施氏鲟的性腺分化及养殖水温对其性腺分化的影响

利用组织学方法观察了养殖施氏鲟(Acipenser schrenckii)原始生殖细胞(PGCs)的起源、迁移和原始生殖腺的形成及分化。结果表明,施氏鲟PGC最早出现于1.5 dph(days post-hatching)的仔鱼中肾管(又称吴尔夫氏管)原基一侧的中胚层中。3~7 dph,PGCs数目增多,并聚集成细胞索,17~31 dph,PGCs沿肠系膜向肾管区下方迁移,并与生殖嵴细胞一起形成原始性腺;40～60 dph时成群的小细胞团由肾区向原始性腺迁移,原始性腺上皮细胞柱状;90~120 dph,原始性腺中出现生殖上皮和性细胞,脂肪细胞和血管增多。180 dph以后,施氏鲟幼鱼性腺在组织学水平上出现光滑型(精巢)和褶皱型(卵巢)2种性腺组织结构,标志施氏鲟幼鱼性腺在解剖学水平上已经分化。本实验结果表明,孵化后31～180 d为其性腺分化的关键期。将孵化后56 d的仔鱼分别在不同的水温(27℃±0.5℃、24℃±0.5℃、21℃±0.5℃、18℃±0.5℃和15℃±0.5℃)中培育至210 d,组织学观察其性腺的发育和分化,结果发现,温度可影响施氏鲟幼鱼性腺分化的时间,但不影响其性腺分化的方向。高温(24℃和27℃)可使施氏鲟幼鱼提前性腺分化,其中27℃组较对照组(21℃)提前50 d,24℃组提前30 d;低温(15℃组)则延长幼鱼性腺分化的时间。施氏鲟幼鱼的性别分化不属于温度依赖型。     

虹鳟性腺分化的研究

对新安江人工养殖虹鳟性腺的自然分化进行了系统组织学研究。刚孵化出膜仔鱼的生殖嵴巳形成,原始生殖细胞成单行排列于生殖嵴上,并正在进行有丝分裂。水温10—11℃时,孵出后6—8周仔鱼的性腺出现解剖学分化。紧接着卵巢中的卵原细胞开始向初级卵母细胞过渡。精巢中的精原细胞向初级精母细胞过渡的时间比卵巢约晚2—3周。性腺的细胞学分化时间,雌雄鱼表现不一致。     

西伯利亚鲟性腺早期发生、分化、发育的组织学观察

利用组织学方法研究人工繁育、养殖的西伯利亚鲟(Acipenser baerii)仔、幼、成鱼性腺发育过程中原始生殖细胞(Primordial germ cells,PGC)的迁移,生殖褶的生成,性腺雌雄分化和发育过程。结果表明,西伯利亚鲟于3dph(Days post-hatch)时,PGC以单细胞的形式存在于仔鱼卵黄囊上方两中肾管之间,10dph时其迁移到背侧的腹膜上皮上,13dph时其沿腹膜上皮朝肾管区下方迁移,22dph时生殖嵴形成,31dph时原始性腺形成,40～60dph时原始性腺发育形成裂腔,PGCs沿腔壁排列,裂腔经过进一步发育并从背离腹膜上皮的底端慢慢愈合成实体,90dph时原始性腺中出现生殖上皮和性细胞,150dph时脂肪细胞和血管增多,210dph时性腺出现2种不同的表面结构,性腺开始分化。本研究利用组织学方法借鉴鱼类性腺分期标准,将西伯利亚鲟性腺分化划分为0期和Ⅰ～Ⅵ期,并描述了各期精巢、卵巢的结构变化以及细胞形态学特征。     

大黄鱼性腺性别分化的组织学观察

利用组织学方法研究了大黄鱼的性腺分化和发育规律。大黄鱼受精卵于2009年9月22日开始孵化,孵化水温26℃,育苗水温22.0~25.8℃,养殖水温11.5~25.6℃。20日龄稚鱼(体长17.6~19.2 mm)腹腔中一对原始性腺已经形成。55日龄幼鱼(体长27.5~37.0 mm)半数个体性腺中形成成簇发育的卵原细胞群,标志着卵巢解剖学分化开始。减数分裂和卵巢腔的形成同时开始于60日龄(体长28.0~37.2 mm)。120日龄幼鱼(体长39.2~51.0 mm)性腺中,初级卵母细胞出现,标志着卵巢细胞学分化的开始。精巢分化开始于第95日龄(体长38.0~48.0 mm),其解剖学标志为生精导管的形成及体细胞在性腺中的散布方式。145～195日龄,由于水温过低,鱼苗停止生长,期间性腺发育水平没有明显变化。215日龄幼鱼(体长44.0~59.2 mm)性腺中精母细胞的出现标志着精巢细胞学分化的开始。精小叶于230日龄(体长56.2~72.8 mm)形成。由此可见,大黄鱼雌性性腺发育早于雄性性腺,且大黄鱼性腺分化类型属于分化型雌雄异体型。     

黑脊倒刺鲃性腺早期分化的研究

对黑脊倒刺鲃(Spinibarbus caldwelli)早期性腺分化的过程进行了显微和亚显微研究。结果显示:在5日龄仔鱼中,观察到生殖嵴出现;在28日龄稚鱼中,观察到精巢的早期分化;在32日幼鱼中,观察到明显分化;而直到42日龄幼鱼中,才观察到早期卵巢的早期分化;在90日龄幼鱼中,观察到明显分化。结果表明,黑脊倒刺鲃的精巢分化要明显早于卵巢的分化。     

黄颡鱼(Pelteobagrus fulvidraco)性腺分化的组织学观察

每天采样观察3~60日龄人工养殖的黄颡鱼Pelteobagrus fulvidraco性腺分化和发育的组织学。结果表明:在水温(20±4)℃下,黄颡鱼卵巢的分化明显早于精巢,性腺的分化型为雌性异体型。孵出后14d卵巢开始分化,原始性腺在横切面出现组织突起,逐步形成卵巢腔;雄性分化始于孵出后54d,主要标志是形成输精管原基,进而形成输精管。本研究成果为黄颡鱼的人工繁殖提供了理论依据。     

花鲈性腺分化组织学及性别相关基因cyp11b和cyp19a1a的表达分析

以花鲈(Lateolabrax maculatus) 1~214 dph (day post hatching)的仔稚鱼、幼鱼以及18月龄的雌鱼和雄鱼为研究对象,研究了花鲈早期性腺发生、发育和分化情况;分析了性腺分化过程中性别相关基因(cyp11b和cyp19a1a)的表达及与性别之间的关系。结果显示,在30 dph [全长为(1.28± 0.10) cm],首次在中肾管前端的腹腔膜周围观察到原始生殖细胞(primordial germ cells, PGCs),说明30 dph前是花鲈胚后PGCs迁移至生殖嵴的关键时期;在55 dph [全长为(2.45±0.19) cm],观察到一对呈对称分布的原始性腺已经形成,说明花鲈幼鱼的原始性腺在30~55 dph (全长为1.28~2.45 cm)之间发生;55~180 dph时(全长为2.45~12.28 cm),原始性腺不断发育变大,并且一直处于未分化状态;180 dph后性腺开始分化;在195 dph [全长(14.54±1.54) cm]观察到精巢开始分化,卵巢于205 dph [全长为(15.86±0.94) cm]开始分化,且性腺的解剖学分化要早于细胞学分化;18月龄的花鲈幼鱼性腺发育到Ⅱ期。性别分化相关基因cyp19a1a在花鲈卵巢中的表达量高于同期精巢,说明其在卵巢的分化及维持中发挥更关键的作用,而cyp11b在18月龄幼鱼Ⅱ期精巢中的表达量显著高于同时期的卵巢及Ⅰ期精巢,说明其主要在精巢的分化及维持中扮演重要角色。本研究结果不仅可以丰富花鲈的繁殖生理学资料,也为其性别调控技术的研究提供了科学依据。     

黑尾近红鲌胚胎发育和原始生殖细胞的迁移研究

用胚胎固定脱膜方法对黑尾近红鲌胚胎发育进行连续观察,用整胚原位杂交技术对原始生殖细胞的起源和迁移进行研究.试验结果表明,黑尾近红鲌受精卵为粘性,淡黄色,圆形,直径(0.94±0.05) mm.在水温24.5 ℃的孵育条件下,受精卵经25 min开始胚胎发育,45 min进入2细胞期,5 h 15 min进入囊胚期,7 h 25 min进入原肠期,10 h 15 min进入神经胚期;50 h,50%以上的仔鱼孵出.刚出膜的仔鱼长约(4.10±0.02) mm.黑尾近红鲌原生殖细胞最早发现于原肠期;在原肠中期和神经胚期,其位于胚盾靠近卵黄囊的内胚层;在胚层分化期,原生殖细胞迁移到脏壁中胚层;在孵化期,原生殖细胞迁移到体两侧的生殖嵴中,与生殖嵴共同组成胚胎的未分化性腺.     

半滑舌鳎性腺分化的组织学观察

运用组织学方法,对半滑舌鳎性腺的发育和分化过程进行了观察。半滑舌鳎的性腺发育为雌雄异体的分化类型,可以分为3个阶段:性腺的原始阶段、性腺分化前期阶段和性腺分化完成阶段。在50日龄以前,原始性腺形成但未分化。62日龄,个体达到40.5mm时,一种性腺形成了成簇发育的原始细胞群,性腺迅速增大变宽,后来发育成为卵巢;另一种仍处于休止阶段,相对较小,后来发育为精巢。雌鱼在120日龄形成明显的卵巢腔,开始了细胞学的分化;精巢在100日龄精原细胞开始快速增殖,体细胞有丝分裂,形成输精管原基,在150日龄左右开始细胞学的分化,并形成精小叶,190日龄左右出现初级精母细胞,性腺分化基本完成。雌鱼性腺分化时期早于雄鱼。     

菊黄东方鲀性腺分化的组织学观察

运用组织切片法,对菊黄东方鲀性腺分化过程进行了观察。在40日龄前,原始性腺未分化。40日龄(21.60～24.71mm),约半数个体开始形成卵巢腔,分化为卵巢;其他约半数个体的性腺仍未分化。雌鱼在60日龄(35.51～43.60mm),形成封闭的卵巢腔;70日龄(46.30～54.75mm),卵原细胞形成卵母细胞;120日龄(94.83～107.29mm),产卵板充分发育,卵巢结构已近完善。雄鱼在60日龄(35.51～43.60mm),形成精小囊及精原细胞,开始分化;90日龄(71.59～80.54mm),形成精小管和精小叶;120日龄(94.83～107.29mm),初级精母细胞形成,性腺分化基本完成。雌鱼性腺分化时期早于雄鱼。     

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).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

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.     

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.