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The use of a single diet with a well defined composition to feed fish throughout their life cycle is an oversimplification that probably does not respond to their metabolic requirements. For example, the seasonal reproduction that characterizes most fish species demands changes in nutritional requirements. Bearing this in mind, the macronutrient selection pattern was studied from January to August in twelve individually housed sea bass exposed to a constant photoperiod (12L:12D h) and temperature (23+/-0.5 degrees C). The endogenous "seasonal" effect on food and energy intake regulation and macronutrient selection was determined, using protein (P), carbohydrate (CH), and fat (F) packaged separately into gelatine capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Energy intake changed monthly, the highest values being recorded in May and June and the lowest values in March and April. The preliminary results illustrated "seasonal" changes in the sea bass macronutrient selection pattern with, which showed a predominantly proteinic selection during April (53% P, 21% CH, 25% F) and lipidic in July (35% P, 19% CH, 42% F); the increase in fat selection from May to July being statistically significant. This is the first evidence supporting the existence of an endogenous rhythm in the "seasonal" energy regulation and macronutrient selection in fish through post-ingestive mechanisms and probably involving chemosensory detection in the gut and/or post-absorptive mechanisms, although the exact mechanisms involved have yet to be clarified.

Teleost fish are able to adjust their energy intake when fed on pure macronutrient sources, although the exact mechanisms regulating macronutrient selection remain unknown. Since cholecystokinin (CCK) has been reported to modify macronutrient selection patterns in mammals, we explored the effect of CCK administered orally to European sea bass on the selection of separately encapsulated macronutrients. CCK doses of 0.05, 0.15 and 0.25 mg/kg BW administered in gelatine capsules for 5 consecutive days produced a significant inhibition of total food intake (21, 28 and 51%, respectively) at highest doses, evenly reducing the quantity of all the macronutrients ingested and, without affecting their relative proportions in the diet. Oral administration of proglumide, a non-specific CCK receptor antagonist, at doses of 5, 15 and 25 mg/kg BW, induced a quantitative total food intake increase of 2, 18 and 44%, respectively, and an increase of 52% in CH and 43% in P quantity ingested at highest dose. Co-administration of proglumide (25 mg/kg BW) and CCK (0.25 mg/kg BW) in a single preload capsule blocked the effects observed with CCK alone. In conclusion, orally administered CCK induced an anorexigenic effect on both total food and single macronutrient intake, an effect that is counteracted by the CCK antagonist proglumide.

Melatonin is an effective antioxidant, immunostimulant, gonadal maturating regulator and antistress indoleamine that may be potentially useful for fish farmers. We have explored two possible ways of increasing plasma melatonin levels through the diet: direct melatonin supplementation (ME diet) and supplementation with the melatonin precursor tryptophan (TRP diet). To this end, a group of sea bass was fed a commercial diet (STD diet) at a regular time for 16 days, after which plasma, intestine, and bile samples were taken at four different time points: 120 min before, and 15, 180 and 480 min after feeding. Locomotor activity, intestinal and biliary melatonin, and plasma melatonin, serotonin and cortisol levels were measured. This same sampling process and analyses were also carried out after feeding sea bass TRP diet or ME diet for 1 week. Our results show that melatonin, but not tryptophan supplementation of the diet increases plasma, intestine and bile levels of melatonin. Plasma serotonin levels, on the other hand, were increased by dietary tryptophan, but not by melatonin, confirming the availability of supplemented tryptophan for serotonin synthesis. Both treatments were equally effective in reducing the high cortisol levels observed with the STD diet.

The effect of restricted feeding schedule was investigated on the seasonal shifting of daily demand-feeding pattern and food anticipatory activity in European sea bass (Dicentrarchus labrax) held under natural environmental conditions in an outdoor laboratory. To that end, demand-feeding behavior was continuously monitored for approximately one year in four groups of 15 fish each exposed to natural fluctuations of water temperature (from 13.2 degrees C to 27.4 degrees C) and photophase (from 9.5 h to 14.5 h of light). When the animals were subjected to a time-restricted feeding schedule, the demand-feeding rhythm rapidly synchronized to the three periods of food availability: the first meal (FM) from 08:00 to 09:00 h, the second meal (SM) from 16:00 to 17:00 h, and the third meal (TM) from 00:00 to 01:00 h. The occurrence of demand-feeding activity into the three periods of food availability displayed a double seasonal shift: fish that self-fed mostly during the daytime periods of feeding availability (FM and SM) in summer and autumn changed to nocturnal feeding (TM) from December to April, returning to diurnal preferences in April. Food-demands appeared to be predominantly associated with feed availability, reaching its maximum levels during the hours of reward. In addition, feeding anticipatory activity (FAA) was observed. A relationship was detected between the duration of FAA and feeding-time, with shortest FAA (30-60 min) when mealtime occurred just after sunrise (FM) or sunset (TM). These findings demonstrate the ability of sea bass to self-feed under time-restricted schedules, and show a seasonal-phase inversion in demand-feeding activity in spite of the restrictions in their feeding availability. Sea bass can use external signals as reference to anticipate the time of feed availability. This information may be useful for designing new feeding strategies for European sea bass fish farming.

The sea bass is a teleost that is able to regulate its energy intake by selecting from pure macronutrient sources, although the regulatory mechanisms involved in this selection are unknown. Nutrient preloads are known to reduce food intake and modify macronutrient selection patterns in mammals, but no information is available on its effects in fish. The aim of the present work was to determine the effect of orally administered macronutrient preloads of protein (P), fat (F) or carbohydrate (CH) on the subsequent macronutrient selection, using for the purpose feed consisting of CH, P or F packaged separately in gelatin capsules. The macronutrient preloads left the total food intake unaltered, but caused differential changes in the pattern of macronutrient selection. The CH preload increased the selection of CH (39%) and decreased that of P (20%), independently of the fish's previous nutritional preferences. The F preload induced an F increase (32%) and a P decrease (18%) in P-preferring fish, but not in F-preferring fish in which the macronutrient selection pattern remained unaffected. The P preload stimulated F selection by 42% in P-preferring fish, but left the macronutrient selection pattern unchanged in F-preferring fish. In conclusion, oral macronutrient preloads affected the pattern of macronutrient selection in fish, acting by post-ingestive mechanisms. The effect was influenced by the fish's previous nutritional preference and/or status, which could depend on its metabolic capacity.

Teleost fish are able to regulate their energy intake selecting from pure macronutrients sources, but the regulatory mechanisms involved in macronutrients selection remain unknown. Serotonin (5-HT) reduces food intake in mammals and fish and modifies the macronutrients selection pattern in mammals; however, no information is available about its role on macronutrients selection in fish. The aim was to determine the effect of orally administered 5-HT (0.1, 0.5 and 2.5 mg kg BW(-)(1)) into gelatine capsules on the subsequent macronutrient selection of sea bass, using for this purpose gelatine capsules including carbohydrates, protein, or lipids separately. The voluntary ingested 5-HT was released into the plasma of fish, reaching a level two times greater than the controls, 45 min after the ingestion of a capsule containing 2.5 mg kg BW(-1) of 5-HT. The indoleamine, at doses of 0.1, 0.5 and 2.5 mg kg BW(-1), produced a reduction in total food intake of 31%, 49% and 37%, respectively, compared to the baseline, modifying the macronutrient selection pattern. The percentage of fat selected was significantly reduced whereas the percentage of protein significantly increased after administration of highest dose, but no changes were observed in the proportion of carbohydrate for any 5-HT doses. In conclusion, oral administration of 5-HT affected both amount of food intake and pattern of macronutrients selected. This is the first evidence supporting a role of 5-HT as a neurohumoral mediator involved in macronutrients selection in fish.

Recent reports describe teleosts as being able to regulate energy intake by selecting from pure macronutrient sources, although the regulatory mechanisms involved in this selection remain unknown. The aim of the present work was to determine the effect of selective macronutrient deprivation on energy regulation and macronutrient selection, using for this purpose carbohydrate (CH), protein (P), and fat (F) packaged separately into gelatin capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Twenty-four individually housed sea bass (Dicentrarchus labrax) were subjected to two experiments: (a) two-macronutrient deprivation, and (b) one-macronutrient deprivation. In two-macronutrient deprivation, fish were fed sequentially with P, CH, or F, and in one-macronutrient deprivation, they were fed sequentially with two separately packaged macronutrients (P and CH, CH and F, or P and F). There was a rapid reduction of macronutrient intake in two-macronutrient deprivation, reaching 80% inhibition after 4, 5, and 7 days of P, CH, and F intake, respectively. In one-macronutrient deprivation, the energy intake was significantly reduced during selective F deprivation, but not with P or CH deprivation. Although the fish were being fed with only two macronutrients, the relative proportions of these macronutrients in each selective deprivation phase were the same as the baseline. These results show that in deprivation studies fish need at least F plus one other macronutrient to regulate their energy intake, and that their macronutrient selection is stable even when one is absent. In summary, fish seem to regulate energy and macronutrient selection through post-ingestive mechanisms probably involving chemosensory detection in the gut, and/or post-absorptive mechanisms.

Melatonin is synthesized, with a circadian rhythm, in the pineal organ of vertebrates, high levels being produced during the scotophase and low levels during the photophase. The retina also produces melatonin, although in the case of the European sea bass, its secretion pattern appears to be inverted. In the study described here, radioreceptor assay techniques were used to characterize the melatonin binding sites, their regional distribution and their daily variations. Brain and retina membrane preparations were used in all the binding assays and 2-[125I]iodomelatonin ([125I]Mel) as radioligand at 25 degrees C. The specific binding of [125I]Mel was seen to be saturable, reversible, specific and of high affinity. In all the tissues assayed, the power of the ligands to inhibit [125I]Mel binding decreased in the following order: melatonin>>4-P-PDOT>luzindole> or =N-acetylserotonin, which points to the presence of Mel1-like receptors. The inhibition curves of 4-P-PDOT suggested the presence of two different binding sites in the brain areas, but only one type of site of low affinity in the neural retina. No daily variations in [125I]Mel binding capacity (Bmax) or affinity (Kd) were detected in the brain areas, while a clear rhythm in Kd melatonin receptor affinity and Bmax binding capacity was observed in the retina. Kd and Bmax retinal rhythms were out of phase with the lowest Kd and the highest Bmax occurring at scotophase. This result suggests that retinal melatonin is a paracrine factor able to control receptor desensitization during photophase when ocular melatonin is higher in this species.

Seasonal and circadian changes have been observed in dietary selection and feeding behavior of fish. It is known that the light-dark cycle is the principal mediator of the production of pineal-derived melatonin in fish, and also that the digestive tract synthesizes and secretes melatonin and indeed is the principal extrapineal source of this secretor product, suggesting that melatonin could be involved in the processes of feeding and/or digestion. Fish are capable of regulating their energy intake from separate sources of macronutrients, but the mechanisms of this selection process are unknown. In the present paper, we explored the effect of melatonin administered orally to European sea bass on their selection of encapsulated macronutrients. Melatonin doses of 0.1, 0.5, and 2.5 mg/kg body weight were administered in gelatin capsules. The voluntarily ingested melatonin was absorbed into the fish's plasma, 45 min after the administration reaching a level depending on the dose that was up to 26 times greater than the controls with the highest dose of melatonin. The indole produced a dose-dependent inhibition of total food intake of 9, 26, and 34%, respectively, and also modified the pattern of macronutrient selection. Carbohydrate intake was significantly reduced (by 17, 33, and 42% for the three doses, respectively), but the observed reductions in fat and protein intake were not statistically significant. On the contrary, fat percentage significantly increased for the highest dose, but no changes were observed in the protein or carbohydrate percentages for any of the melatonin doses. In conclusion, orally administered melatonin affected both the amount of food consumed and the pattern of macronutrients selected. This is the first evidence for the existence of neurohumoral mediators involved in the selection of macronutrients in fish.

Reproduction in fish is cyclical and timed to guarantee the survival of the offspring. Seasonal variations in reproductive hormones of fish have been deeply investigated in fish over the last years. However, there are few studies regarding the daily changes in reproductive hormone profiles in teleosts. The aim of the present research was to investigate the effects of photoperiod manipulation on melatonin and reproductive hormones (pituitary sbGnRH, pituitary LH and plasma LH, testosterone [T], and 11-ketotestosterone [11KT]) daily rhythms in male sea bass, kept in net cages under farming conditions in winter (9L:15D). Fish were distributed in two groups, one under constant long photoperiod (18L:6D) and the other under natural photoperiod. The photoperiod strongly influenced the daily melatonin profile, so that the duration of the nocturnal melatonin rise was longer in the control group than in the group exposed to the artificial photoperiod (18L:6D). A daily rhythm was observed in the pituitary sbGnRH profile in both groups, showing the lowest levels during the dark period. A daily rhythm of pituitary LH was detected in the control group, which was suppressed in the group under long photoperiod. Daily variations in plasma LH were observed, the highest levels being found in the dark phase in both groups, although this profile was significantly altered by artificial light, maintaining a fixed relationship between the first nocturnal rise of melatonin and the nocturnal peaks of plasma LH in both groups. Plasma T levels showed significant fluctuations in their daily cycle following a sinusoidal pattern with an acrophase around sunrise in both groups, without any influence of light regime. No significant daily variations in plasma levels of 11-KT were observed in none of the groups. Our results provide the first evidence of the presence of daily variations in pituitary sbGnRH content, pituitary and plasma LH, and plasma T in sea bass. Artificial lights suppressed the circulating melatonin and significantly affected the daily rhythm of LH storage and release.

The role of the food orosensory properties on protein (P), fat (F) and carbohydrate (CH) self-selection was investigated in fish fed gelatine capsules containing pure macronutrients. A total of 40 sea bass (39.6+/-6.2 g initial body weight) distributed in eight 75-l tanks were used. In a sequence of experimental phases, sea bass were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. Our results demonstrate that fish are able to regulate food intake, so as to balance their energy intake, when they are fed a complete encapsulated diet and, therefore, without using the orosensory properties of the diet. Moreover, sea bass learn to discriminate and select among colour-coded, pure macronutrient capsules to compose a complete and balanced diet using colour as the only external cue. The composition of selected diet was 55% P, 23% CH and 22% F in terms of macronutrient percentage. The diet orosensory properties do not seem to be necessary to regulate macronutrient intake either, suggesting that an associative learning between capsule colour and content can be established through monitoring of macronutrient intake by postingestive and/or postabsorptive mechanisms. These results provide the first insight into energy and macronutrient self-selection by fish fed on gelatine capsules containing separate macronutrients.

Melatonin is involved in the transduction of light information and the photoperiodic control of many important physiological functions in fish. Although artificial photoperiods have been used to improve fish growth and manipulate reproduction, there is little information about the characteristics of light 'quality'. In this paper we describe the effects of a light pulse in the middle of the dark phase on plasma and ocular melatonin in European sea bass. We first determined the light intensity necessary to elicit a melatonin response using white light of varying intensities (0.6-600 mu W/cm(2), experiment 1). Secondly, we tested the effect of the light spectrum on melatonin production using three differently coloured lights (half-peak bandwidth=434-477, 498-575 and 610-687 nm for the blue, green and red lamp, respectively, experiment 2) and, finally, we determined the effect of light orientation (downwards directed versus upwards directed, experiment 3). The results show that the minimum light intensity needed to inhibit or stimulate melatonin levels in both plasma and the eye was 6.0 mu W/cm(2). A linear correlation was found between the logarithm of light intensity and the relative inhibition. In addition, the blue wavelength was more effective in decreasing melatonin levels in the former and increasing the levels in the latter. Nevertheless, red light at sufficient intensity proved effective at significantly suppressing circulating melatonin. Downwards light had a greater effect than upward-directed illumination in suppressing plasma melatonin. In conclusion, the results point to the importance of giving proper consideration to the characteristics of light, to adequately control melatonin production and its related physiological processes.

The transduction of seasonal information from the environment (i.e., photoperiod and water temperature) into melatonin rhythms was studied in sea bass. Plasma and ocular melatonin (N-acetyl-5-methoxytryptamine) was determined in autumn, winter, spring and summer (experiment 1) under natural culture conditions, and in the summer and winter solstices under both natural and "6-month out-of-phase" photoperiods (experiment 2). At each sampling, 48 sea bass were sacrificed at a rate of 6 fish every 3 hr and the level of melatonin was determined in plasma and eye cup samples by ELISA. In experiment 1, significant diel changes were observed in plasma melatonin, with nocturnal melatonin varying from 144 pg/mL (summer) to 23 pg/mL (autumn), while diurnal melatonin remained low, around 8 pg/mL throughout the year. In experiment 2, the photoperiod length was shown to control the duration of the nocturnal melatonin rise, while the water temperature determined the amplitude of the melatonin rhythm. Ocular melatonin peaked during daytime in autumn and winter, but no significant changes were detected in summer and spring. In conclusion, plasma melatonin rhythms in sea bass reflect the pineal capacity to integrate seasonal information and supply precise calendar information, which may synchronize different physiological processes such as annual reproduction and feeding rhythms.

The influence of two different fasting periods on macronutrient self-selection during refeeding was studied in sea bass. Twelve groups of three sea bass were kept under laboratory conditions with a 12:12 light-dark cycle and at 22.5 degrees C. Each aquarium was provided with three self-feeders containing three different diets made up of two macronutrients: protein-fat, protein-carbohydrate, and carbohydrate-fat. The fish were allowed to acclimatise to the diets and laboratory conditions in 4 weeks, during which they selected a baseline diet of 51% protein, 16.5% carbohydrate, and 32.5% fat, in terms of digestible energy. The fish were then deprived of food for 6 days, after which they were permitted to refeed. When food demands stabilised, they were subjected to total food deprivation for another 2 weeks. The total energy demanded from all the macronutrients increased significantly during the first day after both fasting periods. The proportion of macronutrient self-selection after fasting periods remained unchanged in respect to the baseline, except for protein energy which remained high in the two first days of refeeding after the second fasting period (the 51% of total demanded energy from protein during the baseline period rising to 60%). In short, after two short fasting periods of different length, sea bass showed a compensatory ingestion of energy, with a slightly increased demand from protein after the longest fasting period.

In this paper, we show the results from four different experiments in which sea bass, maintained under laboratory conditions, could choose between two or three different diets through self-feeders, which gradually increased the complexity and potential range of selection, to design their own diet in accordance with their requirements. At first, sea bass were allowed to select between two complete diets differing in the proportion of protein (52-58%): this showed their capacity to distinguish between two diets made of the same ingredients. Next, two incomplete diets, containing a fixed amount of protein (56%) and lacking either fat or carbohydrate, were made available. Three mixed diets made up of pairs of macronutrients (protein-carbohydrate, protein-fat or fat-carbohydrate) were tested in the next experiment and, finally, three diets containing only one macronutrient (protein fat or carbohydrate) were made available to fish. Taking into account selection made by the fish in the first three experiments, in which macronutrient selection was statistically different, protein was the main macronutrient chosen by fish (278.15 kJ/kgBW/day, on average), followed by fat and carbohydrate (162.85 and 64.56 kJ/kgBW/day, respectively). In conclusion, the results reveal the ability of sea bass to select an appropriate diet from experimental diets containing two or three macronutrients and suggest that the proposed methodology is a powerful tool for studying the differing nutritional needs of different species of fish.

Daily variations of insulin, cortisol, and glucose are studied in animals adapted to two different photoperiodic regimes, with the intervals between feeding times and photoperiodic events kept constant. Data support the existence of a daily rhythm of plasma glucose which seems to be photoperiodic. In contrast, the daily patterns of insulin in sea bass seem to be mainly influenced by feeding time; however, an effect of photoperiod can not be excluded. When the digestive tract is absent of food, insulin levels are generally minimal at feeding times and maximal during the inter-meals periods, suggesting the central control of insulin secretion during short-term food deprivation. Contrarily, the nadir values of plasma cortisol were reached at midday during the inter-meal period and peak plasma levels were evident at both light onset and offset. Disruption between metabolite and hormone patterns suggest that they are under different controls. Such results could be explained under the existence of a multioscillator system, including a food entrainable oscillator in addition to the master light entrainable oscillator.

Regulation by light and darkness of melatonin rhythms in the plasma and eye of the European sea bass (Dicentrarchus labrax) was studied. During light-dark cycles, plasma and ocular melatonin exhibited day-night changes with higher levels at mid-dark and at mid-light, respectively. Circulating melatonin levels were low in constant light but high in constant darkness (DD); ocular melatonin levels showed the reverse pattern. Plasma melatonin exhibited circadian rhythm for 1 cycle but the rhythm was no longer apparent on day 2. There was no circadian rhythm in ocular melatonin. Acute light exposure in DD decreased plasma melatonin but increased ocular melatonin. These results suggest that circulating melatonin may be used as a signal for darkness but ocular melatonin is used as a signal for the light phase.

Sea bass, a fish species characterized by its dualistic feeding pattern, was investigated to study the synchronizing effect of light and food on the demand-feeding rhythm. Nocturnal and diurnal sea bass, both in groups and individually, were exposed to restricted-feeding (RF) and light-dark (LD) cycles of different periods. The phase relationship between both zeitgebers was also studied. The results show that food-demand rhythms synchronize to periodic food availability under constant light conditions (DD) and that there is a partial coupling between food-entrained and light-entrained activity under conflicting zeitgebers (LD 13:13 h and RF 4:20 h), suggesting the existence of a feeding entrainable oscillator (FEO) in addition to the master light entrainable oscillator (LEO). In some cases, food availability restricted to the light or dark phase contrary to that of the previous feeding phase changed a diurnal feeding pattern into nocturnal and viceversa, suggesting that food can be one of the switching factors that decides whether the circadian system of sea bass is nocturnal or diurnal. However, the fact that the feeding pattern of some fish was unrelated with the phase in which food was available suggests that other internal and/or external factors could be involved in the temporal flexibility of sea bass.

The nocturnal versus diurnal feeding patterns of sea bass under controlled experimental conditions were studied in order to investigate the existence of such a dualistic feeding behavior. The animals (six groups of 4 animals and 8 single fish) were held in tanks filled with recirculating salt water and installed in a "chronolab" under constant conditions (23.5 degrees C and 2.4% salinity). The fish were given access to self-demand feeders and first exposed to a photoperiod regime of 12:12 (12 h light, 70 lx, and 12 h dark, complete darkness) and then to light:dark (LD) pulses (40 min light, 40 min dark). The LD 12:12 cycle was reversed by doubling the light period in day 16, and reversed again in day 27 by doubling the dark period. The circadian rhythm of food demand was strongly synchronized with the LD cycle, and fish exhibited both diurnal and nocturnal patterns. In most fish, the shift of the feeding rhythm to the new LD cycle was very fast for each reversal (bringing forward or delaying their feeds 12 h), indicating a weak participation of an endogenous circadian rhythm. However, when submitted to LD pulses, fish began to free-run with a periodicity of about 23 h and kept feeding in the light or dark phase according to their prior behavior. The existence of a dualism in the diel feeding pattern in sea bass was thus clearly demonstrated and it appeared that the diurnal and nocturnal behavior did not depend exclusively on a circadian phase inversion of the feeding rhythms as this pattern of behavior was enhanced under ultradian LD pulses. The ecological implications of such dual capacity and the switch from one type of phasing to another are hitherto unknown and need further field and laboratory investigation.

A new demand feeder was designed for the automatic and accurate feeding of fish. The feeding device is microcomputer controlled and permits the continuous recording of feeding behaviour. The electromechanical feeder consists of a sliding door powered by a solenoid which provides only one weighed pellet when activated by the fish. Some preliminary results on feeding behaviour in sea bass are presented to test the efficiency of the feeder.