温度极限对银大麻哈鱼胚胎及仔鱼死亡率和发育率的影响

本文查明了水温极限对两个种群的银大麻哈鱼Oncorhynchus kisutch胚胎及仔鱼发育率的影响，以及孵化温度的上限和下限。在稳定的孵化温度下，胚胎能经受1．3～12．4℃的温度范围，在14℃以上或1．3℃以下几乎100％死亡。最大的死亡率发生在胚胎发育早朝，尤其是在胚孔封闭前。除了在最高孵化温度(10．2℃)下，一时性的温度突然变化时间持续8小时，变幅从增加8．4℃到减少6．2℃(包括下降到0．1℃)其胚胎死亡率很少增加甚至不增加。温度对发育时间的影响不是呈直线相关的，有时温度微小的变动会导致发育率明显的变化，尤其在孵化温度的上限和下限时。文中列出的一组公式表明温度与达到50％孵化率和完成100％卵黄吸收所需积温的相关关系；男一组公式则表明温度与达到50％孵化率和100％卵黄吸收的天数之问的相关关系。     

虾夷扇贝三倍体诱导与培育技术的研究

取繁殖期虾夷扇贝（Patinopecten yessoensis)亲体，采用阴干、流水和升温海水催产以及小水体、高密度、雌雄分池排放、人工授精的方法，可获得批重受精卵。分别使用浓度为0．05，0．10，0．15，20，0．30，0．50和0．70mmol/L的6－DMAP诱导虾夷扇贝三倍体，实验水温分别为12℃和14℃。结果表明，水温12℃，6－DMAP0．05mmol/L时，在授精后67min处理胚胎45min诱导三倍体效果较好，三倍体率达83．3％，面盘幼虫相对孵化率达69．5％；水温12℃和14℃时，6－DMAP可诱导17．3％－100．0％的三倍体面盘幼虫；6－DMAP处理组胚胎发育至面盘幼虫的时间比对照组慢，故应推迟1d选优；处理组幼虫初始壳长、壳高及日生长速度皆高于对照组；6－DMAP的残留人影响处理幼虫的成活率，用0．2ml/L的DMSO海水浸洗处理可减轻6－DMAP的副作用。     

淡水养殖鱼类胚胎低温保存的初步研究

以低温冰箱和液氮作为冷源,对四种淡水养殖鱼类的胚胎进行低温保存试验。采用10%二甲亚砜(DMSO)为抗冻剂,鲤鱼胚胎冷冻至-5℃、-10℃和-20℃,复温后的最高成活率分别为96%、90%和31%;团头鲂胚胎冷冻至-10℃、-15℃和-20℃,复温后的最高成活率分别为43%、20%和12.5%;青鱼胚胎冷冻至-5℃、-10℃和-11℃,复温后的最高成活率分别为100%、40%和0%;白鲢胚眙冷冻至-7℃,复温后的最高成活率为19.6%。造成胚胎损伤、致死的主要原因是胚胎在降温过程中细胞严重失水。     

泥鳅胚胎玻璃化液超低温冷冻保存研究

泥鳅胚胎（胚孔封闭期）用10＃玻璃化液作为低温保护剂，经低温（－196℃）冷冻保存17h后，38℃水浴解冻，解冻后的胚胎获得了成活。在稀释液B1、B2、B3中分别有26．7％，15．0％，3．0％成活的胚胎。解冻后的胚孔封闭期胚胎经过50h的培养，胚胎从胚孔封闭期发育至尾鳍出现期。     

温度和盐度对大泷六线鱼仔鱼存活与生长的影响

研究了温度和盐度变化对大泷六线鱼Hexagrammos otakii Jordan et Starks仔鱼存活与生长的影响.结果表明,大泷六线鱼初孵仔鱼及10日龄仔鱼在温度4～16℃范围内都能存活与生长,存活率均在84％以上,温度为20℃试验组的存活率均低于50％.盐度渐变条件下,盐度10～30范围内存活率都大于90％,在盐度为5及以下时仔鱼的存活率降为0,仔鱼对高盐度的适应能力较差.盐度突变条件下,盐度25和30的试验组仔鱼的存活率都在95％以上,盐度10～20的试验组仔鱼出现不同程度的死亡,存治率可达56％～75％.大泷六线鱼仔鱼的最适生长温度为12～16℃,最适生长盐度为25～30.     

温度对云纹石斑鱼(Epinephelus moara)胚胎发育和仔鱼活力的影响

研究了不同孵化温度(16℃、18℃、20℃、22℃、24℃、26℃、28℃和30℃)对云纹石斑鱼(Epinephelus moara)胚胎发育的影响,记录并分析不同温度处理组受精12h后的胚胎发育时期、受精卵的孵化周期、孵化率和初孵仔鱼的畸形率,并对初孵仔鱼进行不同温度下的饥饿耐受性实验,测定其每天的存活率和生存活力指数(Survival activity index,SAI).结果显示,当温度在18-28℃范围内时,胚胎均可孵化出仔鱼,且孵化时间随温度的升高而缩短,孵化时间y与温度x呈极显著的负相关关系,y=12139x-1869,R2=0.993(P＜0.01).22℃时受精卵孵化率最高,为71.01％,该温度下对应最低畸形率(9.70％),其次为24℃时,受精卵孵化率为65.37％,畸形率为13.07％;温度低于22℃时,孵化率逐渐降低,18℃时的孵化率为24.39％;温度高于22℃时,孵化率亦明显降低,28℃孵化率最低,为16.11％,而畸形率的变化趋势与孵化率相反.温度为16℃时,受精卵发育至高囊胚期后不再继续发育;温度为30℃时,胚胎停止于胚体形成期.仔鱼的SAI值随着温度的升高先升高后降低,在20℃时,SAI值最高(25.97),仔鱼的半数死亡时间最长为7 d;22℃时,SAI值有所降低(22.38),仔鱼在该温度下半数死亡时间为6d.分析结果可知,云纹石斑鱼受精卵培育的最适温度为22-24℃,仔鱼孵化的最适温度为20-22℃.     

史氏鲟人工繁殖及养殖技术（连载）

(二 )孵化管理 　　1.水温管理 　　对不同水温下孵化胚胎的出膜率统计表明，孵化水温 13～ 15℃时，出膜率为 25％左右， 17～ 19℃时为 65％， 20～ 22℃时为 30％左右。可见，在人工孵化条件下， 17～ 19℃时，胚胎死亡率低，发育正常，为孵化的适宜温度范围。 13～ 15℃发育缓慢，时间延长，坏死的胚胎数增多。 9～ 11℃时，胚胎发育至大卵黄栓期，几乎全部停滞。 24～ 25℃时，开始发育较快，同时畸变率也较高，约有 1/3，最后接近 100％，最终导致全部死亡。因此史氏鲟的胚胎发育水温应尽可能控制在 17～ 19℃之间。 　　如果是在一…     

温度对海捕野生大黄鱼精子活力的影响

给7尾体质量325~520g的成熟海捕野生大黄鱼人工催产后采精液,在20℃(室温)和4℃(冰箱)下保存0、1、3、6、12、24、36h,以砂滤海水为激活剂,观察大黄鱼精子活力,以精子的激烈运动、摇尾运动、缓慢运动和寿命作为评价指标。试验结果表明,4℃和20℃下随保存时间的延长,精子活力逐渐减弱;4℃下保存24h、20℃下保存12h,精子仍具有激烈运动的能力;4℃下保存的3个运动阶段和寿命较20℃下均有所延长,4℃保存大黄鱼精子更佳。     

日本鬼鲉胚胎及卵黄囊仔鱼发育过程中脂肪及脂肪酸特性变化

以野生日本鬼鲉人工催产获得的受精卵为实验材料,定量检测了日本鬼鲉胚胎及卵黄囊仔鱼发育过程中脂肪组成及脂肪酸含量.结果表明:日本鬼鲉胚胎及卵黄囊仔鱼的总脂肪含量为13.85％～ 11.66％,极性脂肪占总脂肪含量为75.39％～ 72.20％.总脂肪及极性脂肪含量在胚胎发育阶段无显著变化,在卵黄囊仔鱼阶段随发育而显著下降.中性脂肪含量在胚胎发育阶段有显著变化,在卵黄囊仔鱼阶段其含量相对稳定.野生日本鬼鲉胚胎及卵黄囊仔鱼总脂肪的主要脂肪酸为DHA(22∶6n-3),16∶0,ARA(20∶4n-6),EPA(20∶5n-3),18∶0和18∶1n-9.总脂肪及极性脂肪的DHA,ARA,EPA含量(mg/gDW)均随胚胎和卵黄囊仔鱼的发育而显著下降,且DHA和ARA含量均在胚胎囊胚期至尾芽期大幅降低.中性脂EPA和DHA含量随发育呈先升后降,其峰值分别出现在初孵仔鱼和2日龄(2DPH)卵黄囊仔鱼.中性脂ARA含量随发育逐步升高,峰值出现在3日龄(3DPH)卵黄囊仔鱼.在胚胎发育前期,总脂肪DHA和ARA相对EPA被选择性消耗,饱和脂肪酸(SFAs)和多不饱和脂肪酸(PUFAs)相对单不饱和脂肪酸(MUFAs)被机体选择性消耗,N-6PUFA相对N-3 PUFA被选择性消耗;在胚胎发育后期及卵黄囊仔鱼阶段,总脂肪EPA相对DHA和ARA被选择性消耗.在胚胎和卵黄囊仔鱼发育过程中,SFAs中16∶0相对18∶0被选择性消耗.胚胎发育后期阶段和卵黄囊仔鱼阶段,总脂肪MUFAs中16∶1相对18∶1被选择性消耗.实验表明日本鬼鲉胚胎及卵黄囊仔鱼发育阶段极性脂肪中DHA、ARA和EPA可以向中性脂肪中转移,胚胎和卵黄囊仔鱼对不同类别脂肪中的重要脂肪酸的消耗具有选择性,且其选择性与发育阶段相关.     

温度、盐度对似刺鳊鮈胚胎发育过程的影响

设置10℃、15℃、20℃、24℃、27℃、30℃6个温度组和0‰、2.5‰、5‰、10‰、15‰、太湖湖水组6个盐度组,探讨了温度和盐度对似刺鳊鮈(Paracanthobrama guichenoti)胚胎发育的影响。温度实验结果表明:似刺鳊鮈胚胎发育的起始温度(生物学零度)为9.5℃,适宜的发育水温为20～24℃。水温10℃时胚胎卵裂异常,未能进入原肠期;水温升至30℃时胚胎在发育至原肠期前后死亡。水温度为24℃时孵化时间仅为73.5 h,孵化率高达80%,畸形率为0,为最适孵化水温;水温对胚胎发育速率的影响后期较前期大。盐度实验表明:似刺鳊鮈胚胎对盐度有一定的耐受力,各盐度梯度下的胚胎发育速率差异不大,但孵化率的差异极显著。在水温18℃、盐度为2.5‰的水体中胚胎的孵化率为81.67%,高于太湖湖水组的孵化率,此盐度应为似刺鳊鮈胚胎发育较适盐度。     

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.     

Effects of salinity on the ions important and sodium‐potassium ATPase in osmoregulation,cortisol, amino acids,digestive and immune enzymes in Scylla paramamosain during indoor overwintering

Overwintering is an important part of the conservation of Scylla paramamosain, and salinity has an important effect on the conservation of S. paramamosain during overwintering. Three salinities (4‰, 12‰ and 25‰) were selected as the overwintering salinities to reveal the effects of different salinities on the relevant important ions in osmotic pressure regulation, plasma cortisol, digestive enzymes, immune enzymes and amino acids of S. paramamosain during indoor overwintering. Results indicated that after overwintering, Cl^‐, Na⁺ and the osmotic pressure of serum have the highest salinity at 25‰, and the lowest salinity at 4‰. Na⁺/K⁺‐ATPase activity and cortisol were found to increase with decreasing salinity. The activity of digestive and immune enzymes was highest at 25‰, and was the lowest at 4‰. The amount of total amino acids (TAA), umami amino acids (UAA) and essential amino acids (EAA) in 25‰ were significantly higher than in 4‰ and 12‰. After overwintering, the essential amino acid index (EAAI) in the salinity range of 12–25‰ was 54.04–59.00, compared to 48.56–54.04 in the salinity range of 4–12‰. As a result, S. paramamosain at 25‰ had higher digestion and immunity than at 4‰ and 12‰, due to requiring more energy for osmotic pressure adjustment. In addition, S. paramamosain at 25‰ had the best meat quality. The results of this study are helpful for aquaculture production for indoor overwintering of S. paramamosain.     

乌克兰鳞鲤精子超低温冷冻保存方法研究

为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4 ℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种“3步冷冻法”以及3种解冻温度(20、30、40 ℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12 ℃/min,解冻温度为30 ℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。     

史氏鲟精子超低温冷冻保存研究

比较了史氏鲟精子在3种不同配比浓度稀释液的保存效果。试验结果表明,配方Ⅲ作为稀释液,8%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存,5h后取出,38℃水浴解冻取得最好的冻后激活率,解冻后激活率为(52.3±3.5)%。解冻精子分别采用井水和激活液D(10mmol/L Tris+10mmol/L NaCl+25mmol/L Glu,pH 8.0)激活,进行人工授精。结果显示配方Ⅲ冻精采用激活液D激活授精获得最高受精率为68.56%,最高孵化率为52.91%。本次试验表明,1～2mmol/L范围内,低浓度K+比高浓度K+对史氏鲟精子保存有利;52～82mosmol/kg范围内,高渗稀释液有利于史氏鲟精子的保存;且激活授精方法是影响冻精受精率和孵化率的关键因素之一。     

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.     

脊尾白虾水通道蛋白基因4和11在碱度胁迫过程中的作用

水通道蛋白(aquaporin)是一种细胞膜上特异性转运水分子及其他中性代谢分子的膜蛋白家族,对生物的细胞内外渗透压稳定具有重要的调节作用。为了了解水通道蛋白在脊尾白虾(Exopalaemon carinicauda)应对碱度胁迫中的作用,本研究利用RACE技术成功克隆了脊尾白虾水通道蛋白4 (aquaporin 4, EcAQP4)与水通道蛋白11 (aquaporin 11, EcAQP11)基因cDNA全长,EcAQP4基因的开放阅读框为621 bp,编码206个氨基酸,预测分子量为21.673 kDa,理论等电点为8.30,为疏水性蛋白,具有5个跨膜结构域;EcAQP11基因的开放阅读框长度为783 bp,编码260个氨基酸,预测蛋白分子量为28.490 kDa,理论等电点为5.40,为疏水性蛋白,具有4个跨膜结构域。序列比对结果显示,EcAQP4基因与罗氏沼虾(Macrobrachium rosenbergii)同源性最高,为94.63%;EcAQP11与斑节对虾(Penaeus monodon)同源性最高,为81.47%。为验证水通道蛋白的功能,利用RNA干扰技术特异性沉默EcAQP4和EcAQP11基因,结果显示,碳酸盐碱度胁迫后,注射干扰后的脊尾白虾死亡率显著升高,EcAQP4干扰组72 h死亡率达到45%,EcAQP11干扰组72 h死亡率达到55%,与对照组差异显著(P<0.05)。同时发现,EcAQP4干扰组在碳酸盐碱度胁迫24、48与72 h时的血液渗透压变化幅度显著高于对照组(P<0.05),72 h时渗透压显著升高(P<0.05);EcAQP11干扰组血液渗透压在3个时间点均显著升高(P<0.05)。以上结果表明,水通道蛋白在脊尾白虾应对碱度胁迫过程中起到了调节渗透压、维持体内外离子平衡的作用。     

Long-term cryopreservation of spermatophore of the giant freshwater prawn, Macrobrachium rosenbergii (de Man)

Long‐term cryopreservation of the giant freshwater prawn, Macrobrachium rosenbergii, spermatophores using glycerol (Gly) and ethylene glycol (EG) as cryoprotective agents (CPAs) was studied. The tolerance of sperm to cryopreservation was evaluated on the basis of sperm survival and fertilizing ability. The survival of the sperm was determined by trypan blue staining, while the fertilizing ability was assessed from artificial insemination of the cryopreserved spermatophores. The rates of embryo survival on day 5 after spawning and of spermatophores capable of producing embryos survived to hatching were determined. Storage of spermatophores at ?20°C without CPA for a short period of up of 1–5 days decreased the sperm survival significantly and did not preserve fertilizing ability. Preservation at ?20°C in the presence of 10% or 20% Gly or of 10% or 20% EG offered a simple and efficient short‐term storage up to 10 days. For a long‐term storage, cryopreservation in the presence of 20% EG at ?196°C was more efficient than at ?20°C. High sperm survival rates and high fertilizing ability were recorded from those cryopreserved at ?196°C for up to 150 days. High sperm survival rates with moderate levels of fertilizing ability were obtained from those cryopreserved at ?20°C for not more than 30 days. The results indicate that preservation at ?196°C with 20% EG is a suitable procedure for long‐term storage of the giant freshwater prawn spermatophores.     

鱼类配子和胚胎冷冻保存研究进展及前景展望

种质资源是水产养殖生产、优良品种培育及水产养殖业可持续发展的重要物质基础。我国是一个水生生物种质资源较为丰富的国家 ,丰富多样的水生生物种质资源和遗传多样性对于我国水产养殖业的快速发展起到了非常重要的作用。然而 ,由于渔业资源的过度捕捞、无序利用及人工放流等 ,造成了某些鱼类资源的衰退和濒临灭绝 ,如不及时采取保护措施 ,若干年后 ,在自然界中将难以找到上述鱼类原种、良种的遗传资源 ;由于忽视鱼类种质保护及品种选育的工作 ,养殖鱼类近亲交配越来越严重 ,造成种质退化 ,遗传多样性减少 ,生长速度减缓 ,品质下降 ,对病害…     

中华绒螯蟹胚胎的玻璃化冷冻保存

研究了中华绒螯蟹(Eriocheir sinensis)4个不同发育时期胚胎对A号玻璃化液的耐受性和玻璃化冷冻保存。结果表明,不同时期的胚胎对玻璃化液的耐受能力不同,其中卵裂期胚胎对玻璃化液的耐受能力较差(20~30 min),前无节幼体期和原溞状幼体期胚胎在玻璃化液中的适应时间较长(40~60 min);随着平衡时间的延长,中华绒螯蟹各个时期的胚胎成活率逐渐下降。中华绒螯蟹前无节幼体期胚胎在A号玻璃化液中平衡40 min,0.25mol/L的蔗糖分别洗脱5、10、15、20 min后,胚胎成活率无显著性差异(P>0.05)。前无节幼体期胚胎在A号玻璃化液中平衡40 min,在–196℃冷冻40 min,快速解冻后用0.25 mol/L的蔗糖洗脱10 min,有8个胚胎成活,成活率为(9.3±2.5)%,胚胎培养至第4天死亡;原溞状幼体期胚胎在A号玻璃化液中平衡40 min,在–196℃冷冻35 min,经相同浓度的蔗糖洗脱相同的时间,有7个胚胎成活,成活率(11.3±3.6)%,培养至第6天时,1个胚胎孵化出膜,出膜胚胎成活1d后死亡。     

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。