2. Materials and methods
2.1. Production and use of freeze-dried algae N. gaditana was grown in 50-cm-deep, 2000-l tanks under natural radiation in a
green house. Cells were harvested by continuous centrifugation at cell densities between
6 y1
Ž
y1
. 50 and 70 = 10 cells ml
average biomass from 420 to 590 mg dry weight l . I.
galbana was grown in 20-cm-diameter, 50-l acrylic tubes, placed in an algal room at Ž
y1
. 208C under continuous lighting photon flux density of 150 mmol s
. It was also concentrated by continuous centrifugation at an average cell density of 22 = 10
6
cells
y1
Ž
y1
. ml
440 mg dry weight l . Algal pastes were frozen at y808C prior to freeze-dry-
ing for 48 h. Dry algae powder was kept vacuum packaged until its use in larval rearing. The required amount of cells was resuspended in a small volume of seawater by mixing
for 1 min in a kitchen blender.
2.2. LarÕal rearing conditions Experiments were replicated by using three batches of larvae from different spawning
tanks. Eggs were obtained from captive broodstock kept under constant temperature Ž
.
3
208C and natural photoperiod. After hatching, 2-day-old larvae were stocked in 1 m round tanks to achieve an initial larval density of between 80 and 100 larvae l
y1
. The tanks were supplied with seawater pumped from the San Pedro Channel, which is
Ž .
located in an estuary area connected to the Bay of Cadiz SW Spain . Water was filtered
´
through sand filters and 10 and 3 mm nominal retention cartridges. There was no water exchange until day 15 of culture. Then, there was gradually increased water exchange
from 20 to 80 daily up to day 45. Gentle aeration was always provided in the center of the larval tanks. Temperature and salinity in the larval tanks ranged from 198C to
218C and from 25 to 33 ppt, respectively. The light was turned off during yolk sac absorption and a permanent illumination of 1500 lux on water surface was used from
initial exogenous feeding throughout the cultures. The feeding experiments lasted until larvae reached an age of 43 days, as shown in Table 1.
In each replicate, a group of three tanks received 2 g m
y3
of freeze-dried N. gaditana, whereas another three tanks were supplied with an equivalent amount of live
Table 1 Feeding sequence and daily amounts of the different diets provided in tanks with seabream larvae during the
mass rearing trials Type of diet
y3 y1
y1 y3
Ž .
Ž .
Ž .
Ž .
Days Algae g m
Rotifers ind ml Artemia ind ml
Dry feed g m 3–10
2 10
– –
11–15 2
15 –
– 16–30
2 15
1 –
31–40 –
– 3
– 41–45
– –
3 10
Table 2 Experimental treatments followed in the evaluation of freeze-dried microalgae in the mass rearing of S. aurata
larvae. F: Freeze-dried N. gaditana. I: Freeze-dried I. galbana. L: Live N. gaditana. P: Commercial enricher, Ž
. DHA Protein Selco INVE Aquaculture, Belgium
Treatment LF
LI LP
FF FI
FP State of N. gaditana added to larval tanks
L L
L F
F F
Type of rotifer enrichment F
I P
F I
P
algae in terms of dry weight. Both treatments were tested with different types of rotifer enrichment, as detailed in Table 2. When live N. gaditana was added to the larval tanks,
algal cultures were previously concentrated by 500-fold in order to avoid any possible variation in water quality due to the addition of algal medium. The daily amount of
Ž .
6
algae supplied to larval tanks Table 2 provided an average cell density of 0.25 = 10 cells ml
y1
. A strain of Brachionus plicatilis ranging from 120 to 230 mm in size was used. Rotifer enrichment lasted 16 h, and 200 mg l
y1
of enricher was used in all cases. Artemia were used as food from day 16, and rotifer and Artemia density in the rearing
Ž .
tanks was monitored daily and adjusted to desired values Table 1 . 2.3. Analysis of growth and culture eÕaluation
Initial larval density was estimated by counting the number of larvae present in 10 different samples of 500 ml. A gentle shaking of the rearing medium with a plastic sheet
allowed a uniform larval distribution to be obtained before sampling. Final density was calculated by counting fish in sub-samples after concentrating the whole population in
each tank prior to being moved to larger growing tanks.
Larval dry weight was calculated by weighing a number of larvae on pre-washed and pre-weighed fiberglass filters. These filters were rinsed with distilled water and dried at
608C for 48 h. They were then weighed to the nearest 10 mg and a mean value of the individual larval dry weight was calculated. Three replicates were used at every
Ž . sampling. Specific growth rate G was calculated from the slope of the linear regression
Ž . of log-transformed dry weight against age. Final biomass b was calculated from dry
weight and the larval density obtained at the end of the experiments. Oxygen and pH were monitored daily, and ammonia and nitrite were analyzed as described in Grasshoff
Ž .
et al. 1983 . Data from the different treatments were compared by multifactor ANOVA. When
significant differences were detected, the Tukey multiple-range procedure was used to determine statistical differences between treatments and the Student’s-t test for slope
comparison. A probability level of P - 0.05 was used to judge whether any effects were significant. Before the statistical analyses, the dry weight data were log-transformed and
the percentage survivals were arcsine transformed.
3. Results
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