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1), 157161

Phosphorus requirements of gilthead sea bream (Sparus aurata L.) juveniles

A M Pimentel-Rodrigues & A Oliva-Teles
ncias do Porto, 4099002 Porto, Portugal Departamento de Zoologia e Antropologia and CIMAR, Faculdade de Cie

ncias do Porto, 4099002 Porto, Portugal. Correspondence: Aires Oliva Teles, Departamento de Zoologia e Antropologia, Faculdade de Cie E-mail: aoteles@fc.up.pt

Abstract A trial was conducted to evaluate the phosphorus requirements of gilthead sea bream juveniles. Seven semipuried diets were formulated to contain 54% crude protein and 15% lipids and phosphorus (dicalcium phosphate) levels ranging from 0.37% to 1.5% (DM basis). Each diet was fed to duplicate groups of 25 sh with an initial average weight of 5 g over 42 days. The sh were fed by hand to visual apparent satiety twice daily, six days a week. At the end of the trial growth rate was not signicantly different in groups fed diets containing 0.75% phosphorus or higher, while growth rate of sh fed the 0.37% phosphorus diet was signicantly lower than all the other diets. Feed efciency ratio, protein efciency ratio and N retention were signicantly lower in sh fed the 0.37% phosphorus diet than in those fed diets with 0.75% and higher phosphorus levels. Phosphorus content of the sh averaged 2% (on a dry weight basis) and was not signicantly affected by dietary phosphorus content. Phosphorus retention averaged 6.6 g kg1 weight gain and was not statistically different among groups. Phosphorus retention (% phosphorus intake) decreased with the increase of dietary phosphorus content, being even higher than 100% in groups fed diets with 0.37% and 0.57% phosphorus, indicating phosphorus absorption from the water. The results of this study indicate that phosphorus requirements of gilthead sea bream juveniles were satised with a diet including 0.75% phosphorus. Keywords: nutrition, phosphorus requirement, gilthead sea bream.
2001 Blackwell Science Ltd

Introduction Phosphorus is one of the major minerals essential to sh. Besides being a major constituent of the skeleton, nucleic acids and cell membranes, it is directly involved in all energy-producing cellular reactions (NRC 1993). Although sh are able to absorb phosphorus from water, food is the major source of this mineral since its concentration is low both in fresh water and in seawater (Lall 1991). Phosphorus requirements have been determined for only a small number of sh species (NRC 1993; Chavez-Sanchez Martinez-Palacios, Martinez-Perez & Ross 2000). Until recently, the lack of accurate data on phosphorus requirements was of minor relevance in practical diet formulations that were mainly based in sh meal, as phosphorus concentration in these diets considerably exceeded the estimated requirements (Rodehutscord & Pfeffer 1995). However, excess of dietary phosphorus is responsible for the environmental impact caused by surplus phosphorus discharges in the efuents. Therefore, in recent years there has been a trend towards the reduction of dietary phosphorus to levels that satisfy, but do not exceed phosphorus requirements (Lall 1991; Oliva-Teles, Pereira, Gouveia & Gomes 1998; Bureau & Cho 1999). Thus, estimation of dietary phosphorus requirements becomes a priority. The aim of this study was to estimate dietary phosphorus requirements of gilthead sea bream juveniles. Materials and methods The trial was conducted at the Marine Zoological Station at Oporto, with sea bream (Sparus aurata L)
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Requirements of sea bream A M Pimentel-Rodrigues & A. Oliva-Teles Table 1 Composition of the experimental diets
Diet 1 Ingredients (% dry weight) Casein Gelatin CPSP1 Yellow dextrin Cod sh oil Vitamin premix2 Choline chloride (50%) Mineral premix3 Dicalcium phosphate Total Proximate analysis Moisture (%) Ash (%) Protein (%) Lipids (%) Energy (kJ g1) Phosphorus (%)
1 2

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400.0 100.0 50.0 247.9 150.0 10.0 10.0 30.0 2.1 1000 8.7 3.0 54.2 14.3 21.8 0.37

400.0 100.0 50.0 236.8 150.0 10.0 10.0 30.0 13.2 1000 8.6 4.3 54.3 14.1 21.7 0.57

400.0 100.0 50.0 225.7 150.0 10.0 10.0 30.0 24.3 1000 8.2 5.3 54.2 14.2 21.5 0.75

400.0 100.0 50.0 214.6 150.0 10.0 10.0 30.0 35.4 1000 6.9 6.3 53.9 14.4 21.6 0.95

400.0 100.0 50.0 203.5 150.0 10.0 10.0 30.0 46.5 1000 7.2 7.2 54.4 14.8 21.3 1.1

400.0 100.0 50.0 192.4 150.0 10.0 10.0 30.0 57.6 1000 7.0 8.4 53.9 14.7 21.2 1.32

400.0 100.0 50.0 181.2 150.0 10.0 10.0 30.0 68.8 1000 7.2 9.4 54.3 14.9 20.9 1.5

che, France. Fish Protein Concentrate Solubles, Soprope Vitamins (mg kg1 diet): retinol, 18000 (IU kg1 diet); calciferol, 2000 (IU kg1 diet); a-tocopherol, 35; menadion sodium bisulfate, 10; thiamin, 15; riboavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbic acid (stay C), 50; inositol, 400. 3 Minerals (mg kg1 diet): calcium carbonate, 1125; sodium chloride, 5640; potassium sulphate, 7500; magnesium sulphate, 3750; iron sulphate, 2640; manganese sulphate, 114; zinc sulphate, 207; copper sulphate, 60; cobalt chloride, 9.9; potassium iodide, 6; sodium selenite, 0.9.

juveniles obtained from a commercial hatchery and adapted to the experimental conditions over 2 weeks. The experimental facilities consisted of a thermoregulated recirculation water system equipped with 14 tanks of approximately 55 L water capacity, designed according to the Guelph system (Cho, Slinger & Bayley 1982). Fourteen groups of 25 sh with an average individual weight of 5 g were assembled and duplicate groups of these sh were assigned one of the experimental diets whose composition is presented in Table 1. Seven semipuried diets were formulated to be isonitrogenous (55% crude protein) and isoenergetic (21 kJ g1 dry matter) and to include graded levels of phosphorus (dicalcium phosphate) ranging from 0.37% to 1.5%. The dietary ingredients were thoroughly mixed and dry pelleted in a laboratory pellet mill (CPM) through a 2-mm dye. During the trial the sh were hand fed twice a day, six days a week, to apparent visual satiation.
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The growth trial lasted 42 days and during the trial the sh were bulk weighted every 2 weeks after a mild anaesthesia (ethyleneglycol monophenyl ether 0.3 mg L1). Eight sh from the initial stock population and 10 sh from each tank at the end of the trial were sampled for whole-body composition analysis. During the trial water temperature averaged 23.9 6 0.8C and salinity averaged 32.4 6 0.7. Fresh seawater was continuously being introduced in the system allowing a complete water renewal twice a day. Ammonia and nitrite levels were always kept within the acceptable limits for the species, and phosphorus levels in the water averaged 0.08 6 0.01 mg L1. Proximate analysis of sh and diets were performed according to the usual procedures: dry matter by drying the samples in a oven at 104C until constant weight; ash by incinerating the samples in an oven at 450C for 16 h; crude protein by the Kjeldhal method, using a Kjeltec

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Aquaculture Research, 2001, 32 (Suppl. 1), 157161

Requirements of sea bream A M Pimentel-Rodrigues & A. Oliva-Teles

Table 2 Growth and feed utilization by sea bream juveniles fed the experimental diets
Diet 1 Initial weight (g) Final weight (g) Mortality (%) SGR (%)1 Feed intake (g kg1 ABW day1) Weight gain: feed intake Weight gain: protein intake N retention (% NI) Energy retention (% EI)
1

2 5.1 11.4ab 12.0 2.01b 20.0ab 0.90ab 1.67ab 27.5b 34.7

3 5.1 13.1bc 6.0 2.35bc 22.5bc 0.92b 1.70b 26.8ab 31.7

4 5.1 12.5bc 8.0 2.26bc 20.8abc 0.98b 1.80b 28.5b 30.0

5 5.1 13.2bc 4.0 2.38bc 21.7bc 0.99b 1.82b 30.5b 34.1

6 5.1 14.4c 6.0 2.59c 22.9c 0.99b 1.83b 31.0b 34.1

7 5.1 14.3c 12.0 2.57c 21.3bc 1.02b 1.87b 30.7b 30.6

SEM 0.01 0.41 2.83 0.08 0.48 0.03 0.06 1.39 1.78

5.1 9.2a 4.0 1.47a 18.2a 0.74a 1.36a 22.3a 29.0

Specic growth rate: (ln initial weight ln nal weight) 3100/time (days); Values in the same row followed by different letters are signicantly different (P < 0.05).

(Tecator) digestion and distillation units; crude lipids by extraction with petroleum ether in a Soxhlet extractor; total phosphorus by colorimetric process, using the vanado-molybdate reagent; and gross energy by direct combustion of the samples in an adiabatic calorimetric bomb (PARR Instruments, model 1261). Statistical analysis of the data was done by oneway analysis of variance (ANOVA) using the probability level of 0.05 for rejection of the null hypothesis. Signicant differences among means were determined by the Tukey's multiple range test. Statistical analyses were performed using the Statgraphics Plus for Windows 4.0 software package. Tank mean values were considered the unit of observation. Results At the end of the trial growth rate of sh fed Diet 1 was signicantly lower than for all the other diets (Table 2), whereas growth rates of groups fed Diets 37 were not signicantly different from each other. Voluntary feed intake increased with the dietary phosphorus level from 0.37% to 0.75% (Table 2). Feed efciency ratio, protein efciency ratio and N retention (% N intake) were signicantly lower in sh fed the 0.37% phosphorus diet than in those fed 0.75% and higher levels of phosphorus (Table 2). At the end of the trial there were no differences in whole-body composition of the sh, except for the ash and energy content (Table 3). Phosphorus content of the sh averaged 2% (on a dry weight

basis) and was not signicantly affected by dietary phosphorus content. Phosphorus intake signicantly increased with the dietary phosphorus level (Table 4). Phosphorus retention averaged 6.6 g kg1 weight gain or 0.16 g kg1 day1 and was not statistically different among groups. As a percentage of intake, phosphorus retention decreased with the increase of dietary phosphorus content, and was higher than 100% in groups fed diets with 0.37% and 0.57% phosphorus, indicating phosphorus absorption from the water (Table 4). Discussion Reduced growth and low feed efciency ratios as well as poor bone mineralization are the main phosphorus deciency signs observed in most sh species (Lall 1991; NRC 1993). In this trial, growth and feed utilization of sea bream juveniles improved with the increase of dietary phosphorus level up to 0.75%; from that point onwards a plateau was reached, indicating that phosphorus requirement for growth was satised. This requirement value falls within the range of values reported for several other sh species (NRC 1993). In marine sh, phosphorus requirements were determined for red sea bream (Sakamoto & Yone 1973) and estimated to be 0.68% of the diet, a value similar to that determined in this study. The ash content of whole sh signicantly increased with the increase of dietary phosphorus, although phosphorus content was unaffected by
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Requirements of sea bream A M Pimentel-Rodrigues & A. Oliva-Teles

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Table 3 Phosphorus utilization by sea bream juveniles fed the experimental diets
Diet 1 P P P P intake (g/kgABW/d) retention (% PI) retention (g kg1 day1) retention (g kg1 weight gain) 0.07a 142.0c 0.10 7.1 2 0.11b 103.7bc 0.12 6.4 3 0.17c 68.2ab 0.11 5.5 4 0.20d 65.9ab 0.13 6.4 5 0.24e 60.2ab 0.14 8.7 6 0.30f 57.1a 0.17 7.6 7 0.32f 44.7a 0.14 6.6 SEM 0.005 13.2 0.02 0.97

Values in the same row followed by different letters are signicantly different (P < 0.05).

Table 4 Whole-body composition (dry weight basis) of sea bream juveniles at the start and at the end of the experiment
Diet Final Initial Dry matter (%) Protein (%) Lipid (%) Ash (%) Energy (kJ g1) Phosphorus (%) 26.9 61.3 23.8 18.6 21.7 2.0 1 28.7 57.3 29.9 13.8a 24.4cd 2.2 2 29.0 56.7 31.2 13.9a 24.5d 2.0 3 28.3 56.8 30.3 14.7ab 23.8bcd 1.9 4 27.2 59.0 29.4 16.6c 23.1ab 2.2 5 28.6 58.2 29.8 15.5b 23.6bc 2.2 6 28.5 58.6 31.0 15.3b 23.7bc 2.4 7 27.4 60.0 31.1 17.5c 22.3a 2.2 SEM 0.39 0.86 0.70 0.34 0.25 0.19

dietary phosphorus level. A positive correlation between either carcass or bone ash content and dietary phosphorus level was also observed in other studies (Andrews, Murai & Campbell 1973; Ketola 1975; Ogino & Takeda 1978; Sakamoto & Yone 1978; Baeverfjord, Asgard & Shearer 1998; ChavezSanchez, Martinez-Palacios, Martinez-Peres & Ross 2000). However, contrary to our results a positive correlation between carcass or bone phosphorus and dietary phosphorus level was found in some studies (Ogino & Takeda 1978; Sakamoto & Yone 1978; Watanabe, Murakami, Takeuchi, Nose & Ogino 1980; Brown, Jaramillo & Gatlin 1993; Schafer, Koppe, Meyer-Burgdorff & Gunther 1995; Baeverfjord et al. 1998; Chavez-Sanchez et al. 2000). An increase of lipid content has been reported in sh fed phosphorus decient diets (Sakamoto & Yone 1978; Chavez-Sanchez et al. 2000). In this trial this was not observed, although energy content of whole sh signicantly increased in sh fed the phosphorus decient diets. Feed intake of sh fed the phosphorus decient diets was lower than in groups fed adequate
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phosphorus diets, indicating that sh responded to this nutrient unbalance but were not able to regulate phosphorus intake by increasing feed intake. Therefore, as sh eat to satisfy their energy intake (Cho & Kaushik 1990), it seems more adequate to express dietary phosphorus levels in relation to the dietary energy rather than as a percentage or in g kg1 diet, as improvements of feed efciency or increase of dietary energy density may lead to increased requirements (Baeverfjord et al. 1998). Expressed per unit of weight gain phosphorus retention was unaffected by dietary phosphorus level, but as a percentage of intake phosphorus retention was negatively related to dietary phosphorus level. Phosphorus retention even exceeded the intake value in sh fed phosphorus decient diets, denoting phosphorus absorption from the water. However, this uptake was not enough to satisfy requirements, conrming that water uptake of phosphorus is not sufcient to satisfy the nutritional requirements of this mineral (NRC 1993). Contrary to these ndings, in other studies

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Requirements of sea bream A M Pimentel-Rodrigues & A. Oliva-Teles (Salmo gairdneri). World Review of Nutrition Dietetics 61, 132172. Cho C.Y., Slinger S.J. & Bayley H.S. (1982) Bioenergetics of salmonid shes: energy intake, expenditure and productivity. Comparative Biochemistry and Physiology 73B, 2541. Ketola H.G. (1975) Requirement of Atlantic salmon for dietary phosphorus. Transactions of the American Fisheries Society 3, 548551. Kim J.D., Kim K.S., Song J.S., Lee J.Y. & Jeong K.S. (1998) Optimum level of dietary monocalcium phosphate based on growth and phosphorus excretion of mirror carp, Cyprinus carpio. Aquaculture 161, 337344. Lall S.P. (1991) Digestibility, metabolism and excretion of dietary phosphorous in sh. In: Nutritional Strategies and Aquaculture Wastes (ed. by C.B. Cowey & C.Y. Cho), pp. 2136. University of Guelph. NRC (1993) Nutrient Requirements of Fish. National Academy Press, Washington DC, pp. 114. Ogino C. & Takeda H. (1978) Requirements of rainbow trout for dietary calcium and phosphorus. Bulletin of the Japanese Society of Scientic Fisheries 44, 10191022. Oliva-Teles A., Pereira J.P., Gouveia A. & Gomes E. (1998) Utilisation of diets supplemented with microbial phytase by seabass (Dicentrarchus labrax) juveniles. Aquatic Living Resources 11, 255259. Rodehutscord M. & Pfeffer E. (1995) Requirement for phosphorus in rainbow trout (Oncorhynchus mykiss) growing from 50 to 200 g. Water Science and Technology 31, 137141. Ronsholdt B. (1995) Effect of size/age and feed composition on body composition and phosphorus content of rainbow trout, Oncorhynchus mykiss. Water Science and Technology 31, 175183. Sakamoto S. & Yone Y. (1973) Effect of dietary calcium/ phosphorus ratio upon growth, feed efciency and blood serum Ca and P level in red sea bream. Bulletin of the Japanese Society of Scientic Fisheries 39, 343348. Sakamoto S. & Yone Y. (1978) Effect of dietary phosphorus level on chemical composition of red sea bream. Bulletin of the Japanese Society of Scientic Fisheries 44, 227229. Schafer A., Koppe W.M., Meyer-Burgdorff K.H. & Gunther K.D. (1995) Effects of P-supply on growth and mineralization in mirror carp (Cyprinus carpio L.). Journal of Applied Ichthyology 11, 397400. Watanabe T., Murakami A., Takeuchi L., Nose T. & Ogino C. (1980) Requirement of chum salmon held in freshwater for dietary phosphorus. Bulletin of the Japanese Society of Scientic Fisheries 46, 361367.

phosphorus retention efciency (as a percentage of intake) in sh fed phosphorus decient diets did not exceed the intake values (Rodehutscord & Pfeffer 1995; Schafer et al. 1995; Kim, Kim, Song, Lee & Jeong 1998). This study showed that phosphorus requirement for growth of gilthead sea bream juveniles was satised with a diet including 0.75% phosphorus from di-calcium phosphate. However, as body composition and phosphorus content of sh changes with the increase in sh size (Ronsholdt 1995), and as mineral availability is variable among dietary sources of phosphorus (Lall 1991), this requirement value deserves to be re-evaluated for bigger sh and other phosphorus sources. Acknowledgements This study was partially sponsored by PRAXIS XXI and FEDER (project PRAXIS/3/3.2/AQ/2009/95). References
Andrews J.W., Murai T. & Campbell C. (1973) Effects of dietary calcium and phosphorus on growth, food conversion, bone ash and hematocrit levels of catsh. Journal of Nutrition 103, 766771. Baeverfjord G., Asgard T. & Shearer K.D. (1998) Development and detection of phosphorus deciency in Atlantic salmon, Salmo salar L., parr and post-smolts. Aquaculture Nutrition 4, 111. Brown M.L., Jaramillo F. & Gatlin D.M. (1993) Dietary phosphorus requirement of juvenile sunshine bass, Morone chrysops female X M.saxatilis male. Aquaculture 113, 355363. Bureau D.P. & Cho C.Y. (1999) Phosphorus utilization by rainbow trout (Oncorhynchus mykiss): estimation of dissolved phosphorus waste output. Aquaculture 179, 127140. Chavez-Sanchez C., Martinez-Palacios C.A., Martinez-Perez G. & Ross L.G. (2000) Phosphorus and calcium requirements in the diet of the American cichlid Cichlasoma urophthalmus (Gunther). Aquaculture Nutrition 6, 19. Cho C.Y. & Kaushik S.J. (1990) Nutritional energetics in sh: energy and protein utilization in rainbow trout

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