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Effect of microalgal and inert (cornmeal and cornstarch) diets on growth performance and biochemical composition of Ruditapes decussatus seed
Aquaculture 160 Ž1998. 89–102
Effect of microalgal and inert žcornmeal and cornstarch / diets on growth performance and biochemical composition of Ruditapes decussatus seed A. Perez ´ Camacho a
a,)
b , M. Albentosa a , M.J. Fernandez-Reiriz , ´ b U. Labarta
´ Instituto Espanol S N, 15080 A Coruna, ˜ de Oceanografıa, ´ Muelle de Animas ˜ Spain b Instituto de InÕestigacions ´ Marinas, ˜ Eduardo Cabello 6, 36208, Vigo, Spain Accepted 20 October 1997
Abstract Research was carried out into the effect of phytoplankton, cornmeal and cornstarch diets on growth and biochemical composition of the seed of the little-neck clam, Ruditapes decussatus. The seed of R. decussatus, fed on daily rations of Isochrysis galbana Žorganic weight. of 0.5 and 1% of live weight of the seed, showed an improvement in growth rate when cornstarch, which is 99% carbohydrate, was added to these diets. Thus in the case of a daily ration of 0.5%, daily growth rates increased by between 33.5 and 32.3%, depending on whether we are referring to organic weight, dry weight or live weight, when 1.5% cornstarch was added. In the case of a ration of 1% I. galbana, the addition of another 1% cornstarch lead to an improvement in daily growth rates, depending on the different weight class in question, of between 14.1 and 15.5%. When compared to a daily ration consisting of 2% phytoplankton, which was considered to be the optimal ration for growth in the seed of these clams, the replacement of half the quantity of I. galbana by a quantity of cornstarch of equivalent weight gave a growth rate in terms of organic weight of 87.9% that of the phytoplankton diet, while the rates for dry weight and live weight were 89.6 and 87.9%, respectively. These results improved noticeably when cornmeal, consisting of 10% protein and 90% carbohydrate, was used instead of cornstarch. In the case of a 2% phytoplankton diet, if we substituted an equivalent quantity of cornmeal for 50% of the
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Corresponding author. Fax: q34-81229077; e-mail: [email protected].
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 7 . 0 0 2 3 2 - 9
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phytoplankton, the growth rate in organic matter was the same Ž99.0%. as those for the diet consisting of phytoplankton alone, while growth rates in dry weight and live weight were 6.2 and 5.9% higher, respectively, than those of the phytoplankton diet. It would therefore appear that cornmeal Žand to a lesser extent cornstarch. can be successfully used as a partial substitute for phytoplankton in diets for the seed of R. decussatus and its use in hatcheries and nurseries devoted to the culture of this species would lead to a considerable reduction of production costs. q 1998 Elsevier Science B.V. Keywords: Ruditapes decussatus; Seed culture; Inert food; Cornmeal; Cornstarch
1. Introduction
The little-neck clam, Ruditapes decussatus, is one of the most important species in Spanish aquaculture, commanding high prices in the market. Possibilities for production are limited due to the scarcity of naturally-occurring seed and the high cost of seed production in commercial hatcheries. Any saving in this cost is therefore crucial for the viability of this type of industry and would be a major contribution to the expansion of aquaculture production. The basis for the development of the bivalve mollusc culture industry was established at the mid-point of this century in works by Quayle Ž1952.; Loosanoff and Davis Ž1963. and Walne Ž1956., among others. Ever since then the production of live phytoplankton has been seen as one of the major contributory factors to the costs borne by hatcheries and nurseries ŽPersoone and Claus, 1980; De Pauw, 1981; Lucas and Gerad, 1981., accounting for 30% of the total seed production cost in the former ŽCoutteau and Sorgeloos, 1992. and up to 85% of production costs in the latter ŽBolton, 1982.. Various authors have attempted to find a substitute for live phytoplankton in the diet of bivalve molluscs and have experimented, among other products, with spin-dried and freeze-dried algae ŽLaing et al., 1990; Laing and Gil Verdugo, 1991; Laing and Millican, 1992., yeasts ŽEpifanio, 1979; Urban and Langdon, 1984; Nell, 1985., modified yeasts ŽAlbentosa et al., 1989; Coutteau et al., 1991., microcapsules ŽLangdon and Waldock, 1981; Jones et al., 1984; Langdon and Bolton, 1984; Langdon et al., 1985; Laing, 1987. and different varieties of cereal starch ŽHaven, 1965; Ingle, 1967; Dunathan et al., 1969; Castell and Trider, 1974; Wisely and Reid, 1978; Nell and Wisely, 1983, 1984; Langdon and Bolton, 1984; Langdon and Siegfried, 1984; Urban and Kirchman, 1992.. In none of these studies is R. decussatus mentioned and their results vary considerably according to the characteristics of the foodstuff and the species for which it is intended; until now, no total substitute has been found for live phytoplankton, the best results having been obtained with 50% substitution or less. The purpose of this study is to assess the performance of cornmeal and cornstarch in the diet of the seed of the little-neck clam R. decussatus, whether in diets exclusively composed of these products or in mixed diets with varying proportions of phytoplankton. Seed growth rates and biochemical composition were used to assess the results obtained from the different diets.
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2. Materials and methods 2.1. Seed The experiments were carried out with seed of R. decussatus obtained from broodstock conditioned in the Instituto Espanol ˜ de Oceanografıa. ´ The methods used to induce spawning and to cultivate the larvae and the seed were those described by Perez ´ Camacho et al. Ž1977.. 2.2. Diet The basal diet consisted of the microalga Isochrysis galbana, which was replaced in varying proportions by cornmeal or cornstarch. Preliminary trials were carried out to define the sedimentation rates of the corn products concerned in the culture vessels and equivalent amounts of the products were then added to the daily ration to compensate for losses caused by sedimentation. The microalgae were grown in 6-l glass flasks in a temperature-controlled chamber, at a constant temperature of 188C and with constant illumination at 9900 lux. Salinity was maintained at a constant 33‰. The culture medium used was that described by Walne, 1956 and the microalgae were harvested in the initial stationary growth phase. The cornstarch contained 99% carbohydrate, while the cornmeal contained 90% carbohydrate and 10% protein. Both products were suspended in seawater by shaking and sieved through a 20 m m sieve. A Coulter Counter Multisizer was used to measure the concentration of the foodstuff in the seawater. 2.3. Experimental design The base diet for the different experiments was an optimal daily ration of 2% organic seed weight of the microalga I. galbana ŽAlbentosa et al., 1996.. The experiments were carried out in triplicate for each diet. Each replica contained an initial biomass of 200 mg of clams Žlive weight.. 2.3.1. Experiment 1 This experiment was used to compare the growth rates achieved with the basal diet F100, consisting of a daily ration of 2% I. galbana, with the two mixed diets M50 and M25, in which 50% and 75%, respectively, of the phytoplankton was replaced by an equivalent weight of cornstarch. Three control diets were established: F50, consisting of a daily ration of 1% I. galbana; F25, consisting of a daily ration of 0.5% I. galbana; and CS, consisting of 2% cornstarch. Percentages are expressed in terms of the live weight of the clams. Initial figures for the seed were as follows: mean length, 2.10 . 0.20 mm; mean live weight, 2.46 . 0.04 mg; mean dry weight, 1.58 . 0.04 mg; mean organic weight, 0.20 . 0.01; and mean organic matter 12.5 . 0.1%. The experiment lasted 5 weeks.
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2.3.2. Experiment 2 Cornmeal was used in place of cornstarch in this experiment. Four diets were tested, consisting of varying proportions of phytoplankton and cornmeal, as follows: Diet F100, 2% I. galbana; diet M50, 50% phytoplankton and 50% cornmeal; diet M25, 25% phytoplankton and 75% cornmeal; and diet CM, 2% cornmeal. The seed used in this experiment was of similar size to that used in experiment 1 Žmean lengths 1.76 . 0.16 mm, mean live weights 1.34 . 0.06 mg, mean dry weights 0.85 . 0.04 mg, mean organic weights 0.12 . 0.01, mean organic matters 13.2 . 0.1%.. The experimental period was 3 weeks. 2.4. Experimental conditions The seed was placed on the bottom of 6-l plastic vessels which were fitted with an air point in order to reduce the sedimentation rates of the foodstuff. The experimental cultures were kept in a temperature-controlled chamber at a constant temperature of 208C. The water in the vessels was changed daily, using seawater filtered through a 1 m m sieve and sterilised by UV. The experimental period was from 3 to 5 weeks. The cornmeal and cornstarch were suspended in seawater, in 6-l round-bottomed flasks with strong aeration in order to keep the food in suspension. The food was supplied over 15-min periods at 4-h intervals by means of a 12-channel Ismatec MV-CA pump. Each diet was assayed in triplicate and a fourth culture vessel was used to determine the sedimentation rate. The initial volume in the culture vessels was 3-l. In order to avoid the production of pseudofaeces the concentration of the food in the vessels was kept below 1.6 m g mly1 of organic matter ŽAlbentosa et al., 1996.. The initial volume of water in the culture vessels was increased weekly, according to the increase in biomass of the clams during the same period, in order to maintain constant conditions throughout the experiment. 2.5. Diet assessment parameters The live weight ŽLW. of the clams in each experimental batch was obtained weekly, after a 10-min draining period on absorbent paper. At the end of the experimental period half of the clams in each batch were used to obtain the length of the seed Ž L., the dry weight ŽDW., the weight of inorganic matter ŽIW., the organic weight ŽOW s DW y IW. and the percentage of organic matter Ž%OM s ŽOWrDW= 100.. DW was determined by drying the clams at 1008C for 24 h, while IW was obtained by heating to 4508C for 12 h. The other half of the batch was used for biochemical analysis. The daily growth rate ŽDGR. was calculated according to the equation )
DGR: Ž Ž LnW1 y LnW0 . rt . 100
where W1 and W0 are the values of the different variables ŽOW, DW, LW and L. at the end and beginning, respectively, of each experiment and t is the number of days.
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2.6. Analytical methods The organic content of the phytoplankton was determined by filtering the algal culture through Whatman GFrC glass-fibre filters which had previously been rinsed and ashed at 4508C. The filters were rinsed in a 0.5 M ammonium formate solution to eliminate salt residues, dried to a constant weight and ashed at 4508C. The organic content and water of the corn products were determined at 1008C and 4508C respectively. The method described by Lowry et al. Ž1951. was used to determine the protein content, following alkaline hydrolysis with NaOH 0.5 Nr24 hr308C. Total carbohydrates were quantified as glucose by the phenol–sulphur method ŽStrickland and Parsons, 1968.. Lipids were extracted by a modification of the method of Bligh and Dyer Ž1959. ŽFernandez-Reirız ´ ´ et al., 1989. as follows: lipids were extracted by means of chloroform:methanol Ž1:2. and then centrifuged, after which the sediment was once again extracted with chloroform:methanol Ž2:1.. The resulting extract was purified by rinsing both supernatants in a mixture of chloroform, methanol and water Ž8:4:3. as described by Folch et al. Ž1957.. Total lipids were gravimetrically determined through evaporation of the solvent in the purified extract on aluminium sheets at 60–808C. 2.7. Statistical methods The statistical package Statgraphics was used to analyse the results. For each experiment growth was compared by examining the daily increase for each of the variables listed above, while the daily growth rate ŽDGR. was used to compare the results of different experiments. Comparison between the different variables used to evaluate the diets was performed by an ANOVA with a significance level of P - 0.05. Angular transformation was used for values expressed as percentages and logarithmic transformation for the comparison of weight increases. The Bartlett test was used to test for variance homogeneity. Multiple comparisons were carried out with the Newman– Keuls multiple range test ŽSnedecor and Cochran, 1971; Zar, 1974..
3. Results 3.1. Cornstarch 3.1.1. Experiment 1 When the daily diet of 2% I. galbana Ždiet F100. was reduced to 1% Ždiet F50., the growth rate of the seed of R. decussatus decreased to 43% of that obtained with diet F100 and decreases still further to 24% if the diet was reduced to 0.5% phytoplankton Ždiet F25.. If cornstarch, which is 99% carbohydrate ŽTable 1., was added to diets F50 and F25, growth rates increased considerably. Thus, in the case of the 0.5% ration, DW growth increased by 80.1% when 1.5% cornstarch was added, while OW increased by 55.0%,
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Table 1 Mean daily increase in organic weight ŽOW., dry weight ŽDW., live weight ŽLW. and shell length Ž L., and % organic matter ŽOM. of clams fed on different diets Žaverage values of 3 replicates"stand. error.. Diet F100: 2% I. galbana ŽOW. of clam LW daily; diet F50: 50% F100; diet F25: 25% F100; diet M50: 50% F100 and 50% cornstarch; diet M25: 25% F100 and 75% cornstarch; diet CS: 2% cornstarch ŽOW. of clam live weight. Initial figures: mean length, 2.10.0.20 mm; mean live weight, 2.459.0.041 mg; mean dry weight, 1.578.0.038 mg; mean organic weight, 0.198.0.005; and mean organic matter 12.5.0.1%.
F100 F50 F25 M25 M50 CS
OW Ž m g indy1 .
DW Ž m g indy1 .
LW Ž m g indy1 .
L Žmm.
OM Ž%.
45.3"4.8 19.5"0.4 10.9"0.4 31.4"1.5 16.9"0.9 2.6"0.9
375.7"21.9 201.4"0.7 102.7"6.3 263.6"6.1 185.0"0.9 46.4"2.4
593.8"33.2 321.9"11.1 170.9"7.0 433.9"12.4 288.4"2.6 65.3"3.6
85.1"0.9 59.2"2.1 38.9"3.1 70.1"1.0 55.4"1.3 12.7"1.0
12.6"0.7 10.2"0.1 10.6"0.3 12.0"0.4 9.8"0.4 10.2"0.4
Table 2 Mean daily increase in organic matter ŽOW., dry weight ŽDW., live weight ŽLW. and shell length Ž L., and % organic matter ŽOM. of clams fed on different diets Žaverage values of 3 replicates"SE.. Diet F100: 2% I. galbana Žin OW. daily with regard to clam live weight; diet M75: 75% F100 and 25% cornmeal; diet M50: 50% F100 and 50% cornmeal; diet M25: 25% F100 and 75% cornmeal; diet CM: 2% cornmeal Žin OW. with regard to clam live weight. Initial figures: mean length, 1.76.0.16mm; mean live weight, 1.341.0.059 mg; mean dry weight, 0.854.0.039 mg; mean organic weights 0.119.0.005; mean organic matter, 13.2.0.1%.
F100 M75 M50 M25 CM
OW Ž m g indy1 .
DW Ž m g indy1 .
LW Ž m g indy1 .
L Žmm.
OM Ž%.
17.6"1.2 20.6"1.0 17.5"2.8 8.6"1.2 1.8"0.3
131.0"1.0 173.0"4.1 151.3"4.3 89.4"5.1 26.2"3.1
207.6"2.4 261.3"2.7 235.6"4.7 136.8"2.9 28.1"2.7
57.6"0.6 69.0"1.0 61.4"1.0 43.3"2.0 4.8"1.1
14.1"0.3 12.1"0.2 12.0"0.5 10.9"0.7 10.7"0.3
LW by 68.8% and L by 42.4%. For 1% rations of I. galbana, when a 1% supplement of cornstarch was added, DW increased by 30.9%, OW by 61.0% and L by 18.4%. In comparison with diet F100, when half the quantity of I. galbana was replaced by an equivalent weight of cornstarch Ždiet M50. the growth rate for OW was 69.3% of that obtained with a phytoplankton only Ž I. galbana. diet; DW was 73.7%, LW was 73.1% and L was 82.4%. A 25% phytoplanktonr75% starch diet Ždiet M25. gave growth rates of 37.4, 51.7, 48.6 and 65.1% for OW, DW, LW and L, respectively, when compared to the growth rates obtained with diet F100.
Fig. 1. Variation in daily growth rates of organic matter, dry weight, live weight and seed length for the seed of the little-neck clam R. decussatus fed different proportions of phytoplankton, cornstarch ŽA. and cornmeal ŽB.. Initial figures: mean length, 2.10.0.20 ŽA. and 1.76.0.16 mm ŽB.; mean live weight, 2.459.0.041 ŽA. and 1.341.0.059 mg ŽB.; mean dry weight, 1.578.0.038 ŽA. and 0.854.0.039 mg ŽB.; mean organic weight, 0.198.0.005 ŽA. and 0.119.0.005 mg ŽB.; mean organic matter, 12.5.0.1 ŽA. and 13.2.0.1% ŽA.. Vertical lines represent 95% confidence intervals.
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The ANOVA gave significant differences Ž P - 0.001. between daily growth rates for OW, DW, LW and L. The NK test detected significant differences Ž P - 0.05. in OW, DW, LW and L between all groups with the exception of diets F50 and M25, where there was no significant difference Ž P ) 0.05.. Where the %OM of the seed is concerned, the NK test showed two homogeneous groups, one formed by diets F100 and M50, with a %OM of between 12.0 and 12.6% and the other by diets F50, F25, M25 and CS, with a %OM ranging from 9.8 to 10.6%. There were significant differences between the two groups Ž P - 0.05.. 3.2. Cornmeal 3.2.1. Experiment 2 The growth rates of clams on inert diets obtained in experiment 1 were considerably improved if we substitute cornmeal, which consisted of 90% carbohydrate and 10% protein, for cornstarch ŽTable 2.. Thus, compared with a base daily ration of 2% organic weight of I. galbana with respect to the live weight of the clams Ždiet F100., clams fed on a diet consisting of 75% phytoplankton and 25% cornmeal Ždiet M75. achieved OW growth rates of 116.6% compared to those of the basal diet, while growth rates for DW, LW and L were 32.1, 25.9 and 19.8% higher, respectively, than those corresponding to the basal diet ŽTable 2.. When 50% of the basal diet was replaced by cornmeal Ždiet M50., OW growth rates were the same Ž99.3%. as those for the diet composed of phytoplankton only and growth in DW, LW and L were higher by 15.5, 13.6 and 6.6%, respectively. When the proportion of phytoplankton was reduced to 25% and the remaining 75% replaced by cornmeal Ždiet M25., the growth rates for OW, DW, LW and L were 48.6, 68.2, 65.9 and 75.2%, respectively, of the growth rates for diet F100. The ANOVA showed significant differences between the diets Ž P - 0.001. and the NK test gave significant differences Ž P - 0.05. between DW and LW among all the diets, while the only differences for L which were not considered significant Ž P ) 0.05. were those between diets F100 and M50. In the case of OW, the differences between diets F100 and M50, as well as those between M50 and M75, were not significant. Diet had a significant effect on the percentage of organic matter in the seed ŽANOVA, P - 0.001., although this effect was not the same as that on growth. Thus the highest %OM corresponded to diet F100, followed by M75, M50 and M25, with the diet consisting exclusively of cornmeal showing the lowest %OM. The NK test detected significant differences Ž P - 0.05. between all diets, except between M75 and M50, as well as between CM and M25, where the differences were not significant. 3.3. Comparison of cornstarch and cornmeal diets The use of cornmeal in place of cornstarch in mixed diets had a notably positive effect on growth rates of the seed of R. decussatus. Thus, with cornstarch diet M25 the daily growth rate ŽDGR. in OW was 3.9, as opposed to 4.5 for the cornmeal diet M25 ŽFig. 1., which was significantly different ŽANOVA, P - 0.05.. Equally significant was the difference in DGR between the two different M50 diets, which gave values of 6.7 for cornmeal and 5.3 for cornstarch.
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So positive was the effect on growth when cornstarch was substituted for cornmeal that, as can be seen in Fig. 1, the DGRs for the M25 cornmeal diet were comparable to those of the F50 phytoplankton and M50 cornstarch diets, with no significant differences between them when compared by an ANOVA Ž P ) 0.05.. The differences between diets Table 3 Biochemical composition Ž%. of the different cornstarch and cornmeal diets used in the experiments Biochemical composition of diets Ž%. Diets
Protein
Carbohydrate
Lipid
Diets
Protein
Carbohydrate
Lipid
F100 M25 M50 M75 CM
33.4 16.1 21.9 27.7 10.3
30.9 74.3 59.8 45.3 88.8
35.7 9.6 18.3 27.0 0.9
F100 F50 F25 M25 M50 CS
33.4 33.4 33.4 8.7 17.0 0.5
30.9 30.9 30.9 82.3 65.1 99.4
35.7 35.7 35.7 9.0 17.9 0.1
Table 4 Cornstarch experiments. Biochemical composition Ž m g indy1 and %. of the clams at the beginning and at the end of the experimental period Initial biochemical composition of clams Protein Ž m g indy1 .
%
Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
106.5"10.1
53.2
53.7"12.4
26.8
39.9"10.9
19.9
Final biochemical composition of clams Diets Protein Ž m g indy1 . % Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
F100 F50 F25 M25 M50 CS
31.1 34.0 27.0 32.3 35.3 48.0
298.1"73.3 123.3"17.1 83.2"11.0 137.4"72.0 226.0"70.0 37.3"12.0
16.7 13.9 14.2 17.4 17.4 12.8
931.0"64.6 460.8"23.9 343.3"10.9 398.3"42.4 615.1"18.0 114.0"11.0
52.2 52.1 58.8 50.3 47.5 39.2
555.3"60.3 300.6"40.3 157.9"8.0 255.2"42.2 458.0"13.0 139.5"24.1
Table 5 Cornmeal experiments. Biochemical composition of the clams at the beginning and at the end of the experimental period Ž m g indy1 and %. Initial biochemical composition of clams Protein Ž m g indy1 .
%
Carbohydrate ( m g indy1 .
%
Lipid Ž m g indy1 .
%
54.4"4.1
46.1
37.8"3.9
32.0
25.8"2.5
21.9
Final biochemical composition of clams Diets Protein Ž m g indy1 . % Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
F100 M25 M50 M75 CM
31.5 31.4 32.6 33.4 44.7
99.0"11.6 60.4"6.2 98.7"27.1 104.4"5.5 20.8"2.3
20.2 20.2 20.3 18.9 14.1
236.6"30.4 145.2"7.1 229.4"12.0 262.9"15.0 61.0"4.3
48.3 48.4 47.1 47.7 41.2
154.4"15.9 94.1"6.6 159.1"17.7 184.3"8.2 66.2"4.4
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CS and CM, consisting wholly of cornstarch and cornmeal, were not significant Ž P ) 0.05. and neither were those between the F100 diets in experiments 1 and 2. 3.4. Biochemical composition Tables 3–5 show the biochemical composition of each of the diets studied in experiments 1 and 2, as well as the initial and final biochemical composition of the clams fed these diets. As can be seen in these tables, the degree to which food was assimilated depended to a large extent on the initial biochemical composition of the clams: in experiment 1, for example, carbohydrate content was low at the beginning of the experiment and this component was therefore stored, as an energy reserve, to a greater extent than were lipids, thus giving rise to a relatively lower percentage of the latter in the total content. The composition of the diet only produced major variations in the biochemical composition of the clams in the case of extremely unbalanced diets such as those consisting exclusively of cornmeal and cornstarch. In these cases the clams maintained their initial quantities of protein and lipid and only assimilated carbohydrate, leading to a relatively large increase in the proportion of this component and a corresponding decrease in the proportions of protein and lipid.
4. Discussion The results of the present study demonstrate that cornmeal and to a lesser extent cornstarch, constitute a good complement to live phytoplankton in diets for the seed of the little-neck clam R. decussatus. Cornstarch has been used to complement natural food in order to improve the condition index of adult oysters ŽHaven, 1965; Ingle, 1967; Dunathan et al., 1969., thus emphasizing the importance of the accumulation of reserves in the form of glucogen in gametogenesis ŽGabbott, 1976.. The use of artificial diets for juvenile oysters containing carbohydrate in the form of starch has also been described ŽCastell and Trider, 1974; Wisely and Reid, 1978; Langdon and Bolton, 1984, Langdon and Siegfried, 1984; Nell and Wisely, 1984; Urban and Langdon, 1984.. Growth rates achieved with these diets were on the whole lower than those for natural diets. Other authors have examined freeze-dried algae, dried- heterotrophically grown algae and various microparticles as substitutes for live phytoplankton. The results of these studies vary widely: freeze-dried and dried-heterotrophically grown phytoplankton can only be used as partial substitutes for live phytoplankton, with very similar production costs ŽCuratolo et al., 1993; Laing et al., 1990; Laing and Gil Verdugo, 1991, Laing and Millican, 1992., while microcapsules, apart from their very high cost, suffer from the disadvantages of sedimentation, bacterial degradation, leaching of nutrients and poor digestibility ŽLangdon and Bolton, 1984; Chu et al., 1987.. Epifanio Ž1979., using diets consisting of 50% dried yeast Candida utilis and 50% phytoplankton, achieved growth rates in length and dry weight of soft tissue comparable to those of a 100% algal ration for Argopecten irradians, Mytilus edulis and Mercenaria mercenaria, while on the other hand the growth rate of Crassostrea Õirginica was in inverse proportion to the quantity of yeast in the diet. Albentosa et al. Ž1989., working
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with R. philippinarum and Coutteau et al. Ž1994. with M. mercenaria, found that ‘‘replacing 50% of the algal ration with manipulated yeast did not result in a significant decrease in growth rate relative to the algal-fed controls and the substitution of 80% of the algal diets resulted in growth rates reaching 90%’’. Experiments carried out in our laboratories have shown that the behaviour of R. decussatus resembles that of C. Õirginica described above, since growth rates for mixed diets of yeast and phytoplankton were considerably lower than those for both the phytoplankton control diets and the cornstarch and cornmeal diets described in the present study. In this study, the replacement of 50% phytoplankton by cornmeal leads to DGRs for DW that were comparable to those for purely phytoplankton diets and when 75% phytoplankton was replaced by cornmeal, DGRs for DW of 67% those of the control diet were attained. When the results are expressed in terms of LW, the DGRs for the M50 and M25 diets are 106 and 81%, respectively, of the DGRs for the control diet, which underlines the importance of determining the variation in OW in this kind of trial, since it provides a better indication of the quality of the diet than LW alone. The results of our study, together with those on the use of wheatgerm ŽFernandez´ Reirız ´ et al., 1998., are the only ones which consider the use of artificial diets for R. decussatus. According to these results, cornmeal gives growth rates that are comparable to those of the best quality artificial diets that have been examined to date, with the added advantage of ease of handling and lower cost, which are common to all cereal flours. If we consider that at present live phytoplankton is the usual food for broodstock and seed of molluscs in bivalve hatcheries and nurseries and its production accounts for 30% of total seed production cost in the former ŽCoutteau and Sorgeloos, 1992. and up to 85% of the cost of production in the latter ŽBolton, 1982., the use of cornmeal as a partial substitute for phytoplankton would lead to a considerable reduction of production costs in those hatcheries that produce R. decussatus. No clear definition of the nutritional requirements of bivalves is forthcoming from the various studies that are available. Enright et al. Ž1986. achieved good growth rates in juvenile Ostrea edulis with diets containing 15.5% protein and 59.4% carbohydrate, while Wisely and Reid Ž1978. and Nell and Wisely Ž1983, 1984., in their study of artificial diets for the fattening of Saccostrea commercialis, indicate that the optimal level of carbohydrate and protein in the diet were 74 and 18%, respectively, while lipid levels of around 10% total organic matter of the diet were sufficient to ensure optimal indices of condition. On the other hand, Kreeger and Langdon Ž1993. achieved optimal growth rates in juvenile Mytilus trossulus with diets that contain over 40% protein. A comparison between the M50 diets in experiments 1 and 2 of this study, which both contained similar amounts of lipid, showed that an increase in protein from 17 to 22% leads to a large increase in DGR. It may equally be true that the lower growth rate for the M25 diet in experiment 2 compared to that of the M50 diet in experiment 1 is related to the greater lipid content of the latter diet Ž18% as opposed to 9%., since both diets contained a similar amount of protein, while postlarvae and see in the first months after metamorphosis have a high lipid requirement ŽHolland and Hannant, 1974.. The results of experiment 2 show that a substitution of up to 50% phytoplankton for cornmeal allows growth rates similar to those of wholly phytoplankton diets to be achieved, suggesting that in the case of the seed of R. decussatus minimum protein and
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lipid requirements are of the order of 22% and 18%, respectively ŽTable 5.. The fact that there are only minimal differences between diets CM and CS, composed entirely of cereals, in spite of the higher protein content of the former, may indicate a lack of essential amino acids in these proteins, together with an almost total absence of lipid in both diets.
Acknowledgements We would like to thank C. Fernandez Pena, J. Dıaz, ´ ´ L. Nieto and B. Gonzalez ´ for their helpful technical assistance. This study was funded by CICYT-IEO-CSIC project I q D number AGFC-1003-CO2-01.
References Albentosa, M., Naessens, P., Coutteau, P., Lavens, P., Sorgeloos, P., 1989. Promising results in the seed culturing of the Manila clam Tapes semidecussata with a manipulated yeast product as a partial substitute for algae. In: Aquaculture Europe ’89 Bordeaux, France European Aquaculture Society Sp. Pub. 1019897–8. Albentosa, M., Perez-Camacho, A., Beiras, R., 1996. The effect of food concentration on the scope for growth ´ and growth performance of Ruditapes decussatus seed, reared in an open-flow system. Aquacult. Nutr. 2, 213–220. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bolton, E.T., 1982. Intensive Marine Bivalve Cultivation in a Controlled Recirculating Seawater Prototype System. Univ. Delaware Sea Grant Publication DEL-SG-07-82, 165 pp. Castell, J.D., Trider, D.J., 1974. Preliminary feeding trials using artificial diets to study the nutritional requirements of oysters Ž Crassostrea Õirginica.. J. Fish. Res. Board Can. 31, 95–99. Chu, F.L.E., Webb, K.L., Hepworth, D.A., Casey, B.B., 1987. Metamorphosis of larvae of Crassostrea Õirginica fed microcapsulated diets. Aquaculture 64, 185–199. Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirements for live algae in the hatchery and nursery rearing of bivalve molluscs: an international survey. J. Shellfish Res. 11 Ž2., 467–476. Coutteau, P., Hadley, L., Manzi, J., Sorgeloos, P., 1991. Manipulated diets as a partial algal substitute for the nursery culture of the hard clam, Mercenaria mercenaria. In: Aquaculture Europe ’91: Aquaculture and the Environment European Aquacultulture Society Sp. Pub. 14199177–78. Coutteau et al., 1994. Curatolo, A., Ryan, M.M., Mercer, A.P., 1993. An evaluation of the performance of Manila clam spat ŽTapes philippinarum. fed on different rations of spray-dried algae ŽTetraselmis suecica.. Aquaculture 112, 179–186. De Pauw, N., 1981. Use and production of microalgae as food for nursery bivalves. In: Claus, C., De Pauw, N., Jaspers, E. ŽEds.., Nursery Culturing of Bivalve Molluscs. European Mariculture Society. Special Publication No. 7. Oostende, Belgium, pp. 35–69. Dunathan, J.P., Ingle, R.M., Havens, W.K., 1969. Effects of artificial foods upon oyster fattening with potential commercial applications. Flo. Bd. Conserv. Mar. Res. Lab. Tech. Ser. 58, 1–38. Epifanio, C.E., 1979. Comparison of yeast and algal diets for bivalve molluscs. Aquaculture 16, 187–192. Enright, C.T., Newkirk, G.F., Craigie, J.S., Castell, J.D., 1986. Growth of juvenile Ostrea edulis L. fed Chaetoceros gracilis Schutt ¨ of varied chemical composition. J. Exp. Mar. Biol. Ecol. 96, 15–26.
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Fernandez-Reirız, ´ ´ M.J., Perez ´ Camacho, A., Ferreiro, M.J., Blanco, J., Planas, M., Campos, M.J., Labarta, U., 1989. Biomass production and variation in the biochemical profile Žtotal protein, carbohydrates, RNA, lipids and fatty acids. of seven species of marine microalgae. Aquaculture 83, 17–37. Fernandez-Reirız, ´ ´ M.J., Albentosa, M., Labarta, U., Perez ´ Camacho, A., 1998. Effect of microalgal and inert diets on the lipid profile and growth of the seed of Ruditapes desussatus. Comp. Biochem. Physiol. 119 Ž2.: in press. Folch, J., Lees, M., Slone-Stanley, G.G., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 496–503. Gabbott, P.A., 1976. Energy metabolism. In: Bayne, B.L. ŽEd.., Marine mussels, Their Ecology and Physiology. Cambridge Univ. Press, Cambridge, pp. 293–355. Haven, D.S., 1965. Supplemental feeding of oysters with starch. Chesapeake Sci. 6 Ž1., 43–51. Holland, D.L., Hannant, P.J., 1974. Biochemical changes during growth of the spat of the oyster, Ostrea edulis L. J. Mar. Biol. Assoc. U.K. 54, 1007–1016. Ingle, R.M., 1967. Artificial food for oysters. Sea Frontiers 13, 296–303. Jones, D.A., Holland, D.L., Jabborie, S.S., 1984. Current status of microencapsulated diets for aquaculture. Appl. Biochem. Technol. 10, 275–288. Kreeger, D.A., Langdon, C.J., 1993. Effects of dietary protein content on growth of juvenile mussels, Mytilus trossulus ŽGould, 1850.. Biol. Bull. 185, 123–139. Laing, I., 1987. The use of artificial diets in rearing bivalve spat. Aquaculture 65, 243–249. Laing, I., Gil Verdugo, C., 1991. Nutritional value of spray-dried Tetraselmis suecica for juvenile bivalves. Aquaculture 92, 207–218. Laing, I., Millican, P.F., 1992. Indoor nursery cultivation of juvenile bivalve mollusc using diets of dried algae. Aquaculture 102, 231–243. Laing, I., Child, A.R., Janke, A., 1990. Nutritional value of dried algae diets for larvae of Manila clam ŽTapes philippinarum.. J. Mar. Biol. Assoc. U.K. 70, 1–12. Langdon, C.J., Bolton, E.T., 1984. A microparticulate diet for a suspension-feeding bivalve molluscs Crassostrea Õirginica ŽGmelin.. J. Mar. Ecol. 82, 239–258. Langdon, C.J., Siegfried, C.A., 1984. Progress in the development of artificial diets for bivalve filter feeders. Aquaculture 39, 135–153. Langdon, C.J., Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty composition of Crassostrea gigas spat. J. Mar. Biol. Assoc. U.K. 61, 431–448. Langdon, C.J., Levine, D.M., Jones, D.A., 1985. Microparticulate feeds for marine suspension-feeders. J. Microencapsulation 2, 1–11. Loosanoff, V.L., Davis, H.C., 1963. Rearing of bivalve molluscs. Adv. Mar. Biol. 1, 1–138. Lowry, O.H., Rosebrough, N.H., Fair, A.L., 1951. Protein measurement with the folin–phenol reagent. J. Biol. Chem. 193, 265–275. Lucas, A., Gerad, A., 1981. Space requirement and energy cost in some types of bivalve nurseries. In: Claus, C., De Pauw, N., Jaspers, E. ŽEds.., Nursery Culturing of Bivalve Molluscs. European Mariculture Society. Special Publication No. 7. Oostende, Belgium, pp. 151–170. Nell, J.A., 1985. Comparison of some single cell proteins in the diet of the Sydney rock oyster Ž Saccostrea commercialis.. Prog. Fish-Cult. 47 Ž2., 110–113. Nell, J.A., Wisely, B., 1983. Experimental feeding of Sydney rock oyster Ž Saccostrea commercialis.: II. Protein supplementation of artificial diets for adult oysters. Aquaculture 32, 1–9. Nell, J.A., Wisely, B., 1984. Experimental feeding of Sydney rock oyster Ž Saccostrea commercialis.: III. Food concentration and fattening procedures. Aquaculture 37, 197–208. Perez ´ Camacho, A., Roman, ´ G., Torre, M., 1977. Experiencias en cultivos de larvas de tres especies de moluscos bivalvos: Venerupis pullastra ŽMontagu., Venerupis decussata ŽLinnaeus. y Ostrea edulis ŽLinnaeus. ŽExperiences on larvae culture of three species of Bivalve Molluscs.. Bol. Inst. Espa. Oceano. 235, 1–61. Persoone, G., Claus, C., 1980. Mass culture of algae, a bottleneck in the nursery culturing of molluscs. In: Shelef, G., Soeder, C.J. ŽEds.., Algal Biomass. Elsevier, Amsterdam, pp. 265–285. Quayle, D.B., 1952. Structure and biology of the larva and spat of Venerupis pullastra ŽMontagu.. Trans. R. Soc. Edinb. 62, 255–297.
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ŽEstatistical Methods.. Cıa. Snedecor, G.W., Cochran, W.G., 1971. Metodos estadısticos ´ ´ ´ Ed. Continental, Buenos Aires. Strickland, J.D., Parsons, T.R., 1968. A practical handbook of seawater analysis. Bull. Fish. Res. Bd. Can. Urban, E.R., Kirchman, D.L., 1992. Effect of kaolinite clay on the feeding activity of the eastern oyster Crassostrea Õirginica ŽGmelin.. J. Exp. Mar. Biol. Ecol. 160, 47–60. Urban, E.R., Langdon, C.J., 1984. Reduction in costs of diets for the American oyster, Crassostrea Õirginica ŽGmelin., by the use of non-algal supplements. Aquaculture 38, 277–291. Walne, P.R., 1956. Experimental rearing of the larvae of Ostrea edulis L. Fishery Invest., Ser. II, 20 Ž9.. Wisely, B., Reid, B.L., 1978. Experimental feeding of Sydney rock oysters Ž Crassostrea commercialiss Saccostrea cucullata.: I. Optimum particle sizes and concentrations. Aquaculture 15, 319–331. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ.
Effect of microalgal and inert žcornmeal and cornstarch / diets on growth performance and biochemical composition of Ruditapes decussatus seed A. Perez ´ Camacho a
a,)
b , M. Albentosa a , M.J. Fernandez-Reiriz , ´ b U. Labarta
´ Instituto Espanol S N, 15080 A Coruna, ˜ de Oceanografıa, ´ Muelle de Animas ˜ Spain b Instituto de InÕestigacions ´ Marinas, ˜ Eduardo Cabello 6, 36208, Vigo, Spain Accepted 20 October 1997
Abstract Research was carried out into the effect of phytoplankton, cornmeal and cornstarch diets on growth and biochemical composition of the seed of the little-neck clam, Ruditapes decussatus. The seed of R. decussatus, fed on daily rations of Isochrysis galbana Žorganic weight. of 0.5 and 1% of live weight of the seed, showed an improvement in growth rate when cornstarch, which is 99% carbohydrate, was added to these diets. Thus in the case of a daily ration of 0.5%, daily growth rates increased by between 33.5 and 32.3%, depending on whether we are referring to organic weight, dry weight or live weight, when 1.5% cornstarch was added. In the case of a ration of 1% I. galbana, the addition of another 1% cornstarch lead to an improvement in daily growth rates, depending on the different weight class in question, of between 14.1 and 15.5%. When compared to a daily ration consisting of 2% phytoplankton, which was considered to be the optimal ration for growth in the seed of these clams, the replacement of half the quantity of I. galbana by a quantity of cornstarch of equivalent weight gave a growth rate in terms of organic weight of 87.9% that of the phytoplankton diet, while the rates for dry weight and live weight were 89.6 and 87.9%, respectively. These results improved noticeably when cornmeal, consisting of 10% protein and 90% carbohydrate, was used instead of cornstarch. In the case of a 2% phytoplankton diet, if we substituted an equivalent quantity of cornmeal for 50% of the
)
Corresponding author. Fax: q34-81229077; e-mail: [email protected].
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 7 . 0 0 2 3 2 - 9
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phytoplankton, the growth rate in organic matter was the same Ž99.0%. as those for the diet consisting of phytoplankton alone, while growth rates in dry weight and live weight were 6.2 and 5.9% higher, respectively, than those of the phytoplankton diet. It would therefore appear that cornmeal Žand to a lesser extent cornstarch. can be successfully used as a partial substitute for phytoplankton in diets for the seed of R. decussatus and its use in hatcheries and nurseries devoted to the culture of this species would lead to a considerable reduction of production costs. q 1998 Elsevier Science B.V. Keywords: Ruditapes decussatus; Seed culture; Inert food; Cornmeal; Cornstarch
1. Introduction
The little-neck clam, Ruditapes decussatus, is one of the most important species in Spanish aquaculture, commanding high prices in the market. Possibilities for production are limited due to the scarcity of naturally-occurring seed and the high cost of seed production in commercial hatcheries. Any saving in this cost is therefore crucial for the viability of this type of industry and would be a major contribution to the expansion of aquaculture production. The basis for the development of the bivalve mollusc culture industry was established at the mid-point of this century in works by Quayle Ž1952.; Loosanoff and Davis Ž1963. and Walne Ž1956., among others. Ever since then the production of live phytoplankton has been seen as one of the major contributory factors to the costs borne by hatcheries and nurseries ŽPersoone and Claus, 1980; De Pauw, 1981; Lucas and Gerad, 1981., accounting for 30% of the total seed production cost in the former ŽCoutteau and Sorgeloos, 1992. and up to 85% of production costs in the latter ŽBolton, 1982.. Various authors have attempted to find a substitute for live phytoplankton in the diet of bivalve molluscs and have experimented, among other products, with spin-dried and freeze-dried algae ŽLaing et al., 1990; Laing and Gil Verdugo, 1991; Laing and Millican, 1992., yeasts ŽEpifanio, 1979; Urban and Langdon, 1984; Nell, 1985., modified yeasts ŽAlbentosa et al., 1989; Coutteau et al., 1991., microcapsules ŽLangdon and Waldock, 1981; Jones et al., 1984; Langdon and Bolton, 1984; Langdon et al., 1985; Laing, 1987. and different varieties of cereal starch ŽHaven, 1965; Ingle, 1967; Dunathan et al., 1969; Castell and Trider, 1974; Wisely and Reid, 1978; Nell and Wisely, 1983, 1984; Langdon and Bolton, 1984; Langdon and Siegfried, 1984; Urban and Kirchman, 1992.. In none of these studies is R. decussatus mentioned and their results vary considerably according to the characteristics of the foodstuff and the species for which it is intended; until now, no total substitute has been found for live phytoplankton, the best results having been obtained with 50% substitution or less. The purpose of this study is to assess the performance of cornmeal and cornstarch in the diet of the seed of the little-neck clam R. decussatus, whether in diets exclusively composed of these products or in mixed diets with varying proportions of phytoplankton. Seed growth rates and biochemical composition were used to assess the results obtained from the different diets.
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2. Materials and methods 2.1. Seed The experiments were carried out with seed of R. decussatus obtained from broodstock conditioned in the Instituto Espanol ˜ de Oceanografıa. ´ The methods used to induce spawning and to cultivate the larvae and the seed were those described by Perez ´ Camacho et al. Ž1977.. 2.2. Diet The basal diet consisted of the microalga Isochrysis galbana, which was replaced in varying proportions by cornmeal or cornstarch. Preliminary trials were carried out to define the sedimentation rates of the corn products concerned in the culture vessels and equivalent amounts of the products were then added to the daily ration to compensate for losses caused by sedimentation. The microalgae were grown in 6-l glass flasks in a temperature-controlled chamber, at a constant temperature of 188C and with constant illumination at 9900 lux. Salinity was maintained at a constant 33‰. The culture medium used was that described by Walne, 1956 and the microalgae were harvested in the initial stationary growth phase. The cornstarch contained 99% carbohydrate, while the cornmeal contained 90% carbohydrate and 10% protein. Both products were suspended in seawater by shaking and sieved through a 20 m m sieve. A Coulter Counter Multisizer was used to measure the concentration of the foodstuff in the seawater. 2.3. Experimental design The base diet for the different experiments was an optimal daily ration of 2% organic seed weight of the microalga I. galbana ŽAlbentosa et al., 1996.. The experiments were carried out in triplicate for each diet. Each replica contained an initial biomass of 200 mg of clams Žlive weight.. 2.3.1. Experiment 1 This experiment was used to compare the growth rates achieved with the basal diet F100, consisting of a daily ration of 2% I. galbana, with the two mixed diets M50 and M25, in which 50% and 75%, respectively, of the phytoplankton was replaced by an equivalent weight of cornstarch. Three control diets were established: F50, consisting of a daily ration of 1% I. galbana; F25, consisting of a daily ration of 0.5% I. galbana; and CS, consisting of 2% cornstarch. Percentages are expressed in terms of the live weight of the clams. Initial figures for the seed were as follows: mean length, 2.10 . 0.20 mm; mean live weight, 2.46 . 0.04 mg; mean dry weight, 1.58 . 0.04 mg; mean organic weight, 0.20 . 0.01; and mean organic matter 12.5 . 0.1%. The experiment lasted 5 weeks.
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2.3.2. Experiment 2 Cornmeal was used in place of cornstarch in this experiment. Four diets were tested, consisting of varying proportions of phytoplankton and cornmeal, as follows: Diet F100, 2% I. galbana; diet M50, 50% phytoplankton and 50% cornmeal; diet M25, 25% phytoplankton and 75% cornmeal; and diet CM, 2% cornmeal. The seed used in this experiment was of similar size to that used in experiment 1 Žmean lengths 1.76 . 0.16 mm, mean live weights 1.34 . 0.06 mg, mean dry weights 0.85 . 0.04 mg, mean organic weights 0.12 . 0.01, mean organic matters 13.2 . 0.1%.. The experimental period was 3 weeks. 2.4. Experimental conditions The seed was placed on the bottom of 6-l plastic vessels which were fitted with an air point in order to reduce the sedimentation rates of the foodstuff. The experimental cultures were kept in a temperature-controlled chamber at a constant temperature of 208C. The water in the vessels was changed daily, using seawater filtered through a 1 m m sieve and sterilised by UV. The experimental period was from 3 to 5 weeks. The cornmeal and cornstarch were suspended in seawater, in 6-l round-bottomed flasks with strong aeration in order to keep the food in suspension. The food was supplied over 15-min periods at 4-h intervals by means of a 12-channel Ismatec MV-CA pump. Each diet was assayed in triplicate and a fourth culture vessel was used to determine the sedimentation rate. The initial volume in the culture vessels was 3-l. In order to avoid the production of pseudofaeces the concentration of the food in the vessels was kept below 1.6 m g mly1 of organic matter ŽAlbentosa et al., 1996.. The initial volume of water in the culture vessels was increased weekly, according to the increase in biomass of the clams during the same period, in order to maintain constant conditions throughout the experiment. 2.5. Diet assessment parameters The live weight ŽLW. of the clams in each experimental batch was obtained weekly, after a 10-min draining period on absorbent paper. At the end of the experimental period half of the clams in each batch were used to obtain the length of the seed Ž L., the dry weight ŽDW., the weight of inorganic matter ŽIW., the organic weight ŽOW s DW y IW. and the percentage of organic matter Ž%OM s ŽOWrDW= 100.. DW was determined by drying the clams at 1008C for 24 h, while IW was obtained by heating to 4508C for 12 h. The other half of the batch was used for biochemical analysis. The daily growth rate ŽDGR. was calculated according to the equation )
DGR: Ž Ž LnW1 y LnW0 . rt . 100
where W1 and W0 are the values of the different variables ŽOW, DW, LW and L. at the end and beginning, respectively, of each experiment and t is the number of days.
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2.6. Analytical methods The organic content of the phytoplankton was determined by filtering the algal culture through Whatman GFrC glass-fibre filters which had previously been rinsed and ashed at 4508C. The filters were rinsed in a 0.5 M ammonium formate solution to eliminate salt residues, dried to a constant weight and ashed at 4508C. The organic content and water of the corn products were determined at 1008C and 4508C respectively. The method described by Lowry et al. Ž1951. was used to determine the protein content, following alkaline hydrolysis with NaOH 0.5 Nr24 hr308C. Total carbohydrates were quantified as glucose by the phenol–sulphur method ŽStrickland and Parsons, 1968.. Lipids were extracted by a modification of the method of Bligh and Dyer Ž1959. ŽFernandez-Reirız ´ ´ et al., 1989. as follows: lipids were extracted by means of chloroform:methanol Ž1:2. and then centrifuged, after which the sediment was once again extracted with chloroform:methanol Ž2:1.. The resulting extract was purified by rinsing both supernatants in a mixture of chloroform, methanol and water Ž8:4:3. as described by Folch et al. Ž1957.. Total lipids were gravimetrically determined through evaporation of the solvent in the purified extract on aluminium sheets at 60–808C. 2.7. Statistical methods The statistical package Statgraphics was used to analyse the results. For each experiment growth was compared by examining the daily increase for each of the variables listed above, while the daily growth rate ŽDGR. was used to compare the results of different experiments. Comparison between the different variables used to evaluate the diets was performed by an ANOVA with a significance level of P - 0.05. Angular transformation was used for values expressed as percentages and logarithmic transformation for the comparison of weight increases. The Bartlett test was used to test for variance homogeneity. Multiple comparisons were carried out with the Newman– Keuls multiple range test ŽSnedecor and Cochran, 1971; Zar, 1974..
3. Results 3.1. Cornstarch 3.1.1. Experiment 1 When the daily diet of 2% I. galbana Ždiet F100. was reduced to 1% Ždiet F50., the growth rate of the seed of R. decussatus decreased to 43% of that obtained with diet F100 and decreases still further to 24% if the diet was reduced to 0.5% phytoplankton Ždiet F25.. If cornstarch, which is 99% carbohydrate ŽTable 1., was added to diets F50 and F25, growth rates increased considerably. Thus, in the case of the 0.5% ration, DW growth increased by 80.1% when 1.5% cornstarch was added, while OW increased by 55.0%,
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Table 1 Mean daily increase in organic weight ŽOW., dry weight ŽDW., live weight ŽLW. and shell length Ž L., and % organic matter ŽOM. of clams fed on different diets Žaverage values of 3 replicates"stand. error.. Diet F100: 2% I. galbana ŽOW. of clam LW daily; diet F50: 50% F100; diet F25: 25% F100; diet M50: 50% F100 and 50% cornstarch; diet M25: 25% F100 and 75% cornstarch; diet CS: 2% cornstarch ŽOW. of clam live weight. Initial figures: mean length, 2.10.0.20 mm; mean live weight, 2.459.0.041 mg; mean dry weight, 1.578.0.038 mg; mean organic weight, 0.198.0.005; and mean organic matter 12.5.0.1%.
F100 F50 F25 M25 M50 CS
OW Ž m g indy1 .
DW Ž m g indy1 .
LW Ž m g indy1 .
L Žmm.
OM Ž%.
45.3"4.8 19.5"0.4 10.9"0.4 31.4"1.5 16.9"0.9 2.6"0.9
375.7"21.9 201.4"0.7 102.7"6.3 263.6"6.1 185.0"0.9 46.4"2.4
593.8"33.2 321.9"11.1 170.9"7.0 433.9"12.4 288.4"2.6 65.3"3.6
85.1"0.9 59.2"2.1 38.9"3.1 70.1"1.0 55.4"1.3 12.7"1.0
12.6"0.7 10.2"0.1 10.6"0.3 12.0"0.4 9.8"0.4 10.2"0.4
Table 2 Mean daily increase in organic matter ŽOW., dry weight ŽDW., live weight ŽLW. and shell length Ž L., and % organic matter ŽOM. of clams fed on different diets Žaverage values of 3 replicates"SE.. Diet F100: 2% I. galbana Žin OW. daily with regard to clam live weight; diet M75: 75% F100 and 25% cornmeal; diet M50: 50% F100 and 50% cornmeal; diet M25: 25% F100 and 75% cornmeal; diet CM: 2% cornmeal Žin OW. with regard to clam live weight. Initial figures: mean length, 1.76.0.16mm; mean live weight, 1.341.0.059 mg; mean dry weight, 0.854.0.039 mg; mean organic weights 0.119.0.005; mean organic matter, 13.2.0.1%.
F100 M75 M50 M25 CM
OW Ž m g indy1 .
DW Ž m g indy1 .
LW Ž m g indy1 .
L Žmm.
OM Ž%.
17.6"1.2 20.6"1.0 17.5"2.8 8.6"1.2 1.8"0.3
131.0"1.0 173.0"4.1 151.3"4.3 89.4"5.1 26.2"3.1
207.6"2.4 261.3"2.7 235.6"4.7 136.8"2.9 28.1"2.7
57.6"0.6 69.0"1.0 61.4"1.0 43.3"2.0 4.8"1.1
14.1"0.3 12.1"0.2 12.0"0.5 10.9"0.7 10.7"0.3
LW by 68.8% and L by 42.4%. For 1% rations of I. galbana, when a 1% supplement of cornstarch was added, DW increased by 30.9%, OW by 61.0% and L by 18.4%. In comparison with diet F100, when half the quantity of I. galbana was replaced by an equivalent weight of cornstarch Ždiet M50. the growth rate for OW was 69.3% of that obtained with a phytoplankton only Ž I. galbana. diet; DW was 73.7%, LW was 73.1% and L was 82.4%. A 25% phytoplanktonr75% starch diet Ždiet M25. gave growth rates of 37.4, 51.7, 48.6 and 65.1% for OW, DW, LW and L, respectively, when compared to the growth rates obtained with diet F100.
Fig. 1. Variation in daily growth rates of organic matter, dry weight, live weight and seed length for the seed of the little-neck clam R. decussatus fed different proportions of phytoplankton, cornstarch ŽA. and cornmeal ŽB.. Initial figures: mean length, 2.10.0.20 ŽA. and 1.76.0.16 mm ŽB.; mean live weight, 2.459.0.041 ŽA. and 1.341.0.059 mg ŽB.; mean dry weight, 1.578.0.038 ŽA. and 0.854.0.039 mg ŽB.; mean organic weight, 0.198.0.005 ŽA. and 0.119.0.005 mg ŽB.; mean organic matter, 12.5.0.1 ŽA. and 13.2.0.1% ŽA.. Vertical lines represent 95% confidence intervals.
A. Perez ´ Camacho et al.r Aquaculture 160 (1998) 89–102
95
96
A. Perez ´ Camacho et al.r Aquaculture 160 (1998) 89–102
The ANOVA gave significant differences Ž P - 0.001. between daily growth rates for OW, DW, LW and L. The NK test detected significant differences Ž P - 0.05. in OW, DW, LW and L between all groups with the exception of diets F50 and M25, where there was no significant difference Ž P ) 0.05.. Where the %OM of the seed is concerned, the NK test showed two homogeneous groups, one formed by diets F100 and M50, with a %OM of between 12.0 and 12.6% and the other by diets F50, F25, M25 and CS, with a %OM ranging from 9.8 to 10.6%. There were significant differences between the two groups Ž P - 0.05.. 3.2. Cornmeal 3.2.1. Experiment 2 The growth rates of clams on inert diets obtained in experiment 1 were considerably improved if we substitute cornmeal, which consisted of 90% carbohydrate and 10% protein, for cornstarch ŽTable 2.. Thus, compared with a base daily ration of 2% organic weight of I. galbana with respect to the live weight of the clams Ždiet F100., clams fed on a diet consisting of 75% phytoplankton and 25% cornmeal Ždiet M75. achieved OW growth rates of 116.6% compared to those of the basal diet, while growth rates for DW, LW and L were 32.1, 25.9 and 19.8% higher, respectively, than those corresponding to the basal diet ŽTable 2.. When 50% of the basal diet was replaced by cornmeal Ždiet M50., OW growth rates were the same Ž99.3%. as those for the diet composed of phytoplankton only and growth in DW, LW and L were higher by 15.5, 13.6 and 6.6%, respectively. When the proportion of phytoplankton was reduced to 25% and the remaining 75% replaced by cornmeal Ždiet M25., the growth rates for OW, DW, LW and L were 48.6, 68.2, 65.9 and 75.2%, respectively, of the growth rates for diet F100. The ANOVA showed significant differences between the diets Ž P - 0.001. and the NK test gave significant differences Ž P - 0.05. between DW and LW among all the diets, while the only differences for L which were not considered significant Ž P ) 0.05. were those between diets F100 and M50. In the case of OW, the differences between diets F100 and M50, as well as those between M50 and M75, were not significant. Diet had a significant effect on the percentage of organic matter in the seed ŽANOVA, P - 0.001., although this effect was not the same as that on growth. Thus the highest %OM corresponded to diet F100, followed by M75, M50 and M25, with the diet consisting exclusively of cornmeal showing the lowest %OM. The NK test detected significant differences Ž P - 0.05. between all diets, except between M75 and M50, as well as between CM and M25, where the differences were not significant. 3.3. Comparison of cornstarch and cornmeal diets The use of cornmeal in place of cornstarch in mixed diets had a notably positive effect on growth rates of the seed of R. decussatus. Thus, with cornstarch diet M25 the daily growth rate ŽDGR. in OW was 3.9, as opposed to 4.5 for the cornmeal diet M25 ŽFig. 1., which was significantly different ŽANOVA, P - 0.05.. Equally significant was the difference in DGR between the two different M50 diets, which gave values of 6.7 for cornmeal and 5.3 for cornstarch.
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So positive was the effect on growth when cornstarch was substituted for cornmeal that, as can be seen in Fig. 1, the DGRs for the M25 cornmeal diet were comparable to those of the F50 phytoplankton and M50 cornstarch diets, with no significant differences between them when compared by an ANOVA Ž P ) 0.05.. The differences between diets Table 3 Biochemical composition Ž%. of the different cornstarch and cornmeal diets used in the experiments Biochemical composition of diets Ž%. Diets
Protein
Carbohydrate
Lipid
Diets
Protein
Carbohydrate
Lipid
F100 M25 M50 M75 CM
33.4 16.1 21.9 27.7 10.3
30.9 74.3 59.8 45.3 88.8
35.7 9.6 18.3 27.0 0.9
F100 F50 F25 M25 M50 CS
33.4 33.4 33.4 8.7 17.0 0.5
30.9 30.9 30.9 82.3 65.1 99.4
35.7 35.7 35.7 9.0 17.9 0.1
Table 4 Cornstarch experiments. Biochemical composition Ž m g indy1 and %. of the clams at the beginning and at the end of the experimental period Initial biochemical composition of clams Protein Ž m g indy1 .
%
Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
106.5"10.1
53.2
53.7"12.4
26.8
39.9"10.9
19.9
Final biochemical composition of clams Diets Protein Ž m g indy1 . % Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
F100 F50 F25 M25 M50 CS
31.1 34.0 27.0 32.3 35.3 48.0
298.1"73.3 123.3"17.1 83.2"11.0 137.4"72.0 226.0"70.0 37.3"12.0
16.7 13.9 14.2 17.4 17.4 12.8
931.0"64.6 460.8"23.9 343.3"10.9 398.3"42.4 615.1"18.0 114.0"11.0
52.2 52.1 58.8 50.3 47.5 39.2
555.3"60.3 300.6"40.3 157.9"8.0 255.2"42.2 458.0"13.0 139.5"24.1
Table 5 Cornmeal experiments. Biochemical composition of the clams at the beginning and at the end of the experimental period Ž m g indy1 and %. Initial biochemical composition of clams Protein Ž m g indy1 .
%
Carbohydrate ( m g indy1 .
%
Lipid Ž m g indy1 .
%
54.4"4.1
46.1
37.8"3.9
32.0
25.8"2.5
21.9
Final biochemical composition of clams Diets Protein Ž m g indy1 . % Carbohydrate Ž m g indy1 .
%
Lipid Ž m g indy1 .
%
F100 M25 M50 M75 CM
31.5 31.4 32.6 33.4 44.7
99.0"11.6 60.4"6.2 98.7"27.1 104.4"5.5 20.8"2.3
20.2 20.2 20.3 18.9 14.1
236.6"30.4 145.2"7.1 229.4"12.0 262.9"15.0 61.0"4.3
48.3 48.4 47.1 47.7 41.2
154.4"15.9 94.1"6.6 159.1"17.7 184.3"8.2 66.2"4.4
98
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CS and CM, consisting wholly of cornstarch and cornmeal, were not significant Ž P ) 0.05. and neither were those between the F100 diets in experiments 1 and 2. 3.4. Biochemical composition Tables 3–5 show the biochemical composition of each of the diets studied in experiments 1 and 2, as well as the initial and final biochemical composition of the clams fed these diets. As can be seen in these tables, the degree to which food was assimilated depended to a large extent on the initial biochemical composition of the clams: in experiment 1, for example, carbohydrate content was low at the beginning of the experiment and this component was therefore stored, as an energy reserve, to a greater extent than were lipids, thus giving rise to a relatively lower percentage of the latter in the total content. The composition of the diet only produced major variations in the biochemical composition of the clams in the case of extremely unbalanced diets such as those consisting exclusively of cornmeal and cornstarch. In these cases the clams maintained their initial quantities of protein and lipid and only assimilated carbohydrate, leading to a relatively large increase in the proportion of this component and a corresponding decrease in the proportions of protein and lipid.
4. Discussion The results of the present study demonstrate that cornmeal and to a lesser extent cornstarch, constitute a good complement to live phytoplankton in diets for the seed of the little-neck clam R. decussatus. Cornstarch has been used to complement natural food in order to improve the condition index of adult oysters ŽHaven, 1965; Ingle, 1967; Dunathan et al., 1969., thus emphasizing the importance of the accumulation of reserves in the form of glucogen in gametogenesis ŽGabbott, 1976.. The use of artificial diets for juvenile oysters containing carbohydrate in the form of starch has also been described ŽCastell and Trider, 1974; Wisely and Reid, 1978; Langdon and Bolton, 1984, Langdon and Siegfried, 1984; Nell and Wisely, 1984; Urban and Langdon, 1984.. Growth rates achieved with these diets were on the whole lower than those for natural diets. Other authors have examined freeze-dried algae, dried- heterotrophically grown algae and various microparticles as substitutes for live phytoplankton. The results of these studies vary widely: freeze-dried and dried-heterotrophically grown phytoplankton can only be used as partial substitutes for live phytoplankton, with very similar production costs ŽCuratolo et al., 1993; Laing et al., 1990; Laing and Gil Verdugo, 1991, Laing and Millican, 1992., while microcapsules, apart from their very high cost, suffer from the disadvantages of sedimentation, bacterial degradation, leaching of nutrients and poor digestibility ŽLangdon and Bolton, 1984; Chu et al., 1987.. Epifanio Ž1979., using diets consisting of 50% dried yeast Candida utilis and 50% phytoplankton, achieved growth rates in length and dry weight of soft tissue comparable to those of a 100% algal ration for Argopecten irradians, Mytilus edulis and Mercenaria mercenaria, while on the other hand the growth rate of Crassostrea Õirginica was in inverse proportion to the quantity of yeast in the diet. Albentosa et al. Ž1989., working
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with R. philippinarum and Coutteau et al. Ž1994. with M. mercenaria, found that ‘‘replacing 50% of the algal ration with manipulated yeast did not result in a significant decrease in growth rate relative to the algal-fed controls and the substitution of 80% of the algal diets resulted in growth rates reaching 90%’’. Experiments carried out in our laboratories have shown that the behaviour of R. decussatus resembles that of C. Õirginica described above, since growth rates for mixed diets of yeast and phytoplankton were considerably lower than those for both the phytoplankton control diets and the cornstarch and cornmeal diets described in the present study. In this study, the replacement of 50% phytoplankton by cornmeal leads to DGRs for DW that were comparable to those for purely phytoplankton diets and when 75% phytoplankton was replaced by cornmeal, DGRs for DW of 67% those of the control diet were attained. When the results are expressed in terms of LW, the DGRs for the M50 and M25 diets are 106 and 81%, respectively, of the DGRs for the control diet, which underlines the importance of determining the variation in OW in this kind of trial, since it provides a better indication of the quality of the diet than LW alone. The results of our study, together with those on the use of wheatgerm ŽFernandez´ Reirız ´ et al., 1998., are the only ones which consider the use of artificial diets for R. decussatus. According to these results, cornmeal gives growth rates that are comparable to those of the best quality artificial diets that have been examined to date, with the added advantage of ease of handling and lower cost, which are common to all cereal flours. If we consider that at present live phytoplankton is the usual food for broodstock and seed of molluscs in bivalve hatcheries and nurseries and its production accounts for 30% of total seed production cost in the former ŽCoutteau and Sorgeloos, 1992. and up to 85% of the cost of production in the latter ŽBolton, 1982., the use of cornmeal as a partial substitute for phytoplankton would lead to a considerable reduction of production costs in those hatcheries that produce R. decussatus. No clear definition of the nutritional requirements of bivalves is forthcoming from the various studies that are available. Enright et al. Ž1986. achieved good growth rates in juvenile Ostrea edulis with diets containing 15.5% protein and 59.4% carbohydrate, while Wisely and Reid Ž1978. and Nell and Wisely Ž1983, 1984., in their study of artificial diets for the fattening of Saccostrea commercialis, indicate that the optimal level of carbohydrate and protein in the diet were 74 and 18%, respectively, while lipid levels of around 10% total organic matter of the diet were sufficient to ensure optimal indices of condition. On the other hand, Kreeger and Langdon Ž1993. achieved optimal growth rates in juvenile Mytilus trossulus with diets that contain over 40% protein. A comparison between the M50 diets in experiments 1 and 2 of this study, which both contained similar amounts of lipid, showed that an increase in protein from 17 to 22% leads to a large increase in DGR. It may equally be true that the lower growth rate for the M25 diet in experiment 2 compared to that of the M50 diet in experiment 1 is related to the greater lipid content of the latter diet Ž18% as opposed to 9%., since both diets contained a similar amount of protein, while postlarvae and see in the first months after metamorphosis have a high lipid requirement ŽHolland and Hannant, 1974.. The results of experiment 2 show that a substitution of up to 50% phytoplankton for cornmeal allows growth rates similar to those of wholly phytoplankton diets to be achieved, suggesting that in the case of the seed of R. decussatus minimum protein and
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lipid requirements are of the order of 22% and 18%, respectively ŽTable 5.. The fact that there are only minimal differences between diets CM and CS, composed entirely of cereals, in spite of the higher protein content of the former, may indicate a lack of essential amino acids in these proteins, together with an almost total absence of lipid in both diets.
Acknowledgements We would like to thank C. Fernandez Pena, J. Dıaz, ´ ´ L. Nieto and B. Gonzalez ´ for their helpful technical assistance. This study was funded by CICYT-IEO-CSIC project I q D number AGFC-1003-CO2-01.
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