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Survival and growth of larval striped bass (Morone saxatilis) fedArtemia enriched with highly unsaturated fatty acids (HUFA)
Aquaculture, 99 ( I991 ) 117-126 Elsevier Science Publishers B.V., Amsterdam
117
Survival and growth of larval stri (Mmne saxatilis) fed Artemia em highly unsaturated fatty acids ( C.A. Lemm and D.P. Lemarie U.S. Fish and WildlifeService, National FisheriesResearchCenter, Fish Culture and Ecology Branch, Box 700, Kearneysville, WV 25430, USA (Accepted 24 February I99 1)
ABSTRACT Lemm, C.A. and Lemarie, D.P., 1991, Survival and growth of larval striped bass (Morone saxatilis) fed Artemia enriched with highly unsaturated fatty acids (HUFA). Aquaculture,99: 117-126. The nutritional value of Artemia nauplii from the Great Salt Lake was effectively improved for larval striped bass (Morone silxaii/is) by incorporation of unsaturated fatty acids (20: 5n-3 and 22: 6n3 ) into the nauplii by the direct method of enrichment. Survival at 24 days post-hatch increased from 23% to 64% when fish were fed nauplii containing 8.24% lipid as 20: 5n-3 and 3.10%as 22: 6n-3 fatty acids. Growth of the larval fish was significantly improved by a diet of fatty acid enriched nauplii. Fatty acid composition of the larvae reflected the composition of the fed nauplii. Results suggested a requirement by larval striped bass for long-chain highly unsaturated fatty acids and an inability, at this stage of development, to elongate and desaturate the shorter chain fatty acids in sufftcient amounts to meet this requirement.
INTRODUCTION
Newly hatched Artemia (brine shrimp ) are widely used as a first food for larval fishes. Striped bass (Morone saxatiks) readily consume Artemia. However, highly variable survival of striped bass during the first 35 days (less than l%-53%) is evidence that Artemia alone do not provide adequate nutrition through metamorphosis (Rhodes and Merriner, 1973; Braid, 198 1; Westin et al., 1983: Carlberget al., 1984; Webster, 1989 ). Recent studies indicated that essential fatty acids (fiFA), specifically the 0~3series ( n-3 ) polyunsaturated fatty acids, are the essential nutritional component supplied by live foods (Watanabe et al., 1978; Fujita et al., 1980; Schauer et al., 1980; Leger et al., 1986; Navarro et al., 1988). Natural prey organisms of marine fish larvae contain substantial concentrations of highly unsaturated 20: 5n-3 and 22 : 6s3 fatty acids. A lack of these EFA is suggested as the reason some Artemia can fail to provide all the nutrition required for 0044-8486/9 l/$03.50
0 I99 1 Elsevier Science Publishers B.V. All rights reserved.
118
C.A. LEMM ANQ D.P. LEMARIE
the early growth of larval fish (Watanabe et al., 1983). Marine fish larvae experience shock syndrome beginning about 6- 10 days after having been put on a diet of Avtemia deficient in EFA (Fujita et al., 1980; Watanabe et al., 1980; Navarro et al., 1988). Concentrations of 20: k-3 and 22: 6n-3 are low or variable in some sources of Artemia (Schauer et al., 1980; Leger et al., 1986; Webster, 1989) and these fatty acids are now recognized as the single most important indicator of nutritional quality of Artemia for marine fish larvae (Watanabe et al., 1980; Sorgeloos et al., 1988). Little information is available on EFA requirements of marine fish larvae during early stages of development. However, research is addressing formulation of adequate feeds, both enriched live organisms and inert diets, containing these dietary components. Enrichment of Artemia nauplii, by feeding highly unsaturated fatty acid (HUFA) emulsions from refined fish oils, has been tested and adopted by some culturists to avoid nutritional deficiencies from live feeds with inadequate EFA (Watanabe et al., 1982; Van Ballaer et al., 1985; Leger et al., 1986). The objective of our study was to evaluate the effects of feeds with increased concentrations of highly unsaturated fatty acids on the growth, survival and swim bladde-I il;fiation of sir&cd bass duripg;_the early rearing period and to gather information on the qualitative requirements for fatty acids by striped bass. .MATEP iALS AND METHODS
Four-day post-hatch Tennessee striped bass larvae (Eagle Bend Fish Hatchery, Clinton, TN ) were held in a 160-l conical bottom incubator at 17 ‘C and fed newly hatched 24,h-old Artemia. After 3 days, 45 larvae/l were placed in each of 12 ex%perimentalrearing units. Rearing units were 19-l polyethylene cylindrical containers, 27 x 37 cm, with two 23 x23-cm windows that were covered with 500~pm mesh nylon. The units were placed in 1.2-m diameter tanks by suspending them through holes in a plywood tank cover and immersed to a depth of 27 cm, resulting in a rearing volume of 15 1.The interior solid surfaces of the units were dark gray. Each rearing unit received 1 l/min water flow. A single airstone placed near the bottom of each unit provided aeration and additional water movement to keep the live food in suspension. Mean water temperature and dissolved oxygen concentrations in the rearing units during the study were 16.8 + 0.1 OC (range 16.4- 17.1) and 7.8 If:0.2 mg/l (range 6.5-8.6), respectively. Total dissolved gas was < 101% (range 100.6100.1; mean oxygen saturation was 80% and mean nitrogen saturation 105%) during the study. Lighting was not measured but continuously maintained at a low level. Siphons and squeeze bulbs were used to remove accu-
SURVIVAL AND GROWTH OF LARVAL STRIPED t3ASSFED ENRICHED ARTEMIA
119
mulated dead Artemia and larvae from the bottoms and screened areas of the units. Mortality was estimated daily. Three groups of larvae were randomly assigned to receive each of the Artemia treatments. The treatments (Table 1) consisted of Artemia nauplii enriched by having been fed for 24 h with fatty acid emulsions containing no added HUFA (LOW ), a medium concentration of HUFA (MEDIUM), or a high concentration of HUFA (HIGH). A fourth treatment consisted of nauplii held for 24 h without feeding (UNENRICHED). The Great Salt Lake Artemia cysts (BioMarine, Hawthorne, CA) used in this study were the freshwater variety (Watanabe et al., 1978) with a high concentration of the fatty acid 18 : 3n-3 and a relatively low concentration of HUFA. Cysts (2 g/l) were incubated in lo-12 ppt salinity at 28 “C under continuous illumination and aeration in a 160-l conical bottom tank. After 24 h incubation, freshly hatched nauplii were separated from the hatching debris and washed with salt water. The nauplii (500 000 naupliill) were enriched for 24 h with self-emulsifying HUFA concentrates (Artemia Reference Center, State University of Ghent, Belgium) according to the techniques described by Leger et al. ( 1987). Following enrichment, meta-nauplii were harvested, thoroughly rinsed with salt water and dispensed at lO=min intervals 24 h/day via live food feeders, similar to those described by Nicholson et al. ( 1985). The feeding regime provided an estimated level of 1500-2000 Artemia nauplii per 1per day. The study was terminated after 14 days when only a few fish survived in the groups fed the UNENRICHED Artemia. All remaining larvae in each unit were counted, and individuals of a random sample of 5!! larvae (or all if less than 50 remained) were individually measured (total length, mm), weighed (0.00 1g ) and examined for swim bladder inflation. The HUFA emulsion concentrates, each group of Artemia enriched with the emulsions, and the larvae fed the enriched Artemia were analyzed for fatty acid composition following the procedures described by Schauer and Simpson (1978). Larval weights and lengths were transformed to log, values and inflation rates of swim bladders to arcsine values prior to analysis by one-way ANOVA. Significant differences between means were determined by Duncan’s multiple range test (Ott, 1988) (P10.05 unless otherwise stated). Survival data were not analyzed statistically because of heterogeneous variances of the replicates. Untransformed data are reported to aid in the comparison of results from other studies. RESULTS
Fatty acid composition of Artemia nauplii reflected the fatty acid profile of the emulsion received during the enrichment process (Table 1). Concentra-
‘Composition
Artemia
-
0”:; 0.0
:: 0:o 0.0 0.0 0.0 0.0
::: 0.0 10.96 0.0 0.0 2.57 9.12 0.0 2.74 0.0 0.0 0.0 0.0 0.0
7.89 8.72 39.35 17.89
:: 0:o 2.60 1.75
:: 2:19 0.0 0.0 0.70 2.60 2.60 0.0 0.0
x.: 13:82 7.17 1.40 5.91 0.0 28.57 0.0 7.46 i8.52
0.0 0.0 5.48 5.38 0.0
for comparison.
-
z: 3125 4.37 0.49 0.0 9.68 2.93 0.37 1.12 7.79 0.0 0.70
K3 28154 0.0 8.98 0.0 3.93
X:: 10.49 7.77
6.53 0.0
:?I 0:o
Emulsion
MEDRJM
LOW HUFA
Emulsion
Treatment
1
Treatment
of newly hatched Artemia included
24:l Total n-3 HUFA Lipid
22:6n-3 22 : 6n-6
8:0 IO:0 12:o 14:o 114:1 a5:o IS: 1 16:O 16: I 16:2n-4 18:O 18:l 18: In-9 18:2 18:2n-6 18:3 18:3n-3 18:4 18:4n-3 20:! 20: 3n-3 20:4 20: 5n-3 22:l 22:4n-6 22 : Sn-3
Fatty acid
o”:R 8.24 1.12 0.0 0.0 3.:0 0.0 0.0 11.34 1.84
8i 1:01 0.41 0.13 0.0 11.60 5.34 0.0 3.47 0.0 35.10 0.0 6.94 19.64 0.0 0.0 1.60 0.0
0.0
A Hernia
HUFA
2
3
0.0 0.0 0.0 0.55 0.0 0.0 0.0 1.8 0.88 0.0 4.62 12.12 0.0 2.12 0.0 4.11 0.0 0.0 0.0 1.75 1.65 0.0 24.98 2.57 1.17 2.98 36.90 0.0 1.70 -
Emulsion
HIGH HUFA
Treatment
Fatty acid composition (% of methyl esters) and total lipid content (% wet weight) OfArtemia treatments the emulsion and the Artemia fed on the emulsion for 24 h post-hatch
TABLE I
z: 12:48 1.24 0.0 0.97 9.36 0.0 0.0 21.84 t-54
:.: 2:01 (3.0
0.0 0.0 0.0 0.6 I 0.66 0.44 0.0 10.20 4.72 0.78 4.74 0.0 25.70 0.0 4.89 19.2
Artemia
8.8 0:o 3.83 1.75
0.0 0.0 0.0 0.77 0.92 0.21 0.0 12.23 5.53 0.0 5.02 0.0 33.80 0.0 6.43 27.14 0.0 0.0 2.28 0.0 0.0 1.07 3.83 0.26 0.0 0.0
0.0 0.0 0.0 0.75 0.84 0.17 0.0 12.70 5.01 0.0 3.15 0.0 32.27 0.0 9.02 28.25 0.0 0.0 3.72 0.0 0.0 0.61 2.68 0.18 0.0 0.0 0.0 0.0 0.0 2.68 2.28
Newly hatched
Artemia ’
Other
UNENRICHED
4
is shown for
Artemia
Treatment
and newly hatched Arlemia. Composiiion
is c,
121
SURVIVAL AND GROWTH OF LARVAL STRIPED BASSFED ENRICHEDARTEMA
tions of n-3 HUFA (20 :k-3 and 22 :6n-3) were increased from 2.68%~in newly hatched nauplii to 11.34% and 21.84% in nauplii fed the MEBIUM and HIGH HUFA emulsions. Total lipid concentrations decreased by at least 20% in all groups of Artemia cultured for 24 h post-hatch. Survival of striped bass on the UNENRICHED Artemia was 5% (Table 2 ) . Larvae began to die by the 5th day of this treatment ( 14- 15 days post-hatch) and continued to die steadily. Survival was 23% in fish fed Artemia with 2.6% HUFA (LOW treatment, 2.6% 20: 5n-3) and increased to 64% in fish fed Artemia with a total HUFA of 11.34% (MEDIUM HUFA treatment, 8.24% 20 : k-3 and 3.10% 22 : 6n-3 ) . No further improvement in survival (Table 2 ) was apparent when the n-3 HUFA was elevated to 2 1.8% of the lipid (HIGH HUFA treatment, 12.48% 20:5n-3 and 9.36% 22:6n-3). Growth (as measured by mean final length, Table 2 ) was lowest (9.3 mm) in fish fed the UNENRICHED treatment and followed by fish fed the LOW HUFA treatment (9.8 mm). The difference in length between fish fed the MEDIUM ( 10.3 mm) and HIGH HUFA treatments ( 10.2 mm) was not significant. The difference in ‘mean final weight between the groups of fish was significant only for fish fed the UNENRICHED Artemia. Enrichment of Artemia did not affect the rate of swim bladder inflation in this study. At 7 days post-hatch, approximately 30% of the fish had inflated swim bladders and the rate did not substantially change during the study. The percentage of fish with inflated swim bladders at termination ranged from 26 to 33% and was not significantly different among the various treatments (Table 2). The fatty acid composition of the larvae clearly reflected the composition of the Artemia fed (Table 3). The 20 : 5n-3 content of the larvae at 7 days TABLE 2 Growth, survival, and O/oswim bladder inflation of 24-day-old larval striped bass on enhanced or unenhanced Artemiu. Means within a column sharing a letter in common are not significanily different (PIO.05) Artcmia
treatment
Final length (mm)’
Final weight (gl’
Swim bladder inflation (0~12 . I
Survival (%I3
LOW HIJFA MEDIUM HUFA HIGH HUFA UNENRICHED
9.8 f 0.9” 10.3 k0.8b 10.2f0.8b 9.3 k l.OC
0.005 + 0.002” 0.006 k 0.002” 0.006 f 0.002” 0.004 + 0.002b
26 33 32 (291
23 (1,36,32) 64 (66,67,59 1 48 (7,66,72 1 5 (6,7,Il
‘Length and weight of larvae at-7 days post-hatch were 5.5 + 0.2 mm and about 0.00 f 2. respectively. *Percentage is mean of three replicates of 50 fish at 24 days, except for the unenhanced group. Only 100 fish remained, this figure represents the % of the remaining fish with an inflated swim bladder. ‘Survival is the number of larvae at 24 days expressed as a I of the initial number. Survival of fish in individual replicates in parentheses.
C.A. LEMM AND D.P. LEMARIE
122 TABLE 3 Fatty acid composition larvae’ Fatty acid
lo:o !2:0 14:o 14: 1 15:o 15: I
16:0 16: I 16:2n-4 18:O 18: 1 18: ln-9 I 8 : 211-6 18:3n-3 is:4 18:4n-3 2O:l 20: 3n-3 20:4 20: 5n-3 22:l 22 : 4n-6 22 : 50-3 22 : 6n-3 22 : 6n-6
24:l % Lipid
(% of methi
esters) and total lipid levels (% wet weight) of striped bass
Larvae at initiation 7 days
Larvae fed HUFA enhanced Arfemia LOW
MEDIUM
0.0 0.0 3.09 0.0 0.0 0.0 8.97 17.54 2.80 3.14 28.95 0.0 8.45 6.54 i .40 0.0 0.0 0.0 2.19 3.21 0.7 I 0.43 I.21 8.1 I 1.31 0.0 1.87
0.0 0.43 3.30 0.0 0.0 0.0 15.68 5.13 0.43 7.26 27.72 0.0 5.60 15.73 0.0 1.58 0.0 0.0 0.0 5.60 1.89 0.0 0.0 8.94 0.0 0.36 1.50
0.0 0.0 0.5 0.0 0.0 0.0 4.17 5.15 0.0 5.95 30.40 0.0 5.91 15.96 0.0 1.44
0.0 0.0 0.0 10.57 2.17 0.0 0.91 5.75 0.0 0.31 2.01
‘No analysis was conducted on the larvae fed the UNENRICHED remained at termination.
HIGH 0.0 0.10 0.65 0.0 0.0 0.0 13.33
8.07 1.60 8.20 24.74 0.0 7.29 16.48 0.0 0.41 0.0 0.0 0.0 8.49 2.09 0.0 0.68 6.22 0.0 0.46 2.80 treatment as insufficient larvae
post-hatch was 3.2 1% of the total lipids. Concentration of this fatty acid increased during the study to 5.694, 10.57% and 8.49% in larvae fed the LOW, MEDIUM and HIGH HUFA treatments, respectively. The concentration of 22 : 6n-3 was 8.11% in 7-day-old larvae, remained near this concentration (8.94%) in the larvae fed the LOW HUFA treatment, and decreased in larvae fed the MEDIUM (5.75%) and HIGH HUFA (6.22%) treatments. All of the Artemia treatments contained high concentrations of 18 : 3n-3 ( 18.52%-19.64%; Table 1). This fatty acid increased in all groups of larvae from an initial low concentration of 6.54% at 7 days to 15.73%, 15.96% and 16.48% of the lipid for larvae fed the LOW, MEDIUM and HIGH HUFA treatments (Table 3 ) .
SURVlVAL
AND GROWTH
OF LARVAL
STRIPED
BASS FED ENRICHED
ARTEMlA
123
DISCUSSION
Numerous studies have reported the importance of dietary HUFA to early survival of marine fish (Watanabe et al., 1982; Van Ballaer et al., 1985; Sorgeloos et al., 1988 ). Our study confirms this for larval striped bass. We effectively improved the dietary value of Great Salt Lake Artemia (low in HUFA) by elevating the n-3 HUFA content through enrichment with HUFA emulsions. Survival was low (5% and 23%) when the HUFA content was < 4% (UNENRICHED and LOW HUFA treatments) and increased dramatically to 64% when the HUFA content of the food was 11.34% (MEDIUM HUFA treatment ). Our results agree with those of Webster ( 1989) who reported that both growth and survival of striped bass larvae were affected by the HUFA concentration (as 20: 5n-3) in the source of Artemia fed to the fish. Larvae fed UNENRICHED Artemia had the lowest survival (5%). The nauplii fed to these fish were, in effect, starved for 24 h after hatch. Other authors demonstrated that starved nauplii are nutritionally inferior to freshly hatched nauplii as feed for several species of fish larvae (Morris, 1956; Ablett and Richards, 1980). Changes in fatty acid profile (Watanabe et al., 1978; Claus et al., 1979), amino acid profile (Claus et al., 1979; Dabrowski and Rusiecki, 1983), and total energy content (Benijts et al., 1975; Ableti and Richards, 1980) may be responsible for the reduced nutritional value of the starved organisms. Webster ( 1989) and the results of this study suggest that striped bass, at least in the larval growth phase, are unable to elongate and desaturate 18 : 3n3 into the longer chain fatty acids of the 20 carbon series. Those larvae fed the LOW HUFA or UNENRICHED nauplii (containing 18 : 3n-3 as the primary EFA source) did not survive and grow at a rate equivalent to those fed treatments with equal concentrations of 18 : 3n-3 but additional HUFA (MEDIUM and HIGH HUFA). The slight increase in the concentration of 20: 5n3 in the larval lipids of the LOW HUFA larvae from 3.21% to 5.6% may indicate a limited conversion of 18 : 312-3to 20 : 5n-3, but not in sufficient quantity to meet the HUFA requirement. Striped bass exhibiting better growth and improved survival had concentrations of at least 8.49% 20: 5n-3 in the body lipids, Leger et al. ( 1986) concluded from an extensive examination that Artemia for rearing marine larvae should contain more than 4% of the total fatty acid methyl esters as 20 : 5n-2. Batches with less than 3% 20: 5n-3 consistently yielded poor growth and survival of marine organisms. The fatty acid content of Artemia varies by geographic locality, and also from year-to-year at the same location (Watanabe-et al., 1978, 1983; Fujita et al., 1980; Schauer et al., 1980; Leger et al., 1986). Our results suggest that Artemin with less than 4% of the lipid as 20: 5n-3 would not meet the EFA requirements of larval striped bass for good growth and survival.
124
C.A. LEMM AND IX’. LEMARIE
Failure to fully inflate the swim bladder is a serious problem in the intensive culture of striped bass (Doroshev, 1970; Hadley et al., 1987). Kanazawa et al. ( 1982 ) suggested the fatty acid 20 : 5n-3 plays a role in the development of the swim bladder in red sea bream because this fatty acid was readily incorporated into swim bladder tissue in this species. Other research suggests involvement of early nutrition in the proper functioning of the swim bladder (Kitajima et al., 198 1; Katavic, 1986 ). In our study, differences in the rate of swim bladder inflation were not significant among larvae. However, enriched diets were not fed during the 5-7-day post-hatch period when striped bass initiate inflation of the swim bladder (Doroshev and Cornacchia, 1979). Further research is warranted on the possible connection between EFA nutrition and swim bladder inflation. In conclusion, larval striped bass, similar to many other marine fish, require n-3 HUFA in the diet for survival and gowth during early developmental stages and appear to have a limited ability to elongate and desaturate shorter chain fatty acids to meet this requirement. Significant improvements in the successful intensive rearing of this species may result from feeding a marine Artemiu that is naturally high in 20: 5n-3 or a HUFA enriched freshwater type Artemia. ACKNOWLEDGEMENTS
We thank members of the International conference on Exp!oration of the Sea Working Group for allowing us to participate in the study. We thank P. Sorgeloos, Artemia Reference Center, State University of Ghent, Belgium, for providing the standard HUFA emulsions. We are indebted to Mike Smith, Tennessee Wildlife Resources Agency, for providing the striped bass fry, to H. Leibovitz at the University of Rhode Island, for the fatty acid analyses, and to D. Bengston, University of Rhode Island, and C. Webster, Kentucky State University, for review of the manuscript. We gratefully acknowledge our technicians D. Weller and J. Howe for care of the experimental fish and assistance in the collection of data.
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SURVIVAL AND GROWTH OF LARVAL STRIPED BASS FED ENRICHED ARTEMlA
i25
Braid, M.R., 198 1. Incidence of cannibalism among striped bass fry in an intensive culture system. Prog. Fish-Cult., 43: 2 IO-2 12. Carlberg, J.M., Van Olst, J.C., Massingill, M.J. acd Hovanec, T.A., 1984. Intensive culture of striped bass: a review of recent technological developments. In: J.P. McCraren (Editor ). The Aquaculture of Striped Bass: a Proceedings. Maryland Sea Grant Publication UM-SG-MAP84-OI, University of Maryland, College Park, MD, pp. 89-l 27. Claus, C., Benijts, F., Vandeputte, G. and Gardner, “%.,1979. The biochemical composition of the larvae of two strains of Arfemia safina (L.) reared on two different algal foods. J. Exp. Mar. Biol. Ecol., 36: 17 l-183. Dabrowski, K. and Rusiecki, M., 1983. Content of total and free amino acids in zooplanktonic food for fish larvae. Aquaculture, 30: 3 l-42. Doroshev, S.I., 1970. Biological features of the eggs, larvae and young of the striped bass Roccus saxatilis (Walbaum) in connection with the problem of its acclimatization in the USSR. J. Ichthyol., IO: 235-248. Doroshev, S.I. and Cornacchia, J.W., 1979. Initial swim bladder inflation in the larvae of ‘T/lapia mossambica(Peters) and Morone saxariks ( Walbaum ), Aquaculture, 16: 57-66. Fujita, S., Watanabe, T. and Kitajima, C., 1980. Nutritional quality of Artemia salina from different locations as living feed from the viewpoint of essential fatty acids for marine fish. In: G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers (Editors), The Brine Shrimp, Arfemia. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium, pp. 277290. Hadley, C.G., Rust, M.B., Van Eenennaam, J.P. and Doroshov, S., 1987. Factors influencing initial swim bladder inflation by striped bass. Am. Fish. See. Symp., 2: 164-l 69. Kanazawa, A., Teshima, S., Imatanaka, N., Imada, 0. and Inoue, A., 1982. Tissue uptake of radioactive eicosapentaenoic acid in red sea bream. Bull. Jpn. Sot. Sci. Fish., 48: 144 l- 1444. Katavic, I., 1986. Diet involvement in mass mortality of sea bass (Dicentrarchuslabrax) larvae. Aquaculture, 58: 45-54. Kitajima, C., Tsukshima, Y., Fujita, S., Watanabe, T. and Yone, Y., 198 1. Relationship between uninflated swimbladders and lordotic deformity in hatchery reared red sea bream (Pagrus major). Bull. Jpn. Sot. Sci. Fish., 47: 1289-l 294. Leger, P., Bengtson, D.A., Simpson, K.L. and Sergeloos, P., 1986. The use and nutritional value ofrlrtemia as a food source. Gceanogr. Mar. Biol., Annu. Rev., 24: 52 l-623. Leger, P., Naessens-Foucquaert, E. and Sorgeloos, P., 1987. International study on Aricmia. XXXV. Techniques to manipulate the fatty acid profile in Arremia nauplii and the effect on its nutritional effectiveness for the marine crustacean Mysidupsisbahia (M ). In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and its Applications, Vol. 3. Universa Press, Wetteren, Belgium, pp. 4 1l-424. Morris, R.W., 1956. Some aspects of the problem of rearing marine fishes. Bull. Inst. Oceanogr. (Monaco), 1082: l-62. Navarro, J.C., Hontoria, F., Varo, I. and Amat, F., 1988. Effects of alternate feeding witqa poor long-chain polyunsaturated fatty acid Arfemia strain and a rich one for sea bass and prawn larvae. Aquaculture, 74: 307-3 17. Nicholson, L.C., Christmas, J.Y., 111and Lukens, R.R., 1985. A low cost live food feeder. Prog. Fish-Cult., 47: 245-248. Ott, L., 1988. An Introduction to Statistical Methods and Data Analysis, 3rd edition. PWS-Kent Publishing Company, Boston, MA, 835 pp. Rhodes, W. and Merriner, J.V., 1973. A preliminary report on closed system rearing ofstriped bass sac fry to fingerling size. Prog. Fish-Cult., 35: 199-20 1. Schauer, P.S. and Simpson, K.L., 1978. Effects of diets on the fatty acid composition ofjuvenile Atlantic silverside (Menidiu menidkz). In: I.W. Avault, Jr. (Editor), Proceedings of9th An-
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117
Survival and growth of larval stri (Mmne saxatilis) fed Artemia em highly unsaturated fatty acids ( C.A. Lemm and D.P. Lemarie U.S. Fish and WildlifeService, National FisheriesResearchCenter, Fish Culture and Ecology Branch, Box 700, Kearneysville, WV 25430, USA (Accepted 24 February I99 1)
ABSTRACT Lemm, C.A. and Lemarie, D.P., 1991, Survival and growth of larval striped bass (Morone saxatilis) fed Artemia enriched with highly unsaturated fatty acids (HUFA). Aquaculture,99: 117-126. The nutritional value of Artemia nauplii from the Great Salt Lake was effectively improved for larval striped bass (Morone silxaii/is) by incorporation of unsaturated fatty acids (20: 5n-3 and 22: 6n3 ) into the nauplii by the direct method of enrichment. Survival at 24 days post-hatch increased from 23% to 64% when fish were fed nauplii containing 8.24% lipid as 20: 5n-3 and 3.10%as 22: 6n-3 fatty acids. Growth of the larval fish was significantly improved by a diet of fatty acid enriched nauplii. Fatty acid composition of the larvae reflected the composition of the fed nauplii. Results suggested a requirement by larval striped bass for long-chain highly unsaturated fatty acids and an inability, at this stage of development, to elongate and desaturate the shorter chain fatty acids in sufftcient amounts to meet this requirement.
INTRODUCTION
Newly hatched Artemia (brine shrimp ) are widely used as a first food for larval fishes. Striped bass (Morone saxatiks) readily consume Artemia. However, highly variable survival of striped bass during the first 35 days (less than l%-53%) is evidence that Artemia alone do not provide adequate nutrition through metamorphosis (Rhodes and Merriner, 1973; Braid, 198 1; Westin et al., 1983: Carlberget al., 1984; Webster, 1989 ). Recent studies indicated that essential fatty acids (fiFA), specifically the 0~3series ( n-3 ) polyunsaturated fatty acids, are the essential nutritional component supplied by live foods (Watanabe et al., 1978; Fujita et al., 1980; Schauer et al., 1980; Leger et al., 1986; Navarro et al., 1988). Natural prey organisms of marine fish larvae contain substantial concentrations of highly unsaturated 20: 5n-3 and 22 : 6s3 fatty acids. A lack of these EFA is suggested as the reason some Artemia can fail to provide all the nutrition required for 0044-8486/9 l/$03.50
0 I99 1 Elsevier Science Publishers B.V. All rights reserved.
118
C.A. LEMM ANQ D.P. LEMARIE
the early growth of larval fish (Watanabe et al., 1983). Marine fish larvae experience shock syndrome beginning about 6- 10 days after having been put on a diet of Avtemia deficient in EFA (Fujita et al., 1980; Watanabe et al., 1980; Navarro et al., 1988). Concentrations of 20: k-3 and 22: 6n-3 are low or variable in some sources of Artemia (Schauer et al., 1980; Leger et al., 1986; Webster, 1989) and these fatty acids are now recognized as the single most important indicator of nutritional quality of Artemia for marine fish larvae (Watanabe et al., 1980; Sorgeloos et al., 1988). Little information is available on EFA requirements of marine fish larvae during early stages of development. However, research is addressing formulation of adequate feeds, both enriched live organisms and inert diets, containing these dietary components. Enrichment of Artemia nauplii, by feeding highly unsaturated fatty acid (HUFA) emulsions from refined fish oils, has been tested and adopted by some culturists to avoid nutritional deficiencies from live feeds with inadequate EFA (Watanabe et al., 1982; Van Ballaer et al., 1985; Leger et al., 1986). The objective of our study was to evaluate the effects of feeds with increased concentrations of highly unsaturated fatty acids on the growth, survival and swim bladde-I il;fiation of sir&cd bass duripg;_the early rearing period and to gather information on the qualitative requirements for fatty acids by striped bass. .MATEP iALS AND METHODS
Four-day post-hatch Tennessee striped bass larvae (Eagle Bend Fish Hatchery, Clinton, TN ) were held in a 160-l conical bottom incubator at 17 ‘C and fed newly hatched 24,h-old Artemia. After 3 days, 45 larvae/l were placed in each of 12 ex%perimentalrearing units. Rearing units were 19-l polyethylene cylindrical containers, 27 x 37 cm, with two 23 x23-cm windows that were covered with 500~pm mesh nylon. The units were placed in 1.2-m diameter tanks by suspending them through holes in a plywood tank cover and immersed to a depth of 27 cm, resulting in a rearing volume of 15 1.The interior solid surfaces of the units were dark gray. Each rearing unit received 1 l/min water flow. A single airstone placed near the bottom of each unit provided aeration and additional water movement to keep the live food in suspension. Mean water temperature and dissolved oxygen concentrations in the rearing units during the study were 16.8 + 0.1 OC (range 16.4- 17.1) and 7.8 If:0.2 mg/l (range 6.5-8.6), respectively. Total dissolved gas was < 101% (range 100.6100.1; mean oxygen saturation was 80% and mean nitrogen saturation 105%) during the study. Lighting was not measured but continuously maintained at a low level. Siphons and squeeze bulbs were used to remove accu-
SURVIVAL AND GROWTH OF LARVAL STRIPED t3ASSFED ENRICHED ARTEMIA
119
mulated dead Artemia and larvae from the bottoms and screened areas of the units. Mortality was estimated daily. Three groups of larvae were randomly assigned to receive each of the Artemia treatments. The treatments (Table 1) consisted of Artemia nauplii enriched by having been fed for 24 h with fatty acid emulsions containing no added HUFA (LOW ), a medium concentration of HUFA (MEDIUM), or a high concentration of HUFA (HIGH). A fourth treatment consisted of nauplii held for 24 h without feeding (UNENRICHED). The Great Salt Lake Artemia cysts (BioMarine, Hawthorne, CA) used in this study were the freshwater variety (Watanabe et al., 1978) with a high concentration of the fatty acid 18 : 3n-3 and a relatively low concentration of HUFA. Cysts (2 g/l) were incubated in lo-12 ppt salinity at 28 “C under continuous illumination and aeration in a 160-l conical bottom tank. After 24 h incubation, freshly hatched nauplii were separated from the hatching debris and washed with salt water. The nauplii (500 000 naupliill) were enriched for 24 h with self-emulsifying HUFA concentrates (Artemia Reference Center, State University of Ghent, Belgium) according to the techniques described by Leger et al. ( 1987). Following enrichment, meta-nauplii were harvested, thoroughly rinsed with salt water and dispensed at lO=min intervals 24 h/day via live food feeders, similar to those described by Nicholson et al. ( 1985). The feeding regime provided an estimated level of 1500-2000 Artemia nauplii per 1per day. The study was terminated after 14 days when only a few fish survived in the groups fed the UNENRICHED Artemia. All remaining larvae in each unit were counted, and individuals of a random sample of 5!! larvae (or all if less than 50 remained) were individually measured (total length, mm), weighed (0.00 1g ) and examined for swim bladder inflation. The HUFA emulsion concentrates, each group of Artemia enriched with the emulsions, and the larvae fed the enriched Artemia were analyzed for fatty acid composition following the procedures described by Schauer and Simpson (1978). Larval weights and lengths were transformed to log, values and inflation rates of swim bladders to arcsine values prior to analysis by one-way ANOVA. Significant differences between means were determined by Duncan’s multiple range test (Ott, 1988) (P10.05 unless otherwise stated). Survival data were not analyzed statistically because of heterogeneous variances of the replicates. Untransformed data are reported to aid in the comparison of results from other studies. RESULTS
Fatty acid composition of Artemia nauplii reflected the fatty acid profile of the emulsion received during the enrichment process (Table 1). Concentra-
‘Composition
Artemia
-
0”:; 0.0
:: 0:o 0.0 0.0 0.0 0.0
::: 0.0 10.96 0.0 0.0 2.57 9.12 0.0 2.74 0.0 0.0 0.0 0.0 0.0
7.89 8.72 39.35 17.89
:: 0:o 2.60 1.75
:: 2:19 0.0 0.0 0.70 2.60 2.60 0.0 0.0
x.: 13:82 7.17 1.40 5.91 0.0 28.57 0.0 7.46 i8.52
0.0 0.0 5.48 5.38 0.0
for comparison.
-
z: 3125 4.37 0.49 0.0 9.68 2.93 0.37 1.12 7.79 0.0 0.70
K3 28154 0.0 8.98 0.0 3.93
X:: 10.49 7.77
6.53 0.0
:?I 0:o
Emulsion
MEDRJM
LOW HUFA
Emulsion
Treatment
1
Treatment
of newly hatched Artemia included
24:l Total n-3 HUFA Lipid
22:6n-3 22 : 6n-6
8:0 IO:0 12:o 14:o 114:1 a5:o IS: 1 16:O 16: I 16:2n-4 18:O 18:l 18: In-9 18:2 18:2n-6 18:3 18:3n-3 18:4 18:4n-3 20:! 20: 3n-3 20:4 20: 5n-3 22:l 22:4n-6 22 : Sn-3
Fatty acid
o”:R 8.24 1.12 0.0 0.0 3.:0 0.0 0.0 11.34 1.84
8i 1:01 0.41 0.13 0.0 11.60 5.34 0.0 3.47 0.0 35.10 0.0 6.94 19.64 0.0 0.0 1.60 0.0
0.0
A Hernia
HUFA
2
3
0.0 0.0 0.0 0.55 0.0 0.0 0.0 1.8 0.88 0.0 4.62 12.12 0.0 2.12 0.0 4.11 0.0 0.0 0.0 1.75 1.65 0.0 24.98 2.57 1.17 2.98 36.90 0.0 1.70 -
Emulsion
HIGH HUFA
Treatment
Fatty acid composition (% of methyl esters) and total lipid content (% wet weight) OfArtemia treatments the emulsion and the Artemia fed on the emulsion for 24 h post-hatch
TABLE I
z: 12:48 1.24 0.0 0.97 9.36 0.0 0.0 21.84 t-54
:.: 2:01 (3.0
0.0 0.0 0.0 0.6 I 0.66 0.44 0.0 10.20 4.72 0.78 4.74 0.0 25.70 0.0 4.89 19.2
Artemia
8.8 0:o 3.83 1.75
0.0 0.0 0.0 0.77 0.92 0.21 0.0 12.23 5.53 0.0 5.02 0.0 33.80 0.0 6.43 27.14 0.0 0.0 2.28 0.0 0.0 1.07 3.83 0.26 0.0 0.0
0.0 0.0 0.0 0.75 0.84 0.17 0.0 12.70 5.01 0.0 3.15 0.0 32.27 0.0 9.02 28.25 0.0 0.0 3.72 0.0 0.0 0.61 2.68 0.18 0.0 0.0 0.0 0.0 0.0 2.68 2.28
Newly hatched
Artemia ’
Other
UNENRICHED
4
is shown for
Artemia
Treatment
and newly hatched Arlemia. Composiiion
is c,
121
SURVIVAL AND GROWTH OF LARVAL STRIPED BASSFED ENRICHEDARTEMA
tions of n-3 HUFA (20 :k-3 and 22 :6n-3) were increased from 2.68%~in newly hatched nauplii to 11.34% and 21.84% in nauplii fed the MEBIUM and HIGH HUFA emulsions. Total lipid concentrations decreased by at least 20% in all groups of Artemia cultured for 24 h post-hatch. Survival of striped bass on the UNENRICHED Artemia was 5% (Table 2 ) . Larvae began to die by the 5th day of this treatment ( 14- 15 days post-hatch) and continued to die steadily. Survival was 23% in fish fed Artemia with 2.6% HUFA (LOW treatment, 2.6% 20: 5n-3) and increased to 64% in fish fed Artemia with a total HUFA of 11.34% (MEDIUM HUFA treatment, 8.24% 20 : k-3 and 3.10% 22 : 6n-3 ) . No further improvement in survival (Table 2 ) was apparent when the n-3 HUFA was elevated to 2 1.8% of the lipid (HIGH HUFA treatment, 12.48% 20:5n-3 and 9.36% 22:6n-3). Growth (as measured by mean final length, Table 2 ) was lowest (9.3 mm) in fish fed the UNENRICHED treatment and followed by fish fed the LOW HUFA treatment (9.8 mm). The difference in length between fish fed the MEDIUM ( 10.3 mm) and HIGH HUFA treatments ( 10.2 mm) was not significant. The difference in ‘mean final weight between the groups of fish was significant only for fish fed the UNENRICHED Artemia. Enrichment of Artemia did not affect the rate of swim bladder inflation in this study. At 7 days post-hatch, approximately 30% of the fish had inflated swim bladders and the rate did not substantially change during the study. The percentage of fish with inflated swim bladders at termination ranged from 26 to 33% and was not significantly different among the various treatments (Table 2). The fatty acid composition of the larvae clearly reflected the composition of the Artemia fed (Table 3). The 20 : 5n-3 content of the larvae at 7 days TABLE 2 Growth, survival, and O/oswim bladder inflation of 24-day-old larval striped bass on enhanced or unenhanced Artemiu. Means within a column sharing a letter in common are not significanily different (PIO.05) Artcmia
treatment
Final length (mm)’
Final weight (gl’
Swim bladder inflation (0~12 . I
Survival (%I3
LOW HIJFA MEDIUM HUFA HIGH HUFA UNENRICHED
9.8 f 0.9” 10.3 k0.8b 10.2f0.8b 9.3 k l.OC
0.005 + 0.002” 0.006 k 0.002” 0.006 f 0.002” 0.004 + 0.002b
26 33 32 (291
23 (1,36,32) 64 (66,67,59 1 48 (7,66,72 1 5 (6,7,Il
‘Length and weight of larvae at-7 days post-hatch were 5.5 + 0.2 mm and about 0.00 f 2. respectively. *Percentage is mean of three replicates of 50 fish at 24 days, except for the unenhanced group. Only 100 fish remained, this figure represents the % of the remaining fish with an inflated swim bladder. ‘Survival is the number of larvae at 24 days expressed as a I of the initial number. Survival of fish in individual replicates in parentheses.
C.A. LEMM AND D.P. LEMARIE
122 TABLE 3 Fatty acid composition larvae’ Fatty acid
lo:o !2:0 14:o 14: 1 15:o 15: I
16:0 16: I 16:2n-4 18:O 18: 1 18: ln-9 I 8 : 211-6 18:3n-3 is:4 18:4n-3 2O:l 20: 3n-3 20:4 20: 5n-3 22:l 22 : 4n-6 22 : 50-3 22 : 6n-3 22 : 6n-6
24:l % Lipid
(% of methi
esters) and total lipid levels (% wet weight) of striped bass
Larvae at initiation 7 days
Larvae fed HUFA enhanced Arfemia LOW
MEDIUM
0.0 0.0 3.09 0.0 0.0 0.0 8.97 17.54 2.80 3.14 28.95 0.0 8.45 6.54 i .40 0.0 0.0 0.0 2.19 3.21 0.7 I 0.43 I.21 8.1 I 1.31 0.0 1.87
0.0 0.43 3.30 0.0 0.0 0.0 15.68 5.13 0.43 7.26 27.72 0.0 5.60 15.73 0.0 1.58 0.0 0.0 0.0 5.60 1.89 0.0 0.0 8.94 0.0 0.36 1.50
0.0 0.0 0.5 0.0 0.0 0.0 4.17 5.15 0.0 5.95 30.40 0.0 5.91 15.96 0.0 1.44
0.0 0.0 0.0 10.57 2.17 0.0 0.91 5.75 0.0 0.31 2.01
‘No analysis was conducted on the larvae fed the UNENRICHED remained at termination.
HIGH 0.0 0.10 0.65 0.0 0.0 0.0 13.33
8.07 1.60 8.20 24.74 0.0 7.29 16.48 0.0 0.41 0.0 0.0 0.0 8.49 2.09 0.0 0.68 6.22 0.0 0.46 2.80 treatment as insufficient larvae
post-hatch was 3.2 1% of the total lipids. Concentration of this fatty acid increased during the study to 5.694, 10.57% and 8.49% in larvae fed the LOW, MEDIUM and HIGH HUFA treatments, respectively. The concentration of 22 : 6n-3 was 8.11% in 7-day-old larvae, remained near this concentration (8.94%) in the larvae fed the LOW HUFA treatment, and decreased in larvae fed the MEDIUM (5.75%) and HIGH HUFA (6.22%) treatments. All of the Artemia treatments contained high concentrations of 18 : 3n-3 ( 18.52%-19.64%; Table 1). This fatty acid increased in all groups of larvae from an initial low concentration of 6.54% at 7 days to 15.73%, 15.96% and 16.48% of the lipid for larvae fed the LOW, MEDIUM and HIGH HUFA treatments (Table 3 ) .
SURVlVAL
AND GROWTH
OF LARVAL
STRIPED
BASS FED ENRICHED
ARTEMlA
123
DISCUSSION
Numerous studies have reported the importance of dietary HUFA to early survival of marine fish (Watanabe et al., 1982; Van Ballaer et al., 1985; Sorgeloos et al., 1988 ). Our study confirms this for larval striped bass. We effectively improved the dietary value of Great Salt Lake Artemia (low in HUFA) by elevating the n-3 HUFA content through enrichment with HUFA emulsions. Survival was low (5% and 23%) when the HUFA content was < 4% (UNENRICHED and LOW HUFA treatments) and increased dramatically to 64% when the HUFA content of the food was 11.34% (MEDIUM HUFA treatment ). Our results agree with those of Webster ( 1989) who reported that both growth and survival of striped bass larvae were affected by the HUFA concentration (as 20: 5n-3) in the source of Artemia fed to the fish. Larvae fed UNENRICHED Artemia had the lowest survival (5%). The nauplii fed to these fish were, in effect, starved for 24 h after hatch. Other authors demonstrated that starved nauplii are nutritionally inferior to freshly hatched nauplii as feed for several species of fish larvae (Morris, 1956; Ablett and Richards, 1980). Changes in fatty acid profile (Watanabe et al., 1978; Claus et al., 1979), amino acid profile (Claus et al., 1979; Dabrowski and Rusiecki, 1983), and total energy content (Benijts et al., 1975; Ableti and Richards, 1980) may be responsible for the reduced nutritional value of the starved organisms. Webster ( 1989) and the results of this study suggest that striped bass, at least in the larval growth phase, are unable to elongate and desaturate 18 : 3n3 into the longer chain fatty acids of the 20 carbon series. Those larvae fed the LOW HUFA or UNENRICHED nauplii (containing 18 : 3n-3 as the primary EFA source) did not survive and grow at a rate equivalent to those fed treatments with equal concentrations of 18 : 3n-3 but additional HUFA (MEDIUM and HIGH HUFA). The slight increase in the concentration of 20: 5n3 in the larval lipids of the LOW HUFA larvae from 3.21% to 5.6% may indicate a limited conversion of 18 : 312-3to 20 : 5n-3, but not in sufficient quantity to meet the HUFA requirement. Striped bass exhibiting better growth and improved survival had concentrations of at least 8.49% 20: 5n-3 in the body lipids, Leger et al. ( 1986) concluded from an extensive examination that Artemia for rearing marine larvae should contain more than 4% of the total fatty acid methyl esters as 20 : 5n-2. Batches with less than 3% 20: 5n-3 consistently yielded poor growth and survival of marine organisms. The fatty acid content of Artemia varies by geographic locality, and also from year-to-year at the same location (Watanabe-et al., 1978, 1983; Fujita et al., 1980; Schauer et al., 1980; Leger et al., 1986). Our results suggest that Artemin with less than 4% of the lipid as 20: 5n-3 would not meet the EFA requirements of larval striped bass for good growth and survival.
124
C.A. LEMM AND IX’. LEMARIE
Failure to fully inflate the swim bladder is a serious problem in the intensive culture of striped bass (Doroshev, 1970; Hadley et al., 1987). Kanazawa et al. ( 1982 ) suggested the fatty acid 20 : 5n-3 plays a role in the development of the swim bladder in red sea bream because this fatty acid was readily incorporated into swim bladder tissue in this species. Other research suggests involvement of early nutrition in the proper functioning of the swim bladder (Kitajima et al., 198 1; Katavic, 1986 ). In our study, differences in the rate of swim bladder inflation were not significant among larvae. However, enriched diets were not fed during the 5-7-day post-hatch period when striped bass initiate inflation of the swim bladder (Doroshev and Cornacchia, 1979). Further research is warranted on the possible connection between EFA nutrition and swim bladder inflation. In conclusion, larval striped bass, similar to many other marine fish, require n-3 HUFA in the diet for survival and gowth during early developmental stages and appear to have a limited ability to elongate and desaturate shorter chain fatty acids to meet this requirement. Significant improvements in the successful intensive rearing of this species may result from feeding a marine Artemiu that is naturally high in 20: 5n-3 or a HUFA enriched freshwater type Artemia. ACKNOWLEDGEMENTS
We thank members of the International conference on Exp!oration of the Sea Working Group for allowing us to participate in the study. We thank P. Sorgeloos, Artemia Reference Center, State University of Ghent, Belgium, for providing the standard HUFA emulsions. We are indebted to Mike Smith, Tennessee Wildlife Resources Agency, for providing the striped bass fry, to H. Leibovitz at the University of Rhode Island, for the fatty acid analyses, and to D. Bengston, University of Rhode Island, and C. Webster, Kentucky State University, for review of the manuscript. We gratefully acknowledge our technicians D. Weller and J. Howe for care of the experimental fish and assistance in the collection of data.
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SURVIVAL AND GROWTH OF LARVAL STRIPED BASS FED ENRICHED ARTEMlA
i25
Braid, M.R., 198 1. Incidence of cannibalism among striped bass fry in an intensive culture system. Prog. Fish-Cult., 43: 2 IO-2 12. Carlberg, J.M., Van Olst, J.C., Massingill, M.J. acd Hovanec, T.A., 1984. Intensive culture of striped bass: a review of recent technological developments. In: J.P. McCraren (Editor ). The Aquaculture of Striped Bass: a Proceedings. Maryland Sea Grant Publication UM-SG-MAP84-OI, University of Maryland, College Park, MD, pp. 89-l 27. Claus, C., Benijts, F., Vandeputte, G. and Gardner, “%.,1979. The biochemical composition of the larvae of two strains of Arfemia safina (L.) reared on two different algal foods. J. Exp. Mar. Biol. Ecol., 36: 17 l-183. Dabrowski, K. and Rusiecki, M., 1983. Content of total and free amino acids in zooplanktonic food for fish larvae. Aquaculture, 30: 3 l-42. Doroshev, S.I., 1970. Biological features of the eggs, larvae and young of the striped bass Roccus saxatilis (Walbaum) in connection with the problem of its acclimatization in the USSR. J. Ichthyol., IO: 235-248. Doroshev, S.I. and Cornacchia, J.W., 1979. Initial swim bladder inflation in the larvae of ‘T/lapia mossambica(Peters) and Morone saxariks ( Walbaum ), Aquaculture, 16: 57-66. Fujita, S., Watanabe, T. and Kitajima, C., 1980. Nutritional quality of Artemia salina from different locations as living feed from the viewpoint of essential fatty acids for marine fish. In: G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers (Editors), The Brine Shrimp, Arfemia. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium, pp. 277290. Hadley, C.G., Rust, M.B., Van Eenennaam, J.P. and Doroshov, S., 1987. Factors influencing initial swim bladder inflation by striped bass. Am. Fish. See. Symp., 2: 164-l 69. Kanazawa, A., Teshima, S., Imatanaka, N., Imada, 0. and Inoue, A., 1982. Tissue uptake of radioactive eicosapentaenoic acid in red sea bream. Bull. Jpn. Sot. Sci. Fish., 48: 144 l- 1444. Katavic, I., 1986. Diet involvement in mass mortality of sea bass (Dicentrarchuslabrax) larvae. Aquaculture, 58: 45-54. Kitajima, C., Tsukshima, Y., Fujita, S., Watanabe, T. and Yone, Y., 198 1. Relationship between uninflated swimbladders and lordotic deformity in hatchery reared red sea bream (Pagrus major). Bull. Jpn. Sot. Sci. Fish., 47: 1289-l 294. Leger, P., Bengtson, D.A., Simpson, K.L. and Sergeloos, P., 1986. The use and nutritional value ofrlrtemia as a food source. Gceanogr. Mar. Biol., Annu. Rev., 24: 52 l-623. Leger, P., Naessens-Foucquaert, E. and Sorgeloos, P., 1987. International study on Aricmia. XXXV. Techniques to manipulate the fatty acid profile in Arremia nauplii and the effect on its nutritional effectiveness for the marine crustacean Mysidupsisbahia (M ). In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and its Applications, Vol. 3. Universa Press, Wetteren, Belgium, pp. 4 1l-424. Morris, R.W., 1956. Some aspects of the problem of rearing marine fishes. Bull. Inst. Oceanogr. (Monaco), 1082: l-62. Navarro, J.C., Hontoria, F., Varo, I. and Amat, F., 1988. Effects of alternate feeding witqa poor long-chain polyunsaturated fatty acid Arfemia strain and a rich one for sea bass and prawn larvae. Aquaculture, 74: 307-3 17. Nicholson, L.C., Christmas, J.Y., 111and Lukens, R.R., 1985. A low cost live food feeder. Prog. Fish-Cult., 47: 245-248. Ott, L., 1988. An Introduction to Statistical Methods and Data Analysis, 3rd edition. PWS-Kent Publishing Company, Boston, MA, 835 pp. Rhodes, W. and Merriner, J.V., 1973. A preliminary report on closed system rearing ofstriped bass sac fry to fingerling size. Prog. Fish-Cult., 35: 199-20 1. Schauer, P.S. and Simpson, K.L., 1978. Effects of diets on the fatty acid composition ofjuvenile Atlantic silverside (Menidiu menidkz). In: I.W. Avault, Jr. (Editor), Proceedings of9th An-
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nual Meeting of the World Mariculture Society, Louisiana State University, Baton Rouge, LA, pp. 175-187. Schauer, P.S., Johns, D.M., Olney, C.E. and Simpson, K.L., 1980. International study on Avtcmia. IX. Lipid level, energy content and fatty acid composition of the cysts and newly hatched nauplii from five geographical strains of Artemiu. In: G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers (Editors), The Brine Shrimp, Artemia. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium, pp. 365-374. Sorgeloos, P., Leger, P. and Lavens, P., 1988. Improved larval rearing of European and Asian sea bass, sea bream, mahi-mahi, siganid and milkfish using enrichmer t diets for Brachianus and Artemia. World Aquaculture, 19: 77-88. Van Ballaer, E., Amat, F., Hontoria, F., Leger, P. and Sorgeloos, P., 1985. Preliminary results on the nutritional evaluation of n-3 HUFA (highly unsaturated fatty acid) enriched Artemiu nauplii for larvae of the sea bass, Dicentrarchus labrax. Aquaculture, 49: 223-229. Watanabe, T.. Oowa, F., Kitajima, C. and Fujita, S., 1978. Nutritional quality of brine shrimp, .-Wwia sulina, as a living feed from the viewpoint of essential fatty acids for fish. Bull. Jpn. Sot. Sci. Fish., 44: 1115- 12 1. Watanabe, T., Oowa, F., Kitajima, C. and Fujita, S., 1980. Relationship between dietary value of brine shrimp Artemia salina and their content of n-3 highly unsaturated fatty acids. Bull. Jpn. Sot. Sci. Fish., 46: 35-41. Watanabe, T.. Ohta, M., Kitajima, C. and Fujita, S., 1982. Improvement of dietary value of brine shrimp Artemia sulina for fish larvae by feeding them on n-3 highly unsaturated fatty acids. Bull. Jpn. Sot. Sci. Fish., 48: 1775-l 782. Watanabe, T., Kitajima, C. and Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34: 115- 143. Webster, C.D., 1989. Nutritional value of brine shrimp naupiii for striped bass larvae. Doctoral Thesis, Auburn University, Auburn, AL, 117 pp. Westin, D.T.. Olney, C.E. and Rogers, B.A., 1983. Effects of parental and dietary PCBs on survival, growth and body burden? of larval striped bass. Bull. Environ. Contam. Toxicol., 30: 50-57.