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。     

斧文蛤精子超微结构与受精过程的细胞学变化

采用电镜和荧光显微镜技术,对斧文蛤精子超微结构与受精过程的细胞学变化进行了系统研究。电镜观察表明,斧文蛤精子为鞭毛型,全长45.2~47.7 μm,由头部、中段和尾部三部分组成。头部呈稍弯曲的狭茧形,长约2.5 μm,由顶体和细胞核组成,顶体呈圆锥形,内部中轴处有亚顶体腔;细胞核为长锥形,有核前窝和核后窝。中段由线粒体围绕中心粒复合体构成,5个近球形的线粒体呈单层梅花状排列;中心粒复合体由近端中心粒和远端中心粒组成,远端中心粒延伸出尾部的轴丝。尾部为细丝状鞭毛,内部的轴丝呈典型的“9+2”结构,外周有波浪状质膜包被。用荧光显微镜观察了斧文蛤受精及早期卵裂过程的细胞学变化,结果发现,斧文蛤成熟未受精卵呈圆球形,核相处于第一次成熟分裂中期;在水温27~28 ℃条件下受精,受精后6 min,精子入卵并膨胀成球形;受精后12~15 min、20~25 min受精卵排出第一、第二极体;30 min左右,精、卵核膨胀形成雄、雌原核;35 min,两性原核在卵子中央发生染色体联合;40~45 min,受精卵进行第一次卵裂,形成2个大小不等的分裂球;55~60 min,第二次卵裂结束,形成1大3小4个卵裂球,核相变化与第一次卵裂基本相同;75~80 min,第三次卵裂完成,自此次起开始进行螺旋分裂。另外,在斧文蛤受精过程中发现了约1%的多精入卵现象,其对成熟分裂和早期卵裂过程影响很大,常造成成熟分裂紊乱和第一次卵裂染色体分离异常。     

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。     

大菱鲆受精过程的细胞学观察

采用人工授精和组织切片技术对大菱鲆（Scophthalmus maximus L．）受精过程进行细胞学观察。大菱鲆成熟卵处于第二次成熟分裂的中期。精子入卵后，卵子被激动，第二次成熟分裂继续进行，同时发生皮层反应；受精后15min出现精子星光；受精后20min雄原核早于雌原核形成，然后两性原核逐渐靠拢；受精后30min两原核相互靠拢，结合线清晰；受精后40min两原核结合线逐渐消失联合成合子核，之后合子核核膜消失；受精后50min合子核处于第一次有丝分裂中期；受精后60min第一次卵裂完成。[中国水产科学，2006，13（4）：555—560]     

中国对虾受精过程的细胞学研究

通过光镜和电镜切片技术,对中国对虾的受挤过程进行了观察。结果表明,中国对虾成熟卵子处于第1次成熟分裂前期,卵子激活后产生胶质层,精子在卵子的诱导下产生顶体反应后进入卵子。中国对虾为多精入卵、单精受精。精子入卵后,卵子完成成熟分裂并举起围卵膜,但精子的存在并非此过程所必需。在卵子进行第1次成熟分裂时,其纺锤体旋转90°。卵子完成成熟分裂后,精卵原核原核化并向卵子中央移动,最后,精卵原核结合形成合子核,卵子完成受挤过程。     

彭泽鲫雌核发育的细胞学研究

通过对彭泽鲫(♀)×彭泽鲫(♂)的受精细胞学观察,彭泽鲫卵子受精后,具有与两性生殖鱼类相同的精子入卵过程和排出极体,但精子入卵后精核始终不解凝,也不与雌性原核融合。对精母细胞成熟分裂中期染色体观察表明,彭泽鲫精母细胞在进行成熟分裂时未发生同源染色体配对,全部为单价体形式,其精子形成过程中第一次成熟分裂异常;对精子与体细胞DNA含量进行比较,体细胞DNA含量与精子DNA含量比为2.218:1,精子DNA含量发生了减半。以上实验结果说明彭泽鲫为雌核发育鱼类。同时,作者对卵核染色体倍性保持稳定的方式及精卵不同的形成机制进行了初步的分析。     

栉孔扇贝（♀）×虾夷扇贝（♂）精子入卵过程的电镜观察

采用扫描电镜和透射电镜技术对栉孔扇贝(Chlamys farreri,)×虾夷扇贝(Patinopecten yessoensis,)的精子入卵过程进行观察。结果表明,这2种扇贝杂交与亲本自交的精子入卵过程没有本质的区别。成熟的精卵相遇时,相互激活,产生一系列胞间反应,卵子的激活集中表现为皮层反应、受精膜举起、成熟分裂重新启动;精子激活集中表现为顶体反应和受精锥形成。在16～18℃条件下,精子入卵的时间集中在精卵混合后的第6 min。8 min后,精子的线粒体随精核一起进入卵子中。精子入卵后精核略微膨胀,卵细胞中的线粒体在精核附近出现聚集现象。杂交中有多精入卵现象。本研究目的旨为优化扇贝种间杂交技术及探讨种间受精生物学过程提供基础依据。     

栉孔扇贝正常发育和人工雌核发育二倍体早期胚胎核行为的细胞学观察

采用Bouin氏液固定、石蜡切片和苏木精-伊红染色方法，在光镜下对栉孔扇贝正常发育和6-DMAP第二极体抑制型雌核发育二倍体卵在受精和第一、二次卵裂过程中的核相变化进行了详细观察。结果表明，正常发育卵内的雌、雄原核能融合为合子核，而雌核发育卵内的精核、雌核变化相当复杂。精核至少存在两种状态：（1）精核只在刚入卵时发生轻微膨胀，以后保持固缩状态，不膨大形成雄原核；（2）在第二次成熟分裂之后，精核再次去浓缩膨胀，但其体积并未达到正常雄原核的大小。在第一、第二次卵裂过程中，此精核不参与核分裂，以致密的染色质体（DCB）存在于两组分开的母本染色体之间，卵裂结束时滞留于两卵裂球的分裂沟上或进入其一的细胞质中。受精卵经6-DMAP处理，雌核形成第二极体的过程受到有效抑制，导致雌核二倍化。此外，作者对实验中出现的多精入卵和多极分离现象进行了观察和分析。     

菲律宾蛤仔受精及早期胚胎发育过程的细胞学观察

利用HOECHST 33258荧光染料对已固定的菲律宾蛤仔受精卵及早期胚胎样品进行染色的方法,连续观察记录了菲律宾蛤仔受精及早期胚胎发育的细胞生物学过程。结果表明,菲律宾蛤仔的成熟未受精卵子处于第一次成熟分裂中期;精卵混合后精子迅速结合于卵子周围并开始进行顶体反应;精子人卵后去致密并且激发卵子恢复两次成熟分裂,排放出第一及第二极体,完成成熟分裂;受精卵完全排放出第二极体的同时早期的精卵原核形成,随后二者相互靠拢,体积扩散膨大,在细胞中央位置形成各自的染色体组后完成联合;菲律宾蛤仔早期胚胎发育速度进程较快;在受精过程中存在极少数的多精受精现象。     

管角螺受精过程的荧光显微观察及保育卵形成分析

为了研究管角螺受精卵在受精和早期卵裂过程中的核相变化以及保育卵的形成,实验通过Hoechst 33258染色、荧光显微观察方法,对管角螺受精和早期卵裂过程中核行为的细胞学变化进行了详细观察,并对其胚胎发育过程中的数量变化进行统计分析。结果显示,管角螺的成熟未受精卵呈卵圆形,平均卵径(0.33±0.03)mm,核相处于第一次成熟分裂中期,在水温为(26±2)°C条件下,1 h精子入卵,2~3 h受精卵先后排出第1、第2极体,完成第一次和第二次成熟分裂,6 h雌、雄性原核形成,靠近融合,第一次卵裂形成2细胞,12~24 h时2细胞继续分裂生成4细胞。研究表明,管角螺受精卵在成熟分裂和卵裂过程中表现出发育的不同步性,同个卵荚内多个发育阶段共存,受精率为43.14%,而受精卵正常发育概率仅5.70%。卵荚内多数卵子不受精或者受精卵停止发育,成为营养卵(保育卵),为正常可育卵消化利用,这两种来源的保育卵比率约为3∶2。管角螺胚胎发育过程中,1~6 d内胚胎数减少明显,担轮幼虫发育率为0.48%,之后数量稳定,直至稚螺出膜。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.