The Open Access Israeli Journal of Aquaculture – Bamidgeh

As from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published exclusively as an on-line Open Access (OA) quarterly accessible by all AquacultureHub (http://www.aquaculturehub.org) members and registered individuals and institutions. Please visit our website (http://siamb.org.il) for free registration form, further information and instructions. This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible.

Editor-in-Chief Published under auspices of Dan Mires The Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), Editorial Board University of Hawaii at Manoa Library Sheenan Harpaz Agricultural Research Organization and Beit Dagan, Israel University of Hawaii Aquaculture Zvi Yaron Dept. of Zoology Program in association with Tel Aviv University AquacultureHub Tel Aviv, Israel http://www.aquaculturehub.org Angelo Colorni National Center for Mariculture, IOLR Eilat, Israel

Rina Chakrabarti Aqua Research Lab Dept. of Zoology University of Delhi

Ingrid Lupatsch Swansea University Singleton Park, Swansea, UK

Jaap van Rijn The Hebrew University Faculty of Agriculture Israel

Spencer Malecha Dept. of Human Nutrition, Food and Sciences University of Hawaii

Daniel Golani The Hebrew University of Jerusalem Jerusalem, Israel

Emilio Tibaldi Udine University Udine, Italy ISSN 0792 - 156X

 Israeli Journal of Aquaculture - BAMIGDEH. Copy Editor Ellen Rosenberg PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH - Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAEL Phone: + 972 52 3965809 http://siamb.org.il The Israeli Journal of Aquaculture Ð Bamidgeh 60(4), 2008, 261-267. 261

Optimal Dietary Protein Levels in Juvenile Electric Blue ( fryeri)

Kenan Gullu1, Derya Guroy2, Ihsan Celik2 and Ahmet Adem Tekinay2* 1 Department of Fisheries, Faculty of Agriculture, Yuzuncu Yil University, Van 65080, Turkey 2 Department of Aquaculture, Faculty of Fisheries, Canakkale Onsekiz Mart University, Canakkale 17100, Turkey

(Received 17.8.08, Accepted 11.9.08)

Key words: electric blue cichlid, , ornamental fish, protein requirements, growth performance

Abstract A feeding trial was conducted to determine the dietary protein requirement of juvenile electric blue (Sciaenochromis fryeri). Groups of fish (0.50±0.01 g) were fed one of four isocaloric diets containing protein levels ranging 35-50% for 12 weeks. According to the broken-line model, the dietary protein requirement was estimated as 38.8%. The feed conversion ratio ranged from 1.73 in the 50%-protein group to 2.16 in the 35% group. There were statistically significantly dif- ferences in feed intake and protein efficiency ratio. In general, the protein efficiency ratio decreased as the dietary protein level increased. For optimum growth, feed conversion, and pro- tein utilization, a diet containing 39-40% protein, 11% crude lipid, and 20.7 MJ gross energy/kg diet is recommended for juvenile S. fryeri.

Introduction The live aquarium animal trade is a global Ornamental fish obtain most of their ener- multi-million dollar industry. Cichlid species gy from dietary lipid. However, to avoid hepat- represent approximately 95% of the world’s ic lipidosis, lipids should not exceed 15% of ornamental fish. The electric blue cichlid, the daily intake. Therefore, determination of Sciaenochromis fryeri, is an African cichlid the protein requirement of ornamental fish is cultured throughout the world (Smith, 2000). It important. Dietary protein plays a major role in is a bright blue freshwater cichlid and one of determining the rate of fish growth (NRC, the most commercially valuable ornamental 1993). Since protein is the most expensive fish species. component of formulated fish feeds, the pre-

* Corresponding author. Fax: +90-286-2180543, e-mail: [email protected] 262 Gullu et al. cise protein requirement of cultured fish must recirculating system annexed to 210-l sand fil- be known for economic and environmentally- ter system at a rate of 8 fish per tank, three sound aquaculture production. tanks for each dietary treatment. Water flow Like other fish species, the reported pro- was 1 l/min. Temperature (26.5±0.8¡C), pH tein requirement of ornamental fish is relative- (7±0.5), and dissolved oxygen (7.2±0.4 mg/l) ly high compared to terrestrial (NRC, were measured daily. The system was 1993). Optimum dietary protein levels for installed in a climate-controlled laboratory with ornamental fishes depend on species, age, an artificial photoperiod of 12 h light:12 h dark. feeding level, quality of diet ingredients, pro- Experimental groups were fed three times a tein-energy ratio, diet composition, etc. (NRC, day (8:00, 12:00, 16:00) by hand to visual sati- 1993; Sales and Janssens, 2003). For exam- ation for 12 weeks. ple, dietary protein requirements vary from Chemical analyses. Proximate analyses of around 50% for growing carnivorous discus, feedstuffs and diets were performed using Symphysodon aequifasciata (Chong et al., standard methods (AOAC, 2000). Dry matter 2000) to 30% for the omnivorous goldfish, was measured by drying at 105¡C until a con- Carassius auratus (Lochmann and Phillips, stant weight was reached, crude protein by 1994). the Kjeldahl method after acid digestion using The energy requirement of electric blue the Gerhardt system, crude fat by ether cichlids has recently been investigated extraction, and crude ash by incineration at (Royes et al., 2006). The purpose of this study 525¡C for 12 h in a muffle furnace. Crude fiber was to determine the optimum protein require- was determined by acid alkali hydrolysis and ment of juvenile S. fryeri in a semi-recycled ignition of the dried sample for 3 h. Nitrogen- water system. free extracts (NFE) were calculated as: NFE = 100 - (%protein + %lipids + %ash + %fiber). Materials and Methods Gross energy was estimated using the follow- Diets. Four isoenergetic (21 MJ/kg) experi- ing values: 23.7 kJ/g for protein, 39.5 kJ/g for mental diets were formulated to supply crude lipids, and 17.2 kJ/g for carbohydrates (Brett protein levels of 35%, 40%, 45%, and 50% and Groves, 1979). To analyze essential using fishmeal and soybean meal as sources amino acids (excluding tryptophan), about 50 of protein, wheat as the carbohydrate source, mg of diet was hydrolyzed in 10 ml 6 N HCl at and fish oil as the lipid source (Table 1). Diets 110¡C for 24 h. After removal of the HCl by were prepared by thoroughly mixing the dry evaporation under vacuum, the amino acids ingredients with oil and water in a kitchen were separated by ion-exchange chromato- mixer and extruding the mixture through a 1- graphy on a Shimadzu RF-10AXL sodium col- mm die. The moist pellets were fan-dried, umn and detected following postcolumn ground in a food blender, and stored frozen at derivatization with ninhydrin, by measuring -20¡C until needed. absorbance at 350-450 nm. Rearing systems, fish, experimental Evaluation of fish performance and pro- design. Juvenile electric blue cichlids duction costs. Growth was determined (Sciaenochromis fryeri) were obtained from a biweekly by collectively weighing the fish from commercial aquarium in Istanbul and trans- each aquarium. Feed intake was recorded ported to the Aquarium Unit of the Fisheries daily to calculate the feed conversion ratio Faculty in Canakkale Onsekiz Mart University. (FCR). The specific growth rate was calculat- Prior to the start of the feeding trial, the fish ed as SGR = 100 x [(ln final fish wt) - (ln initial were kept in a 100-l aquarium and fed a com- fish wt)]/no. experimental days, the feed con- mercial diet (38% protein; 6% fat) for 2 weeks version ratio as FCR = feed intake (g)/wt gain to adjust to experimental conditions. (g), the protein efficiency ratio as PER = wt At the start of the trial, the fish were ran- gain (g)/dietary protein intake (g) x 100, and domly distributed amongst twelve 30-l glass the economic conversion ratio as ECR = feed aquaria (30 x 30 x 40 cm) inside a 750-l semi- cost x FCR. Dietary protein levels in juvenile electric blue cichlids 263

Table 1. Formulation, chemical composition, and amino acid contents of experimental diets.

Dietary protein level (%) 35 40 45 50 Ingredient (%) Fishmeal 38.0 47.0 56.0 64.0 Soybean meal 10.0 10.0 10.0 10.0 Wheat meal 42.0 34.0 26.0 19.0 Fish oil 7.0 6.0 5.0 4.0 Vitamin 1.0 1.0 1.0 1.0 Mineral 0.8 0.8 0.8 0.8 Binder (guar gum) 1.2 1.2 1.2 1.2 Chemical composition Crude protein (%) 35.1 40.8 45.2 50.1 Crude lipid (%) 11.1 11.0 10.9 11.2 Crude ash (%) 7.4 8.7 9.9 11.1 Crude fiber (%) 1.3 1.1 1.0 0.9 Nitrogen free extract (%) 45.1 38.4 33 26.7 Gross energy (MJ/kg) 20.5 20.6 20.7 20.9 Amino acid content (% of protein) Arginine 1.9 2.2 2.5 2.7 Cystine 0.4 0.4 0.4 0.5 Histidine 0.8 0.9 1.0 1.1 Isoleucine 1.8 2.1 2.4 2.6 Leucine 2.3 2.7 3.1 3.4 Lysine 2.3 2.8 3.2 3.6 Methionine 0.8 0.9 1.1 1.2 Phenylalanine 1.4 1.6 1.8 2.0 Threonine 1.2 1.4 1.6 1.8 Valine 1.6 1.9 2.2 2.4

Statistical analysis. Data were subjected requirement was made from nonlinear regres- to one-way analysis of variance (ANOVA). sion of the SGR against the dietary protein When a significant difference was found level using broken-line model analysis (SAS among treatments, Duncan’s multiple range statistical software) as per Robbins et al. test was performed to rank the groups using (2006) and second order polynomial analysis Statgraphics 4.0 statistical software using SigmaPlot (Lovell, 1989). All values (Manugistics Inc., Rockville MD) as per Zar were considered significant at a 5% level of (2001). Estimation of the dietary protein confidence. 264 Gullu et al.

Results than for other carnivorous ornamental fish All the experimental diets were well accepted such as 45% for swordtails (Xiphorus helleri; by the fish. No pathological signs were Kruger et al., 2001) and discus observed during the trial and no losses (Symphysodon spp.; Chong et al., 2000), but occurred. Final weight, SGR, FCR, feed higher than for herbivorous or omnivorous fish intake (%), PER, feed cost, and ECR are pre- such as 29% for goldfish (Carassius auratus; sented in Table 2. The optimal dietary protein Lochmann and Phillips, 1994) or 25% for requirement for growth was estimated at dwarf gourami (Colisa lalia; Shim et al., 1989). 38.8% (Fig. 1). Second order polynomial In addition to the difference in species, the regression analysis showed that the maxi- high protein requirement of the electric blue mum SGR response point (Ymax) occurred at may be due to its small size, as protein 46.4% (Fig. 2). requirements of fish decrease with increasing size and age (NRC, 1993). The optimum Discussion dietary protein level for fish is influenced by This study indicates that 40.0% dietary protein the dietary protein:energy ratio, the essential is the optimal level for weight gain in juvenile amino acid composition, digestibility of the electric blue cichlids under the culture condi- dietary protein, and the amount of non-protein tions used in this study. The broken-line energy sources in the diet (Akiyama et al., model has been used to determine protein 1997; Wilson, 2002). In this study, dietary requirements of several fish species (Ng et essential amino acids were influenced by the al., 2001; Kim et al., 2001). Broken-line analy- protein level as they increased with the dietary sis of the SGR in our study shows that 38.8% protein level. dietary protein was optimum when fishmeal Our growth performance results compared was the main protein source and the dietary well with African cichlids in similar experimen- energy value was 20.7 MJ/kg diet. tal conditions (Royes et al., 2005, 2006). For Comparison of protein requirements instance, Royes et al. (2005) reported that among fish species is complicated by differ- increasing the protein level for juveniles of the ences in fish size, diet formulation, and culture omnivorous Pseudotropheus socolofi from conditions among studies. The optimum 42.5% to 58.9% did not enhance growth per- dietary protein level for juvenile electric blue formance. Similarly, Royes et al. (2006) did cichlids determined by this study was lower not obtain significant increases in weight gain

Table 2. Growth and feed utilization of electric blue fed experimental diets.

Dietary protein level (%) 35 40 45 50 Initial mean wt (g) 0.57 0.56 0.56 0.57 Final mean wt (g) 2.97±0.03a 3.56±0.09b 3.39±0.08b 3.57±0.22b Specific growth rate 2.01±0.01a 2.25±0.04b 2.19±0.01b 2.24±0.08b Feed intake (%) 6.07±0.45b 5.01±0.01a 5.67±0.72a 4.92±0.26a Feed conversion ratio 2.16±0.15b 1.82±0.06a 1.85±0.02a 1.73±0.09a Protein efficiency ratio 1.16±0.09b 1.23±0.04b 1.07±0.01a 1.07±0.06a Feed cost (€/kg) 0.70 0.79 0.87 0.94 Economic conversion ratio (€/kg) 1.52 1.43 1.61 1.63 Dietary protein levels in juvenile electric blue cichlids 265

2.5 Y= 2.23 - 0.057 (R - XLR), R = 38.83±0.23

2.0 SGR

1.5 30 35 40 45 50 55 Dietary protein level (%)

Fig. 1. Broken line analysis of specific growth rate (SGR) in juvenile electric blue cichlids (Sciaenochromis fryeri) fed diets with different protein levels.

2.5 Y = -5.2905 + 0.3811X - 0.041X2

2.4

2.3

2.2

2.1 SGR

2.0

1.9 Xmax = 46.4 1.8 30 35 40 45 50 55

Dietary protein level (%)

Fig. 2. Second order polynomial relationship between specific growth rate (SGR) and dietary protein levels for juvenile electric blue cichlids (Sciaenochromis fryeri) fed diets with different protein levels.

in carnivorous S. fryeri fed increasing levels of Our SGR was lower than in Royes et al. dietary protein and lipid levels (36-55% pro- (2006). The slower growth rate seems to be tein and 10-20% lipid). They suggest protein due to a difference in body weight. In Royes et and lipid levels of 36% and 10%, respectively. al. (2006), fish with an average weight of In our experimental conditions, the optimum about 2.0 g were fed experimental diets for 8 dietary protein level was determined to be weeks and gained more than a twofold in final 40% without significant pathological changes weight in all treatment groups. In the present to the liver. study, fish of 0.5 g were fed experimental 266 Gullu et al. diets for 12 weeks and attained 5.2 to 6.4-fold 17th ed. Assoc. Official Analytical Chemists, increases in final weight. Arlington, VA. Fish regulate feed intake to satisfy their Brett J.R. and T.D.D. Groves, 1979. Physio- energy requirements (Wilson, 2002). In this logical energetics. Fish Physiol., 8:280-351. feeding trial, fish were fed to visual satiation Chong A.S.C., Hashim R. and A.B. Ali, and achieved maximum growth by controlling 2000. Dietary protein requirements for discus feed intake. In general, the feed intake (Symphysodon spp.). Aquac. Nutr., 6:275- dropped as the dietary protein level 278. increased. This is in agreement with results Elangovan A. and K.F. Shim, 1997. Growth for discus (Chong et al., 2000). Likewise, the response of juvenile Barbodes altus fed FCR improved as the protein level increased, isocaloric diets with variable protein levels. well-known in ornamental fish (Kruger et al., Aquaculture, 158:321-329. 2001; Royes et al., 2005). The FCR in this Kim K.W., Wang X.J. and S.C. Bai, 2001. study was excellent (1.73-2.16) and quite Reevaluation of the optimum dietary protein lower than reported for similar small size level for the maximum growth of juvenile African cichlids (Royes et al., 2006). The FCR Korean rockfish, Sebastes schlegeli for omnivorous African cichlids P. socolofi (Hilgendorf). Aquac. Res., 32(Suppl. 1):119- was 2.75-3.35 (Royes et al., 2005). 125. The PER tended to decrease as the Kruger D.P., Britz P.J. and J. Sales, 2001. dietary protein level increased, as reported for Influence of varying dietary protein content at other ornamental fish species (Elengovan and three lipid concentrations on growth charac- Shim, 1997; Royes et al., 2005). The low PER teristics of juvenile swordtails (Xiphophorus in the groups fed the 45% and 50% protein helleri Heckel 1848). Aquarium Sci. Conserv., diets, together with the lack of significant 3:275-280. improvement in growth, indicates that exces- Lochmann R.T. and H. Phillips, 1994. sive protein in those diets was used for meta- Dietary protein requirement of juvenile golden bolic purposes rather than growth. PER in this shiners (Notemigonus crysoleucas) and gold- study increased until the dietary protein fish (Carassius auratus) in aquaria. requirement was met. Aquaculture, 128:277-285. Based on growth performance, PER, and Lovell T., 1989. Nutrition and Feeding of Fish. ECR, 39-40% protein in diets with 11% crude Van Nostrand Reinhold, New York. lipid and 20.7 MJ gross energy/kg is recom- Ng W.K., Soon S.C. and R. Hashim, 2001. mended for juvenile electric blue cichlids The dietary protein requirement of a bagrid under the experimental conditions used in this catfish Mystus nemurus (Cuvier & study. Valenciennes), determined using semipurified diets of varying protein level. Aquac. Nutr., Acknowledgements 7:45-51. The authors wish to thank Dr. O. Ozen, NRC, 1993. Nutrient Requirements of Fish. Faculty of Fisheries, Canakkale Onsekiz Mart Natl. Res. Council, Natl. Acad. Press, University, for his help in statistical analysis. Washington DC. This work was supported by the Council of Robbins K.R., Saxton A.M. and L.L. Scientific Research Projects of Canakkale Southern, 2006. Estimation of nutrient Onsekiz Mart University, Project No. 2005/19. requirements using broken-line regression analysis. J. Anim. Sci., 84 (E Suppl.):155-165. References Royes J.B., Murie D.J. and R. Francis Akiyama T., Oohara I. and T. Yamamoto, Floyd, 2005. Optimum dietary protein level for 1997. Comparison of essential amino acid growth and protein efficiency without hepato- requirements with A/E ratio among fish cyte changes in juvenile African cichlids species. Fish. Sci., 63:963-970. (Pseudotropheus socolofi). North Am. J. AOAC, 2000. Official Methods of Analysis, Aquac., 67:102-110. Dietary protein levels in juvenile electric blue cichlids 267

Royes J.B., Murie J.D. and R. Francis dwarf gourami, Colisa lalia (Hamilton). J. Floyd, 2006. Effects of varying dietary protein Aquac. Trop., 4:111-123. and lipid levels on growth performance and Smith M.P., 2000. Cichlids. hepatocyte changes in juvenile African cich- Barron’s Educational Ser. Inc., NYC. lids (Pseudotropheus socolofi and Haplo- Wilson R.P., 2002. Amino acids and protein. chromis ahli). J. World Aquac. Soc., 37:48-59. pp. 143-179. In: J.E. Halver, R.W. Hardy Sales J. and G.P.J. Janssens, 2003. (eds.). Fish Nutrition, 3rd ed. Academic Press, Nutrient requirements of ornamental fish. San Diego, CA. Aquat. Living Resour., 16:533-540. Zar J.H., 2001. Biostatistical Analysis, 4th ed. Shim K.F., Landesman L. and T.J. Lam, Prentice-Hall Inc., Upper Saddle River, NJ. 1989. Effect of dietary protein on growth, 931 pp. ovarian development and fecundity in the