Variation in fatty acid composition of Artemia salina nauplii enriched with microalgae and baker's yeast for use in larviculture

Article abstract of DOI:10.1021/jf063654l

The high content of the essential fatty acids in some microalgae and baker's yeast has made them excellent diets for boosting the fatty acid content of livefood Artemia. The influences of baker's yeast (Saccharomyces cerevisiae) and three microalgae, viz., Chlorella salina, Chaetoceros calcitrans, and Nannochloropsis salina, were tested as diet components in marine livefeed brine shrimp Artemia salina nauplii to improve the polyunsaturated fatty acid (PUFA) composition. Artemia nauplii submerged in these diets for four different enrichment intervals (3, 6, 8, and 24 h) were found to incorporate essential fatty acids, and the percentage composition of different fatty acids was measured in the enriched Artemia nauplii and enrichment diets. N. salina produced higher levels of arachidonic acid (AA, 20:4n6, 9.50%), eicosapentaenoic acid (EPA, 20:5n3, 25.80%), and docosahexaenoic acid (DHA, 22:6n3, 4.18%) as compared to other diets. The total PUFA content of the enriched Artemia by N. salina increased by 56.50% with enrichment periods up to 8 h, followed by a significant reduction in the final 24 h. N. salina yielded Artemia nauplii with considerable EPA (8.05%), AA (14.15%), and DHA (1.85%) after 8 h of enrichment, which are significantly higher levels than in nauplii fed with the other three diets (p = 0.05). The DHA/EPA values in Artemia enriched for 6 h by N. salina and C. calcitrans were found to be, respectively, 88.46 and 25% higher than freshly hatched Artemia. Artemia enriched by C. salina and baker's yeast exhibited a reduction in PUFA content even at 6 h of enrichment. Significant relative decreases in DHA, EPA, and total PUFA in Artemia enriched with all of the diets were apparent, with a corresponding increase in the total saturated fatty acid content (26.95 ± 9.75%) in the final stages (24 h) of enrichment (p = 0.05).

Full text of DOI:10.1021/jf063654l

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J. Agric. Food Chem. 2007, 55, 4043−4051 4043
Variation in Fatty Acid Composition of Artemia salina Nauplii
Enriched with Microalgae and Baker’s Yeast for Use in
Larviculture
REKHA D. CHAKRABORTY,† KAJAL CHAKRABORTY,*,‡ AND
†
E. V. RADHAKRISHNAN
Crustacean Fisheries Division and Physiology Nutrition and Pathology Division, Central Marine
Fisheries Research Institute, Ernakulam North P.O., P.B. No. 1603, Cochin-682018, Kerala, India
The high content of the essential fatty acids in some microalgae and baker’s yeast has made them
excellent diets for boosting the fatty acid content of livefood Artemia. The influences of baker’s yeast
(Saccharomyces cerevisiae) and three microalgae, viz., Chlorella salina, Chaetoceros calcitrans, and
Nannochloropsis salina, were tested as diet components in marine livefeed brine shrimp Artemia
salina nauplii to improve the polyunsaturated fatty acid (PUFA) composition. Artemia nauplii submerged
in these diets for four different enrichment intervals (3, 6, 8, and 24 h) were found to incorporate
essential fatty acids, and the percentage composition of different fatty acids was measured in the
enriched Artemia nauplii and enrichment diets. N. salina produced higher levels of arachidonic acid
(AA, 20:4n6, 9.50%), eicosapentaenoic acid (EPA, 20:5n3, 25.80%), and docosahexaenoic acid (DHA,
22:6n3, 4.18%) as compared to other diets. The total PUFA content of the enriched Artemia by N.
salina increased by 56.50% with enrichment periods up to 8 h, followed by a significant reduction in
the final 24 h. N. salina yielded Artemia nauplii with considerable EPA (8.05%), AA (14.15%), and
DHA (1.85%) after 8 h of enrichment, which are significantly higher levels than in nauplii fed with the
other three diets (p ) 0.05). The DHA/EPA values in Artemia enriched for 6 h by N. salina and C.
calcitrans were found to be, respectively, 88.46 and 25% higher than freshly hatched Artemia. Artemia
enriched by C. salina and baker’s yeast exhibited a reduction in PUFA content even at 6 h of
enrichment. Significant relative decreases in DHA, EPA, and total PUFA in Artemia enriched with all
of the diets were apparent, with a corresponding increase in the total saturated fatty acid content
(26.95 ( 9.75%) in the final stages (24 h) of enrichment (p ) 0.05).
KEYWORDS: Polyunsaturated fatty acid (PUFA); eicosapentaenoic acid (EPA); docosahexaenoic acid
(DHA); arachidonic acid (AA); Artemia salina; microalgae; Baker’s yeast
INTRODUCTION
20:4n6), docosahexaenoic acid (DHA, 22:6n3), and eicosapen-
tanoic acid (EPA, 20:5n3) in particular. Nearly all mariculture
production systems predominantly rely on Artemia nauplii,
which is naturally deficient in 20:5n3, and no known strains of
Artemia contain significant levels of 22:6n3, making n3 PUFA
enrichment necessary. Research on enriching Artemia nauplii
in increasing the amounts of long-chain PUFAs, viz., DHA,
EPA, and AA, prior to their use as live prey have received
considerable attention. These PUFAs, which are usually low in
abundance in Artemia, are regarded as essential for finfish and
crustaceans and must be supplied in this livefeed. The impor-
tance of PUFAs in larval finfish/shellfish nutrition has been
extensively investigated during the past 20 years (5-10). DHA
is one of the important PUFAs, which maintains structural and
functional integrity in larval cell membranes in addition to the
neural development and function, while AA and EPA are
involved in, respectively, the production and modulation of
eicosanoids (11). AA has been an essential function of producing
Artemia salina, the brine shrimp, are the most widely used
aquaculture live food organism for marine larvae primarily
because they are very convenient to use and are readily available
(1). The biochemical composition of Artemia is regarded as
important in optimizing larval nutrition for survival and growth
of aquaculture species like finfish and shellfish (2). Although
Artemia are not the natural prey of these animals, they are simple
to prepare and have been used effectively as the major or sole
component to culture aquaculture larvae, presently making them
a diet of choice (3, 4). However, Artemia nauplii are an
incomplete food source for larvae of marine finfish and
crustaceans, because of their paucity of essential n3 and n6
polyunsaturated fatty acids (PUFAs), viz., arachidonic acid (AA,
* To whom correspondence should be addressed. Tel: 91-484-2394867.
Fax: 0091-0484-2394909. E-mail: kajal_iari@yahoo.com.
† Crustacean Fisheries Division.
‡ Physiology Nutrition and Pathology Division.
10.1021/jf063654l CCC: $37.00
© 2007 American Chemical Society
Published on Web 04/04/2007

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4044 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Chakraborty et al.
MATERIALS AND METHODS
eicosanoids, making it an essential fatty acid in marine fish,
which has to be provided in larval and broodstock diets because
prostaglandins (PGF2R) are produced from 20:4n6, and has roles
in the natural shedding of eggs, synchronizing ovulation, and
spawning. AA is the basis for cyclo-oxygenase (COX) action
to produce PGF2R. AA, being a major component of phosphoi-
nositol, was reported to have a vital role in the transduction
signal mechanism in larvae. The roles of live food in aquaculture
and enrichment of live food by PUFA-rich sources are well-
established facts. However, a majority of the research works
have been concentrated in increasing PUFA levels and DHA:
EPA ratios in Artemia nauplii and other live prey species that
are fortified with PUFA-rich commercial oil emulsions (e.g.,
DHA Selco from INVE) (2, 12). The success and convenience
of this technique have led to commercially produced n3 PUFA
enrichment emulsions being widely used in marine hatcheries
(13). The essential n3 PUFA content is very small in TAG
micelles generated in enrichment procedures, and there is a
potential chance of autoxidation of PUFAs, resulting in the
formation of potentially toxic oxidation products (14, 15).
Moreover, the cost-benefit ratio in enrichment emulsion is fairly
high.
Artemia Production and Enrichment with Diets. The encapsulated
Artemia cysts obtained from the Red Top brand, United States (0.5-
1.0 g), were hatched to nauplii following established procedures (19,
20). The nauplii were left in sterile filtered seawater and were enriched
with four different enrichment diets, viz., three species of marine
microalgae, C. calcitrans, C. salina, and N. salina, and baker’s yeast,
S. cereVisiae. Newly hatched Artemia served as a control. The
microalgae were cultivated in sterile Mequil’s/Conway’s medium
following established procedures (21). The microalgae were used in
feeding experiments when they reached the end of the logarithmic
phase, i.e., after 5-7 days of growth. The ratio of microalgae to Artemia
was kept at about 6000-6500 algal cells per Artemia. Three replicates
were carried out for each treatment. The enrichment diets were added
at the concentration of 0.3 g/L. Artemia nauplii were enriched by
microalgal culture (2 L) each day at 25 °C, taking into account the
consumption of these microalgae by Artemia. Samples of Artemia were
randomly taken at four different intervals (3, 6, 8, and 24 h) throughout
the enrichment period. During feeding experiments, triplicate cultures
of Artemia (200 mL) were collected. Uneaten microalgae and baker’s
yeast were separated from Artemia by washing with sterile filtered
seawater on 120 mesh screen, rinsed with ammonium formate (NH4-
COOH, 0.5 M), freeze-dried, and stored at -20 °C prior to chemical
analysis.
As alternatives to fish oil fractions, there is a growing interest
in the fatty acid composition of marine microalgae and baker’s
yeast that produce PUFAs (16, 17). The importance of microal-
gae in aquaculture is large because they start the food chain.
The n3 and n6 fatty acids with more than 18 carbon atoms are
made primarily by aquatic phytoplankton and are transferred
via the food chain to the higher animals like Artemia (18). In
addition, to provide protein (essential amino acids) and energy,
they supply other key nutrients such as vitamins, pigments, and
sterols, which are transferred through the food chain. It is
significant that microalgae contain high contents of essential
PUFAs and are major contributors to the marine food web as a
renewable source. Earlier studies demonstrated the use of
microalgae of the genera Nannochloropsis, Chaetoceros, and
Chrolella as potential diets for livefeed Artemia for PUFA
enrichment. Also, there are reports of yeast being used to enrich
the copepods (19). Little work has been carried out regarding
in-depth biochemical and metabolic studies of PUFA enrichment
in livefeed A. salina by different tropical microalgal species
like Nannochloropsis salina, Chaetoceros calcitrans, and
Chrolella salina. Interest in the culture of tropical microalgal
species has increased over recent years in response to the growth
of tropical mariculture and the resulting need for nutritious
microalgae that are tolerant to tropical culture conditions. PUFAs
in live organisms like microalgae are much more stable than in
commercially available emulsion formulations. It is significant
that the levels and ratios of 22:6n3 : 20:5n3 : 20:4n6 in live
microalgal cells more closely resemble larval natural diets, and
the probabilities of natural protection of PUFAs by natural
antioxidants in microalgae are advantageous.
Extraction and Derivatization of Fatty Acids to Fatty Acid
Methyl Esters (FAME) and N-Acyl Pyrrolidides. The freeze-dried
samples of Artemia, microalgae, and baker’s yeast were analyzed for
different fatty acids. The lipids were extracted following the Bligh and
Dyer method (22). The lipid extracts were saponified with 0.3 N KOH
in CH3OH. After removal of the nonsaponifiable material with n-hexane
and acidification with 1 N HCl, the saponifiable materials were extracted
with petroleum ether-diethyl ether (1:1, v/v) and transesterified into
their methyl esters by reaction (30 min under reflux) with methylating
mixture (14% BF3/MeOH, 5 mL) (23). This anhydrous reaction was
performed under an inert atmosphere, and the setup was assembled
and cooled under dry N2. The product was added with distilled water
(20 mL) and extracted with n-hexane. The organic layer was concen-
trated under an inert atmosphere of N2. The resulting FAMEs
concentrate thus obtained was dissolved in n-hexane and purified by
silica gel column chromatography with n-hexane-diethyl ether (10:1,
v/v) as the eluant and by thin-layer chromatography (TLC) using the
same eluant. The PUFAs were further separated by preparative
argentation-TLC (1.0 mm, 20 cm × 20 cm) by double-developing
with n-hexane-diethyl ether-acetic acid (94:4:3, v/v/v). The purified
methyl esters were reconstituted in petroleum ether, flushed with N2
in glass vials, and stored in a deep freeze (-20 °C) until required for
gas chromatography (GC)/GC-mass spectrometry (MS) analyses.
N-acyl pyrrolidides were synthesized by reaction between pyrrolidine
(1 mL) and FAME (1 mg) in acetic acid (0.1 mL) under reflux (100
°C, 2 h) (24), and the product was solubilized in n-hexane-diethyl
ether (1:1, v/v) and washed with distilled water (4 mL × 3). The organic
phase was dried with anhydrous MgSO4, filtered, and concentrated to
furnish N-acyl pyrrolidides, which were homogeneous, by TLC.
The crude extracted lipids were separated into different classes on
a small column packed with silica gel (30 mm × 5 mm i.d., Kieselgel
60, 80-200 mesh, E-Merck), previously heated to 350 °C overnight
for deactivation. The first fraction was eluted with CH2Cl2-n-hexane
(1:2, v/v) to remove the steryl esters. This was followed by CH2Cl2
(100%) eluting the triacylglycerols (TAGs), CH2Cl2-diethyl ether (40:
1, v/v) eluting the sterols, and CH2Cl2-CH3OH (9:2, v/v) eluting the
free fatty acids. The fractions were monitored by TLC on silica gel
using n-hexane-diethyl ether-acetic acid (80:20:1, v/v/v) as the
developing solvent. TLC was performed on 250 µm silica gel G plates
with 10% gypsum as binder, preactivated at 120 °C for 2 h, using 2,7-
dichlorofluorescein (0.2% in ethanol, w/v) as the visualizing agent. The
TAG was recovered from the column eluate followed by evaporation
under N2 until dryness. The resulting product was saponified and
transesterified into their methyl esters to be analyzed by GC/GC-MS
as illustrated earlier.
In this study, various enrichment diets that have been used
to improve the nutritional composition of Artemia fall into two
groups: (i) live microalgae, viz., the diatom C. calcitrans, the
eustigmatophyte N. salina, and C. salina, and (2) baker’s yeast
Saccharomyces cereVisiae. We report the diet-induced changes
in the fatty acid composition of enriched Artemia with consid-
eration for the initial content of the nauplii and possible
metabolic changes of the fatty acids for various durations, viz.,
3, 6, 8, and 24 h, during the time-course enrichment. The
objective of the feeding trials was to study the value of
microalgae as live food enrichment diets for Artemia and to
compare their efficacy with S. cereVisiae.

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Fatty Acid Composition of Enriched Artemia salina
J. Agric. Food Chem., Vol. 55, No. 10, 2007 4045
Analysis of Fatty Acids by GC and GC-MS. Quantitative and
qualitative analyses of FAME obtained by transesterification were
performed on a Perkin-Elmer AutoSystem XL, gas chromatograph
(Perkin-Elmer, United States) equipped with an Elite-5 (crossbond 5%
diphenyl-95% dimethyl polysiloxane) capillary column (30 m × 0.53
mm i.d., 0.50 µm film thickness, Supelco, Bellfonte, PA) using a flame
ionization detector. The oven temperature ramp was 110 °C for 1.0
min, followed by an increase of 45 °C/min to 210 °C, where it was
held for 1.0 min, followed by an increase of 30 °C/min to 250 °C,
where it was held for 1.0 min, followed by an increase of 25 °C /min
to 285 °C, where it was held for 2.0 min, until all peaks had appeared.
For N-acyl pyrrolidide derivatives, the oven temperature was pro-
grammed to hold 160 °C for 1 min, and then increased at the rate of
10 °C/min up to 220 °C, and thereafter 5 °C/min up to 290 °C. Ultrahigh
purity He (99.99% purity) was used as the carrier gas at a flow rate of
1 mL/min. The injector temperature was maintained at 285 °C. It was
equipped with a split/splitless injector, which was used in split (1:15)
mode. The flow rate of He and air were maintained at 50 psi. The
injection volume was 1 µL. The detector was isothermal at 290 °C.
FAMEs were identified by comparison of retention times with the
known standards (37 component FAME Mix, PUFA-3, PUFA-1, and
BAME; Supelco). The shorthand notation used in fatty acid nomen-
clature is L:Bnx, where L is chain length, B is number of double bonds,
and nx is the position of the ultimate double bond from the terminal
methyl group. The fatty acid composition was expressed as weight
percent of the total fatty acids (TFA) of each fraction. Chromatograms
were analyzed using Perkin-Elmer Chromstation (version 4.02) soft-
ware.
prior to statistical analyses of FAME data expressed in percentages.
All measurements were performed in triplicate, and values were
averaged.
RESULTS
Spectroscopic Analyses of FAMEs and N-Acyl Pyrrolid-
ides. The molecular ions for saturated fatty acid (SFA) and
monounsaturated fatty acid (MUFA)-FAME were conspicuous
and as obvious from the mass spectra; hence, the molecular
weight was discernible from the EI-MS spectra. An ion at (M
- 31)+ represents the loss of the -OCH3 group, thus confirming
the molecular structure as methyl ester. In the mass fragmenta-
tion pattern of FAME, the base peak was assigned to be the
1-methoxyethenol moiety (m/z ) 74) obtained by McLafferty
rearrangement per se with the loss of the McLafferty ion (m/z
) 222). The results are similar to those obtained in earlier
studies (24). The molecular ion undergoes further rearrangement
to furnish 1-methoxyprop-2-en-1-ol (m/z ) 88). The spectra
further contained lower m/z fragment ions at a difference of
m/z ) 14, definitely supporting the general structure of FAME,
viz., [-CH3OCO(CH2)nCH3]+ in the homologous series. The
abundance of fragment ions in SFA and MUFA-FAME was
found to be higher than in the EI-MS spectra of PUFAs. The
EI-MS spectra of methyl octadeca-9,12-dienoate or methyl
linoleate have an abundant molecular ion (m/z ) 294) and base
peak as the McLafferty ion (m/z ) 74). In methyl octadeca-9,-
12,15-trienoate or methyl R-linolenate (18:3n3), a fragment ion
at m/z ) 150 was obvious, which is characteristic for fatty acids
with an n3 terminal moiety, while one at m/z ) 108 defines an
n3 terminal group as in methyl methyl eicosa-5,8,11,14,17-
pentaenoate or methyl eicosapentaenoate and methyl docosa-
4,7,10,13,16,19-hexaenoate or methyl docosahexaenoate. In the
mass fragmentation pattern of methyl eicosa-5,8,11,14-tet-
raenoate or methyl arachidonate, the molecular ion is barely
distinguishable, but the characteristic ion for the n3 moiety, at
m/z ) 108, is apparent. In the mass fragmentation pattern of
PUFAs with more double bonds (n g 4), the tropylium ion (m/z
) 91) does stand out as the diagnostic fragment ion. In the
mass fragmentation pattern of N-acyl pyrrolidides, the base peak
was assigned to be the McLafferty rearrangement ion, 1-(pyr-
rolidin-1-yl)-ethenol (m/z ) 113), with the concurrent loss of
the (M - 113) fragment ion. The molecular ion undergoes
further rearrangement to furnish 1-(pyrrolidin-1-yl)-prop-2-en-
1-ol (m/z ) 127). In the mass spectrum of 1-(pyrrolidin-1-yl)
octadeca-9,12,15-trien-1-one, the double bonds in positions 9
and 12 are located by the gaps of m/z ) 12 units between ions
at m/z ) 196, 208 and 236, 248, respectively.
Fatty Acid Composition of Microalgae and Baker’s Yeast.
The abundance of the fatty acid content of the logarithmic phase
N. salina was 20:5n3 > 16:1n7 > 16:0 > 18:1n9 > 20:4n6 >
22:6n3, in decreasing order (Table 1). Among SFAs, 16:0 was
found to be the most dominant contributing 21% of TFAs in
N. salina. Among n3 fatty acids, EPA was found to be the most
abundant in the logarithmic phase of N. salina (25.8% TFA)
and C. calcitrans (18.7% TFA). The high DHA (4.18%) levels
in N. salina caused it to have a higher DHA:EPA ratio (0.16)
than C. calcitrans diet (Table 1). The highest levels of PUFAs
in N. salina were influenced by high percentages of EPA and
AA, with the EPA/AA ratio of 2.72. The C18 fatty acids were
present in small amounts in C. calcitrans (6.62% TFA), although
significant C16 PUFAs (like 16:2n4, 22.5% TFA) were apparent.
Chlorella salina had higher proportions of shorter carbon chain
PUFAs (18:2n6, 18:3n3) together with lower amounts of C16
fatty acids (Table 1). The highest levels of MUFAs were found
The qualitative GC-MS analyses were performed using electronic
impact (EI) ionization mode in a Varian GC (CP-3800) interfaced with
a Varian instrument 1200L single quaduple Mass Spectrometer for
confirmation of fatty acid identification. The GC apparatus was
equipped with WCOT fused silica capillary column of high polarity
(DB-5; 30 m × 0.25 mm i.d., 0.39 mm o.d., and 0.25 µm film thickness;
Varian). The polymeric stationary phase was nonpolar (VF-5MS, 5%
phenyl-substituted methylsiloxane). The carrier gas was ultrahigh purity
He (99.99% purity) with a constant flow rate of 1 mL/min. The injector
(type 1079) and detector temperatures were maintained at 300 °C. The
injection volume was 1 µL. Samples were injected in split (1:15) mode
at 300 °C into the capillary column similar to that used for the GC
analyses, and the oven was identically programmed. The ion source
and transfer line were kept at 300 °C. Electron ionization (E.I.) was
produced by accelerating electrons from a hot filament through a
potential difference at the standard value of 70 eV. The mass
spectrometer conditions were as follows: ion source temperature, 200
°C; scan, 10-800 atomic mass units (amu); centroid scan mode; scan
rate, 1250 amu/s; and dwell time, 1.5 s. Mass spectra were analyzed
using Varian Workstation (version 6.2) software. For example, the mass
spectrometric data of methyl eicosa-5,8,11,14,17-pentaenoate/methyl
eicosapentaenoate were as follows. EI-MS m/z (rel. int. %): 315 (M+,
1.67), 175 (6.67), 161 (8.33), 145 (11.67), 131 (18.33), 119 (31.67),
108 (31.67), 91 (70.00), 79 (100), 67 (68.33), 55 (48.33). The data for
methyl docosa-4,7,10,13,16,19-hexaenoate/methyl docosahexaenoate
were as follows. EI-MS m/z (rel. int. %): 342 (M+, 0.60), 145 (4.20),
131 (6.60), 119 (10.80), 108 (11.40), 91 (28.20), 79 (100), 67 (20.40).
The following are the mass spectrometric data of 1-(pyrrolidin-1-yl)
eicosa-5,8,11,14,17-pentaen-1-one. EI-MS m/z (rel. int. %): 355 (M+,
3.85), 286 (7.69), 232 (7.69), 126 (13.46), 113 (100), 85 (17.31), 72
(26.92), 55 (21.15). The data for 1-(pyrrolidin-1-yl) octadeca-9,12-dien-
1-one are as follows. EI-MS m/z (rel. int. %): 381 (M+, 3.91), 312
(7.05), 272 (7.29), 232 (16.22), 218 (15.76), 192 (8.24), 166 (23.67),
153 (22.85), 113 (100), 98 (46.62), 72 (21.98).
Statistical Analyses. The percentage composition of individual
FAMEs in the diets and Artemia sampled at different enrichment times
were subjected to a one-way analysis of variance (ANOVA), and
significant means were compared by Tukey’s multiple range tests.
Correlations between Artemia and microalgal fatty acids were inves-
tigated using SPSS (version 10.0) software. A significance level of
95% (p ) 0.05) was used throughout. Arc sin transformation was used

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4046 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Chakraborty et al.
C. calcitrans (14.94% TFA), C. salina (18.6% TFA), and
baker’s yeast (21.14% TFA) (Tables 2 and 3). The content of
18:0 was found to decline by 54.38% in Artemia enriched by
N. salina up to 8 h (Table 2). Other SFAs like 16:0 exhibited
an increase of 24.66 and 76.58% in Artemia fed with N. salina
and C. calcitrans for 24 h. In Artemia enriched with C. salina
and baker’s yeast, the corresponding values were recorded to
be 100.74 and 24.91%, respectively. The fatty acid 14:0
increased from 0.32% in Artemia nauplii to 3.25 and 4.12%,
respectively, in Artemia enriched with N. salina and C.
calcitrans for 24 h. Artemia enriched with baker’s yeast recorded
the highest value of 14:0 (18.69% TFA) of all of the feeds
throughout the enrichment period (24 h).
Table 1. Fatty Acid Content (% TFA) of the Four Enrichment Diets
(Three Microalgaea and Baker’s Yeast) for Artemia Nauplii
fatty acids (% TFA)
C. salina
C. calcitrans
N. salina
S. cerevisiae
SFAs
6.60 a
0.32 b
6.90 b
1.50 b
ND
14:0
15:0
16:0
18:0
24:0
0.25 c
ND
21.49 a
2.13 a
ND
0.39 c
3.10 a
21.0 a
0.94 b
ND
0.76 b
0.28 b
7.09 b
3.10 a
0.85
∑
SFA
23.87 a
15.32 b
25.43 a
12.08 c
MUFAs
7.50
14:1n6
16:1n7
16:1n9
18:1n7
18:1n9
ND
22.18 a
ND
ND
12.11 b
34.29 b
ND
20.10 b
ND
0.50 b
9.40 c
30.00 c
ND
19.20 b
3.10 b
1. 50 a
4.22 c
34.02 b
25.58 a
10.46 a
1.78 a
41.43 a
79.25 a
MUFAs. Artemia enriched by N. salina for 8 h showed a
decrease in 16:1n7 and 16:1n9 by 47.19 and 38.77%, respec-
tively, whereas 18:1n7 and 18:1n9 exhibited an increase of 71.95
and 28.98% after 24 h of enrichment (Table 2). C. calcitrans
followed a similar trend, although the percent increase of MUFA
in the enriched Artemia was found to be marginally higher
(Table 2). The total MUFA in Artemia enriched by baker’s yeast
for 8 h was found to be highest after it exhibited a decline to
37.49% after 24 h of enrichment (Table 3).
∑
MUFA
PUFAs
3.60 a
18.90 a
ND
0.60 c
0.30 a
ND
16:2n4
16:3n4
16:3n6
18:2n6
18:3n6
18:3n3
18:4n3
18:4n6
20:4n6
20:5n3
22:5n3
22:6n3
ND
ND
ND
17.20 a
ND
21.04 a
ND
ND
0.92 c
ND
ND
0.05 b
0.05 b
0.31
2.10 b
ND
0.40 b
0.15 a
0.32
9.50 a
25.80 a
0.35 a
4.18 a
43.21 a
0.16 a
2.72 b
2.52 a
ND
ND
ND
4.83 b
0.28 a
0.15 b
0.10 a
ND
0.08 c
0.25 c
ND
0.05 c
5.74 c
0.20 a
3.13 b
0.11 c
PUFAs. n3 PUFAs. The amount of total PUFA in N. salina
enriched A. salina nauplii increased from 31.64% at 3 h to
43.93% at 8 h of enrichment (Table 2). No significant
differences were apparent in the percentage of 18:3n3, 18:4n3,
16:3n4, and 16:2n4 in Artemia nauplii enriched by microalgal
and baker’s yeast diets for 48 h (p ) 0.05). The n3 PUFAs
were found to be highest in Artemia enriched with N. salina
ranging from 12.38 (for 3 h of enrichment) to 16.75% (for 8 h
of enrichment). Among n3 PUFAs, EPA was found to increase
from 3.18% in Artemia nauplii to 8.05% after 8 h of enrichment
with N. salina. By 24 h, EPA in C. calcitrans fed Artemia was
reduced by 8.13%. Artemia enriched by baker’s yeast and C.
salina exhibited a significant decrease of n3 fatty acids,
particularly EPA after 24 h of enrichment (Table 3). Except N.
salina, none of the dietary treatments significantly increased
the proportion of DHA in enriched Artemia (Tables 2 and 3).
The DHA/EPA values in Artemia enriched for 6 h by N. salina
and C. calcitrans were found to be, respectively, 88.46 and 25%
higher than freshly hatched Artemia. Artemia enriched by C.
salina and baker’s yeast exhibited reduction in PUFA content
even at 6 h of enrichment.
ND
ND
2.80 b
18.70 b
0.19 b
1.80 b
46.89 a
0.10 b
6.68 a
1.85 b
ND
39.16 b
∑
PUFA
DHA/EPA
EPA/AA
∑
n3:
∑
n6
1.16 b
a The three microalgae are C. salina, C. calcitrans, and N. salina as illustrated
in the table. The individual fatty acid is expressed as the percentage of total
identifiable fatty acids. Some of the fatty acid could not be identified on the GC
trace; the GC-MS analysis suggested the identification.
total MUFAs; and PUFA, total PUFAs. Data are the mean values of triplicate
samples. Statistical comparison of some major fatty acids (%) was as outlined in
the Materials and Methods. Row values with different letters (a c) are significantly
different (p 0.05); ND, not determined.
∑SFA, total SFAs; ∑MUFA,
∑
−
)
in S. cereVisiae (79.25% TFA), which was mostly contributed
by 16:1n7 (25.6% TFA) and 18:1n9 (91.4% TFA). Baker’s yeast
was found to contain negligible total PUFA contents (5.74%
TFA).
n6 PUFAs. The content of AA exhibited enrichment by all
diets during enrichment up to 8 h, attaining 14.15% of TFA in
Artemia enriched by N. salina to 2.38% TFA in Artemia
enriched by baker’s yeast. It was accompanied by a decrease
of 18:2n6 in the enriched livefood (Tables 2 and 3). Artemia
enriched by C. calcitrans exhibited an increase of 20:4n6 and
18:4n6 up to 8 h of enrichment (10.16 and 57.14% TFA,
respectively) with the simultaneous reduction of 18:2n6 from
8.50% TFA in newly hatched Artemia to as low as 0.26% TFA
in 8 h enriched Artemia (Table 2).
Fatty Acid Composition of A. salina Enriched with
Microalgae and Baker’s Yeast for Different Duration.
Artemia is well-known for its value as one of the major live
foods used for the feeding of larvae of decapod crustaceans and
finfish (25, 26). Freshly hatched nauplii of Artemia in our
laboratory contain a fatty acid pattern of herbivorous zooplank-
ton. Among SFAs, 16:0 was found to be the highest (8.07%),
whereas 18:1n9 and 16:1n9 recorded higher shares (24.26 and
16.30% TFA, respectively) among MUFAs (Table 2). Among
PUFAs, high concentrations of 18:2n6 (8.50% TFA), 18:3n3
(4.10% TFA), 20:5n3 (3.18% TFA), and a negligible amount
of 22:6n3 (0.1% TFA) were apparent, thus confirming earlier
reports as proportions of TFAs (4, 26). Tables 2 and 3 have
given the fatty acid composition of A. salina fed with four
different diets separately for various enrichment periods (3, 6,
8, and 24 h).
As evident from Table 2, the artemial PUFA content was
found to be highly correlated with the n6 and n3 PUFAs of N.
salina and C. calcitrans and indicate a negative correlation
between 16:1n7, 16:1n9, and SFAs. It is apparent that increasing
the time of enrichment by N. salina or other microalgae (from
6 to 24 h) did not appreciably increase the content of AA, EPA,
or DHA. Enrichment with C. calcitrans for 24 h resulted in
negligible PUFA content (22.61% TFA) as compared to 8 h of
enriched Artemia (34.8% TFA).
SFAs. There appeared to be no significant correlation between
microalgal and artemial SFA contents (p ) 0.05). The SFA level
of Artemia fed with N. salina for 8 h was found to be lower
(12.36% TFA) as compared to those observed in Artemia fed
Other PUFAs and Their Interaction. The acid 16:2n4 was
gradually accumulated up to 0.92% TFA in Artemia nauplii