21 - - .001 .001 .001 -
22 .001 .001 .001 .001 .001 .001
23 .001 .001 .001 .002 .002 .001
24 .001 .002 .002 .002 .002 .002
25 .002 .002 .003 .003 .003 .002
26 .002 .002 .003 .003 .003 .003
27 .003 .003 .004 .004 .004 .004
28 .003 .003 .004 .005 .005 .004
29 .004 .004 .005 .005 .005 .005
30 .004 .004 .005 .006 .006 .006
31 .004 .005 .006 .006 .006 .006
32 .005 .006 .006 .007 .007 .007
33 .005 .007 .007 .007 .007 .007
34 .006 .007 .007 .008 .008 .008
35
add correction to measured density
.006 .007 .008 .008 .008 .008

4.3. Use of nauplii and meta-nauplii

4.3.1. Harvesting and distribution

4.3.2. Cold storage
4.3.3. Nutritional quality

4.3.4. Enrichment with nutrients
4.3.5. Enrichment for disease control
4.3.6. Applications of Artemia for feeding different species


As the live food is suspected to be a source of bacterial infections eventually causing
disease problems in larval rearing, microbial contamination should be kept to a minimum.
During the hatching of Artemia cysts, bacterial numbers increase by 10
3
to 10
5
compared
to the initial population before the breaking of the cysts. This bacterial population
remains well established and cannot be removed from the nauplii by rinsing with
seawater or freshwater; rinsing only having a diluting effect on the water surrounding the
nauplii. However, hatching nauplii from cysts that have been submitted to a disinfection
procedure successfully reduces the bacterial numbers after harvesting compared to
standard hatching techniques using non-disinfected cysts (Fig. 4.3.3.); in particular Vibrio
levels are reduced below 10
3
CFU.g
-1
. At the moment of writing a new disinfected cyst
product has become commercially available (namely DC-cysts, INVE Aquaculture NV,
Belgium) which has proved to result in low bacterial numbers after hatching.
Since instar I nauplii completely thrive on their energy reserves they should be harvested
and fed to the fish or crustacean larvae in their most energetic form, (i.e. as soon as
possible after hatching). For a long time farmers have overlooked the fact that an Artemia
nauplius in its first stage of development can not take up food and thus consumes its own
energy reserves. At the high temperatures applied for cyst incubation, the freshly-hatched
Artemia nauplii develop into the second larval stage within a matter of hours. It is
important to feed first-instar nauplii to the predator rather than starved second-instar
meta-nauplii which have already consumed 25 to 30% of their energy reserves within 24
h after hatching (Fig. 4.3.4.). Moreover, instar II Artemia are less visible as they are

can be reduced and hence growth of the Artemia in the culture tank can be minimized.
For example, applying one or maximum two feedings per day, shrimp farmers often
experienced juvenile Artemia in their larviculture tanks competing with the shrimp
postlarvae for the algae. With poor hunters such as the larvae of turbot Scophthalmus
maximus and tiger shrimp Penaeus monodon, feeding cold-stored, less active Artemia
furthermore results in much more efficient food uptake.
4.3.3. Nutritional quality
The nutritional effectiveness of a food organism is in the first place determined by its
ingestibility and, as a consequence by its size and form. Naupliar size, varying greatly
from one geographical source of Artemia to another, is often not critical for crustacean
larvae, which can capture and tear apart food particles with their feeding appendages. For
marine fish larvae that have a very small mouth and swallow their prey in one bite the
size of the nauplii is particularly critical. For example, fish larvae that are offered
oversized Artemia nauplii may starve because they cannot ingest the prey. For at least
one species, the marine silverside Menidia menidia, a high correlation exists between the
naupliar length of Artemia and larval fish mortality during the five days after hatching:
with the largest strains of Artemia used (520 µm nauplius length), up to 50% of the fish
could not ingest their prey and starved to death whereas feeding of small Artemia (430
µm) resulted in only 10% mortality (Fig. 4.3.5.). Fish that produce small eggs, such as
gilthead seabream, turbot and grouper must be fed rotifers as a first food because the
nauplii from any Artemia strain are too large. In these cases, the size of nauplii (of a
selected strain) will determine when the fish can be switched from a rotifer to an Artemia
diet. As long as prey size does not interfere with the ingestion mechanism of the predator,
the use of larger nauplii (with a higher individual energy content) will be beneficial since
the predator will spend less energy in taking up a smaller number of larger nauplii to
fulfill its energetic requirements. Data on biometrics of nauplii from various Artemia
strains are presented in Table 4.1.2. (see chapter 4.1.) and ranges given in Fig 4.3.6.
Figure 4.3.5. Correlation of mortality rate of Menidia menidia larvae and nauplii
length of Artemia from seven geographical sources offered as food to fish larvae
(modified from Beck and Bengtson, 1982).

Table 4.3.1. Intra-strain variability of 20:5n-3 (EPA) content in Artemia. Values
represent the range (area percent) and coefficient of variation of data as compiled
by Léger et al. (1986).
Cyst source 20:5n-3 range
(area %)
Coefficient of variation
(%)
San Francisco Bay, CA-USA 0.3-13.3 78.6
Great Salt Lake (South arm), UT-USA 2.7-3.6 11.8
Great Salt Lake (North arm), UT-USA 0.3-0.4 21.2
Chaplin Lake, Canada 5.2-9.5 18.3
Macau, Brazil 3.5-10.6 43.2
Bohai Bay, PR China 1.3-15.4 50.5

The levels of essential amino acids in Artemia are generally not a major problem in view
of its nutritional value, but sulphur amino acids, like methionine, are the first limiting
amino acids (Table 4.3.2.).
The presence of several proteolytic enzymes in developing Artemia embryos and Artemia
nauplii has led to the speculation that these exogenous enzymes play a significant role in
the breakdown of the Artemia nauplii in the digestive tract of the predator larvae. This
has become an important question in view of the relatively low levels of digestive
enzymes in many first-feeding larvae and the inferiority of prepared feeds versus live
prey.
Table 4.3.2. Amino acid composition of Artemia nauplii (mg.g
-1
protein) (modified
from Seidel et al., 1980).

Macau, Brazil Great Salt Lake, UT-USA San Pablo Bay, CA-USA
aspartic acid 110 113 141

-1
), pantothenic acid (56-72 µg.g
-1
) and retinol (10-48 µg.g
-1
). A
stable form of vitamin C (ascorbic acid 2-sulphate) is present in Artemia cysts. This
derivative is hydrolysed to free ascorbic acid during hatching, the -ascorbic acid levels in
Artemia nauplii varying from 300 to 550 µg g
-1
DW. The published data would appear to
indicate that the levels of vitamins in Artemia are sufficient to fulfill the dietary
requirements recommended for growing fish. However, vitamin requirements during
larviculture, are still largely unknown, and might be higher due to the higher growth and
metabolic rate of fish and crustacean larvae.
4.3.4. Enrichment with nutrients
As mentioned previously, an important factor affecting the nutritional value of Artemia as
a food source for marine larval organisms is the content of essential fatty acids,
eicosapentaenoic acid (EPA: 20:5n-3) and even more importantly docosahexaenoic acid
(DHA: 22:6n-3). In contrast to freshwater species, most marine organisms do not have
the capacity to biosynthesize these EFA from lower chain unsaturated fatty acids, such as
linolenic acid (18:3n-3). In view of the fatty acid deficiency of Artemia, research has
been conducted to improve its lipid composition by prefeeding with (n-3) highly
unsaturated fatty acid (HUFA)-rich diets. It is fortunate in this respect that Artemia,
because of its primitive feeding characteristics, allows a very convenient way to
manipulate its biochemical composition. Thus, since Artemia on molting to the second
larval stage (i.e. about 8 h following hatching), is non-selective in taking up particulate
matter, simple methods have been developed to incorporate lipid products into the brine
shrimp nauplii prior to offering them as a prey to the predator larvae. This method of
bioencapsulation, also called Artemia enrichment or boosting (Fig. 4.3.7.), is widely

carotenoids. Upon dilution in seawater, finely dispersed stable microglobules are formed
which are readily ingested by Artemia and which bring about EFA-enrichment levels
which largely surpass the values reported in the literature (Léger et al., 1986). For
enrichment the freshly-hatched nauplii are transferred to an enrichment tank at a density
of 100 (for enrichment periods that may exceed 24 h) to 300 nauplii.ml
-1
(maximum 24-h
enrichment period); the enrichment medium consisting of disinfected seawater
maintained at 25
°
C. The enrichment emulsion is usually added in consecutive doses of
300 mg.l
-1
every 12 h with a strong aeration (using airstones) being required so as to
maintain dissolved oxygen levels above 4 mg.l
-1
(the latter being necessary to avoid
mortalities). The enriched nauplii are harvested after 24 h (sometimes even after 48 h),
thoroughly rinsed and then fed directly or stored at below 10
°
C so as to minimize the
metabolism of HUFA prior to administration, i.e. HUFA levels being reduced by 0-30%
after 24 h at 10
°
C, Fig. 4.3.9. By using these enrichment techniques very high
incorporation levels of EFA can be attained that are well above the maximal
concentrations found in natural strains. These very high enrichment levels are the result
not only of an optimal product composition and presentation, but also of proper
enrichment procedures: i.e. the nauplii being transferred or exposed to the enrichment
medium just before first feeding, and opening of the alimentary tract (instar II stage).

during storage at 10 and 25°C (modified from Dhont et al., 1993).