超低温冷冻对中华绒螯蟹胚胎形态结构的影响

为了解超低温冷冻对胚胎形态结构的影响,以中华绒螯蟹为研究对象,运用石蜡切片、扫描电镜和透射电镜观察细胞分裂期、原肠期和原溞状幼体期胚胎,分别用玻璃化液处理和经超低温冷冻后外部形态和内部结构的变化。结果发现:(1)显微观察表明,细胞分裂期胚胎在玻璃化液中用二步平衡法处理后吸水膨胀明显,三步平衡后胚胎形态无明显变化,超低温冷冻后,卵黄物质从细胞中溢出,细胞破损严重;原肠期胚胎在玻璃化液中用二步平衡法处理后,外部形态与鲜胚无明显差异,经过冷冻后,所有胚胎内部变成粉红色,胚体由原来的透明变成不透明状,细胞膜边缘模糊似绒毛状;(2)扫描电镜观察,玻璃化液处理后的所有原肠期胚胎表面褶皱呈沟壑状,形成一层网状结构;透射电镜观察,处理后胚胎细胞内出现白色团块,细胞边缘变得粗糙有突起,细胞内冰腔清晰可见,空泡形成,80%以上线粒体解体,细胞破裂明显;(3)组织切片观察,原肠期胚胎细胞外面的膜脱落破损,胚层内有大小不一的冰腔,细胞内出现明显的空泡。原溞状幼体期胚胎经过玻璃化液处理后,外部形态与鲜胚间无明显区别,但经过超低温冷冻后,95%以上胚胎组织呈弥散状,部分卵黄物质碎裂成颗粒状,胚胎的细胞膜脱落,胚层内出现大量冰腔和空泡,90%胚胎表面皱缩凹陷,但仍有10%的胚胎表面保持光滑完整,表明原溞状幼体期胚胎是适合进行冷冻保存的时期。     

一株中华绒螯蟹幼体益生菌的筛选及其种属特性的初步研究

通过单株细菌感染中华绒螯蟹Ⅰ期溞状幼体的方法筛选出2株病原菌,它们对幼体的72 h致死率为100%;获得有益菌1株,与对照组相比提高中华绒螯蟹Ⅰ期溞状幼体的变态率近50%,该菌株有望成为中华绒螯蟹幼体的益生菌株.根据16S rRNA序列同源性分析结果,将其初步鉴定为节杆菌属的一种.     

卤虫胚胎和无节幼体超低温冷冻保存的研究↑（*）

用玻璃化液作为低温保护剂，对卤虫胚胎和无节幼体进行超低温冷冻保存实验。通过实验筛选出５种适宜卤虫胚胎保存的玻璃化液，找出了影响胚胎成活的降温温层和升温温层，使胚胎经－１９６℃保存后的成活率稳定在９０％。建立了较为完善的卤虫胚胎冷冻保存的程序。卤虫无节幼体经超低温冷冻保存后，获得３－５％的成活率。     

中华绒螯蟹胚胎发育及几种代谢酶活性的变化

采用实验室内小规模实验的方法,在水温(16.2±1.5)℃和盐度20的条件下,对中华绒螯蟹胚胎发育过程中的形态学变化和几种重要代谢酶的活性变化进行了系统性研究。实验结果表明,中华绒螯蟹的胚胎发育过程可以分为9个主要阶段:受精卵、卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、原溞状幼体期、出膜前期和孵化期;水温16℃的条件下,整个胚胎发育过程需40 d左右,有效积温达到10 758 h.℃。乳酸脱氢酶(LDH)、总ATPase和苹果酸脱氢酶(MDH)的活性都随胚胎发育时期的变化而变化,LDH活性呈现出先上升后下降的趋势,在囊胚期中活性最高;总ATPase活性在原溞状幼体期最高,在原肠期最低;MDH活性在原溞状幼体期最高,在囊胚期最低。     

0~#柴油中的水溶性石油烃对中华绒螯蟹溞状幼体的急性毒性

以中华绒螯蟹溞状幼体为实验对象,通过48 h半静水式急性毒性试验探究0~#柴油中的水溶性石油烃对中华绒螯蟹(Eriocheie Sinensis)溞状幼体的急性毒性效应。结果发现,水溶性石油烃对5期(Z_1～Z_5)溞状幼体的24 h半致死浓度分别为9.902、10.542、11.596、13.441和15.684 mg/L; 48 h半致死浓度分别为6.249、7.193、8.022、9.889和12.948 mg/L;安全浓度分别为0.813、1.005、1.247、1.606和2.480 mg/L。参照《环境监测技术规范》生物监测(水环境部分),可以看出0~#柴油的水溶性石油烃对中华绒螯蟹各期溞状幼体为高毒。本研究可为科学评价水溶性石油烃对中华绒螯蟹各期溞状幼体的毒性提供依据,同时为河蟹养殖环境风险预警监测提供数据参考,对生产实践具有良好的指导意义。     

中华绒螯蟹幼体及仔蟹阶段肝胰腺发育的组织学研究

通过对中华绒螯蟹幼体及仔蟹进行连续采样和组织切片,系统地研究了该阶段中华绒螯蟹肝胰腺的发育情况。结果表明,中华绒螯蟹的肝胰腺前体出现于心跳期胚胎,由卵黄囊特化而来,为1对囊状结构,此阶段首次出现肝胰腺细胞(柱状上皮细胞)。胚胎孵化后,随着幼体的发育,肝胰腺由最初的1对囊状结构逐渐分叶生长。从Ⅰ期溞状幼体至Ⅴ期溞状幼体,肝胰腺分叶数量分别为4-6-8-12-12,肝胰腺细胞高度由(12.63±4.91)μm增至(44.16±13.57)μm。肝胰腺中的细胞组成也随着幼体的发育逐渐完善,Ⅱ期溞状幼体的肝胰腺中首次出现E细胞和B细胞,Ⅲ期溞状幼体肝胰腺中首次出现F细胞,而R细胞直到Ⅱ期仔蟹才首次出现。从大眼幼体到Ⅲ期仔蟹,肝胰腺细胞高度由(44.93±18.27)μm增至(56.38±5.69)μm。     

对中华绒螯蟹生态育苗池中桡足类控制的初步研究

比较了蛛虫速灭、鱼虫恨和强力混杀3种渔药对桡足类和中华绒螯蟹溞状幼体的急性毒性。试验结果表明,蛛虫速灭对桡足类的96h半致死添加量为0.011mL/m3,鱼虫恨为0.036mL/m3,强力混杀精为0.014mL/m3,蛛虫速灭对桡足类的杀灭能力最强;对中华绒螯蟹溞状幼体Ⅰ期的急性毒性鱼虫恨最大,蛛虫速灭最小。蛛虫速灭在0.18～0.20mL/m3的剂量下,对溞状幼体毒性低,幼体能正常变态,可用于蟹苗池桡足类的控制。强力混杀精对桡足类和中华绒螯蟹溞状幼体的毒性很接近,鱼虫恨对中华绒螯蟹溞状幼体的毒性大于对桡足类的毒性,两者均不适宜在中华绒螯蟹生态池控制桡足类。     

六株海洋微藻强化的酵母轮虫对中华绒螯蟹溞状幼体的影响

实验用6株海洋微藻来强化用酵母预培养的褶皱臂尾轮虫,并将强化后的轮虫投喂中华绒螯蟹的溞状幼体,以研究使用经不同食物强化后的轮虫投喂溞状幼体对溞状幼体变态时间及成活率的影响。结果表明：经过直链藻强化的轮虫投喂的溞状幼体的成活率及变态时间均优于其他海洋微藻强化的褶皱臂尾轮虫,海水蒜头藻与海水小球藻次之,酵母轮虫最差。     

杭州湾咸淡水河蟹人工育苗技术探讨

1995年,用杭州湾咸淡水和池塘养殖的长江水系中华绒螯蟹亲蟹,在水温10~14℃、海水比重1.0125条件下催产,加温孵化;溞Ⅰ幼体放养密度为20万~40万/m³,以丰年虫无节幼体为主要饵料,水温24.5~25.5℃。390m³育苗水体中,两茬生产大眼幼体110.4kg,每立方水体出苗量0.28kg。     

鲈鱼胚胎程序化冷冻保存的研究

对鲈鱼(Lateolabrax japonicus)胚胎的程序降温冷冻保存进行了研究。通过稀释液的筛选实验，选择了一种培养效果好的稀释液DSl液。研究了不同发育阶段胚胎对抗冻液的毒性耐受力，表明肌肉效应期胚胎的耐受力最强。测定了心跳期胚胎在抗冻液A1、A2、B1、B2中的平衡处理时间，为进行鲈鱼胚胎冻前平衡提供了参考时间。比较了植冰和不植冰对鲈鱼胚胎冷冻保存结果的影响及不同洗脱方法洗脱胚胎的效果。结果表明，在冷冻过程中诱导植冰的胚胎成活率高于未植冰组，两步法洗脱效果优于一步法和三步法。用不同降温速率进行了鲈鱼胚胎的超低温冷冻保存实验，结果表明，采用1．5℃／min的降温速率降温，在液氮中冷冻保存30min的72粒心跳期胚胎中有3粒成活，成活率为4．2％。     

Survival of zebrafish, Brachydanio rerio (Hamilton-Buchanan), embryo after immersion in methanol and exposure to ultrasound with implications to cryopreservation

This study examined the viability of embryos after immersion in highly concentrated methanol solutions (40–60%) and exposing embryos to ultrasound to enhance efficient transport of the cryoprotectant. The exposure to ultrasound, methanol concentrations, duration of treatment and the stages of embryonic development was found to have measurable effects on embryo viability. The effect of ultrasound was more evident at high voltage (>440 V) settings and at early developmental stages (30 and 60% epiboly stage). Older embryos were more resistant to cryoprotectant toxicity and ultrasound‐induced mortality. The high concentration of methanol (60%) was more toxic to embryos than the low concentration (40%). When methanol treatment and ultrasound were applied simultaneously the optimum concentration was found to be 45% methanol (45% survival; P<0.05) in a 3 min treatment. Although there was no significant difference between the 2 and 3 min treatments, embryos treated for 4 min had a significantly lower survival rate (P<0.05). These findings provide initial results to select the developmental stage of the embryo, the concentration of methanol for the preparation of a vitrification solution and duration of ultrasound treatment for cryopreservation. Furthermore, it indicates the potential use of ultrasound to enhance the transport of methanol intracellularly with minimum mortality of the developing embryos.     

Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus maximus) embryos: Toxicity and protein distribution

Fish embryo cryopreservation has not been achieved. Different methods and alternative cryoprotective agents (CPAs) should be explored in order to succeed in this purpose. Antifreeze proteins (AFPs) are naturally expressed in sub-arctic fish species, and they inhibit the growth of ice crystals as well as recrystallization during thawing. Therefore, their introduction into embryos can be highly beneficial for vitrification purposes. In this study, AFP type III was introduced into turbot embryos, by microinjection into the yolk sac and the perivitelline space at F stage (tail bud).Toxicity and distribution of protein in microinjected embryos were established before testing the protein effect on embryo cryopreservation. AFP-FITC distribution within the embryo was analyzed by confocal microscopy at 5 min and 24 h after microinjection in F stage embryos. To test the sensitivity of microinjected embryos to CPAs, embryos were subjected to a protocol for the incorporation of a vitrifying solution that was specially designed for turbot embryos. Hatching rates after CPA incorporation were determined. Results indicate that embryos at late developmental stages are more resilient to microinjection, with embryo survival rates between 60 and 82%. Confocal microscopic images demonstrated that the protein was homogeneously distributed within the microinjected embryo compartment, but did not enter any other compartment. On the other hand, microinjected embryos successfully surmounted their incubation in the CPAs. This study explores new alternatives for cryopreservation suggesting the use of natural cryoprotectants (AFPs) in the protection of intra-embryo compartments, which are usually unprotected with the conventional cryopreservation protocols for fish embryos.     

Cryogenic and Refrigerated Storage of Rainbow Smelt Osmerus mordax Spermatozoa

The effects of extender composition, cryoprotectant, and freezing rate on post-thaw rainbow smelt Osmerus mordax sperm motility were examined, and the fertilization capacity of fresh and post-thaw sperm were compared. The highest post-thaw motility (75%) was obtained when milt was diluted 1:3 with an extender containing 600 mM sucrose supplemented with 10% dimethyl sulfoxide and 1.5% bovine serum albumin and frozen at a rate of –20 C/min. Post-thaw motility for sperm stored in this extender was similar to fresh sperm and did not change after 90 d of storage. Furthermore, there were no differences in fertilization rate or embryo survival to the eyed stage between fresh and post-thaw sperm frozen in this extender. The lowest post-thaw motility was observed when sperm were frozen with methanol at a rate of -30 C/min. Refrigerated sperm diluted 1:3 with the 600 mM sucrose extender remained motile for 30 d. These data demonstrate that rainbow smelt spermatozoa can be effectively used following short and long-term storage using a simple, sucrose-based extender.     

Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me₂SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me₂SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me₂SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me₂SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrations of CPAs into the yolk sac without deleterious effects on the embryo, but did not provide a significant level of protection for the whole embryo against chilling injury.     

鱼类胚胎冷冻保存前几个因子对其成活率影响的研究

1.稀释液渗透压升高是影响胚胎成活的主要因素,在达到480mOsm/L时胚胎全部死亡。2.在室温和0℃条件下几种抗冻剂对胚胎的极限浓度分别为二甲亚砜16％和20％,甘油4％和5％,乙二醇12％和12％,甲醇0℃下为20％。3.原肠期以前的胚胎为敏感期胚胎,不宜进行低温冷冻保存。4.冻前处理超过180分钟后胚胎出现畸形变化。     

牙鲆淋巴囊肿病毒纯化及不同来源病毒免疫特性比较

比较了3种不同的牙鲆淋巴囊肿病毒纯化方法，优化后的方法如下：剥离囊肿表面薄膜，收集内容物，匀浆后再用超声波细胞破碎仪破碎，反复冻融，650×g、1800×g差速离心，30%（W/W）蔗糖垫底超速离心（78500×g）浓缩病毒，最后蔗糖密度梯度超速离心（78500×g）纯化病毒。电镜观察发现，出现在47%～52%蔗糖密度区域的病毒带含有多量、纯净和结构一致的病毒粒子。此外，利用制备的兔抗血清对不同地区的病毒进行了免疫特性分析，Western blotting检测显示来自威海、青岛及秦皇岛3个地区的淋巴囊肿病毒反应结果是一致的，均有3条蛋白带发生反应，其分子量分别为125、66和55kDa。     

Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity,cooling rate and thawing temperature

Aquaculture of hard clams Mercenaria mercenaria is a $65 million industry along the east coast and Gulf of Mexico coast in the United States. The goal of this study was to develop a preliminary protocol to cryopreserve trochophore larvae of hard clams. The objectives were to evaluate the: 1) toxicity of cryoprotectants, including dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol and glycerol, at 5, 10, 15 and 20% for exposure time of 1, 15, 30, 45, 60 and 75 min; 2) effects of cooling rates (5, 10, 20 and 30°C/min for the first trial; and 1, 3 and 5°C/min for second trial from 4 to ?80°C), thawing temperature (30, 40 and 50°C) and their interactions on post‐thaw viability. A basic protocol was concluded as: 15‐hr trochophore larvae mixed with DMSO or propylene glycol (5, 10%), equilibrated for 15 min, cooled in a programmable freezer from 4 to ?80°C at a cooling rate of 5°C/min and thawed at 50°C for 6 s. With this protocol, the immediate post‐thaw trochophore survival was 23 ± 14%, and survival to D‐stage was 27 ± 14%. This is the first report on larval cryopreservation in the hard clam and would have application for genetic breeding and seed production.     

Preliminary studies on the cryopreservation of gilthead seabream (Sparus aurata) embryos

Vitrification could provide a promising tool for the cryopreservation of fish embryos. In order to achieve successful cryopreservation, several parameters should be taken into account in the design of a vitrification protocol. In the present study some relevant factors were investigated (permeable and non-permeable cryoprotectant toxicity, toxicity of vitrificant solutions, adequate container for embryo loading and temperature of thawing) using two gilthead seabream embryonic development stages (tail bud and tail-bud-free). Permeabilized embryos were incubated in dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and 1,2-propanediol (PROH) in concentrations ranging from 0.5 to 6 M for 10 and 30 min and in 5%, 10% and 15% polyvinyl pyrrolidone (PVP), 10%, 15% and 20% sucrose or 0.1%, 1% and 2% X1000® for 2 min. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. EG was best tolerated by the embryos. Exposure to non-permeable cryoprotectants did not affect the hatching rate except at F stage. Six vitrificant solutions (DMSO—V1, V2 and V3 and EG—V1, V2 and V3) were tested using a stepwise incorporation protocol. The DMSO-based solutions contained 5 M DMSO + 2 M MeOH + 1 M EG plus 5% PVP, 10% sucrose or 2% X1000® and the EG-based solutions contained 5 M EG + 2 M MeOH + 1 M DMSO plus 5% PVP or 10% sucrose. Before loading the embryos into 0.5 ml straws or 1 ml macrotubes, toxicity tests were effected with these solutions. Our results demonstrated that DMSO-based solutions were better tolerated by seabream embryos than EG-based solutions. After thawing (water bath, 0 or 25 °C), embryos were evaluated by stereoscopic microscopy and the percentage of embryos with intact morphology was registered. The highest percentage of embryos with intact morphology (28%) was observed in samples frozen in macrotubes and thawed at 25 °C. Several malformations associated with ice crystal formation inside the embryos were detected. None of these embryos achieved hatching. Our results suggest that the absence of a proper incorporation of cryoprotectants prior to vitrification is the main problem that must be overcome. This procedure should be optimized in order to avoid ice crystal formation inside embryo compartments.     

紫外线照射对中华锯齿米虾胚胎发育的影响

自抱卵的中华锯齿米虾雌虾腹部剥离取卵裂期、囊胚期、原肠胚期和无节幼体Ⅰ期的胚胎置于培养皿中,暴露在紫外线下,照射剂量分别为0、1、2、4、8、16 W·s/cm^2,人工振荡培养箱中以速度55 r/min、温度25℃、湿度60%培养。每隔24 h在解剖镜下观察胚胎发育至48 h后进行数据分析,研究紫外线照射剂量对中华锯齿米虾胚胎发育的影响。结果表明,胚胎的存活率对紫外线具有明显的剂量依赖关系。随着紫外线照射剂量的增加,胚胎发育延缓。紫外线暴露后48 h的半数致死量分别为:卵裂期11.5 W·s/cm^2、囊胚期8.6 W·s/cm^2、原肠胚期12.0 W·s/cm^2、无节幼体Ⅰ期14.2 W·s/cm^2。相同的暴露剂量下,暴露时间对胚胎发育也有明显影响。     

雌核发育与正常二倍体条斑星鲽胚胎发育比较研究

在用牙鲆精子诱导条斑星鲽雌核发育的同时,对条斑星鲽单倍体、雌核发育胚胎发育过程进行了观察,并与普通胚胎发育作了比较.观察结果表明,(1)单倍体胚胎,卵裂开始至64细胞期,发育速度较普通胚胎发育发育速度明显缓慢,至多细胞期发育速度相同,历时14 h;到达高囊胚期比普通二倍体慢1.5 h,到达低囊胚期发育速度相同,历时31 h;到达原肠末期、神经胚期、晶体形成期历时相同,其他发育时期一直处于缓慢状态;孵化仔鱼具有明显的单倍体综合症.(2)雌核发育与普通胚胎的发育的特征相比无明显区别,在卵裂期发育速度明显迟缓,但在囊胚期以后,所用时间基本相同;孵化时间比普通胚胎长约1 h.(3)与普通胚胎相比,雌核发育胚胎、单倍体胚胎孵化率较低,仔鱼畸形率高.