Performance of juvenile turbot ( Scophthalmus maximus ) fed varying dietary L-carnitine levels at different stocking densities

Commercial farming of turbot (Scophthalmus maximus) at high stocking densities may lead to growth depression and increasing production costs. Moreover, the high levels of accumulated waste in an intensive system may cause rapid deterioration of water quality, which may undermine the production. Lcarnitine is known as a growth-enhancer which shows promise as mitigator of crowding effects. The effects of stocking densities (4, 8, 11 and 14 kg m) on growth performance, feed utilization and body composition were evaluated during 75 days on turbot (75.6 ± 2.8 g) fed two dietary L-carnitine levels (40 or 240 mg kg). At the end of the feeding trial, total ammonia excretion (TAN) was measured postprandially for 24h. Specific growth rate and weight gain decreased with increasing stocking density. Fish held at 4 kg m had higher final body weight (94-96 g) than fish held at higher densities (80-87 g). Protein efficiency ratio was higher in fish held at 4 kg m (1.33-1.36), in comparison to fish stocked at 8 kg m (0.98) or 14 kg m (0.45). Voluntary feed intake decreased from 0.70 to 0.56% BW with increasing stocking density. Dietary L-carnitine supplementation did not affect growth performance and body composition, except for body L-carnitine content which increased from 75 to 128 mg kg BW with supplementation. Fish fed 240 mg L-carnitine supplements had lower TAN that the ones fed 40 mg L-carnitine (p < 0.05).


Introduction
The turbot, (Scophthalmus maximus) is recognized by European consumers as one of the most valued species.The commercial farming of turbot has grown steadily in the Atlantic region (FAO, 2009;Garza-Gil et al., 2009) fostered, in recent years, by improvements in selective breeding, improved larval rearing methods and nutrition and husbandry practices in the grow-out phase (Brown, 2002;Person-Le Ruyet, 2002).Although environmental factors influencing fish growth have been significantly studied, little attention has been devoted to rearing conditions (e.g.stocking density, culture system, husbandry) leading to the establishment of social interactions in confined stocks.
Social interactions, such as development of hierarchical groups through competition for food and/or space, can negatively affect biomass accumulation (Irwin et al., 1999;Aksungur et al., 2007).Both safe and detrimental interactions between stocking density and Sci.Agric.(Piracicaba, Braz.), v.67, n.2, p.151-157, March/April 2010 growth have been reported, and the pattern of this interaction appears to be species specific (Irwin et al., 1999).In many cultured fish species, growth is inversely related to stocking density and growth reduction or "stunting" of fish in high densities is a ordinarily cited disadvantage in intensive culture of marine fish, although this is not universally true (Webb Jr. et al., 2007).Moreover, the intensive production of turbot generates size disparities during the grow-out phase that could be partly attributed to poor husbandry practices and stocking density conditions.In many cases, this may lead to the development of slow growing individuals, increasing production costs.Imsland et al. (1998) showed that social hierarchies may lead to higher size variation and to lower growth responses.
In recent years, L-carnitine, a multi-physiological, bioactive and pollution-free additive has attracted the interest of many investigators after its potential positive effect on growth and lipid metabolism in fish (Harpaz, 2005).Carnitine plays important physiological roles, shuttling the long-chain fatty acids across the inner mitochondrial membrane for oxidation and ATP production in peripheral tissues (Gulçin, 2006).As a matter of fact, there is solid evidence that carnitine has a growthpromoting effect in a number of fish species (Santulli and D'Amelio, 1986a;Torreele et al., 1993;Chatzifotis et al., 1995;Keshavanath and Renuka, 1998;Jayaprakas et al., 1996;Becker et al., 1999;Twibell and Brown, 2000).It has also been shown that carnitine can reduce lipid content of muscle in sea bass (Santulli and D'Amelio, 1986b) and seabream (Sparus macrocephalus) (Ma et al., 2008).In addition, Mohseni et al. (2008) showed that growth performance of sturgeon (Beluga beluga) was improved when fish was fed 300 mg L-carnitine kg -1 diet.Nevertheless, little or no effect of L-carnitine supplementation on growth was observed in other species (Burtle and Liu, 1994;Ji et al., 1996;Chatzifotis et al., 1997;Harpaz et al., 1999;Gaylord and Gatlin, 2000;Dzikowski et al., 2001;Ozório et al., 2001;Schlechtriem et al., 2004).The contradictory results may be an effect of different experimental conditions, such as the duration of experiments, level of L-carnitine supplementation, fish size, leaching of carnitine, etc. (Harpaz, 2005).
There is little evidence that L-carnitine can play a protective role against stress response to confinement (Harpaz et al., 1999;Harpaz, 2005) and ammonia toxicity (Santulli and D'Amelio, 1986b;Tremblay and Bradley, 1992).This study hypothesizes that the addition of carnitine to aquafeeds may promote wellbeing of turbot, decreasing stress-related pitfalls induced by intensive aquaculture practices, and thus investigates interaction effects of dietary L-carnitine and stocking density on growth performance, body composition and nitrogen excretion of juvenile turbot.

Experimental animals and facilities
The experiment was conducted in Porto, Portugal using full siblings turbot juveniles with identical nutritional history.Fish were acclimated to the rearing system conditions for one week before start of the trials.Fish (75.6 ± 2.8 g) were distributed into 12 tanks divided into two identical compartments (0.25 m W × 0.40 m L × 0.20 m H), at four densities: 4, 8, 11 and 14 kg m -2 or 40, 80, 120 and 140 kg m -3 , equivalent to 10, 20, 30 and 35 fish per compartment, in a 2 × 4 × 3 factorial scheme, totally randomized experimental design.All tanks were connected to a recirculation system (3.5 m 3 ) with identical husbandry conditions.The water conditioning structure (TMC 5000 seawater recirculation system) was equipped with a mechanic filter (100 µ), two biological filters, UV sterilization unit and refrigeration system.Dissolved oxygen (6.17 ± 0.97 mg L -1 ), water temperature (19.7 ± 4.5ºC) and salinity (29.4 ± 3.6 mg L -1 ) were checked daily; NH 4 (1.0 ± 0.7 mg L -1 ) and NO 2 (1.2 ± 0.9 mg L -1 ) were checked once a week.Fish were kept under a natural photoperiod cycle, spring season.

Diets, feeding and experimental conditions
Fish were fed twice a day (9h30 and 15h30) for 75 days with an iso-nitrogenous (39% CP) and isoenergetic (19.5 Kj g -1 ) fishmeal-based diet (Table 1), containing 40 or 240 mg kg -1 L-carnitine.Feed consumption was monitored carefully to avoid wastes.Carnitine supplement and carnitine analysis were provided by LONZA Switzerland).At the end of the feeding trial, a complementary study was conducted in which Total Ammonia Nitrogen (TAN = NH 3 -N + NH 4 + ) postprandial excretion was measured along a 24h cycle, in a way that enabled fish to have enough time to develop a different L-carnitine profile between both dietary L-carnitine levels.Fish were fed at fixed level (0.56 ± 0.17% body weight -BW) and a tank without fish was used as control.TAN was determined by Palintest® Photometer 7000 (Palintest Ltd, Tyne and Wear, England) and expressed as mg TAN kg -1 body weight.

Sampling
Fish were individually weighed and measured at days 20, 40, 60 and 75.Pooled samples of carcass were taken from five fish from the initial stock and 15 fish per treatment (five fish per tank) at the end of the experiment, and stored at -20ºC for subsequent proximate analyses.

Statistical analyses
Each tank represented one experimental unit, three tanks per treatment.Statistics of all the data was tested in fasting fish (excepted for ammonia which, tested at pre-and posprandially level) by two-way ANOVA, followed by Tukey's multiple range test.

Results
Mortality rates were below 5%, occurred with no systematic trend and did not differ among experimental groups.Growth performance and protein efficiency ratio (PER) were affected by stocking density (Table 2).Fish held at the lower stocking density (4 kg m -2 ) had higher final body weight (94-96g) than the other groups (80-87g).Protein efficiency ratio was higher in fish held at 4 kg m -2 (1.33-1.36), in comparison to fish stocked at higher densities, in which PER varied between 0.45 (14 kg m -2 ) and 0.98 (8 kg m -2 ).Fish held at 4 kg m -2 had the highest voluntary feed intake (VFI, p < 0.05) and lower feed conversion ratio (p < 0.05).Dietary carnitine supplements did not affect growth performance, although an interaction between L-carnitine and stocking density was observed for VFI.Specific growth rates and weight gain decreased with increasing stocking density (Figure 1).
The whole body dry matter and lipid contents were affected by stocking density (Table 3).Dietary L-carnitine supplementation did not affect body composition, except for body L-carnitine content which increased from 75 to 128 mg kg -1 BW with supplementation.Hepatossomatic index was not affected by diets and stocking density (p > 0.05).The coefficient of variation (CV) for final body weight increased during the experimental period in all groups with no systematic trend (Figure 2, p > 0.05).The postprandial excretion of TAN (Figure 3) peaked at approximately 4 hours after the meal (35 mg kg -1 BW), and slowly decreased below the prefeeding level (2.4 mg kg -1 BW).Fish fed 240 mg L-carnitine supplements had lower TAN excretion in comparison to fish fed 40 mg L-carnitine (p < 0.05).TBARS were higher in muscle of fish fed 240 mg L-carnitine in comparison to fish fed 40 mg L-carnitine, but only when held at 4 kg m -2 (Figure 4).

Discussion
Turbot can be reared at very high stocking densities.Howell (1998) evaluated effects of stocking density on turbot and demonstrated that growth was unaffected un- til the combined area of the fish exceeded 200% of tank bottom coverage.In this study the number of fish per tank was kept constant throughout the trial so stocking density increased with time.Because there are evidences that removing fish may disrupt hierarchies, resulting in high levels of interactions between individuals and affecting growth rates (Baardvik and Jobling, 1990), a decision was made not to remove fish from the tanks.
Based on reports by Torreele et al. (1993), Keshavanath and Renuka (1998), Becker et al. (1999) and Twibell and Brown (2000) it was expected that juvenile turbot fed a 240 mg carnitine kg -1 and subjected to increased rearing density, would show faster growth and/ or have better feed conversion than fish fed 40 mg carnitine kg -1 .This was not observed in the current study, even though the "apparent" stress conditions were brought in by the extreme high densities.This is especially relevant because densities 11 and 14 kg m -2 , corre-  Data are shown as the mean±standard deviation (n = 6 fish per treatment).C, carnitin effect; D, density effect; C × D, interaction effect.NS, not statistically different.
Table 3 -Mean values of proximate composition of the turbots stocked at four densities and fed with different diets (g kg -1 , unless otherwise stated).(Howell, 1998) or recent investigation (Labatut and Olivares, 2004;Ma et al., 2006;Aksungur et al., 2007).Thus, although the density had been deliberately increased to expose fish to extreme stress, as suggested by Harpaz (2005), L-carnitine did not improve fish growth or feed conversion efficiency.Some authors also reported that carnitine did not elicit any measurable effect on growth for a range of warmwater (Burtle and Liu, 1994;Harpaz et al., 1999;Dias et al., 2001;Dzikowski et al., 2001;Ozorio, 2001;Schlechtriem et al., 2004) and coldwater fish species (Rodehutscord, 1995;Ji et al., 1996;Chatzifotis et al., 1997;Gaylord and Gatlin, 2000).Harpaz (2005) concluded that these contradictory results are associated with different factors such as age, fish size, experimental period, feed composition and levels of supplement.The fact that the efficiency of carnitine reabsortion decreases with increasing levels of circulating carnitine and, as a result, more carnitine is excreted, as demonstrated by Berger and Sachan (1991) in rats, may be an additional explanation for the registered results.Moreover, the carnitine effect is more visible in fish prone to high fat accumulation (Ozório, 2001), which is not the case of most flatfish, turbot included.

Sci
SGR decreased with increasing stocking density (Figure 1) and the recorded values represent half of the values previously found for fish of the same size (Strand and Øiestad, 1997;Aksungur et al., 2007).The negative impact of stocking density on growth and SGR may have resulted from "overcrowding", which increased social interactions between individuals.These interactions lead to heterogeneity of individual feed intake and to size variation within experimental groups.In those cases, growth suppression is inevitable (Jobling, 1985;Irwin et al., 1999;Lambert and Dutil, 2001).Furthermore, an increased CV for body weight within population is considered an indicative of establishment of hierarchies (Irwin et al., 1999;Lambert and Dutil, 2001).Some species have very specific behaviour, so hierarchies of dominance and submission result in stressful condition for both dominant and subordinate fish, thus compromising of any given group.In this work, CV increased with time, regardless of stocking densities and dietary L-carnitine levels.
The negligible increase in the CV for body weight (Figure 2) during the trial indicates low level of social interactions in all experimental groups; hierarchy dominance associated with changes in density was not observed.Stocking density and growth rates are often reported to be related, however, the relationships between both parameters may not be uniformly, positively or negatively, linear for a given species.For instance, Baker and Ayles (1990) suggested that growth of Arctic charr increased with stocking density up to a threshold of 40-50 kg m -3 and then declined at higher densities.Bjørnsson (1994) reported that stocking density affects the growth of halibut only above a threshold level corresponding to approximately 100% coverage of the tank bottom.
Ammonia is the major excretory nitrogenous compound of teleost fish.Since there is very little energy  cost involved in the conversion of protein nitrogen to ammonia, production and excretion will always be energetically advantageous in aquatic environments.However, production of ammonia also implies production of the highly toxic non-ionized ammonia (NH 3 ).Ammonia toxicity is one of the ordinary stressors in fish culture.Carnitine has been shown to provide increased lipid oxidation for energy production (Ozório et al., 2005) resulting in a protein-sparing effect (Ozório, 2009), consequently reducing nitrogen excretion.
In this study, there was an immediate rise in TAN excretion after feeding.The rates of postprandial TAN excretion recorded for juvenile turbot rest within the range reported for other species (Robaina et al., 1999;Ismiño-Orbe et al., 2003;Gómez-Requeni et al., 2003;Webb Jr. and Gatlin III, 2003).TAN excretion peaked at approximately 4 h after a single meal and slowly decreased to values lower than the prefeeding level.Postfeeding pulses of nitrogen excretion have also been reported for many other marine species, such as European sea bass (Dicentrarchus labrax), grouper (Epinephelus areolatus) and snaper (Lutjanus argentimaculatus) (Robaina et al., 1999;Leung et al., 1999).In this experiment, decreased TAN excretion was registered for fish fed 240 mg carnitine suggesting a more efficient utilization of ingested protein following the addition of extra carnitine to the diet.
To estimate how carnitine supplementation may affect lipid peroxidation, the muscle concentration of TBARS was determined.The sensitivity of measuring TBARS has made this assay the method of choice for monitoring lipid peroxidation in fish (Oakes and Van Der Kraak, 2003), a major indicator of oxidative stress.According to previous studies (Oakes and Van Der Kraak, 2003;Zhang et al., 2008), carnitine acts as an antioxidant, that is, TBARS would be reduced with increasing carnitine contents in the diet.In this study, no effect of carnitine on TBARS value was recorded.In fact, there was a tendency of increasing instead of decreasing TBARS in fish fed 240 mg carnitine, in comparison to fish fed 40 mg carnitine.On the other hand, stocking density noticeably affected TBARS values.Animals stocked at 4 and 14 kg m -2 showed the highest levels, eliciting to infer that the oxidative stress is influenced by stocking density.However this effect has to be considered very carefully and should not be generalised to any peroxidation process.The factors responsible for lipid peroxidation and the protection mechanism may vary between species and for a given tissue according to the nature of the factors inducing peroxidation as suggested by Iliou et al. (1992).
In brief, size disparities in the grow-out stage of turbot reared in recirculation systems can be minimized by stocking fish at lower densities than previously reported in literature.A sizable decrease in TAN excretion was registered for fish fed diets supplemented with L-carnitine, which may suggest a more efficient utilization of ingested nitrogen.If this effect could be confirmed, nitrogen loads from fish farms to the aquatic systems could be reduced by extra addition of L-carnitine to fish diets.

Figure 1 -Figure 2 -Figure 3 -
Figure 1 -Mean values (n = 3 tanks treatment -1 ) of the changes in specific growth rate (SGR) and weight gain is juvenile turbot fed to dietary L-carnitine levels and subjected to stocking densities (mean ± standard deviation).Different letters are different, p < 0.0001.40 mg kg -1 carnitine 240 mg kg -1 carnitine

Figure 4 -
Figure 4 -Effect of stocking density and dietary L-carnitine on the muscle content of thiobarbituric acid reactive substances (TBARS, mean ± standard deviation, n = 6 replications per treatment).C, carnitine effect; D, density effect; C × D, interaction effect.

Table 2 -
Mean values (n = three replications per treatment) of performance parameters of turbot fed the experimental diets and held at four stocking densities, after 75 days of feeding trial.