Cryopreservation of sperm of common carp, Cyprinus carpio and silver barb, Barbonymus gonionotus for genetically improved seed production

Experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of common carp, Cyprinus carpio and also for using the cryopreserved sperm for fertilization of eggs. Nine extender solutions as Alsever's solution, kurokura-1, kurokura-2, urea egg-yolk, egg-yolk citrate, 0.6% glucose, 0.9% NaCl, Ma and Mb, and five cryoprotectants namely ethanol, methanol, dimethylsulfoxide (DMSO), dimethylamine (DMA) and glycerol were tested. The cryoprotectants were mixed at 10% concentration of the extenders (v/v) to make the cryodiluents. Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever's solution, kurokura-1, kurokura-2, 0.6% glucose and 0.9% NaCl, 1:4 for urea egg-yolk, egg-yolk citrate, Ma and Mb. Among the cryodiluents Alsever's solution mixed with either ethanol or methanol was found to be suitable and it produced more than 90% and 80% spermatozoan motility at equilibrium and post-thaw periods, respectively. Kurokura-1 and kurokura-2 when mixed with the same cryoprotectants showed good spermatozoan motility at equilibrium period (80-90%) but the motility was reduced (30-55%) at post-thaw state. Other extenders did not produce acceptable sperm-motility and in some cases the frozen milt became clotted. Different dilution ratios (1:1, 1:2, 1:4, 1:5, 1:7, 1:9, 1:12, 1:15, 1:20) were formulated for obtaining a suitable milt dilution, the dilution ratio of 1: 9 (milt : cryodiluent) demonstrated the highest post-thaw spermatozoan motility (80%) in Alserver's solution. The optimum concentration of cryoprotectants in the cryodiluents was determined, 10% concentration level was found to be effective to produce the highest number of spermatozoan motility in comparison to the other concentrations (5%, 15%, 20% 30%). Sperm preserved with the cryodiluent Alsever's solution along with either methanol or ethanol was found to be effective to fertilize eggs and produce hatchlings. The hatching rates ranged between 1.48% and 14.76%, compare to control. The fish produced through use of cryopreserved sperm and normal sperm were found to grow well and no significant (P<0.05) growth difference was observed between them. In case of silver barb, Barbonymus gonionotus, sperm tested against six extenders such as egg-yolk citrate, urea-egg-yolk, kurokura-1, kurokura-2, 0.9% NaCl and modified fish ringer (MFR) solution. Cryoprotectants used were the same as those of C. carpio. Milt was diluted with the cryodiluent at a ratio of 1:4 for egg-yolk citrate and urea-egg-yolk, 1:5 for kurokura-1 and 1:9 for 0.9% NaCl, MFR and kurokura-2. The cryoprotectant concentration was maintained at 10% of the extender (v/v) in all the cases. Among the extenders, egg-yolk citrate and urea-egg-yolk mixed with 10% DMSO, methanol and ethanol produced 50% post-thaw spermatozoan motility, whereas DMA and glycerol provided only 10% motility. Trials on milt dilution ratio and cryoprotectant concentration are being conducted. Fertilization trials are also underway.

Optimisation of stocking density of Thai silver barb (Barbodes gonionotus Bleeker) in the duckweed-fed four species polyculture system

The optimisation of stocking density of Thai silver barb (Barbodes gonionotus) in the polyculture with Labeo rohita, Catla cat/a and Cyprinus cmpio was investigated in seasonal ponds. Three different stocking densities of Thai silver barb i.e., 5,000, 6,000 and 7,000 fingerlings ha-1 were tested with stocking density of carps fixed at the rate of 10,000 fingerlings ha-1 Duckweed was applied to all ponds supplemented with rice bran and oil cake. There were no significant variations on either water quality parameters or abundance of planktonic organisms due to the different stocking densities of silver barb. A significantly higher fish production (p

Size related feeding patterns and electivity indices of silver barb (Barbodes gonionotus Bleeker) from a pond, Bangladesh

The feeding patterns with respect to quality and quantity of food of silver barb, Barbodes gonionotus varied with their size and development. The results indicated that the fish in the size group I (7-25 mm TL) were fairly omnivore with particular liking for rotifera, green and blue-green algae while the size group II (25.1-44 mm TL) and III (44.1-55 mm TL) were omnivore with higher tendency of feeding on debris, aquatic plants, green algae, blue-green algae and rotifera. However, the fish of the size group IV (55.1-80 mm TL) were found to be herbivore with feeding preference for aquatic plants, green and blue-green algae. In all the size groups, debris was the most dominant food item. Feeding preference of the fish showed clear ontogenetic shift. The electivity indices revealed that the fish were selective feeder.

Performance of genetically improved farmed tilapia (GIFT) under mono and mixed cui ture with silver barb (Barbodes gonionotus) in south-west Bangladesh

A participatory on-farm trial was carried out to evaluate the production performance of GIFT (genetically improved farmed tilapia) strain of Oreochmis sp., either alone or with silver barb (Barbodes gonionotus), in six rain-fed freshwater ponds of coastal area. There vvere two treatments; (i) GIFT alone at a stocking density of 24,700/ha (T1) and (ii) 1:1 combination of GIFT and silver barb (T1). Each of the treatments had three replications. A significantly (p

Growth and food availability of silver and bighead carps: evidence from stable isotope and gut content analysis

A 2-year investigation of growth and food availability of silver carp and bighead was carried out using stable isotope and gut content analysis in a large pen in Meiliang Bay of Lake Taihu, China. Both silver carp and bighead exhibited significantly higher delta 13C in 2005 than in 2004, which can probably be attributed to two factors: (i) the difference between isotopic compositions at the base of the pelagic food web and (ii) the difference between the compositions of prey items and stable isotopes. The significantly positive correlations between body length, body weight and stable isotope ratios indicated that isotopic changes in silver carp and bighead resulted from the accumulation of biomass concomitant with rapid growth. Because of the drastic decrease in zooplankton in the diet in 2005, silver carp and bighead grew faster in 2004 than in 2005. Bighead carp showed a lower trophic level than silver carp in 2005 as indicated by stable nitrogen isotope ratios, which was possibly explained by the interspecific difference between the prey species and the food quality of silver carp and bighead.

Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

Morphological and molecular phylogenetic analysis of two Saprolegnia sp (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates under-went repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (1) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species. (C) 2009 Published by Elsevier Ltd on behalf of The British Mycological Society.

Plasmonic silver nanoparticles for improved organic solar cells

In the present work we compare the performance of organic solar cells, based on the bulk heterojunction system of P3HT:PCBM when adequate silver nanoparticles (NPs) are incorporated in two distinct places among the device structure. Introduction of NPs on top of the transparent anode revealed better overall performance with an increased efficiency of 17%. Alternatively, placing the NPs on top of the active photovoltaic layer resulted to 25% higher photo-current generation albeit with inferior electrical characteristics (i.e series and shunt resistance). Our findings suggest that enhanced scattering to non-specular directions from NPs site is maximized when penetrating light meets the particles after the polymer blend, but even this mechanism is not sufficient enough to explain the enhanced short circuit current observed. A second mechanism should be feasible; that is plasmon enhancement which is more efficient in the case where NPs are in direct contact with the polymer blend. J-V characteristics measured in the dark showed that NPs placed on top of the ITO film act as enhanced hole conducting sites, as evident by the lower series resistance values in these cells, suggesting this mechanism as more significant in this case. © 2012 Elsevier B.V. All rights reserved.

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 mu g g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 mu g g(-1) dry weight at 48 h, indicating a deputation of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis. (C) 2007 Elsevier Inc. All rights reserved.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.

Long-term changes of Copepoda community (1957-1996) in a subtropical Chinese lake stocked densely with planktivorous filter-feeding silver and bighead carp

In contrast to the relatively well documented impact of particulate-feeding fish on zooplankton communities, little attention has been devoted to the impact of filter-feeding fish. Filter-feeding silver and bighead carp are the most intensively cultured fish species in Asia and comprise much of the production of Chinese aquaculture. However, little information is known about the impact of either fish on the zooplankton community. Long-term changes in the Copepoda community (1957-1996) were studied at two sampling stations of a subtropical Chinese lake (Lake Donghu) dominated by silver and bighead carp. For both calanoids and cyclopoids, the littoral station (I) was much more resource profitable than the pelagic station (II). There has been a tremendous increase in the annual fish catch over the past 30 years due to the increased stocking with fingerlings of the two carp species. There was a notably higher fish density at Station I than at Station II. Cyclopoid abundance was notably higher at Station I than at Station II during the 1950s to the 1980s, while the reverse became true in the 1990s. This is probably because when fish abundance increased to an extremely high level, the impact of fish predation on the cyclopoids became more important than that of food resources at the littoral station. At both stations, cyclopoid abundance was relatively low in spite of the presence of abundant prey. Similarly, calanoid density did not differ significantly between the two stations in the 1950s and 1960s, but was significantly lower at Station I than at Station II during the 1980s and 1990s. Such changes are attributed to the gradient of fish predation between the stations and an increasing predation pressure by the fish. The increased fish predation also correlated with a shift in summer-dominant calanoids from larger species to smaller ones. In conclusion, the predaceous cyclopoids are affected by fish predation to a much lesser extent than the herbivorous calanoids, and therefore increased predation by filter-feeding fish results in a definite increase in the cyclopoid/calanoid ratio. Predation by filter-feeding fish has been a driving force in shaping the copepod community structure of Lake Donghu during the past decades.

Enhanced photocatalytic activity of TiO2 nano-structured thin film with a silver hierarchical configuration

TiO2 sol-gels with various Ag/TiO2 molar ratios from 0 to 0.9% were used to fabricate silver-modified nano-structured TiO2 thin films using a layer-by-layer dip-coating (LLDC) technique. This technique allows obtaining TiO2 nano-structured thin films with a silver hierarchical configuration. The coating of pure TiO2 sol-gel and Ag-modified sol-gel was marked as T and A, respectively. According to the coating order and the nature of the TiO2 sol-gel, four types of the TiO2 thin films were constructed, and marked as AT (bottom layer was Ag modified, surface layer was pure TiO,), TA (bottom layer was pure TiO,, surface layer was Ag modified), TT (pure TiO, thin film) and AA (TiO, thin film was uniformly Ag modified). These thin films were characterized by means of linear sweep voltammetry (LSV), X-ray diffraction (XRD), scanning electron microscopy (SEM), electrochemical impedance spectroscopy and transient photocurrent (I-ph). LSV confirmed the existence of Ago state in the TiO, thin film. SEM and XRD experiments indicated that the sizes of the TiO,, nanoparticles of the resulting films were in the order of TT > AT > TA > AA, suggesting the gradient Ag distribution in the films. The SEM and XRD results also confirmed that Ag had an inhibition effect on the size growth of anatase nanoparticles. Photocatalytic activities of the resulting thin films were also evaluated in the photocatalytic degradation process of methyl orange. The preliminary results demonstrated the sequence of the photocatalytic activity of the resulting films was AT > TA > AA > TT. This suggested that the silver hierarchical configuration can be used to improve the photocatalytic activity of TiO2 thin film.

Type I collagen-mediated synthesis of noble metallic nanoparticles networks and the applications in Surface-Enhanced Raman Scattering and electrochemistry

In this paper, we demonstrated an effective enviromentally friendly synthesis route to prepare noble metallic (Au, Ag, Pt and Pd) nanoparticles (NPs) networks mediated by type I collagen in the absence of any seeds or surfactants. In the reactions, type I collagen served as stabilizing agent and assembly template for the synthesized metallic NPs. The hydrophobic interaction between collagen and mica interface as well as the hydrogen bonds between inter- and intra-collagen molecules play important roles in the formation of collagen-metallic NPs networks. The noble metallic NPs networks have many advantages in the applications of Surface-Enhanced Raman Scattering (SERS) and electrochemistry detection. Typically, the as-prepared Ag NPs networks reveal great Raman enhancement activity for 4-ATP, and can even be used to detect low concentration of DNA base, adenine.