microalgae. The spent animals were distributed among 18 tanks which were divided into two groups and placed in separate temperature-controlled laboratories, one at 16
8C and the other at 20
8C. Three groups in each laboratory were fed with a mixture of Ž
. microalgae
50 Isochrysis galbana q 50 of Chaetoceros gracilis , three other groups received 70 of the same mixture of microalgae plus 30 carbohydrates
Ž .
commercial potato’s starch , and the other tanks received 70 microalgae and 30 of a Ž
lipid emulsion. The experimental ICES Internationational Council for the Exploration .
of the Sea lipid emulsion was provided by Artemia Reference Center from Belgium and Ž
. corresponded to the EmDHA one whose composition is described in Caers et al. 1999 .
This experiment was run twice, in different years. For the first experiment animals were fed for 72 days at 3 of the dry biomass per day. The second experiment lasted three
months and the diet amounted to 6 of the dry biomass per day. The duration of the experiments was determined according the progress of gonadal ripening; when about
40 of scallops appeared mature in any of the tanks, the experiment was ended to avoid spontaneous spawning which would have made controlled spawning and fertilization
impossible.
Before beginning the conditioning experiment, five spent scallops were sampled to Ž
evaluate gonadal condition using the gonadal index percentage of the total tissue mass .
of the animal that consisted of gonad and histological analysis. Periodically, similar determinations were performed for three animals from each experimental treatment
Ž .
each animal from a different tank to ascertain the course of gonadal recovery. These animals were chosen after visual inspection of all the animals, which were then
separated into three groups according to their degree of apparent maturation. Then, one animal from the largest group in each tank was sampled. The duration of the experi-
ments was determined according the progress of gonadal ripening; when about 40 of scallops appeared mature in any of the tanks, the entire experiment was ended to avoid
spontaneous spawning which would have made controlled spawning and fertilization impossible.
Ž .
On ending the experimental time, animals that looked ripe turgid, orange gonad in each tank were counted, and induced to spawn. The number of scallops that spawned
from each experimental tank was registered. Oocytes obtained from each group were fertilised by the corresponding sperm and the percentage of fertilization was calculated
by counting the number of eggs that were in cleavage stage after two h. The resulting
Ž .
larvae were left in tanks with seawater for 48 h, after which swimming larvae larva D were counted to calculate the percentage survival.
3. Results
3.1. Effect of diet and temperature on gonadal recoÕery 3.1.1. First experiment
For all diets and at both temperatures, the gonadal indexes of the scallops did not Ž
. show marked variations before day 58 Fig. 1 . From this day on, individuals fed all
diets at 16 8C, showed sharp increases of these indexes. Gonadal indexes of the animals
conditioned at 20 8C and fed microalgae–carbohydrates increased slightly after day 58.
Fig. 2. Gonadosomatic indexes of A. purpuratus broodstock during conditioning with different diets at 16 8C
and 20 8C. Microalgae–CHO: mixture of microalgae and carbohydrates; Microalgae–lipids: mixture of
Ž .
Ž .
microalgae and lipids. Second experiment 1998 . MeansS.E. N s 3 are shown.
When the experiment was ended, the highest percentage of scallops that looked ripe Ž
. was obtained by feeding the microalgae–lipids diet and at 16
8C results not shown . From the animals conditioned at 16
8C, the highest percent spawning was obtained with scallops fed microalgae alone. After fertilization of gametes, the highest survival of
larvae D was obtained from the gametes obtained from the scallops conditioned with the Ž
. mixture microalgae–lipids results not shown .
Although this first experiment gave us good information about how to condition scallops for reproduction, many individuals never became ripe. This meant that we could
Ž .
not make enough replicates in the final assays spawning and fecundation of gametes to allow statistical analysis. As this might have been caused by low food levels, the
experiment was repeated using the same diets and temperatures, but at a higher ration.
3.1.2. Second experiment The results of the second experiment confirmed that the mixture microalgae–lipids is
the best diet to condition scallops among the diets assayed. The lowest temperature Ž
. 16
8C also proved to be more appropriate for this conditioning than 208C. As evidenced by the gonadal index, gonadal recovery of the scallops, for all diets and
Ž .
both temperatures, progressed rather slowly until day 80 of conditioning Fig. 2 . During the first 10 days, the gonadal index increased more rapidly for the animals conditioned
at 20 8C. After the 10th day, the increases were greater at 168C for all diets. At 168C,
animals fed pure microalgae changed little between days 10 and 80. Within this period of time, the highest value was attained for animals fed the mixture microalgae–lipids
Ž .
Fig. 2, upper . The gonadal indexes of animals conditioned at 20 8C and fed the mixed
diets also oscillated, but the increases to complete gonadal recovery were smoother than those of animals conditioned at 16
8C. The animals fed only microalgae, never showed Ž
. marked increases of their gonadal indexes at 20
8C Fig. 2, lower . The histological Ž
. analysis not shown confirmed the time course of gonadal ripening.
Ž .
A two-way analysis of variance for the percentage transformed to arcsin values of mature scallops at the end of the experiment showed that both diet and temperature, but
Ž .
not their interaction, significantly affected these percentages Table 1 . To better
understand the effect of diet and considering the lack of interaction between diet and temperature, one-way analyses of variance, followed by Tukey a posteriori multiple
comparisons, were run for each experimental temperature. The percentage mature scallops differed significantly between the three diets at 16
8C, but not at 208C.
Table 1 Two-factor analysis of variance examining the effect of diet and temperature upon the maturity of A.
purpuratus Diet and temperature were controlled as explained in Section 2.
df F
p Diet
2 3.908
0.049 Temperature
1 7.956
0.015 Interaction
2 0.327
0.727 Residual
17
Table 2 Percent of mature and spawned A. purpuratus broodstock after conditioning under different diets and
temperatures Ž
. Each value is a meanS.D. ns 3 .
Ž .
Ž .
Different letters in superscript indicate significantly different values Tukey’s test, p- 0.05 . Ž .
Diet Mature scallops
Spawnedrinduced animals 16
8C
a
Microalgae 27.643.24
3r3 4r4
3r3
b
Microalgaeqcarbohydrates 19.173.61
3r3 2r2
3r3
c
Microalgaeqlipids 42.644.68
4r4 5r5
4r4 20
8C Microalgae
12.9211.20 2r2
0r2 Microalgaeqcarbohydrates
18.5912.26 0r1
2r2 2r3
Microalgaeqlipids 29.135.77
3r3 3r4
2r3
At 16 8C, the highest percentage of maturation was obtained for scallops fed microal-
gae–lipids, while the least maturation was found for scallops fed microalgae–carbo- Ž
. hydrates Table 2 . At 20
8C, the least gonadal recovery was obtained for scallops fed microalgae alone. In fact, no animal fed this diet became mature.
All the mature scallops that had been conditioned at 16 8C spawned when induced
Ž .
Table 2 . In contrast, not all of the animals conditioned at 20 8C responded to spawning
stimuli. 3.2. Effect of diet and temperature on larÕal yield
No differences were detected between the percentage fertilization of gametes ob- tained from scallops conditioned at the two temperatures or the three diets. Survival of
larvae D obtained from fertilised eggs from scallops conditioned at 16 8C was higher
Table 3 Fertilization success and larval survival for A. purpuratus broodstock conditioned under different diets and
temperature Ž
. Each value is a meanS.D. ns 3 .
Ž .
Different letters in superscript indicate significantly different values at a given temperature. Ž .
Ž . Diet
Fertilized eggs Larvae D survival
16 8C
a,b
Microalgae 88.523.11
80.023.88
a
Microalgaeqcarbohydrates 65.3820.66
50.1226.49
b
Microalgaeqlipids 88.672.24
87.803.49 20
8C
1 1
Microalgae 86.84
47.37 Microalgaeqcarbohydrates
81.4616.53 24.378.15
Microalgaeqlipids 81.119.93
42.4515.71
1
Only scallops from one tank spawned.
Ž .
than that for animals maintained at 20 8C Table 3 . For the animals conditioned at 168C,
those fed with microalgae–carbohydrates yielded the least larvae D.
4. Discussion
