Effect of Dietary Supplementation with Taurine, B-Alanine and GABA on The

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Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682003 Blackwell Science Asia Pty Ltd 69 614 Effect of taurine on ﬂounder growth S-K Kim et al. 10.1046/j.0919-9268.2002.00614.x Original Article242248BEES SGML

FISHERIES SCIENCE 2003; 69: 242–248

Effect of dietary supplementation with taurine, bb-alanine and GABA on the growth of juvenile and ﬁngerling Japanese ﬂounder Paralichthys olivaceus

Shin-Kwon KIM,1 Toshio TAKEUCHI,1* Masahito YOKOYAMA2† AND Yuko MURATA2

1Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato, Tokyo 108-8477 and 2National Research Institute of Fisheries Science, Yokohama, Kanagawa 235-8648, Japan

ABSTRACT: Three diets supplemented with taurine, b-alanine and GABA and a control diet were fed to juvenile and fingerling Japanese flounder to investigate the effects of the diets on growth and metabolic changes of free amino acids in whole body and tissues. In experiment I, three diets supplemented with 1% each of taurine, b-alanine and GABA and a control diet were fed to juvenile Japanese flounder with an initial mean body weight of 0.4 g for 4 weeks at 20∞C. In experiment II, the taurine-supplemented diet and a control diet were fed to fingerling Japanese flounder with an initial mean body weight of 15 g for 4 weeks at 20∞C. Only supplementation of taurine in the diet of juvenile flounder improved their growth performance in experiment I, but fingerling growth performance of experiment II was not significantly related to taurine supplementation in the experimental diet. These results suggest that there is a greater requirement for taurine for the growth of juvenile Japanese flounder than fingerling Japanese flounder.

KEY WORDS: bb-alanine, ﬁngerling, GABA, growth, Japanese ﬂounder, juvenile, taurine.

INTRODUCTION and retina.5 In cats, the offspring of taurine-deficient females have a large number of neurological Taurine (2-aminoethanesulfonic acid) is a small defects, including tapetal and retinal degeneration, sulfur-containing b-amino acid present in large delayed cerebellar granule cell division and migra- quantities in the intracellular space of the brain, tion and abnormal cortical development.5 The retina, liver, kidney, heart and muscle in vertebrate offspring of taurine-deficient female cats have species. In mammals, taurine is known to serve low cysteinesulfinate decarboxylase activity, which many important biological functions, including is a rate-limiting enzyme in taurine biosynthesis. osmoregulation, bile acid conjugation, membrane Consequently, taurine is an essential amino acid stabilization, antioxidation and Ca2+ modulation.1,2 in cats.6 Taurine serves as an important organic osmolyte in In wild juvenile Japanese flounder, mysids are the brain and kidney. It contributes to the process the most important food organisms. Juveniles fed of cell volume regulation that is especially critical mysids have been shown to grow faster than those following hypo- or hyperosmolar stresses. This fed a formula diet.7 Juvenile Japanese flounder fed osmoregulatory role is important in the develop- on mysids were found to contain higher levels of ment of the central nervous system and retina.3,4 taurine than those fed a formula diet.8 Park et al.9 Taurine must have a remarkable and specific phys- reported that the fish fed a taurine-supplemented iological role that accounts for its accumulation in diet showed significantly better growth than those cells against a gradient.3,4 Taurine that is related to fed a non-supplemented control diet. This indi- mammalian development during the immature cates that supplementary taurine in the diet period is found in high concentrations in the brain improves the growth of juvenile Japanese flounder. Although taurine improved the growth and feed efficiency of juvenile Japanese flounder, the phys- *Corresponding author: Tel: 81-3-5463-0545. iological role of taurine in the growth and feed effi- Fax: 81-3-5463-0545. Email: take@tokyo-u-fish.ac.jp †Present address: Japan International Research Center for ciency of juvenile Japanese flounder is not yet Agriculture Sciences, Ohwashi, Tsukuba, Ibaraki 305-8686, clear. Japan. The present study was conducted to determine Received 9 April 2002. Accepted 24 September 2002. the effect of dietary taurine on the growth and tau-

Effect of taurine on ﬂounder growth FISHERIES SCIENCE 243

rine accumulation in tissues of juvenile Japanese Experimental conditions flounder fed diets supplemented with 1% each of taurine, b-alanine, GABA and a control diet in Two feeding experiments were conducted in the (experiment I) and fingerling Japanese flounder National Research Institute of Fisheries Science, fed a diet supplemented with 1% taurine and a Yokohama, Japan. Juvenile Japanese flounder used control diet (experiment II). It is well known that b- in this experiment were obtained from the Nisshin alanine is one of the b-amino acids, as taurine, and Marin Tech, Aichi, Japan. Before starting the exper- that GABA is a neurotransmitter in the central iment, juvenile Japanese flounder were fed on nervous system, as taurine.10 commercial diets for 7 days in experiment I (Higashimaru, Kagoshima, Japan) and for 35 days in experiment II (Nippon Formula Feed, Ehime, MATERIALS AND METHODS Japan). In experiment I, 50 fish (average body weight 0.4 g) were randomly placed in each of eight Experimental diets 60-L aquariums. After 2 weeks, the numbers were reduced to 30 fish/aquarium. In experiment II, 10 The formulation and content (mg/g) of taurine, b- fish (average weight 15 g) were placed randomly alanine and GABA and the percentage of crude into each of four 60-L aquariums. Fish were fed protein and lipid in the experimental diets used in three times a day to satiation for 4 weeks at a water experiments I and II are shown in Table 1. Four temperature of 20∞C. After a 4-week feeding trial, experimental diets using fish meal as the main pro- fish were weighed and stored at -80∞C for free tein source were prepared: a control diet and three amino acid analysis. After the 4-week feeding trial diets supplemented with 1% each of taurine, b- in experiment II, three fish from each group were alanine or GABA (Wako Pure Chemical Industries, fed the respective experimental diet for an addi- Osaka, Japan). All ingredients were mixed together tional 2 days, were placed immediately in separate with distilled water, mixed to make a mash, pel- plastic flasks containing 1 L water at 20∞C and then leted with a press machine and then dried for 24 h starved for 24 h. Water samples were then collected in a freeze-dryer (Nissei, Tokyo, Japan). Crude pro- to determine the taurine conent as an index of tau- tein and crude lipid levels were adjusted to 50 and rine excretion using the method of Yokoyama and 9%, respectively. Nakazoe.11

Table 1 Composition and taurine, b-alanine, GABA, crude protein and lipid content in experiment and control diets for Japanese ﬂounder Ingredients Taurine (1%) b-Alanine (1%) GABA (1%) Control diet Brown ﬁsh meal 77.0 77.0 77.0 77.0 Taurine 1.0 – – – b-Alanine – 1.0 – – GABA – – 1.0 – a-Starch 8.0 8.0 8.0 8.0 Dextrin 5.0 5.0 5.0 5.0 Cellulose 1.4 1.4 1.4 2.4 n-3 HUFA 2.0 2.0 2.0 2.0 Mineral mixture1 4.0 4.0 4.0 4.0 Vitamin mixture2 1.0 1.0 1.0 1.0 Choline chloride 0.5 0.5 0.5 0.5 Vitamin E3 0.1 0.1 0.1 0.1 Chemical analysis Taurine (mg/g) 10.2 ± 0.2 1.8 ± 0.0 1.8 ± 0.1 1.8 ± 0.0 b-Alanine (mg/g) 0.0 ± 0.0 8.9 ± 0.2 0.1 ± 0.0 0.2 ± 0.1 GABA (mg/g) 0.0 ± 0.0 0.0 ± 0.0 9.4 ± 0.6 0.0 ± 0.0 Crude protein (%) 49.4 ± 0.4 50.1 ± 0.3 50.7 ± 0.2 49.2 ± 0.4 Crude lipid (%) 8.8 ± 0.2 9.3 ± 0.1 9.3 ± 0.1 9.3 ± 0.1

n-3HUFA, n-3 highly unsaturated fatty acids (EPA, 7.6%; DHA, 38.5%; Nippon Chemical Feed, Chiba, Japan). 1 From Ogino et al.19 2 From Ogino and Yang.20 3 DL-a-Tocopheryl acetate, purity 50% (Nippon Roche, Tokyo, Japan). Where appropriate, data are the mean ± SD (n = 5).

244 FISHERIES SCIENCE S-K Kim et al.

Free amino acid analysis RESULTS

Frozen fish from each group were dissected and Experiment I the brain, liver and muscle were used for free amino acid (FAA) analysis. The whole body and Growth performance in the feeding experiments is tissues (n = 5 in experiment I; n = 3 in experiment shown in Table 2. The final average body weight of II) were homogenized with 2% sulfosalicylic acid juvenile Japanese flounder was improved with and centrifuged at 2300 ¥ g for 15 min at 5∞C. For taurine supplementation. Fish fed the taurine- determination of excreted amino acids, water sam- supplemented diet showed significant effects on ples were deproteinized by the addition of 10% percent gain and feed efficiency compared with sulfosalicylic acid, followed by centrifugation at fish fed any other diet in the second period. The 2300 ¥ g for 15 min at 5∞C. Free amino acid levels final body weights of fish fed the control diet were determined individually with an automatic and the b-alanine-supplemented diet were higher amino acid analyzer (model L-8500A; Hitachi, than the body weight of fish fed the GABA- Tokyo, Japan). supplemented diet. The taurine, b-alanine and GABA contents of the Taurine retention rate and statistical analysis whole body and tissues are shown in Fig. 1. The taurine content of the whole body and tissues The taurine retention rate was calculated using val- increased only in the taurine-supplemented diet ues for the total amount of taurine fed and the group; the cystathionine content of the whole taurine content of whole body at the beginning body and muscle was decreased by the taurine- and end of the experiment. supplemented diet (Fig. 2). No changes were Statistical analysis of growth performance and observed in other FAA (data not shown). The reten- FAA accumulation in fish fed different diets was tion rates for taurine were calculated to be 65% for performed using one-way ANOVA AND Tukey’s the taurine-supplemented group and 48% for the multiple-range test. control group (Table 3).

Table 2 Results of the 4-week feeding trial in experiments I and II Mean body weight (g) Percent Feed Mortality Type of diets Initial Final gain (%) efﬁciency (%) Experiment I First period (2 weeks; n = 50) Taurine (1%) 0.40 2.46 ± 0.82a 669.5a 1.76a 9 b-Alanine (1%) 0.40 2.11 ± 0.72b 577.9b 1.66b 4 GABA (1%) 0.37 2.12 ± 0.81b 579.5b 1.47d 7 Control diet 0.37 2.29 ± 0.82ab 570.9b 1.57c 4 Second period (2 weeks; n = 30) Taurine (1%) 2.73 ± 0.61 8.41 ± 1.57a 308.6a 1.63a 0 b-Alanine (1%) 2.42 ± 0.48 7.24 ± 1.28b 298.9ab 1.60a 0 GABA (1%) 2.34 ± 0.46 6.49 ± 1.14c 276.8b 1.43b 0 Control diet 2.56 ± 0.56 7.22 ± 1.54b 282.4b 1.49b 0 Experiment II First period (2 weeks; n = 10) Taurine (1%) 14.76 ± 1.21 27.75 ± 4.41a 188.1a 1.43a 0 Control diet 14.78 ± 1.27 27.40 ± 4.93a 185.4a 1.47a 0 Second period (2 weeks; n = 10) Taurine (1%) 27.75 ± 4.41 42.84 ± 7.30a 154.4a 1.34a 0 Control diet 27.40 ± 4.93 40.20 ± 8.58a 146.7a 1.12b 0

Means with different superscripts within the same column are signiﬁcantly different (P < 0.05). Effect of taurine on ﬂounder growth FISHERIES SCIENCE 245

Fig. 2 Cystathionine content in the liver, brain, muscle and whole body of juvenile Japanese ﬂounder in experiment I (n = 5). Means with different superscripts within the same solumn are signiﬁcantly diffeent (P < 0.05).

content of the whole body and tissues of fish in the taurine-supplemented diet group were higher than those of fish fed the control diet (Fig. 3). In the excretion experiment, taurine excretion was quite small compared with total taurine content in fish fed the taurine-supplemented diet (Table 4). The retention rate was calculated to be 60% for the taurine-supplemented group and 53% for the control group in experiment II (Table 3). The change in taurine content in tissues is shown in Fig. 4. As fish grew, the taurine content in tissues decreased in the taurine-supplemented diet group and, conversely, increased in fish fed the control diet.

DISCUSSION

In the present study, only supplementation of taurine to the diet of juvenile flounder improved their growth performance in experiment I, but fingerling growth performance in experiment II was not significantly related to taurine supplementation in the experimental diet. Conceicao et al.12 have reported that the relative content of taurine is Fig. 1 Taurine, b-alanine and GABA content in the liver, brain, muscle and whole body of juvenile Japanes floun- higher for larvae fed zooplankton than for larvae der in experiment I (n = 5). Means with different super- fed Artemia and suggested a correlation between scripts within the same column are significantly different taurine levels and the growth rate of turbot larvae. (P < 0.05). Park et al.8 reported that juvenile Japanese flounder fed a mysid meal diet had higher levels of taurine (above 20 mg/g) than juvenile Japanese flounder fed a fish meal diet. Park et al. also suggested that Experiment II the taurine content in the diet affects growth rates and the metabolism of sulfur amino acids in juve- The growth of fish fed the taurine-supplemented nile Japanese flounder and concluded that the tau- diet was not significantly greater than that of fish rine requirement in the diet is 15–20 mg/g.9 In the fed the control diet in experiment II (Table 2). present study, the taurine content of tissues However, the feed efficiency of fish fed the taurine- decreased with the growth period of fish in the tau- supplemented diet was higher than the feed effi- rine-supplemented diet group and, conversely, the ciency of fish fed the control diet. The taurine taurine content in tissues increased with the 246 FISHERIES SCIENCE S-K Kim et al.

Table 3 Retention rates for taurine in the body in experiments I and II Total taurine Taurine content Taurine content Retention rate in diet in initial fish in final fish of taurine Diet (g) (g) (g) (%) Experiment I Control 0.48 ± 0.01 0.02 ± 0.00 0.25 ± 0.01 47.9 Taurine (1%) 2.87 ± 0.06 0.02 ± 0.00 1.88 ± 0.08 64.8 Experiment II Control 1.12 ± 0.02 0.37 ± 0.02 0.97 ± 0.04 53.2 Taurine (1%) 6.15 ± 0.13 0.37 ± 0.02 4.07 ± 0.24 60.1

Data are the mean ± SD (n = 2).

Table 4 Taurine and NH3 excretion of Japanese floun- that juvenile Japanese flounder fed fish meal con- der fed a diet supplemented with taurine and a control trol and cystine-supplemented diets showed diet in experiment II higher tissue cystathionine contents than juvenile Diets Japanese flounder fed a taurine-supplemented diet. Yokoyama et al.15 reported that cysteinesulfi- Control diet Taurine (1%) nate decarboxylase activity was different among Taurine (mmol/g body ND 0.18 ± 0.1 the species and the activity in flounder was very weight per 24 h) low. This means that cystathionine may accumu- NH3 (mmol/g body 9.2 ± 2.0 7.2 ± 2.0 late in the tissues of juvenile flounder in the case weight per 24 h) of fish fed a diet without taurine supplementation and the accumulated cystathionine of these fish Data are the mean ± SD (n = 3). ND, not detected. may be used for taurine biosynthesis. In the present study, only taurine, and not b- alanine and GABA, was accumulated in the whole growth period of fish in the control diet group. This body and tissues of juvenile Japanese flounder. means that taurine has an important role during This result supports the existence of a specific tau- the juvenile period (post-metamorphosis period) rine transport channel in the flounder. Taurine is of Japanese flounder, but not in the fingerling known to be an important osmolyte involved in period of Japanese flounder and that there was a cell volume regulation in fish.16 Taurine comprises possible taurine insufficiency in the control diet more than 50% of intracellular FAA in flounder tis- group of experiment I. The retention rates of tau- sues.17 Perlman et al.18 suggested that net secretion rine were approximately 50–60% in both the tau- of taurine was achieved by the presence of two dif- rine-supplemented group and the control group in ferent carriers on opposite sides of the epithelium: experiments I and II. Little taurine was excreted (i) cellular uptake of taurine (and other b-amino by fingerling Japanese flounder in the taurine- acids) is achieved by a Na+ cotransporter located supplemented group in experiment II. Park et al.13 on both the basolateral and luminal sides; and (ii) reported that taurine excretion was very small the luminal carrier for taurine (which does not in juvenile Japanese flounder fed a taurine- transport other b-amino acids, such as b-alanine) supplemented diet. This implies that a part of tau- facilitates the flux of taurine from the cell to the rine may be changed and/or conjugated to other lumen by changes in the extracellular taurine materials that were not determined in the present concentration and osmolality.18 This indicates that study. the renal epithelium of flounder can apparently The concentration of cystathionine in muscle maintain its cell volume while regulating the extra- and whole body decreased with taurine supple- cellular fluid volume through taurine secretion.18 mentation in the diet. The major pathway for tau- This means that flounder have a specific trans- rine synthesis from methionine in mammals is port channel for taurine that does not transport b- believed to involve the transformation of methion- amino acids, including b-alanine. The growth- ine to cystathionine by cystathionine synthetase, promoting effect of taurine in juvenile Japanese the transformation of cystathionine to cysteine by flounder found in the present study may be related cystathionase, the oxidation of cysteine to cys- to this specific taurine transport channel. teinesulfinate, the decarboxylation of cysteine- Takeuchi et al.19 suggested that cells of fish sulfinate to hypotaurine and further oxidation of embryos are directly exposed to the osmotic pres- hypotaurine to taurine.14 Park et al.13 suggested sure of the environmental water because the Effect of taurine on flounder growth FISHERIES SCIENCE 247

Fig. 3 Taurine and cystathionine content in the liver, muscle and whole body and tauine and GABA content in the brain of fingerling Japanese flounder in experiment II (n = 3). Means with different superscripts within the same column are significantly different (P < 0.05). 248 FISHERIES SCIENCE S-K Kim et al.

Fig. 4 Taurine content in the liver, brain and muscle of Japa- nese flounder in experiments I and II. The values were significantly different between experiment I and experiment II, except for the taurine content of the liver in the taurine-supplemented group. Means with different superscripts within the same column are significantly different (P < 0.05, n = 5 in experiment I; n = 3 in experiment II). osmoregulatory organs are not well developed. of juvenile Japanese flounder Paralichthys olivaceus. Nip- Takeuchi et al.19 suggested that an increase in the pon Suisan Gakkaishi 2000; 66: 697–704. ambient osmotic pressure increased the taurine 9. Park GS, Takeuchi T, Seikai T, Yokoyama M. The effects of transporter mRNA level and that taurine has a sig- dietary taurine on growth and taurine levels in whole body nificant role as an osmolyte in carp cells. This of juvenile Japanese flounder Paralichthys olivaceus. Nip- pon Suisan Gakkaishi 2001; 67: 238–243. means that the osmoregulatory role of taurine may 10. Davidson N. Neutotransmitter Amino Acids. Academic be more important in larvae or during the juvenile Press, London. 1976. period than in fingerling fish or during the adult 11. Yokoyama M, Nakazoe J. Effects of dietary protein levels on period of fish, just as taurine supplementation in free amino acid and glutathione contents in the tissues of the diet in the present study improved the growth rainbow trout. Comp. Biochem. Physiol. 1991; 99A: 203– of juvenile but not fingerling Japanese flounder. 206. The osmotic stabilization provided by taurine 12. Conceicao LEC, Meeren VD, Verreth JAJ, Evjen MS, Houli- may be related to the effect of taurine on the han DF, Fyhn HJ. Amino acid metabolism and protein turn- growth of juvenile Japanese flounder. However, over in larval turbot (Scophthalmus maximus) fed natural the physiological role of taurine in fish remains zooplankton or Artemia. Mar. Biol. 1997; 129: 255–265. 13. Park G-S, Takeuchi T, Yokoyama M, Seikai T. Optimal undetermined. Further studies are needed to dietary taurine level for growth of juvenile Japanese floun- determine the physiological role of taurine in fish. der Paralichthys olivaceus. Fish. Sci. 2002; 68: 824–829. 14. Worden JA, Stipanuk MH. A comparison by species, age and REFERENCES sex of cysteinesulfinate decarboxylase activity and taurine concentration in liver and brain of animals. Comp. Bio- 1. Huxtable RJ. Physiological actions of taurine. Physiol. Rev. chem. Physiol. 1985; 82B: 233–239. 1992; 72: 101–163. 15. Yokoyama M, Takeuchi T, Park GS, Nakazoe J. Hepatic cys- 2. Sturman JA. Taurine in development. J. Nutr. 1988; 118: teinesulphinate decarboxylase activity in fish. Aquacult. 1169–1176. Res. 2001; 32 (Suppl. 1): 216–220. 3. Bitoun M, Tappaz M. Taurine down-regulates basal and 16. Schrock H, Forster RP, Goldstein L. Renal handling of tau- osmolarity-induced gene expression of its transporter, but rine in marine fish. Am. J. Physiol. 1982; 242: R64–R69. not the gene expression of its biosynthetic enzymes, in 17. King PA, Goldstein L. Organic osmolytes and cell volume astrocyte primary cultures. J. Neurochem. 2000; 75: 919–924. regulation in fish. Mol. Physiol. 1983; 4: 53–66. 4. Lombardini JB. Taurine: Retinal function. Brain Res. Rev. 18. Perlman DF, Renfro JL, Goldstein L. Taurine secretion in 1991; 16: 151–169. cultured winter flounder renal epithelium. Am. J. Physiol. 5. Chesney RW, Helms RA, Christensen M, Budreau AM, Han 1991; 261: R1155–R1163. X, Sturman JA. The role of taurine in infant nutrition. Adv. 19. Takeuchi K, Toyohara H, Sakaguchi M. A hyperosmotic Exp. Med. Biol. 1998; 442: 463–476. stress-induced mRNA of carp cell encodes Na+- and Cl–- 6. Knopf K, Sturman JA, Armstrong M, Hayes KC. Taurine. An dependent high affinity taurine transporter. Biochim. Bio- essential nutrient for the cat. J. Nutr. 1978; 108: 773–778. phys. Acta 2000; 1464: 219–230. 7. Seikai T, Takeuchi T, Park GS. Comparison of growth, feed 20. Ogino G, Takeuchi L, Takeda H, Watanabe T. Availability of efficiency, and chemical composition of juvenile flounder dietary phosphorus in carp and rainbow trout. Nippon fed live mysids and formula feed under laboratory condi- Suisan Gakkaishi 1979; 45: 1527–1532. tions. Fish. Sci. 1997; 63: 520–526. 21. Ogino G, Yang G-Y. Requirement of rainbow trout for 8. Park GS, Takeuchi T, Seikai T, Yoshinaga T. The effects of dietary zinc. Nippon Suisan Gakkaishi 1978; 44: 1015– residual salts and free amino acids in mysid meal on growth 1018.