Changes in Lipid and Fatty Acid Composition of Wild Freshwater
Zooplankton during Enrichment and Subsequent Starvation
S.E Lochmann,* K.J.Goodwin, AND C.L Racey
Aquaculture Fisheries Center, University of Arkansas at Pine Bluff.
1200 North University Drive, Mail Slot 4912, Pine Bluff, Arkansas 71601, USA
Concentrated wild zooplankton harvested from freshwater ponds have been used to feed the larvae
of hybrid striped bass (female white bass Morone chrysops X male striped bass M.saxatilis) in tanks.
However, larval growth and survival have been superior when cultured rotifers and brine shrimp Artemia
spp. nauplii have been offered as first feeds. We hypothesized that enrichment with highly unsaturated
fatty acids (HUFAs), which is common for cultured zooplankton, would enhance the nutritional value of
wild freshwater zooplankton were enriched for 24 h with Super Selco, a formula rich in HUFAs. Lipid and
fatty acid composition changes in wild freshwater zooplankton were monitored during the e..richment
period and the subsequent 72 h. Total lipids in wild zooplankton increased from a preenrichment level of
38 mg/g dry weight (DW) to 72 mg/g DW after enrichment. Although wild zooplankton were initially
deficient in HUFA level after enrichment was 10.41 mg/g DW, which was above the recommended level
for good growth and survival of hybrid striped bass larvae. The nutritional state of wild zooplankton
returned to the preenrichment level 24 h after enrichment was terminated. Therefore, enriching wild
zooplankton from culture ponds for 24 h after harvesting and concentration offers producers another
nutritional option for hybrid striped bass larvae the early life history stages.
Phase-one fingerling hybrid striped bass (female white bass Morone chrysops X male striped bass
M.saxatilis) are produced in ponds (Brewer and Rees 1990) and tanks (Ludwig 1994; Denson and Smith
1997). Both production techniques eventually rely on formulated feeds. However, fingerlings produced in
ponds initially rely on wild zooplankton for nutrition (Geiger and Turner 1990), whereas fingerlings
produces in tanks initially rely on cultured rotifers and brine shrimp Artemia spp. nauplii for nutrition.
Culturing rotifers can labor intensive and unreliable (Dhert et al 2001) because the process requires either
fresh algae (Dhert et al. 2001) or algal pastes (Lee 2003). Decapsulated brine shrimp nauplii are not
difficult to prepare but can be expensive because of market fluctuations (Dhont and Van Stappen 2003;
Lee 2003).
* Corresponding author: slochmann@uaex.edu
Concentrated wild zooplankton harvested from freshwater ponds have been used to feed hybrid
striped bass larvae in tanks. Ludwig and Lochmann (2000) used a rotating drum filter to collect and
concentrate wild zooplankton. Hybrid striped bass larvae initially fed wild zooplankton and then weaned to
formulated feed averaged 8.2 mm total length (TL) and experienced 24% survival at 26 d postsocking. By
comparison. Deson and Smith (1997) reported an average of 15.4 mm TL and a survival rate of 62% at 23
d poststocking. Denson and Smith used Brachionus plicatilis cultured with Isochrysis spp. and enriched
with Cultures Selco (INVE Aquaculture, Baasrode, Belgium) as an initial feed and transitioned larvae to
Artemia nauplii before weaning them to prepared feed. Ludwig (2003) achieved an average of 12.3 mm TL
and 53% survival at 26 d poststocking. Ludwig used B.plicatilis cultured with Nannochloropsis spp. and
enriched with Super Selco (INVE Aquaculture) as an initial feed, transitioned larvae to Artemia nauplii,
and weaned them to prepared feed.
Clearly, growth and survival were superior when cultured rotifers and Artemia nauplii were offerd
as first feeds. Wild freshwater zooplankton have high levels of saturated fatty acids in addition to moderate
level of n-3 and n-6 highly unsaturated fatty acids (HUFAs) (Domaizon et al. 20001).1 It is unclear whether
the superior growth and survival of fingerling hybrid striped bass rely specifically on B.plicatilis and
Artemia nauplii. The superior growth and survival are more likely due to enrichment with products high in
n-3 and n-6 HUFAs (Clawson and Lovell 1992; Tuncer and Harrell 1992) than to use of cultured rather
than wild zooplankton. Numerous fish species have been successfully cultured after enrichment has been
incorporated into the production process (Izgiierdo et al. 1992; Tuncer et al. 1993; Hernandez-Cruz et al.
1999). Furthermore, cladocerans Daphnia spp. and Ceriodaphmia spp. and rotifers Keratella spp.
responded with increased abundance to the addition of fatty acid emulsions in enclosure experiment
(Boersma and Stelzer 2000)
We hypothesized that wild freshwater zooplankton could be enriched with HUFAs by a method
similar to that used for cultured zooplankton and that wild zooplankton so enriched would have a
nutritional value similar to that of cultured rotifers or Artemia nauplii. The specific objectives of this
research were to determine (1) the degree of change in the lipid and fatty acid composition of wild
zooplankton enriched with Super Selco and (2) the interval during which enriched wild zooplankton would
maintain a heightened lipid and fatty acid condition.
1
In this paper , fatty acids are designated by notation of the sort 20:4(n-6). Where the number to the left of
the colon is the number of carbon atoms. The number immediately to the right of the colon is the number
of double bonds. And the number after the hyphen indicates the position of the first double bond from the
methyl end.
Methods
The experiment was conducted during the spring at the University of Arkansas at Pine Bluff
Aquaculture Research Station. A Hydrotech rotating drum filter (Ludwig and Lochmann 2000) with a 60µm mesh was used to collect wild zooplankton from an earth pond. Water containing the concentrated
zooplankton from the drum filter again through a 150-µm mesh net to remove Volvox spp., larger
cladocerans, and adult copepods. A Sedgewick-Rafter counting cell and a compound microscope were
used to determine zooplankton compound microscope were used to determine zooplankton composition
and concentration. Three counts were taken and averaged. The zooplankton concentration was diluted to
250 zooplankton/mL, and 3 L of the solution was added to each of 16 McDonald hatching jars. The jars
were gently aerated during the entire experiment to provide oxygen and to maintain the zooplanktons in
suspension, and the initial temperature in the jars was recorded.
Samples of wild zooplankton were preserved at the beginning of the experiment. Two jars were
randomly selected for determination of initial conditions. Zooplankton from both jars were filtered with an
80-µm sieve, rinsed with tap water, and preserved at 700C for lipid analysis. The water quality variables of
the filtered water from the jars were measured. A dissolved oxygen (DO) meter (YSI. Inc., Yellow
Springs, Ohio; Model 52) was used to measure DO, the Nessler method was used to measure total
ammonia nitrogen (TAN) by means of a spectrophotometer (Hach Co., Loveland, Colorado; Model
DR/2000). and a pH meter (Orion Model 290A) was used to measure pH.
Super Selco was added to the remaining 14 jars to enrich the zooplankton with lipids. A solution
of 50 g Super Selco/L carbon-filtered water was prepared in a blender. Then approximately 24 mL of this
solution was added to added to each jar to obtain a final concentration of approximately 0.4 g/L. After 6
h, the temperature was measured and zooplankton were collected from two randomly select jars with the
80-µm sieve and rinsed with carbon-filtered water remove excess Super Selco. Each sample was
preserved at -700 C. This process was repeated after 12 h , zooplankton were enriched a second time by
adding 50 g Super Selco/L carbon-filtered water to remaining 10 jars, giving a final concentration in the
jars of approximately 0.4g/L. At 18 and 24 h, the zooplankton collection process used at 6 and 12 h was
repeated. Water quality variables were measured at 24 h.
The enrichment process was terminate at 24 h. Water in the remaining six jars was exchanged to
remove the unconsumed Super Selco. Zooplankton were collected with the 80-µm sieve, rinsed with
carbon-filtered water, and transferred back to the jars. The volume was brought to 3 L with carbon-filtered
water. The zooplankton collection and preservation process was repeated at 48, 72 , and 96 h. Water
quality variables were measured again at 96 h.
All zooplankton samples were freeze-dried before lipid analysis. Samples previously frozen at
-700C. were rinsed into freeze-dryer flasks with distilled water. Flasks were covered with Parafilm and
frozen at -700C. Sample were then freeze-dried (Labconco Crop., Kansas City, Missouri; Freeze Dryer 3,
Model 75200). The dry weight (DW) of each zooplankton sample was determined to the nearest 0.1 mg.
Lipids were extracted with chloroform : methanol mixtures (2:1 by volume; Folch et al. 1957). Total lipids
were compared among times with one-way analysis of variance (ANOVA) and Tukey’s Studentized
honestly significant difference (HSD) range test.
Each total lipid extract was analyzed for lipid class composition with an Iatroscan thin-layer
chromatography-flame ionization detector. A 100-µm aliquot was places into an individual vial and dried
under nitrogen. Lipids and solvent were immediately removed with a microsyringe and sported at the
origins of the S-III Chromarods. In repeat runs, the amount of total lipid extract used was adjusted
depending on the first scan to ensure accuracy of the measurement of lipid classes.
For each run , a rack of 10 Chromarods was subjected to three developments 1 and 2 were partial
scans , and development 3 was a complete scan. In development 1, the rods were developed for 33 min in a
hexane : diethy1 ether : formic acid (67.9:2.1:0.04 by volume) solution , dried for 2 min at 600C , then
placed into the Iatroscan detector. In a 60% partial rod scan , each rod was sequentially burned and the
ionized lipids were detected. Development 1 yielded estimates of aliphatic hydrocarbons (NONs) and
sterol esters (SEs). For development 2 , rods were developed for 33 min in a hexane : diethyl ether : formic
acid (47.9:21.2:2:0.06 by volume) solution , dried for 2 min at 600C , and rescanned. The second scan was
a partial rod scan of 700C. This development yielded concentration estimates of triacylglycerides (TAGs),
free fatty acids (FFAs), diacylglycerides (DAGs), sterols (STs), and monoacylglycerides (MAGs). In
development 3 , rods were developed for 33 min in a dichlormethane : methanol : water (42.2:25.3:2.5 by
volume) solution , dried for 2 min at 600C , and scanned. The third scan was a 1000C rod scan and yielded
concentration estimates of polar lipids (PLs).
Lipid classes were identified according to their retention time relative to known lipid standards for
those classes. Calibration curves using known standards were set up with PeakSimple for Windows
software (SRI , Inc.) and used to determine the relative lipid class quantities. Standards (and lipid classes)
were 3-hexadecanone (NON) , cholesteryl palmitate (SE) , tripalmitin (TAG) , palmitic acid (FFA) ,
dipalmitin (DAG) , cholesterol (ST) , 1-monopalmitoyl-rac-glycerol (MAG), and a phospholipids mix
(PL;Sigma Chemicals) containing phosphatidylcholine , phosphatidylethanolamine , phosphatidylinositol ,
and lysophosphatidylcholine. Lipid classes were reported as relative percentages of the total lipid
measured on the latroscan for each sample. Chi-square tests for equal proportions were used to compare
distributions of lipid classes among times.
An aliquot of the total lipid extract from each sample was transesterified with boron
trifluoride , and fatty acids were analyzed with a gas chromatograph with a flame-ionization detector
(Woodson-Tenent Laboratories , Des Moines. Iowa). Fatty acids were identified by comparing retention
times with those of known standards and individually compared among times with one-way ANOVA and
Tukey’s Studentized range test.
Results
Wild zooplankton were composed of 5% cladocerans Ceriodaphnia pulchella. 5% adult copepods
Cyclops vernalia, and 30% copepods nauplii. The remaining 60% of the sample included the rotifers
Brachionus havanensis valga, and Kellicottia longispina. Average temperature in the jars during the study
was 22.90C (SD. 1.4). Dissolved oxygen averged 7.4 mg/L (1.0), and TAN averaged 5.9 mg/L (1.4). The
average pH was 8.0 (0.3).
FIGURE 1.- Total lipid content of wild freshwater zooplankton before enrichment (0 h), during
enrichment (6,12,18, and 24 h), and after enrichment (48, 72, and 96 h) with Super Selco. The error bars
represent SDs.
Wild zooplankton had an initial total lipid level of 38.3 mg/g DW (6.7). The lipid level increased
significantly (F = 13.25; df = 15; P = 0.0008) by 6 h to 82.7 mg/g DW (2.9) and remained at a constant
level until enrichment was terminated (Figure 1). By 72 h, the total lipid level had returned to a
preenrichment level.
The lipid class composition of wild zooplankton changed during the enrichment and starvation
processes. The proportion of polar lipids changed at 18 h postenrichment (Figure 2) and returned to
preenrichment level at 48 h. The proportion of MAGs ranged from 12% to 16% during most of the
experiment but SEs consistently increased during the enrichment period but fell to preenrichment level at
48 h. The proportions of STs and DAGs varied little during the experiment. The lipid class composition at
0 h was significantly different from the composition was not significantly different between any other
times.
The fatty acid composition of wild zooplankton showed clear indications of the enrichment
process. The proportion of palmitic acid (16:0) nearly doubled by 18 h (Table 1:F = 24.24: df = 15 : P
<0.0001). The proportions of the unsaturated fatty acids with 18 carbon atoms approximately doubled by
24 h. The proportions of arachidonic acid (AA, 20:4[n-6]) and docosahexaenoic acid (DHA, 22:6[n-6])
were undetectable in wild zooplankton, but enrichment increased the levels of AA (F = 13.39; df =15;
P =0.0530) and DHA (F = 13.39; df = 15;P = 0.0008) to 0.36 and 3.89 mg/g DW, respectively. The most
striking significant change (F = 18.79; df =15; P = 0.0002) occurred in the proportion of eicosapentaenoic
acid (EPA, 20:5[n-3]), which was 23 times higher following 24 h of enrichment. The radio of EPA to AA
raged from 15.24 to 30.73, but AA was undetectable until 12 h and again after 24 h. The DHA to EPA
radio ranged from 0.17 at 6 h to 0.94 at 48 h. The radio of n-3 to n-6 HUFAs increased monotonically
during enrichment but declined to below the original radio by 48 h.
FIGURE 2. – Proportions of sterols (STs), diacylgycerides (DAGs), free fatty acids (FFAs),
monoacylglycerides (MAGs), and polar lipids (PLs) in the total lipids of wild freshwater zooplankton
before enrichment (0 h), during enrichment (6, 12, 16, and 24 h), and after enrichment (48, 72, and 96 h)
with Super Selco.
Discussion
The lipid level of wild zooplankton was lower than that of rotifers and Artemia nauplii from other
studies. When cultured on baker’s yeast, B. plicatilis had a lipid level of 105 mg/g DW (Fernandez-Reiriz
et al. 1993). Artemia nauplii had a lipid level of 123 mg/g DW (McEvoy et al. 1996). The lipid level of
wild zooplankton after enrichment with Super Selco increased as much as it did when rotifers were
enriched and more than when Artemia nauplii were enriched. After enrichment. The lipid levels of B.
plicatilis (Fernandez-Reiriz et al. 1993) and Artemia nauplii (McEvoy et al. 1996) were 1.9 and 1.4 times
as great , respectively , as they were before enrichment. Fernandez-Reiriz et al. (1993) enriched rotifers for
6 h , and McEvoy et al. (1996) enriched Artemia nauplii for 18 h. Wild zooplankton enriched for 24 h had
1.9 times as much lipid after enrichment as they did before enrichment. Enrichment increased the amount
of
TABLE 1.- Fatty acid composition (mg/g dry weight) of wild zooplankton before (0 h) , during
(6, 12,18, and 24 h), and after (48, 72, and 96 h) enrichment with Super Selco (mean [SD] of two replicate
groups). Elements within a row with the same letter are not significantly (P > 0.05)
The lipid class composition of wild zooplankton differed from that of B. plicatilis
(X2 = 54.77; df = 6; P < 0.0001) and Artemia nauplii (X2 = 107.19; df = 6; P < 0.0001) before enrichment.
Wild zooplankton had a higher proportion of PLa than rotifers or Artemia nauplii before enrichment.
About 50% of the lipid in unenriched B. plicatilis was polar and approximately 25% of the lipid in Artemia
nauplii was polar (Table 2). Polar lipid are components of cellular membranes and are generally
considered structural in nature. The lipid classes TAG and SE constituted a much greater proportion of
total lipids in B. plicatilis and Artemia nauplii than in wild zooplankton. These lipid classes are associated
with short-term energy storage, suggesting that rotifers cultured on baker’s yeast and recently hatched
Artemia nauplii had more stored excess energy, but wild zooplankton had little excess energy, for storage.
The lipid class composition of wild zooplankton also differed from that of B. plicatilis (X2 = 33.39; df = 6;
P < 0.0001) and Artemia nauplii (X2 = 98.60; df = 6; P < 0.0001) following enrichment. Enrichment with
Super Selco increased the neutral lipid classes of all three groups, but B. plicatilis changed the lipid class
composition least. Enriched wild zooplankton had about 12% less PLs and considerably more TAG and
SEs. Artemia nauplii had about 9% more TAGs following enrichment. The proportion of PLs in B.
plicatilis fell 8% and the FFAs in B. plicatilis doubled during enrichment.
TABLE 1.- Extended.
The fatty acid profile of wild zooplankton was different from that of B. plicatilis and Artemia
nauplii before enrichment. Unlike the results of Domaizon et al.(2000), the wild zooplankton from this
study had less n-3 HUFAs and more n-6 HUFAs. Wild zooplankton had lower levels of 18:1 fatty acid,
AA, and total monoenes than B. plicatilis or Artemia nauplii (Table 3). Wild zooplankton also had EPA
levels lower than those of B. plicatilis. Enrichment increased the amount of certain fatty acids in all three
groups, including C unsaturated fatty acids with 18 carbon atoms, AA, EPA, and DHAs (Table 3). Wild
zooplankton were enriched in AA and EPA more than B. plicatilis and Artemia nauplii. No significant
difference was observed in the level of n-3 HFAs between wild zooplankton and B. plicatilis or Artemia
nauplii following enrichment. Overall, there were fewer differences between wild and cultured
zooplankton following enrichment.
Hybrid striped bass larvae fed diets low in HUFAs displayed essential fatty acid deficiency
syndrome (rapid swimming and subsequent fainting) and experienced poor survival at metamorphosis
(Tuncer and Harrell 1992). Tuncer and Harrell (1992) recommended a dietary HUFAs level of at least 5.7
mg/g DW for larval hybrid striped bass. Clawson and Lovell (1992) used oil from Gulf menhaden
Brevoortia patronus to enrich Artemia nauplii (Great Salt Lake strain). The HUFA level in their enriched
Artemia nauplii was 16 mg/g DW. This HUFA level resulted in significantly faster growth and better
survival than in hybrid striped bass larvae reared on unenriched Artemia nauplii. Rotifers enriched with
Super Selco had a HUFA level of 30.9 mg/g DW (Fernandez-Reiriz et al. 1993). Hence, it is not surprising
that Ludwig (2003) found good growth and survival of hybrid striped bass fed B. plicatilis enriched with
Super Selco. Unenriched wild zooplankton had only about 2.03 mg HUFAs/g DW. This HUFA level is
low and probably explains the slow growth and poor survival reported by Ludwig and Lochmann (2000).
After enrichment, wild zooplankton had 10.41 mg HUFA/g DW. This level should be sufficient for good
growth and survival of hybrid striped bass larvae. Sargent et al. (1999) suggested an optimal DHA to EPA
ratio of two for most marine fish. Nematipour and Gatlin (1993) showed that the essential fatty acid
requirements of hybrid striped bass are similar to those of marine fish. The DHA to EPA ratio of wild
zooplankton after enrichment was only 0.7, which is lower than ideal. Tacon (1987) suggested that the
dietary requirement of fish for n-6 fatty acids. Hence, ratio of n-3 to n-6 HUFAs greater than one is
favorable, and the postenrichment ratio of the wild zooplankton in this study was 1.89.
TABLE 2.- Lipid class composition (% of total lipids) of wild zooplankton Brachionus plicatilis
(Fernandez-Reiriz et al 1993). And brine shirp Artemia nauplii (McEvoy et al. 1996) before and after
enrichment with Super Selco.
Ammonia can be toxic to aquatic invertebrates if levels are high and a large proportion is in the
unionized form. At the temperature and pH from our study, approximately 4.7 % (0.3 mg/L) of the
ammonia would be in the un-ionized ammonia in our study was below the 96-h concentration lethal to
50% of the test animals reported by Williams et al. (1986) for a suite of aquatic invertebrates. Denser
concentrations of zooplankton could result in higher levels of un-ionized ammonia. This water quality
parameter should be closely monitored during enrichment of wild zooplankton.
TABLE 3.-Fatty acid composition (mg/g dry weight) of wild zooplankton, Brachionus plicatilis
(Fernandez-Reiriz et al. 1993), and brine shrimp Artemia nauplii (McEvoy et al.1993) before and after
enrichment with Super Selco. The fatty acid composition of Super Selco (reported in Fernandez-Reiriz et
al 1993) is included for comparison. The fatty acids of wild zooplankton before and after enrichment were
compared with the fatty acids of B. plicatilis or Artemia nauplii. Before or after enrichment using twotailed t-tests assuming unequal variances (x = 0.05). The asterisks indicate that fatty acid composition of
Brachionus plicatilis or Artemia nauplii was significantly different from that of unenriched wild
zooplankton;a plus sign indicates that fatty acid composition was significantly different from that of
enriched wild zooplankton.
Enriched wild zooplankton were nutritionally superior to unenriched wild zooplankton. A 24-h
enrichment period was required to maximize the nutritional state of wild zooplankton compared with just 6
h for B. plicatilis and 18 h for Artemia nauplii. After 24 h of starvation, the nutritional state of wild
zooplankton had returnd to a preenrichment level. Enrichment with Super Selco resulted in a HUFA level
above that recommended for good growth and survival of hybrid striped bass larvae. Therefore, enriching
wild zooplankton from culture ponds for 24 h after harvesting and concentration offers producers another
nutritional option for hybrid striped bass larvae during the early life history states.
Acknowledgments
The authors would like to thank R.T. Lochmann, G. M. Ludwig, and S.D.Rawles for their review
of the manuscript during preparation. This research was funded in part by the U.S. department of
Agriculture Evans-Allen 1890 Research Program.
References
Boersma, M., and C.P.Stelzer. 2000. Response of a zooplankton community to the addition of unsaturated
fatty acids: an enclosure study. Freshwater Biology 45:179-188.
Brewer, D.L., and R.A. Rees. 1990. Pond culture and phase I striped bass fingerlings. Pages 99-120 in
R.M Harrell, J.H. Kerby, and R.V. Moniton, editors. Culture and propagation of striped bass and
its hubrids. American Fisheries Society, Southern Division, Bethesda, Maryland.
Clawson, J. A., and R.T. Lovell. 1992. Improvement of nutritional value of Artemia for hybrid. Striped
bass/white bass (Morone saxatilis x M. chrysops) larvae by n-3 HUFA enrichment of nauplii with
menhaden oil. Aquaculture 108:125-134.
Denson, M.R., and T.I.J. Smith . 1997. Tank culture of larval sunshine bass. Progressive Fish-Culturist
59:59-63.
Dhert, P., G. Rombaul, G. Suantika, and P.Sorgeloos. 2001. Advancement of rotifer culture and
manipulation technique in Europe. Aquaculture 200:129-146.
Dhont, J., and G. Van Stappen. 2003. Biology, tank production. And L.A. McEvoy, editors. Live feeds in
marine aquaculture. Blackwell Scientific Publications, Oxford, UK.
Domaizon, I., C. Desvilettes, D. Debroas, and G. Bourdier. 2000. Influence of zooplankton and
phytoplankton on the fatty acid composition of digesta and tissue lipids of silver carp: mesocosm
experiment. Journal of Fish Biology 57:417-432.
Fernandez-Reiriz. M.J.,U Labarta, and M.J. Ferreiro. 1993. Effects of commercial enrichment diets on the
nutritional value of the rotifer (Brachionus plicatilis). Aquaculture 112:195-206.
Folch, J.,N. Lees,and G.H. Sloane-Stanley. 1957. A simple method for the isolation and purification of
total lipids from animal tissues. Journal of Biology and chemistry 226:497-509.
Geiger, J. G., and C.J. Turner. 1990. Pond fertilization and zooplankton management techniques for
production of fingerling striped bass and hybrid striped bass. Page 79-98 in R.M. Harrell, J.H.
Kerby, and R.V. Minton, editiors. Culture and propagation of striped bass and its hybrids.
American Fisheries Society. Southern Division. Committee, Bethesda, Maryland.
Hernandez-Cruz, C.M. M. Salhi, M. Bessonart, M.S. Izquierdo, M. M. Gonzales, and H. FernandezPalacios. 1999. Rearing techniques for red porgy (Pagrus pagrus) during larval development.
Aquaculture 179:489-497.
Izquierdo, M.S.,T. Arakawa, T. Takeuchi, R. Haroun, and T. Watanabe. 1992. Effect of n-3 HUFA levels
in Artemia on growth of larval Japanese flounder (Paralichthys olivaceus). Aquaculture 105:7382.
Lee, C. 2003 Biotechnological advances in finish hatchery production: a review. Aquaculture 227:439458.
Ludwig, G.M. 2003. Tank culture of larval sunshine bass. Morone chrysops x M. saxatilis fry with
freshwater rotifers Brachionus calyciplorus and salmon starter meal as first food sources. Journal
of the World Aquaculture Society 31:1-9.
Ludwig, G.M. 2003. Tank culture of larval sunshine bass. Morone chrysops (Rafinesque) x M.saxatilis,
(Walbaum), at three feeding level. Aquaculture Research 34:1277-1285.
Ludwig, G. M., and S.E. Lochmann. 2000. Culture of sunshine bass, Morone chrysops x M.saxatilis, fry in
tanks with zooplankton cropped from ponds with a drum filter. Journal of Applied Aquaculture
10(2): 11-26.
McEvoy, L. A.,J.C. Navarro, F. Hontoria, F. Amat, and J.R. Sargent. 1996. Two novel Artemia enrichment
diets containing polar lipid. Aquaculture 144:339-352.
Nematipour, G.R., and D.M. Gatlin, III. 1993. Requirment of hybrid striped bass for dietary (n-3) highly
unsaturated fatty acids. Journal of Nutrition 123:744-753.
Sargent, J., G.Bell, L.McEvoy,D. Tocher, and A. Estevez. 1999. Recent developments in the essential fatty
acid nutrition of fish. Aquaculture 177:191-199.
Tacon, A. G.J. 1987. The nutrition and feeding of farmed fish and shrimp: a training manual. 1. The
essential nutrients. Food and Agriculture Organization of the United Nations,
GCP/RLA/075/ITA.FAO Field Document 2/E. Brasilia, Brazil.
Tuncer, H., R. M. Harrell. 1992. Essential fatty acid nutrition of larval striped bass (Morone saxatilis) and
palmetto bass (M.saxatilis x M. chrysops). Aquaculture 101:105-121.
Tuncer, H., R. M. Harrell, and T.chai. 1993. Beneficial effects of n-3 HUFA enriched Artemia as food for
larval palmetto bass (M.saxatilis x M. chrysops). Aquaculture 110:341-359.
Williams, K.A.,D.W.Green, and D. Pascoe. 1986. Studies on the acute toxicity of pollutants to freshwater
macroinvertebrates, 3. Ammonia. Archiv fuer Hydrobiologie 106:61-70.