Weaning of Senegalese sole (Solea senegalensis) postlarvae to an inert diet with a co-feeding regime

Destete de postlarvas del lenguado senegalés (Solea senegalensis) a una dieta inerte utilizando un régimen de coalimentación

Laura Ribeiro*, Sofia Engrola and Maria Teresa Dinis

^* Centro de Ciências do Mar, Universidade do Algarve Campus de Gambelas, 8000-117 Faro, Portugal. * E-mail: mribeiro@ualg.pt

Recibido en octubre de 2003;
aceptado en octubre de 2004.

Abstract

The objective of this study was to evaluate the weaning success of Senegalese sole, Solea senegalensis, postlarvae when co-fed a compound diet, by means of the analysis of growth parameters (relative growth rate [RGR] and condition factor [K]), survival, protein and lipid content. Total ammonia in the rearing water was also measured. The experiment lasted for 46 days (36 to 82 days posthatching). One group was fed only with enriched Artemia sp. metanauplii (Artemia treatment), whereas in another group, the Artemia sp. metanauplii were gradually replaced by the International Council for the Exploration of the Sea (ICES) diet over a period of 39 days (ICES treatment). Postlarvae were sampled on rearing days 0, 9, 23, 30, 39 and 46 for growth and biochemical analysis. Water was sampled on rearing day 30 during a 24-h cycle in both treatments. Postlarvae from the ICES treatment were weaned, though by the end of the experiment the Artemia treatment exhibited significantly higher values for growth, RGR, K and survival rates (P < 0.05). No significant differences were observed concerning total lipid content. Protein content was significantly higher for the Artemia treatment after 46 days of rearing (P < 0.05). The total ammonia nitrogen concentration in the water increased after each meal and remained below a harmless level for postlarvae.

Key words: flatfish, growth, inert diet, lipid, protein, weaning.

Resumen

El objetivo de este estudio consistió en evaluar el éxito del destete de postlarvas del lenguado senegalés, Solea senegalensis, utilizando un régimen de coalimentación. El estudio se realizó mediante el análisis de parámetros de crecimiento (tasa de crecimiento relativa [RGR] y factor de condición [K]), supervivencia y contenido de proteínas y lípidos. La concentración del amonio total en el agua de cultivo también fue analizada. El experimento se realizó durante 46 días (de 36 a 82 días posteclosión). Un grupo de postlarvas se alimentó únicamente con metanauplios de Artemia sp. enriquecidos (tratamiento con Artemia), mientras que en otro grupo los metanauplius de Artemia sp. fueron gradualmente substituidos por la dieta del International Council for the Exploration of the Sea (tratamiento ICES) durante un periodo de 39 días. Las postlarvas se muestrearon durante los días 0, 9, 23, 30, 39 y 46 de cultivo para el estudio de crecimiento y el análisis bioquímico Las muestras de agua se tomaron el día 30 de cultivo durante un ciclo de 24 h en ambos tratamientos. Las postlarvas del tratamiento ICES fueron destetadas, aunque al final del experimento se registraron valores significativamente mayores en relación al crecimiento, RGR, K y tasas de supervivencia (P < 0.05) en el tratamiento con Artemia. No se observaron diferencias significativas en el contenido total de lípidos. El contenido de proteínas fue significativamente mayor en el tratamiento con Artemia a los 46 días de cultivo (P < 0.05). La concentracion de amonio total en el agua se incrementó después de cada toma de alimento pero se mantuvo por debajo del nivel tóxico para las postlarvas de peces marinos.

Introduction

The need for diversification of cultured species increased the interest of fish farmers for Senegalese sole, Solea senegalensis, a highly economical species commonly exploited in southern Europe (Dinis et al., 1999). The high growth and survival rates observed during larval stages (Dinis et al., 1999) and the recent advances in weaning (Day et al., 1997; Cañavate and Fernández-Díaz, 1999; Engrola et al., 2001) provided encouraging perspectives for sole aquaculture.

Live food provides higher growth and survival rates in the early life stages when compared with compound diets (Person-Le-Ruyet et al., 1993; Blair et al., 2003). Nevertheless, some disadvantages have been pointed out concerning the use of live feeds, since they may act as vectors of diseases, their nutritional value can be variable, their nutritional quality is difficult to manipulate and they are time-consuming to produce and, consequently, expensive (Hart and Purser, 1996). These reasons led to the research of suitable diets in order to replace live food in the early stages of marine fish larvae. Until now only sea bass larvae have been reared successfully on an artificial diet from the time of mouth opening (Cahu and Zambonino-Infante, 2001).

Co-feeding of fish has been shown to enhance larval performance (Kanazawa et al., 1989; Cañavate and Fernández-Díaz, 1999; Baskerville-Bridges and Kling, 2000), and to allow weaning in a shorter time (Person-Le-Ruyet et al., 1993).

The aim of this study was to evaluate the weaning success of S. senegalensis postlarvae when co-fed with a compound diet, through the analysis of growth, survival, and protein and total lipid contents. To assess the effect of the diet on water quality, total ammonia nitrogen (TAN; sum of ionized, NH₄⁺, and unionized, NH3, form of ammonia) was also determined.

Materials and methods

Postlarval rearing

The standard International Council for the Exploration of the Sea diet (ICES Laboratory of Aquaculture and Artemia Reference Center, Belgium) was specially formulated for marine fish larvae in order to allow comparisons of data on fish nutrition from different laboratories (Coutteau et al., 1995). Diet formulation is reported in Coutteau et al. (1995) and the batch used was ICES895. From preliminary experiments with Senegalese sole (Ribeiro et al., 1999a), higher survival and growth rates were obtained when feeding postlarvae the ICES diet with 3% less lipids on the coating than the standard diet.

The moment to start the weaning was based on the fact that Senegalese sole exhibits a histologically differentiated stomach around 30 dph (Ribeiro et al., 1999b).

The experiment lasted 46 rearing (R) days: R0 (36 dph) to R46 (82 dph). In three randomly selected tanks, fish received the ICES diet with 3% less lipids on the coating (ICES treatment), while in the remaining three tanks, fish were fed with enriched Artemia sp. metanauplii (Artemia treatment). Postlarvae were fed twice a day (11:00 and 17:00 h) and the quantity of food was determined based on the predicted maximum growth attainable, which takes into consideration the postlarval growth rate and the feeding conversion efficiency. The weekly wet weight determinations of postlarvae allowed adjustments to the amount of food offered the post-larvae. During the co-feeding period, the ICES diet was gradually introduced, resulting in the total substitution of Artemia metanauplii at day R39 (percentage of ICES diet: 10% at day R0, 50% at day R19 and 100% at day R39).

Tanks were cleaned every day and dead postlarvae were removed and counted for survival rate determination.

Postlarvae were sampled before feeding at days R0, R9, R30, R39 and R46 for length and weight measurements (15 individuals per tank) and for biochemical analysis (a pool of 30 postlarvae per tank).

Total length was determined by measuring 15 postlarvae. After careful rinsing with distilled water and dry blotting with a paper towel, these postlarvae were weighed. Postlarvae were then individually frozen in liquid nitrogen and subsequently freeze-dried; dry weight was determined in a microbalance at the laboratory.

The condition factor (K) was calculated according to Jobling (1994):

The relative growth rate (RGR) was calculated according to Ricker (1958):

For the biochemical analysis (protein and lipid content), samples were carefully rinsed with distilled water and immediately frozen in liquid nitrogen. Protein content was determined according to the method of Lowry et al. (1951), with an adaptation of Rutter (1967). Total lipids were determined gravimetrically after extraction with chloroform/ methanol based on the method of Bligh and Dyer (1959). A micro- balance was used to gravimetrically determine total lipids.

The TAN concentration in the tank water was monitored over a period of 24 h on day R30, when the compound diet comprised 60% of the daily ration in the ICES treatment. Water was sampled the morning before and after the purge (point AP in figs. 1 and 2) in the tanks. Samples were also collected during the morning meal (point 0 in figs. 1 and 2) and one, two, two and a half, three, three and a half and four hours after this meal, and during the afternoon meal (point 0Af in figs. 1 and 2) and one hour after this meal. The next morning, water was again sampled before (BP24 in figs. 1 and 2) and after purging the tanks. Sampling was carried out in two tanks: one tank from the Artemia treatment and the other from the ICES treatment. At every sampling point, water was also sampled from the inlet to control the background ammonia levels in the water.

Samples were analyzed in triplicate for TAN concentrations in the water using the indophenol method (Koroleff, 1983).

The length and weight data were log-transformed, while the data expressed as a percentage (protein, lipid, RGR, K, survival) were arcsin-transformed (Sokal and Rohlf, 1995). Variance homogeneity was verified by Levene's test. The effect of the treatment was analyzed through the comparison of means by one-way ANOVA. Differences were considered significant for an α = 0.05 level. The statistical analysis was done using the SPSS software for Windows version 10.0 (SPSS Inc.).

Growth parameters

Fish fed with the Artemia diet exhibited higher survival rates than the fish fed with the ICES diet (P < 0.05) (table 1).

Fish growth adjusted to an exponential curve for both treatments, although the Artemia treatment exhibited a better fit to this curve (r = 0.97) than the ICES treatment (r = 0.81) (fig. 3). From day R23 onwards, postlarvae from the Artemia treatment exhibited a significantly higher dry weight value than those from the ICES treatment (table 1). This difference was concomitant with the increase in the quantity of ICES diet in the daily ration to more than 50%, resulting in a 30% decrease in weight at day R39. From this moment onwards, the fish were able to recover their weight. Size dispersion was more evident in the Artemia treatment, and increased during the experimental period. A similar pattern was observed for the ICES treatment, although a decrease was observed on day R39.

The RGR presented a strong variation during the experimental period, decreasing after day R23 and increasing again at the end of the experiment. The decrease was more evident in the ICES treatment, especially on days R30 and R39 when negative RGR values were obtained (table 1). Nevertheless, ICES-fed postlarvae were able to recover, exhibiting a higher value for RGR on day R46 than the Artemia-fed postlarvae. The overall RGR was significantly higher for the Artemia treatment than the ICES treatment (P < 0.05), being 3.7 ± 1.02 and 1.7 ± 0.19, respectively.

Throughout the experimental period, K exhibited a similar pattern of variation in both treatments (table 1). This value was persistent during the first three samplings. After this period, a decrease in K was observed, being more evident in the ICES treatment; however, the difference between treatments was significant only on day R46 (P < 0.05).

Biochemical content

Protein content exhibited some variability at first sampling in both treatments (table 2). After that point, a small decrease was observed for the Artemia treatment until day R23, followed by an increase until the end of the experiment. For the ICES-fed group, the protein content was more stable during the first 23 days of rearing, but after day R30 it decreased considerably (around 14%), resulting in significant differences for both treatments on day R46 (P < 0.05).

Ammonia experiment

The background level of TAN in the water was almost negligible, and was subtracted to the TAN values obtained from the experimental tanks analyzed.

At food addition, TAN concentrations were 0.37 ± 0.01 and 0.36 ± 0.02 mg L^-1 for the Artemia and ICES treatments, respectively (fig. 1). Thereafter, the variation in TAN level was different for each treatment. In the ICES treatment, TAN concentration increased one hour after the morning and afternoon meals to 0.56 ± 0.07 and 0.4 ± 0.01 mg TAN L^-1, respectively, decreasing gradually afterwards. The Artemia treatment presented an increase in TAN concentration one hour after the morning meal to 0.85 ± 0.004 mg TAN L^-1; afterwards, this value kept decreasing and increasing, reaching the highest value of 1.28 ± 0.001 mg TAN L^-1 when the afternoon meal was given and decreasing to 0.90 ± 0.004 mg TAN L^-1 one hour after the afternoon meal. Seventeen hours after the last meal, the TAN concentration had decreased to 0.11 ± 0.016 and 0.06 ± 0.021 mg TAN L^-1 in the Artemia and ICES treatments, respectively.

Since fish biomass was different for each treatment, the TAN values were also expressed relative to fish biomass (mg TAN L^-1 kg^-1 fish). The pattern observed in TAN variation taking into account fish biomass in the tanks was similar to that previously described, although the differences were not so evident between treatments until three hours after the first meal (fig. 2).

Discussion

Senegalese sole postlarvae from the ICES treatment adapted to the diet at the end of the experiment, but postlarvae from the Artemia treatment exhibited significantly higher values for growth, survival, RGR, K, and protein and total lipid contents.

The survival rate of Senegalese sole postlarvae fed Artemia sp. was higher than values previously reported for this species (Dinis, 1992; Marin-Magan et al., 1995; Cañavate and Fernández-Díaz, 1999), while growth rate was identical to that reported by Marin-Magan et al. (1995), but lower than the values obtained by Cañavate and Fernández-Díaz (1999).

In the treatment where fish were fed a compound diet, the survival rate obtained was higher than the values reported by Dinis (1992), Marin-Magan et al. (1995) and Cañavate and Fernández-Díaz (1999), except compared with Engrola et al. (2001). On the other hand, the growth rate results were similar to those obtained by Dinis (1992), but much lower than the values reported by other authors (Marin-Magan et al., 1995; Cañavate and Fernández-Díaz, 1999; Engrola et al., 2001).

The age of sole larvae at the beginning of weaning was similar among studies, but the dry weight at that moment was substantially different in all the studies that reported weight (Marin-Magan et al., 1995; Cañavate and Fernández-Díaz, 1999; Engrola et al., 2001). In this case, we observed a positive correlation between the initial dry weight and the RGR obtained (y = 0.12x + 3.52, r = 0.68; P < 0.05; n = 12). Nevertheless, the relation between initial dry weight and survival rate was not significant (r = 0.31; P = 0.325; n = 12).

From previous experience with this species it seems that the acquisition of new structures during larval development and the beginning of metamorphosis is more related to fish larval size than to fish age, within the same spawn. Fernández-Díaz et al. (2001) observed that Senegalese sole larvae with lower growth rate started metamorphosis a few days later than those exhibiting higher growth rates. Some authors (Verreth, 1994; Roselund et al., 1997) previously reported the high accuracy of larval weight rather than age as an indicator of the physiological and development status of larvae, suggesting that for the same age, bigger larvae will be more developed than smaller larvae. The fact that Senegalese sole postlarvae exhibited a lower weight, independently of age, when compared with other studies on this species, suggests that these postlarvae could be less developed and consequently less prepared to adapt to a compound diet.

Fish density in the tanks may have contributed to the low growth observed during the experiment. Due to the high number of larvae needed for the analytical purposes of this study, the initial density was 2000 larvae m^-2, which was reduced to 1200 larvae m^-2 within three weeks. This initial value is 33% lower than that reported by Cañavate and Fernández-Díaz (1999) that used an initial value of 3000 larvae m^-2, and it is 45% lower on a weight basis (13.2 g dry weight m^-2) than the value used by those authors (Cañavate and Fernández-Díaz, 1999; 24 g dry weight m^-2). Both values represent lower density values when compared with the values used for Dover sole, Solea solea (Day et al., 1997; 58 g of fish dry weight m^-2, with 50-60 days).

The lower values obtained for growth parameters (growth, RGR, K) for the ICES treatment postlarvae were observed after day R23, coinciding with the increase above 50% of the ICES diet in the daily ration. This fact suggests that postlarvae had difficulties in adapting to the compound diet, which led to the increase in mortality rate from this moment onwards; however, ICES-fed larvae were able to start feeding on the compound diet as verified by the increase in RGR by the end of the experiment. The slow introduction of the diet at this stage of postlarval development, resulting in a long period of co-feeding, did not appear to be a suitable weaning strategy, since postlarvae get used to ingest live feed in their daily ration. Roselund et al. (1997) observed that fish larvae became preconditioned to live feed as a consequence of co-feeding. Though the ICES treatment exhibited lower RGR values, post-larvae from the Artemia treatment also exhibited a delay in growth, suggesting that there were other factors influencing postlarval condition apart from the nutritional ones, or that both diets were suboptimal.

No significant effect of the ICES diet was observed on the postlarval body composition. The long period of co-feeding when ICES postlarvae were mainly eating Artemia sp. can justify this observation. In fact, the decrease in RGR after the concomitant increase of the ICES diet in the daily ration also reinforces this observation. Even if the amount of live food was insufficient to fulfill postlarval energetic requirements, leading to a decrease in growth, a proportional mobilization of body components would have maintained the protein and lipid contents at the same value. Total lipid content of ICES postlar-vae at day R23 decreased although protein content and dry weight increased, suggesting that total lipids were mobilized for energetic purposes. At the following sampling points, the decrease in dry weight was more evident and protein content also decreased, reflecting a situation of food scarcity. Biological and environmental factors influence the pattern of nutrient depletion in body composition (Hung et al., 1997).

Ammonia may become a limiting factor for growth and even survival of fish (Person-Le-Ruyet et al., 1997). The TAN concentration in water was kept lower than 1 mg L^-1 for both treatments, although postlarvae from the Artemia treatment were exposed for two hours above this value (1.28 mg TAN L^-1). Parra and Yúfera (1999) observed that total mortality was attained when Senegalese sole larvae were exposed to 80 mg L^-1 of ammonia (TAN). Based on water temperature, oxygen and pH, these authors calculated a 24-h LC50(NH3) of 1.32 mg L^-1 for this species (1.6% of TAN).

According to Person-Le Ruyet (1997), unionized ammonia (NH₃) corresponds to 2-4% of TAN in saltwater. Using these reference values, the concentration of NH3 obtained for the higher value observed in the present study corresponds to 0.03-0.05 mg NH₃ L^-1, well below the value of 0.13 mg L^-1 recommended by Parra and Yúfera (1999) to prevent deaths.

Food degradation and organic detritus may contribute to increase the level of ammonia in water; however, TAN present in the rearing water mainly originates from excretion (Jobling, 1994; Person-Le-Ruyet et al., 1997). Some authors report that the quantity of TAN can be related to the amount of food eaten (Jobling, 1994; Lawson, 1995). The TAN values and the sharp decrease in growth exhibited by the ICES treatment postlarvae when 60% of the ICES diet was added to the daily ration, reinforces the idea that postlarvae were not eating the ICES diet properly.

The strong variation in TAN concentration observed in the Artemia treatment may be justified by the feeding behavior of Soleidae species of eating small amounts for a long period of time (De Groot, 1971), and also by the high postlarval size dispersion observed, which can lead to several moments of ammonia excretion. According to Parra and Yúfera (1999), fluctuating levels of TAN may have a more toxic effect than a continuous exposure.

Postlarvae from the ICES treatment were weaned by the end of the experimental period; however, the Artemia treatment postlarvae exhibited a better performance. The adoption of a long period of co-feeding seemed to have delayed postlarval adaptation to an inert diet, leading to a decrease in their condition and making weaning more difficult. A long period of co-feeding seems to be advantageous for larval stages (Person-Le-Ruyet et al., 1993; Cañavate and Fernández-Díaz, 1999; Baskerville-Bridges and Kling, 2000), but appears not to be the most suitable for Senegalese sole postlarvae.

Acknowledgements

Laura Ribeiro acknowledges receiving a Ph.D. grant (Praxis XXI: BD/5057/95). This study was supported by the project Praxis XXI 3/3.2/Aq/2023/95.

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