Aquaculture 285 (2008) 184–192

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Effect of zinc and manganese supplementation in Artemia on growth and vertebraldeformity in red sea bream (Pagrus major) larvae

Van Tien Nguyen a,c, Shuichi Satoh a,⁎, Yutaka Haga a, Hiroshi Fushimi b, Tomonari Kotani b

a Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japanb Department of Marine Biosciences, Fukuyama University, Onomichi, Hiroshima 722-2101, Japanc Department of Applied Biosciences, Research Institute for Aquaculture No.1, Tu Son, Bac Ninh, Viet Nam

⁎ Corresponding author. Tel.: +81 3 5463 0557; fax: +E-mail address: ssatoh@kaiyodai.ac.jp (S. Satoh).

0044-8486/$ – see front matter © 2008 Elsevier B.V. Adoi:10.1016/j.aquaculture.2008.08.030

a b s t r a c t

a r t i c l e i n f o

Article history:

Feeding trials were carried o Received 27 May 2008Received in revised form 19 August 2008Accepted 21 August 2008

Keywords:Pagrus majorRed sea breamZincManganeseSkeletal deformityArtemia enrichment

ut to determine the effects of zinc (Zn) and manganese (Mn) supplementation inArtemia on growth, survival, body composition and skeletal deformity of red sea bream larvae. Triplicategroups of red sea bream larvae from 15–30 day post-hatching (dph) were fed four types of Artemia enrichedwith Zn (Z), Mn (M), both Zn and Mn (ZM) and without Zn or Mn (control). At 30 dph, significantly higher(Pb0.05) growth performance of the fish was recorded in M group (TL=15.60±0.45 mm) compared to that ofthe control (TL=14.90±0.41 mm). Fish fed Artemia supplemented with only Zn and with both Zn and Mnshowed similar growth performance compared to that of the control. Survival of the fish was not affectedeither by Zn or Mn supplementation. Increased Mn or both Zn and Mn in Artemia nauplii significantlyelevated (Pb0.05) crude lipid content in 30dph juvenile compared to that in the Z group. At 30 dph, Mncontent in juvenile of M and ZM groups was significantly higher (Pb0.05) compared to that in the othergroups. Similarly, Zn content in the Z group was significantly higher (Pb0.05) compared to that in the M andcontrol groups. Skeletal deformities in the experimental fish at 30 dph were highest (Pb0.05) in the controlgroup and were significantly improved by supplementation with Zn and Mn. The major skeletal deformitieswere observed in the vertebral column, neural and hemal spines. In the vertebral column, occurrence ofdeformities in the neck, hemal and preural were higher than in other regions. The results of the present studydemonstrated that maintenance of Mn level in Artemia nauplii from 12 to 42.8 µg g−1 (dry-matter basis)improved growth performance of red sea bream larvae. Zn and Mn supplementation in Artemia promotednormal skeletal development of red sea bream larvae.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Zinc (Zn) is an essential nutrient for normal growth and skeletaldevelopment inhumansandotheranimals includingfish (Calhounet al.,1974, 1975; Yamaguchi et al., 1987; Watanabe et al., 1997; Yamaguchi,1998; Ovesen et al., 2001; Yamaguchi and Fukagawa, 2005). Zinc hasbeen demonstrated to have a stimulatory effect on bone formation andmineralization (Yamaguchi et al., 1987). The metal directly activatesaminoacyl-tRNA synthetase in osteoblastic cells, and it stimulatescellular protein synthesis (Yamaguchi, 1998). Supplementation of Zn inthe diet of growing rat increased their bone strength (Ovesen et al.,2001). Dietary Zn deficiency causes retardation of ectopic bone for-mation (Calhoun et al., 1975). In pregnant animals, Zn deficiency causedcongenital bone deformities in their offspring (Calhoun et al., 1974). Infish, Zn deficiency caused growth retardation, dwarfism, cataract, ero-sion of fins and skin and reducedwoundhealing in rainbow trout (Satohet al., 1983; Watanabe et al., 1997). Therefore, supplementation of Zn to

81 3 5463 0553.

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fish larvae is necessary for normal development and reduces structuraldeformities caused by Zn deficiency.

Dietary manganese (Mn) is important, even in small quantity, fornormal growth of animals.Mn functions as a cofactor for a large numberof enzymes and forms metal-enzyme complexes or acts as an integralpart of certain metalloenzymes in carbohydrate, lipid and proteinmetabolism (Watanabe et al., 1997; Lall, 2002). In vertebrates, Mn isessential for development of the organic matrix of the bone, which islargely composed of mucopolysaccharides. Manganese-deficient bonesare considerably shortened and thickened (McDowell,1992). Satoh et al.(1983, 1987b, 1989) reported that dietary Mn deficiency caused growthretardation in rainbow trout and common carp fed white fish meal-based diets. Other Mn deficiency signs such as dwarfism linked todisturbances in bone formation and cataracts of eye lens have beenobserved in rainbow trout and carp (Satoh et al., 1987b, 1991). A reduc-tion of Mn content in bone corresponding to insufficient dietary supplyof Mn has also been reported (Satoh et al., 1983).

Skeletal deformities are major problems in hatchery-reared larvaeof marine fish. These problems have serious impacts on the industry'sproductivity, on product quality, animal welfare and causes significanteconomic loses (Divanach et al., 1996). The skeletal deformities in fish

185V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

associated with exposure to harmful or sub-optimal conditions suchas physical, chemical, environmental variables, infection organisms orinbreeding have been proposed. Effect of various nutrients on skeletaldeformities in fish have been reviewed by Cahu et al. (2003) and Lalland Lewis-McCrea (2007). These authors reported that lipid fractions(phospholipid, DHA) in live prey, protein fractions, vitamin A and Cwere the major nutrients affecting skeletal development in marinefish. Pathogenic signs in skeletal development linked to mineral defi-ciency have been well documented in terrestrial animals but havereceived only limited attention in marine fish larvae.

The whole body Zn and Mn contents decrease rapidly as a con-sequence of the deletion of Zn and Mn in diets fed to rainbow troutswim up fry for 40 weeks (Satoh et al., 1987d), suggesting that Zn andMn are essential for fish larvae and juveniles. In nature, zooplanktoncontains higher Zn content than that in newly hatched Artemia. Zncontents (in dry-matter basis) were about 700 µg g−1 in Acartia clausi(Watanabe et al., 1978), 123.8 µg g−1 in Calanus finmarchicus and161.0 µg g−1 in Calanus cristatus (Fujita, 1972). In our experiment, nonenriched Artemia contained about 104 µg Zn g−1, lower than that innatural zooplankton. Therefore, it is necessary to improve the Zn inArtemia through enrichment. As Zn and Mn are both important forskeletal development and growth, supplementation of these elementsin Artemia is necessary for red sea bream larvae.

Skeletal deformities are observed more frequently in early de-velopmental stage than later, especially during skeletal formation.Marine fish larvae undergo different changes in skeletal and organsformation during its development. Supplementation of Zn and Mn tolive prey for feeding larvae during their skeletal formation and ossi-fication may be a solution to the problem of Zn and Mn deficiencies,such as skeletal deformities and reduced growth. Hence, this studywas carried out to investigate the effects of Zn and Mn supplementa-tion in Artemia on growth, body composition and skeletal deformitiesof red sea bream larvae.

2. Materials and methods

2.1. Experimental fish

Fertilized eggs of red sea bream (Pagrus major) were obtained fromBio Ehime Co, Ltd., Japan and maintained at the Laboratory of Aqua-culture and Stock Enhancement, Department of Marine Science,Fukuyama University, Hiroshima, Japan. After acclimatization, 22,000fertilized eggs were stocked in each treatment tank with a volume of1000 L.Hatchingpercentagewas around90%based on core samplings at0 and 3 dph (n=5). The stocking density was adjusted to approximately20,000 larvae per tank.

Fig. 1. Feeds, feeding schedule and water exchange rate for red sea bream larvae during 0–30and moisture 7.9%. Minerals (dry-matter basis): zinc 198.8 µg g−1, manganese 88.4 µg g−1.Belgium): crude protein 6.2%, crude lipid 2.0%, moisture 89.7%, and ash 1.4%. Minerals (dry-

2.2. Experimental design and Artemia enrichment method

Four Artemia enrichment treatments were designated for thefeeding trial. Where, an Artemia group enriched with marine ω A®

(Nisshin Marine Tech Co., Ltd., Japan) but without added Zn or Mnserved as the control. In the treatment groups, Artemia nauplii en-riched with marine ω A® added with 0.1 mg Zn mL−1 served as the Ztreatment; withmarineω A® added 0.24 mgMnmL−1 served as theMtreatment; Artemia enriched with marine ω A® added both 0.1 mg Znand 0.24 mg Mn mL−1 served as the ZM treatment.

Prior to enrichment, the marine ω A® was added along with Znand Mn to prepare the 4 treatments (Z, M, ZM and control) andincubated for 2 h. Newly hatched Artemia franciscana nauplii (E.G.grade, INVE, Belgium) were stocked into 60 L containers at a density of100–150 nauplii mL−1, and then enriched with each prior-preparedmarineω A® (4.5mLmarineω A® per 1 L enrichmentmedia) for 32 h at25 °C. Dissolved oxygen in the enrichment containers was maintainednearly 110% saturation by vigorous aeration. The Artemia nauplii werepartially harvested 3 times at 24, 29 and 32 h after enrichment and thenfed to the red sea bream larvae.

2.3. Feeding trial

Red sea bream larvae were reared in 1000 L flow-through blackpolyethylene tanks equipped with a drain pipe in the center and 4 airstones. Water temperature was controlled from 21 to 22 °C by 1.0 KWheaters placed in each tank during the experiment period. Cartridge-filtered seawater (mesh size 10 µm) was supplied continuously. Dailywater exchange was from 30 to 300% (Fig. 1). Photoperiod was 12 hdark and 12 h light by using a 20 W neon lights located above eachtank.

Feed and feeding schedule is shown in Fig. 1. The euryhaline rotiferBrachionus plicatilis (L strain) was used as the initial larval feed from2–20 dph. The rotifers were enriched with 25 g DHA protein Selco(INVE Aquaculture, Inc., Belgium) per 100 L media at a density of about1000 rotifermL−1 at 25 °C for 6–8h. The enriched rotiferswere fed to thelarvae2 times daily at 08:00 and 16:00 h, at 5 rotifersmL−1. The enrichedArtemiawere fed 3 times daily at 08:00; 14:00 and 17:00 h to the larvaefrom 15 to 30 dph. The feeding rate of Artemia varied with the day afterhatchingof the larvae. This feeding ratewas 12 nauplii per a larva during15–17 dph; 28 nauplii during 18–24 dph; 56 nauplii during 25–27 dphand 28 nauplii during 28–30 dph (Fig. 1). During the co-feeding ofrotifer and Artemia period, the feeding times of larvae fed rotiferswere 09:00 and 15:00 h. Weaning of the larvae was accomplished byusing NRD larval diet (INVE Aquaculture, Inc., Thailand) fed every 2 hfrom 21–30 dph.

dph. Compositions of NRD ½ (INVE Thailand) diet: crude protein 55.2%, crude lipid 9.1%,Compositions of rotifer (L strain of B. plicatilis, enriched with DHA protein selco (INVEmatter basis): zinc 55.2 µg g−1, manganese 11.4 µg g−1.

Fig. 2. Change in survival (%) of red sea bream larvae from started feeding with enrichedArtemia from 15 to 31 days post-hatching. Data represent mean of the results in 3triplicate tanks, and values indicatemean±S.D. (n=3) at the end of the rearing experiment.

186 V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

2.4. Air-dive challenge test

At 30 dph, 20 fish from each tank were air-dived for 2 min by usinga scope net then returned to a 5 L bucket filled with seawater. Survivalafter the challenge test was calculated based on the number of sur-vivors at 24 h after the challenge.

2.5. Sampling and analytical procedures

Newly hatched Artemia nauplii were sampled prior to enrichment.After enrichment, Artemia were sampled at 24, 29 and 32 h by aplankton net mesh size 150 µm. The samples were rinsed withseawater, labelled and then stored at −30 °C. Before analysis, thesamples were thawed to room temperature and then centrifuged at2000 rpm for 20 min. The samples were used for moisture, ash andmineral analysis after the supernatant was discarded.

To measure growth performance, 20 fish from each tank wererandomly sampled every 5 days from hatching to 25 dph (0, 5, 10, 15,20, 25) and 50 fish at 30 dph for measuring total and standard bodylength. The fish were magnified by a profile projector (V-12B Nikon)and measured by using a digital calliper. The specimens were thenfixed in 5% neutralized formalin and stored at 4 °C for skeletal exami-nation. Fish samples for proximate composition, fatty acid andmineralanalysis were sampled at 30 dph. The fish were starved for 24 h foremptying of gut contents and faeces before sampling. About 100 g

Table 1Moisture, ash and mineral compositions (dry-matter basis) of Artemia before and after 24 h

Parameters Initial(⁎) 24 h enrichment 29 h enrichmen

Control Z M ZM Control Z

Moisture (%) 85±0.0 89±0.1a 90±0.1a 90±0.1a 90±0.2a 89±0.0⁎ 8Ash (%) 9±0.4 14±0.7a 13±0.4a 15±0.5a 14±0.2a 13±0.8⁎ 1Zn (µ g−1) 104±1.5 119±0.4a 306±6.0c 126±2.1a 284±0.9b 114±0.3⁎ 34Mn (µ g−1) 8±0.2 10±1.0a 9±1.0a 35±3.3c 13±1.0b 12±3.3⁎ 1P (mg g−1) 5±0.8 17±1.2a 16±1.1a 18±1.8a 17±0.3a 19±2.7⁎ 1Fe (µ −1) 107±1.4 767±8.4c 593±2.9a 772±10.7c 630±2.7b 901±6.0⁎⁎⁎ 74Ca (mg −1) 1±0.0 2±0.0a 2±0.1a 2±0.0a 2±0.1a 2±0.0⁎Mg (mg −1) 4±0.0 5±0.1a 5±0.1a 5±0.0a 5±0.1a 5±0.0⁎Na (mg −1) 18±0.5 33±0.6b 31±0.5a 34±0.7b 32±0.9a 29±0.1⁎ 3K (mg −1) 14±0.1 15±0.5a 16±0.3a 15±0.9a 15±0.2a 15±0.1⁎ 1Cu (µ −1) 6±0.4 12±1.7a 13±1.6a 14±0.3a 12±0.7a 14±1.0⁎ 1

Control=neither Zn norMn supplementation, Z=supplementedwith Zn 0.1 mgmL−1 marineZn 0.1 mg g, Mn 0.24 mg mL−1 marine ω A®, ⁎initial=newly hatched Artemia nauplii. Resultsdonewith four treatments: Control, Z, M, and ZM at each sampling point: 24, 29 and 32 h fromdifferent (Pb0.05).

pooled samples from each tank were minced and homogenized. Thehomogenates were labelled and stored at −30 °C until analysis.

Survival of fish during the Artemia feeding stage was calculatedbased on the number of survivors that were individually counted atthe end of the experiment and dead fish from each tank counted eachday. Initial fish numbers in each tank at 15 dph were calculated as asum of survivors at the end of the experiment plus dead fish duringthe 16 to 30 dph period. Percent survival during the Artemia feedingstage (15–30 dph) was calculated daily and depicted in Fig. 2.

Samples for mineral analysis were digested in 5 mL nitric acidusing a MLS-1200 mega microwave digestion system (Nihon GeneralCo. Ltd., Japan), cooled under flowing tap water for 30 min thendiluted with de-ionized water to 50 mL. Concentrations of Zn, Mn, Fe,Ca, Mg, Na, K and Cu were determined by Polarized Zeeman AtomicAbsorption Spectrophotometer (Hitachi Z-500, Tokyo, Japan) (Satohet al., 1987a,b,c). Phosphorus was analyzed by using a UV spectro-photometer (Shimadzu, UV 265FW, Kyoto, Japan) at 750 nm.

Moisture and ash were measured by gravimetric methods. Nitrogenwas analyzed by an automated Kjeldahl technique, and crude proteinwas calculated as N×6.25. Total lipid content was determined gra-vimetrically by themethod of Folch et al. (1957). Fatty acidmethyl esterswere prepared from the total lipid by saponification and methylesterification (AOCS, 1990). Fatty acids composition was identified bygas liquid chromatography (GC 15A; Shimadzu, Kyoto, Japan). Individualfatty acids were identified by comparison with a fish oil standard andrepresented as a percentage area of themethyl ester (Kiron et al., 2004).

To determine skeletal deformity, 50 juveniles at 30 dph from eachtank were randomly sampled, anesthetized in ice water, fixed in 5%neutralized formalin and then stored in 4 °C storage until analysis.Before staining, specimens were dehydrated by placing in 50% ethanolsolution for 1 day, followed by absolute ethanol (99.9%) for 1 day. Thespecimens were then cleared and double stained with alcian blue andalizarin red, respectively, by the method of Potthoff (1984) with minorrevisions. To prevent ossified bone from decalcification, cartilagestaining was conducted within 4 h. Cleared and stained specimenswere examined for skeletal deformities by using a stereomicroscope.Criteria for skeletal deformity in red sea bream larvae were accordingto Matsuoka (1987).

2.6. Statistical analysis

Analytical data are represented as mean±standard deviations. Sta-tistical analyses were carried out using Statistica release 6.0 (StatSoft,2001). Each experimental group was run in triplicate, resulting in n=3for each treatment. The data were examined using one-way ANOVA.Differences among treatments were determined by Duncan's test.

, 29 h and 32 h enriched with marine ω A®

t 33 h enrichment

M ZM Control Z M ZM

9±0.0⁎ 89±0.1⁎ 89±0.1⁎ 90±0.01 89±0.31 90±0.11 90±0.11

4±0.3⁎ 14±0.1⁎ 13±0.4⁎ 14±0.91 13±0.11 13±0.71 13±0.41

7±1.0⁎⁎⁎ 121±6.1⁎ 317±1.0⁎⁎ 121±0.41 423±1.04 148±0.52 363±4.33

2±1.0⁎ 35±3.4⁎⁎⁎ 19±2.2⁎⁎ 16±0.42 15±1.01 43±5.24 25±1.93

9±0.5⁎ 18±0.8⁎ 17±1.7⁎ 21±1.21 20±1.31 20±0.41 18±0.41

2±5.2⁎⁎ 905±8.0⁎⁎⁎ 634±9.7⁎ 1059±3.64 917±8.32 995±2.53 684±2.81

2±0.2⁎ 2±0.2⁎ 2±0.0⁎ 2±0.02 2±0.02 2±0.12 1±0.01

5±0.1⁎ 5±0.1⁎ 5±0.1⁎ 5±0.11 4±0.11 5±0.11 4±0.11

1±0.4⁎⁎ 30±0.2⁎ 30±1.0⁎ 30±1.32 28±0.11 30±0.32 28±0.31

5±0.8⁎ 15±0.9⁎ 15±0.6⁎ 15±0.41 15±0.51 15±0.21 16±0.11

4±1.2⁎ 13±1.2⁎ 13±1.4⁎ 13±0.11 13±0.81 13±0.81 13±1.01

ω®, M=supplementedwith Mn 0.24mgmL−1 marineω A® and ZM=supplemented withare means from three samples±with their standard deviation. One-way ANOVAs wereenrichment. Means in the same raw not sharing a common superscript are significantly

Table 2Growth performance of red sea bream larvae during 30 days nursing

Treatments Significance

Control Z M ZM Zn Mn Zn×Mn

Total length (mm)0 day (n=50) 2.5±0.10 2.5±0.10 2.5±0.10 2.5±0.105 day (n=20) 3.5±0.22 3.6±0.11 3.6±0.10 3.5±0.1110 day (n=20) 4.9±0.11 4.9±0.13 4.9±0.28 4.9±0.1315 day (n=20) 6.3±0.33 6.1±0.19 6.1±0.16 6.2±0.19 n.s. n.s. n.s.20 day (n=20) 8.9±0.28 8.8±0.30 8.7±0.60 9.5±0.55 n.s. n.s. n.s.25 day (n=20) 14.1±0.43 14.5±0.35 14.2±0.30 14.5±0.51 n.s. n.s. n.s.30 day (n=50) 18.0±0.26a 18.7±0.31ab 19.1±0.54b 18.8±0.33ab n.s. b0.05 n.s.

Standard body length (mm)0 day (n=50) 2.5±2.47 2.5±2.47 2.5±2.47 2.5±2.475 day (n=20) 3.4±0.23 3.4±0.11 3.4±0.17 3.4±0.1010 day (n=20) 4.7±0.12 4.7±0.12 4.7±0.25 4.7±0.1415 day (n=20) 5.9±0.32 5.8±0.15 5.8±0.13 5.9±0.14 n.s. n.s n.s.20 day (n=20) 7.4±0.16 7.6±0.54 7.9±1.64 8.0±0.31 n.s. n.s n.s.25 day (n=20) 11.5±0.28 11.7±0.30 11.4±0.28 11.7±0.36 n.s. n.s n.s.30 day (n=50) 14.9±0.14a 15.2±0.12ab 15.6±0.45b 15.3±0.29ab n.s. b0.05 n.s.

Control=neither Zn nor Mn supplementation, Zn=zinc supplementation, Mn=manganese supplementation, ZM=zinc and manganese supplementation, Zn×Mn=interactionbetween Zn and Mn supplementation. Results are means from three tanks±with their standard deviation (n.s.=not significant). Means in the same raw not sharing a commonsuperscript are significantly different (Pb0.05).

187V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

Values were considered significant at Pb0.05. Subsequently, two-wayANOVAs were carried out to determine the effects of Zn and Mn or aninteraction effect between Zn andMn supplementation. Homogeneityof variances was tested using Levene's test, and was considered to behomogenous when P wasN0.05.

3. Results

3.1. Moisture, ash and mineral contents in Artemia

Moisture, ash and mineral content of newly hatched and Artemianauplii at 24, 29 and 32 h enrichment are summarised in Table 1. Ateach sampling point, no significant difference in moisture or ashcontent of enriched nauplii among all treatments was observed. Sup-plementation of Zn markedly elevated the Zn in nauplii after enrich-ment. Where, the Zn content in Artemia of the Z and ZM groups wasaround 3 to 4 folds of that in the control and M groups. Similarly, theMn content in Artemiawas significantly higher (Pb0.05) in the M andZM groups compared to that of the control and Z groups. The Zncontent in Artemia nauplii was higher (Pb0.05) in the Z group than in

Table 3Proximate and mineral composition of red sea bream juvenile 30 day post-hatching fed Zn

Treatments

Control Z M

Proximate composition (%)Moisture 81.4±0.32 82.0±0.61 82.9±0.57Crude protein 13.0±0.51 12.3±0.54 12.9±0.26Crude lipid 2.3±0.11ab 2.1±0.11a 2.3±0.12ab

Ash 3.0±0.17 2.7±0.11 2.9±0.10

Mineral composition in dry basisZn (µg g−1) 74.0±2.91a 81.7±2.36b 72.7±2.12a

Mn (µg g−1) 6.8±0.26ab 6.4±0.44a 7.5±0.24b

P (mg g−1) 24.4±2.41 24.7±3.35 24.4±4.00Ca (mg g−1) 29.3±1.31 30.5±1.98 29.9±0.89Mg (mg g−1) 2.9±0.32 3.2±0.26 3.1±0.26Na (mg g−1) 20.4±1.87 22.8±2.29 22.2±1.70K (mg g−1) 22.6±1.52 23.1±0.16 22.5±1.07Fe (µg g−1) 58.1±1.95c 48.5±3.27ab 53.0±4.39bc

Cu (µ g−1) 2.8±0.10b 2.6±0.24ab 3.3±0.25c

Control=neither Zn nor Mn supplementation, Zn=zinc supplementation, Mn=manganesbetween Zn and Mn supplementation. Results are means from three tanks±with their stasuperscript are significantly different (Pb0.05).

the ZM group. Zn supplementation significantly reduced (Pb0.05) theaccumulation of iron (Fe) in enriched Artemia. At 3 sampling points,the Fe content in Artemia of the Z and ZM groups was significantlylower (Pb0.05) compared to that in the M and control groups.

3.2. Growth performance and survival

The growth performances as total and body length of the fish arepresented in Table 2. No significant difference in total length or bodylength among the larvae groups at 15, 20 and 25 days post-hatchingwas observed. At 30 dph, the fish fed nauplii enriched with Mn alone(M treatment) had significant higher (Pb0.05) growth performancecompared to that of the control group. Supplementation of Mn in Ar-temia significantly improved growth performance of the red sea breamlarvae, while Zn supplementation did not improve growth perfor-mance compared to that of the control group. No interactive effectbetween Zn and Mn on growth of the fish was observed.

Survival of red sea bream larvae during the Artemia feeding stage(from 15 to 30 dph) ranged from 61.1% in the ZM group to 68.5% inthe Z group (Fig. 2). No significant difference in survival among the

and Mn enriched Artemia

Significance

ZM Zn Mn Zn×Mn

81.9±0.32 n.s. n.s. n.s.12.8±0.24 n.s. n.s. n.s.2.4±0.05b n.s. b0.05 b0.052.7±0.06 n.s. n.s. n.s.

76.8±2.08ab b0.05 n.s. n.s.7.5±0.02b n.s. b0.05 n.s.

26.6±1.07 n.s. n.s. n.s.27.7±2.38 n.s. n.s. n.s.2.8±0.15 n.s. n.s. n.s.

19.6±1.51 n.s. n.s. n.s.24.1±1.62 n.s. n.s. n.s.43.9±0.69a b0.05 b0.05 n.s.2.2±0.08a b0.05 n.s. b0.05

e supplementation, ZM=zinc and manganese supplementation, Zn×Mn=interactionndard deviation (n.s.=not significant). Means in the same raw not sharing a common

Table 4Major fatty acid composition in whole body of red sea bream juvenile (at 30 dph)

Fatty acid (% area) Treatment Significance

Control Z M ZM Zn Mn Zn×Mn

14:0 0.5±0.0 0.6±0.1 0.5±0.0 0.6±0.0 n.s. n.s. n.s.16:0 17.6±0.1 18.0±0.0 17.9±0.4 17.7±0.5 n.s. n.s. n.s.16:1n-7 2.4±0.0 2.5±0.1 2.5±0.1 2.5±0.0 n.s. n.s. n.s.18:0 10.0±0.2 10.0±0.3 10.2±0.3 9.6±0.2 n.s. n.s. n.s.18:1n-(9+7) 19.5±0.3 19.0±0.1 19.7±0.5 19.3±0.0 n.s. n.s. n.s.18:2n-6 10.7±0.0 10.9±0.2 10.7±0.1 10.7±0.1 n.s. n.s. n.s.18:3n-3 9.8±0.1 7.9±0.2 9.5±1.0 9.8±0.4 n.s. n.s. n.s.18:4n-3 0.9±0.0 0.8±0.0 0.8±0.1 1.0±0.1 n.s. n.s. n.s.20:0 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 n.s. n.s. n.s.20:1n-(11+9) 0.9±0.0 1.0±0.0 1.0±0.0 0.9±0.1 n.s. n.s. n.s.20:2n-6 0.5±0.0 0.5±0.0 0.5±0.0 0.5±0.0 n.s. n.s. n.s.20:3n-6 2.5±0.2 2.5±0.1 2.3±0.1 2.2±0.0 n.s. n.s. n.s.20:4n-3 0.7±0.0 0.7±0.0 0.7±0.0 0.7±0.0 n.s. n.s. n.s.20:4n-6 0.8±0.0 0.7±0.0 0.8±0.0 0.8±0.0 n.s. n.s. n.s.20:5n-3 (EPA) 6.2±0.6 6.4±0.3 6.2±0.3 5.8±0.0 n.s. n.s. n.s.22:1n-(11+13+9) 0.2±0.0 0.3±0.1 0.3±0.0 0.3±0.0 n.s. n.s. n.s.22:4n-6 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 n.s. n.s. n.s.22:4n-9 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 n.s. n.s. n.s.22:5n-3 (DPA) 1.5±0.0 1.6±0.1 1.4±0.1 1.5±0.1 n.s. n.s. n.s.22:5n-6 0.4±0.0 0.5±0.0 0.4±0.0 0.4±0.0 n.s. n.s. n.s.22:6n-3 (DHA)a 9.0±0.5a 10.9±0.7b 8.9±0.7a 10.1±1.0ab b0.05 n.s. n.s.Saturates 28.3±0.3 28.7±0.3 28.9±0.6 28.1±0.7 n.s. n.s. n.s.Monounsaturates 23.0±0.5 22.7±0.3 23.4±0.5 22.9±0.0 n.s. n.s. n.s.Polyunsaturates 43.1±1.3 43.4±0.3 42.3±0.3 43.5±0.8 n.s. n.s. n.s.Total n-3 PUFA 28.0±1.1 28.2±0.2 27.5±0.2 28.8±0.7 n.s. n.s. n.s.Total n-6 PUFA 15.1±0.2ab 15.2±0.1b 14.8±0.2ab 14.7±0.2a n.s. b0.05 n.s.n-3 HUFA 17.3±1.1 19.6±0.4 17.1±1.2 18.1±1.1 n.s. n.s. n.s.

Control=neither Zn nor Mn supplementation, Zn=zinc supplementation, Mn=manganese supplementation, ZM=zinc and manganese supplementation, Zn×Mn=interactionbetween Zn and Mn supplementation. Results are means from three tanks±with their standard deviation (n.s.=not significant). Means in the same raw not sharing a commonsuperscript are significantly different (Pb0.05).HUFA n-3:≥20:3 n–3; HUFA n-6:≥20:2 n-6.

Fig. 3. Survival of red seabream juvenile at24h afterair-dive challenge test.Data representmeans of three replicates. Error bars represent standard deviations. Treatments sharingdifferent superscript letter are significantly different (Pb0.05).

188 V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

experimental groups was observed. Fig. 2 shows the increased mor-tality in all groups during the 20 to 24 dph period. The mortality wasa consequence of cannibalism and occurred in all treated and thecontrol groups.

3.3. Proximate composition and mineral content of 30 dph red sea breamjuvenile

At the end of experiment, no significant difference in moisture,crude protein or ash content in whole body samples was observed inthe experimental groups (Table 3). The fish fed Artemia enriched withboth Zn and Mn showed significantly higher (Pb0.05) crude lipid intheir whole body compared to those supplemented only with Zn.Significant effects of Mn and an interaction between Zn andMn on theincreased crude lipid content in the juvenile were observed.

Zinc and Mn supplementation significantly increased (Pb0.05)whole body Zn and Mn content at 30 dph. Zn content of 30 dphjuveniles was higher (Pb0.05) in the Z group compared to that of thenon-supplemented groups. Similarly, whole body Mn content wassignificantly higher in Mn supplemented groups (M and ZM) than thatof the non-supplemented groups (Z and control). Iron content in thelarvae was significantly lower (Pb0.05) as a result of Zn supplementa-tion. Whereas, the whole body Fe content was significantly lower(Pb0.05) in fish fed enriched nauplii supplemented with Zn (Z and ZMgroups) compared to that in the control group.

3.4. Fatty acid content of red sea bream larvae at 30 dph

Major fatty acid composition in the 30 dph red sea bream juvenilesis presented in Table 4. Significantly higher (Pb0.05) DHA content wasobserved in the fish fed Zn supplemented nauplii compared to theother groups. Even though not significantly different, total n-3 HUFAtended to be higher in the Zn supplemented groups: 19.6 and 18.1% inthe Z and ZM groups, respectively, compared to that of the control

(17.3%) and M (17.1%) groups. The total n-6 PUFA was significantlyaffected (Pb0.05) by Zn supplementation. Total n-3 PUFA, saturates,monounsaturates and polyunsaturates contents were not differentamong all groups.

3.5. Vitality in air-dive challenge test

Survival during the air-dive challenge test of the fish at 30 dphis shown in Fig. 3. Significantly higher survival (Pb0.05) at 24 hafter the challenge test was observed in the ZM group. After thechallenge test, no difference in survival among the Z, M and controlgroups was observed. In the control group, a large variation in sur-vival was observed. Results demonstrated that fish fed Artemia nau-plii enriched with both Zn and Mn improved their ability againststress condition.

189V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

3.6. Skeletal deformities

Typical skeletal deformities observed in 30 dph specimens(TL=18–20 mm) are presented in Fig. 4. Typical skeletal deformitieswere jaw deformity (A), pugheadness (B), lordosis and kyphosis (C),fused vertebral centrum (D), deformity in interneural spine (E) anddeformity in preural centra (F). Those deformities were observed inall fish groups. However, the occurrence of skeletal deformities variedamong the treatments.

A total of 38.7–52.7% of fish had at least one skeletal deformity inthe fish groups at 30 days post-hatching (Table 5). Fish fed Artemiaenriched with only Zn, only Mn, and both Zn and Mn showedsignificantly lower (Pb0.05) skeletal deformities compared to that ofthe control group. A higher frequency of deformities in the vertebralcolumn, neural spines and arches and dorsal fin rays regions wasobserved. Significant lower (Pb0.05) deformities in neural spines and

Fig. 4. Photographs showing typical skeletal deformities in cleared and stained specimensdeformity, (B) pugheadness, (C) lordosis and kyphosis, (D) fusion of vertebral centrum, (E) d

arches was recorded in the groups fed Zn orMn supplemented nauplii.Zinc supplementation lowered (Pb0.05) the incidence of malforma-tion in dorsal fin rays.

Deformities of the vertebral column in juvenile red sea bream at30 dph are shown in Fig. 5. Significantly higher frequency of vertebraldeformities (Pb0.05) was observed in the control group than in theother groups in all regions of the vertebral centrum. Higher incidenceof deformities in the vertebral column was observed between the 1–2nd and 10–14th vertebrae and in the preural centra (21–23rd). In theabdominal vertebrate, the incidence of winding (Fig. 4C) was higherthan vertebral fusion (Fig. 4D).

4. Discussion

In the present study, Mn supplementation (0.24 µg mL−1 marineω A®) to Artemia nauplii significantly improved growth performance of

of red sea bream juveniles at 30 days post-hatching, (average TL=18–20 mm). (A) jaweformity in interneural spine and (F) deformity in preural centra.

Table 5Frequency of occurrence of major skeletal deformities of the red sea bream juvenile at30 day post-hatching (%) and average percentage of fish with at least 1 skeletaldeformity

Deformities Treatments Significance

Control Z M ZM Zn Mn Zn×Mn

1. Jaw deformity 0.0 0.7 0.0 2.0 n.s n.s n.s2. Vertebral column 38.7 27.3 27.3 22.0 n.s n.s n.s3. Neural spine and arches 18.0b 8.0a 8.0a 12.7ab n.s n.s b0.054. Hemal spines and arches 4.0 3.3 2.0 4.0 n.s n.s n.s5. Dorsal fin ray andfin support

12.0b 2.7a 8.7ab 1.3a b0.05 n.s n.s

6. Ventral fin ray andfin support

0.0 0.0 0.0 0.0 n.s n.s n.s

7. Hypural 3.4 3.8 4.1 3.3 n.s n.s n.s8. Epural 2.1 1.8 2.7 1.7 n.s n.s n.sAverage deformity fish 52.7b 40.0a 41.3a 38.7a b0.05 b0.05 b0.05

Control=neither Zn nor Mn supplementation, Zn=zinc supplementation, Mn=manganesesupplementation, ZM=zinc and manganese supplementation, Zn×Mn=interactionbetween Zn and Mn supplementation. Results are means from three tanks. 50 specimensfrom each tank were examined. Means in the same raw not sharing a common superscriptare significantly different (Pb0.05), (n.s=not significant).

190 V.T. Nguyen et al. / Aquaculture 285 (2008) 184–192

red sea bream larvae. This finding suggests that Mn supplementation isrequired for red sea bream during larval stages. The importance ofdietaryMn in fish nutrition has been intensively reviewed byWatanabeet al. (1997). Satoh et al. (1983, 1987b, 1989, 1991) reported thatsupplementation of Mn was required for rainbow trout fed white fishmeal-based diets and common carp fed various types of fish meal dietsfor normal growth. Fish fed diets without Mn supplementation showedreduced growth and dwarfism. The requirement of common carp fordietary Mn was estimated to be 12–13 μg g−1 diet (Satoh et al., 1987b)and 15 μg g−1 diet for rainbow trout (Satoh et al., 1991). In the currentstudy, theMn content in newly hatchedArtemiawas 7.81 μg g−1 on a drybasis. This is much lower than the requirement of common carp orrainbow trout. The higher level of Mn in the enriched nauplii in the Mand ZM treatments (12–42.8 μg g−1) would act as a stimulating factorfor better growth performance in the experimental fish. Even thoughslightly better growth in total lengthoffish fed themineral supplementswas seen at 25dph, but the differencewasnot significant. The differencein total length became significant at 30 dph (Table 2). During 20–30 dph,the red seabreamgrew faster (average daily length increment=1.94mmday−1) compared to that at the 15–20 dph stage (average daily length

Fig. 5. Vertebral deformity in juvenile red sea bream at 30 days post-hatching.

increment=0.56 mm day−1). During this time, ossification of the ske-leton occurs rapidly. Supplementation of Mn and Zn during this periodeffectively improved growth performance and promoted normal skel-etal development of red sea bream larvae.

Water contains Zn and other trace elements but they are notabsorbed in sufficient amounts to meet the requirement of fish, thusdietary supplementation is considered a more significant source oftrace elements (NRC, 1993). Maage (1994) reported that Zn supple-mentation of 57–97 mg kg−1 diet improved growth performance ofAtlantic salmon fry, and Zn supplementation up to 1000 mg kg−1 dietshowed similar growth as fish fed diets supplemented with 57–97 mgZn kg−1. Satoh et al. (1987a,c) concluded that supplementation of Zngreater than 40 μg g−1 to white fish meal-based diets was necessaryfor normal growth of rainbow trout without the appearance ofdwarfism or cataracts. In juvenile Nile tilapia fed a plant ingredientbased diet, the optimal dietary Zn content was 79.51mg kg−1 (Carmo eSá et al., 2004). In the current study, the Zn content in enriched Ar-temia ranged from 118.6 μg g−1 in control group to 423.08 μg g−1 in theZ group after 32 h enrichment. Within this range, growth performanceof the fish groups fed nauplii supplemented with Zn (Z) and with bothZn and Mn (ZM) was not significantly different from that of the con-trol. The advantage of Zn supplementation therefore could not con-clusively be demonstrated considering growth data alone.

Carcass Zn andMn content of red sea bream at 30 dph reflected thedifference of Zn and Mn content in the enriched Artemia nauplii. Ofwhich, the higher Zn and Mn content in Artemia resulted in higher ZnandMn in the 30 dphfish. Lorentzen andMaage (1999) showed similarresults where the Zn in fish was increased as a result of increaseddietary Zn. Furthermore, results of the current study showed that Mnin Artemia nauplii in the Zn supplemented group was lower than thatof the control. A negative effect of Zn supplementation on the accu-mulation of Mn in rotifers has been reported by Matsumoto (2006).However, supplementation of both Zn andMn (ZMgroup) elevated theMn content in Artemia nauplii which was equal or higher than that ofthe control group. Carcass Mn content of juveniles at 30 dph in the ZMand M group was also significantly higher than that of the control andthe Z groups. It is recommended based on these findings is that Znshould be supplemented to Artemia nauplii together with Mn to avoidthe possibility of a decrease in whole body Mn of the fish larvae.

Significantly lower Fe content in enriched Artemia and in wholebody of the experimental fish as a consequence from Zn supplementa-tionwas observed, suggesting that Zn supplementation may influence

Data are means of three replicates. Each replicate included 50 specimens.

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Fe accumulation in Artemia andwhole body Fe in red sea bream larvae.This finding was in agreement with Wieringa et al. (2007) whoreported that Zn supplementation in infants negatively affected theirFe status. The authors noted that combined supplementation of Fe andZn was safe and effective in reducing the high prevalence of anaemiaand Fe and Zn deficiency.

After the air-dive challenge test, higher survival of the fish wasobtained in the ZM group suggesting that stress tolerance of the fishcan be improved by supplementationwith both Zn and Mn. As fish fryare usually exposed to various handling activities such as grading andtransportation, the air-dive challenge test is one of the useful methodsto evaluate their vitality. Healthy fry normally show higher survivalduring the air-dive challenge test. It has been shown that DHA en-hances the vitality of larvae and juvenile of red sea bream (Watanabeet al., 1989; Furuita et al., 1996). In the current study, significantlyhigher DHA content inwhole body red sea bream in the ZM group wasobserved than in the M group. But whole body DHA content was notsignificantly different among the ZM, Z and control groups. Thus,higher survival of the fish in the ZM group compared to the othergroups during the challenge test was not totally caused by the DHAstatus of the fish. Additionally, slightly higher whole body lipid con-tent in the ZM group, suggests that lipid status of the fish may alsoaffect their ability to with stand the stress condition.

In the current study, a markedly lower frequency of fish with atleast one skeletal deformity in the red sea bream larvae fed naupliisupplemented with Zn and Mn, suggesting that Zn and Mn may pro-mote normal skeletal development in the larvae. Mn deficiency signssuch as dwarfism linked to disturbance of bone formation in rainbowtrout and common carp has previously been reported (Watanabeet al., 1997). The results from the current study showed that sup-plementation of Mn could increase the Mn content in Artemia naupliito more than 12 µg g−1. This concentration is required for normalgrowth and skeletal development of rainbow trout and common carp(Satoh et al., 1987b, 1991). Moreover, significantly lower occurrence ofskeletal deformities in the Zn supplemented groups illustrates thebenefit of Zn supplementation for normal skeletal development of redsea bream larvae. A stimulatory effect of Zn on bone formation andmineralization in vitro and in vivo has been reported (Yamaguchiet al., 1987). The possible mechanism by which Zn stimulates bonegrowth is that Zn directly activates aminoacyl-tRNA synthetase inosteoblastic cells and it stimulates cellular protein synthesis (Yama-guchi, 1998). Moreover, Zn inhibits osteoclastic bone re-sorption byinhibiting osteoclast-like cell formation from marrow cells (Yamagu-chi, 1998). Findings from the current study revealed that supplemen-tationwith Zn andMn through Artemia to red sea bream larvae duringthe bone ossifying stage (TL=6–19 mm) was more effective inpromoting normal skeletal development than in the rotifer feedingstage, when most bones are still in the cartilage form.

Vertebral columndefection due to un-inflation of the swimbladderas reported by Boglione et al. (1995) and Lall and Lewis-McCrea (2007)was not apparently observed in our study since approximately 100% ofthe fish in all treatments at 30 dph (n=600) developed normal swimbladder.

Recent studies on European sea bass larvae have demonstrated arelationship between dietary PUFAs, particularly dietary EPA and DHAlevels and vertebral malformations (Villeneuve et al., 2005, 2006).They showed that larval feed based on 17% lipid (12 to 13% phos-pholipid frommarine lipid sources), containing phospholipids with 1.1to 2.3% of EPA and DHA, supported optimum larval growth and sur-vival with low incidence of vertebral and cephalic deformities. Theyalso found that similar levels of EPA and DHA provided by the neutrallipid fractions of the lipid were teratogenic and lethal. However,dietary phospholipids with 4.8% EPA and DHA induced cephalic (8.5%)and vertebral column deformities (35.3%) adversely affecting fishgrowth and survival. Moreover, an excess amount of PUFA acceleratedthe osteoblast differentiation process through the upregulation of

RXRα and (BMP)4, leading to supernumerary vertebra. In our study,the total DHA and EPA in the whole body comprised about 1.3% of thelipid (dry-matter basis). That is comparable to the level of DHA andEPA that supported normal growth and skeletal development ofEuropean sea bass. Furthermore, no significant difference in total n-3HUFA in whole body of the fish groups was detected, suggesting thatthere was no correlation between total n-3 HUFA and skeletal de-formities in the current experiment.

Regarding the occurrence of skeletal deformities in the experi-mental groups, the percentage of fish with at least one skeletal defor-mity (38.7–52.7%) was relatively high. The deficiency or excessmicronutrient such as vitamin A, Zn or Mn in weaning diets shouldbe further considered as possible causes of skeletal deformities in redsea bream larvae.

In conclusion, findings from the present study suggest that Mnsupplementation in Artemia improves growth performance of red seabream larvae. Zn and Mn supplementation in Artemia promotes nor-mal skeletal development of the fish larvae. This study confirmed thatmaintenance of Mn level in enriched Artemia from 12–42.8 µg g−1

(dry-matter basis) is required for better growth and normal skeletaldevelopment of the larvae. Zn and Mn should be supplemented to-gether to avoid a decrease in the Mn content of enriched Artemia.Further study is necessary to investigate the safe concentration of Znand Mn in live feeds and the function or mechanism by which Zn andMn promotes normal skeletal development to improve the quality ofhatchery-reared marine fish larvae for aquaculture.

Acknowledgements

The authors gratefully acknowledge the financial assistance pro-vided by the Ministry of Education, Culture, Sport, Science and Tech-nology (Monbukagakusho), Japan for this research. The authors thankImoto Tatsuhiro, Matsumura Keisuke, Kitamoto Eri and Miyajima Akifor their technical support during larvae culture.

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