The influence of the dietary carbohydrate: lipid ratio on the chronic toxicity of sodium pentachlorophenate to rainbow trout (Salmo gairdneri Richardson)

The influence of diets varying in carbohydrate: lipid ratio on the chronic toxicity of waterborne sodium pentachlorophenate (0 or 50 g NaPCP.l^–1) to rainbow trout (Salmo gairdneri) was examined over a 12 week period. The three diets used were practical salmonid formulations, equivalent in energy and protein content, with cerelose: fish oil ratios of 18.24.7% (high carbohydrate, HC), 9.39.1% (intermediate, INT), and 0.613.8% (low carbohydrate, LC). With increasing levels of dietary carbohydrate trout exhibited enlarged livers, elevated liver glycogen content, reduced liver protein content, reduced body lipid content, reduced weight gain, and elevated feed: gain ratio. For all diet groups, exposure of trout to NaPCP resulted in reduced liver glycogen content, higher liver protein content, reduced levels of plasma total triiodo-L-thyronine and L-thyroxine, increased thyroid epithelial cell height, and increased feed: gain ratios relative to their respective dietary control group. NaPCP exposure led to nominal reductions in weight gain and body lipid content of fish reared on the HC and INT diets. NaPCP exposed fish on the LC diet gained significantly less weight but had a higher body lipid content than control fish. The increasing NaPCP bioconcentration factor (BCF) associated with increasing lipid content of fish (log BCF=2.75 [log % lipid] +0.30; r=0.95) may explain the differences in response. Correction of bioconcentration data for the lipid content of fish reduced the range in BCF from 5- to 2.8-fold.     

Cytochrome P-450-dependent metabolism in isolated liver cells from fed and starved rainbow trout,Salmo gairdneri

The cytochrome P-450 content in liver cells from rainbow trout was not affected by starvation for 12 weeks whereas the rate of cytochrome P-450-dependent deethylation of 7-ethoxycoumarin in liver cells from 6 or 12 weeks starved fish was 60% of the rate in fed fish. Treatment of fish with -naphthoflavone increased the 7-ethoxycoumarin metabolism several-fold in both starved and fed fish.Optimal cytochrome P-450 monooxygenation in liver cells from fed or starved fish was not affected by addition of glucose or 2-bromooctanoate, an inhibitor of fatty acid -oxidation which is the main source of metabolic fuel in trout liver. The cellular content of NADPH, an obligatory cofactor for cytochrome P-450 monooxygenation, was not affected by addition of substrate to cytochrome P-450, inhibition of fatty acid -oxidation or inhibition of the oxidative phosphorylation. This indicates a great capacity of rainbow trout liver cells to retain high NADPH/NADP⁺ ratios. These results suggest that the cytochrome P-450 mediated metabolism of xenobiotics in liver cells from fed or starved trout is not limited by the availability of reducing equivalents.     

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in β-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss)

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or -naphthoflavone (NF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the NF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with NF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and NF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by NF- and ISF treatments in the rainbow trout liver.Part of this work was presented at the 6th International Conference on Biochemistry and Biophysics of Cytochrome P-450, Vienna, Austria, July 3–8, 1988.     

Metabolic effects of feeding a high protein/low carbohydrate diet as compared to a low protein/high carbohydrate diet to rainbow troutSalmo gairdneri

Groups of trout were fed either a high protein/low carbohydrate (HP/LC) or a low protein/high carbohydrate (LP/HC) diet for 16 weeks. Fish fed the HP/LC diet had significantly higher levels of protein, ninhydrin positive material and tryptophan in plasma as well as lipid and protein in liver, but lower levels of glucose in plasma, glycogen in liver and a lower hepatosomatic index compared with fish fed the LP/HC diet. There were no differences concentrations of tryptophan or serotonin in the brain of fish fed the different diets. Fish fed the HP/LC diet had significantly lower activities of glycolytic enzymes but higher activities of gluconeogenic enzymes compared with the group fed the LP/HC diet. Those enzymes measured concerned with the TCA cycle or initiating amino acid catabolism were generally unaffected by diet although activities of threonine dehydrogenase, alanine aminotransferase and glutamate dehydrogenase were higher in those fish fed the HP/LC diet. The data suggest that rainbow trout show little adaptation in activities of amino acid catabolising enzymes to variations in dietary protein intake.     

Variations in hepatic metallothionen,zinc and copper levels during an annual reproductive cycle in rainbow trout,Salmo gairdneri

The normal variations in hepatic levels of metallothionein, zinc and copper were studied during an annual reproductive cycle in rainbow trout of both sexes. In female fish, the total hepatic zinc levels closely followed the estradiol-17 and the LSI levels. Hence, the zinc levels rose in September, peaked in December and dropped in January. No distinct peaks were, however, observed in the whole-liver copper content. The hepatic metallothionein levels in female fish began to increase at the onset of exogenous vitellogenesis. Maximum levels were reached after estradiol-17 and LSI levels had dropped in January. In male fish no distinct peaks in either zinc or copper levels were observed. The metallothionein levels increased somewhat during the time of spermatogenesis. It is suggested that metallothionein may regulate the hepatic zinc distribution during the annual reproductive cycle in female rainbow trout, thereby ensuring the organism of a control mechanism to keep the pool of available zinc at an appropriate level.     

Does oral 3,5,3 ′-triiodo-l-thyronine affect dietary glucose utilization and plasma insulin levels in rainbow trout (Salmo gairdneri)?

A factorial experiment was conducted to determine the effect and interaction of dietary carbohydrate level and triiodo-L-thyronine (T₃) supplementation on the growth, physiological response and plasma insulin and cortisol levels of rainbow trout. The oral administration of T₃ significantly increased the growth, protein efficiency ratio and feed efficiency of trout, indicating an increased protein and perhaps energy utilization in these fish. However, T, administration did not significantly increase the utilization of dietary glucose as an energy source by the trout. Similarly, the administration of T₃ did not significantly affect plasma insulin levels in either the fed or the fasted trout. Plasma insulin levels were significantly higher in fed trout reared on the non-T₃ supplemented high carbohydrate diet in comparison to trout reared on the low carbohydrate diets. This indicates that increased dietary carbohydrate stimulates increased insulin secretion in the trout. Therefore, although rainbow trout are not insulin-deficient, they can still be considered a diabetic-like animal due to their poor glucose tolerance. Plasma cortisol levels were not affected by diet composition and altered plasma glucose levels.     

Reproduction related immunoglobulin changes in rainbow trout

Annual changes in plasma immunoglobulin (IgM) levels were investigated in three strains of rainbow trout Oncorhynchus mykiss which have different spawning periods, i.e., September–October, November–December, and January, reared under constant water temperature and natural day length. Plasma IgM levels decreased during the spawning season in all strains tested. The IgM changes became reversed in response to significant increases in plasma testosterone (T) and estradiol-17 in females and T and 11-ketotestosterone in males. Though the IgM decline showed a connection with suppressed immunocompetence, since many mature fish caught fungal diseases, no clear differences were observed in the plasma IgM levels between infected and noninfected fish during the spawning season. Incidentally, plasma IgM levels in infection prone fish were higher than in noninfection prone fish prior to the spawning season, whereas coincident differences in the plasma steroid levels were observed. Immature fish reared under lower water temperatures showed lower IgM levels. The effect of water temperature may have to be considered when analyzing the defense mechanism during the spawning season in rainbow trout.     

Cytochrome P-450 mediated O-dealkylation of 7-alkoxycoumarins in liver microsomes from rainbow trout (Salmo gairdneri)

Evidence has recently been presented for variation in the inducibility of various 7-alkoxycoumarin-O-dealkylase activities in liver microsomes from a number of mammalian species by -naphthoflavone (NF). In the present study we have investigated the inducibility of hepatic microsomal 7-methoxycoumarin-O-demethylase, 7-ethoxycoumarin-O-deethylase, 7-propoxycoumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities in rainbow trout by NF. O-demethylase activity was increased approximately 17-fold, O-deethylase and O-depropylase activities approximately 9-fold and O-debutylase activity approximately 25-fold. The kinetics of the various hepatic microsomal 7-alkoxycoumarin-O-dealkylase activities were investigated in control and NF-treated rainbow trout. The O-demethylase-, O-depropylase- and O-debutylase activities exhibited monophasic Michaelis-Menten kinetics in liver microsomes from both control and NF-treated rainbow trout, whereas the O-deethylase activity exhibited biphasic Michaelis-Menten kinetics in control liver microsomes and monophasic Michaelis-Menten kinetics in liver microsomes from NF-treated rainbow trout.     

Cortisol does not affect hepatic α- and β-adrenoceptor properties in rainbow trout (Oncorhynchus mykiss)

Distribution and function of hepatic - and -adrenoceptors were examined in rainbow trout (Oncorhynchus mykiss) injected with slow release hydrogenated coconut oil implants alone (sham) or containing cortisol. - and -Adrenoceptors were assayed on purified hepatic membranes 10–14 days post-implantation using ³H-prazosin () and ³H-CGP (). At 10–14 days, plasma cortisol values were significantly elevated to approximately 220 compared with 35.0 ng ml^-1 in cortisol implanted vs. sham trout. No significant differences were found between any of the experimental groups for either the affinity (K_d) or maximal number of binding sites (B_max) for either receptor type. Epinephrine significantly stimulated glucose release from hepatocytes isolated from sham injected trout, but not from cortisol-treated fish. Epinephrine-induced glucose release was blocked by both - and -antagonists. These studies do not support the hypothesis that rainbow trout exposed to chronic cortisol alter properties of hepatic adrenoceptors.     

Influence of oral administration of estradiol-17β and testosterone on growth,digestion, food conversion and metabolism in the underyearling red sea bream,Chrysophrys major

Estradiol-17 (E2) administered in the diet to the red sea bream Chrysophrys major did not affect appetite, food conversion efficiency, protein efficiency ratio and specific growth rate. Serum concentrations of total protein, albumin, globulin, vitellogenin, -amino acids, total lipid, free fatty acids, cholesterol and calcium were elevated. The hepatosomatic index was also increased. Activities of hepatic enzymes including lactate dehydrogenase, isocitrate dehydrogenase and glutamate-pyruvate transaminase were higher than found in untreated control fish. Intestinal activity of leucine aminopeptidase was augmented. However, there were no changes in muscle water, protein, lipid and glycogen content. In contrast, testosterone (T) given by the same route increased appetite, food conversion efficiency, protein efficiency ratio and specific growth rate. There were no alterations in serum protein and calcium concentrations but serum glucose, ammonia and triglyceride levels were elevated. Hepatic glycogen content was increased. The activities of hepatic fructose- 1,6-diphosphatase, glucose-6-phosphatase and glycogen synthetase and intestinal activities of alkaline phosphatase and -glutamyltransferase were higher than noted on control fish. The results reveal that estradiol-17 and testosterone exerted different metabolic effects in the red sea bream and they suggest that testosterone exerts its anabolic actions by increasing appetite, food conversion efficiency and activities of digestive enzymes.     

Effect of increased liver glycogen and liver weight on liver function in rainbow trout, Salmo gairdneri Richardson: recovery from anaesthesia and plasma 35S-sulphobromophthalein clearance

Abstract. Juvenile rainbow trout (initial mean body weight 17 g) reared on practical trout diets containing different levels of available carbohydrate for 8–24 weeks were assayed for liver function based on the time required to recover from the anaesthetics tricaine methanesulphonate (MS222), 2-phenoxyethanol (2PE) and tertiary-amyl alcohol (TAA), as well as plasma clearance of ³⁵S-sulphobromo-phthalein (BSP), Liver/body weight ratios (LBW) and percentage liver glycogen content (LG) increased in a linear manner with increased available carbohydrate. The mean recovery time (MRT) of trout anaesthetized with 2PE and TAA increased linearly with increased LBW and LG. No difference was observed in the MRT of trout anaesthetized with MS222 which is not metabolized in the trout liver. There was a significant linear regression of the biological half-life of plasma BSP on LBW (MR²= 95.7) and LG (MR²= 99.6). The results indicate that increased liver glycogen and liver weight due to higher levels of available carbohydrate affect the liver function of rainbow trout. These liver function tests may be applicable in determining acceptable levels of available carbohydrate in practical trout diets.     

Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss

The immunosuppressive effects of steroid hormones were evaluated as the response against implanted steroid hormones, cortisol (F), testosterone (T), estradiol-17 (E2), and 11- ketotestosterone (11-KT), in juvenile rainbow trout. In long term experiments (5 weeks), fish were given a single intraperitoneal implant of F or T. A clear suppressive effect of plasma IgM levels with F and T was not necessarily obtained, although mucus IgM levels were reduced corresponding to the elevated plasma steroid hormone levels. In short term experiments (1 week), intraperitoneal implantation of T, 11-KT and E2 suppressed plasma and mucus IgM levels, although the effects were not dose-dependent. When administered through diet, F and T caused a suppression of plasma IgM levels; F administration at both high and low dosages caused a significant decrease in plasma IgM levels, while only a high dose of T caused the suppression. These results suggest that sex steroid hormones, as well as F, have immunosuppressive functions in rainbow trout.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

Insulin-receptor binding in skeletal muscle of trout

Two hundred rainbow trout (Oncorhynchus mykiss) age 0 +, weight range 11.3 – 11.5 g, were distributed randomly in two groups and maintained for five weeks on either 10% dextrin, or 20% dextrin diet. The fish were sampled 3–5 h and 18–20 h after the last feeding and insulin binding to partially purified insulin receptors in white and red skeletal muscles and to liver plasma membranes was assessed. Plasma insulin, plasma glucose, and liver glycogen content were analyzed in the same fish.Fish fed a diet with higher carbohydrate content (HC) had elevated insulin and glucose levels in peripheral blood, but lower liver glycogen contents compared to the fish fed a diet with lower carbohydrate content (LC). No growth retardation was observed in the fish from HC group.Three to five hours after the last feeding, insulin-receptor binding in white skeletal muscles was higher in HC group of fish, mostly because of an increase in number of high affinity binding sites. Eighteen to twenty hours after the last feeding this difference disappeared. In contrast, the specific binding of insulin to the liver plasma membranes appeared to be lower in the HC group of fish. The lower insulin binding to the liver plasma membranes observed 3–5 h after feeding, could be attributed to the lower quantity of binding sites, while the same phenomenon 18 h after feeding was likely a result of affinity changes. We conclude that higher glycemic levels observed in trout fed a HC diet as compared to LC group of fish, are not a consequence of impaired binding of insulin to its receptors in skeletal muscles.Presented in part at the Annual Meeting of American Society of Zoologists, December 26–30, 1989, Boston, U.S.A. Supported by a grant (Personal investigador en el extranjero) of Ministerio de Educacion y Ciencia, (CYCIT) Spain to J.G. and by grants of the U.S. National Science Foundation (DCB 8615551 and DCB 8915935 to E.P.) and North Atlantic Treaty Organization (NATO) 0926/87 to C. Ottolenghi and E.P.     

Role of exogenous glutathione in teleost fish and its effects on antioxidant defense responses in rainbow trout exposed to 3,3',4,4'-tetrachlorobiphenyl

Intraperitoneal injection of glutathione (GSH) is shown to effectively increase tissue glutathione content in two teleost species, the rainbow trout and the American eel. GSH uptake into teleost tissues is a distinct piscine feature of GSH biochemistry compared with mammalian species where little or not uptake occurs of exogenously added GSH. Enhancement and depletion of tissue GSH status was used as a tool to examine the effects of altered GSH content on other cellular antioxidants in 3,3,4,4-tetrachlorobiphenyl (TCB) exposed rainbow trout. -Tocopherol content was unchanged and thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, changed little in liver and muscle of trout exposed to TCB and the various GSH treatments. However, -tocopherol levels in muscle and superoxide dismutase activities in the various tissues were significantly higher in controls and TCB-saline treated fish than in GSH deficient and lipoate supplemented TCB exposed trout. GSH status thus appears to affect some cellular antioxidant defenses in TCB exposed trout.     

Stress response of juvenile rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)to chemical cues released from stressed conspecifics

The objective of this study was to determine whether exposure of rainbow trout (Oncorhynchus mykiss) to water containing a stressed trout or skin extract from stressed and non-stressed trout would elicit a stress response in conspecifics. Juvenile rainbow trout were exposed for 1 hour to water containing a stressed fish, homogenized skin extracts from a non-stressed fish, skin extract from a stressed fish and water with none of these factors. The stress response was measured over a 24-h period (1, 6, 12, 24 h after exposure). Plasma cortisol levels increased at 12 h in fish exposed to water from a stressed fish and skin extract from a stressed fish. Plasma glucose and hepatic hsp70 levels were not affected by treatments. The results suggest that rainbow trout elicit a stress response when exposed to stress-related alarm cues released from conspecifics.     

Accumulation of gamma-oryzanol in teleost

Japanese people consume crop bran, which contains relatively high amounts of gamma-oryzanol (ORZ), as foodstuffs and food materials. We have recently confirmed that ORZ inhibits NF-κB activation, activates peroxisome proliferator-activated receptor gamma (PPARγ), and increases plasma adiponectin levels. ORZ is therefore expected to improve lipid and carbohydrate metabolisms and various inflammatory diseases, i.e., metabolic syndrome including type 2 diabetes. ORZ administration also allowed fish, such as rainbow trout, yellowtail, and red sea bream, to accumulate protein through enhancement of lipid and carbohydrate metabolisms. In the present study, we have investigated ORZ accumulation levels in mouse and rainbow trout administered ORZ-containing feed. Although mouse muscle and liver hardly contained ORZ, muscle tissues of every fish species accumulated higher amounts of ORZ. These findings suggest that rainbow trout, red sea bream, and yellowtail accumulate ORZ in muscle.     

Oxidative effect of l -carnitine on energy metabolism in diploid and triploid rainbow trout ( Oncorhynchus mykiss ): impact on metabolites

l-Carnitine plays a key role in the regulation of energy metabolism and growth in fish. The aim of this study was to determine the effects of dietary l-carnitine levels on carnitine homeostasis and energy metabolism in diploid and triploid trout (Oncorhynchus mykiss). Diploid and triploid trout (29.5 ± 0.6 and 31.8 ± 0.5 g, respectively) were fed with three diets supplemented with 15, 200 or 530 mg l-carnitine/kg, respectively, for 56 days. Compared to 15 mg l-carnitine, total carnitine content increased in the liver by 182% in diploid and by 154% in triploid trout fed 530 mg l-carnitine. In muscle, total carnitine content increased by 60 and 73% in diploid and by 34 and 75% in triploid with the increase in dietary l-carnitine levels. l-Carnitine caused no significant change in the plasma concentrations of metabolites like proteins (ammonia, urea and protein), lipid (triglycerides), carbohydrate (glucose, lactate) and enzyme activities (lipase, lactate, alanine transaminase, lactic acid dehydrogenase). Triploid trout had significantly lower plasma ammonia (P = 0.003), lipase (P = 0.005) and triiodothyronine (T3; P = 0.003) levels than diploid trout. In conclusion, ploidy significantly affected the energy metabolism in rainbow trout, dietary l-carnitine levels altered the l-carnitine homeostasis, but not influence nutritional metabolism.     

Effects of food deprivation on white muscle energy reserves in rainbow trout (Oncorhynchus mykiss): the relationships with body size and temperature

The relationship between body size and the depletion of white muscle metabolites (e.g., PCr, ATP, glucose and glycogen) was examined in two sizes (30 or 700 g) of rainbow trout deprived of food for one, four or seven days at either 5 or 15 °C. Following 7 days of food deprivation at 15 °C, the levels of muscle glycogen decreased by approximately 50% in small fish relative to control values (i.e., day 1). In comparison, small fish acclimated to cold temperatures did not exhibit a significant reduction of muscle glycogen over the seven day fasting regime. In contrast to small fish, the levels of white muscle glycogen in large fish remained unchanged after food deprivation, regardless of acclimation temperature. A seven day deprivation of food also resulted in a 50% depletion of white muscle glucose concentrations in small and large fish acclimated to warm temperatures, but there were no significant changes in this variable in fish acclimated to cold temperatures. In contrast to the negative effects of food deprivation on white muscle glycogen and glucose levels, the concentrations of white muscle PCr and ATP were not greatly affected by food deprivation under any of the experimental conditions. Taken together, these results clearly show that food deprivation can have an important influence on the storage of energy metabolites for anaerobic energy production, particularly in small fish at warm temperatures. In the future, it may be very important to consider the physiological effects of short-term food deprivation when interpreting results from studies in which fish have been fasted prior to treatments such as exercise.     

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.