An acute increase in water temperature can increase free amino acid concentrations in the blood, brain, liver, and muscle in goldfish (Carassius auratus).

Water temperature directly affects the body temperature in fish, so increasing water temperatures in oceans and rivers will lead to increases in fish body temperatures. Whilst a range of responses of fish to increases in water temperature have been measured, amino acid metabolism in a fish under high water temperature (HT) conditions has not been investigated. The aim of this study was to determine the effects of an acute increase in water temperature on oxygen consumption, plasma cortisol concentrations, and free amino acid concentrations in plasma and several tissues in goldfish (Carassius auratus). Oxygen consumption and plasma cortisol concentrations were increased in goldfish exposed to HT (30 ± 1 °C) for 200 min compared with goldfish at a control water temperature (CT 17 ± 1 °C). Oxygen consumption and plasma cortisol concentrations in both groups of fish combined were positively correlated. When goldfish were exposed to HT for 300 min oxygen consumption and plasma concentrations of 15 free amino acids were increased compared with goldish at CT. Concentrations of several free amino acids were increased to varying extents in the brain, liver, and muscle tissues. In conclusion, an acute increase in water temperature affected amino acid metabolism differently in the brain, liver, and muscle tissues. Goldfish will be a useful species for further studies of the possible roles of various amino acids in the brain, muscle, and liver during acute increases in water temperature in fish.

High levels of circulating prostaglandin F2α associated with ovulation stimulate female sexual receptivity and spawning behavior in the goldfish (Carassius auratus).

This study tested the hypothesis that blood-borne prostaglandin F2α (PGF2α) produced at the time of ovulation by female goldfish, a typical scramble-spawning, egg-laying cyprinid fish, functions as a hormone which stimulates female sexual receptivity, behavior, and pheromone release, thereby synchronizing female mating behavior with egg availability. We conducted 5 experiments. First, we tested whether PGF2α is found in the blood of female fish and if it increases at the time of ovulation. Using gas chromatography-mass spectrometry, we found that circulating PGF2α was approximately 1 ng/ml prior to ovulation, increased over 50-fold within 3 h of ovulation and returned to preovulatory values after spawning and egg release. Ovulated fish also released over 2 ng/h of PGF2α and 800 ng/h of 15-keto-PGF2α, a metabolite of PGF2α - both compounds with known pheromonal function. Second, we tested how closely levels of circulating PGF2α tracked the timing of ovulation by sampling fish at the time of ovulation and discovered that PGF2α increased within 15 min of ovulation, peaked after 9 h, and fell to basal levels as fish spawned and released their eggs. Third, we tested whether an interaction between eggs and the reproductive tract serves as a source of circulating PGF2α and its relationship with female sexual receptivity by injecting ovulated eggs (or an egg-substitute) into the reproductive tract of females stripped of ovulated eggs. We found both of these treatments elicited measurable increases in plasma PGF2α as well as female sexual behavior. A fourth experiment showed that indothemacin, a PG synthase inhibitor, blocked both PGF2α increase and female sexual behavior in egg-substitute-injected fish. Finally, we tested the relationship between the expression of female behavior and PGF2α in PGF2α-injected fish and found that circulating PGF2α levels closely paralleled behavior, rising within 15 min and peaking at 45 min. Together, these experiments establish that PGF2α functions as a behavioral blood-borne hormone in the goldfish, suggesting it likely has similar activity in other related, externally-fertilizing fishes.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

Effect of sub-chronic exposure to lead (Pb) and Bacillus subtilis on Carassius auratus gibelio: Bioaccumulation, antioxidant responses and immune responses.

Lead (Pb) poisoning in humans and fish represents a significant global problem. Bacillus subtilis (B. subtilis) is a widely used probiotic in aquaculture. Carassius auratus gibelio (C. gibelio) is one of the most important aquaculture species with great commercial value. The objective of this study was to evaluate the potential of B. subtilis in ameliorating lead-induced toxicity in C. gibelio. The fish were exposed for 60 days to waterborne Pb at 0, 0.05, 0.5 and 1 mg/L and/or dietary B. subtilis at 109 cfu/g. After 30 and 60 days, the fish were sampled and bioaccumulation, antioxidant activity and immune responses were assessed. The results revealed that B. subtilis confers significant protective effects against lead toxicity by preventing alterations in the levels of bioaccumulation, superoxide dismutase, catalase and glutathione. B. subtilis also assists in the recovery of blood δ-aminolevulinic acid dehydratase, lysozyme, and IgM levels while regulating the expression of immune-related genes including IL-10, lysozyme, TNF-α, IgM and Hsp70 after 60 days of lead exposure. Our results suggest that administration of B. subtilis (109 cfu/g) has the potential to combat lead toxicity in C. gibelio.

Pharmacokinetics of cefquinome in crucian carp (Carassius auratus gibelio) after oral, intramuscular, intraperitoneal, and bath administration.

The pharmacokinetics (PK) of cefquinome (CEQ) was studied in crucian carp (Carassius auratus gibelio) after single oral, intramuscular (i.m.), and intraperitoneal (i.p.) administration at a dose of 10 mg/kg body weight and following incubation in a 5 mg/L bath for 5 hr at 25°C. The plasma concentration of CEQ was determined using high-performance liquid chromatography (HPLC). PK parameters were calculated based on mean CEQ concentration using WinNonlin 6.1 software. The disposition of CEQ following oral, i.m., or i.p. administration was best described by a two-compartment open model with first-order absorption. After oral, i.m., and i.p. administration, the maximum plasma concentration (Cmax ) values were 1.52, 40.53, and 67.87 µg/ml obtained at 0.25, 0.23, and 0.35 hr, respectively, while the elimination half-life (T1/2ß ) values were 4.68, 7.39, and 6.88 hr, respectively; the area under the concentration-time curve (AUC) values were 8.61, 339.11, and 495.06 µg hr/ml, respectively. No CEQ was detected in the plasma after bath incubation. Therapeutic blood concentrations of CEQ can be achieved in the crucian carp following i.m. and i.p. administration at a dosage of 10 mg/kg once every 2 days.

Pharmacokinetics of florfenicol and its metabolite florfenicol amine in crucian carp (Carassius auratus) at three temperatures after one single intramuscular injection.

The pharmacokinetic profiles of florfenicol (FF) or florfenicol amine (FFA) in crucian carp were compared at different water temperatures after single intramuscular administration of FF at 10 mg/kg bodyweight. The concentrations of FF and FFA were determined by a high-performance liquid chromatography method, and then, the concentration versus time data were subjected to compartmental analysis using a one-compartment open model. At the water temperatures of 10, 20, and 25°C, the peak concentrations (Cmax s) of FF were 2.28, 2.29, and 2.34 µg/ml, respectively, while those of FFA were 0.42, 0.71, and 0.82 µg/ml, respectively. And the absorption half-life (t1/2ka ) of FF was 0.21, 0.19, and 0.21 hr, while the elimination half-life (t1/2kel ) was 31.66, 24.77, and 21.48 hr, respectively. For FFA, the formation half-life (t1/2kf ) was 3.85, 8.97, and 12.43 hr, while the t1/2kel was 58.34, 30.27, and 21.22 hr, respectively. The results presented here demonstrated that the water temperature had effects on the elimination of both FF and FFA and the formation of FFA. Based on the T > MIC values calculated here, to treat the infections of bacterial with MIC value ≤ 0.5 µg/ml, FF intramuscularly given at 10 mg/kg bodyweight with a 72-hr interval is sufficient at the water temperature of 10°C, while the intervals of 60 and 48 hr were needed at 20 and 25°C, respectively. But to treat bacterial with higher MIC values, more FF or FF at 10 mg/kg BW but with shorter intervals should be intramuscularly given to the infected fish.

Pharmacokinetics of orbifloxacin in crucian carp (Carassius auratus) after intravenous and intramuscular administration.

The pharmacokinetics of orbifloxacin was studied after a single dose (7.5 mg/kg) of intravenous or intramuscular administration to crucian carp (Carassius auratus) reared in freshwater at 25°C. Plasma samples were collected from six fish per sampling point. Orbifloxacin concentrations were determined by high-performance liquid chromatography with a 0.02 µg/ml limit of detection, then were subjected to noncompartmental analysis. After intravenous injection, initial concentration of 5.83 µg/ml, apparent elimination rate constant (λz ) of 0.039 hr-1 , apparent elimination half-life (T1/2λz ) of 17.90 hr, systemic total body clearance (Cl) of 75.47 ml hr-1  kg-1 , volume of distribution (Vz) of 1,948.76 ml/kg, and volume of distribution at steady-state (Vss) of 1,863.97 ml/kg were determined, respectively. While after intramuscular administration, the λz , T1/2λz , mean absorption time (MAT), absorption half-life (T1/2ka ), and bioavailability were determined as 0.027 hr-1 , 25.69, 10.26, 7.11 hr, and 96.46%, respectively, while the peak concentration was observed as 3.11 ± 0.06 µg/ml at 2.0 hr. It was shown that orbifloxacin was completely but relatively slowly absorbed, extensively distributed, and slowly eliminated in crucian carp, and an orbifloxacin dosage of 10 mg/kg administered intravenously or intramuscularly would be expected to successfully treat crucian carp infected by strains with MIC values ≤0.5 µg/ml.

Effect of long term exposure to sublethal concentration of imidacloprid on some biochemical and haematological parameters of Grass carp and Goldfish.

During the present research, C. idella and C. auratus fish were exposed to 2 ppm concentration of imidacloprid for 28 and 24 days, respectively, and the effect on biochemical and haematological parameters was investigated. During the study of biochemical parameters, there occurred significant increase (P<0.05) in the serum levels of ALT and creatinine of imidacloprid exposed groups of both species of fish. The level of serum albumin of imidacloprid exposed groups of both fish species was significantly lower as compared to control group (P<0.05). Serum globulin level in imidacloprid exposed group of C. idella was insignificantly lower as compared to control group, however the serum globulin level of C. auratus was significantly lower than the control group (P < 0.05). The level of total proteins in serum of imidacloprid exposed groups of both fish species was insignificantly lower as compared to control groups (P>0.05). During the study of haematological parameters, TLC of C. idella was insignificantly (P>0.05) higher than control group but the TLC of C. auratus was significantly (P<0.05) higher than control. There was also observed increasing trend in the percentage of neutrophils and lymphocytes of imidacloprid exposed group of each fish species. The platelets count of imidacloprid exposed group of each fish species was significantly (P<0.05) lower than control group. The haemoglobin concentration of imidacloprid exposed group of C. idella was significantly lower than control group (P<0.05). In case of C. auratus, the haemoglobin level of imidacloprid exposed group was insignificantly lower than control group (P>0.05). From the finding of the present research it was concluded that 28 days exposure of C. idella and 24 days exposure of C. auratus to 2 ppm concentration of imidacloprid does not cause mortality however the exposure causes alteration in the normal level of biochemical and haematological parameters.

Rates of hypoxia induction alter mechanisms of O2 uptake and the critical O2 tension of goldfish.

The rate of hypoxia induction (RHI) is an important but overlooked dimension of environmental hypoxia that may affect an organism's survival. We hypothesized that, compared with rapid RHI, gradual RHI will afford an organism more time to alter plastic phenotypes associated with O2 uptake and subsequently reduce the critical O2 tension (Pcrit) of the rate of O2 uptake (MO2 ). We investigated this by determining Pcrit values for goldfish exposed to short (∼24 min), typical (∼84 min) and long (∼480 min) duration Pcrit trials to represent different RHIs. Consistent with our predictions, long duration Pcrit trials yielded significantly lower Pcrit values (1.0-1.4 kPa) than short and typical duration trials, which did not differ (2.6±0.3 and 2.5±0.2 kPa, respectively). Parallel experiments revealed these time-related shifts in Pcrit were associated with changes to aspects of the O2 transport cascade that took place over the hypoxia exposures: gill surface areas and haemoglobin-O2 binding affinities were significantly higher in fish exposed to gradual RHIs over 480 min than fish exposed to rapid RHIs over 60 min. Our results also revealed that the choice of respirometric technique (i.e. closed versus intermittent) does not affect Pcrit or routine MO2 , despite the significantly reduced water pH and elevated CO2 and ammonia levels measured following closed-circuit Pcrit trials of ∼90 min. Together, our results demonstrate that gradual RHIs result in alterations to physiological parameters that enhance O2 uptake in hypoxic environments. An organism's innate Pcrit is therefore most accurately determined using rapid RHIs (<90 min) so as to avoid the confounding effects of hypoxic acclimation.

Calorespirometry reveals that goldfish prioritize aerobic metabolism over metabolic rate depression in all but near-anoxic environments.

Metabolic rate depression (MRD) has long been proposed as the key metabolic strategy of hypoxic survival, but surprisingly, the effects of changes in hypoxic O2 tensions (PwO2 ) on MRD are largely unexplored. We simultaneously measured the O2 consumption rate (MO2 ) and metabolic heat of goldfish using calorespirometry to test the hypothesis that MRD is employed at hypoxic PwO2  values and initiated just below Pcrit, the PwO2 below which MO2  is forced to progressively decline as the fish oxyconforms to decreasing PwO2 Specifically, we used closed-chamber and flow-through calorespirometry together with terminal sampling experiments to examine the effects of PwO2  and time on MO2 , metabolic heat and anaerobic metabolism (lactate and ethanol production). The closed-chamber and flow-through experiments yielded slightly different results. Under closed-chamber conditions with a continually decreasing PwO2 , goldfish showed a Pcrit of 3.0±0.3 kPa and metabolic heat production was only depressed at PwO2  between 0 and 0.67 kPa. Under flow-through conditions with PwO2  held at a variety of oxygen tensions for 1 and 4 h, goldfish also initiated MRD between 0 and 0.67 kPa but maintained MO2  to 0.67 kPa, indicating that Pcrit is at or below this PwO2 Anaerobic metabolism was strongly activated at PwO2 ≤1.3 kPa, but only used within the first hour at 1.3 and 0.67 kPa, as anaerobic end-products did not accumulate between 1 and 4 h exposure. Taken together, it appears that goldfish reserve MRD for near-anoxia, supporting routine metabolic rate at sub-PcritPwO2  values with the help of anaerobic glycolysis in the closed-chamber experiments, and aerobically after an initial (<1 h) activation of anaerobic metabolism in the flow-through experiments, even at 0.67 kPa PwO2.

Identification of the putative goldfish (Carassius auratus) magnesium transporter SLC41a1 and functional regulation in the gill, kidney, and intestine in response to dietary and environmental manipulations.

While magnesium requirements for teleost fish highlight the physiological importance of this cation for homeostasis, little is known regarding the molecular identity of transporters responsible for magnesium absorption or secretion. The recent characterization of the vertebrate magnesium transporter solute carrier 41a1 (SLC41a1) in the kidney of a euryhaline fish has provided a glimpse of possible moieties involved in piscine magnesium regulation. The present study obtained a novel SLC41a1 coding sequence for Carassius auratus and demonstrated ubiquitous expression in all tissues examined. Transcriptional regulation of SLC41a1 in response to dietary and environmental magnesium concentrations was observed across tissues. Specifically, decreased environmental magnesium correlated with decreased expression of SLC41a1 in the intestine, whereas the gill and kidney were unaffected. Dietary magnesium restriction correlated with decreased expression of SLC41a1 in the intestine and gill, while again no effects were detected in the kidney. Finally, elevated dietary magnesium correlated with increased expression of SLC41a1 in the kidney, while expression in the intestine and gill remained stable. Plasma magnesium was maintained in all treatments, and dietary assimilation efficiency increased with decreased dietary magnesium. Consumption of a single meal failed to impact SLC41a1 expression, and transcript abundance remained stable over the course of digestion in all treatments. Transcriptional regulation occurred between 7 and 14days following dietary and environmental manipulations and short-term regulation (e.g. <24h) was not observed. Overall the data supports transcriptional regulation of SLC41a1 reflecting a possible role in magnesium loss or secretion across tissues in fish.

An in vivo assay performed using multiple biomarkers related to testosterone synthesis and conversion for assessing the androgenic potency of refuse leachate.

Refuse leachate is likely an important source of androgens. However, common in vitro bioassays underestimate the potential androgenic activity of leachate, owing to non-receptor-mediated mechanisms that modify the balance of sex hormones and promote the accumulation of endogenous androgens. This study aimed to develop an in vivo assay by using multiple biomarkers related to testosterone synthesis and conversion for assessing the potential androgenic activity of refuse leachate sampled from a municipal solid waste treatment plant in Qingdao, China. The results indicated that exposure to leachate increased the levels of testosterone and luteinizing hormone, but decreased those of 17ß-estradiol in both male and female goldfish (Carassius auratus), suggesting a potential androgenic activity. Further, Leydig cell hyperplasia and decreased gonadal P450 aromatase mRNA levels were observed; these alterations might promote the biosynthesis of testosterone and hinder the conversion of testosterone to 17ß-estradiol, which in turn enhance testosterone accumulation. Exposure to leachate also resulted in reproductive impairments, including decreased gonadosomatic index and plasma vitellogenin levels of female goldfish, as well as decreased testicular enzyme activities in male goldfish. The integrated use of biochemical, molecular, and histological markers not only improved our understanding of the androgenic effects of leachate but also verified the reliability and validity of the results. Therefore, the in vivo bioassay described in this study might allow the investigation of the androgenic effects of other complex contaminant mixtures in the future.

Plasma pharmacokinetics and muscle residue dynamics of mebendazole in Carassius auratus.

Mebendazole is approved for use in aquatic animals and is widely used in Chinese aquaculture. We developed a pharmacokinetic and residue analysis for mebendazole levels in the goldfish (Carassius auratus). Plasma and muscle samples of C. auratus were taken after oral administration of 10 mg/kg mebendazole. The maximal drug plasma concentration of 0.55 mg/L was achieved at 48 hr and then declined with the elimination half-life (T1/2ß ) of 7.99 hr. Administration of 10 mg/kg by oral gavage for 5 successive days resulted in a peak mebendazole concentration of 0.70 mg/kg in muscle at 96 hr after the last dose. The drug was then eliminated at a relatively slow rate from muscle with T1/2ß of 68.41 hr. There was no detectable mebendazole in any muscle samples at 24 days postadministration. The AUClast in plasma and muscle was 19.42 and 105.33 mg hr/L, respectively. These data provide information for dosage recommendations and withdrawal time determinations for mebendazole use in aquariums.

Diel cyclic hypoxia alters plasma lipid dynamics and impairs reproduction in goldfish (Carassius auratus).

Diel cyclic hypoxia occurs with varying frequency and duration in freshwater habitats, yet little is known about its effects on reproduction of freshwater fishes. The present study shows that long-term exposure of goldfish (Carassius auratus) to cyclic hypoxia (0.8 ± 0.2 mg/l dissolved oxygen) for 9 h or more, per day, altered plasma lipid and sex steroid profiles, which in turn directly or indirectly suppressed ovarian growth and viable spermatozoa production. Hypoxia decreased total cholesterol and high density lipoprotein (HDL p < 0.05) and elevated triglycerides (TG; p < 0.05) in both sexes. Plasma steroid concentrations particularly of 17α-hydroxyprogesterone (17-HP), estradiol (E2), testosterone (T) in females, and T and 11-ketotestosterone (11-KT) in males were attenuated under diel hypoxic conditions. Intriguingly, both diel and continuous hypoxia elevated plasma E2 and vitellogenin levels in males. However, neither lipid nor steroid profiles recorded any variation in a dose-dependent manner in response to diel hypoxia. The reduced GSI, decreased number of tertiary oocytes, and motile spermatozoa in hypoxic fish clearly indicate suppression of gametogenesis. Thereby, prolonged diel cyclic hypoxia may affect valuable fishery resources and fish population structure by impairing reproductive performances and inducing estrogenic effects in males.

BIOCHEMISTRY PANEL REFERENCE INTERVALS FOR JUVENILE GOLDFISH (CARASSIUS AURATUS).

Reference intervals for diagnostic tests are vitally important for clinical decision making. Despite the popularity of pet goldfish (Carassius auratus), reference intervals have not been generated for routine biochemistry panel analytes in this species. This study establishes de novo reference intervals for packed cell volume and total solids, using 47 apparently healthy immature goldfish, and for 11 common chemistry panel analytes (albumin, aspartate aminotransferase, calcium, creatine kinase, globulin, blood glucose, sodium, potassium, phosphorous, total protein, and uric acid) using 39 immature goldfish. Robust reference intervals were generated following recommendations of the American Society for Veterinary Clinical Pathology. Linear regression was used to demonstrate a statistically significant relationship between body weight and calcium, albumin, total protein, potassium, packed cell volume, and total solids. The results of this study serve as a useful baseline for future reference interval generation in goldfish.

Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus.

We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m(2)). We measured the change in the levels of plasma cortisol and H2O2 and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H2O2) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time. The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina.

Effects of melatonin and green-wavelength LED light on the physiological stress and immunity of goldfish, Carassius auratus, exposed to high water temperature.

This study investigated the effects of increasing water temperature (22-30 °C) on the physiological stress response and immunity of goldfish, Carassius auratus, and the ability of green light-emitting diode (LED) irradiation or melatonin injections to mitigate this temperature-induced stress. To evaluate the effects of either green-wavelength LED light or melatonin on stress in goldfish, we measured plasma triiodothyronine (T3), thyroxine (T4), and thyroid hormone receptor (TR) mRNA expression; plasma cortisol and glucose; and immunoglobulin M (IgM) and lysozyme mRNA expression. The thyroid hormone activities, TR mRNA expression, and plasma cortisol and glucose were higher in goldfish exposed to high-temperature water, but were lower after exposure to melatonin or green-wavelength LED light. Lysozyme mRNA expression and plasma IgM activity and protein expression were lower after exposure to high water temperatures and higher after melatonin or green-wavelength LED light treatments. Therefore, high water temperature induced stress and decreased immunity; however, green-wavelength LED light and melatonin treatments mitigated the effects of stress and enhanced immunity. The benefits of melatonin decreased with time, whereas those of green-wavelength LED treatment did not.

Genotoxicity of Water Contaminants from the Basin of Lake Sevan, Armenia Evaluated by the Comet Assay in Gibel Carp (Carassius auratus gibelio) and Tradescantia Bioassays.

Combination of bioassays and chemical analysis was applied to determine the genotoxic/mutagenic contamination in four different sites of the basin of Lake Sevan in Armenia. Water genotoxicity was evaluated using the single cell gel electrophoresis technique (comet assay) in erythrocytes of gibel carp (Carassius auratus gibelio), Tradescantia micronucleus (Trad-MCN) and Tradescantia stamen hair mutation (Trad-SHM) assays. Significant inter-site differences in the levels of water genotoxicity according to fish and Trad-MCN bioassays have been revealed. Two groups of locations with lower (south-southwest of the village Shorzha and Peninsula of Lake Sevan) and higher (estuaries of Gavaraget and Dzknaget rivers) levels of water genotoxicity were distinguished. Correlation analysis support the hypothesis that the observed genetic alterations in fish and plant may be a manifestation of the effects of water contamination by nitrate ions, Si, Al, Fe, Mn and Cu. Increase of DNA damage in fish also correlated with content of total phosphorus.

Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide.

Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17ß-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.

Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus).

Goldfish (Carassius auratus) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17ß-estradiol (E2)-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82ng/mL, and the working range was 3.9-250ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E2-induced goldfish. The surface mucus Vtg level in E2-induced males was significantly higher than that in the control males and E2-induced females, and was much closer to the plasma Vtg level in E2-induced males than that in E2-induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment.