诱导虹鳟鱼四倍体的初步研究

利用静水压法和热休克法对虹鳟鱼(Oncorhynchus mykiss)受精卵进行处理,诱导四倍体.对发育至原肠后期的胚胎进行染色体制备并计数,结果显示热休克处理组的四倍体诱导率为5.5%,静水压处理组的诱导率为22.8%,静水压的诱导效果明显优于热休克.最佳的开始处理时间为受精后340min,静水压力为650 kg/cm2,处理持续时间为5min.     

四倍体草鱼的诱导和存活

采用热和静水压刺激草鱼受精卵诱导四倍体。在21C受精后36、39和42分钟,对受精卵进行9000和10000psi(磅/平方寸)静水压处理,鱼苗孵出后一天测定四倍体的诱导率为25%～100%(平均52.5%)。最适处理时间一般在卵裂前15～20分钟,这时能抑制核分裂。在受精后65、66和70分钟,对多细胞受精卵进行压力刺激(9000和10000psi),也可以诱导四倍体,受精后33～60分钟,对受精卵进行1.25和1.5分钟42C热刺激,四倍体的诱导率为0～100%;而同样温度的1分钟热刺激则不能产生四倍体。总之,大多数处理组的孵化率完全不同,且直接和四倍体转化有关。四倍体鱼苗存活天数没有超过50日龄。可以认为死亡鱼苗中有部分为非整倍体(次四倍体)和2n～4n嵌合体所引起。     

半滑舌鳎四倍体鱼苗的诱导与鉴定

采用静水压法处理半滑舌鳎(Cynoglossus semiliaevis)受精卵以抑制其卵裂并进行染色体加倍,筛选出有效的静水压处理起始时间、处理强度及其持续时间。结果表明,孵化水温(23±0.2)℃时,授精后21.5 min,采用40 MPa的静水压压力,休克处理4.5 min,四倍体诱导效果最好,鱼苗四倍体率达到68.3%。采用流式细胞仪分析了四倍体鱼苗细胞DNA含量,表明四倍体鱼苗细胞DNA含量为二倍体对照鱼苗的2倍。通过染色体制作分析表明四倍体鱼苗的染色体数为84条,而二倍体对照鱼苗的染色体数为42条。本研究采用静水压方法,在国内外首次建立了半滑舌鳎四倍体诱导方法。本项技术的建立旨在为大量生产半滑舌鳎三倍体不育群体奠定基础。     

热休克法诱导日本沼虾四倍体的初步研究

以热休克抑制受精卵第一次卵裂，进行日本沼虾四倍体诱导实验，热休克温度在３８、３９、４０和４１℃，于产卵２１０￣２３０分钟后，处理１￣２分钟均可获得四倍体胚胎，４０℃处理１．５分钟，四倍体胚胎诱导率达３６．８％，３８和３９℃处理２分钟后所获得的嵌合体胚胎比率大于四倍胚胎比率，而热休克温度４０℃时，四倍体率显著高于合体比率，４１℃处理时由于有丝分裂异常导致胚胎死亡率高，更适宜的处理时间有待进一步研究，     

近江牡蛎受精过程、胚胎及早期幼虫发育特征

为了解近江牡蛎(Crassostrea ariakensis)从受精到早期幼虫发育阶段的发育特性，在水温22～24℃、盐度20～22的实验室环境中对采自山东东营丁字湾海域的近江牡蛎进行人工授精，利用普通光学显微镜和荧光显微镜对近江牡蛎受精、早期胚胎和幼虫发育过程中的形态和核行为变化进行观察，并对这一过程中牡蛎的壳长变化进行了统计分析。结果表明，近江牡蛎成熟的未受精卵呈梨形，卵径为50μm左右，核相处于第一次成熟分裂中期。受精卵在受精后15～20 min、25～30 min,先后排出第一、第二极体，完成第一次和第二次成熟分裂，在受精后30 min左右先后形成雄原核和雌原核；雌、雄原核各自形成染色体组，在卵子中央发生联合，染色体排列于赤道板上，形成第一次有丝分裂的中期分裂相；受精后45 min左右，受精卵进行第一次卵裂，形成2个体积不等的卵裂球；受精75 min后，受精卵开始第二次卵裂，90 min后形成4个卵裂球，进入4细胞时期；6 h后受精卵发育至桑葚期，8～10 h进入囊胚期，12～15 h后发育至担轮幼虫，27～30 h左右发育成D形幼虫，5 d左右进入壳顶幼虫时期，17 d后幼虫...     

刺参受精及早期胚胎发育过程的细胞学观察

采用HOECHST 33258染色荧光显微方法,对刺参成熟未受精卵以及受精过程中精子入卵、极体排放、雌雄原核的形成与结合、早期卵裂以及多精入卵等细胞学进行了研究。结果显示,刚产出的刺参成熟未受精卵呈圆形,核相处于第一次成熟分裂中期;在水温22~23 ℃、盐度29条件下进行受精,受精后12 min,完成第一次成熟分裂,释放第一极体;受精后20 min,大部分受精卵完成第二次成熟分裂,放出第二极体。受精后35 min,雌、雄原核开始在卵中央发生染色体联合;受精后80 min,部分受精卵完成第一次卵裂,受精后100 min,部分受精卵完成第二次卵裂。刺参在受精过程中存在极少数的多精入卵现象。     

斑马鱼雄核发育技术研究

为探索人工诱导斑马鱼雄核发育二倍体克隆鱼的途径和方法,运用紫外线照射卵子使其遗传物质失活,利用斑马鱼体表花纹形态作为遗传标记,使用热休克技术阻止受精卵的第一次卵裂加倍染色体。试验结果表明总辐射剂量为120mJ cm2紫外线照射斑马鱼卵可以使雌核遗传物质完全失活;体表花纹为豹斑花纹被证实是由纯合隐性基因控制的性状,可以作为雄核发育遗传标记;遗传失活卵受精后经2min,(41 5±0 5)℃的热休克后出现两个染色体加倍的高峰期,分别为受精后的11min和23min,且后一时期效果优于前一时期,最高二倍体诱导率达46 15%;经遗传分析,共获得正常存活的雄核发育二倍体克隆鱼19尾,说明经过优化的紫外线结合热休克等染色体操作技术,可以成为诱导雄核发育二倍体克隆鱼的有效手段。     

兴凯湖大白鱼人工繁殖及胚胎发育研究

2019年6月,通过人工催产获得兴凯湖大白鱼受精卵,系统地观察了胚胎发育的全过程。结果表明,在池塘养殖的条件下,人工养殖可获得成熟的亲鱼,人工催产获得的兴凯湖大白鱼的受精卵为圆球型,呈青灰色或黄绿色。平均卵直径为0.78 mm(0.65～1.02 mm),吸水后平均卵直径为4.02 mm。胚胎发育过程可分为23个阶段。在24.5～26.8℃范围内,受精42 min后开始第一次卵裂,受精10 h 45 min后开始形成器官。胚胎发育的总积温为486.98℃·h。     

彭泽鲫胚胎发育观察

通过干法人工授精获得彭泽鲫受精卵,在水温为(28±0.5)℃条件下连续观察彭泽鲫胚胎发育过程。结果显示,彭泽鲫卵子呈浅黄色,卵膜透明,近似圆形,黏性、沉性卵,卵径为(1.65±0.15)mm。发育过程中,受精后53 min进入卵裂期,受精后3 h 16 min进入囊胚期,受精后5 h 41 min进入原肠期,受精后8 h 55 min进入神经胚期,受精后12 h 03 min进入器官形成期,受精后37 h 26 min仔鱼孵化出膜,从受精到孵化出膜总积温1050.90℃·h。     

施氏鲟精子诱导匙吻鲟雌核发育

采用照射强度为10 188 μW/(cm2·s),照射距离为15 cm的紫外线对施氏鲟(Acipenser schrenckii)精子进行4～7 min的照射,用照射后的精子对匙吻鲟(Polyodon spathula)卵进行人工授精,以探索精子的最佳紫外线照射时间.设计热休克起始时间、持续时间和休克温度3因子、3水平的正交试验,以探索人工诱导匙吻鲟雌核发育的理想热休克条件.结果表明:用紫外线照射5 min处理的施氏鲟精子,与匙吻鲟卵受精所得胚胎经染色体观察均为单倍体(n=60),表明照射精子遗传失活的有效性和可靠性;若热休克处理前受精卵保持在(18±013)℃,在一定的休克温度(35～39℃)条件下,于不同时间(受精后16～20 min)起始热休克,持续处理2 min均能诱导受精卵染色体加倍.对热休克处理条件的优化结果表明,将受精卵于受精后18 min,在37℃的水中热休克处理2 min,二倍体诱导率最高,达18.8%.染色体鉴定显示,该条件下处理的胚胎均为二倍体(2n=120),未发现单倍体和非整倍体.本研究为进一步分析异源精子诱导匙吻鲟雌核发育机制以及匙吻鲟性别决定的分子机制奠定了基础.     

Hydrostatic pressure treatment during the first mitosis does not suppress the first cleavage but the second one

Suppression of cell division causes chromosome set doubling. Some chemical agents or physical shocks such as temperature or hydrostatic pressure are effective tools for suppression of cell division. As spindles are obviously inactivated or disorganized by these treatments, it has been supposed that inactivation or disassembly of spindles blocks the anaphase movement of chromosomes and a duplicated nucleus is formed without cell division. The present study demonstrated that hydrostatic pressure treatment (650 kg/cm² for 6 min) around the time of metaphase of the first cell cycle of the rainbow trout embryos did not suppress the first cleavage but the second one. Spindles disassembled by the hydrostatic pressure or heat shock regenerated soon after treatment, resulting in the occurrence of the first mitosis. Interestingly, a monopolar spindle was assembled in each blastomere in the second cell cycle, and disjunction of duplicated chromosomes and the cleavage was prevented, leading to the chromosome set doubling. From the third cell cycle, normal cell division resumed. No significant difference was found between the area of the nucleus plate of the treated embryos and twice the area of the nucleus plate of control embryos in the third cell cycle, meaning that the chromosome sets had been doubled at the end of the second cell cycle. The process of chromosome set doubling caused by heat shock seemed to be fundamentally the same as that caused by hydrostatic pressure. To the best of our knowledge, this is the first time that the mechanism of chromosome set doubling in animal eggs treated with hydrostatic-pressure or heat shock has been clarified.

Haploid–diploid or diploid–tetraploid mosaics sometimes occur among individuals treated for cleavage inhibition. The mechanism of such occurrence of mosaicism is, however, not clear. In this study, we found interesting two-cell stage embryos, which had a monopolar spindle in one blastomere and a bipolar spindle in the other during the second mitosis in a batch subjected to tetraploidization treatment. These embryos have a high potential of developing diploid–tetraploid mosaics. This paper also discusses the mechanism of occurrence of these aberrant embryos and discusses their relationship to diploid–tetraploid mosaicism.     

Induction of Triploidy and Tetraploidy in Nile Tilapia, Oreochromis niloticus (L.)

Abstract.— Induction of triploidy and tetraploidy in Nile tilapia, Oreochromis niloticus , was investigated by heat shock, cold shock, hydrostatic pressure, and/or chemicals (cytochalasin A, B, and D). Additionally, efficacy of combined protocols was determined. Heat shock 10 min after fertilization induced triploidy when incubation temperature was 24 C but not when incubation temperature was 31 C. Heat shock of 40–41 C at 4–6 min after fertilization was effective in inducing up to 100% triploidy with hatchability similar to controls. Cold shock at 13 C for 45 min five min after fertilization induced 85–100% triploids. Heat shock and multiple heat shocking were the most effective treatments for the induction of tetraploidy. Two heat treatments of 41 C applied at 65 and 80 min after fertilization for 5 min each produced approximately 80% tetraploidy in hatched fry. Immersion of fertilized eggs in cytochalasin A, B, or D at concentrations up to 10 μg/L applied at various times and durations was ineffective in inducing triploidy or tetraploidy.     

静水压休克法诱导三倍体鲶鱼（silurus asotus L）的研究

采用静水压休克法诱导三倍体鲶鱼。通过对受精时间、静水压力及持续施压处理时间三方面进行筛选试验的结果表明，鲶鱼卵受精4－5min，用600－649kg/cm^2的静水压力处理3min，可以获得100％的三倍体鲶鱼，而且胚胎存活率也较高，孵化率达对照组的90％以上，是静水压休克法诱导三倍体鲶鱼的最佳条件。三倍体鲶鱼的倍性用细胞遗传学方法验证。     

Double heat shock more potently suppresses cleavage of fertilized willow minnow (Gnathopogon caerulescens) eggs than single heat‐shock treatment

This study examined the effects and optimal conditions of double heat‐shock treatment (HST) for the suppression of cleavage in fertilized willow minnow (Gnathopogon caerulescens) eggs. In addition, we performed a cytological analysis of the nucleus and mitotic apparatus dynamics before and after HST. Tetraploidy occurred at the highest frequency (45.4%) when the first HST was delivered 25 min after fertilization and the second 15 min later. In this group, the spindle atrophied immediately after completion of the second HST; subsequently, three distinct patterns were observed in the nucleus and mitotic apparatus of the first cell cycle. In the first pattern, one pole disappeared and a monopolar spindle formed; chromosomes were unable to separate and accumulated at one pole where they formed a large nucleus. In the second pattern, the poles were dispersed or localized to one side of the nucleus, resulting in the failure of chromosomes to separate and the formation of a single nucleus. In the third pattern, the first cleavage was completed normally. Thus, double HST can block the first cleavage division by eliminating one centrosome or bringing two poles close together, giving rise to a subset of tetraploid embryos.     

Polypolar spindle formation during first cell cycle in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> embryos after heat-shock treatment

ABSTRACT:   Heat shock has been used to inhibit cleavage for the induction of monogenic diploids or tetraploids in animals, but usually the success rate is low. Heat-shocked rainbow trout Oncorhynchus mykiss embryos were used in this histological study to clarify the causes of this low success rate. Embryos treated with hydrostatic pressure were used for comparison. After heat shock had disorganized the spindles, polypolar (tripolar or tetrapolar) spindles in addition to bipolar spindles were often reassembled soon after treatment. The embryos then completed tripolar or tetrapolar division at the first mitosis, and directly turned into three- or four-cell embryos as a result of the first cleavage. During the second mitosis, a monopolar spindle was formed in each blastomere of four-cell embryos and approximately 60% of three-cell embryos. In the remaining three-cell embryos, two of the three blastomeres formed a monopolar spindle, and the third one formed a bipolar spindle. The formation of polypoles is assumed to be caused by insufficient disorganization of daughter centrioles and splitting from the mother centriole by heat shock. Polypolar division is considered to be the cause of aneuploidy and the low success rate of chromosome set doubling. In the case of hydrostatic pressure treatment, the regenerated spindles were bipolar in almost all embryos.     

Production of all-female triploid rainbow trout

Triploid rainbow trout were produced by heat shock treatment of eggs soon after fertilization with either normal sperm or sperm from masculinised females. The proportion of triploid fry, as judged from red blood cell nuclear volume, varied between 75% and 100% in three experiments using different batches of eggs from an autumn-spawning strain of trout while a single batch of eggs from a winter-spawning strain yielded 50% triploids.

A microscopic examination of the gonads was made on 5-month-old fish weighing between 1 and 3 g. In female controls the ovaries were packed with oocytes while those from female triploids, although showing the typical lamellar structure of an ovary, contained no oocytes, thus indicating that female triploids are sterile. The testes from triploid males appeared to be developing normally.

The use of masculinized females combined with heat shock treatment of eggs to produce triploids, allowed the production of sterile all-female triploids. This should have considerable potential for aquaculture.     

三倍体大黄鱼的诱导及其对生长、性腺发育的影响

自20世纪50年代以来,采用染色体操作技术人工诱导多倍体在鱼类遗传育种领域已得到广泛的应用。国内外已先后在三棘刺鱼[1]、鲤[2]、水晶彩鲫[3]等30多种鱼类成功获得三倍体。在理论上,由于三倍体性腺发育受阻,其用于性腺发育的能量可全部用于生长。因此鱼类育种学家期望通过诱导三倍体,使经济鱼类生长更快,经济效益更高。但历经30余年的研究,诸家看法仍未统一。一些学者认为三倍体鱼比二倍体生长快[4,5],另一些学者则认为三倍体鱼并不比二倍体生长快[6,7]还有一些学者的研究表明三倍体鱼在性成熟以后比二倍体生长稍快[8,9]。对于三倍体…     

Induction of triploidy and tetraploidy in stinging catfish, Heteropneustes fossilis (Bloch), using heat shock

Induction of triploidy and tetraploidy was attempted in Heteropneustes fossilis using heat shock. The optimal age of zygote, temperature level and duration of thermal shock required for effective induction of triploidy and tetraploidy was investigated in a series of experiments. A maximum of 82±7% triploids (3n=87) were obtained when fertilized eggs (2.5‐min old) were heat shocked at 40°C for 4‐min duration. A maximum of 40±8% tetraploids (4n=116) was obtained when the fertilized eggs (30‐min old) were heat shocked at 40°C for 4‐min duration. The triploid and tetraploid red blood cells (RBCs) nucleus volumes were 1.4 and 2.1 times greater, respectively, than that of the diploid RBC nucleus.     

缢蛏受精和早期卵裂过程的细胞学变化观察

采用普通光镜、荧光显微镜和石蜡切片技术,对缢蛏受精和早期卵裂过程中的精子入卵、减数分裂、雌雄原核形成与结合、早期卵裂以及多精入卵等细胞学事件进行了显微观察。结果表明,缢蛏成熟未受精卵多呈圆球形或卵圆形,少数呈梨形,卵径为82～88μm,核相处于第一次成熟分裂中期;精子为典型的原生型,全长55～58μm,头部呈保龄球形,顶体前端的顶体杆呈特别细长的花丝状;在水温21～22℃、盐度10的条件下进行人工授精,精、卵混合后,精子迅速附着于卵子表面,启动卵子发育;受精后4～6 min,精子的头部已进入卵内并明显膨胀,卵子外形变圆,卵外附着的精子量明显减少;在受精后12～15 min、20～25 min,受精卵先后排出第一极体、第二极体,完成两次成熟分裂;第二次成熟分裂结束以后,精、卵核体积迅速膨胀,雄原核的膨胀早于雌原核,核膜重建,在受精后约30 min形成雌、雄原核;雌、雄原核均向卵子中央移动,雄原核旁边的精子星光清晰可见,随后二者在卵子中央以染色体联合的方式结合,联合核的染色体共同排列在纺锤体的赤道板上,形成第一次有丝分裂的中期分裂相;受精后40 min左右,受精卵进行第一次卵裂,染色体在纺锤丝的牵引下向两极移动,结果形成2个大小不等的卵裂球;受精后45～50 min,卵子进行第二次卵裂,核相变化与第一次卵裂相同,在与第一次卵裂垂直的纵轴方向上发生不等全裂,最终形成3小1大4个卵裂球;受精后60～70 min,胚胎进行第三次卵裂,仍为不等全裂,但自此次卵裂起已开始进行螺旋分裂。此外,对实验中发现的缢蛏极少量多精入卵、多极分离等异常细胞学现象进行了分析,并探讨了海洋贝类卵子阻止多精入卵发生的机制。