Enrichment of Artemia nauplii with n-3 HUFAs prior to feeding
the nauplii to larval fish and shrimp is a common procedure in the aquaculture
industry (Jones et al., 1993). These fatty acids are essential
for the normal development of larval fish and shrimp, but most of the
commonly available strains of Artemia used as food for these larvae
have only a very small amount of these HUFAs (Watanabe et al.,
Immanuel et al. (2007) enriched A. franciscana with marine
trash fish liver oil for enhancement of HUFA.
Rekha et al. (2007) enriched Artemia salina nauplii with
microalgae and baker`s yeast for use in larviculture. The importance of
DHA, 22:6(n-3), facilitating the normal development of larval fish and
oyster spat have been noted by many investigators including (Watanabe,
1993; Ozkizilcik and Chu, 1994). However, few of the existing enrichment
methods produce significant increases in the DHA content of Artemia
nauplii. Menhaden oil, the fish oil most commonly used in microencapsulated
or emulsified oils, generally has a DHA content of less than 12% total
fatty acids (Ozkizilcik and Chu 1994).
Much focus has been placed on the essential role of long-chain n-3 fatty
acids especially in early nervous system development of fish and shrimp,
but the n-6 HUFA, arachidonic acid is also important as the precursor
of some prostaglandins and other biologically active compounds which regulate
growth and reproductive functions (Stanley-Samuelson, 1987; De Petrocellis
and Di Marzo, 1994).
The purpose of this study was to determine the effectiveness of enriching
Artemia nauplii with long chain fatty acids by using different
concentrations of ICES30/4 as a commercial emulsion, rich in both n-3
and n-6 long chain fatty acids.
MATERIALS AND METHODS
This study was conducted in 2007 in Artemia Research Center, Urmia University.
Artemia urmiana cysts were hatched and divided into twenty one
25 L tanks filled with filtered 30 g L-l salinity sea water
at a density of approximately 300 individuals mL-1 and enriched
by the direct method described by Watanabe et al. (1982). The culture
was strongly aerated and the water temperature ranged between 27-29 °C.
Lipid emulsions were prepared by mixing 100 mL of sea water, 1 g of raw
egg yolk, 0.5 g of coconut lecithin and sufficient oil to obtain concentrations
of 100, 200 and 300 ppm ICES30/4 in Artemia medium.
For each concentration of oil, samples of Artemia were taken after
12 and 24 h. A sample of Artemia exclusively fed on ICES/0/0 as
control, was also taken. Artemia were collected with a 100 ppm
scoop mesh, thoroughly washed with freshwater, carefully dried with filter
paper and stored at-40 °C until analysis.
Analytical procedures: Total lipid and fatty acid composition
of each sample were determined. Total lipid was extracted after homogenization
in chloroform-methanol as described by Folch et al. (1957) and
determined according to standard AOAC method (AOAC, 1997). Fatty acid
mixtures were prepared from the crude lipids by saponification with KOH.
Unsaponifiable matter was determined gravimetrically. The fatty acid were
analyzed in a Varian GC-14A gas chromatograph (Shimadzu) equipped with
a flame ionization detector (250 °C) and a Supelcowax-I0 fused-silica
capillary column (30 mx 0.32 mm I.D., Supelco, Bellefonte, PA, USA) using
helium as carrier gas. The initial temperature was 180 °C for 10 min
followed by a thermal gradient to 215 °C at 2.5 C min and then maintained
for 12 min. Individual fatty acid was identified by reference to authentic
standards and to well characterize emulsion oil.
Statistical analysis: All biochemical results are given as means
and were subjected to a two factor factorial analysis of univariate, SPSS
Ver. 14. Differences between means were studied using Tukey`s multiple
range test (p<0.05).
ICES0/0 and ICES30/0 were analyzed with GC and showed in Table
1. Although the total lipid content of Artemia increased, in
general terms, over time and with the elevation of the amount of oil present
in the culture medium, but statistical analysis showed there are no any
differences among enrichment periods and also among combination between
concentrations and periods (p>0.05), Only there are significant differences
among concentrations in lipid percentage of Artemia nauplii (p<0.05).
Comparing the fatty acid composition of the treatments and control (Table
2), the content of C18:0 increased to high level in treatment ICES30/4
with 200 ppm concentration and 24 h enriching period. According to statistical
analysis, there are no any differences among concentrations (p>0.05)
but there are significant differences among enrichment period in C18:0
of Artemia nauplii (p<0.05).
The content of C18:1n7 increased to high level in treatment ICES30/4
with 100 ppm concentration and 24 h enriching period. Although, there
are significant differences between 12 and 24 h treatments (p<0.05),
but there are no any differences between treatments of 100, 200 and 300
ppm in 24 h enriching periods (p>0.05) . The content of C18:2n6-cis
decreased after all enrichment. It seems ICES30/4 could not improved this
fatty acid in the nauplii compare to control. Regarding to the content
of C20:4n6 (ARA), there were an increasing trend in 100 ppm to 300 ppm
concentration treatments and analysis of variance shows, there are differences
between concentration and period treatments (p<0.05) but their combinations
have not any differences (p>0.05).
|| Ingredients of the ICES0/0 and ICES30/4 emulsion
||Total lipid (DW %) and fatty acid composition (mg g-1
DW) of Artemia nauplii enriched with ICES30/4 in different
concentrations and periods Average ± SD
The content of C20:5n3 (EPA) and C22:6n3 (DHA) increased to high level
in treatment ICES30/4 with 300 ppm concentration and 24 h enriching period.
The results of statistical analysis showed that there are significant
differences among concentration (p<0.05), period (p<0.05) and their
combinations (p<0.05) treatments in both EPA and DHA contents. Table
2 showed the highest ration of DHA/EPA obtain when Artemia nauplii
enrich with 300 ppm concentration of ICES30/4 during 24 h period (1.22)
while this ratio in control which is 0.00.
The rapid increase in total lipid content of Artemia observed
during the enrichment process was similar to that reported by other authors
in experiments carried out with lipid emulsions (Rainuzzo et al.,
This study is in agreement with Rodriguez et al. (1996), which
studied the improvement of rotifer nutritional value with varying the
type and concentration of oil and the enrichment period, suggested it
is more effective to increase the enrichment time than the amount of oil
present in the culture medium.
Certain patterns can be observed in the incorporation of the different
groups of fatty acids, probably due to their metabolic activities or enzymatic
affinities. The behavior of Artemia with respect to n-6 and n-3
fatty acids seemed to differ from the saturated and monoenes. In this
case, after 24 h of enrichment, DHA attained values more than 5 times
and EPA less than 2 times greater than that of the initial Artemia
and, as other authors have pointed out, n-6 and n-3 HUFA fatty acids were
also assimilated to a great extent in Artemia after 24 h of enrichment
(Rainuzzo et al., 1994).
To achieve increments of n-6 and n-3 HUFA in Artemia, not only
it is more effective to increase the enrichment time but also the amount
of oil present in the culture medium.
Walford and Lam (1987) used microcapsules as enrichment materials, recommended
enrichment times over 5 h and observed maximum assimilation of n-3 HUFA
after 12 h. Watanabe (1993) considered the optimum enrichment period to
be 12 h for rotifers and nauplii of Artemia fed on lipid emulsions.
Mcevoy et al. (1995) showed that enrichment periods of 24 h increased
the risk of perioxidation of polyunsaturated fatty acids in Artemia
enrichment medium. It seems that the difference between the present works
with the results of above authors is due to differences in species of
This study in agreement with other researchers, Leger et al. (1987),
Rodríguez et al. (1996) and Watanabe (1993) suggested 300
ppm concentration of the enrichment oil for Artemia nauplii. Oil
concentrations above 300 ppm are not recommended for the well being of
the Artemia from an economic standpoint. This could be due to the
fact that an excess of oil in the medium could lead to water quality degradation
and to enhanced bacterial growth (Dabrowski and Poczycznski, 1988).
In the present study, ICES containing 300 ppm oil generated increased
DHA/EPA ratio after 24 h of enrichment (DHA/EPA =1.2). The literature
indicates that this amount should be sufficient to meet the requirements
of marine fish larvae (Snther and Jobling, 2001). Previous studies have
shown that the EPA/DHA ratio in the diet is important for larval development
(Mourente et al., 1993; Reitan et al., 1994).
In summary, the results of the present study indicate that ICES30/4 can
improve the nutritional quality of lipid and some fatty acid in Artemia
urmiana nauplii and in order to obtain increased n-3 and n-6 HUFA
content in Artemia, prolong the enrichment period and increase
the amount of oil present in the culture medium have the same effective
value but for saturated and monoen fatty acid, prolong the time is more
effectiveness than high concentration. The best results in terms of nutritive
value were obtained with ICES 30/4, with concentration 300 ppm and an
enrichment period of no longer than 24 h.
This project was funded by the Iranian Fisheries Research Organization,
and performed in the Artemia and Aquatic Animal Research Center,
Urmia University. We are most grateful to all staff of AAARC for helping
us and providing the experimental oils used in this study.