development of Gadus macrocephalus Tilesius YU Dao-de 1, LIU Ming 1, LIU Hong-jun 1, JIANG Yun-rong 2, 3,GUAN Shu-guang 1 (1. Mariculture Institute of Shandong Province, Qingdao , China; 2. Shanghai Ocean University, Shanghai , China; 3. Yellow sea fisheries research institute chinese recedemy of fishery sciences, Qingdao266071, China) Received: Jun., 28, 2013 Key words: Gadus macrocephalus Tilesius; embryonic development; Kupffer s vesicle Abstract: The fertilized eggs of wild broodstock, Gadus macrocephalus Tilesius wereacquired through two methods, natural fertilization and artificial insemination. The naturallyfertilized eggs collected by flooding assay. The fertilized eggs were incubated in filtered sea water at 6~8, salinity 32~33, ph7.8~8.2, with subtle aerated. The ontogenetic embryos were observed using a Olympus SZ-61 microscope.the total development time of every stage was noted and calculated and the characteristic images were taken using a charge-coupled device(ccd). The fertilized eggs were globular, single oil globule and the egg diameter was 0.95~1.12mm(n=30). The embryonic developmentexperienced 336h including blastodisc stage, cleavage stage, blastula stage, gastrula stage, neurula stage, organogenesis stage, tail bud stage, heart beating stage and hatching stage.we found there were significant differences between Gadus macrocephalus Tilesiusand other teleost in some stages:1) unregular blastomeres in early cleavage; 2) devoid of Kupffer s vesicle in most embryos during development, only 10% embryos could be observed and occurred later and disappeared earlier than other teleost; 3) the newly-hatched larvae were not related with certain developing stage, and there were three major ontogenetic patterns when the larvae hatched; 4) it has a high death rate during all the development stages, especially in gastrula stage. ( 本文编辑 : 梁德海 ) 86 / 2014 / 38 / 3

Yellow sea fisheries research institute chinese recedemy of fishery sciences, Qingdao266071, China) Received: Jun.

人工催产干法授精孵化胚胎发育观察

人工催产干法授精孵化胚胎发育观察金万昆俞丽杨建新张慈军高永平赵宜双王春英人工催产干法授精孵化胚胎发育观察胚盘隆起细胞期细胞期细胞期细胞期细胞期多细胞期囊胚早期囊胚中期囊胚晚期原肠早期原肠中期原肠晚期神经胚期胚孔封闭期眼泡出现期尾芽期晶体出现期肌肉效应期心脏搏动期孵出期日龄仔鱼日龄仔鱼日龄仔鱼日龄仔鱼感谢中国水产科学院黑龙江水产研究所沈俊宝研究员的悉心指导