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Aquaculture 219 (2003) 645 653

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Diet development and evaluation for juvenile


abalone, Haliotis asinina: animal and plant
protein sources
Myrna N. Bautista-Teruel a,*, Armando C. Fermin a,
Shunsuke S. Koshio b,1
a

Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines
Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20,
Kagoshima 890-0056, Japan

Received 4 March 2002; received in revised form 6 August 2002; accepted 7 August 2002

Abstract
Growth studies were conducted to determine the suitability of animal and plant protein sources in
the diet of abalone, Haliotis asinina. Juvenile abalone with mean initial weight and shell length of
0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were fed practical diets for 84 days at a temperature
range of 28 31 jC. The practical diets contained 27% crude protein from various sources such as
fish meal (FM), shrimp meal (SM), defatted soybean meal (DSM), and Spirulina sp. (SP). A
formulated diet (diet 1) served as the control. The diets were fed to abalone at 2 5% body weight
once daily at 1600 h. Weight gain (WG), increase in shell length (SL), specific growth rate (SGR),
protein efficiency ratio (PER) and feed conversion ratio (FCR) were evaluated. Highest weight gain
(WG: 454%) was attained with abalone fed diet 2 with protein sources coming from a combination
of FM, SM, and DSM. This value was, however, not significantly different ( P < 0.05) from those fed
diets 4 and 1 (Control diet) with protein sources coming from FM, SM, SP and FM, DSM, SM,
respectively. Abalone fed diet 3, which used both plant protein sources, DSM and SP, showed
significantly lower WG (327%). Survival was generally high ranging from 85% to 100% for all
treatments. The SGR showed the same trend as the percent weight gain. The FCR and PER obtained,
however, were not significantly different for all treatments. The amino acid profile of diets 1, 2, and 4
simulated that of the abalone protein, which could have been a contributing factor to the higher
growth rate of abalone fed these diets. Diet 3, which contained only plant protein sources, showed

Corresponding author. Tel.: +63-33-3362965; fax: +63-33-3351008.


E-mail addresses: mbt@aqd.seafdec.org.ph (M.N. Bautista-Teruel), koshio@fish.kagoshima-u.aac.jp
(S.S. Koshio).
1
Tel.: +81-99-286-4182; fax: +81-99-286-4184.
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 4 1 0 - 6

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relatively lower methionine values compared with the abalone muscle tissue. Although abalone are
considered herbivorous animals, results of this study indicate that a combination of dietary plant and
animal protein sources was necessary to attain the best growth rate.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Protein; Haliotis asinina; Spirulina sp.; Abalone; Defatted soybean meal

1. Introduction
The decreasing commercial catch and the high market demand for abalone in both the
domestic and export markets have stimulated great interest in the development of its
aquaculture. As early as 1881, Japanese scientists led the initiation of biological studies
that became the basis for the successful controlled reproduction attempts for Haliotis
gigantea and Haliotis discus. In the United States, studies on controlled seed production
and culture were particularly focused on the red abalone Haliotis rufescens (Hahn, 1989).
The aquaculture of tropical and subtropical abalone species such as the Haliotis asinina
and the Haliotis diversicolor supertexta begun in Thailand and Taiwan (Jayarabhand and
Paphavasit, 1996). Interest on the other species like Haliotis varia and Haliotis ovina were
not as developed as the former two species mainly because of the smaller sizes of the latter
that keeps them less attractive to consumers or export buyers. H. asinina is the largest
among the tropical species (Singhagraiwan and Doi, 1993).
Artificial feeding for abalone culture has been practiced in countries like Japan, USA,
and Australia. Feeding experiments in Taiwan showed that growth of juveniles fed
artificial diets was 65% greater than those juveniles fed macroalgae. Further, animals
fed artificial diets had higher body weight per shell length and a relatively higher protein
content in their meat compared to animals fed seaweed.
Since abalone are generally characterized by slow and heterogenous growth rates,
proper nutrition must be provided to make a successful culture. Artificial feed for abalone
must contain sufficient protein and essential amino acids in order to satisfy their nutrient
requirements. Protein, which is essential for growth of abalone, is the most expensive
component in their diet (Fleming et al., 1996). It is necessary that the protein supplied in
the diet be palatable, digestible and possess an appropriate balance of amino acids, before
the inclusion levels are determined. Thus, protein sources should first be evaluated before
incorporation into practical diets.
The most commonly used protein sources in abalone feed diets are fish meal, defatted
soybean meal, and casein (Guzman and Viana, 1998). Fish meal is the only sole protein
source that can support good growth performance whereas soybean meal and casein needs
to be fed in combination with other protein sources in order to support good growth
(Fleming et al., 1996). A combination of abalone viscera silage and soybean meal as
protein sources in abalone, Haliotis fulgens diet was found to be effective in supporting
good growth (Guzman and Viana, 1998). Uki et al. (1985a,b), in their work with various
protein sources for abalone, found casein to be the most suitable protein. The reason for
better growth was attributed to protein quality due to differences in digestibility of each
protein source by juvenile abalone. However, because of its relatively high cost, casein is

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647

not practical for use in abalone feeds. Spirulina, likewise, was shown to have a very good
potential as a protein source for abalone Haliotis midae diets (Britz et al., 1994). Plant
protein sources such as field peas, faba beans, yellow lupins defatted soyflour and vetch
were tested and used by Vandepeer et al. (1999) for Haliotis laevigata. Their results
showed that all these legumes were well digested by various abalone species.
This study focused on the development and evaluation of practical diets for the culture
of juvenile abalone, H. asinina, with emphasis on determining the suitability of protein
sources, such as fish meal, shrimp meal, defatted soybean meal, and Spirulina sp. for
incorporation in formulated diets.

2. Materials and methods


2.1. Experimental animals
Abalone, H. asinina, juveniles with initial mean weight and shell length of
0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were used as the experimental animals.
They were harvested from natural spawnings at the mollusc hatchery of the SEAFDEC,
Aquaculture Department, Tigbauan, Iloilo, Philippines. The abalone were acclimated in
the laboratory for a week in a 500-l fiberglass tank filled with seawater and fed with
commercial pellets.
2.2. Diet preparation
Four practical diets were formulated to contain either FM, SM, DSM (diet 1); FM,
DSM (diet 2); DSM, SP (diet 3); FM, SM, SP (diet 4) at 27% crude protein. The source of
energy from carbohydrate came from wheat flour while that of lipid was from a 1:1
mixture of squid oil and soybean oil. Mineral and vitamin mixes were added to the diet.
Seaweed and wheat flour served as binders. Composition of experimental diets is shown in
Table 1. Diet 1, a slightly modified formulation used and tested to give good growth in a
previous experiment, served as the control. Procedures for diet preparation were patterned
after Bautista-Teruel and Millamena (1999).
2.3. Proximate and amino acid analysis
Proximate analysis of the different diets and abalone carcasses (Table 2) were
conducted using standard methods (AOAC, 1985). Moisture was determined using a
moisture balance, crude protein by semimicro Kjeldahl, crude fat by soxhlet extraction,
and crude fiber was obtained in a fat-free material sample by dilute acid and alkali
treatment. Ash content was determined in a muffle furnace at 550 jC and nitrogen-free
extract (NFE) was calculated by difference.
Amino acid analysis was done on the various diets and abalone muscle tissue using
an HPLC amino acid analyzer employing a Shimadzu Shim-pack ISC-071/S1504; 4.0
mm  15 cm column packing with a flow rate of 0.5 ml/min. The flow rate of the
reaction reagent was 0.3 ml/min; column temperature at 550 jC with wavelength of ex

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Table 1
Percentage (dry weight basis) composition of the formulated diets (g/100 g diet)
Ingredients (g/100 g diet)

Diet number
1

Fish meal
Shrimp meal
Defatted soybean meal
Spirulina
Rice bran
Wheat flour
Seaweed
Squid oil
Soybean oil
Vitamin mixa
Mineral mixa
Dicalcium phosphate
Butylatedhydroxy toluene

10.00
15.00
20.00

15.00
20.00
9.00
0.50
0.50
3.00
4.00
2.95
0.05

7.50
7.50
35.00

10.00
20.00
9.00
0.50
0.50
3.00
4.00
2.95
0.05

35.00
20.00
5.00
20.00
9.00
0.50
0.50
3.00
4.00
2.95
0.05

10.00
10.00

20.00
20.00
20.00
9.00
0.50
0.50
3.00
4.00
2.95
0.05

a
Vitamin and mineral mixes, commercial brand. h-carotene (3.0 MIU kg  1), cholecalciferol (0.6 MIU
kg  1), thiamin (3.60 g kg  1), riboflavin (7.20 g kg  1), pyridoxine (6.60 g kg  1), cyanocobalamine (0.02 g
kg  1), a-tocopherol (16.50 g kg  1), menadione (2.40 g kg  1), niacin (14.40 g kg  1), pantothenic acid (4.00 g
kg  1), biotin (0.02 g kg  1), folic acid (1.20 g kg  1), inositol (30.00 g kg  1), Stay C (100.00 g kg ). Mineral
mix: P (12.00%), Ca (12.00%), Mg (1.50%), Fe (0.15%), Zn (0.42%), Cu (0.21%), K (7.50%), Ge (0.0001%), Co
(0.011%), Mn (0.160%), Se (0.001%), Mo (0.0005%), Al (0.0025%), I (0.04%), B (0.0001%), Ni (0.0001%).

348 nm and em 450 nm. Reaction reagents used were o-pthalaldehyde/sodium carbonate
buffer and sodium hypochlorite. The HPLC system consisted of the following: pump
LC-3A; step gradient unit, SGR-1A; oven CTO-2A; fluorescence detector, FLD; and
chromatopac, C-RIB. The following buffers were used: 0.07 M sodium citrate (pH
3.19); 0.07 M sodium citrate (pH 3.80); 0.20 M sodium citrate (pH 8.48); and 0.50 M
sodium hydroxide.
2.4. Feeding experiment
Twenty abalone juveniles were stocked in 12 60-l fiberglass tanks containing 40-l of
filtered seawater. Shelters of two halved polyvinyl chloride pipes (65 mm diameter) were
Table 2
Proximate composition (%) of diets
Diet number

Crude protein
Crude fat
NFEa
Estimated metabolizable energy(kcal/kg)b
a

27.84
3.13
43.55
3137

27.30
3.27
42.30
3078

27.72
2.81
43.68
3109

26.97
3.04
42.62
3057

NFEnitrogen free extract, given by difference.


Computed based on standard physiological fuel values of 9 kcal/g lipid and 4 kcal/g protein and
carbohydrate (Brett and Groves, 1979).
b

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649

provided in each tank. Four dietary treatments with three replications were randomly
allocated to different tanks. A flow-through water system was maintained at approximately
300 350 l/h with gentle aeration provided. Excess feed that settled on the tank bottom was
siphoned out, dried and weighed to get the true value of the feed consumed by the abalone.
Water temperatures ranged from 28 to 31 jC and salinity varied from 28xto 32x
.
Ranges of dissolved oxygen, nitrite, ammonia, and pH in the rearing tanks were at 5.0 5.6
mg/l; 0 0.05 mg/l; 0 0.40 and 8.3 8.4, respectively.
Diets were fed to abalone at 2 5% of their body weight once in the late afternoon at
about 1600 h for 90 days. Shell length increase (SL), weight gain (WG), survival (SURV),
and feed conversion ratio (FCR) were determined every 20 days until the 84th day. Water
stability of the diets was conducted according to the method of Hastings et al. (1971). Diet
stability was determined at 6, 12, and 24 h interval. Percent water stability was computed
as:
%water stability B  A%dry matter=A%dry matter  100
where B is the final weight of feed and A is the initial weight of feed.
2.5. Statistical analysis
All data were analyzed by one-way analysis of variance (ANOVA) and Duncans
Multiple Range Test on the SAS Package for the IBM-PC (SAS Institute, 1988).
Differences among treatment means were considered significant at P < 0.05.

3. Results
At the end of the 84-day feeding trial, the highest weight gain (453.8%) was attained
with abalone fed diet 2 with protein sources coming from a combination of FM, SM, and
DSM. This value was, however, not significantly different from those fed diets 4 (420.6%)
with protein sources from FM, DSM, SP and the control diet (400%) containing protein
sources from FM, SM, and DSM. Abalone fed diet 3, which contained DSM and SP, had
significantly lower weight gain of 326.5%. Similarly, the specific growth rates and shell
Table 3
Initial and final weight, shell length, and SGR of abalone fed formulated diets for 84 daysa
Treatment

Initial weight
(average, g)

Initial SL
(average, mm)

Final weight
(average, g)

Final SL
(average, mm)

Weight
gain (%)b

SGRc

1
2
3
4

0.73 F 0.08
0.65 F 0.03
0.68 F 0.03
0.68 F 0.01

11.2 F 0.27
11.5 F 0.48
11.5 F 0.28
11.4 F 0.37

3.65 F 0.19
3.60 F 0.10
2.90 F 0.28
3.54 F 0.17

35.4 F 0.31
34.5 F 0.58
33.2 F 0.91
35.4 F 0.86

400.0 F 49.8a
453.8 F 42.8a
326.5 F 392b
420.6 F 20.6a

0.83 F 0.09a
0.89 F 0.02a
0.75 F 0.07b
0.86 F 0.03a

a
Means of three replicate groups with the same superscript in each column are not significantly different
( P < 0.05).
b
Weight gain: [(final weight  initial weight)/initial weight]  100.
c
SGR (specific growth rate) = 100 (ln average final weight  ln average initial weight)/number of days.

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Table 4
Shell length increase, total feed intake, FCR, percent survival of abalone fed formulated diets for 84 days and
percent water stability of pellets
Treatment

Shell length
increase (%)

Total feed
intake (g)

FCRa

PERb

SURV (%)

Percent water stability


of pellets (24 h)c

1
2
3
4

216 F 2.45a
200 F 3.52a
188 F 2.12b
210 F 2.38a

365.36
391.68
285.06
329.16

1.01
1.00
0.97
0.86

3.90
4.17
4.14
4.58

95
95
85
85

64
63
65
65

Means of replicate groups F S.E.M. with the same superscript in each column are not significantly different
( P < 0.05).
a
FCR = dry weight of feed (g)/wet weight gain (g).
b
PER = wet weight gain/protein intake.
c
%Water stability=(final dry weight of feed/initial dry weight  %dry matter)/100.

length increase of abalone fed diets 1, 2, and 4 were significantly higher compared to those
fed diet 3 (Table 3). No significant differences were observed in the efficiency of protein
(PER) and feed conversion (FCR) for all treatments (Table 4). Survival was generally high,
ranging from 85% to 95%. All diets prepared were found to be water stable retaining at
64% of dry mass after the 24 h test for stability (Table 4). Amino acid composition of diets
1, 2, and 4 had profiles that were very similar to that of abalone tissue. Diet 3, which
contained all plant protein sources, showed some lower level of methionine (Table 5) as
compared to other diets.

Table 5
Amino acid composition of formulated diets and abalone meat (g/100 g protein)a
Amino acid

Alanine
Arginine
Aspartic
Cystine
Glutamic
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
a

Diet number

Abalone tissue

2.68
5.28
7.05
0.35
8.99
3.65
1.35
2.05
5.09
6.14
4.40
2.42
2.27
1.74
3.60
0.98
2.34
4.26

2.79
5.12
6.11
0.59
8.10
2.70
1.25
1.97
5.60
5.99
4.32
2.72
2.12
1.10
3.59
0.65
2.29
4.28

2.38
5.09
6.07
0.36
8.72
2.99
1.24
1.86
5.17
5.78
2.97
2.18
2.55
1.21
2.79
0.43
1.52
3.41

2.40
5.22
7.08
0.11
7.98
3.15
1.38
2.35
5.42
5.22
4.42
2.35
2.70
1.42
3.38
1.01
2.05
4.48

Means of two replicate samples.

3.72
5.48
6.23
0.42
9.56
3.71
1.78
2.51
5.95
5.90
4.09
3.29
3.41
2.30
3.69
0.55
2.97
4.90

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651

4. Discussion
Abalone fed formulated diets with protein sources from a combination of animal and
plant protein origin such as fish meal, shrimp meal, defatted soybean meal, and
Spirulina sp. showed better performance in terms of growth rate per day, weight gain,
and increase in shell length compared to those fed diets with protein sources of plant
origin alone. Since all diets were made isonitrogenous at 27%, the results would signify
that the quality and physiological availability of protein in the combined animal and
protein sources may have contributed significantly to the positive effect of these diets on
the growth of abalone. The amino acid profile of the various diets tested (Table 5)
showed some low levels of certain amino acids such as methionine in the formulated
diet, which contained only the plant protein sources. The low level of this essential
amino acid (methionine) in diet 3 with protein sources coming solely from plant origin
might have contributed to the lower growth rate of abalone fed this diet. The use of
plant protein source alone in a formulated diet may need some supplementation of
crystalline amino acids or may be used in combination with other animal protein sources
to compensate for the low levels of some essential amino acids such as methionine.
Murai et al. (1986, 1989) reported that supplementing soybean meal diets with either
coated and uncoated methionine significantly improved growth and feed efficiency of
common carp fingerlings. Guzman and Viana (1998) have successfully demonstrated the
effectivity of the use of a combination of soybean meal and abalone viscera silage as
protein sources in the diet of abalone, H. fulgens. In the study conducted by Britz
(1996), abalone, H. midae fed fish meal and Spirulina spp. based diets produced a
significantly higher length increment and specific growth rate compared to those fed
diets containing soya oil cake, torula yeast, and casein alone. Faster growth rate of
abalone with formulated diets containing combination of plant and animal protein
sources has also been documented in studies for other Haliotis species (Hahn, 1989;
Morrison and Whitington, 1991; Nie, 1992).
Another concern in the use of plant protein source is the presence of antinutritional
factors, which if not removed or processed may likewise contribute to the low growth of
fish. In this study, however, plant protein sources used were properly processed and were
assumed not to contain any of these antinutritional factors.
The higher growth rate obtained when abalone were fed diets containing animal protein
sources in combination with plant protein sources may be attributed to the fact that some
limiting amino acids in plant protein source may be compensated by the availability of that
amino acid in the animal protein source, resulting in a better profile of the diet formulation
and better growth for the abalone. The amino acid profile of a diet is at its best when it
simulates that of the tissue muscle of the animal being fed. The profile of animal protein
source like fish meal is considered to be an excellent one, which can support good growth
performance used alone or in combination with other plant protein sources. Generally,
animal protein sources like fish meal are highly digestible and contain attractants that
make them an attractive ingredient for any fish diet. However, the high demand for fish
meal may soon result in an unaffordable price and availability of good quality sources may
also be a problem. It is best therefore that fish meal or any animal protein source be used
in combination with plant protein source to compensate for the limiting amino acids of

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M.N. Bautista-Teruel et al. / Aquaculture 219 (2003) 645653

each other, thus may result in faster growth rate of the animal. Plant protein sources are
cheaper but are generally deficient in some of the sulfur-containing amino acid such as
methionine. Thus, these plant protein sources are best used in combination with animal
protein sources.
The values obtained for the feed conversion ratio for all dietary treatments were
generally low ranging between 0.9 and 1.0 and were not significantly different ( P > 0.05).
This would indicate that there was high efficiency of feed conversion to body weight in all
treatments. The more efficient feed conversion but lower growth rate of abalone fed the all
plant protein-based diet compared to those fed the fish meal based diet or the animal
plant combination diet can be explained perhaps by the lower feed consumption of
abalone fed diet 3. However, this finding does not conform with the results of Uki et al.
(1986) wherein they reported poorer growth and high FCR values for H. discus hannai fed
fish meal based diets compared with casein-based diets. The source and processing of fish
meals used in these two different studies probably may account for the difference in
results. The ability of H. discus hannai and H. asinina to digest fish meal cannot be
discounted. Viana et al. (1993), on the other hand, reported that fish meal fed abalone had
similar growth rates and FCR values with those abalone fed casein as protein source. This
is likewise in conformity with the work of Britz (1996), wherein abalone, H. midae fed
fish meal and Spirulina-based diet exhibited good growth and high efficiency of feed
conversion to body weight.
The efficiency of protein utilization as evidenced by the PER values (4.0 4.6) was not
significantly different for all treatments. This would indicate that the proteins from each
diet were efficiently utilized by abalone regardless of the protein source.
The lower growth rates obtained with abalone fed diets with plant origin as the sole
protein sources might be due to lower levels of some essential nutrients that could not have
been sufficient for deposition of tissue muscle. Although the essential amino acid content
was used as a determining factor for the nutritive value of the different protein sources,
further work has to be done to test the availability of these amino acids to abalone. It has
been reported that apparent amino acid availability and true amino acid availability vary
within and among the various protein sources (Wilson, 1989).

Acknowledgements
The authors would like to thank Ms. Mae F. Mallare and Mr. Narciso Entusiasmo for
their assistance in the conduct of the study, Ms. Florence Jarder for the proximate analyses
and Dr. O.M. Millamena for the review of the manuscript.

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