Dietary vitamin C improves the quality of yellowtail (Seriola quinqueradiata) seedlings

Aquaculture 161 Ž1998. 427–436

Dietary vitamin C improves the quality of yellowtail žSeriola quinqueradiata / seedlings Yoshitaka Sakakura a , Shunsuke Koshio b,) , Yoshisuke Iida c , Katsumi Tsukamoto a , Takao Kida d , Jozef H. Blom b a

c

Ocean Research Institute, UniÕersity of Tokyo, Nakano, Tokyo 164, Japan b Faculty of Fisheries, Kagoshima UniÕersity, Kagoshima 890, Japan Freshwater Fish Branch, Hiroshima Prefectural Fisheries Experiment Station, Shobara, Hiroshima 727, Japan d Vitamin and Food DiÕision, Takeda Chemical Ind., Osaka 532, Japan

Abstract Several parameters were investigated to determine the effects of vitamin C ŽAsA. on improvement of juvenile yellowtail quality for restocking. The dry pellets with different levels of L-ascorbyl-2-phosphate-Mg ŽAPM. Ž0, 400, 1000, and 2000 mgrkg diet. were formulated and fed to 500 juveniles Žmean total length ŽTL.: 3.6 cm. per treatment with duplicate tanks Ž500 l round type.. Test diets were fed three times a day at 258C water temperature for 20 days. After the 20-day trial, 10 to 20 juveniles from each treatment group were randomly sampled for a test of ‘air dive’ and schooling behavior. The ‘air dive’ was conducted to determine the number of survivors after exposure to air while the schooling behavior such as schooling pattern, swimming activity and individual distance were recorded using the video system and analyzed by the computer graphic system. Survival was over 90% and there was no sign of AsA deficiency in any group during the 20-day trial. There was no effect of APM intake on growth. Liver AsA increased with increasing APM intake whereas that of brain AsA levelled off at 1000 mg APM. The tolerance against air exposure seemed poor in fish fed on APM-free diet although statistical significance was not detected Ž P ) 0.05.. The schooling rate was significantly higher in fish fed on diets containing 1000 or 2000 mg APM. It is speculated that juvenile yellowtail fed on diet containing

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C orresponding author. T el.: [email protected].

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0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 7 . 0 0 2 9 0 - 1

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more than 1000 mg APM would be able to show better growth and survival under natural conditions after release. q 1998 Elsevier Science B.V. Keywords: Vitamin C; L-ascorbyl-2-phosphate-Mg; Yellowtail; Seriola quinqueradiata; Fish quality; Restocking effectiveness; Behavior

1. Introduction A critical phase in marine ranching programs is the transition of artificially produced seedlings and juvenile fish for restocking from the hatchery to the wild condition ŽSvasand, 1993.. Seedlings should be expected to have some characteristics such as ˚ normal morphology, low standard metabolism, good health, developed escape behavior, and high recapture with species-specific normal behavior to maintain high survival and growth rates under wild conditions after release ŽNakano, 1993.. Recently, several studies on the relationship between the quality and behavior of seedlings have been carried out to determine or improve the quality of seedlings. It has been suggested that efficiency of release could be more affected by the behavioral patterns of fish rather than the ability of fish seedlings ŽTsukamoto, 1993.. For example, in red sea bream seedlings, higher survival after release was obtained from fish which showed the typical flight response, ‘tilting behavior’ ŽUchida et al., 1993; Yamaoka et al., 1994., and the ability of predator avoidance in Japanese flounder was higher in wild fish than in cultured seedlings ŽFuruta, 1996.. Therefore, it is necessary to produce fish which develop normal feeding and anti-predator behavior similar to their wild counterparts during the course of seedling production. Since fish behavior is controlled by the brain and central nervous system, behavior may be affected by intake of certain micronutrients which accumulate in the brain. Vitamin C Žascorbic acid, AsA. is one of the nutrients which accumulates in brain and its content in brain is higher than that in other organs or tissues of fish ŽIkeda et al., 1963.. Previous studies ŽKoshio et al., 1997. have indicated that brain AsA accumulation and behavior, such as schooling pattern and aggressive behavior, of juvenile ayu were similar to those in wild fish when the fish were fed diets containing high levels of AsA. However, there is only limited information on the effects of AsA on altering behavior, which can lead to improvement of the quality of marine fish seedlings. The present study was conducted to determine the effects of AsA intake on improvement of seedling quality of yellowtail, focusing on schooling behavior as an index of anti-predator behavior. Yellowtail was chosen as the experimental fish due to the recent efforts on stock enhancement in Japan ŽImaizumi, 1993., and interest in their behavioral patterns ŽSakakura and Tsukamoto, 1996.. 2. Materials and methods 2.1. Test diets The stable form of ascorbic acid, L-ascorbyl-2-phosphate Mg ŽAPM., was used as the AsA source in this experiment. APM was added to other ingredients by mixing with

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Table 1 Basal composition of the test diet Ingredient

grkg

Fish meal Žbrown. a Krill meal a Potato starcha Torula yeast a Vitamin mix b Mineral mix b Glutenc APM d qwheat powder c Total

520 180 90 20 10 30 50 100 1000

a

Supplied from Sakamoto Feed, Japan. Koshio et al. Ž1997.. c Hourai Chemicals. d Supplied from Takeda Food Vitamin, Japan. b

wheat powder. Basal composition of the test diets is presented in Table 1. Dry ingredients and lipids with fat-soluble vitamins were well mixed with warm water Žca. 708C. and they passed through a disc pellet machine Ž1.2 mm in diameter.. As shown in Table 2, four concentrations of APM Ž0, 400, 1000, and 2000 mgrkg diet. were added to the test diets. Protein, lipid, moisture, and ash analyses of the test diets were conducted according to the methods of the Association of Official Analytical Chemists Ž1990.. Test diets were stored at y208C until used. 2.2. Feeding trial Yellowtail juveniles, Seriola quinqueradiata, which were artificially raised from egg stage at Goto Station of Japan Sea Farming Association ŽJASFA., were reared in a 90

Table 2 AsA and APM contents Žmgrkg diet. and proximate composition Ž%. of test diets Components

Diet number 1

2

3

4

APM added APM analyzeda AsA analyzeda AsA estimatedb

0 0 0.7"0.2 0.7

400 360"8 2.1"0.1 157

1000 1106"109 4.7"0.3 480

2000 2035"39 7.4"0.3 882

Moisture Crude proteinc Crude lipid c Crude ashc

18 57 7 14

18 55 7 14

19 55 7 13

16 56 8 14

a

Mean"S.D. Ž ns 3.. APM analyzed=0.43qAsA analyzed. c Dry weight basis. b

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m3 concrete pond and fed a commercial diet ŽC-1000; Kyowa. at 258C water temperature until ready for use in the experiment. A total of 500 fish Žmean total length of 3.6 " 0.4 cm, wet weight 0.4 " 0.1 g. per dietary treatment were randomly distributed in 500 l cylindrical tanks and two tanks per dietary treatment were used. Fish were acclimated for 6 days by feeding the APM-free diet three times a day. Filtered seawater was continuously supplied to the tanks at a flow rate of 500 lrh, while the trial was conducted at 25.2 " 1.18C water temperature over 20-day period under natural lightrdark conditions. Fish were fed to satiation Žabout 11% of body weight. three times a day at 0900, 1300, and 1800 hours, respectively. The following parameters were used to evaluate fish growth: final total length ŽTL, cm.; final wet weight ŽFW, g.; percentage weight gain ŽWG. s ŽW1 y W0 . = 100rW0 ; feed conversion efficiency ŽFCE. s ŽW1 y W0 .rD; and condition factor ŽCF. s W1 = 1000rTL3 ŽW1 is the final wet weight Žg., W0 is the initial wet weight Žg., and D is the dry diet intake.. 2.3. AsA analysis The quantification of dietary APM ŽKoshio et al., 1997. was conducted by high-performance liquid chromatography ŽHPLC. using a reversed-phase column ŽCOSMOSIL 5C18, 10 = 250 mm, Nacalai Tesque.. Mobile phase Žflow rate, 1.2 mlrmin. was an aqueous solution of 0.1 M KH 2 PO4 , 0.5% metaphosphoric acid. The pH was adjusted to 3.7 with NaOH. The HPLC system used was as follows: column oven ŽShimadzu COP-6A, temperature 408C., detector ŽShimadzu SPO-6AV, UV 257 nm., pump ŽShimadzu LC-9A., and chromatopak ŽShimadzu CRG-A.. In extraction of APM, samples Ž0.05 to 0.1 g wet. were added to 5 ml of mobile phase, and homogenized with an ultrasonic cell disrupter for 1 min, and then centrifuged at 3000 rpm for 40 min. The supernatant was filtered using Sep-pak C 18 cartridge Žpore size filter of 0.45 m m., and 20 m l filtrate was injected into the HPLC system. On the other hand, AsA of diets and fish organs such as liver and brain were quantified by the method of Speek et al. Ž1984. with a slight modification. The analysis was conducted by HPLC using a reversed-phase column ŽDevelosil ODS-UG-5, 4.6 mm = 150 mm, Nomura Kagaku.. Mobile phase Žflow rate: 1 mlrmin. was an aqueous solution of 0.08 M KH 2 PO4 : methanol, 80:20 and 5 ml of 3% metaphosphoric acid. The instruments used in the HPLC system were as follows; column oven ŽShimadzu CTO-6A, temperature 408C., fluorescence HPLC monitor ŽShimadzu RF-535, Ex 345 nm, Em 420 nm., pump ŽShimadzu LC-9A., and Chromatopak ŽShimadzu CRG-A.. In extraction of AsA, two or three fish from each replicate tanks were taken and dissected, then five tissue samples per treatment were used for the analysis. Tissue samples Ž0.3 g wet weight. were added to 5 ml mobile phase, and homogenized with an ultrasonic cell disrupter for 1 min, and then centrifuged at 12,000 rpm for 10 min. The supernatant was mixed with 0.04 ml 0.2% 2,6-sodium dichloroindophenol solution, 0.04 ml 3% thiourea solution Ž50% ethanol., 0.4 ml 50% sodium acetate, and 0.4 ml 0.1% o-phenylene diamine solution, and then filtered using Sep-pak C-18 cartridge Žpore size filter of 0.45 m m.. Finally, 20 m l filtrate was injected into the HPLC system. All samples for the analysis were stored in a freezer at y858C until used.

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2.4. ‘ Air diÕe’ test Tolerance against exposure to air was examined according to Koshio et al. Ž1997.. A black vinyl sheet with small holes Ž40 holesr100 cm2 . was spread out with a slight sag on a plastic container Ž36 cm = 70 cm = 25 cm.. A total of 10 fish from each treatment group were placed on the vinyl sheet for 4 min. After exposure to the air, fish were immediately transferred to a net cage in a flow through system for 2 h, and survival Ž% s survivorr10 fish. was recorded. The ‘air dive’ test was conducted four times on each tank, and the survival values for each treatment group were pooled Žeight data per treatment. and then mean values were calculated. 2.5. BehaÕioral obserÕation The schooling behavior such as schooling pattern, spontaneous activity, and distance to the nearest neighbors were observed. After the growth trial, 10 fish ŽTL: 8.7 " 0.9 cm. were randomly chosen from each rearing tank and acclimated for 1 h in the experimental tank before the observation. Fish behavior in the tank was recorded by an 8 mm video camera ŽHandycam CCD, Sony. for 10 mins. Eight green cylindrical polycarbonate tanks 88 cm in diameter, containing 250 l seawater Ž30 cm depth. in a 258C water bath, were used as experimental tanks. Behavioral observations were conducted two times at each rearing tank, then the parameters within the following three categories of each treatment groups Žfour times in total. were pooled and mean values were calculated. 2.5.1. Schooling pattern Two schooling patterns were defined according to Tsukamoto and Uchida Ž1990.. These were ‘aggregation’; a mass of fish aggregating without parallel orientation to neighbors, and ‘schooling’; a maneuverous swimming with parallel orientation to neighbors. These patterns were determined by applying each 1 min frame for 30 s in 10 min. Video images accounted for a total of 10 times and 300 s for each observation. Schooling rates, expressed as % of schooling against 10 times analyses, were determined. 2.5.2. Spontaneous actiÕity All 10 individuals from each experimental tank were discriminated by the video image analysis system ŽLA525, PIAS.. This system can recognize fish from background color by threshold brightness and therefore both swimming distance and distance between heads of nearest neighbors can be measured for each fish on each consecutive frame of video record ŽSakakura and Tsukamoto, 1996.. The activity, expressed in cm, was measured by analyzing continuous frames of 30 s video record Ž30 frames in 1 s. for each 1 min of video image. Mean values of swimming speed were obtained from the analysis of 9000 video frames, and spontaneous activity determined from swimming distance for 1 min was calculated for each fish and pooled for each treatment group. 2.5.3. Distance to the nearest neighbor (DNN) DNN Žcm. in terms of the distance between heads of nearest neighbors was determined from the same video image used in the analysis of spontaneous activity, and

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the mean DNN value of each fish in 30 s was calculated from the video image analysis system ŽSakakura and Tsukamoto, 1996.. 2.6. Statistical analysis One-way analysis of variance ŽANOVA, Super Anova, Abacus Concepts, Berkeley, CA. was applied to the data and the Tukey–Kramer comparison ŽSuper Anova, Abacus Concepts. was used to determine significant differences between individual treatments when significance Ž P - 0.05. of factors was detected by ANOVA.

3. Results 3.1. Growth, surÕiÕal and AsA accumulation in liÕer and brain Survival during the growth trial was very high Žover 90%; Table 3. and no AsA deficiency signs were observed during the trial. There was no significant difference in the growth parameters such as TL, body weight, WG, FCE, and CF as shown in Table 3. The analytical results of AsA concentration in liver and brain for yellowtail fed on test diets is shown in Fig. 1. AsA concentration in liver increased with increasing dietary APM content. On the other hand, AsA concentration per unit weight in brain was higher than that in liver of any of the test groups. Brain AsA seemed to level off at 1000 mg APMrkg diet since the concentration was not significantly different between fish fed 1000 mg and 2000 mg APMrkg diet. As shown in liver, AsA concentration in fish fed on APM-free diet was the lowest Ž P - 0.05.. 3.2. ‘ Air diÕe’ and behaÕior It is likely that tolerance against air exposure, determined by the ‘air dive’ test, increased with increasing dietary APM and peaked at 1000 mg APMrkg diet although no statistical significance was detected ŽFig. 2Ž1...

Table 3 Results of growth trial Diet ŽAPM mgr kg diet.

Survival Ž%. a

Final mean total length Žcm. b

Final mean weight Žg. b

Weight gain Ž%. a,c

Feed conversion efficiency a,c

Condition factor b,c

Free 400 mg 1000 mg 2000 mg

93.7 91.8 91.7 91.7

8.3"0.1 8.2"0.3 7.9"0.3 7.9"0.5

5.2"0.2 5.3"0.5 4.7"0.4 4.9"0.9

1105 1124 987 1035

1.1 1.1 1.0 1.1

8.8"0.1 9.3"0.1 8.9"0.2 9.2"0.1

a

Mean value of duplicate tanks. Mean value"S.D. of duplicate tanks. c Weight gains Žfinal wet weight Žg.yinitial wet weight Žg..=100rinitial wet weight Žg.. Feed conversion efficiency s wet weight gain Žg.rdry diet intake Žg.. Condition factor s final wet weight Žg.=1000rŽtotal length Žcm.. 3 . b

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Fig. 1. AsA concentration Ž m grg wet weight. in liver and brain of yellowtail fed diets containing different contents of APM for 20 days. For liver and brain, each bar with different letters represent significantly different mean values among dietary treatments Ž P - 0.05..

Schooling rates were significantly higher Ž P - 0.05. at groups of 1000 and 2000 mg APMrkg diet, and those fish had uniform schooling ŽFig. 2Ž2... In the APM-free group, the schooling rate was under 50% and typical aggregation was observed, whereas the

Fig. 2. Ž1. Percent survival Žrecovery. after air exposure when yellowtail fed diets containing different contents of APM for 20 days. Ž2. Schooling rate Ž%. of yellowtail fed diets containing different contents of APM for 20 days. Bars with different letters represent significantly different mean values among dietary treatments Ž P - 0.05.. Ž3. Spontaneous activity Žcmrmin. of yellowtail fed diets containing different contents of APM for 20 days. Ž4. DNN Žcm. of yellowtail fed diets containing different contents of APM for 20 days.

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schooling rate at 400 mg APM was an intermediate value between the APM-free and high-APM supplemented groups. Both spontaneous activity and DNN of the APM-free group showed the lowest values although statistical significance was not detected ŽFig. 2Ž3.Fig. 2Ž4... 4. Discussion The present study demonstrated that dietary AsA affects the schooling behavior of yellowtail as reported in ayu ŽKoshio et al., 1997.. Although there were no deficiency signs of AsA or growth differences in any treatment group during the 20-day trail, fish fed on higher dietary APM formed normal schooling pattern but those fed on APM-free diet showed an aggregated one. DNN data also indicated that fish fed on APM supplemented diets kept an optimal distance in schooling, which is the same distance as their body length ŽPitcher and Partridge, 1979.. Both spontaneous activity and DNN in fish fed on APM-free diet seemed to be lower than APM-fed fish. These results demonstrate that fish fed on APM-free diet have a trend of aggregating together and therefore could not form a normal schooling pattern. Therefore, it seems that the onset of schooling behavior may be correlated with AsA storage in their body, reflecting that AsA accumulations in both brain and liver of fish fed on APM-free diet were the lowest whereas fish fed on APM supplemented diets showed significantly higher AsA accumulations in both brain and liver. Furthermore, the AsA accumulation in brain appears to satiate when fed on diet containing more than 1000 mg APMrkg diet, the level which corresponded to the highest schooling rate. As pointed out by the study of Koshio et al. Ž1997., ascorbate might accelerate transmission of substances in relation to the central nervous system for fish. Several studies on mammals reported that ascorbate acts as a neuromodulator of noradrenergic, dopaminergic and cholinergic neurotransmission ŽKuo et al., 1979; Gardiner et al., 1985; Rebec and Pierce, 1994., and is a possible factor which can control cognitive, emotional and sensomotory behaviors. In fish, Johnston et al. Ž1989. reported in the study of rainbow trout that in very young fish AsA deficiency, brain serotonin levels and weight gain were related. Further investigation is necessary to clarify the interaction between AsA and neurotransmitters for fish. Under wild conditions, juvenile yellowtail form schools and aggregate around drifting seaweed ŽUchida et al., 1958; Anraku and Azeta, 1965. with individuals of similar size and age ŽSafran, 1990; Sakakura and Tsukamoto, 1997.. Since normal schooling behavior is recognized as a functional avoidance against predators ŽPitcher and Parrish, 1993., it is likely that yellowtail seedlings fed high dosage of AsA might be able to avoid predators in the wild after release. One of the major causes for high mortality of recently released fish is predation since fish just after release are in a panic condition and are, therefore, caught easily by predators ŽTsukamoto et al., 1989.. This incidence can be reduced by normal onset of the tilting behavior of red sea bream ŽUchida et al., 1993. and the schooling behavior of yellowtail as a protection behavior against the predators. Although the result of the ‘air dive’ in our 20-day trial statistically failed to prove that the stress tolerance enhances by increasing dietary AsA level, we could expect much

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clear difference on the stress tolerance when the longer feeding period was applied. Therefore, the high AsA intake might be necessary to produce seedlings with a stronger tolerance ability against handling stress such as transfer, release, and the temporal rearing before release. Several workers have studied the effect of dietary AsA level on stress ŽLovell and Lim, 1978; Mazik et al., 1987; Dabrowska et al., 1991.. The requirement of AsA increased in channel catfish stocked at high densities ŽLovell and Lim, 1978. and the same species fed AsA-free diets were more sensitive to physiological stress such as ammonia and oxygen level in rearing water than those fed AsA supplemented diets ŽMazik et al., 1987.. Thus, by controlling dietary AsA levels, it is possible to improve the quality of yellowtail for release in terms of the resistance against stress as well as schooling behavior. Although Kanazawa et al. Ž1992. reported that the optimal level of dietary APM for sustaining good growth and survival, and for preventing deficiency signs of juvenile yellowtail ranged from 30 to 60 mg APMrkg diet, the present study demonstrated that these levels were not high enough to develop the onset of a normal schooling pattern for yellowtail juveniles. They started the growth trial using fish with about 2.7 g Ž6 cm in TL., and fish fed APM-free diet all died within 44 days. On the other hand, the initial weight of yellowtail and rearing period in the present study was 0.4 g Ž3.6 cm in TL., and 20 days, respectively. Therefore, 30 to 60 mg APMrkg diet is not adequate to improve the stocking effectiveness but enough for optimal growth and prevention of deficiency signs. Since it is likely that the AsA requirement depends on the developmental stages, such as pre-larval, post-larval, early juvenile, young, and matured stages, more detail investigations of the AsA requirement on this species will be required to determine the optimal dietary AsA for improving fish quality. Acknowledgements The present study was partly supported by the Japan–US cooperative science program, Japan Society for the Promotion of Science. We are grateful to Prof. K. Dabrowski at Ohio State University for his critical discussion. We express our sincere gratitude to Mr. K. Maruyama, Dr. M. Arimoto, Mr. S. Shiozawa and all the staff at Goto Station of JASFA for providing the experimental fish and rearing facilities. Thanks are also due to Mr. Y. Hidaka and Mr. Y. Nukutani for their support of the experiments. Finally, many thanks are given to Sakamoto Feed for providing ingredients of yellowtail diets. Y. Sakakura was partly supported by Fellowships for Japanese Junior Scientists. References Anraku, M., Azeta, M., 1965. The feeding habits of larval and juveniles of yellowtail, Seriola quinqueradiata Temminck et Schlegel, associated with floating seaweeds. Bull. Seikai Reg. Fish. Res. Lab. 33, 14–15. Association of Official Analytical Chemists, 1990. Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Arlington, VA, 1141 pp. Dabrowska, H., Dabrowski, K., Meyer-Burgdorff, K., 1991. The effect of large doses of vitamin C and magnesium on stress responses in common carp, Cyprinus carpio. Comp. Biochem. Physiol. A Physiol. 99, 681–685.

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