Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

Motion of integrated CdS nanoparticles by phase separation of block copolymer brushes

A new method of reversibly moving US nanoparticles in the perpendicular direction was developed on the basis of the phase separation of block copolymer brushes. Polystyrene-b-(poly(methyl methaerylate)-co-poly(cadmium dimethacrylate)) (PS-b-(PMMA-co-PCdMA)) brushes were grafted from the silicon wafer by surface-initiated atom transfer radical polymerization (ATRP). By exposing the polymer brushes to H2S gas, PS-b-(PMNlA-co-PCdNlA) brushes were converted to polystyrene-b-(poly(methyl methacrylate) -co-poly(methacrylic acid)(CdS)) (PS-b-(PMMA-co-PMAA(CdS))) brushes, in which US nanoparticles were chemically bonded by the carboxylic groups of PMAA segment. Alternating treatment of the PS-b-(PMMA-co-PMAA(CdS)) brushes by selective solvents for the outer block (a mixed solvent of acetone and ethanol) and the inner PS block (toluene) induced perpendicular phase separation of polymer brushes, which resulted in the reversible lifting and lowering of US nanoparticles in the perpendicular direction. The extent of movement can be adjusted by the relative thickness of two blocks of the polymer brushes.

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e-5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs. (C) 2009 Elsevier Ltd. All rights reserved.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Cryopreservation of flounder (Paralichthys olivaceus) sperm with a practical methodology

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5 +/- 3.6, 79.17 +/- 4.5 and 13.25 +/- 4.7%, respectively. The fertilization rates of this sperm were 67.06 +/- 15.1, 76.20 +/- 10.0 and 44.93 +/- 22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40 +/- 8.3, 48.18 +/- 25.7 and 23.35 +/- 10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm. (C) 2003 Elsevier Science Inc. All rights reserved.

Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Effect of ultraviolet irradiation on sperm of the left-eyed flounder, Paralichthys olivaceus

In this study, at proper dosage of ultraviolet (UV) irradiation (180 sec: 36,000 erg/mm(2)), sperm chromosomes of left-eyed flounder, Paralichthys olivaceus, were inactivated, while spermatozoa maintained ability to move and inseminate eggs. Gynogenetic haploids were detected by morphological observation, chromosome counting, and flow cytometer analysis. The ultrastructure of treated sperm was observed under scanning electronic microscope (SEM) and transmission electronic microscope (TEM). The results showed that after being irradiated at lower dosage of irradiation (0-180 sec: 0-36,000 erg/mm(2)), the surface structure of spermatozoa was not affected by UV irradiation, while the inner structures including membrane system and karyoplasm denseness of treated spermatozoa were little changed. However, obvious changes were observed in their membrane system, mitochondria, and nucleus if the dosage of irradiation increased to 240 sec: 48,000 erg/mm(2) or 300 sec: 60,000 erg/mm(2). The sperm survival rates did not change at the lower dosages of the UV irradiation (0-180 sec: 0-36,000 erg/mm(2)) but decreased as the irradiation dosage increased. The motility of treated sperm was lower than that of control group in general but did not change with UV irradiation dosage increasing at the certain range of 0-300 sec: 0-60,000 erg/mm(2).

大西洋庸鲽Hippoglossus hippoglossus和大西洋鲑Salmo salar配子质量研究

种质问题是养殖健康发展的基础。在鱼类养殖中，卵子和精子的质量直接关系到受精、胚胎发育，仔稚鱼发育以及幼鱼生长等一系列过程。本论文针对大西洋庸鲽和大西洋鲑的配子质量进行研究。研究内容涉及大西洋庸鲽精子冷冻保存方法；促性腺激素释放激素类似物（GnRHa）使用对其精子冷冻保存效果、以及脂肪酸组成的影响；野生和驯养大西洋鲑卵子在脂肪酸、类胡萝卜素、矿物盐方面的差异比较。 精子冷冻保存通过提高对精子的利用效率，进而对于种质改良，推进鱼类养殖科研和生产具有重要意义。本实验建立了大西洋庸鲽精子大容量冷冻保存方法。八种抗冻剂冷冻保存实验结果表明：10% 及15% DMSO配以 HBSS 或KS 的抗冻剂组合冷冻保存效果最佳，4 mL体积冷冻保存可获得与1.6 mL同样的保存效果。 在繁殖季节后期注射GnRHa激素缓释剂，可获得质量稳定的大西洋庸鲽精液，将激素注射方法与精子冷冻保存方法相结合对于提高雄鱼利用率，扩大生产规模具有重要实用价值。本项研究分三个时间采集注射GnRHa激素后的雄鱼精子以及同期未注射激素的雄鱼精子，对所有精子样品使用同样的方法进行冷冻保存，检测冷冻保存后解冻精子的受精率与活力。结果表明，激素注射与否对于冷冻保存后精子的受精率和活力无显著影响，两类冷冻精液均达到鲜精水平。实验结果还表明，注射激素14天后的精子的密度显著的降低。说明GnRHa激素的使用可以显著降低精子密度，但不会影响精子的冷冻保存效果。 本相研究同时对注射GnRHa 缓释激素和未注射GnRHa 缓释激素的大西洋庸鲽精液脂肪酸成分进行分析，以检测该激素使用对精子生化组分的影响。结果表明激素的使用对在DHA (22:6n-3,二十二碳六烯酸)、EPA(20:5n-3,二十碳五烯酸)、AA(20:4n-6，花生四烯酸)等重要脂肪酸，不饱和脂肪酸、饱和脂肪酸以及n-3、n-6等重要种类的脂肪酸总量及其比例没有显著影响。精液脂肪酸中DHA含量最高，约占25%；PUFA约为44%。 作为世界性的重要养殖品种，野生和驯养大西洋鲑在形态、生化组成以及遗传 等方面表现出的差异被广泛关注。本论文，对野生和驯养大西洋鲑受精卵关键生化成分进行分析，通过与野生受精卵比较阐明驯养受精卵的质量状况，为亲鱼营养需求提供指导依据。本实验中野生配子和驯养配子的受精率没有显著差异，但重要脂肪酸组成、类胡萝卜素以及矿物盐含量都存在多方面显著差异。两类受精卵脂肪酸中含量最高的依次为18:1n-9（油酸）、DHA（二十二碳六烯酸）、16:0（棕榈酸）、EPA（二十碳五烯酸）。野生受精卵的单不饱和脂肪酸总量显著高于驯养受精卵，而多不饱和脂肪酸（PUFA）比例显著低于驯养的受精卵。在主要必需不饱和脂肪酸（EFA）中，DHA和EPA在野生受精卵中的比例高于驯养受精卵，AA（花生四烯酸）低于驯养受精卵。野生受精卵虾青素（Ax）的含量低于驯养受精卵而鸡油菌素（Cx）含量高于驯养受精卵。野生受精卵中多种矿物盐的含量（铝、铜、铁、硒和锌）含量显著高于驯养的受精卵。差别最大的为铜。诸多方面的差异表明，野生亲鱼与驯养亲鱼产出的卵子确实存在显著差异，因此关注亲鱼的营养极为重要。

A preliminary study on the mechanism of harmful algal bloom mitigation by use of sophorolipid treatment

in order to investigate a new method of mitigating the deleterious effect of harmful algal blooms (HABs), the inhibition of the glycolipid biosurfactant sophorolipid on three common harmful algae Alexandrium tamarense, Heterosigma akashiwo and Cochlodinium polykrikoides was studied. The optimum preparation condition for sophorolipid, the inhibition capability of sophorolipid and the interaction mechanism of sophorolipid on the three algal species were investigated. Results showed that sophorolipid prepared by extraction with ethyl acetate exhibited the most prominent inhibition effect and that storage time of one year had little influence on the inhibition effect of sophorolipid. The optimum concentration of 10-20 mg/l sophorolipid inhibited the motility of about 90% of the tested harmful algal cells without recovery. Investigation of the algicidal process revealed that sophorolipid induced ecdysis of A. tamarense, quick lysis of H. akashiwo and swelling of C. polykrikoides in a relatively short time. Investigation of the nucleotides showed that more than 15% of the nucleotides were released from the cytoplasm under the effect of 10-20 mg/l sophorolipid, indicating the irreversible damage on the cellular membrane, which resulted in the disintegration of the harmful algal cells. (C) 2004 Elsevier B.V. All rights reserved.

Screening of surfactants for harmful algal blooms mitigation

Screening experiments were conducted in order to find promising synthetic surfactants for harmful algal blooms (HABs) mitigation. The chemically synthesized surfactant cocamidopropyl betaine (CAPB) showed characteristics of relatively high inhibition efficiency, high biodegradability and low cost. The motility inhibition ratios of 10 mg/L CAPB on Cochlodinium polykrikoides and Alexandrium tamarense were about 60% after 5 min. The biodegradation test indicated that the half-life of CAPB in seawater was shorter than one day and 90% was biodegraded after five days under the initial concentration of 100 mg/L at 25degreesC. Further cell lysis experiments revealed the selective lysis effect of CAPB on different HAB organisms. More than 90% of C. polykrikoides lysed at the concentration of 10 mg/L CAPB after 24 h and at 15 mg/L CAPB after 4 h, whereas the lysis effect of CAPB on A. tamarense was slight, no more than 10% after 2 h interaction with 50 mg/L CAPB. This research provided preliminary data for CAPB as a candidate in harmful algal blooms mitigation and pointed out unresolved problems for its practical application in the meantime. (C) 2003 Elsevier Ltd. All rights reserved.

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabrearn (Pagrus major). Mean (+/- S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0 +/- 5.4, 92.8 +/- 1.9, and 91.8 +/- 5.2%, respectively; for fresh sperm, they were 87.5 +/- 7.7, 95.8 +/- 2.4, and 93.8 +/- 4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P < 0.05), there was no effect (P > 0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8 +/- 5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0 +/- 7.2% had normal morphology, 20.6 +/- 3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4 +/- 4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased. (c) 2007 Elsevier Inc. All rights reserved.

Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream (Pagrus major)

In the present study, the quality of post-thaw sperm of red seabream Pagrus major frozen with 6-24% DMSO was investigated. The motility, average path velocity and fertilizing capacity of fresh and their corresponding post-thaw sperm were examined for evaluation of the post-thaw sperm motion characteristics and its association with fertilizing capacity. An analysis of sperm motility before and after cryopreservation has been performed using computer-assisted sperm analysis (CASA). For post-thaw sperm frozen with 12-21% DMSO, the percentages of motile sperm were not significantly (P > 0.05) changed 10 s after activation. Moreover, the main motility pattern and swimming velocity of the motile post-thaw sperm were not significantly (P > 0.05) changed and the progressive linear motion was still the dominant pattern. However, the total motility of post-thaw sperm (72.3 +/- 6.3%) 30 s after activation was (P < 0.05) lower than the corresponding fresh sperm (82.7 +/- 7.2%). Additionally, the fertilizing capacity of post-thaw sperm was investigated with a standardized sperm to egg ratio 500:1. There is a linear regression relationship between the percentage of motile post-thaw sperm and fertilizing capability. These data demonstrate that 12-21% DMSO can provide good protection to the sperm during the freezing-thawing process. (c) 2006 Elsevier B.V. All rights reserved.

Effect of long-term cryopreservation on physiological characteristics, antioxidant activities and lipid peroxidation of red seabream (Pagrus major) sperm

The aim of this study was to determine the effect of long-term cryopreservation on physiological characteristics, the antioxidant activities and lipid peroxidation of red seabream sperm which were respectively cryopreserved with 15% dimethylsulfoxide (Me2SO) for 1 month, 13 months, 26 months, 48 months and 73 months. The motility and fertility of post-thaw sperm decreased with the storage time going on. The highest motility (87.67 +/- 2.52%) was obtained in sperm cryopreserved for 1 month and the lowest (50.67 +/- 5.31%) was in sperm for 73 months. There were no significant differences (p < 0.05) in fertilization rates of sperm cryopreserved for 1 month (71.33 +/- 8.84%), 13 months (69.22 +/- 1.02%) and 26 months (60.33 +/- 2.33%); however, the sperm fertility decreased significantly for 48 months (47.22 +/- 3.89%) and 73 months (39.56 +/- 0.69%) storage. In addition, superoxide dismutase (SOD) activities of sperm were at a stable level for less than 26 months storage, then, decreased significantly after 48 months storage. Catalase (CAT) activities of sperm cryopreserved for 13 months, 26 months, 48 months and 73 months were significantly lower than that for 1 month. There were no significant differences in the malondialdehyde (MDA) level of sperm for less than 13 months storage. After 26 months storage, the concentration of MDA increased significantly, and the highest concentration (3.22 +/- 0.05 nmol/mgprot) was obtained in 73 months storage sperm. (C) 2010 Elsevier Inc. All rights reserved.

An efficient methodology for cryopreservation of spermatozoa of red seabream, Pagrus major, with 2-mL cryovials

The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2-mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 +/- 4.7% to 88.6 +/- 8.0%), fertilization rates (89.6 +/- 2.9 to 95.6 +/- 1.9%), and hatching rates (85.3 +/- 5.1% to 91.4 +/- 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.