静水压法诱导虾夷扇贝多倍体的研究

为了探究采用静水压法诱导虾夷扇贝多倍体的适宜条件,研究了在不同的处理时机和压力强度条件下,静水压处理对虾夷扇贝受精卵多倍体诱导效果的影响。试验结果表明:(1)在授精后80 min内处理,所得的多倍体以三倍体为主。在授精后75 min,以60 MPa持续处理5 min,所得的三倍体率最大,达100%。(2)在授精后150~190 min处理,既能获得三倍体也能获得四倍体,但获得的四倍体率要高于三倍体率。其中,在授精后170 min,以65 MPa持续处理3 min时得到的四倍体率最大(54.39%)。     

半滑舌鳎三倍体鱼苗的人工诱导与鉴定

针对半滑舌鳎雌雄个体生长差异过大、雌性性腺发育成熟后腹部凸起影响舌鳎生长和商品鱼质量等问题,开展了人工诱导半滑舌鳎三倍体的研究。采用静水压处理抑制半滑舌鳎受精卵第二极体排出进行染色体加倍,筛选出有效的静水压处理强度及其持续时间。结果表明,孵化水温23 ℃左右时,授精后5 min,采用36 MPa的静水压压力,休克处理4 min,三倍体诱导率最高,达到100%。采用该诱导条件,大批量获得了半滑舌鳎三倍体鱼苗。采用流式细胞仪分析了三倍体鱼苗细胞DNA含量,表明三倍体鱼苗细胞DNA含量为二倍体对照鱼苗的1.5倍。通过染色体分析表明,三倍体鱼苗的染色体数为63条,而二倍体鱼苗的染色体数为42条。     

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。     

冷、热休克法诱导黄颡鱼三倍体的比较研究

分别采用冷、热休克抑制第二极体释放的方法诱导黄颡鱼三倍体。结果表明,在卵受精后2min,5℃处理20min,胚胎时期的三倍体率达70%左右,孵化率50%左右,幼鱼时期三倍体(含嵌合体)的检出率为25%,此条件为冷休克处理的优化参数;在卵受精后2min,40℃处理2min,胚胎时期的三倍体诱导率达58%,孵化率为39%,幼鱼时期三倍体(含嵌合体)的检出率为40%,此条件为热休克处理的优化参数。正交分析得出,冷休克条件下起始休克时间是原肠期三倍化率和孵化率的重要影响因子,温度对畸形个体的产生有重要影响;热休克条件下,参考三倍体率、畸形率、孵化期相对存活率三者而言,休克温度均是重要因素。比较观察到冷休克处理组的胚胎受损情况严重,后期的成活率较热休克处理组要低,总体诱导效果逊于热休克处理组。     

热休克诱导全雌虹鳟三倍体

以虹鳟二倍体的伪雄鱼为父本（遗传型为xx），探讨了采用热休克方法阻止第二极体排放诱导全雌虹鳟三倍体的适宜条件。结果表明：虹鳟三倍体诱导率明显受处理温度、起始时间以及持续时间等因子的影响。在孵化水温为6．5℃，卵子受精后20min经26℃热处理20min，孵化率为64．62％，三倍体诱导率为86．67％；卵子受精后lOmin经26℃热处理20min，三倍体诱导率为100％，但孵化率仅为3．87％。受精卵经26℃处理的诱导效果好于24℃和28℃的（P〈0．01）。     

紫外辐射灭活大黄鱼和黄姑鱼精子及其激活大黄鱼卵子发育为胚胎的效果

为探讨大黄鱼(Larimichthys crocea)和黄姑鱼(Nibea albiflora)精子的紫外辐射灭活适宜剂量及其激活大黄鱼卵子发育为胚胎的效果,以Ringer氏液为稀释液,按1:30稀释大黄鱼和黄姑鱼精子,采用自制紫外灭活装置[紫外辐射强度2200μW/(cm~2·s),紫外波长254 nm]对这2种鱼的精子进行紫外照射处理及活力测定,然后与正常大黄鱼卵子进行人工授精,授精后一部分卵未作冷休克处理,另一部分卵进行了冷休克处理(受精2 min 30 s,3℃海水,冷休克10 min),并进行了早期胚胎成活率和仔鱼孵化率的测定与比较。结果显示:1)大黄鱼和黄姑鱼精子的激活率与紫外照射处理时间呈负相关,精子的快速运动时间变化呈典型的Hertwig效应。2)未冷休克组中大黄鱼和黄姑鱼诱导的早期胚胎成活率与精子的紫外照射时间总体呈负相关,而仔鱼孵化率呈Hertwig效应。3)冷休克组中大黄鱼和黄姑鱼精子诱导的早期胚胎成活率和仔鱼孵化率随紫外照射时间的增加呈Hertwig效应,分别于2 min 20 s和1min 30 s时达相对峰值,此时,大黄鱼早期胚胎成活率和仔鱼孵化率分别为(38.3±4.3)%和(66.5±5.1)%,黄姑鱼早期胚胎成活率和仔鱼孵化率分别为(43.3±3.3)%和(67.7±6.3)%。分析认为,大黄鱼和黄姑鱼精子遗传失活的紫外辐射剂量分别以308 m J/cm~2和198 m J/cm~2为佳。本研究旨在为大黄鱼雌核发育技术的改进提供依据。     

红鳍东方鲀三倍体诱导的初步研究

通过冷休克的方法诱导红鳍东方鲀的受精卵,孵化后的仔鱼,用PAS-Ⅲ型细胞流速仪测定其倍性,得到较高的三倍体诱导率。受精后8 min,0℃下处理15 min和受精后5 min,2℃下处理15min三倍体率最高,为100%;受精后8 min,4℃下处理10 min倍化率最低,为20%;其余几组的三倍体率为90%。正交试验结果表明,影响三倍体诱导率的主次顺序是处理时间、处理温度、处理起始时间。     

黑鲷精子诱导漠斑牙鲆雌核发育研究

采用紫外线遗传失活的黑鲷精子与漠斑牙鲆卵子授精,获得了漠斑牙鲆雌核发育单倍体受精卵,经冷休克处理后诱导出漠斑牙鲆雌核发育二倍体.试验结果表明,黑鲷精子经紫外线照射后(0～180 J/cm2)与漠斑牙鲆卵子受精,胚胎孵化率呈现明显的Hertwig效应.黑鲷精子遗传失活的最适紫外线照射剂量为120 J/cm2.18℃的水温条件下,冷休克处理的最适时刻为受精后4 min.处理持续时间和处理温度组合试验表明,在相同处理持续时间条件下,3℃休克水温处理效果均优于对应1℃休克水温各组,且45 min处理持续时间诱导效果最佳.综合试验结果,黑鲷精子诱导漠斑牙鲆雌核发育的适合条件为:水温18℃,将黑鲷精子经120 J/cm2紫外线照射后与漠斑牙鲆卵子受精,受精后4min,用3℃冷海水处理45min,可获得46.59％的雌核发育漠斑牙鲆二倍体初孵仔鱼.     

双斑东方鲀三倍体育苗生产性研究

研究了双斑东方鲀三倍体的诱导方法、技术参数的筛选及生产性育苗的模式.采用温度休克方法诱导双斑东方鲀三倍体,在水温18～20℃条件下,卵受精后5 min用40℃的水温处理双斑东方鲀受精卵10 min,三倍体诱导率100%,孵化率相对诱导量达25%;育苗采用前期室内工厂化培育与后期土池培育相结合方法,培育出全为三倍体(体长3 cm以上)苗种5.8万尾,对应孵出苗量,成活率为34.6%.     

大黄鱼与黄姑鱼异源三倍体的诱导和微卫星分析

参照大黄鱼雌核发育诱导程序,应用冷休克抑制大黄鱼(♀)与黄姑鱼(♂)杂交受精卵的第二极体排出,培育了两个异源三倍体家系(PPN1和PPN2)。异源三倍体家系的受精率、孵化率略低于大黄鱼自繁二倍体对照家系(PP),而初孵仔鱼畸形率略高于PP家系。倍性分析显示,异源三倍体家系初孵仔鱼细胞DNA含量约为大黄鱼自繁对照家系的初孵仔鱼细胞DNA含量的1.46倍,且三倍体率达到100%。5个微卫星标记分析结果表明,父本杂合基因在异源三倍体中分离,后代分别得到父本两个等位基因中的一个,分离比符合孟德尔式遗传预期;由于基因的第二次分离被阻断,母本基因在异源三倍体中的传递表现出半四分子的特点,其中部分个体同时保留了母本两个等位基因,表现为杂合基因型。综合倍性分析和微卫星分析结果可以判断,异源三倍体家系成员为含有2个大黄鱼基因组和1个黄姑鱼基因组的异源三倍体。可见,大黄鱼减数分裂雌核发育或三倍体诱导程序中用于抑制第二极体排放的条件,同样适用于诱导异源三倍体。然而,PPN1和PPN2的仔鱼在15日龄后出现生长停滞,陆续死亡,没有个体存活超过1个月,表明母本染色体加倍不能有效提高杂种成活率,但异源三倍体仔鱼可作为遗传作图、基因组比较...     

热休克和水静压刺激诱导三倍体硬头鳟(Oncorhynchus mykiss)对比研究

雌性三倍体鲑鳟鱼已知是不育的,因而有利于水产养殖。本研究旨在对多种温度和水静压刺激处理诱导三倍体硬头鳟(Oncorhynchus mykiss的一种溯河回游类型)的效果加以对比。热休克和水静压刺激在各组硬头鳟卵受精后25分钟开始施行。热休克处理所用温度为26—36℃,持读1.25～20分钟;水静压处理所用压力为5.5～8.3×10~4KPa(1KPa=0.145psi)持续2～6分钟。三倍体的诱导率通过红血球细胞核长径的测定来计算。在26℃温度下持续20分钟的热休克处理和在7.6×10~4kPa压力下持续6分钟的水静压刺激诱导出100％的三倍体比率(triploid rate),但是各组卵的成活率却与处理的强度成负的相关关系。因而三倍体产量(triploid yield)(即三倍体的诱导率与孵出时的成活率的乘积)在26℃下持续10分钟的热休克或6.9×10~4KPa下持续6分钟的水静压刺激处理时为最好,分别是50.3％和49.9％。     

Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

白鱚卵各胚胎发生阶段对高温突变、落下冲击和紫外线照射的感受性

本研究将白鱚卵的胚胎发生过程严格区分为27个阶段,并就各不同发生阶段的卵对高温突变、落下冲击和紫外线照射的感受性进行了详细的比较研究,得出了卵裂早期和原肠晚期至胚孔封闭期的卵对高温突变的感受性高;卵裂晚期至胚体出现期,对落下冲击的感受性高;对紫外线照射的感受性,则以胚体出现期为最高。文中同时分析了这种不同环境因素作用于相同鱼卵的同一发生阶段,产生不同影响的原因;建议在鱼类资源保护和鱼苗生产的孵化管理上,应尽量避免感受性较高的相应环境因素变化对鱼卵的影响。     

Physiological responses of largemouth bass to acute temperature and oxygen stressors

Abstract Temperature and oxygen gradients exist in nearly every water body, but anthropogenic activities can subject fish to rapid changes in these important environmental variables. These rapid changes in temperature and oxygen (generally referred to as temperature or oxygen shock) may have sub‐lethal consequences depending upon the magnitude and the fish species. This study quantified physiological changes in largemouth bass, Micropterus salmoides (Lacepède), exposed to two levels of heat and cold shocks and to two levels of hypoxic and hyperoxic shocks. Following a cold shock from 20 °C to 8 °C, plasma cortisol and glucose increased after 1 h and lactate dehydrogenase activity increased after 6 h. Plasma glucose and K⁺ concentrations increased 1 h after a heat shock from 20 °C to 32 °C but not after 6 h. Bass subjected to a hypoxic shock from 8 to 2 mg O₂ L^?1 showed decreased plasma K⁺ and increased plasma glucose and white muscle lactate. No changes in physiological parameters were observed in bass subjected up to 18 mg O₂ L^?1 hyperoxia. Results from this study suggest that largemouth bass can tolerate a wide range of temperature and oxygen shocks, but temperature decreases of 20 to 8 °C and hypoxia as low as 4 mg O₂ L^?1 should be avoided to minimise physiological perturbations.     

Preliminary study on performance of triploid Thai silver barb, Puntius gonionotus

The triploid chromosome condition was induced in Thai silver barb (Puntius gonionotus) by application of cold shock (2°C) to eggs at time intervals after activation of 0.5 min with a duration of 10 min which resulted in mean triploidy yield of 72.5% at 9 months of age. Growth rate of the 2–9-month-old, cold shock group (0.1–6.2 g/month) did not differ from that of the control (0.1–5.7 g/month). Gonadal somatic indices (GSI) of presumed triploid males and females were lower than that of control (GSI values of the presumed triploids were 35.0–60.2% and 28.7–75.9% of control males and females, respectively). Spermatogenesis and oogenesis were retarded in triploids. However, all stages of spermatogenic cells were observed in triploid males, including few spermatozoa. Oocytes of triploid females did not undergo vitellogenesis while normal oogenesis was observed in diploids. Nuclear volume of red blood cells (RBCs) of triploid fish was 1.63 times larger than that of diploids.     

Induction of triploidy in spotted sand bass (Paralabrax maculatofasciatus Steindachner, 1868) by cold shock

Conditions for the induction of triploidy with cold shock of fertilized eggs of the spotted sand bass Paralabrax maculatofasciatus (Steindachner) were investigated. Different temperatures (12, 8 and 4 °C), timing of cold shock application (5, 10 and 15 min after fertilization) and duration of the shock (5, 10, 15 and 20 min) were tested. Triploidy was determined using flow cytometry at 12 h after larvae hatched. Triploids were produced only when the cold shock treatment was applied 5 min after fertilization. No significant difference was observed in the percentage of triploidy between temperature and the shock duration. At 8 and 4 °C, 100% triploidy was obtained at different durations of cold shock. Survival was significantly lower at 12 or 4 °C than at 8 °C. No significant difference was observed for shock duration at the temperature of 8 or 12 °C; however, at 4 °C, survival was significantly lower at longer durations. We recommend induction of triploidy by applying cold shock at 8 °C for a duration of 15–20 min starting at 5 min after fertilization, in the spotted sand bass.