Aquaculture 233 (2004) 293 – 304 www.elsevier.com/locate/aqua-online
A balanced dietary amino acid profile improves amino acid retention in post-larval Senegalese sole (Solea senegalensis) Cla´udia Araga˜o a,*, Luis E.C. Conceic¸a˜o a, Dulce Martins b, Ivar Rønnestad c, Emı´dio Gomes b, Maria Teresa Dinis a b
a CCMAR, Universidade do Algarve, Campus de Gambelas, 8000-117 Faro, Portugal CIIMAR, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal c Department of Zoology, University of Bergen, Alle´gt 41, N-5007 Bergen, Norway
Received 9 June 2003; received in revised form 16 August 2003; accepted 18 August 2003
Abstract The rearing of most marine fish larvae still relies on live food. Dietary amino acid (AA) imbalances when using live food in the larval rearing of flatfishes have been suggested. The aim of this study was to test if dietary AA supplementation affects AA metabolism in Senegalese sole (Solea senegalensis) post-larvae. This was done by tube-feeding Artemia-fed sole with a dipeptide solution containing two potential limiting AA (leucine and phenylalanine), in order to supplement the larval gut content and to balance the dietary AA profile. Two experiments were done using different 14C-labelled AA as tracers. The first used a 14C-protein hydrolysate to test the effect of balancing the dietary AA profile on the overall AA metabolism. The second experiment analysed the effect of balancing the dietary AA profile on metabolism of threeselected AA: arginine, leucine and glutamate. A set-up to determine the handling of 14C-labelled AA was used, in order to quantify the absorption, catabolism and retention of the test mixtures by the sole post-larvae. The first experiment demonstrated that, when fish were fed the dipeptide supplement, AA catabolism decreased and AA retention increased. This agrees with the hypothesis that balancing the dietary AA profile increases AA retention in fish. In the second experiment, arginine and glutamate catabolism were reduced by the dipeptide supplementation. Leucine supplementation did not reduce leucine catabolism but instead increased arginine and glutamate retention. This result supports earlier studies that fish larvae are able to regulate their
* Corresponding author. Tel.: +351-289-800-900x7374; fax: +351-289-818-353. E-mail address:
[email protected] (C. Araga˜o). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.08.007
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AA metabolism. A balanced dietary AA profile increases the AA retention and may improve growth and nitrogen utilisation. D 2004 Elsevier B.V. All rights reserved. Keywords: Amino acid metabolism; Solea senegalensis; Balanced diets
1. Introduction Young stages of fish have high amino acid (AA) requirements, partly due to their high growth rates that implies high protein synthesis rates and partly since AA are important sources of energy for these early stages (Dabrowski, 1986; Rønnestad and Fyhn, 1993; Conceic¸a˜o et al., 1998; Rønnestad et al., 1999). The ideal dietary AA profile can be defined as the one that will allow for optimal protein growth (Chung and Baker, 1992; Boisen et al., 2000; Conceic¸a˜o et al., 2003b). Therefore, this profile depends on the AA profile of the proteins being synthesized and which AA is used for energy dissipation or for other metabolic purposes (Conceic¸a˜o et al., 1998, 2003a,b). Growth optimisation can be achieved by manipulation of dietary nitrogen profile in order to obtain the ideal profile. Despite some recent encouraging results on the use of formulated diets for fish larvae (Cahu and Zambonino Infante, 2001), the rearing of most marine fish species still relies on live food. Manipulation of the nutritional composition of live prey is difficult, with the exception of lipid components. However, a method of controlled tube-feeding of radio-labelled nutrients developed by Rust et al. (1993) is a mean to overcome some of the limitations in nutritional studies with fish larvae. The method has been modified by Rønnestad et al. (2001a) and permits discrimination between the unabsorbed labelled nutrients that have been emptied by the gut from the labelled molecules that originate from catabolism or metabolism of the absorbed nutrients. Leucine was proposed as one of the AA that were probably limiting growth in turbot larvae (Conceic¸a˜o et al., 1997), and this might be true for other flatfishes since it seems to apply to Senegalese sole (Araga˜o et al., in preparation). Phenylalanine and tyrosine can also be limiting AA, as these aromatic AA have important roles for larval development other than protein synthesis, since they are the precursors of thyroid hormones, melanin, dopamine and cathecolamines (Cowey and Walton, 1989; Bender, 1995). The objective of this study was to test if dietary AA supplementation affects AA metabolism in Senegalese sole post-larvae. The AA supplementation was achieved through tube-feeding post-larvae with dipeptides containing potential limiting AA. Dipeptides were chosen to supplement the diet, since free AA (FAA) are known to be absorbed faster than protein and this might lead to transient AA imbalances (Rønnestad et al., 2000b). A first experiment was done to test the effect of the dipeptide supplement on overall AA metabolism. A second experiment was done in order to examine the effect of the dipeptide supplementation on three-selected individual AA. Arginine and glutamate were chosen as an example of an indispensable and a dispensable AA. In addition, leucine was also chosen since it was one of the potential limiting AA in the diet.
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2. Materials and methods 2.1. Fish Solea senegalensis larvae were reared at C.I.C.E.M. ‘‘El Torun˜o’’ (Ca´diz, Spain) facilities, according to standard procedures. At 18 days after hatching (DAH), larvae were transferred to the University of Algarve facilities, where they were stocked in a recirculating system comprising plastic white trays (23 33 cm; 4 L volume). Water was renewed every hour and maintained at 20.2 F 0.6 jC and 32x . Oxygen was always above 85% saturation and a 12/12-h light/dark cycle was adopted. Post-larvae were fed on frozen Artemia metanauplii enriched for 24 h in DC Super Selco (INVE Aquaculture, Belgium). On the day before experiments, post-larvae were transferred to small white trays and placed in the room where the experiments were conducted. Senegalese sole post-larvae used in the experiments had between 36 and 40 DAH (2.6 F 0.8 mg dry weight, mean F S.D., n = 30). Room temperature during experiments was 23.5 F 1.0 jC. 2.2. Treatments On the day of experiments, fish were fed on frozen enriched Artemia metanauplii as usual. Half an hour later, the post-larvae were transferred with a wide-mouth pipette and tube-fed with 14C-labelled mixtures, as described in Section 2.3. Two treatments were tested: one, which serves as control, consisted in tube-feeding the Artemia-fed fish with a saline solution (seawater/distilled water 1:3). A second treatment consisted in tube-feeding the Artemia-fed fish with a saline solution containing two dipeptides: Leu – Gly and Phe– Ala (both from Sigma-Aldrich, Germany), in order to supplement the larval gut content (supplemented treatment). To calculate the amount of dipeptides that should be tube-fed, 12 post-larvae that have been feeding for half an hour under similar conditions were dissected and the Artemia present in the gut was counted. This procedure showed that in 30 min Senegalese sole were eating 6.7 F 1.3 Artemia metanauplii, representing 5.4 Ag of protein. Based on this, calculations were done in order to compensate the dietary AA profile. Peptide solutions were prepared in order that the pulse delivered by tube-feeding represented 6% of the dietary protein content of a normal Artemia meal (3% from each peptide). This was expected to increase the phenylalanine content in the stomach in 5 times and leucine in 1.5 times than if Artemia alone was in the fish gut. The tube-feeding mixtures were prepared by freezedrying both solutions (saline solution for the control treatment and saline peptide solution for the supplement treatment) and then adding the different 14C-labelled AA tracers. Two experiments were performed in order to understand how balancing the dietary AA profile affects metabolic handling of AA by Senegalese sole post-larvae. The first experiment was done to test the effect of balancing the dietary AA profile on overall AA metabolism. This was done by adding a L-[U-14C]-protein hydrolysate (Amersham Pharmacia, UK) that contains all the 20 AA used for protein synthesis as a tracer. The protein hydrolysate was added to both freeze-dried solutions. Total
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activity of the protein hydrolysate was 1.85 MBq/ml. The two mixtures prepared were tube-fed to the Artemia-fed Senegalese sole post-larvae, as described in Section 2.3. The second experiment was performed to test the effect of balancing the dietary AA profile on the metabolism of selected AA: arginine, leucine and glutamate. L-[U-14C]tracers for arginine (arginine monohydrochloride), leucine or glutamate (Amersham Pharmacia), each with an activity of 1.85 MBq/ml, were added individually to the freeze-dried solutions. Mixtures were tube-fed to the Artemia-fed fish, as described in Section 2.3. 2.3. Tube-feeding set-up An in vivo method for controlled tube-feeding of fish larvae as described by Rust et al. (1993) and modified by Rønnestad et al. (2000a,b) was used. This set-up comprised a stereo-dissecting microscope and a nanoliter injector (World Precision Instruments, Sarasota, USA) fixed firmly to a micromanipulator. A 0.19-mm diameter plastic capillary tube (Sigma-Aldrich) was fastened to the nanoliter injector. Prior to tube-feeding the Senegalese sole, post-larvae were anaesthetized in 0.033 mM MS-222 and then gently placed on a microscope slide in a droplet of clean seawater. One single injection of 13.8 nl of test mixture was then deposited into the stomach lumen. The tube-fed volume was based on a pilot study in which increasing volumes of a solution containing food colorant were tube-fed and the distension of the stomach wall was observed. Volumes above 13.8 nl filled the stomach and passed on to the mid-gut. After capillary withdrawal, the fish were gently rinsed for any spillage through three successive transfers to wells (10 ml) with clean seawater. The fish were transferred with a wide-mouth pipette with as little water as possible and then transferred to single incubation wells with 5 ml of clean seawater. Total transfer time was 1.5 F 0.5 min. Each test mixture was tube-fed to six fish. Visual observations verified that all fish tube-fed had their stomach filled with Artemia. 2.4. Post tube-feeding incubation A set-up to determine the fate of 14C-labelled AA was used (Rønnestad et al., 2001a), in order to quantify the absorption, catabolism and retention of the test mixtures by sole post-larvae. A CO2 trap permitted the separation between the catabolised nutrients (14CCO2) and the label in the water that was due to unabsorbed and evacuated 14C-labelled AA. Glass scintillation vials were used as incubation wells. The incubation wells with the single fish were sealed and a gentle airflow (approx. 2 ml/min) was directed to a KOH trap (5 ml; 0.5 M) to collect the produced CO2. Once the set incubation period was over (6 h), the incubation wells were opened and the fish sampled. The well was then resealed and the CO2 remaining in the incubation water was collected accordingly to the procedure described by Rønnestad et al. (2001a). The incubation period was chosen based on previous experiments that determined that the gut-clearance time was 6 h.
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2.5. Metabolic budget of the amino acids For each tube-fed fish, the following samples were counted (DPM) by liquid scintillation: incubation water (assumed to be unabsorbed evacuated labelled AA), KOH trap (CO2 produced by labelled AA oxidation) and whole fish body (representing labelled AA retention). The post-larvae were transferred to individual 6-ml scintillation vials and 30% H2O2 was used as tissue solubilizer. Ultima Gold scintillation cocktail (Packard Instruments) was used for incubation water, CO2 trap and body samples. All samples were counted using a Beckman LS 6000IC liquid scintillation counter (Fullerton, CA). The metabolic budgets were calculated after subtraction of blanks and correction for counting efficiency. Each fraction (incubation water, CO2, whole fish body) was expressed as a percentage of the tracer fed (i.e. the sum of the DPM in all compartments for a given fish).
3. Results All Senegalese sole post-larvae tube-fed were alive and with a normal appearance at the end of the incubation period (6 h). 3.1. Metabolism of the protein hydrolysate When the mixture of the 20-labelled AA was used as a tracer (Fig. 1), the unabsorbed evacuated fraction (% water) was similar between treatments (around 10%). Fish that
Fig. 1. Proportion (%) of the total tube-fed label ([14C]-protein hydrolysate) found in water (grey columns), CO2 trap (black columns) or body (white columns) of post-larval S. senegalensis, that received or not a dipeptide supplement to balance the dietary amino acid profile. Control = control treatment (tube-fed physiological solution), Suppl. = supplemented treatment (tube-fed dipeptide solution). Values are means F S.D. (n = 6). Different letters in the same compartment indicate significant differences ( p < 0.05), after Student’s t-test.
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received the dipeptide supplement oxidised less labelled AA (%CO2) than fish in the control treatment (18.9 F 2.5% and 29.6 F 5.6%, respectively). The retained fraction (% body) was significantly higher in the supplemented (71.0 F 5.4%) than in the control treatment (60.9 F 7.7%). 3.2. Metabolism of single amino acids Fig. 2 shows the results obtained when selected labelled AA were used as tracers. It can be seen that when the dipeptides were tube-fed to the Senegalese sole post-larvae (supplemented treatment), an increase in the evacuation fraction (% water) of the AA used as tracers was observed. Due to the accentuate differences obtained in the evacuation fraction among the treatments and since the fractions are calculated as a percentage of the total, it is difficult to compare the fate of the absorbed AA between treatments. Therefore, the results from the absorbed fraction (excluding the results from the water fraction) were examined by comparing the AA proportion that was oxidised (CO2 fraction) or retained (body fraction) in the body (Fig. 3). For leucine, there were no differences in oxidation and retention between treatments (around 14.5% and 85.5%, respectively). For arginine and glutamate, fish from the supplemented treatment oxidised less labelled AA than fish in the control treatment: 0% and 9.9 F 6.6 for arginine, 26.8 F 7.3% and 53.8 F 12.1 for glutamate, respectively, for the supplemented and the control treatment. The retained fraction for both
Fig. 2. Proportion (%) of the total tube-fed label ([14C]-amino acid) found in water (grey columns), CO2 trap (black columns) or body (white columns) of post-larval S. senegalensis, that received or not a dipeptide supplement to balance the dietary amino acid profile. Control = control treatment (tube-fed physiological solution), Suppl. = supplemented treatment (tube-fed dipeptide solution). Values are means F S.D. (n = 6). Different letters in the same compartment, within the same amino acid used as a tracer, indicate significant differences ( p < 0.05), after Student’s t-test.
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Fig. 3. Proportion (%) of the absorbed tube-fed label ([14C]-amino acid) that was oxidised (black columns) or retained (white columns) in post-larval S. senegalensis, that received or not a dipeptide supplement to balance the dietary amino acid profile. Control = control treatment (tube-fed physiological solution), Suppl. = supplemented treatment (tube-fed dipeptide solution). Values are means F S.D. (n = 6). Different letters in the same compartment, within the same amino acid used as a tracer, indicate significant differences ( p < 0.05), after Student’s t-test.
labelled AA was higher in the supplemented than in the control treatment: 100% and 90.1 F 6.6% for arginine, 73.2 F 7.3 and 46.2 F 12.1% for glutamate, respectively.
4. Discussion 4.1. Methodology The main problem of the tube-feeding technique used in this study is the variability in the volumes tube-fed. As discussed by Rønnestad et al. (2001a), this seems partly to be caused by rigid gut walls that do not permit the luminal volume increase necessary to retain the pulse, by retrograde flux through the oesophageal sphincter when the capillary is withdrawn, and on occasions also by regurgitation. This variability raises the question if the differences between treatments and different tracers were due to different intestinal loading. The expression of the results in percentage helps to identify a pattern in the metabolism that is independent of the total amount tube-fed. But, in the supplemented treatments, tube-feeding different volumes and consequently different dipeptide amounts, could lead to different AA balances. However, the variation was very similar for the several tracers and also between treatments. This similarity in the variation between treatments is due to the small volume tube-fed and due to the fact that fish were fed. These two factors have been demonstrated to decrease the variation in the amounts tube-fed (Rønnestad et al., 2001a). One question often raised when using this technique is if the stress imposed affects the larval metabolism (Rust et al., 1993). The results from this and previous studies (Rønnestad et al., 2000b, 2001b) have shown that Senegalese sole has low sensitivity
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to handling during this type of experimentation, resulting in 100% survival after the tubefeeding experiments. In order to evaluate if the sole nitrogen metabolism was being affected by the tube-feeding technique, ammonia excretion was determined in fed postlarvae that have or have not been tube-fed (unpublished results). Results showed that ammonia excretion was similar in both groups of fish (0.044 F 0.009 and 0.050 F 0.008 Amol NH4+ larva 1 h 1, for tube-fed and not tube-fed fish, respectively), indicating that nitrogen metabolism does not seem to be affected by the tube-feeding set-up. Rønnestad et al. (2001a), by measuring oxygen consumption, also showed that metabolism in metamorphosed Atlantic halibut was not affected by the handling and anaesthetics used in the set-up of tube-feeding experiments. The results from this study refer to the metabolism of fed fish. Previous works have been done in the metabolism of AA using the tube-feeding technique, but in these studies fasted fish were used (Rust et al., 1993; Rønnestad et al., 2000a,b, 2001b; Conceic¸ a˜o et al., 2002). Some doubts were raised if these studies would reflect the ‘‘normal’’ metabolism that occurs after a meal (Rust et al., 1993). When comparing the results from the present study with fed post-larvae with the ones from Rønnestad et al. (2001b) using fasted postlarvae, it can be seen that the metabolism of arginine and glutamate seems to follow the same pattern in fasted and in fed Senegalese sole post-larvae. In both cases, glutamate (a dispensable AA) is used preferentially as an energy substrate and arginine (an indispensable AA) is spared for growth. Taken together, the results obtained in this study supports that the tube-feeding technique is a valid tool for in vivo studies on AA metabolism. 4.2. Amino acid metabolism Senegalese sole post-larvae that received a supplement of dipeptides retained more AA than the fish that did not receive this supplement (Fig. 1). This supports the hypothesis that when dietary AA profile is balanced through supplementation, an increase in AA retention and a decrease in the catabolic losses of AA are observed. The increase in AA retention is probably reflected in an increase in protein retention. Although this experimental work does not permit to discriminate between protein, lipid and carbohydrate in the retained fraction, previous work with herring larvae has demonstrated that only a small proportion (less than 1.5%) of AA are converted into lipids (Conceic¸a˜o et al., 2002). The glycogen content is four to six times lower than the lipid content in larval turbot (Conceic¸ a˜o, 1997), which suggests that even a smaller fraction of AA is used for gluconeogenesis. Benevenga et al. (1993) by reviewing several works done in AA catabolism suggested that a decrease in protein synthesis should result in an increase in AA catabolism. Since there is no storage nitrogen molecule other than proteins, the incorporation of AA into protein plays an important role in whether or not an AA is catabolised. The free AA (FAA) pool is small and is kept within narrow limits (Houlihan et al., 1995; Conceic¸a˜o et al., 1997), since intracellular accumulation of some AA may be toxic to Vertebrates (D’Mello, 1994). Therefore, when fish are fed an imbalanced diet, the absorbed dietary AA that do not match the profile that is needed for protein synthesis will be deaminated and used in energy production, gluconeogenesis or lipogenesis (Ballantyne, 2001). The preferential pathway is towards energy dissipation through oxidation via the tricarboxylic acid cycle.
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AA imbalanced diets have been shown to increase AA oxidation (Kim et al., 1983; Kaczanowski and Beamish, 1996). In the control treatment, this was the observed result: oxidation was higher, probably due to the dietary AA imbalance that impaired protein synthesis. The control diet might be imbalanced in more AA than what was estimated. The comparison between the dietary and larval indispensable AA profiles is a rough estimation of the fish AA requirements (Conceic¸a˜o et al., 2003a,b). The absorption of individual AA by the different transport systems may proceed at different rates (Dabrowski, 1983) and selective catabolism of individual AA may occur (Rønnestad et al., 2001b; Conceic¸ a˜o et al., 2002). This may reduce or amplify dietary AA imbalances. Thus, the precise knowledge of the ideal AA profile implies taking into account the relative bioavailabilities, i.e. the rates of absorption and catabolism, for the individual AA (Conceic¸a˜o et al., 2003b). In a recent study with larval gilthead seabream larvae (Conceic¸a˜o et al., 2003a), it was shown that the relative bioavailabilities of individual AA vary. Threonine has a low relative bioavailability meaning that is retained less efficiently by seabream larvae compared to other AA. The AA relative bioavailabilities may be species dependent and may change during development. Until now, no information is available for sole. Therefore, in this study, the possible dietary AA imbalances were determined based on a rough estimation. Accurate AA supplementation in order to achieve the ideal AA profile that can optimise growth and feeding efficiencies requires the information on the relative bioavailabilities of individual AA. Nevertheless, the results from this study reinforce the idea that balancing the dietary AA profile enhances AA retention. Considering that any AA imbalance could lead to an increase in the AA catabolism, the choice of the nitrogen form used to compensate the dietary AA imbalance is of importance. FAA are absorbed 3.5 times faster than protein in Senegalese sole post-larvae and their use to supplement dietary protein can lead to transient AA imbalances (Rønnestad et al., 2000b). The end-products of protein digestion are not only FAA, but a mixture of FAA and small peptides, mainly di- and tripeptides (Ganapathy et al., 1994). Visual observations on dissected fish revealed that Artemia digestion already started when post-larvae were tube-fed. So it seems that the choice of dipeptides to supplement the diet was a good option. The tube-feeding of dipeptides does not seem to cause an AA imbalance due to different absorption rates in the fish intestine, since this resulted in increased AA retention and decreased AA catabolism. Analysing the results obtained in the second experiment, an unexpected observation was the increase in evacuation of labelled AA when fish were given the dipeptide supplement (Fig. 2). This was not observed when the tracer was the mixture of 20-labelled AA (protein hydrolysate). Peptides are transported across the brush-border membrane of fish intestine apparently by a saturable transport system that appears to accommodate several dipeptides, but not FAA (Maffia et al., 1997; Bakke-McKellep et al., 2000; Verri et al., 2000). FAA seem to be transported across the brush-border membrane of fish intestine by several mechanisms, including several saturable carrier-mediated transporters and apparent diffusion (Storelli et al., 1989; Vilella et al., 1990; Bakke-McKellep et al., 2000). Carrier-mediated transporters for cationic, anionic, imino and neutral AA have been described for the eel intestine (Storelli et al., 1989). However, these transporters often have overlapping specificities and interactions between AA seem to interfere with
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the uptake of a specific AA. For instance, uptake of lysine in the eel intestine is inhibited by several neutral AA and also by arginine (Vilella et al., 1990; Berge et al., 1999), while proline is inhibited by alanine and phenylalanine (Storelli et al., 1989). Having this in mind, the dipeptides and FAA were probably not competing for the same transporters. However, it is possible that some of the dipeptides tube-fed to the post-larvae had been digested in the stomach. In this case, post-larvae in the supplemented treatment would have FAA from several sources in their intestine: from Artemia, since digestion already started when the fish were tube-fed, from dipeptide hydrolysis and also labelled FAA. The excess of FAA from the dipeptide digestion might have competed or at least interfered with the transport of some AA. However, since this increase in the evacuation was not observed for the mixture of the 20-labelled AA (protein hydrolysate), this does not seem to have been the rule for all AA. The balance of absorbed indispensable AA within the body and not simply their overall availability at the level of the digestive tract is important to sustain efficient protein synthesis. Thus, it is interesting to observe the fate of the AA once absorbed by the gut. When the dipeptide supplement was given to the fish, the oxidation of arginine and glutamate was reduced (Fig. 3). It has been demonstrated before that larval and post-larval fish have the ability to regulate their AA metabolism: dispensable AA are used preferentially as an energy substrate and indispensable AA are spared for growth (Rønnestad et al., 2001b; Conceic¸a˜o et al., 2002). In the present study, the same pattern was observed. Glutamate was more oxidised than arginine or leucine (Fig. 3), indicating that the indispensable AA were being spared for growth. When these results with fed fish are compared with the ones of Rønnestad et al. (2001b) using fasted fish, it seems that fed Senegalese sole post-larvae catabolise less and retain more AA than fasted fish. This tendency suggests that AA tend to be spared for growth in fed fish (81.6 F 12.9 and 85.3 F 8.2 for arginine and 32.9 F 15.3 and 44.6 F 11.8 for glutamate, for fasted and fed fish respectively), while in fasted fish there is a tendency for a higher use of AA as an energy source (15.1 F 10.4 and 9.2 F 6.0 for arginine and 64.9 F 15.9 and 51.9 F 11.8 for glutamate, for fasted and fed fish, respectively). Regarding the results obtained for leucine in the present study, it was observed that oxidation of this AA was similar in both treatments. It remains to be explained why for leucine, oxidation did not decrease in the supplemented treatment as with the other tracers used. Since larval fish are able to regulate their AA metabolism, it might be speculated that if leucine was limiting growth in the control treatment, oxidation of this AA was already at the minimum level possible in order to spare it for growth. The other AA that, due to the lack of leucine, could not be used for protein synthesis were being catabolised. Thereby, when leucine was supplemented to the diet, a further decrease in the oxidation of this AA was not possible. The supplement of leucine did not decrease leucine oxidation but instead increased the retention of the other AA, so the overall retention was increased. This effect has been observed in young pigs (Kim et al., 1983). When these animals were fed a diet with a histidine deficiency, phenylalanine oxidation was elevated. When dietary concentration of histidine was increased, phenylalanine catabolism decreased to a minimum. The results obtained in the present study with Senegalese sole post-larvae reinforce the idea that young fish stages are able to regulate their AA metabolism, as have been pointed out by recent works (Rønnestad et al., 2001b; Conceic¸a˜o et al., 2002). A balanced dietary
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AA profile increases the AA retention and may improve growth and nitrogen utilisation. Thereby, definition of the ideal dietary AA profile taking digestion and absorption into account is paramount. Acknowledgements This work was supported by project POCTI/1999/CVT/34608 (FCT, Portugal, with the support of the European fund FEDER). Cla´udia Araga˜o and Luis Conceic¸a˜o were supported by PRAXIS XXI grants BD/18390/98 and BPD/7149/2001 (FCT, Portugal), respectively. Ivar Rønnestad was supported by NFR grant 141990/120 (ELHMFL Paper# 198). The authors would like to thank C.I.C.E.M. ‘‘El Torun˜o’’ (Ca´diz), and also Dr. J.P. Can˜avate and Dr. V. Anguis, for supplying the fish used in this study.
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