大西洋牙鲆冷冻精子×褐牙鲆卵杂交胚胎的发育及胚后发育

利用大西洋牙鲆冷冻精子与褐牙鲆卵授精，培育出了生长健康的杂交鱼苗。并对杂交后代的胚胎发育、胚后发育和不同温度下的生长特征进行了研究。利用OLIMPUS显微镜连续观察和描述了冷冻精子受精杂交胚胎的发育，在水温15～17.1 ℃，其受精卵经过卵裂、囊胚、原肠胚、胚孔封闭、肌节胚、尾芽胚、心跳胚、出膜胚等时期的发育，经历65 h 57 min完成胚胎发育进入胚后发育，发现胚胎搐动15 min后心脏开始跳动。利用OLIMPUS解剖镜每天观察和记录一次鱼苗的生长形态，并测定其体全长；将杂交鱼苗的胚后发育划分为卵黄囊吸收期（出膜后1～6 d）、变态前期（出膜后7～25 d）、变态期（出膜后25～30 d）和变态后生长期（出膜30 d以后）4个时期。对16～17 ℃、18～19 ℃、20 ℃和22～23 ℃4个温度环境下胚胎发育的研究结果显示，杂交胚胎宜于在16～20 ℃的水温下发育，鱼苗在22～24 ℃孵化出膜后2～3 d大量死亡。对18～19 ℃、20 ℃、22 ℃、和24 ℃ 4个温度下稚鱼的生长研究结果显示，变态期稚鱼在22～24 ℃水温下饲养时变态和生长速度较18～19 ℃快。     

鱼类胚胎冷冻保存试验初报

用不同处理方法对红鲫、泥鳅不同时期胚眙进行冷冻保存试验。结果表明：加葡萄糖、NaCl、DMSO组成的保护液具有较好的防冻效果。红鲫心跳期和眼色素出现期的胚胎解冻后存活率分别为26．8％和18．7％；泥鳅心跳期和出膜前期的胚胎存活率分别为14％和11．1％。红鲫受精卵至神经胚期，泥鳅受精卵至眼基出现期解冻后无存活胚胎。     

罗非鱼精液的冷冻贮存

本文叙述了莫桑比克罗非鱼的精液在以5％的甲醇和15％的奶粉作低温防护剂时的冷冻贮存方法。用此法冷冻精子解冻后精子的运动能力大约是以5％或10％的二甲亚砜(DMSO)代替甲醇作低温防护剂时的二倍。本文还详细描述了低温防护剂的最适浓度,冷却和解冻加温的最适速度以及稀释度和平衡时间对精子的影响。还报导了受精能力的试验:以冷冻贮存的精子受精后的胚胎有64.3％±34.2％可以正常发育到胚孔封闭期而未经冷冻的对照组为57.5±10.5％。     

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。     

热应激处理对不同发育时期大菱鲆胚胎的影响

<正>对大菱鲆5个不同发育时期:桑椹期、低囊胚期、原肠晚期、胚孔关闭期和尾芽期分别进行热应激处理。设计温度为40℃和30℃,热应激处理3分钟。结果表明:40℃处理3分钟,所有胚胎在处理后全部死亡;30℃处理3分钟,无胚胎在处理后死亡,但是尾芽期胚     

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。     

江鳕胚胎发育的观察

研究了人工繁殖时江鳕（Lota lota）的胚胎发育。江鳕的胚胎发育分为胚盘期、卵裂期、囊胚期、原肠期、神经胚期、器官形成期6个阶段,20个发育时期,详细描述了各个发育时期的特征。在0~3℃下,胚胎历时998h孵出仔鱼,从受精卵至孵出的积温为887~1023h.℃。本研究还比较了江鳕自产与人工授精时卵的受精率的差异。结果显示,人工授精卵的受精率（约30%）比自产（19.4%）高10%。江鳕胚胎发育的不同时期对外界环境的敏感性不同,主要经历了3个死亡高峰,包括胚盘期至二细胞期、桑椹胚期至原肠早期、出膜期。     

西昌白鱼雅砻亚种胚胎发育研究

西昌白鱼(Anabarilius liui)为中国特有种,隶属鲌亚科,白鱼属,共记载有16种和亚种,大多分布于云南,在四川省仅分布于凉山州,共有2个种和亚种。西昌白鱼(雅砻亚种)(A. liui yalonggensis)是雅砻江特有种,是增殖放流品种之一,但自命名后多次调查均未采集到,资源量情况不容乐观。通过人工干法授精获得西昌白鱼(雅砻亚种)受精卵,在恒定水温(14.4 0.8)℃条件下进行孵化,对其胚胎的发育过程进行了观察和描述,旨在为该鱼的科学养护提供技术支撑。结果表明：西昌白鱼成熟卵(n=10)卵径为(1.83 0.15)mm,沉性,粘性,接近无色透明,吸水充分膨胀后卵径为(2.53 0.07)mm。在波动水温“昼(21.5 1.7)℃-夜(15.0 1.2)℃”、恒定水温(23.5 2.2)℃条件下胚胎均能不能成功孵化,胚胎均发育至肌肉效应期即全部死亡。在水温(14.4 0.8)℃的条件下,胚胎受精后1h30min胚盘隆起,14h55min和35h45min进入囊胚期和原肠胚期,46h5min胚孔封闭,158h35min开始出膜。胚胎发育积温为2284.4℃?h。初孵仔鱼(n=10)全长(5.77 0.24)mm。西昌白鱼(雅砻亚种)胚胎较鲌亚科其他种类,具有较大的卵径、脊索形成期在胚孔封闭期之前等特征,是其对雅砻江环境的一种适应和进化。     

马氏珠母贝精子的超低温保存

通过比较不同pH值（5．5-9．5）、不同盐度的海水等作为基础液对马氏珠母贝精子保存的影响，选择其中无激活精子作用又对其生理特性（活力，寿命，受精能力等）无影响者，加入二甲亚砜抗冻剂配制成超低温保存的抗冻保护液，精液与保护液按1：10和1：20的比例混合，样品按4组不同的降温程序平衡后转入液氮冷冻，比较其超低温冻存的效果。结果表明：以海水（pH7．5—8．0）为基础液，配制10％DMSO作为抗冻保护液，精液与保护液比例为1：20，在低温（0-4℃）中平衡约30min，在距液氮面15cm、5cm处分别停留5min、10min后转入液氮（-196℃），冻存精子效果良好。冷冻24h、48h及5个月后，在38—40℃下水浴解冻复苏后，用终浓度0．25‰的氨海水刺激，精子存活率可超过60％，受精率可达80％。     

斑马鱼性别决定相关基因的表达分析

本研究选择一些可能参与斑马鱼性别决定相关的基因（cyp19a、cyp19b、gata4、dmrt1和wt1），通过RT-PCR和实时荧光定量PCR技术分析各个基因在斑马鱼发育不同时期（64细胞期(2h)、256-512细胞期(2.5h)、囊胚球体期(4h)、30%外包(4.6h)、50%外包(5.2h)、75%外包(8h)、胚孔封闭期(10h)、10-15体节期(15h)、成型期10-20 (32h)、孵化期(60h)、幼苗期(120h)、7天、10天、12天、14天、16天、18天、20天、22天、24天、26天、28天和30天）的表达情况。结果显示：cyp19a与cyp19b基因随着胚胎的发育表达量逐渐增加，在28天-30天达到最高；gata4基因在胚孔封闭期表达量最高随后降低；dmrt1基因在30%外包期表达量最高随后逐渐递减；wt1基因在10-15体节期表达量最高随后逐渐递减并保持稳定的表达。     

Feeding and gut evacuation of cultured juvenile spiny lobsters, Jasus edwardsii

Low feed consumption has been suggested as a major impediment for the development of effective formulated diets for spiny lobsters. To seek an explanation for the low feed consumption, this study compared the feed consumption, appetite revival, foregut evacuation and fecal production of different sizes of spiny lobster Jasus edwardsii juveniles fed a fresh natural diet (i.e. mussel flesh) and a dry formulated diet. In addition, the effect of feeding frequency was determined by feeding juvenile lobsters the same ration of formulated diet at different time intervals. i.e., same ration delivered 3 week^− 1, 1 day^− 1, 2 day^− 1, 5 day^− 1 and 5 night^− 1. Lobster (10–15 g) growth and feed consumption (1.15% BW day^− 1) was maximized when receiving a single ration daily of the formulated diet. A slow appetite revival (AR > 18 h) on the formulated diet was consistent with the negative effect of increasing feeding frequency on growth and consumption. In addition, there appeared to be no major advantage in dry matter intake by providing the nutrient dense dry formulated diet to Jasus edwardsii. Lobsters fed fresh mussel flesh (79.8% moisture) consumed similar levels of dry matter as those lobsters fed the dry formulated feed (7.3% moisture). This appears to be due to high moisture gain and expansion of the dry feed in the lobster foregut. Foregut evacuation lasted 10 h for the two diets but a delay in initial foregut evacuation and processing of the formulated diet was evident, and may have negatively affected feed consumption, appetite revival and dry matter digestibility (61.2%) of the dry formulated diet compared to the mussel flesh diet (89.2%). Appetite revival on mussel flesh was almost complete by 12 h after a satiation ration and was governed primarily by the rate of foregut evacuation. Differences in the timing of foregut evacuation and fecal production between the two diets suggested that a greater proportion of the formulated diet entered the midgut directly.The results indicate that J. edwardsii cannot achieve high levels of feed consumption on formulated diets because gut throughput is reduced due to a small foregut capacity, expansion of the feed post-ingestion, lengthy foregut filling time (1–2 h) and clearance time (10 h), combined with slow gut throughput time (34–42 h) and appetite revival (> 18 h). A slow appetite revival and difficulties in processing and digestion of formulated diets appear as major issues to be resolved to improve the performance of formulated diets required for the commercial aquaculture of J. edwardsii and possibly other spiny lobsters.     

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.     

达氏鲟精巢细胞消化分离和超低温冷冻保存

通过研究两种酶对精巢细胞的消化效果,探究抗冻剂、降温程序、糖类和卵磷脂对达氏鲟(Acipenser dabryanus)精巢细胞冻存效果的影响,获得消化冻存后高存活率的细胞。实验使用0.25%胰蛋白酶和2 mg/m L胶原酶H+500 U/m L中性酶II的组合酶对达氏鲟精巢细胞进行消化,获得不同消化时间内的活细胞数量。另外,还研究了冷冻稀释液中分别添加10%二甲基亚砜(DMSO)、乙二醇(EG)、甲醇(MET)作为抗冻剂,采用-1℃/min慢速降温以及直接投入液氮中的快速降温方法冻存,冷冻稀释液采用D-海藻糖或同浓度D-蔗糖,以及添加5%、8%、11%卵磷脂对冻存效果的影响。结果显示:两种消化酶在同一时间消化所得的活细胞数和活细胞率无显著差异,并且都在3 h获得最多活细胞。慢速降温的冻存效果极显著地好于快速降温(P=0.01)。不同抗冻剂的保存效果差异显著,复苏后细胞相对存活率EG(51.70%±5.24%)MET(45.09%±3.15%)DMSO(40.18%±3.90%)。不同糖对达氏鲟精巢细胞冻存效果无显著影响;不同浓度的卵磷脂冻存效果有极显著差异。含8%卵磷脂的冻存液对细胞的冻存效果最好,细胞存活率可达(93.55±2.56)%,培养10 d后细胞数目为0 d时的3.19倍。     

黑尾近红鲌精子低温保存方法研究与应用

在4℃条件下,以精子活力为指标,研究了不同浓度的Na~+、K~+、Ca~(2+)、Mg~(2+)、葡萄糖、氨基酸等组成的5种(A、B、C、D、E)精子保存液及其适量添加青霉素对黑尾近红鲌(Ancherythroculter nigrocauda)精子活力的影响。结果显示:1)温度为4℃时,精子活力达80%的保存液A、B、C、D、E的保存时间分别为48、24、60、48、48 h,保存液C的保存时间明显高于其它各组;2)在各保存液中分别添加浓度为2.0×104IU/m L的青霉素,精子活力达80%的保存液A、B、C、D、E的保存时间分别为60、36、156、48、144 h,保存时间延长了12~96 h,保存液C和E的延长时间最长,均为96 h;3)人工授精试验证明,经保存的黑尾近红鲌精子能正常用于人工繁殖,受精率达(90.6±0.8)%~(91.8±0.9)%,与对照组精子受精率(92.4±0.8)%无显著差异。添加青霉素,保存液C的保存效果最好,其次是保存液E。     

鱼类胚胎低温冷冻保存降温速率研究

在低温下采用慢速降温速率，以０．２－０．５℃／分降至－４０℃以上温度，胚胎获得２０％以上成活率。在超低温下采用快速降温速率，以２℃／分降至－４０℃，再以１０℃／分降至－１９６℃，胚胎获得了９０％以上复活率。     

Blocking polar body with cytochalasin B in the fertilized eggs of the small abalone, Haliotis diversicolor supertexta (Lischke), and the development and ploidy of the resultant embryos

The effects of blocking polar body I (PB1) or polar body II (PB2) with four different dosages of cytochalasin B (CB) on the development and ploidy of resultant embryos were studied in the small abalone, Halitis diversicolor supertexta (Lischke). To block the release of PBI, the fertilized eggs were treated with 0.25, 0.5, 1.0 or 2.0 mgL^?1 of CB for 10min beginning at 3 min post-fertilization at 24°C. To block the release of PB2, the fertilized eggs were treated under the same conditions as PB1, except that the treatment was begun 10min post-fertilization. In the control group, only 41.8% of the cells had a diploid number of 32 chromosomes, although spontaneous haploids (9.0%). tripolids (7.5%) and aneuploids (41.7%) were also observed. In CB treatment of PB1 and PB2 groups. 5.0-28.6% of the cells remained as diploid. triploids (10.0-18.9%) and aneuploids (41-1-61.0%). With regard to the development of the resultant embryos, the proportion of normal embryos in the control group was 87%, while in the treatment groups, the proportions of normal embryos in the FBI and PB2 groups were 57-58% and 53-56% in the 0.25 mg L^?1 and 0.5mg L^?1 CB treatments, respectively. From this data on induced triploids and the resultant development of normal embryos, the proportions suggest that 0.25-0.5 mg L^?1 of CB for 10min was sufficient for blocking the release of FB1 or PB2 to produce triploids in the small abalone.     

Effects of developmental stage, seawater concentration and rearing temperature on cryopreservation of Pacific oyster Crassostrea gigas larvae

ABSTRACT: For the development of a stepwise cryopreservation technique for larvae of the Pacific oyster Crassostrea gigas , various conditions were examined. Larvae at 9, 12, 15, 18 and 21 h after insemination were cooled at a rate of −1°C/min (seeding at −8°C for 15 min) and then plunged into liquid nitrogen at −35 or −40°C using 1.5 M dimethyl sulfoxide (DMSO) and 250 mM trehalose as cryoprotectants. Among these larvae, 15 h after insemination (the trochophore stage before formation of the shell gland) showed the highest motility and the best external appearance after thawing. Trochophore larvae were cryopreserved in preservation media containing different dilutions (1/4, 1/6, 1/8, 1/10 and 1/30) of seawater. Larvae preserved in the 1/4 seawater medium showed the highest appearance of shelled larvae 4 days after thawing. Trochophore larvae reared in seawater at 21, 25 or 29°C were cryopreserved for 8 months and then reared at 26°C after thawing. Larvae reared at 25°C showed the highest survival rate and normal larval ratio at day 6 after thawing, although larvae reared at 21°C showed the highest rates until day 4. One larva developed at 25°C succeeded to settle.     

Differences in the effectiveness of reproduction among ten breeding strains of the common carp Cyprinus carpio L. after stimulation of ovulation with carp pituitary homogenate or [(D‐Ala6, Pro9NEt) mGnRH‐a + metoclopramide] (Ovopel)

This study focused on the reproduction effectiveness of 10 breeding strains of common carp (Polish strains 2, 3, 6; Hungarian strains 0, W, 7; Lithuanian strain B; French strain F; Israeli strain D and Yugoslavian strain J) after stimulation of ovulation with carp pituitary homogenate or Ovopel. The percentage of females with recorded ovulation became higher after Ovopel treatment in as many as eight breeding strains (2, 3, 6, 0, W, B, F, D). The interaction between the spawning agent and the breeding strain was statistically significant (P ≤ 0.05) for the mass of spawn and non‐significant for traits determining the quality of eggs. Among the 10 breeding strains, in 4 (3, 7, B, J) the least‐squares means (LSM) for the weight of egg (g) were higher for fish stimulated with Ovopel. Among the strains tested, in seven (3, W, F, J, 2, 7, B), the LSM for the percentage of living embryos after 36 h incubation were higher for fish treated with Ovopel. The evaluation of reproduction effects based on the values of such parameters as the percentage of ovulating females, weight of eggs (g) and the percentage of live embryos (36 h) showed that after Ovopel treatment the poorer effects were obtained only for strains 6, 0 and D.     

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.