Nitrogen excretion and determination of nitrogen and energy budgets in rainbow trout (Oncorhynchus mykiss R.) under different feeding regimes

The postprandial excretion pattern of ammonia in dependence of feeding regime (fasting, 1 ×/day, 2 ×/day, 4 ×/day and continuous feeding), was determined in rainbow trout ( Oncorhynchus mykiss ), using continuous flow analysis. In fed fish ammonia peaked c. 7 h after the first meal with no differences in pattern between treatments. Fasting fish did not show a pattern. Overall production rates (NH₄ and NO₂+ NO₃) ranged from 0.29–0.31 g kg⁻¹ BW/d in fed fish and were around 0.07 g kg⁻¹ BW/d in fasting fish. Additionally determined total N (Kjeldahl) showed much higher values in fed fish (0.78–1.05 g kg⁻¹ BW/d) but only slightly higher values in fasting fish (0.11 g kg⁻¹ BW/d). Budgets of nitrogen (N) and energy (E) showed low recoveries ( c. 50% and between 50% and 70%, respectively). When correcting ammonia excretion (NH₄ and NO₂+ NO₃) using literature data on urea excretion of O. mykiss and assuming that total N partly stemmed from uneaten but undetected feed, both N and E budgets reached a recovery of around 100% in all four fed groups. Implications of this approach are discussed in the light of incomplete budgets as determined in earlier studies.     

Luteinizing hormone and sexual steroid plasma levels after treatment of European sea bass with sustained-release delivery systems for gonadotropin-releasing hormone analogue

Spermiating male European sea bass Dicentrarchus labrax were treated with gonadotropin-releasing hormone agonists (GnRHa), either a GnRHa injection (IN; 25 μg kg⁻¹ body mass) or one of three types of controlled-release GnRHa-delivery systems: fast release implants (EVAc; 1OO μg kg⁻¹), slow release implants (EVSL; lOO μg kg⁻¹) and slow release microspheres (MC; 50 μg kg⁻¹). Luteinizing hormone (LH) release was highly stimulated by all GnRHa treatments, with elevated plasma levels lasting for 2 days in injected fish (IN) and 2, 4 and 6 weeks in controlled-release-treated fish (EVAc, MC and EVSL, respectively), correlating with a 1, 3, 5 and 5 week period of stimulation of milt production, respectively. Plasma levels of the androgens testosterone (T) and 11-ketotestosterone (11-KT), were not significantly affected by the GnRHa treatments. Plasma T was high at early spermiation and declined sharply near the end of this period. Plasma 11-KT levels declined continuously throughout the experiment. Levels of 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β -P), a proposed maturation-inducing steroid (MIS) in European sea bass, fluctuated around 0.2–1 ng ml⁻¹ and were not greatly affected by the treatments. These results indicated a close correlation between sustained stimulation of LH release, achieved by GnRHa-delivery systems, and long-term enhancement of milt production. They also show an absence of changes in the common sex steroids, associated with elevated LH and enhanced spermiation.     

Bioactivity of stored luteinizing hormone-releasing hormone analogue (LHRHa) in sea bass, Lates calcarifer Bloch

The spawning induction activity of dissolved and pelleted (D-Ala⁶, Pro⁹ N ethylamide) luteinizing hormone-releasing hormone analogue (LHRHa) stored for various periods was assessed in mature female sea bass. The spawning response of mature fish was reduced significantly after injection of dissolved LHRHa (20 μg kg⁻¹) stored for more than 90 days in a refrigerator (4–10°C) or for more than 30 days at room temperature (28–30°C). Similar to fish administered fresh preparations of LHRHa, fish spawned successfully after injection of a solution of LHRHa previously frozen, subjected to alternate freezing and thawing, exposed to sunlight or implanted pelleted LHRHa (50 ng kg⁻¹) stored at room temperature for 30–120 days. Loss of hormone bioactivity after prolonged storage may have been due to bacterial growth in solubilized preparations. Injection or implantation of stored LHRHa did not influence egg production among treated sea bass. These results demonstrated the relatively prolonged shelf life of stored LHRHa.     

Orally induced spawning of Thai carp (Puntius gonionotus, Bleeker) following co-administration of des Gly10 (D-Arg6) sGnRH ethylamide and domperidone

Oral delivery of a salmon gonadotropin releasing hormone analogue (50–100 μg kg⁻¹ B.W.) and domperidone (25–50 mg kg⁻¹ B.W.) to Thai carp, induced ovulation in 83–100% of treated animals within 48 h of administration.     

Cortisol stimulates hypo-osmoregulatory ability in Atlantic salmon, Salmo salar L.

Hypo-osmoregulatory ability in juvenile Atlantic salmon, Sulrno salur L., was improved by cortisol treatment. Implantation of a vegetable shortening pellet containing cortisol (50 mg kg⁻¹) resulted in elevated plasma cortisol titres. Maximum cortisol levels (160–170ngml⁻¹) were observed at days 6 and 12 after the implantation and dropped significantly by day 55. Cortisol-implanted fish in fresh water developed a twofold increase in gill Na⁺/K⁺-ATPase activity at days 6 and 12, and a threefold increase by day 55. Intestinal mucosa Na⁺/K⁺-ATPase activity was not affected by cortisol. Cortisol-implanted fish exposed to 28 ppt sea water for 48 h tended to show an improved ability to regulate their plasma osmolarity and reduce their ionic load. The osmo-regulatory ability attained at days 12 and 55 was further evaluated by exposing fish to 37 ppt sea water for 96 h. While all the control fish died relatively early in these tests, cortisol-implanted fish showed a clear reduction in their mortality rate. These results indicate that cortisol can induce biochemical and organismal changes during winter months that typify preadaptive events normally occurring in the spring.     

Development of lamellar epithelial hyperplasia in gills of pantothenic acid-deficient rainbow trout, Salmo gairdneri Richardson

Two semisynthetic diets differing in the levels of pantothenic acid (PA) supplementation (0 or 40 mg kg⁻¹ diet) were fed to rainbow trout, Salmo gairdneri Richardson, initially weighing 0.7 g fish⁻¹. Each diet was fed to two tanks of fish and, at the start of the feeding trial, each tank contained 200 animals. The experiment was conducted for 28 days during which time, every 2 days, gill tissue was sampled and food consumption was determined. Lamellar hyperplasia was first detected in the gills of deficient fish on the 12th day sample, while reduced feed intake was first manifest at 16 days. Hyperplasia first appeared in the distal regions of the gill filaments, but the lesion rapidly progressed in a proximal direction and 35 of 40 fish examined on days 22–28 exhibited hyperplasia on more than 75% of the filament surface. Gill lamellar hyperplasia is a sensitive indicator of PA deficiency in the rainbow trout. Moreover, the lesion is specific to PA deficiency since its developmental pattern differs histologically from the lamellar hyperplasia of the non-nutritional gill diseases.     

Mercury and chlorinated hydrocarbons in the food chain of Lake Päijänne, Finland

The sediments and various organisms in Lake Päijänne were examined for contaminants. The average mercury content of water plants was 9, of plankton 14, of sediment 114, of zoobenthic predators 83, of fish 332–1510 and of birds 240–13685 μg kg⁻¹ (wet weight). The average PCB content of plants was 3, of plankton 21, of the zoobenthos 44, of fish 36-117 and of birds 219–13490 μg kg⁻¹. The average ΔDDT content of plants was 0.5, of plankton 6, of the zoobenthos 14, of fish 7–42 and of birds 144-8262 μg kg⁻¹. Regional differences in mercury content were most pronounced in sediment and fish. PCB concentration was highest near a town. ΔDDT was quite evenly distributed. Water plant species did not differ from each other, nor did the plankton fractions. The zoobenthic predators contained more chlorinated hydrocarbons than did the herbivores. There were clear differences between most species of fish and the chlorinated hydrocarbon content was highest in vendace. In adult birds levels of all residues were significantly higher than in juveniles.
In most cases PCB content was positively correlated with ΔDDT and in birds PCB, ΔDDT and mercury levels were correlated. DDT residues occurred mostly as DDE, but in vendace the proportion of DDT was high. At most trophic levels, ΔDDT/PCB was 0.15-0.40 but in birds it reached 1–2.     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Direct and circulatory paths of Permethrin (NRDC-143) causing histopathological changes in the gills of rainbow trout, Salmo gairdneri Richardson

Rainbow trout were exposed to sublethal concentrations of Permethrin in water (0.09, 0.18 and 0.35 μg l⁻¹) and in food (85, 180 and 350 μg kg⁻¹ dry diet) in 20–0 day experiments. Histological changes in the gills included epithelial separation or necrosis, mucus cell hyperplasia, clubbing of epithelial cells or hyperplasia and fusion of adjacent secondary lamellae. These changes were noted in all fish, suggesting that the pesticide or its metabolites reached the gills not only directly through the water, but also indirectly via the circulation.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Relationship of plasma steroids to germ cell development and the presence of protamine mRNA in rainbow trout during the induction of spermatogenesis with partially purified salmon gonadotropin

The gonadosomatic index (GSI) of pre–pubertal male rainbow trout, which had been injected biweekly with partially purified salmon gonadotropin (sG–G100, 50 μ mUg kg⁻¹ body weight), increased from 0.05 to 1.85 over 21 weeks from injection, while control GSI remained below 0.05. Plasma testosterone (T) increased from 2 to 11.34 ng ml⁻¹ by week 21 in injected fish, while control level remained below 1.5 ng ml⁻¹. In injected fish plasma 11–ketotestosterone (KT) and 17,20–dihydroxyprogesterone (17α20βP) levels increased from 20.2 to 41.9 and 8.9 to 219.7 ng ml⁻¹ respectively. Plasma T, 11–KT, and 17α20βP were all correlated with the GSI ( P <0–001) in injected fish. The most advanced stage of germ cells present in the control fish were spermatogonia. However, in injected fish spermatozoa were present by week 21. Eggs fertilized at this time with spermatozoa from injected fish achieved a 78% fertilization rate, whereas the testicular homogenate was incapable of fertilizing eggs.     

Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout (Oncorhynchus mykiss) following black-box confinement and epinephrine injection

The present study investigates the effect of cannulation and chronic'black-box' confinement, as well as epinephrine administration (4–0 μg kg⁻¹), on the degree and time-course of alterations in trout ( Oncorhynchus mykiss ) catecholamine and cortisol concentrations. Plasma cortisol concentrations in seawater trout acclimated to 3–6° C reached 104 ng ml⁻¹ 1 day after cannulation/confinement and remained elevated above resting levels (8 ng ml⁻¹) until 6 days post-confinement. Although plasma epinephrine and norepinephrine generally declined over the period of confinement (day 1 approx. 12 nM; day 7 approx. 6 nM), norepinephrine titres were usually higher and more variable. Epinephrine injection caused elevations in plasma epinephrine levels but not in norepinephrine levels; epinephrine titres reaching 107 ± 26 nM (range 65–238 nM) at 2 min post-injection and returning to pre-injection levels by 30 min post-injection. Plasma cortisol increased by 20 ng ml⁻¹ following epinephrine administration. Based on the time-course for post-confinement alterations in plasma cortisol, it appears that up to a week may be required before cannulated fish are completely acclimated to 'black-box' confinement. The findings suggest that meaningful results from experiments utilizing epinephrine injection and 'black-box confinement are contingent upon: (1) knowledge of circulating epinephrine levels shortly after injection (i.e. within 2 min post-injection); and (2) an experimental design that takes into account the elevated cortisol titres that are inherent with cannulation/confinement and epinephrine injection.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. II. The effect of altering serum cortisol concentrations on the immune response

The effect of altering serum cortisol concentrations on the immune response was elucidated in endrin- and non-endrin-exposed rainbow trout, Salmo gairdneri. Fish were immunized with 10 μg of Yersinia ruckeri O-antigen following 30 days of treatment. The migration inhibition factor assay (MIF), plaque-forming cell assay (PFC) and serum agglutination titres (SAG) were performed 2, 14 and 30 days post-antigen inoculation. Endrin exposure was continued subsequent to antigen inoculation. Control fish were fed 20 and 35 mg kg⁻¹ body weight day⁻¹ of cortisol and metyrapone, respectively. Endrin-exposed fish received 35 mg kg⁻¹ body weight day⁻¹ of metyrapone in their diet. Control fish receiving cortisol had significantly reduced MIF, PFC and SAG responses. The MIF response was completely restored in endrin-exposed fish receiving dietary metyrapone. The PFC response and SAG titres were partially restored, 61 and 69% respectively, in endrin-exposed fish receiving metyrapone. The results indicate that elevated serum cortisol concentration obtained in endrin-exposed fish has a central role in repression of the immune response.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Amount and distribution of biochemical-genetic variation among wild populations and a hatchery stock of Atlantic salmon, Salmo salar L., from north-east Ireland

The effects of tributyltin (TBT) compounds on gill morphology were examined in the mummichog, Fundulus heteroclitus , in 96-h LC50 and 6-week sublethal exposures. Morphometry was used for the identification and quantification of effects with the light microscope. A 96-h LC50 of 17.2 μg 1 ¹ was determined. Morphometric analysis of gill tissues revealed hypertrophy of the lamellar epithelium in fish exposed to 17.2 μg 1–1. Relative diffusing capacity was significantly decreased (−41 %); ( P <0.05, ANOVA, Bonferroni t -test). At 35.6 μg 1⁻¹, TBT exposure resulted in a significant reduction (− 40%) in the volume of the lamellar blood channels. Both of these observations occurred in fish that showed signs of acute poisoning including loss of equilibrium. In fish exposed to sublethal concentrations of 0.105–2.000 μg TBT 1⁻¹ for 6 weeks, there were no pathological changes in the gill. There were no treatment-related changes in the surface morphology of the gills of fish from both experiments upon scanning electron microscopic examination. Although gill pathology was observed in acutely toxic exposures, it does not appear to be a major mechanism of TBT toxicity.     

Absolute feeding design, a realistic way for fish nutrient requirement determination

The feeding experiment was designed on the basis of food allowance rather than on feed formulation. Food allowances are fixed in an attempt to provide the desired amount of nutrients per body mass of fish per day (g* kg⁻¹* d⁻¹ or J* kg⁻¹* d⁻¹). Therefore, the initial determination of optimum feeding ration is not required, and utilization of neutral feed complement (e.g. cellulose) is unnecessary. Data interpretation for such a design study is presented by a sample experiment for protein-energy requirements of a tilapia species, Sarotherodon melanotheron. The interrelationships of commonly used indices for growth and nutritional efficiency are discussed.     

The effect of humic substances on the toxicity of aluminium to adult rainbow trout, Oncorhynchus mykiss (Walbaum)

Some physiological parameters were measured in adult rainbow trout during a 10-day exposure to 180 μg Al_total l⁻¹ in acid water (pH 4.7) with or without humic substances (10 mg l⁻). The fish were acclimatized to pH 5.0 for 7 days prior to the experimental treatments.
Chemical analyses revealed that, in the presence of human substances, 74–80% of the A1 was organic bound, while in the absence of humic substances most of the Al(987percnt;) occurred in the inorganic form.
Al bound to humic substances (13–150 μg l⁻¹) did not alter the plasma NaCl-concentration, nor the haematocrit value, of rainbow trout during an exposure period of 10 days. This contrasts with the high death rate obtained within 2–3 days when most of the A1 (175 μg l⁻¹) was in the inorganic form. The lethality was accompanied by a 25% decrease in the plasmaconcentration of NaCl and a doubling of the haematocrit value. Bulk analysis revealed that when the metal was present in inorganic forms the total Al content of the gills (75 μg A1 g⁻¹ wet weight) was 15 times higher than when it was present as bound to the humic substances. These experiments showed that the accumulation of A1 at the gills was accompanied by physiological disturbances, both being a function of the chemical speciation of Al.     

Exposure to low concentrations of dissolved ammonia promotes growth rate in walleye Sander vitreus

The objective of the current study was to examine whether sublethal (moderate) levels of dissolved ammonia may be beneficial to growth in juvenile walleye Sander vitreus (recent evidence in juvenile rainbow trout Oncorhynchus mykiss has shown significant increases in protein synthesis in the presence of moderately elevated concentrations of dissolved ammonia). Moderately elevated dissolved ammonia concentrations between 100 and 300 μmol l⁻¹ suppressed routine aerobic metabolic activity by 20% during acute trials (2 h), while promoting specific growth rate (>50%) and elevating whole body soluble protein content by 20% in the early stages (14–42 days) in chronic ammonia exposure experiments. Juvenile S. vitreus held at ammonia concentrations between 107·6 ± 5·5 and 225·5 ± 4·7 μmol l⁻¹ (mean ± s . e .) grew significantly faster than control fish and significantly reduced plasma cortisol levels (<3 μg dl⁻¹). Results from this study suggest that chronic exposure to moderate amounts of dissolved ammonia significantly increase growth rates in juvenile S. vitreus by increasing nitrogen accessible for supplementary protein deposition leading to somatic development.     

Constitutive mineralization of low concentrations of the herbicide linuron by a Variovorax sp. strain

The mineralization of the herbicide linuron at concentrations of μg and mg L⁻¹ was studied in liquid batch experiments with Variovorax sp. strain SRS16. The strain was highly efficient at mineralizing a range of linuron concentrations (0.002–10 mg L⁻¹) with 20–60% of the added ¹⁴C-ring-labeled linuron metabolized to ¹⁴CO₂ within hours to days depending on the initial linuron concentration and incubation period. At mg L⁻¹ linuron concentrations the mineralization activity by SRS16 was inducible and a shift to constitutive mineralization activity was apparent with a reduction in the linuron concentration to μg L⁻¹ levels. This study revealed that strain SRS16 is a promising candidate for bioaugmentation of water or soil resources contaminated with low linuron concentrations.     

More effective induction of spawning with long-acting GnRH agonist in the shi drum, Umbrina cirrosa L. (Sciaenidae, Teleostei), a valuable candidate for Mediterranean mariculture

Three experiments of spawning induction in shi drum, Umbrina cirrosa L., were performed in six different commercial Italian hatcheries from May to August (water temperatures: 19–29 °C; salinity: 21–37 p.p.t.). In the first experiment, 119 females (1–4.7 kg), subdivided into 29 lots, were injected with a single dose (2, 5, 8, 10, 15 and 20  μ g kg⁻¹ body weight) of short-acting gonadotropin- releasing hormone agonist (GnRHa-S), des-Gly10,[D-Ala6]-LH-RH ethylamide. In the other two experiments, 85 females (0.7–5.8 kg), subdivided into 22 and four lots, were treated with one (40 or 80  μ g kg⁻¹) or three doses (40  μ g kg⁻¹) of long-acting GnRHa (GnRHa-L), respectively. GnRHa-S stimulated spawning in 69% of the 29 treated lots; the number of eggs laid reached a maximum of 130 000 and a weighted mean of 29 200 total eggs kg⁻¹. GnRHa-L elicited a spawning response in 95% of the 22 one-dose treated lots; the number of laid eggs was higher than with GnRHa-S, reaching a maximum of 213 100 and a weighted mean of 59 400 total eggs kg⁻¹. The yield of developing embryos in 67% of the single GnRHa-L treatments was higher (sometimes up to three times) than with GnRHa-S. Triple treatments of the four lots of females with GnRHa-L always resulted in spawning responses; the best result corresponded to a number of total laid eggs of 358 900 eggs kg⁻¹ with a yield of 177 300 developing embryos.