Plasma levels of insulin and liver glycogen contents in one- and two-year old Atlantic salmon (Salmo salar L.) during the period of parr-smolt transformation

Plasma levels of insulin were measured by specific radioimmunoassay in 1-year and 2-year old Atlantic salmon (Salmo salar) parr during the period of parr-smolt transformation. The two-year old fish were of two different categories; silvering pre-smolts and previously mature male parr. If insulin plays an important role in parr-smolt transformation and/or subsequent osmoregulatory changes it was expected that the pre-smolts would show a different insulin profile compared to the mature male parr and one-year old parr, both of which show impaired hypoosmoregulatory ability compared to smolts. Measurements were taken during two separate years. Between January and April both categories of two-year old fish had generally higher plasma levels of insulin compared to the non-smolting one-year old parr. In the pre-smolts insulin levels ranged from 4.0 to 7.9 ng ml⁻¹, and from 7.8 to 16.7 ng ml⁻¹ in 1990 and 1992 respectively, while in the previously mature males the same respective values were from 4.3 to 10.0 ng ml⁻¹, and from 6.6 to 24.1 ng ml⁻¹. In the two-year old fish, whether pre-smolts or mature males, plasma insulin levels peaked between 1–2 months before final smoltification, after which insulin titers declined sharply. In 1990, the 1-year old parr showed a dual peak in plasma insulin. Insulin first peaked in February (7.8 ng ml⁻¹), and then again in April–May (7.7 ng ml⁻¹), while in 1992 the 1-year old parr showed a number of smaller transient peaks (5–7 ng ml⁻¹) between March–May, followed by sharp elevation of insulin levels in June. Liver glycogen contents were at their highest (3.5–5.0 g 100 g⁻¹ I liver wet weight) in March in both 1-year and 2-year old fish. Glycogen levels were low during the later stages of parr-smolt transformation, before rising again in June in both the 1-year old and precociously mature parr, but not in the smolts.     

Thyroid hormones in brown trout (Salmo trutta) reproduction and early development

Gravid brown trout (Salmo trutta) females were injected with various doses of a synthetic gonadotropin-releasing hormone analog (GnRHa), given with or without an injection of triiodothyronine (T₃), in order to investigate the potential of T₃ (a) to enhance the stimulatory effect of GnRHa on ovulation, and (b) to enhance the growth and survival of the produced progeny. From the time the hormonal treatments were initiated until ovulation was detected 5–38 days later, endogenous plasma T₃ levels increased from an average of 3.6 to 11.6 ng ml⁻¹. Injection with 20 mg T₃ kg⁻¹ body weight, further elevated plasma T₃ levels at ovulation (16.0 ng ml⁻¹. Mean time to ovulation was reduced significantly in fish injected with 10 μg kg⁻¹ of GnRHa, whereas treatment with lower doses was ineffective. Injection with T₃ did not enhance the ovulatory response of brown trout to GnRHa. Unfertilized eggs obtained from T₃-injected females had a higher T₃ content, suggesting a transfer of T₃ from the maternal circulation into the oocytes. Maternal T₃ injection had no effect on egg fertilization rates, embryo survival to eyeing and hatching, or the prevalence of abnormal larvae at the time of hatching. Length and weight gain of the progeny during yolk absorption was also not influenced by maternal T₃ treatment. At the completion of yolk-sac absorption, progeny from females injected with T₃ had a higher prevalence of skeletal abnormalities than controls. The results suggest that in teleosts like brown trout, which have high endogenous circulating T₃ levels, treatment of females with T₃ does not enhance responsiveness to GnRHa and it has the potential for deleterious effects on their offspring.     

Stress-induced changes in the affinity and abundance of cytosolic cortisol-binding sites in the liver of rainbow trout,Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding

Plasma cortisol levels and the number (N_max) and affinity (K_d) of specific hepatic cortisol-binding sites were determined in rainbow trout subjected to chronic confinement stress for 14 days. Confinement significantly elevated plasma cortisol levels to 47.3 ± 13.5 ng ml^–1 within 24h and although levels declined to 8.0 ± 3.0 ng ml^–1 after 14 days, they were significantly higher throughout than levels in unstressed control fish (< 2.0 ng ml^–1). There was a 60% reduction in cytosolic N_max in stressed fish during the first 24h of confinement (35.8 ± 7.9 cf. 129.0 ± 15.2 fmol mg^–1 protein), a decline which was sustained at 7 days after the onset of stress but, although numbers of binding sites in the liver of stressed fish were still lower than in unstressed fish, the difference was no longer significant after 14 days of confinement. There was an accompanying significant rise in the K_d of cortisol binding in stressed fish during confinement, from 4.0 ± 0.6 nM at time 0 to 8.4 ± 0.8 nM after 24 h confinement. This increment in K_d was sustained at a level significantly higher than in control fish throughout the 14 day confinement period, despite marked reductions in cortisol levels and increases in N_max in stressed fish. Throughout the study, specific binding of cortisol could not be consistently detected in high-salt nuclear extracts from stressed or unstressed fish, suggesting either that high-affinity binding sites for cortisol were absent from these preparations, that receptors were present but unable to interact with ligand because they were occupied, or that receptors were present but not being extracted. These possibilities were investigated using a range of extraction procedures, by varying the temperature of incubation, by employing dexamethasone as ligand and by examining binding in purified, intact, nuclei. Estradiol was employed as a methodological control throughout and substantial amounts of specific estradiol binding were detected in all compartments and preparations. Specific cortisol-binding sites were detected in intact nuclei of both stressed and unstressed fish, at levels an order of magnitude lower than estradiol binding in the same preparations. These data demonstrate that activation of the pituitary-interrenal axis leads to significant changes in the nature of target-tissue binding of cortisol in rainbow trout, and reveal a clear difference in the subcellular distribution of binding-sites for estradiol and cortisol, which reflects the situation in mammalian tissues.     

The purification and development of a quantitative enzyme linked immunosorbent assay (ELISA) for the measurement of vitellogenin in diploid and triploid brook trout (Salvelinus fontinalis)

Vitellogenin (VTG) was purified from the plasma of estradiol-17β (E₂) treated male brook trout (Salvelinus fontinalis; bt) using gel filtration and anion exchange chromatography. An antiserum to bt-VTG was raised in rabbits, then used to detect bt-VTG by Western blot analysis. Purified bt-VTG and its antibody were then used to develop an antibody-capture, competitive enzyme-linked immunosorbant assay (ELISA). Confirmation of the purified protein as bt-VTG was based on the characteristics of high molecular weight (∼562 kDa), dominant plasma protein in vitellogenic females and induction by exogenous E₂ treatment in males. The antiserum recognized a major 184 kDa polypeptide as well as minor bt-VTG degradation products (150–66 kDa) in purified bt-VTG and in plasma from vitellogenic females. Low levels of antibody cross reactivity were shown with plasma from nonvitellogenic females, control rabbit serum, and several other antisera known to not contain VTG. The ELISA had a minimum detection limit of 8 ng ml⁻¹ and intra- and inter-coefficients of variation less than 9% and 15%, respectively. The ELISA demonstrated parallel binding slopes among dilution curves of purified bt-VTG standard and plasma from diploid and triploid brook trout females. Using the ELISA, maximum plasma VTG levels of 93.5±33.6 mg ml⁻¹ were detected in vitellogenic diploid females, whereas only 0.18±0.15 mg ml⁻¹ were detected in triploid females of the same age (n=5 for each). Diploid and triploid males cohabitating with vitellogenic females showed measurable levels of plasma VTG during vitellogenesis in females (i.e., 0.17 and 0.06 mg ml⁻¹, respectively (n=1)). This revised version was published online in August 2006 with corrections to the Cover Date.     

Physiological stress responses in spotted wolffish (Anarhichas minor) subjected to acute disturbance and progressive hypoxia

The spotted wolffish (Anarhichas minor) is considered a promising species for coldwater aquaculture. It is a sedentary, bottom-dwelling fish which exhibits a calm and “non-stressed” behaviour in captivity. There are, however, no reports on the physiological responses to stressors in this species. In the present study we investigated primary (cortisol secretion) and secondary (glucose mobilization) responses to common aquaculture stressors like disturbance and hypoxia. Pre-stress plasma cortisol levels were within those generally considered representative for unstressed fish (~ 10 ng ml^− 1), whereas basal glucose levels were unusually low (0.3–0.4 mM). After exposure to a short, but severe disturbance challenge (emptying the tank of water for 10 min), the increase in plasma cortisol level was slow and relatively weak, reaching a peak level of 25 ng ml^− 1 4 to 8 h after disturbance. When the fish were exposed to a gradually decreasing oxygen level in the tank, a significantly elevated plasma cortisol level (35 ng ml^− 1) was seen in the fish that remained in the tank until oxygen saturation had decreased to 20% oxygen saturation after 2.5 h. However, a two-fold higher plasma cortisol level (~ 70 ng ml^− 1) was seen in all fish exposed to reduced oxygen levels (60, 40 and 20% oxygen saturation) after 3.5 h recovery in normoxic water. Plasma glucose levels showed only moderate increases (~ 70%) following disturbance and hypoxia challenges. An in vivo injection of ACTH caused a strong elevation of plasma cortisol (peak level ~ 170 ng ml^− 1), demonstrating a high capacity for interrenal steroidogenesis in the spotted wolffish. The slow and relatively weak cortisol response to stressors, and low plasma glucose levels, may relate to the sedentary lifestyle of the spotted wolffish. The stress-response is characterised by a passive (reactive) coping style, which is considered adaptive for farming of this species.     

Cortisol and hematological response in Brazilian codling, Urophycis brasiliensis (Pisces, Phycidae) subjected to anesthetic treatment

The effects of benzocaine anesthesia on hematological parameters (erythrocyte and platelet number, hemoglobin concentration and hematocrit), plasma electrolytes (Na⁺, K⁺ and Cl⁻), cortisol, glucose and cholesterol levels in Brazilian codling (Urophycis brasilensis) exposed to a benzocaine bath solution were investigated. Cortisol and glucose levels 1 h after capture from the wild and on days 7 and 14 during the acclimatization period (20 days) were also determined. During the second week, cortisol levels reached to basal values (9 ng ml⁻¹). The use of benzocaine causes a significant increase in the erythrocyte number, in the concentration of hemoglobin and sodium and a decrease in chloride. Plasma cortisol and cholesterol levels were higher in fish exposed to the anesthetic, and glucose levels decreased significantly comparing with the control group, showing strong fluctuations between individuals in all the cases. These results show that the use of benzocaine causes an acute stress response that could be reverted quickly and it has to be considered during the evaluation of stress in fish.     

The biliary accumulation of corticosteroids in rainbow trout,Oncorhynchus mykiss,during acute and chronic stress

The accumulation of immunoreactive corticosteroids in the bile of rainbow trout during stress was monitored by radioimmunoassay and GUMS. Although plasma cortisol levels were elevated by confinement for 1 hour, biliary levels of free and conjugated steroids in the bile were unaffected. However, after 24 hours confinement, in addition to elevated plasma cortisol levels, free and conjugated steroids in the bile were also significantly higher than in control, unstressed fish. The time-course of change in plasma and biliary corticosteroid levels was determined in rainbow trout subject to 96 hours confinement stress. Free steroid levels in the bile of stressed fish were elevated within 2 hours of the onset of stress, while levels of conjugated steroids were significantly elevated within 4 hours of the onset of confinement. Analysis of bile from stressed fish, by GC/MS, established the major conjugated steroids present to be tetrahydrocortisone (230 g ml^–1 bile), tetrahydrocortisol (75 g ml^–1), cortisone (33.5 g ml^–1), cortisol (25 g ml^–1) and -cortolone (5 g ml^–1). The data are discussed with reference to the role of cortisone and conjugating enzymes in the clearance of cortisol, and further data are presented to suggest that the analysis of biliary steroid content may provide a suitable means of identifying stressed fish under conditions in which an additional sampling stress is unavoidable.     

Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost,Stizostedion vitreum

The annual reproductive cycle of walleye (Stizostedion vitreum) was characterized by documenting changes in gonadal development and serum levels of estradiol-17β (E₂), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), and 11-ketotestosterone (11-KT) in wild fish captured from upper midwestern lakes and rivers throughout the year. Fish from the populations used in this study spawn annually in early- to mid-April. Walleye showed group synchronous ovarian development with exogenous vitellogenesis beginning in autumn. Oocyte diameters increased rapidly from ∼ 200 μm in October to ∼ 1,000 μm in November, and reached a maximum of 1,500 μm just prior to spawning. Changes in gonadosomatic indices (GSIs) paralleled changes in oocyte diameters. Serum E₂ levels in females increased rapidly from low values in October (< 0.1 ng ml⁻¹) to peak levels of 3.7 ng ml⁻¹ in November, coinciding with the period of the most rapid ovarian growth. Subsequently, E₂ levels decreased from December through spawning. Serum T levels exhibited a bimodal pattern, increasing to 1.6 ng ml⁻¹ in November, and peaking again at 3.3 ng ml⁻¹ just prior to spawning. We detected 11-KT in the serum of some females at concentrations up to 5.6 ng ml⁻¹, but no seasonal pattern was apparent. In this study (unlike our results in a related study) 17,20-P was not detected. In males, differentiation of spermatogonia began in late August, and by January the testes were filled (> 95% of germ cells) with spermatozoa. Mature spermatozoa could be expressed from males from January through April. GSIs ranged from 0.2% (post-spawn) to 3.2% (pre-spawn). Serum T levels rose from undetectable levels in post-spawn males to 1.6 ng ml⁻¹ by November, remained elevated throughout the winter, and peaked at 2.8 ng ml⁻¹ I prior to spawning. Levels of 11-KT in males remained low (< 10 ng ml⁻¹, from post-spawning through January, then increased significantly by March and peaked just prior to spawning at 39.7 ng ml⁻¹. Our results indicate that vitellogenesis and spermatogenesis are complete or nearly so, in walleye by early winter, and suggest that it may be possible to induce spawning in this species several months prior to the normal spawning season by subjecting fish to relatively simple environmental and hormonal treatments.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Thyroid hormone concentrations in the gonads of wild chum salmon during maturation

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T₄) were less than 5 ng ml⁻¹, and those of 3,5,3′-triiodo-L-thyroxine (T₃) were less than 2 ng ml⁻¹ I in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI). There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T₄ concentrations were undetectable (less than 0.2 ng g⁻¹) or less than 2 ng g⁻¹ when GSI was lower than 1%, but increased thereafter and reached a plateau of 8–10 ng g⁻¹ when GSI became 2%. The ovarian T₃ concentrations were about 5 ng g⁻¹ when GSI was 1%, increased to a maximum level (20 ng g⁻¹) when GSI was about 2%, and decreased to a constant level of 10 ng g⁻¹ thereafter. The T₄ and T₃ content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte. The T₄ concentrations in the testis were 1 ng g⁻¹ or less regardless of the GS1. On the other hand, the T₃ concentrations were highest (15 ng g⁻¹) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g⁻¹ I in testes containing spermatozoa, raising the possibility of a role for T₃ during early gamete and/or gonad maturation of testes.     

Modelling the effects of capture and sea lice [Lepeophtheirus salmonis (Krøyer)] infestation on the cortisol stress response in trout

Measuring baseline cortisol in wild salmonids is problematic because of the stress induced by most capture methods. The cortisol response to different periods of confinement stress was modelled in a laboratory population of rainbow trout [Oncorhynchus mykiss (Walbaum)]. A quadratic model was used to predict baseline (precapture) cortisol levels in these fish. A similar quadratic response pattern of cortisol was observed in wild sea trout (Salmo trutta L.). These were captured in timed sequences on four dates by seine netting in Clew Bay, west coast of Ireland. The estimated precapture cortisol for these fish indicated higher levels soon after migration to sea and a positive correlation with the level of infestation by Lepeophtheirus salmonis (Krøyer). Curvilinearity of the quadratic model was correlated with levels of lice infestation, indicating an enhanced cortisol response to confinement stress during capture when parasitic abundance was high. Estimated precapture cortisol for individual sea trout was positively correlated with numbers of lice although the linear regression had low predictive power. The method provides, for the first time, a means to estimate baseline cortisol levels in wild salmonids, and addresses one of the most difficult problems in working with wild fish. Keywords: Cortisol, trout, Lepeophtheirus salmonis (Krøyer), capture, quadratic model     

Effects of chronic confinement on physiological responses of juvenile gilthead sea bream, Sparus aurata L., to acute handling

Understanding how gilthead sea bream, Sparus aurata L., an important Mediterranean Sea species for aquaculture, respond physiologically to stressors commonly encountered in intensive rearing is important for effective production, as managing for stress is a major factor in maintaining healthy fish stocks. Our objective was to determine whether holding juvenile gilthead sea bream at a high density (HD), as a chronic stressor, would affect their physiological responses to a subsequent acute handling stressor. After acclimation at a low density (LD) of 6 kg m^?3 in 200‐L circular tanks containing 33–36 g L^?1 recirculating seawater at 19°C under a normal photoperiod, juvenile 37‐g gilthead sea bream were confined for 14 days at a HD of 26 kg m^?3 and then subjected to 30‐s aerial emersion in a dipnet. Plasma levels of cortisol, glucose, lactate, osmolality and chloride were determined in fish held in separate lots during LD (control) and HD confinement at 0, 1, 2, 7 and 14 days, and then after handling at 0, 1, 2, 4 and 8 h. Although plasma cortisol levels were similar in LD and HD fish groups after 14 d of confinement (15 and 23 ng mL^?1, respectively), the cortisol response in fish from the HD treatment at 1 and 2 h following acute handling (70 and 37 ng mL^?1, respectively) was only about half of that measured in the control group (139 and 102 ng mL^?1); plasma cortisol was similar in both groups by 4 and 8 h. In contrast, plasma glucose elevations in response to handling were higher at 4 and 8 h in the HD‐held fish (94 and 72 mg dL^?1, respectively) than in those from the LD treatment (59 and 51 mg dL^?1); glucose responses were similar in both groups at 1 and 2 h after handling and throughout confinement. Plasma lactate levels were higher in LD fish than in the HD group at the beginning of the experiment but were similar after 14 d confinement and responses to handling were similar (e.g. 33 and 35 mg dL^?1 at 1 h). Plasma osmolality showed increases during the first 2 h after acute handling but no differences were evident between the two density treatments at any time during confinement or posthandling. Plasma chloride levels did not change throughout the experiment. The reduced plasma cortisol response to acute handling likely resulted from negative feedback of mildly but chronically elevated circulating cortisol caused by the confinement stressor on the hypothalamic–pituitary–interrenal axis. While other post‐handling physiological changes also showed differences between treatment groups, the suppressed cortisol response in the HD‐held fish suggests a reduction in the gilthead sea bream's normal capacity to respond to an acute stressor.     

Composition of ovarian fluid in endangered Caspian brown trout, <Emphasis Type="Italic">Salmo trutta caspius</Emphasis>, and its effects on spermatozoa motility and fertilizing ability compared to freshwater and a saline medium

In this study, ovarian fluid composition and its effects on the motility and fertilizing ability of sperm were studied in endangered Caspian brown trout, Salmo trutta caspius, and were compared with a saline activation medium (125 mM NaCl, 30 mM Glycine, 20 mM Tris–HCl, pH = 9.0) and freshwater as the control. The ovarian fluid was composed of sodium 164.4 ± 4.4 mM l⁻¹, potassium 1.8 ± 0.1 mM l⁻¹, calcium 0.6 ± 0.1 mM l⁻¹, magnesium 0.4 ± 0.02 mM l⁻¹, chloride 127.4 ± 5.9 mM l⁻¹, total protein 389.5 ± 89.6 mg 100 ml⁻¹, cholesterol 9.3 ± 1.2 mg dl⁻¹, and glucose 3.3 ± 0.2 mM l⁻¹. The percentage of motile spermatozoa and the duration of sperm motility were significantly higher in ovarian fluid (62 ± 3%, 74.6 ± 0.8 s) than freshwater (35 ± 4%, 44 ± 1 s), but they did not differ significantly from saline medium (56 ± 3%, 74.3 ± 0.7 s) (P > 0.05). Higher eyeing rates were observed after the activation of sperm in ovarian fluid and saline solution than freshwater when 35,000 or 350,000 spermatozoa per egg were added into the activation media. However, no significant differences were observed at higher concentrations of spermatozoa per egg (730,000) (P > 0.05). Also, this study showed that the ovarian fluid composition can be considered as a species-specific character among salmonid fishes. As a conclusion, the results of this study recommend the use of ovarian fluid or the saline solution as an activation medium in the artificial reproduction of Caspian brown trout.     

Effect of ethanolic extract of propolis on growth performance and plasma biochemical parameters of rainbow trout (Oncorhynchus mykiss)

This study was conducted to evaluate the effect of ethanolic extract of propolis (EEP) on growth performance and plasma biochemical parameters of rainbow trout (Oncorhynchus mykiss). Graded levels of EEP [0 (control), 1, 2, and 4 g kg⁻¹ diet] were fed to trout juveniles (mean weight 7.73 ± 0.17 g) for 10 weeks. Dietary EEP supplementation regardless of inclusion level significantly improved the specific growth rate of fish. Similarly, supplemental EEP generally improved the feed efficiency ratio and protein efficiency ratio, but no significant differences were observed between the 1 g kg⁻¹ EEP group and the control group. In addition, dietary EEP supplementation generally increased the plasma superoxide dismutase, lysozyme, total antioxidant capacity, glutathione peroxidase, and catalase activities, but decreased the plasma malondialdehyde level. The plasma triglycerides level was significantly lower in the 1 or 4 g kg⁻¹ EEP group as compared with the control group. Dietary EEP supplementation generally decreased the plasma aspartate aminotransferase and alanine aminotransferase activities, but increased the hepatic aspartate aminotransferase and alanine aminotransferase activities. These results indicate the potential to use the EEP as a growth promoter, hepatoprotective agent, and immunostimulant for rainbow trout.     

Development of a label-free immunosensor system for detecting oocyte maturation-inducing hormone in fish

A label-free immunosensor for detecting the oocyte maturation-inducing hormone 17, 20β-dihydroxy-4-pregnen-3-one (DHP), was developed. A principle of the sensor system was based on differences in electrochemical activity changed by an immunoreaction in the absence and presence of DHP. For preparation of the immunosensor, anti-DHP IgG was immobilized on an Au working electrode modified with a self-assembled monolayer of 3-mercaptopropionic acid. The sensor was immersed into a sample solution containing DHP. DHP was determined by cyclic voltammetry. The immunosensor showed a specific response to DHP, and the oxidation peak current linearly decreased in the range of 7.8–500 pg ml⁻¹ with a correlation coefficient of 0.996. The sensor system was applied to determine the DHP levels in plasma of goldfish and was compared with the DHP levels of the same samples determined using an ELISA as the conventional method. Good correlation was obtained between values determined using both methods in the range of 0.1–7.7 ng ml⁻¹ (correlation coefficient 0.876). These findings suggest that the proposed label-free immunosensor can be used to analyze DHP levels in fish plasma samples.     

Dietary nucleotide supplements influence the growth,haemato‐immunological parameters and stress responses in endangered Caspian brown trout (Salmo trutta caspius Kessler, 1877)

A feeding trial was conducted to evaluate the effects of Optimûn, a commercial nucleotide (NT) product, on the growth, haemato‐immunological and serum biochemical parameters and stress responses of Caspian brown trout fingerlings (average initial weight of 12.26 g). A basal diet was supplemented with levels of 0 (control), 1.5, 2.5, 3.5 and 5 g NT kg^?1 to formulate five experimental diets. After 8 weeks of feeding trial, fish fed diet with 2.5 g NT kg^?1 had the highest final weight compared with other treatments. The fish fed diets with 2.5 g NT kg^?1 had higher blood protein, albumin, albumin/globulin ratio, red blood cells, white blood cells and lymphocyte content and lower alkaline phosphatase. The lysozyme activity in serum was found to be significantly (P < 0.05) greater in fish fed diet with 2.5 g NT kg^?1. Concerning both stressors (confinement and salinity stress), fish fed diet with 2.5 g NT kg^?1 had lower plasma cortisol and glucose levels. Thus, administration of 2.5 g kg^?1 of the Optimûn dietary nucleotide formula is recommended to promote growth and immunity as well as to enhance stress responses of Caspian brown trout.     

Immunological and physiological validation of enzyme-linked immunosorbent assay for growth hormone of the Asian catfish, Clarias batrachus

This study describes the development and validation of competitive antigen capture enzyme-linked immunosorbent assay (ELISA) for growth hormone (GH) of the catfish, Clarias batrachus. Isolated GH was characterized first through bioassay, amino acid sequencing, immunoblotting and immunocytochemistry, and then used to raise antibody to develop ELISA. The lowest detection limit of the assay system was 0.17 ng ml⁻¹, and the standard curve had an ED₅₀ value of 0.35 ng ml⁻¹. Repeated determination of GH in a plasma pool exhibited intra- and interassay co-efficient of variation of 8.1% and 7.09% (n=5), respectively. Dose–response inhibition curves resulting from dilutions of plasma and pituitary homogenate of C. batrachus were parallel to the standard curve, while such parallelism in the case of Cyprinus carpio and H. fossilis was absent revealing no cross reaction of them in the present ELISA. In vivo effects of sGnRH, testosterone, 5-HT and morphine on plasma GH were also examined to validate the ELISA physiologically. sGnRH, testosterone and morphine increased, while 5-HT decreased GH level in a dose-dependent manner.     

Influence of alginic acid and fucoidan on the immune responses of head kidney leukocytes in cod

The present study investigated the immunomodulatory activities of alginic acid and fucoidan, both derived from brown seaweeds, on selected cellular immune responses and antibacterial activity of head kidney (HK) leukocytes of cod, Gadus morhua. Primary cultures of HK leukocytes were incubated with either 10 or 100 μg ml⁻¹ of the substances and the effects on respiratory burst, cellular proliferation, acid and alkaline phosphatase activity and cellular myeloperoxidase were measured at 3- and 24-h post-incubation. The antibacterial activity of the supernatants collected from the cell cultures incubated with 100 μg ml⁻¹ of the substances were tested against Vibrio anguillarum and Aeromonas salmonicida. Respiratory burst was significantly elevated in cells incubated with either alginic acid or fucoidan in a dose-dependent manner. Incubation with a higher dose of alginic acid and fucoidan resulted in lower cellular proliferation at 3- and 24-h, respectively. Both acid and alkaline phosphatase activities of HK leukocytes were not significantly modulated, except for a slight elevation of acid phosphatase in cells incubated with 100 μg ml⁻¹ of alginic acid for 24-h. Fucoidan, but not alginic acid significantly increased cellular myeloperoxidase activity at a concentration of 100 μg ml⁻¹. The growth of the bacteria in both the treated and control supernatants was significantly lower than what was observed in the bacterial culture medium. However, the supernatants from the treated cells had significantly higher bacterial growth compared with supernatants of the control cells. Taken together, these results showed that at the tested concentrations, both alginic acid and fucoidan are able to differentially stimulate some cellular immune responses of cod HK leukocytes in vitro and the respiratory burst activity was significantly stimulated by these brown algal derivatives. These substances could be tested as potential immunostimulants in future in vivo studies.     

The development of a radioimmunoassay for carp,Cyprinus carpio,vitellogenin

The development of an easily — performed and robust radioimmunoassay (RIA) to carp, Cyprinus carpio, vitellogenin (c-VTG) is described. Purified c-VTG was iodinated using Iodogen. The resulting c-VTG label was useful for up to 60 days. High titre antibodies were raised in rabbits to the purified c-VTG. The practical operating range of the c-VTG RIA was between 2 and 200 ng. ml^-1. VTG was detected in plasma from all female mirror carp investigated, and all plasmas diluted parallel to the standard. The plasma VTG level in female carp increased concomitantly with the GSI; levels increasing from the ng. ml^-1 level in juveniles to a maximum of 1 mg. ml^-1 in fully mature females. VTG level was a far more sensitive index of the degree of sexual development than was GSI. In males, blood levels of VTG were always undetectable. Vitellogenic plasma from all subspecies of C. carpio (e.g. mirror, common, Koi) diluted parallel to the standard, as did blood from most other female cyprinids, such as the roach (Rutilis rutilis), bream (Abramis brama), and dace (Leuciscus leuciscus), but not all. These results suggest that the structure of VTG is highly conserved within this family. However, plasma from vitellogenic salmonids did not cross-react in the RIA. The relationship between plasma VTG and calcium levels was studied in both carp and rainbow trout. In rainbow trout it was found that plasma calcium levels do not rise above basal levels until the VTG level exceeds about 1 mg. ml^-1, and therefore in this species it is only useful as an indicator of the degree of ovarian development in the later stages of the reproductive cycle. In the carp, however, and probably other cyprinids, blood VTG levels do not appear to naturally exceed about 1 mg.ml^-1, and plasma calcium levels are not suitable as an indirect measure of the VTG level.     

Fin condition and growth among rainbow trout reared at different sizes,densities and feeding frequencies in high-temperature re-circulated water

Rainbow trout were studied at different rearing densities, fish sizes and feeding frequencies so that we could evaluate the effect of these parameters on fin condition, growth and feed utilisation. In one study, two sizes of rainbow trout (18–70 g or 48–125 g), fed to near satiation at 17.7°C, were examined at two rearing densities (11–41 kg m⁻³ or 21–92 kg m⁻³). This study showed that the anal fin was healthier (P < 0.05) at low densities. In the second study rainbow trout were again fed to near satiation and grown from 50 g to 125 g in 16.4°C water, and they were fed either once daily or three times daily at two densities (18–45 kg m⁻³ or 54–124 kg m⁻³). Rainbow trout growth and feed utilisation were slightly but significantly (P < 0.05) reduced at high densities, while dorsal fin condition, surprisingly, was better (P < 0.0001) at the high densities. Possible explanations to these findings are discussed. Condition of the left pectoral fin was improved at once daily feeding (P < 0.05) compared with three feedings per day, at which fights for feed possibly are more frequent.