Doxycycline pharmacokinetics and tissue depletion in striped catfish (Pagasianodon hypophthalmus) after oral administration.

The pharmacokinetics and residue depletion of doxycycline (DOX) in striped catfish (Pagasianodon hypophthalmus) after oral dosage were investigated. The pharmacokinetic experiment was conducted in an aquarium, while the experiment of residue depletion was performed in both an aquarium and earth ponds. Medicated feed was administered orally using the gavage method at a dosage of 20 mg/kg body weight. Blood, liver, and kidney from medicated fish samples were collected. In the depletion experiments, fish were fed medicated feed for five consecutive days at a dosage of 20 mg/kg body weight, with samples collected during and after medication. The concentrations of DOX were quantified using an LC-MS/MS system. The pharmacokinetics parameters of DOX in striped catfish included the absorption rate constant (ka), absorption half-life (T1/2abs), maximal plasma concentration (Cmax), time to maximal plasma concentration (Tmax), and area under the plasma concentration-time curve from time 0 to 96 h (AUC0-96 h) which were 0.12 h-1, 5.68 h, 1123.45 ng/mL, 8.19 h, and 25,018 ng/mL/h, respectively. Residue depletion results indicated that the withdrawal times of DOX in muscle (with skin) from fish kept in the aquarium were slightly longer than that in fish raised in earth ponds, corresponding to 194 degree-days compared with 150 degree-days. In conclusion, administration of DOX at the dosage of 20 mg/kg body weight can be used for treatment of bacterial infections in striped catfish, and a withdrawal time of 5 days at 29.4°C will ensure consumer food safety due to the rapid depletion of DOX from muscle and skin.

Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii.

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

Analysis of the economic performance of salmon farming in submerged and surface cages in the Black Sea.

The production period for salmon farming in the Black Sea comprises the winter period and is limited to seven months, due to high water temperatures during the summer time. As an alternative strategy, temporary cage submersion during the summer season might be a solution for salmon grow-out throughout the year. Therefore, this study was conducted for comparative evaluation of economic performance of submerged and surface cages, by analyzing structural costs and returns for Turkish salmon farming in the Black Sea. As a result of the temporary cage submersion strategy, economic profits increased by nearly 70%, granting higher values of financial indicators with increased net profit (685,652.5 $ year-1) and margin of safety (89.6%), compared to the traditional surface cage (397,058.5 $ year-1 net profit and 88.4% margin of safety). The "What-if" analysis showed that profits from both cage systems were sensitive to variations in sale price, and the simulation by 10% reduced export market value may decrease revenues, with less financial profit loss for the submerged cage over the surface once. Hence, temporary cage submersion seems to be an alternative farm management strategy with extended production cycle and higher profits for the sustainable development of Turkish salmon farming in the Black Sea.

Masculinization of Red Tilapia (Oreochromis spp.) Using 17α-Methyltestosterone-Loaded Alkyl Polyglucosides Integrated into Nanostructured Lipid Carriers.

The aim of the present study was to optimize a masculinization platform for the production of all-male red tilapia fry by oral administration of 30 and 60 ppm of MT and alkyl polyglucoside nanostructured lipid carriers (APG-NLC) loaded with MT, respectively, for 14 and 21 days. The characterization, encapsulation efficiency and release kinetics of MT in lipid-based nanoparticles were assessed in vitro. The results showed that the MT-loaded nanoparticles were spherical, ranging from 80 to 125 nm in size, and had a negative charge with a narrow particle distribution. The APG-NLC loaded with MT provided higher physical stability and encapsulation efficacy than the NLC. The release rate constants of MT from MT-NLC and MT-APG-NLC were higher than those of free MT, which is insoluble in aqueous media. There was no significant difference in survival between the fish administered MT or the those fed orally with MT-APG-NLC fish. According to the logistic regression analysis, the sex reversal efficacy of MT-APG-NLC (30 ppm) and MT (60 ppm), resulted in significantly higher numbers of males after 21 days of treatment compared with the controls. The production cost of MT-APG-NLC (30 ppm) after 21 days of treatment was reduced by 32.9% compared with the conventional MT treatment group (60 ppm). In all the treatments, the length-weight relationship (LWR) showed negatively allomeric growth behavior (b < 3), with a relative condition factor (Kn) of more than 1. Therefore, MT-APG-NLC (30 ppm) would seem to be a promising, cost-effective way to reduce the dose of MT used for the masculinization of farmed red tilapia.

Lack of a p16INK4a/ARF locus in fish genome may underlie senescence resistance in the fish cell line, EPC.

Unlike most mammalian cell lines, fish cell lines are immortal and resistant to cellular senescence. Elevated expression of H-Ras contributes to the induction of senescence in a fish cell line, EPC, but is not sufficient to induce full senescence. Here, we focused on the absence of a p16INK4a/ARF locus in the fish genome, and investigated whether this might be a critical determinant of the resistance of EPC cells to full senescence. We found that transfected EPC cells constitutively overexpressing p16INK4a exhibited large size and flat morphology characteristic of prematurely senescent cells; the cells also showed p53-independent senescence-like growth arrest and senescence-associated ß-galactosidase (SA-ß-gal) activity. Furthermore, the mRNA levels of proinflammatory senescence-associated secretory phenotype (SASP) factors increased in EPC cells constitutively overexpressing p16INK4a. These results suggest that the lack of p16INK4a in the fish genome may be a critical determinant of senescence resistance in fish cell lines.

Increased expression of hras induces early, but not full, senescence in the immortal fish cell line, EPC.

In contrast to most mammals including human, fish cell lines have long been known to be immortal, with little sign of cellular senescence, despite the absence of transformation. Recently, our laboratory reported that DNA demethylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induces telomere-independent cellular senescence and senescence-associated secretory phenotype (SASP) in an immortal fish cell line, EPC (Epithelioma papulosum cyprini). However, it is not known how fish derived cultured cells are usually resistant to aging in vitro. In this study, we focused on Ras, which carries out the main role of Ras-induced senescence (RIS), and investigated the role of Ras in the regulation of senescence in EPC cells. Our results show that 5-Aza-dC induced the expression of the ras (hras, kras, nras) gene in EPC cells. EPC cells overexpressing HRas or its constitutively active form (HRasV12) showed p53-dependent senescence-like growth arrest and senescence-associated ß-galactosidase (SA-ß-gal) activity with a large and/or flat morphology characteristic of cell senescence. On the other hand, the SASP was not induced. These results imply that the increased expression of HRas contributes to early senescence in EPC cells, but it alone may not be sufficient for the full senescence, even if HRas is aberrantly activated. Thus, the limited mechanism of RIS may play a role in the senescence-resistance of fish cell lines.

DNA demethylation with 5-aza-2'-deoxycytidine induces the senescence-associated secretory phenotype in the immortal fish cell line, EPC.

Fish cell lines are known to be immortal and do not show the signs of cellular senescence despite the absence of transformation. Furthermore, high telomerase activities responsible for maintenance of telomere length are detected in many organs in live fish, irrespective of fish age. On the other hand, although it is reported that cytosine methylation at CpG island shores decreases as zebrafish age, the relationship between DNA methylation and cellular senescence in fish has not been explored. In this study, we investigated the induction of cellular senescence and senescence-associated secretory phenotype (SASP) in a fathead minnow Pimephales promelas immortal cell line, Epithelioma papulosum cyprini (EPC) treated with the DNA demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). DNA demethylation by 10 µM of 5-Aza-dC caused cell growth arrest, morphological senescence-like phenotypes and induction of senescence-associated ß-galactosidase (SA-ß-gal) activity, likely due to a mitotic catastrophe caused by disruption of chromosome segregation. Furthermore, RT-qPCR analyses revealed significant up-regulation of senescence markers such as p53-p21 and p16-Rb pathways as well as several SASP factors in 5-Aza-dC treated cells. Meanwhile, although DNA demethylation suppressed the transcription of myc and its downstream target, telomerase reverse transcriptase (tert), telomerase activity was no more than modestly decreased. These results suggest that although DNA methylation may be involved in the suppression of cellular senescence, it not critical for the immortalization of the fish cell line.

Human exposure to trace elements via farmed and cage aggregated wild Axillary seabream (Pagellus acarne) in a copper alloy cage site in the Northern Aegean Sea.

Axillary seabream (Pagellus acarne) farmed in a copper alloy mesh pen and wild individuals of P. acarne aggregated near the copper-alloy cages presented higher concentrations of trace metals in the liver, skin and gills than in fish muscle tissues in two batches of small and large fish sizes. Elevated mean levels of metals (mg kg-1) in muscle tissues in both small and large fish size groups were observed in the rank order of Zn(3.43) > Fe(3.01) > Cu(0.59) > Mn(0.13) and Fe(3.82) > Zn(3.32) > Cu(0.62) > Mn(0.17) for copper cage-farmed fish, relative to ranked mean levels for Zn(2.64) > Fe(1.95) > Cu(0.25) > Mn(0.09) and Fe(5.79) > Zn(3.58) > Cu(0.58) > Mn(0.28) for the copper cage-aggregated wild fish. Nevertheless, trace metal concentrations in fish harvested from the copper cage or those of the cage-aggregated wild individuals in both size groups were far below maximum levels of seafood safety recommended by USEPA and FAO/WHO. Target hazard quotients, calculated to estimate the non-carcinogenic health risks of metals by consuming these fish, were below "1″ (THQ < 1), indicating that there were no potential health risks for humans when consuming copper-caged fish or wild-caught individuals aggregated around the copper mesh pen, with respect to the limits suggested by US Food and Drug Administration and EU Regulations for Seafood Consumption.

Comprehensive evaluation of immunomodulation by moderate hypoxia in S. agalactiae vaccinated Nile tilapia.

Streptococcus agalactiae is a major bacterial pathogen in tilapia aquaculture. Vaccines are known to provide protection but S. agalactiae clearance in tilapia can be reduced by marginal environmental conditions. Therefore, the purpose of this study is to examine S. agalactiae clearance in vaccinated Nile tilapia under moderate hypoxic (55± 5% DO) and normoxic (85 ± 5%DO) conditions. Fish were acclimatized to either moderate hypoxia or normoxia and immunized with formalin-inactivated S. agalactiae. Fish were experimentally challenged with S. agalactiae at 30 days post-vaccination. Serum antibody titer was significantly higher in vaccinated fish kept under normoxic condition compared to the moderate hypoxic condition at fifteen and thirty days post-vaccination. The cumulative mortality following challenge was significantly reduced in vaccinated fish kept under normoxic condition compared to those in moderate hypoxic condition reflecting that pre-challenge antibody titer may correlate with survival of fish. Blood and tissue pathogen burden detection of S. agalactiae studies revealed that culturable S. agalactiae cells could not be detected in the blood of normoxic vaccinated fish at all the sampling points. In contrast, fish vaccinated in moderate hypoxic condition had considerable number of culturable S. agalactiae cells in their blood up to 5 days following challenge. Phagocytosis and intracellular reactive oxygen species (ROS) production were lowered by moderate hypoxia in vitro. Furthermore, presence of specific antibodies and higher specific antibody level in the serum increased phagocytosis, ROS production and lowered intracellular survival of S. agalactiae in head kidney leukocytes. Overall this study has highlighted that S. agalactiae clearance in vaccinated Nile tilapia is modulated by moderate hypoxia. One of the possible explanations for this might be less efficient phagocytic activities due to low oxygen availability and lower specific antibody production in vaccinated fish.

On-site Direct Detection of Astaxanthin from Salmon Fillet Using Raman Spectroscopy.

A new technology employing Raman spectroscopy is attracting attention as a powerful biochemical technique for the detection of beneficial and functional food nutrients, such as carotenoids and unsaturated fatty acids. This technique allows for the dynamic characterization of food nutrient substances for the rapid determination of food quality. In this study, we attempt to detect and measure astaxanthin from salmon fillets using this technology. The Raman spectra showed specific bands corresponding to the astaxanthin present in salmon and the value of astaxanthin (Raman band, 1518 cm-1) relative to those of protein/lipid (Raman band, 1446 cm-1) in the spectra increased in a dose-dependent manner. A standard curve was constructed by the standard addition method using astaxanthin as the reference standard for its quantification by Raman spectroscopy. The calculation formula was established using the Raman bands typically observed for astaxanthin (i.e., 1518 cm-1). In addition, we examined salmon fillets of different species (Atlantic salmon, coho salmon, and sockeye salmon) and five fillets obtained from the locations (from the head to tail) of an entire Atlantic salmon. Moreover, the sockeye salmon fillet exhibited the highest astaxanthin concentration (14.2 mg/kg), while coho salmon exhibited an intermediate concentration of 7.0 mg/kg. The Raman-based astaxanthin concentration in the five locations of Atlantic salmon was more strongly detected from the fillet closer to the tail. From the results, a rapid, convenient Raman spectroscopic method was developed for the detection of astaxanthin in salmon fillets.

Influence of moderate hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus (Nile tilapia).

Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55 ± 5% DO) or normoxia (85 ± 5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V. anguillarum were significantly higher in normoxic fish. Serum killing of V. anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 ß, TCR-ß, MHC-II ß), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.

The study on the candidate probiotic properties of encapsulated yeast, Saccharomyces cerevisiae JCM 7255, in Nile Tilapia (Oreochromis niloticus).

Saccharomyces cerevisiae JCM 7255 was tested as a probiotic candidate in tilapia after encapsulating and freeze drying. Viability and morphology during storage and during transit through simulated gut and bile conditions were determined. Growth performance, anti-streptococcal activity and gut mucosal immune parameters were also tested. The viability of encapsulated yeasts was significantly high in simulated gastric and bile conditions and remained high after storage at room temperature for 14 days. The morphology of free S. cerevisiae revealed rough, bumpy, ruptured surface during incubation in gut and bile conditions. Agar spot anti-streptococcal activity showed inhibition of 20 out of 30 strains of Streptococcus agalactiae. Supplementation improved the intestinal structure and growth performance in tilapias. Intraepithelial lymphocytes in the proximal intestine were significantly observed. Lower cumulative mortality after the oral streptococcal challenge was also seen. The results suggest that encapsulated S. cerevisiae JCM 2755 could be a potential probiotic strain in tilapia culture.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish.

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Efficacy of silkworm (Bombyx mori L.) chrysalis oil as a lipid source in adult Wistar rats.

The effects of silkworm chrysalis oil, rich in n-3 α-linolenic acid (ALA), on lipid metabolism in Wistar rats were investigated. The rats were fed diets containing 7% soybean oil (control), silkworm chrysalis oil (SWO), or fish oil (FO) for 8weeks. Plasma triglyceride and glucose levels were significantly lower in the SWO group after 8weeks compared to the control and FO groups. The total cholesterol and blood urea nitrogen levels were higher in the control group than in the SWO and FO groups at 8weeks post-consumption. However, aspartate amino transferase and alanine amino transferase levels were not significantly different among all groups. A higher arachidonic acid (AA) content was detected in the control group, while lower AA levels were observed with the increase in EPA and DHA in the SWO and FO groups. These results suggest that n-3 α-linolenic acid-rich silkworm chrysalis oil can improve hyperlipidaemia and hyperglycaemia.

Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus.

The present study revealed a relationship between the kinetic change of apoptosis and the inflammatory response during experimental intraperitoneal infection with Edwardsiella tarda as a septicemic model. The morphological changes of apoptotic cells including cellular shrinkage, condensed nuclear chromatin, nuclear fragmentation and membrane blebbing were detected by light and transmission electron microscopy. TUNEL and agarose gel electrophoresis confirmed the fragmentation of DNA in the apoptotic cells. Apoptosis was highly detected in lymphoid organs prior to the inflammatory process and gradually decreased after an extensive inflammatory response. Apoptosis in thymus and spleen was extensive and an in vitro study revealed that lymphocytes were the major cell population which underwent apoptosis. The result suggests that E. tarda-induced systemic immunosuppression via lymphocyte apoptosis as determined by suppression of the systemic inflammatory response during an initial step of generalized septicemia.

Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus).

In recent years, probiotics, especially lactic acid bacteria, have been used as dietary supplements to protect fish from various infections. Here, we examined the protective effects of Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Cumulative mortality was significantly lower in probiotic-supplemented fish than in control fish. In a histopathological survey, pyogranulomatous responses were observed at an earlier stage and to a greater extent in the probiotic-supplemented fish than in the control fish. Immunohistochemistry using an anti-E. tarda antibody revealed a larger number of positive signals in pyogranuloma-participating cells, indicating an enhanced phagocytic ability. Alternative complement activity was significantly higher in the probiotic groups than in the control. These results suggest that L. rhamnosus enhanced the alternative complement system of the fish, enabling phagocytic cell aggregation, increasing phagocytic activity and subsequently protecting the fish from acute septicemic death by E. tarda infection. Prevention of thymic necrosis by the probiotic supplement seems to minimize immunosuppression and to initiate an immune response against edwardsiellosis.

A needle-type optical enzyme sensor system for determining glucose levels in fish blood.

A needle-type biosensor system was developed for rapid and simple determination of glucose levels in fish blood. The sensor comprises a needle-type hollow container (18-gauge needle), immobilized enzyme membrane and optic fiber probe with ruthenium complex. The enzyme membrane was prepared from glucose oxidase, azide-unit pendant water-soluble photopolymer and an ultra-thin dialysis membrane. The optic fiber probe was inserted into the rolled enzyme membrane placed in the needle-type hollow container. The calibration curve was linear for glucose levels in fish plasma. One assay was completed within 3 min. A good reproducibility was observed for 60 times without exchange of the enzyme membrane. The sensor was inserted into the caudal vein of fish to measure blood glucose levels. The sensor responded immediately after insertion and glucose levels could be monitored. Good correlations were observed between values determined using the sensor and conventional methods in the range of 48-157 mg dl(-l) (correlation coefficient, 0.9474).

Development of functional fish feed with natural ingredients to control heavy metals.

The effects of two natural ingredients, Chinese parsley (CP) and chitosan (CT), on growth, accumulation, and excretion of cadmium in fish body and preservation of essential trace metals in the body were investigated by using rainbow trout that had been fed cadmium-added diet, low and high concentration, for 3 weeks. This pretest confirmed that cadmium was accumulated in the liver, kidney, and intestine of the test fish. The cadmium level of the fish, fed diet with CP or CT, was decreased by 18% and 24%, respectively, compared to that of the fish given the control diet. But CP and CT did not have an influence on normal growth of test fish and the levels of essential trace metals in the body. In addition, the level of cadmium was higher in liver than kidney in the high-cadmium dietary group, indicating the Cd level in kidney follows that of liver as kidney lies in the final stage of metabolism. The cadmium accumulation in the fish body was supposed to be reduced, by giving CP to increase the solubility of Cd to body fluid by conjugation into metallothioneins (MTs), while CT was supposed to be responsible for the physical adsorption of cadmium ions by glucosamine groups.

Rainbow trout neutrophils are responsible for non-specific cytotoxicity.

This is the first report that rainbow trout (Oncorhynchus mykiss) neutrophils are responsible for non-specific cytotoxicity. A monoclonal antibody (mab) for rainbow trout leucocytes was produced. Using this mab (TTL-5E9), neutrophils (5E9-positive cells) were isolated from the pronephros by a panning technique. The isolated neutrophils showed high viability (approximately 95%) and purity (92-95%), and were functional in cytotoxic activity assays. The neutrophils demonstrated significantly higher cytotoxic activities against YAC-1 target cells than the other cells (5E9-negative cells, predominantly lymphocytes). The number of neutrophils contaminating the 5E9-negative fraction and their non-specific cytotoxicities were positively correlated. These findings demonstrate that rainbow trout neutrophils possess non-specific cytotoxic activities.