Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Changes of ammonia, urea contents and transaminase activity in the body during aerial exposure and ammonia loading in Chinese loach Paramisgurnus dabryanus.

The Paramisgurnus dabryanus was exposed to 30 mmol L-1 NH4Cl solution and air to assessing the change of body ammonia and urea contents and the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST). After 48 h of ammonia exposure, ammonia concentration in the plasma, brain, liver and muscle were 3.3-fold, 5.6-fold, 3.5-fold and 4.2-fold, respectively, those of the control values. Plasma, brain, liver and muscle ammonia concentrations increased to 2.2-fold, 3.3-fold, 2.5-fold and 2.9-fold, respectively, those of control values in response to 48 h of aerial exposure. Within the given treatment (ammonia or aerial exposure), there was no change in plasma, brain and liver urea concentrations between exposure durations. The plasma ALT activity was significantly affected by exposure time during aerial exposure, while the liver ALT activity was not affected by ammonia or aerial exposure. Exposure to NH4Cl or air had no effect on either plasma or liver AST activity. Our results suggested that P. dabryanus could accumulate quite high level of internal ammonia because of the high ammonia tolerance in its cells and tissues, and NH3 volatilization would be a possible ammonia detoxification strategy in P. dabryanus. Urea synthesis was not an effective mechanism to deal with environmental or internal ammonia problem. The significant increase of ALT activity in plasma during aerial exposure, indicating that alanine synthesis through certain amino acid catabolism may be subsistent in P. dabryanus.

[Certain Blood Values of the Siberian Roach Rutilus rutilus in Affected Area of Warm Water from Gusinoozerskaya State District Power Plant (Eastern Siberia)].

The erythron parameters of Siberian roach inhabiting Gusinoe Lake were determined for the first time in an area of warm water discharged from the Gusinoozerskaya State District Power Plant (SDPP) (Eastern Siberia). It was noted that the defects of erythropoiesis identified (degenerative changes in the cytoskeleton, cytoplasm structure, and erythrocytes nuclei and a tendency to the development of hypochromic anemia) were obviously due to the combined impact of chemical and heat pollution of the water area of Gusinoe Lake by water discharged from the SDPP.

Effects of nonylphenol and ethinylestradiol on copper redhorse (Moxostoma hubbsi), an endangered species.

The copper redhorse, Moxostoma hubbsi, is an endangered species endemic to Quebec. The presence of contaminants, in particular endocrine disrupting chemicals (EDCs), in its habitat has been advanced as partly responsible for the reproductive difficulties encountered by the species. In the present study, immature copper redhorse were exposed to the estrogenic surfactant nonylphenol (NP; 1, 10 and 50µg/l) and the synthetic estrogen 17α-ethinylestradiol (EE2; 10ng/l) for 21 days in a flow-through system. The endpoints investigated included general health indicators (hepatosomatic index and hematocrit), thyroid hormones, sex steroids, brain aromatase activity, plasma and mucus vitellogenin (VTG), cytochrome P4501A protein expression and ethoxyresorufin-O-deethylase activity, heat shock protein 70 (HSP70) and muscle acetylcholinesterase. Exposure to 10ng EE2/l significantly increased brain aromatase activity. Exposure to 50µg NP/l resulted in a significant reduction of plasma testosterone concentrations and a significant induction of hepatic HSP70 protein expression. NP at 50µg/l also induced plasma and mucus VTG. The presence of elevated VTG levels in the surface mucus of immature copper redhorse exposed to NP, and its correlation to plasma VTG, supports the use of mucus VTG as a non-invasive biomarker to evaluate copper redhorse exposure to EDCs in the environment and contribute to restoration efforts of the species. The results of the present study indicate that exposure to high environmentally relevant concentrations of NP and EE2 can affect molecular endpoints related to reproduction in the copper redhorse.

cDNA cloning and expression characterization of serum transferrin gene from oriental weatherfish Misgurnus anguillicaudatus.

In this study, the cDNA coding serum transferrin (stf) of Misgurnus anguillicaudatus (mastf) was cloned. mastf cDNA is composed of 2326 bp with a 2007 bp open reading frame encoding 668 amino acids. The deduced Mastf protein consists of a signal peptide, two lobes (N and C-lobes) and signature motifs of transferrin (Tf) family. The results of tissue distribution indicated that mastf mRNA was predominantly expressed in the liver. The results indicate that the mastf expression increased significantly in liver, blood, spleen and head kidney after the challenge with Aeromonas sobria, acting as a positive acute protein, suggesting that mastf is related to the immune response. The cloning and expression analysis of mastf further demonstrates the evolutionary conservation of Stf and immune function in vertebrates.

An inter-laboratory study on the variability in measured concentrations of 17ß-estradiol, testosterone, and 11-ketotestosterone in white sucker: implications and recommendations.

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.

[Ultrastructure of granulocytes of bony fishes (orders Salmoniformes, Cypriniformes, Perciformes)].

Analysis of data on utrastructure of granulocytes of freshwater and marine bony fish of orders Salmoniformes, Cypriniformes, and Perciformes showed that in all studied species there were revealed two types of granulocytes - neutrophils and eosinophils. The exception was the bluefish Pomatomus saltatrix L. whose pronephros hemopoietic tissue was found to contain one type of the granulocytic line - neutrophils. The identification parameters of granular leukocytes are specific granules filling the cytoplasm. The main form of specific granules in neutrophils of bony fish of various phylogenetic groups is an elongated granule with different distribution of fibrils or a granule that has crystalloid formed from fibrils. The main form of eosinophil granules - large, electron-dense, homogenous.

Protein-sparing effect of dietary lipid in practical diets for blunt snout bream (Megalobrama amblycephala) fingerlings: effects on digestive and metabolic responses.

This study aimed to evaluate the protein-sparing effect of dietary lipid on digestive and metabolic responses of fingerling Megalobrama amblycephala. Fish were fed nine practical diets with three protein levels (270, 310 and 350 g kg(-1)) and three lipid levels (40, 70 and 100 g kg(-1)) for 8 weeks. Weight gain was significantly affected only by dietary lipid levels with the highest found in fish fed 70 g kg(-1) lipid. Relative feed intake and whole-body protein content showed little difference among all the treatments. Activities of intestine lipase and amylase increased significantly as dietary lipid levels increased, whereas little difference was observed in protease activities. Liver lipid content was significantly affected only by protein levels with the lowest found in fish fed 310 g kg(-1) protein. Liver aspartate aminotransferase (GOT) activities increased significantly with decreasing lipid levels, whereas the highest GOT activity was obtained in fish fed 310 g kg(-1) protein in terms of dietary protein levels. Activities of liver lipoprotein lipase, total lipase and plasma cholesterol concentration of fish fed 350 g kg(-1) protein were significantly lower than that of the other groups, whereas the same was true for plasma 3, 5, 3'-triiodothyronine level of fish fed 270 g kg(-1) protein. The results indicated that an increase of dietary lipid content from 40 to 70 g kg(-1) can enhance the growth and digestive enzyme activities of this species and reduce the proportion of dietary protein catabolized for energy without inducing hepatic steatosis; meanwhile, decreasing protein level from 350 to 310 g kg(-1) leads to the increase of lipase activities both in intestine and liver coupled with the reduced liver lipid content.

Response of white sucker (Catostomus commersoni) to pulp and paper mill effluent in the Androscoggin River, Maine, USA.

Adverse effects of pulp and paper mill effluent on fish populations have been well documented in many countries over the last two decades. Some of the initial studies were at mills with conventional chlorine bleaching and no secondary effluent treatment. Following installation of secondary treatment, changes in bleaching technology to elemental chlorine-free bleaching, and other process changes, adverse effects on fish were reduced or eliminated at some mills. Because no two mills are exactly alike, it is difficult to predict adverse impacts of any given mill on fish populations. In 1994, a study of female white sucker (Catostomus commersoni) in the Androscoggin River, Maine, USA, showed induction of mixed function oxidase, reductions in gonad size and plasma estradiol, and an increase in plasma testosterone in fish downstream of discharges from three large bleached kraft pulp and paper mills, and host community municipal sewage treatment plants (STP). After all three mills switched to elemental chlorine-free bleaching in the late 1990s, studies from 2001 to 2003 found that the pattern of reproductive impacts on white sucker populations measured in 1994 was not repeated. In addition, population estimates of white sucker from 2002 to 2003 using mark-recapture techniques found that densities and biomass were well within the range of those of a reference population, and of those reported in the literature for unimpacted populations. Detailed studies immediately above and below each mill/sewage treatment plant showed no evidence of reproductive effects. However, a clear pattern of eutrophication was noted, which increased cumulatively downstream below each mill/STP.

[Genetic diversity, origin and patterns of geographic distribution of polyploid spiny loach (Cypriniformes, Cobitidae, Cobitis) in Ukraine].

Only 16 biotypes from 28 possible ones, which could be generated during hybridization of C. elongatoides, on the one hand, and representatives of superspecies C. taenia s. lato (C. taenia, C. tanaitica, C. species-1), on the other hand, were discovered in water areas of Ukraine. Furthermore. two biotypes, presumably C. aff. melanoleuca-tanaitica, were discovered in the north-western regions. Polyploids have compounded 65% of investigated individuals of Cobitis genus, the average quantity of males has compounded less than 1% and it was equal among triploids and tetraploids. Absence of amphidiploids and also lack of sufficiently large number of individuals with recombined genotype and abnormal electrophoretic spectra should be stressed. Two loci of polyploid biotypes formation are marked out: southern (the Lower Danube) and northern (the Upper Danube, Oder and Rhine). C. taenia and non-specified C. species, typical of the north-western basins of Ukraine, participate in formation of polyploids in the last mentioned river alongside with C. elongatoides and C. tanaitica. It is established that in spite of clearly evident expansion capacity, so-called southern biotypes (C. 2 (3) elongatoides-tanaitica, C. elongatoides--2 (3) tanaitica) in comparison with the northern ones, which genome includes chromosomal complement C. taenia or C. species-1, are sharply limited in their spread to the East. The reason of such a situation is unbalanced gynogenetic crossing between females of the southern biotypes and C. taenia males, accompanied by introgressions, genetic instability and reduced posterity viability.

Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody.

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of approximately 425 kDa (copper redhorse) and approximately 450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of approximately 150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.

Comparison of haematology and serum biochemistry of cultured and wild Dojo loach Misgurnus anguillicaudatus.

Haematological and serum biochemistry parameters were studied and compared between cultured and wild ecotypes of Dojo loach Misgurnus anguillicaudatus during the prime fishing season, i.e. May-August. Data were analysed for the impact of feeding regime and other ecological conditions on the physiology of fish. The results revealed that haemoglobin, cholesterol, total protein, creatinine and uric acid levels in the two ecotypes were significantly different (n = 56, df = 54, P < 0.05). In addition, red blood cell, glucose, triglyceride and urea nitrogen levels were significantly higher in cultured individuals (n = 56, df = 54, P < 0.01) than in their wild counterparts. In contrast, the white blood cell level in cultured fish was significantly (n = 56, df = 54, P < 0.01) lower than that in the wild ones. These differences can be attributed to the physiological acclimatization of the fish to their living conditions and feeding regime, which influences the energy metabolism and, consequently, the health of the fish.

Reproductive toxicity of vinclozolin in the fathead minnow: confirming an anti-androgenic mode of action.

The objective of the present study was to characterize responses of the reproductive endocrine system of the fathead minnow (Pimephales promelas) to the fungicide vinclozolin (VZ), using a 21-d reproduction assay, and a shorter-term (approximately two weeks) test in which fish were cotreated with the VZ (a putative anti-androgen) and the androgen 17beta-trenbolone (TB). Effects on fecundity, gonadal histology, secondary sexual characteristics, reproductive hormones, and relative abundance of androgen receptor (AR) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) mRNA transcripts were evaluated in one or both of these studies. Fecundity of VZ-exposed fish was decreased in a concentration-dependent manner in the 21-d test, culminating in complete reproductive failure at a concentration of 700 microg/L. Exposure to VZ decreased expression of male secondary sexual characteristics -- an effect typical of anti-androgens. The finding that exposure of females to TB-induced expression of prominent, male-like tubercles, which could be effectively blocked with VZ, provides powerful evidence of the anti-androgenic activity of VZ in vivo. In the two experiments VZ produced several responses possibly indicative of compensation or adaptation of the fish to the anti-androgen, including increases in gonad weight, AR and 11 betaHSD mRNA transcript abundance, and ex vivo gonadal production of testosterone and 11-ketotestosterone. Overall, our results demonstrate that the model anti-androgen VZ, which also is an environmental contaminant, impairs reproductive success of fathead minnows and elicits endocrine responses consistent with an anti-androgenic mode of action.

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens.

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis.

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.

Purification, crystallization and preliminary x-ray diffraction analysis of carboxyhaemoglobin-II from the fish Piaractus mesopotamicus (pacu).

Carboxyhaemoglobin-II isolated from the pacu (Piaractus mesopotamicus) has been crystallized and X-ray diffraction data were collected to 2.0 A resolution using synchrotron radiation. Crystals were characterized as belonging to the space group I23; preliminary structural analysis reveals the presence of one dimer in the asymmetric unit.

Plasma testosterone and 11-ketotestosterone levels of male pacu Piaractus mesopotamicus (Cypriniformes, Characidae)

The levels of testosterone (T) and 11-ketotestosterone (11-KT) of the South American pacu Piaractus mesopotamicus were determined by radioimmunoassay during two stages of the reproductive cycle, i.e., resting and maturation, and the gonadosomatic index (GSI) was calculated. The highest levels of T and 11-KT were reached during the maturation stage (T= 2400 + 56 pg/ml; 11-KT= 2300 + 60 pg/ml) and lower levels were maintained during the resting period. The rise in androgen levels occurred with the appearance of spermatozoa in the maturation stage, when GSI was highest.

Plasma testosterone and 11-ketotestosterone levels of male pacu Piaractus mesopotamicus (Cypriniformes, Characidae).

The levels of testosterone (T) and 11-ketotestosterone (11-KT) of the South American pacu Piaractus mesopotamicus were determined by radioimmunoassay during two stages of the reproductive cycle, i.e., resting and maturation, and the gonadosomatic index (GSI) was calculated. The highest levels of T and 11-KT were reached during the maturation stage (T = 2400 +/- 56 pg/ml; 11-KT = 2300 +/- 60 pg/ml) and lower levels were maintained during the resting period. The rise in androgen levels occurred with the appearance of spermatozoa in the maturation stage, when GSI was highest.

Effects of temperature and white sucker (Catostomus commersoni) serum supplement on the in vitro multiplication of Cryptobia catostomi in cell-free culture medium.

Cryptobia catostomi, a non-pathogenic haemoflagellate of white suckers (Catostomus commersoni) multiplied rapidly in modified TDL-15 medium supplemented with 10% fetal bovine serum and 1% heatinactivated white sucker serum (WSS) at 10 degrees C and 18 degrees C. The numbers of C. catostomi counted were significantly higher in cultures incubated at 10 degrees C than in those incubated at 18 degrees C beginning at 3 weeks post-incubation. The culture forms (from the eighth subculture at 9 months after isolation) were morphologically similar to blood forms and were infective to laboratory-raised white suckers. The parasite survived for about 4 weeks in the medium without WSS at 18 degrees C. The present study indicates that WSS supplement supports the in vitro multiplication of C. catostomi and that its multiplication is temperature-dependent.

Effects of [D-Arg6, Trp7, Leu8, Pro9NEt]-luteinizing hormone-releasing hormone (sGnRH-A) and [D-Ala6, Pro9NEt]-luteinizing hormone-releasing hormone (LHRH-A), in combination with pimozide or domperidone, on gonadotropin release and ovulation in the Chinese loach and common carp.

The effects of LHRH-A and sGnRH-A alone and in combination with the dopamine receptor antagonists pimozide (PIM) and domperidone (DOM) on stimulation of gonadotropin (GtH) secretion and ovulation in Chinese loach (Paramisgurnus dabryanus) and common carp (Cyprinus carpio) were studied. When tested in the absence of a dopamine receptor antagonist, sGnRH-A was about 10-fold more potent than LHRH-A in stimulating GtH secretion in loach, whereas these peptides were of similar effectiveness in common carp. Both peptides were effective alone in inducing ovulation in loach, but were ineffective in common carp. PIM and DOM stimulate a small but significant increase in serum GtH, and greatly potentiate the effects of LHRH-A and sGnRH-A on GtH secretion and ovulation in both species. DOM was of equal or somewhat greater potency than PIM in potentiating the action of releasing hormone agonists in loach; however, DOM had markedly greater effectiveness in common carp. sGnRH-A in the presence of DOM was more effective than LHRH-A in stimulating GtH secretion and ovulation in common carp. The present studies suggest that there are marked differences in the relative contribution of GnRH and dopamine in the control of GtH secretion in loach and common carp, and indicate in particular that the more intense dopaminergic inhibitory control of GtH release in common carp modifies the responsiveness of the pituitary to GnRH peptides.