Aquaculture Research, 2001, 32 (Suppl.
1), 249±254
Reproductive performance of hatchery-bred
donkey's ear abalone, Haliotis asinina, Linne, fed
natural and arti®cial diets
M N Bautista-Teruel, O M Millamena & A C Fermin
Aquaculture Department, South-east Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines
Correspondence: Myrna N Bautista-Teruel, Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo,
Philippines, 5021. Tel. +63-33 336-2965. Fax: +63-33 335-1008. E-mail: mbt@aqd.seafdec.org.ph
Abstract
Hatchery-bred donkey's ear abalone, Haliotis
asinina, Linne broodstock were given diets consist-
ing of natural food, seaweed (SW), Gracilariopsis
bailinae, D1; combination of SW and arti®cial diet
(AD), D2; and AD alone, D3. Equal numbers of
1 : 1 female and male abalone were stocked in 24
units, 60 L tanks with eight replicate tanks per
dietary treatment. Reproductive performance, e.g.
number of spawnings, instantaneous fecundity
and egg hatching rates, was monitored over
270 days. The mean number of spawnings was
not signi®cantly different among treatments. The
mean instantaneous fecundity and percent hatch-
ing rates were signi®cantly higher in abalone fed
D2 or D3 compared to those given D1. Survival of
abalone broodstock fed D1 was, however, sig-
ni®cantly higher at 88% than those fed either D2
or D3 at 75%. Fatty acid analysis showed that
the n-3/n-6 fatty acid ratios of abalone hepato-
pancreas re¯ected those of their diets. Mature
abalone ovary had n-3/n-6 fatty acid ratio of
1.3. A higher amount of essential nutrients in
the arti®cial diet such as protein, lipid and the
highly unsaturated fatty acids, e.g. 20 : 4n-6,
20 : 5n-3, 22 : 6n-3 in abalone fed D2 or D3,
may have in¯uenced the increased reproductive
performance.
Keywords: broodstock, nutrition, abalone, Haliotis
asinina
ã 2001 Blackwell Science Ltd
Introduction
The decreasing commercial catch, and the high
price that abalone command in both the domestic
and export markets, have stimulated great inter-
est in the development of its aquaculture (FAO
1998). A major constraint to development of
abalone aquaculture is the declining supply of
seed from the wild due to overexploitation and
habitat destruction (Jarayabhand & Paphavasit
1996). Although spontaneous spawning of aba-
lone is reported to occur year-round
(Singhagraiwan & Doi 1992; Singhagraiwan &
Doi 1993; Capinpin, Encena & Bayona 1998),
production of good quality larvae is very incon-
sistent. Thus, there is a need to develop a reliable
technique for abalone broodstock development
through dietary manipulation.
Nutrition of broodstock has a profound effect
on gonadal maturation and fecundity (Watanabe
1988), and plays a major role in reproductive
success in both ®sh and crustaceans (Yu,
Sinnhuber, & Hendricks 1979; Takeuchi, Ishii,
& Ogino 1981; Teshima & Kanazawa 1983;
Watanabe, Itoh, Murakami, Tukashima,
Kitajima, & Fujita 1984). There are no published
studies on the in¯uence of nutrition on the
reproductive performance of abalone broodstock,
despite their importance in abalone aquaculture.
Thus, this study was undertaken to investigate
the effects of natural and arti®cial diets on
reproduction of the donkey's ear abalone,
Haliotis asinina, Linne.
249
Reproductive performance of abalone M N Bautista-Teruel et al. Aquaculture Research, 2001, 32 (Suppl. 1), 249±254
Table 1 Percentage composition of arti®cial abalone
broodstock diet (g kg±1diet)
Ingredients Amount (g kg±1 diet)
Fish meal 100
Shrimp meal 100
Defatted soybean meal 200
Rice bran 119.5
Wheat ¯our 200
Seaweed (Gracilaria sp.) (powdered) 170
Tuna oil 5 1
Soybean oil 5 2
Vitamin mix* 30
Mineral mix** 40
Dicalcium phosphate 30
Butylatedhydroxy toluene 0.5
*Vitamin and mineral mixes, commercial brand. Vitamin
A (3.0 M.I.U kg±1), Vitamin D3 (0.6 M.I.U kg±1), Vitamin
B1 (3.60 g kg±1), Vitamin B2 (7.20 g kg±1), Vitamin B6
(6.60 g kg±1), Vitamin B12 (0.02 g kg±1), Vitamin E
(16.50 g kg±1), Vitamin K3 (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), Stable C (100.00 g kg±1).
**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%).
Materials and Methods
Experimental diets
Dietary treatments consisted of a natural diet,
seaweed (Gracilariopsis bailinae) (D1), combination
of natural and arti®cial diets (D2), and arti®cial diet
(D3). Seaweed was given every 4 days, ad libitum.
The combination diet was given alternately each
day. Arti®cial diet was a modi®cation of a previous
formulation of Bautista-Teruel & Millamena (1999)
in that the amount of some ingredients, e.g. shrimp
meal, tuna and soybean oils, was reduced while
adding more of the others, e.g. rice bran, seaweed.
The composition and proximate composition of the
natural and arti®cial diets are shown in Tables 1
and 2, respectively. Treatments were arranged in a
completely randomized experimental design.
Abalone broodstock fed diets D2 and D3 were
initially fed D1 and gradually switched to the
arti®cial diet by the second week of culture.
Formulated diet was given at 3±5% of broodstock
250 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl. 1), 249±254
Table 2 Proximate composition (%) of natural and
arti®cial diet for broodstock abalone, Haliotis asinina,
Linne
Natural diet1 Arti®cial diet
Crude protein 17.56 25.40
Crude fat 0.48 4.38
Nitrogen-free extract 35.38 47.59
Estimated energy (kcal kg±1)2 2161 3315
Seaweed, Gracilariopsis bailinae.
Computed based on standard physiological fuel values of
9 kcal g±1 lipid and 4 kcal g±1 protein and carbohydrates
(Brett & Groves 1979).
biomass once daily at 16.00±17.00 hours. Excess
diet was removed and the feeding rate was adjusted
based on weight gain after each sampling, which
was done every 2 weeks. Mortalities were recorded
daily.
Abalone culture and rearing system
Experiments were conducted at the wet laboratory
of the SEAFDEC Aquaculture Department in Iloilo,
the Philippines. Abalone, Haliotis asinina, adults
with initial mean body weight of 24.5 6 0.12 g
and shell length of 48.5 6 0.68 mm were obtained
from the mollusc hatchery at SEAFDEC, Tigbauan,
Iloilo. Gonadal maturity of the female abalone was
judged according to the criteria developed by
Singhagraiwan & Doi (1993). Abalone having
immature gonads were used in the study.
Forty-eight male and female abalone were
stocked at a 1 : 1 ratio into 24 units, 60-L ®breglass
tanks with eight replicate tanks per dietary treat-
ment. Halved PVC pipes were provided in each tank
as shelter for abalone. Sand-®ltered seawater was
supplied in a ¯ow-through system equipped with
adequate aeration. Tanks were cleaned of faeces and
excess diet whenever necessary.
Abalone broodstock were checked for spawning
each day in the early morning. Eggs were siphoned
and counted in 40-L ®breglass tanks located just
under the spawning tanks. Instantaneous fecundity
was computed as the total number of spawned eggs
per g body weight of abalone. Percent hatching rate
was computed as the total number of larvae divided
by the total number of spawned eggs multiplied by
100.
Aquaculture Research, 2001, 32 (Suppl. 1), 249±254 Reproductive performance of abalone M N Bautista-Teruel et al.

Table 3 Reproductive performance

of abalone, Haliotis asinina, Linne, Treatment1
broodstock fed natural diet alone,
combination of natural and arti®cial Parameter D1 D2 D3
diets, and arti®cial diet alone
Mean number of spawnings 8.0 6 0.6a 9.0 6 1.8a 9.5 6 1.2a
a
Mean instantaneous fecundity 4077 6 67.8 5478 6 45.8b 5050 6 74.1b
Mean hatching rate (%) 39.49 6 8.2a 46.22 6 7.3b 45.65 6 11.2b
Broodstock survival (%) 88 6 7.6a 75 6 12.5b 75 6 12.5b
Mean frequency of spawning/month 1±2 1±2 1±2
Mean spawning time interval (days) 19 6 1.8 17 6 1.3 16 6 0.7

1
Treatment means with different superscripts are signi®cantly different
(P < 0.005).
D1 = Seaweed, Gracilariopsis bailinae (natural diet) (given alone).
D2 = Seaweed (natural diet) + arti®cial diet given alternately.
D3 = arti®cial diet (given alone)

Table 4 Comparison of fatty acid composition methods (AOAC 1995). Fatty acid composition of
(percentage of total lipid) of natural and arti®cial diets for the diets and abalone muscle tissues were deter-
abalone, Haliotis asinina, Linne, with mature ovary of the mined by gas-liquid chromatography (GLC). Total
wild abalone lipids were extracted using the method of Bligh &
Dyer (1959). Methyl esters of constituent fatty acids
Fatty acid Natural diet Arti®cial diet Mature ovary1 were prepared by the saponi®cation-transesteri®ca-
tion method of Metcalfe, Schmitz, & Pelka (1966).
14 : 0 6.6 16.5 12.9
Fatty acid composition of replicate samples was
15 : 0 2.3 1.5 3.8
16 : 0 29.2 20.1 22.9
analysed using a Shimadzu GC 9A (Shimadzu Co.,
16 : 1n-7 1.4 2.3 1.3 Tokyo, Japan) gas chromatograph with a ¯ame
18 : 0 1.5 1.8 2.4 ionization detector (FID) ®tted with 15% diethylene
18 : 1n-7 15.5 22.8 20.5 glycol succinate column on chromosorb support.
18 : 2n-6 10.9 13.4 10.8
The gas chromatograph was operated isothermally
18 : 3n-3 16.8 7.5 5.9
18 : 4n-3 7.0 1.2 1.5 at 190 °C with a 30 mL min±1 ¯ow of nitrogen. Fatty
20 : 4n-6 2.1 2.4 3.1 acids were identi®ed using authentic standards and
20 : 5n-3 1.8 3.9 5.1 published values for fatty acid marine oils (Ackman
20 : 5n-6 0.9 0.8 1.2
& Burgher 1965) and quanti®ed with an electronic
22 : 5n-3 3.2 0.8 2.3
22 : 6n-3 0.7 3.8 5.2
integrator (Shimadzu Chromatopac CR-2AX).
Total n-3 28.5 17.2 20.0 Results are presented as the weight percentage of
Total n-6 14.4 16.5 15.1 total fatty acids.
n-3/n-6 2.0 1.0 1.3

1
Mature ovary from wild abalone
Statistical analysis

Data were analysed using analysis of variance

Water temperature (26±30 °C) and salinity (32±
35 g L±1) were monitored daily. Ammonia nitrogen
(0±0.08 mg L±1), nitrite nitrogen (0.0±0.17 mg L±1)
and dissolved oxygen (4.6±7.1 mg L±1) were
monitored weekly. Water quality remained within
ranges suitable for abalone culture in tanks.
(Gomez & Gomez 1984) and Duncan's multiple
range test (P < 0.05) to test signi®cant differences
among treatment means (Duncan 1955). Arcsin
transformation of percentage data was done prior to
statistical analysis. Replicate tanks were considered
units of observation for ANOVA testing.

Chemical analyses Results

Natural and formulated diets were analysed for The effects of the different diets on reproductive
proximate composition according to standard performance of broodstock abalone are shown in

ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl. 1), 249±254 251
Reproductive performance of abalone M N Bautista-Teruel et al. Aquaculture Research, 2001, 32 (Suppl. 1), 249±254

Table 5 Fatty acid composition (percentage of total lipid) (HUFA), e.g. 20 : 4n-6, 20 : 5n-3, 22 : 6n-3, were
of hepatopancreas of abalone, Haliotis asinine, Linne, fed present in relatively higher amount in the arti®cial
natural diet alone, combination of natural and arti®cial diets as compared with those found in the natural
diets and arti®cial diet alone diet.

Hepatopancreas
Discussion
Fatty acids D1 D2 D3
The higher reproductive performance in terms of
14 : 0 9.3 12.2 14.0
total number of spawnings, instantaneous fecundity
15 : 0 4.1 7.2 6.3 and hatching rates of abalone Haliotis asinina fed the
16 : 0 21.2 22.1 23.2 combination of natural and arti®cial diets or the
16 : 1n-7 5.8 1.9 2.2 arti®cial diet alone would suggest that the nutri-
18 : 0 5.7 6.4 9.2
tional quality of the broodstock diet in¯uences
18 : 1n-7 17.9 19.2 18.6
18 : 2n-6 9.4 10.5 8.9
reproduction. Although it is possible to mature
18 : 3n-3 13.5 8.3 5.2 and spawn abalone with natural diet, the provision
18 : 4n-3 5.3 0.9 1.2 of an effective arti®cial diet fed alone or in
20 : 4n-6 2.1 3.2 2.4 combination with the natural diet can achieve
20 : 5n-3 1.8 2.8 3.7
better reproductive performance of the abalone
20 : 5n-6 0.8 0.6 0.7
22 : 5n-3 2.8 1.2 0.9
broodstock. This may result in the enhancement
22 : 6n-3 0.7 2.5 3.1 of production of quality seeds for abalone hatch-
Total n-3 24.1 15.7 14.1 eries. Dietary nutrients, especially in terms of
Total n-6 12.3 14.3 12.0 proteins, lipids, and fatty acids, e.g. 20 : 4n-6;
n-3/n-6 2.0 1.1 1.2
20 : 5n-3, 22 : 6n-3, which are essential in repro-
duction and that are insuf®cient in the natural diet,
D1 = Seaweed, Gracilariopsis bailinae (natural diet) (given may have been compensated by feeding the arti®cial
alone).
diet as a supplement or as total food for the abalone.
D2 = Seaweed (natural diet) + arti®cial diet given alter-
Proteins that are associated with essential fatty
nately.
D3 = arti®cial diet (given alone)
acids and are usually mobilized to the gonads are
considered important components for reproductive
functions (Harrison 1991). Highly unsaturated fatty
acids (HUFAs) are important nutrients for repro-
Table 3. Mean number of spawnings ranged from ductive functions of ®sh and crustaceans
8.0 6 0.6 to 9.5 6 1.2 and were not different (Millamena, Primavera, Pudadera & Caballero
among treatments (P > 0.05). Spawning occurred 1986; Millamena 1989; Millamena & Quinitio
once or twice every 4 weeks. The time interval 2000). Watanabe, Takeuchi, Ogino & Kawabata
between spawnings varied from 15 to 35 days. The (1977) have also demonstrated that essential fatty
mean instantaneous fecundity (5478 6 45.8, D2; acids play as important a role in reproductive
5050 6 74.1, D3) and percent hatching rates physiology in ®sh as in higher animals. The results
(46.22 6 7.3, D2; 45.65 6 11.2,D3) were higher obtained in red sea bream fed essential fatty acid-
(P < 0.05) in abalone fed D2 or D3 compared to de®cient diets for 6 months before spawning indi-
those fed D1 (mean instantaneous fecund- cated that the total egg production and hatchability
ity = 4077 6 67.8; percent hatching were signi®cantly in¯uenced by the level of these
rate = 39.49 6 8.2). Survival of abalone brood- essential fatty acids in the diet (Watanabe et al.
stock fed D1 was however, signi®cantly higher 1984).
(88%) than those fed either D2 or D3 (75%). The n-3/n-6 fatty acid ratios of the hepatopan-
Table 4 shows the fatty acid pro®les of the creas re¯ected those of their diets at 2.0 (D1), 1.1
natural and arti®cial diets. The natural diet had (D2) and 1.2 (D3) (Table 5). Cahu, Fauvel, &
higher n-3/n-6 ratio (2.0) compared to arti®cial diet Aquacop (1986) and Millamena (1989) reported a
(1.0). Mature abalone ovary from the wild abalone correlation between dietary fatty acid composition,
had n-3/n-6 ratio of 1.3, whereas those of the tissue fatty acid pattern and quality of eggs in
hepatopancreas were more of a re¯ection of their shrimp, Penaeus monodon. Dietary HUFAs and n-3/
diets. (Table 5). Highly unsaturated fatty acids n-6 ratio that simulate that of mature ovaries have

252 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl. 1), 249±254
Aquaculture Research, 2001, 32 (Suppl. 1), 249±254 Reproductive performance of abalone M N Bautista-Teruel et al.
been reported to improve reproductive performance
and hatchability of eggs in the tiger shrimp
(Millamena 1989). This could partly explain why
better reproductive performance was obtained with
the combination of natural and arti®cial diet or
arti®cial diet given solely. The type of macroalgae
consumed can offer different proportions of nutri-
tionally important PUFA (Dunstan, Baillie, Barrett,
& Volkman 1996; Mai, Mercer, & Donlon 1996). In
this experiment, the amount of PUFA provided by
the natural diet, which is the seaweed Gracilariopsis
bailinae, may not have been as suf®cient as the one
provided in the arti®cial diet. The essential nutrients
such as proteins, lipids, and fatty acids, which are
limiting in the natural diet may have been
compensated by the amounts present in the arti®cial
diet, thus better reproductive performance resulted
in abalone fed D2 or D3. The lower survival of the
abalone fed either D2 or D3 may indicate that more
spawnings that were undergone by the abalone fed
these diets might have contributed to more stressful
activity, thus leading to higher mortalities. Feeding
abalone broodstock with suitable arti®cial diet may
enhance their reproductive performance.
Acknowledgements
The authors wish to acknowledge the assistance of
Ms M. Mallare and Mr N. Entusiasmo in the conduct
of the study; Ms F. Jarder for the proximate analysis;
Ms A. Asutilla for the water analysis; Ms I.
Borlongan for the use of the gas chromatograph;
and Mr R. Sanares for the statistical analysis of the
data. The Organizing Committee of The Ninth
International Symposium on Nutrition and
Feeding in Fish through the Chairman, Dr T.
Watanabe, and South-east Asian Fisheries
Development Center through the Chief, Dr R.
Platon, are likewise gratefully acknowledged for
funding support.
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