Aquaculture research, tập 41, số 1, 2010

Aquaculture Research, 2010, 42, 1^8

doi:10.1111/j.1365-2109.2010.02572.x

REVIEW ARTICLE
Effects of stocking density and algal concentration on
the survival, growth and metamorphosis of Bobu Ivory
shell, Babylonia formosae habei (Neogastropoda:
Buccinidae) larvae
Huaiping Zheng1,2, Caihuan Ke1, Zewen Sun2, Shiqiang Zhou1 & Fuxue Li1
1

State Key Laboratory of Marine Environmental Science, Department of Oceanography, Xiamen University, Xiamen, China

2

Mariculture Research Center for Subtropical Shell¢sh & Algae, Shantou University, Shantou, China

Correspondence: C Ke, State Key Laboratory of Marine Environmental Science, Department of Oceanography, Xiamen University,
Xiamen 361005, China. E-mail:

Abstract
Independent and combined e¡ects of stocking density
and algal concentration on the survival, growth and
metamorphosis of the Bobu Ivory shell Babylonia formosae habei larvae were assessed using a 5 Â 5 factorial design with densities of 0.25, 0.5, 0.75, 1.00 and
1.50 larvae mL À 1 and algal concentrations of 5, 10,
15, 20 and 25 Â 104 cells mL À 1 in the laboratory.
Larval growth, survival and metamorphosis were
signi¢cantly a¡ected by both the independent e¡ects
of stocking density and algal concentration and by

their interaction. The highest per cent survival
(72.5%) and metamorphosis (49.5%), fastest growth
(41.57 mm day À 1) and shortest time to initial metamorphosis (10 days) all occurred at the lowest stocking density and the highest algal concentration. Both
crowding and food limitation had independently negative impacts on the survival, growth and metamorphosis of larvae, and these negative impacts were
further strengthened by the interaction of a higher
stocking density and a lower algal concentration.
Moreover, the results suggest that stocking density
and algal concentration obviously played di¡erent
roles in determining larval survival and growth. To
maximize survival and growth, B. formosae habei larvae should be reared at a lower stoking density of
0.25 larvae mL À 1 and fed a higher algal concentration of 25 Â 104 cells mL À 1 in large-scale hatchery
seed culture.

r 2010 The Authors
Aquaculture Research r 2010 Blackwell Publishing Ltd

Keywords: Babylonia formosae habei larvae, stocking density, Algal concentration, Survival, Growth,
Metamorphosis

Introduction
Temperature, salinity, diet and rearing density are
exogenous factors a¡ecting larval growth, settlement and metamorphosis (for a review, see Crisp
1974). Stocking density and algal concentration are
two more important factors in£uencing the success
of hatchery seed culture for planktotrophic larvae in
marine molluscs, because they are easier to manipulate than other environmental factors in arti¢cial
larval production systems. Therefore, the e¡ects of
stocking density or food concentration on larval survival, growth and metamorphosis have been well
documented in marine molluscs (Fretter & Montgomery 1968; Pilkington & Fretter 1970; Perron & Turner
1977; Aldana-Aranda, Lucas, Brule, Salguero &

Rendon 1989; Pechenik, Eyster, Widdows & Bayne
1990; Hansen 1991; His & Seaman 1992; Pechenik,
Estrella & Hammer 1996; Avila, Grenier,Tamse & Kuzirian 1997; Preece, Shepherd, Clarke & Keesing 1997;
Doroudi & Southgate 2000; Pechenik, Jarrett &
Rooney 2002; Powell, Bochenek, John, Klinck &
Hofmann 2002; Daume, Huchette, Ryan & Day 2003;
Zhao, Qiu & Qian 2003; Zheng, Ke, Zhou & Li 2005;

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E¡ects of density and concentration on larvae H Zheng et al.

Aquaculture Research, 2010, 42, 1^8

Liu, Dong, Tang, Zhang & Xiang 2006; Yan, Zhang &
Yang 2006; Mazo¤n-SuaŁstegui, Ru|¤ z-Ru|¤ z, ParresHaro & Saucedo 2008; Raghavan & Gopinathan
2008; Capo, Bardales, Gillette, Lara, Schmale & Serafy 2009; Rico-Villa & Robert 2009). In general, larvae,
reared under a lower stocking density or higher food
concentration conditions, have higher survival, faster growth and more metamorphosed individuals. In
contrast, larvae, reared crowing or lower food supply
conditions show slower growth and development,
less survival and metamorphic success, and a smaller size or a lower energy content at metamorphosis.
Most of these studies focused on the independent effect of stocking density or food concentration on larval survival, growth and metamorphosis; only a few
studies involved their combined e¡ects (Doroudi &
Southgate 2000; Powell et al. 2002; Mazo¤n-SuaŁstegui
et al. 2008; Capo et al. 2009). In large-scale hatchery
seed culture practice, it is important to de¢ne a realistic strategy for maximizing larval growth and survival under optimal stocking density and diet
concentration conditions.
Members of the gastropod genus Babylonia are

found only in the Indo-Paci¢c region (Altena, Regteren & Gittenberger1981). Bobu Ivory shell B. formosae
habei only exists on the SE coast of China and generally inhabits the muddy/sandy subtidal zone at
depths of 4^20 m (in summer) or 40^60 m (in winter) (Liu & Xiao 1998). The conch is a large marine
gastropod (adult size 50^60 mm), whose meat is an
important source of protein and is of considerable
dietary and economic signi¢cance to the inhabitants
of the coast of southern China. In recent years, the
Bobu Ivory shell has been widely cultured in the
coast of southern China. Larvae are generally reared
at a density of 0.5 larvae mL À 1 and fed an algal concentration of 10^20 Â 104 cells mL À 1 (Ke, Zheng,
Zhu, Zhou & Li 2001; Zheng, Zhu, Ke, Zhou & Li
2001). However, the combined e¡ects of stocking density and algal concentration on larvae have been not
studied. Determination of their combined e¡ects may
be an important step in developing more e⁄cient
large-scale hatchery culture techniques for B. formosae habei larvae culture.
Because the factorial design provides a greater
precision for assessing the interactions between
di¡erent factors and allows the interpolation of interactions at intermediate levels of the factors being
tested (Doroudi & Southgate 2000), a 5 Â 5 factorial
design with densities of 0.25, 0.50, 0.75, 1.00 and
1.50 larvae mL À 1 and algal concentrations of 5, 10,
15, 20 and 25 Â 104 cells mL À 1 was applied in the

laboratory. And the independent and combined
e¡ects of larval density and algal concentration
on the survival, growth and metamorphosis of
B. formosae habei larvae were investigated in the
present study.

2

Materials and methods
Acquisition of larvae
Adults of B. formosae habei were collected from the
coast of Changle, Fujian Province (China), and maintained between 23 and 28 1C in two1m3 aquaria and
fed with the razor clam Sinonovacula constricta (L.).
Egg capsules containing fertilized eggs were deposited after several days, after which the parents were
removed. The resulting embryos were maintained
for development by changing the seawater daily
and by continuous aeration. Most larvae escaped
from their egg capsules after about 1 week. Larvae
were isolated on a 200 mm sieve and transferred to
0.45 mm ¢ltered seawater. All larvae tested in the experiment were released on the same day (day 0), but
not necessarily by the same female.

Experimental design and larval rearing
A 5 Â 5 factorial design with stocking densities
of 0.25, 0.50, 0.75, 1.00 and 1.50 larvae mL À 1 and
algal concentrations of 5, 10, 15, 20 and 25 Â 104
cells mL À 1 was used in the experiment. Two replicate
glass beakers were used for each treatment, and in all
the experimental groups, larvae were initially reared
in 400 mL of seawater. As soon as the newly hatching
veligers (0-day-larvae) were transferred to ¢ltered
seawater, they were collected in pipettes and distributed into ¢fty glass beakers. Each day, seawater was
replaced with fresh 0.45 mm membrane-¢ltered seawater, food was replaced with a fresh diet and beakers were thoroughly cleaned with deionized water
during water changes. Larval density was maintained as in the original beakers from the beginning
to the end of the experiment by adjusting the water
volumes with daily water change. Larvae were reared
at 24^25 1C, 24^26% salinity and a constant photoperiod of 12 L:12 D using a 40 W £uorescent lamp,

these being optimal for larval rearing (Ke et al. 2001;
Zheng et al. 2001).
Larvae were fed a unialgal diet of Dicrateria
zhanjiangensis, this enrichment ensuring the rapid
growth of B. formosae habei with low mortality

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Aquaculture Research, 2010, 42, 1^8

E¡ects of density and concentration on larvae H Zheng et al.

through metamorphosis (Zheng et al. 2001). Algal
concentration was microscopically measured using
a blood cell counter plate. The algae was cultured in
Walne’s medium and used when the stock cultures
were in the exponential phase. To avoid the introduction of algal medium into the larval containers, the
stock cultures were centrifuged and the cells were
suspended in 0.45 mm membrane-¢ltered seawater
before feeding to the larvae (Lucas & Costlow 1979).

cent metamorphosis were examined by ANOVA. Because data on survival and metamorphosis were presented as percentages of the total amount, an
arcsine-transformation was performed before analysis. All statistical analyses were performed on a SAS
System for windows (SAS 8.0, SAS Institute, Cary, NC,
USA), and signi¢cance for all analyses was set to
Po0.05 unless noted otherwise.

Measurement of larval growth and survival

To determine larval growth, shell lengths (longest
dimension) of 10 larvae from each replicate were
randomly measured nondestructively every other
day using a microscope equipped with an ocular
micrometer ( Â 64). Larvae were pipetted onto a
microscope slide in a small volume of water and the
water was quickly removed by a pipette to immobilize
the larvae, and then shell length was measured. After
this, the larvae were immediately returned to their
containers. The average growth rate for each replicate was measured by regressing larval shell length
over time for the ¢rst 10 days of larval life for each
larva.
Larval survival was expressed by a percentage of
numbers of live 10-old-day larvae to the initial numbers of larvae in each replicate.

De¢nition of larval metamorphosis
Metamorphosis of B. formosae habei larvae as described by Zheng et al. (2005) using the de¢nition of
Pechenik (1980) involves: (1) loss of the larval velum,
(2) larval behaviour changing from free swimming to
crawling, (3) shell changing from the larval £attenedelliptic pattern to the adult spiral pattern and (4) siphon being extended.
The time to initial metamorphosis was denoted as
developmental duration from day 0 larvae to the day
the ¢rst juvenile occurred in each replicate.
Per cent metamorphosis was the ratio of the total
numbers of metamorphosed larvae to the initial
numbers of larvae in each replicate.

Statistic analyses
The independent and combined e¡ects of larval density and algal concentration on per cent survival,
growth rate, time to initial metamorphosis and per

Results
Larval survival
Analyses of variance (Table 1) demonstrated that larval per cent survival was signi¢cantly a¡ected by the
independent e¡ects of both stocking density and algal concentration and by their interaction, and estimation of variance components further indicated
that the e¡ect of stocking density was more important than that of algal concentration. Larvae survived less with increasing stocking density at the
same algal concentration and survived more with increasing algal concentration at the same stocking
density. Of 10-old-day larvae, 72.5% survived at the
lowest larval density and the highest algal concentration combination, which only survived 4.0% at the
highest stocking density and the lowest algal concentration combination (Table 2).

Larval growth
The average growth rates of larvae at di¡erent combinations of stocking density and algal concentration
are listed in Table 3. Larvae grew more slowly as
the stocking density increased regardless of the
algal concentration and with decreasing algal concentration regardless of the stocking density. The
average growth rate was up to 41.57 mm day À 1 when
larvae were cultured at the lowest density of
0.25 larvae mL À 1 and fed the highest algal concentration of 25 Â 104 cells mL À 1, whereas larvae only
grew 17.06 mm daily when they were cultured at the
highest density of 1.5 larvae mL À 1 and fed the lowest
algal concentration of 5 Â 104 cells mL À 1. Analyses
of variance (Table 1) showed that the larval growth
rate was not only a¡ected by the independent e¡ects
of stocking density and algal concentration but
also by their interaction, and the e¡ect of algal concentration was more signi¢cant than that of stocking
density.

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8

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E¡ects of density and concentration on larvae H Zheng et al.

Aquaculture Research, 2010, 42, 1^8

Table 1 Analyses of variance for traits in larval survival, growth rate, time to initial metamorphosis and per cent metamorphosis of Babylonia formosae habei larvae

Traits

Source

df

MS

Per cent survival

a
b
aÂb
Error
a
b
aÂb
Error
a
b

aÂb
Error
a
b
aÂb
Error

4
4
16
25

1160.765856
292.964204
10.575150
1.439511
143.984948
336.739468
4.874038
0.329176
34.7026495
104.9052536
5.3123383
2.2554348
535.795754
224.698804
12.519950
0.280943

Growth rate

Time to initial metamorphosis

Per cent metamorphosis

4
4
14
23
4
4
16
25

Variance components
(% of total)

F
806.36ÃÃÃ
203.52ÃÃÃ
7.35ÃÃÃ
437.41ÃÃÃ
1022.98ÃÃÃ
14.81ÃÃÃ
15.39ÃÃÃ
46.51ÃÃÃ
2.36Ã
1907.14ÃÃÃ
799.80ÃÃÃ
44.56ÃÃÃ

115.01907
28.23891
4.56782
1.43951
13.91109
33.18654
2.27243
0.32918
2.98031
9.39207
2.97286
2.25543
52.32758
21.21789
6.11950
0.28094

(77.06%)
(18.92%)
(3.06%)
(0.96%)
(27.99%)
(66.77%)
(4.57%)
(0.66%)
(16.93%)
(53.36%)
(16.89%)
(12.81%)

(65.45%)
(26.54%)
(7.65%)
(0.35%)

a and b represents the stocking density (larvae mL À 1) and the algal concentration (cells mL À 1) respectively.
ÃPo0.05; ÃÃÃPo0.001.
df, degrees of freedom; MS, mean squares.

Table 2 Per cent survival (%) of Babylonia formosae habei
larvae at di¡erent stocking density and algal concentration
combinations

Stocking density
(larvae mL À 1)
0.25
0.50
0.75
1.00
1.50

Algal concentration (Â 104 cells mL À 1)
5

10

15

20

25

35.0
32.0
14.0
8.5
4.0

50.0
38.0
20.0
16.5
7.75

65.0
44.0
26.0
20.0
12.0

70.0
48.5
32.0
26.0
14.0

72.5
52.0
38.0
30.0

20.0

Table 3 Average growth rate (mm day À 1) of Babylonia formosae habei larvae at di¡erent stocking density and algal
concentration combinations

Stocking density
(larvae mL À 1)
0.25
0.50
0.75
1.00
1.50

Algal concentration (Â 104 cells mL À 1)

Percentage metamorphosis

5

10

15

20

25

23.85
21.84
21.26

18.52
17.06

28.39
27.27
24.30
22.74
21.00

34.40
31.48
27.77
25.17
24.82

37.08
35.99
31.25
28.24
26.54

41.57
38.18
34.38
34.52
27.98

Per cent metamorphosis listed in Table 5 decreased
with increasing stocking density regardless of the
algal concentration and increased with increasing

algal concentration regardless of the stocking
density. No larvae survived to metamorphosis when
they were cultured at the highest density of
1.5 larvae mL À 1 and fed the lowest algal concentration combination of 5 Â 104 cells mL À 1, but 49.5%
larvae successfully metamorphosed to juveniles
when they were cultured at the lowest density of
0.25 larvae mL À 1 and fed the highest algal concen-

Time to initial metamorphosis
Table 4 presents the time to initial metamorphosis for
larvae cultured at di¡erent stocking density and algal

4

concentration combinations. Time to initial metamorphosis increased with increasing stocking density at the same algal concentration and reduced
with increasing algal concentration at the same
stocking density. The time to initial metamorphosis
was only 10 days when larvae were cultured at a
density of 0.25 larvae mL À 1 and fed an algal concentration of 25 Â 104 cells mL À 1, whereas the time
to initial metamorphosis was 20^26 days for larvae
cultured at the lowest algal concentration of
5 Â 104 cells mL À 1. Stocking density and algal concentration exerted signi¢cantly independent and
combined e¡ects on the time to initial metamorphosis (Table 1).

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E¡ects of density and concentration on larvae H Zheng et al.

Table 4 Time to initial metamorphosis (day) of Babylonia
formosae habei larvae at di¡erent stocking density and algal
concentration combinations

10%, 9% and 5% respectively (Aldana-Aranda et al.
1989). Of the ¢ve densities tested, 500 larvae L À 1
yielded the least growth, and the most growth was
obtained with 100 larvae L À 1. In the abalone Haliotis
rubra, survival at 64 days followed a negative power
function of density at settlement (Daume et al. 2003).
Larvae of the nudibranch mollusk Hermissenda crassicornis grew 4.30 mm day À 1 at the minimum larval
density (1larvae mL À 1) and 1.60 mm day at the highest larval density (15 larvae À 1), and stocking density
exhibited a clear negative e¡ect on larval growth (Avila et al. 1997). The survival of Crassostrea gigas larvae
through metamorphosis declined drastically when
larval densities were increased above 1larvae mL À 1,
and high density reduced the survival of larvae with
low growth e⁄ciency (Powell et al. 2002). Manila clam
Ruditapes philippinarum larvae survived more and
grew faster at 5 larvae mL À 1 than those at 15 and
20 larvae mL À 1 (Yan et al. 2006). For the California
sea hare Aplysia californica larvae, the growth rates
declined from approximately 16 mm day À 1 at the lowest stocking densities to about 9 mm d À 1 at the highest, time to competency increased by about two-fold
and survival rates declined from about 85% to 5% or
less at a density ranging from 0.5 to 4 larvae mL À 1
(Capo et al. 2009). All results exhibited an adverse relationship between larval survival/growth and stocking density. Therefore, to maximize survival and
growth, B. formosae habei larvae should be cultured
at a lower density in a large-scale hatchery culture.
Basch (1996) pointed out three possible e¡ects of

larval density including: (1) larvae grazing particles
to levels where detection or feeding e⁄ciency is reduced, (2) physical interactions between larvae or
(3) accumulation of soluble wastes, reducing the
feeding times or rates. When the stocking density is
lower, the density-dependent behaviour in relation
to competition for resources such as space and food
is slight (McShane1991; Preece et al.1997), and so larvae show higher survival and faster growth. When
the stocking density is higher, crowding conditions
have a signi¢cant impact on larval survival and
growth through chemical interactions and mechanical/physical interference such as collisions between
swimming larvae (Sprung 1984; Avila et al. 1997) and
becoming entangled in mucus strings (Hansen1991).
Moreover, it is di⁄cult for larvae at a high density to
accumulate energy due to competition for food, social stress, and growth inhibitors, even when food is
abundant (Crump 1981). Therefore, larvae have lower
survival and slower growth when the stocking density is higher.

Stocking density
(larvae mL À 1)
0.25
0.50
0.75
1.00
1.50

Algal concentration (Â 104 cells mL À 1)
5

10

15

20

25

20.0
23.25
26.0
–
–

16.0
20.25
22.0
23.0
22.25

14.75
16.75
17.5
19.0
18.75

12.5
15.0
16.75
16.25
18.5

10.5
13.0
15.25
15.75
16.75

^, no larvae survived to metamorphosis.

Table 5 Per cent metamorphosis (%) of Babylonia formosae
habei larvae at di¡erent stocking density and algal concentration combinations

Stocking density
(larvae mL À 1)
0.25
0.50
0.75
1.00
1.50

Algal concentration (Â 104 cells mL À 1)
5

10

15

20

25

15.0
3.5
0.25
0.0
0.0

35.5
10.0
4.5
3.0
1.75

39.5
13.75
11.75
5.9
2.8

41.0
19.25
17.75
10.3
3.1

49.5
32.5
27.75
14.8
11.3

tration of 25 Â 104 cells mL À 1. Per cent metamorphosis of larvae was a¡ected not only by the independent e¡ects of stocking density and algal
concentration but also by their signi¢cantly combined e¡ect (Table 1).

Discussion
The independent e¡ects of stocking density
on larval survival, growth and metamorphosis
Larval culture density is an important factor in£uencing the success of hatchery seed culture of molluscs.
Although larger larval density may increase the yield
of hatchery-produced spat, it may result in reduced
growth and survival. In the present study, stocking
density had striking e¡ects on the survival, growth
and metamorphosis of B. formosae habei larvae. Under
the same algal concentration conditions, a high stocking density exerted negative e¡ects on larval survival,
growth and metamorphosis. Such negative e¡ects of
crowding or density stress on larvae have been reported in other molluscs. For milk conch Strombus
costatus larvae, the survival rate for ¢ve larval densities tested (from100 to 500 larvae L À 1) was17%,18%,

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E¡ects of density and concentration on larvae H Zheng et al.

The independent e¡ects of algal
concentration on larval survival, growth and
metamorphosis
Algal concentration is another important factor in£uencing the success of hatchery seed culture of molluscs. Particularly, planktotrophic larvae survive less
and grow slower when food is limited or scarce. Such

negative e¡ects of low algal concentration on larval
survival, growth and metamorphosis were also observed in the present study. Dicrateria zhanjiangensis
has been used as a good unialgal diet to feed B. formosae habei larvae, and supported rapid growth and
high survival at a high concentration of
20 Â 104 cells mL À 1 (Zheng et al. 2001, 2005).
Furthermore, no detrimental e¡ect of this alga has
been reported on mollusk larvae. Therefore, the negative e¡ects of low algal concentration on larvae can
be attributed to a de¢ciency in energy gain from food.
The negative e¡ects of food limitation on larvae have
been documented in other molluscs (Fretter & Montgomery 1968; Pilkington & Fretter 1970; AldanaAranda et al. 1989; Pechenik et al. 1990, 1996; His &
Seaman 1992; Strathmann, Fenaux, Sewell & Strathmann 1993; Avila et al. 1997; Rico-Villa & Robert
2009). It is known that planktotrophic larvae feed
on phytoplankton (and possibly other organic material in suspension) and are dependent on a net energy
gain from such food for successful growth and development. From the point of view of the individual’s
physiological energetics, the energy and nutrients
gained by the animal (in this case the larva) from
the environment are distributed among the various
metabolic requirements of maintenance, movement
and growth (Bayne 1983). The energy and nutrients
gained by the larvae from food are not able to meet
the various normal metabolic requirements when
food is scarce, and so it is inevitable that larvae survive less and grow slower. Therefore, to maximize
survival and growth, B. formosae habei larvae should
be fed a higher algal concentration in a large-scale
hatchery culture.

Interaction of stocking density and algal
concentration on larvae
In general, the e¡ects of stocking density or algal concentration on the growth and survival of planktotrophic larvae have been studied for each factor
separately, and reports on their combined e¡ects have

been very scarce. Doroudi and Southgate (2000)
found that the interaction of algal ration and larval

6

Aquaculture Research, 2010, 42, 1^8

density did not a¡ect the growth or the survival of 7or 20-day-old black-lip pearl oyster Pinctada margaritiferia larvae. However, Powell et al. (2002) found that
a number of di¡erent combinations of culture conditions including stocking density, algal concentration,
feeding frequency and food quality generated very
complex interactions for the growth and development
of Paci¢c oyster C. gigas larvae. In the present study,
the combined e¡ects of stocking density and algal
concentration on the growth and survival of B. formosae habei larvae were very common. Larvae survived
more, grew faster and metamorphosed earlier at a
lower stocking density and a higher algal concentration combination, whereas larvae survived less, grew
slower and metamorphosed later at a higher stocking
density and a lower algal concentration combination.
According to the present results, larvae should be cultured at a density of 0.25 larvae mL À 1 and fed an algal
concentration of 25 Â 104 cells mL À 1 in order to
maximize the survival and growth in B. formosae habei hatchery culture practice.

Di¡erence between the e¡ects of stocking
density and algal concentration on larval
survival and growth
A more interesting and important ¢nding from the
present study is that stocking density and algal concentration obviously exert di¡erent impacts on larval
survival and growth, that is, stocking density played
a more important role than algal concentration in determining larval survival, whereas algal concentration played a more important role than stocking
density in determining larval growth. This result

has been not reported in those previous studies. From
an ecological point of view, it is reasonable to draw
such conclusions because larval density and algal
concentration represent two di¡erent types of factors
^ spatial and nutritional. Competition for space in a
crowded living space with an increase in physical
collision or interference may be a major and direct
factor resulting in the lower survival of larvae,
whereas an increase in physical interference in a
crowded living space may be a causal or an indirect
factor resulting in slow larval growth by reducing
feeding e⁄ciency (Rasheed & Bull 1992). This can be
supported by the fact that percentage survival was
very low under higher density conditions even in the
presence of abundant food resources. The energy and
nutrients gained by larvae may be reallocated to
maintain survival ¢rst rather than growth when

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E¡ects of density and concentration on larvae H Zheng et al.

food is scarce (Pechenik et al. 1996). Therefore, competition for nutrition during food limitation may be
the principal factor resulting in slow larval growth.
This can be supported by larvae surviving longer,
combined with very slow growth, at a low food concentration in the present study or when fully deprived of food (Zheng et al. 2005).

In conclusion, both stocking density and algal concentration independently had signi¢cant impacts on
the survival and growth of B. formosae habei larvae;
their combined e¡ects were also signi¢cant. Larvae
survived more, grew faster and metamorphosed earlier at a lower stocking density and a higher algal
concentration combination, whereas larvae survived
less, grew slower and metamorphosed later at a higher stocking density and a lower algal concentration
combination. In a large-scale hatchery seed culture
of B. formosae habei, larvae should be reared at a lower stocking density of 0.25 larvae mL À 1 and fed a
higher algal concentration of 25 Â 104 cells mL À 1 in
order to maximize larval survival and growth.

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Acknowledgments
We thank Dr Haihui Ye and Donghui Guo for their
kind help in this experiment.We also thank Ms Kirsty
A. Mattinson (Cantab MA) and Professor I. J. Hodgkiss for helping to revise the manuscript. This work
was supported in part by the Earmarked Fund for
Modern Agro-industry Technology Research System

(No. nycytx-47) and Research Project of Technical
Exploitation of Fujian Province (No.: 98 À Z À 8).

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8

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8


Aquaculture Research, 2010, 42, 9^13

doi:10.1111/j.1365-2109.2010.02482.x

Growth and survival of juvenile lined seahorse,

Hippocampus erectus (Perry), at different stocking
densities
Dong Zhang1,2,Yinghui Zhang2, Junda Lin2 & Qiang Lin2,3
1

East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Shanghai, China

2

Vero Beach Marine Laboratory, Florida Institute of Technology,Vero Beach, FL, USA

3

Key Laboratory of Tropical Marine Environmental Dynamics, South China Sea Institute of Oceanology, Chinese Academy of

Sciences, Guangzhou, China
Correspondence: D Zhang, East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Shanghai 200090,
China. E-mail: zd_¢

Abstract

The lined seahorse, Hippocampus erectus (Perry), is an
important species in both medicinal and aquarium
trades. The aim of this study was to evaluate the effects of stocking density (1, 3 and 5 individuals L À 1)
on the growth performance and survival of the
early-stage juvenile H. erectus. The height (HT), wet
weight, weight gain (WG) and speci¢c growth rate
(SGR) were a¡ected signi¢cantly by the stocking density during the 40-day study. The HT,WG and SGR of
the seahorse at 1 and 3 juveniles L À 1 were signi¢cantly higher than that at 5 juveniles L À 1. The survival of juveniles at the three stocking densities was
not signi¢cantly di¡erent at day 25 (90.3 Æ 4.5%,
86.7 Æ 4.2% and 86.2 Æ 3.8% for 1, 3 and
5 juveniles L À 1 respectively), but was signi¢cantly
di¡erent at day 40 (87.8 Æ 3.9%, 69.6 Æ 4.2% and
52.9 Æ 2.8% for 1, 3 and 5 juveniles L À 1 respectively).
For the early-stage juvenile H. erectus, we recommend a
stocking density of 3 juveniles L À 1, but the density
should be reduced to1^2 juveniles L À 1 to avoid reduced
and variable growth and high mortality after 25 days.

Keywords: Hippocampus erectus (Perry), density,
growth, survival, seahorse

Introduction
Although there have been attempts at commercial
culturing of seahorses for over 50 years (Zou 1958),

r 2010 The Authors
Aquaculture Research r 2010 Blackwell Publishing Ltd

signi¢cant breakthroughs in culture techniques
have only occurred in the last 10 years. This has in

part been the result of increasing culture e¡orts since
all 33 recognized seahorse species were listed on Appendix II of the Convention on International Trade in
Endangered Species of Wild Fauna and Flora (CITES
2004) due to overexploitation of the wild populations
to meet the growing demand in Chinese medicine
and ornamental market (Vincent 1996; Lourie, Vincent & Hall1999). More than10 seahorse species have
been reared successfully in captivity, such as
Hippocampus abdominalis (Woods 2000a, b; 2005),
Hippocampus comes (Job, Buu & Vincent 2006), Hippocampus erectus (Correa, Chung & Manrique 1989;
Scarratt 1995; Lin, Lin & Zhang 2008), Hippocampus
kuda (Job, Do & Hall 2002; Lin, Lu & Gao 2006; Lin,
Gao, Sheng, Chen, Zhang & Lu 2007), Hippocampus
reidi (Olivotto, Avella, Sampaolesi, Piccinetti, Ruiz &
Carnevali 2008), Hippocampus subelongatus (Payne &
Rippingale 2000) and Hippocampus trimaculatus
(Sheng, Lin, Chen, Gao, Shen & Lu 2006). Research
e¡orts have focused on the e¡ects of food type and
feed regimes and various environmental factors, including temperature, salinity, light intensity and
photoperiods, on the growth and survival of juvenile
seahorses to establish appropriate rearing protocols
(e.g. Payne & Rippingale 2000; Woods 2000b, 2005;
Lin et al. 2006, 2008; Olivotto et al. 2008). However,
the e¡ect of stocking density on the growth and survival of seahorses in the early juvenile stages has received much less attention, although it is recognized

9


Growth of seahorse at di¡erent densities D Zhang et al.

as a common factor a¡ecting growth and survival in

¢n¢sh aquaculture (e.g. Wallace, Kolbeinshaven &
Reinsnes 1988; Christianssen, Svendsen & Jobling
1992; Bj˛rnsson 1994; Hossain, Beveridge & Haylor
1998; Feldlite & Milstein 1999; Irwin, O’Halloran &
FitzGerald 1999; Salas-Leiton, Anguis, Manchado &
Canìavate 2008).
In commercial seahorse culture, low survival, particularly in the early juvenile stages (Scarratt 1995;
Forteath 1996), is still one of the bottlenecks a¡ecting
the economic return. Although high survival of several seahorse species in the early juvenile stages has
been reported, e.g. H. abdominalis (Woods 2000b),
H. erectus (Lin et al. 2008) and H. comes (Job et al.
2006), these have been obtained at stocking densities
around or lower than1individual L À 1, which may be
too low for an economically viable commercial mass
production.
The lined seahorse, H. erectus (Perry), is a highly
valued species in both medicinal and aquarium
trades (Correa et al. 1989; Scarratt 1995; Lourie et al.
1999; Foster, Marsden & Vincent 2003). It is distributed from Nova Scotia along the western Atlantic
coast through the Gulf of Mexico and Caribbean to
Venezuela (Lourie et al. 1999; Fritzche & Vincent
2002; Foster & Vincent 2004). It has been reared successfully in captivity, and is recognized as a good candidate for commercial aquaculture (Correa et al.1989;
Lin et al. 2008). In this study, we investigated the
growth performance and survival of H. erectus at different stocking densities during the early juvenile
stage in order to further develop successful culture
protocols.

Materials and methods
Broodstock seahorses
Twenty (M:F) pairs of F2 seahorses [16^20 cm in

height (HT), the vertical distance from the tip of the
coronet to the tip of the outstretched tail, with the
head held at right angles to the body (Lourie et al.
1999)] were used as the broodstock and maintained
in the £ow-through tanks (90 Â 80 Â 60 cm) at the
Vero Beach Marine Laboratory, Vero Beach, FL, USA,
with sand-¢ltered seawater pumped directly from
the Atlantic Ocean at a rate of 0.5^0.6 L min À 1. The
Salinity, temperature, light intensity and photoperiod
were 35%, 24.0 Æ 0.5 1C (mean Æ SD, the same format throughout this paper), 1000 lx and 14 h L/10 h
D respectively. Plastic plants and corallites were used
as the holdfasts and substrate for the seahorses. The

10

Aquaculture Research, 2010, 42, 9^13

broodstock seahorses were fed twice a day (09:00
and 16:00 hours) with frozen mysis (HikariTM, Hikari
Sales USA, Hayward, CA, USA) at a daily rate of approximately 15% wet body weight, and faeces and
uneaten food in the tanks were siphoned out 2 h after
each feeding. Upon pregnancy being noted, brooding
males were isolated temporarily in a separate £owthrough hatching tank (50 Â 25 Â 30 cm) containing 26 L of seawater with the same environmental
conditions as those for broodstock tanks.

Experimental design
Three stocking densities, 1, 3 and 5 individuals L À 1,
each with three replicate tanks, were tested. Transparent £ow-through glass tanks (50 Â 25 Â 30 cm)
were used. Each tank contained 30 L seawater with
a £ow rate of 0.2 L min À 1 and gentle aeration. Oneday post-hatch juveniles [1.11 Æ 0.02 cm HT, mean

wet weight (WW) 5 2.4 Æ 0.09 mg, n 515] from four
male broodstock seahorses were haphazardly allocated to the tanks. Plastic plants were used as holdfasts for the seahorses. Juveniles were fed with
newly hatched Artemia sinica (350^400 mm in
length) (Bohai strain, China) at approximately
5 nauplii mL À 1 during the ¢rst week, followed by 2day-old Artemia (500^800 mm in length) enriched
with Chlorella sp. (newly hatched Artemia at approximately 30 nauplii mL À 1 were cultured in 50 L tanks
with 50 000^60 000 cells of Chlorella sp. per ml) at
the same food density. Juveniles were fed at 08:00,
14:00 and 20:00 hours each day to maintain the food
density. Before each feeding, the bottom of the tanks
was siphoned to remove faeces and dead food. The
salinity, temperature, light intensity and photoperiod
were 34^35%, 23 Æ 0.5 1C, 1000 lx and 14 L/10 D,
respectively, throughout the experimental period.
Ten seahorses were haphazardly selected from each
tank every 5 days for measuring the HT and returned
to their respective tanks. Because HT in
5 juveniles L À 1 was signi¢cantly lower than that in
1 and 3 juveniles L À 1 at day 25 (see Results), all the
juvenile seahorses were counted and 10 seahorses
from each tank were also weighed. The experiment
continued and was terminated when the di¡erence
in HT was signi¢cant between the treatments of 1
and 3 juveniles L À 1. At the termination, all the juvenile seahorses were counted and 10 seahorses from
each tank were weighed. The weight gain
[WG 5100 Â (¢nal body weight À initial body
weight)/initial body weight (g)], the speci¢c growth

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 9^13

Aquaculture Research, 2010, 42, 9^13

Growth of seahorse at di¡erent densities D Zhang et al.

rate [SGR 5100 Â (ln ¢nalWW À ln initialWW)/time]
and the condition factor [(CF 5100 Â WW(g)/HT
(cm3)] of the juveniles were calculated. Dead seahorses, if any, were removed upon daily inspection
and recorded.

Statistics
One-way analysis of variance (ANOVA) was used to compare the ¢nal HT andWW,WG, SGR and CF of the juveniles among the stocking densities. All the variables
were tested for normality and homogeneity before
one-way ANOVA. If there was a signi¢cant di¡erence
among the stocking densities, a Welsch-up multiple
procedure was applied to compare the di¡erent means
among the densities. The di¡erence in the percentage
survival among the stocking densities was tested
using Kruskal^Wallis ANOVA (Sokal & Rohlf 1995).

Results
The stocking density signi¢cantly a¡ected HT (oneway ANOVA, F2,89 53.963, P 5 0.023), WW (one-way
ANOVA, F2,89 53.147, P 5 0.047), WG (one-way ANOVA,
F2,89 55.745, P 5 0.040) and SGR (one-way ANOVA,
F2,8 57.633, P 5 0.023) of the juvenile seahorses after
25 days (Table 1). A multiple comparison test indicate
that the juveniles at1and 3 individuals L À 1 grew signi¢cantly (Po0.05) faster in HT than those at 5 individuals L À 1 (Table 1). WG and SGR of the juveniles
at 1 and 3 individuals L À 1 were also signi¢cantly
higher (Po0.05) than that at 5 individuals L À 1 (Table1). HT,WG and SGR were not signi¢cantly di¡erent

(P40.05) between 1 and 3 individuals L À 1 (Table 1).
There was no signi¢cant di¡erence in the survival
(Kruskal^Wallis ANOVA, H 51.415, P40.05) and CF

(one-way ANOVA, F2,89 5 0.179, P 5 0.837) among the
stocking densities (Table 1).
At day 40, HT (one-way ANOVA, F2,89 511.520,
Po0.0001), WW (one-way ANOVA, F2,89 57.152,
Po0.001), WG (one-way ANOVA, F2,8 55.790,
P 5 0.039) and SGR (one-way ANOVA, F2,8 57.354,
P 5 0.024) of the juvenile seahorses were still signi¢cantly di¡erent among the treatments (Table 1).
Moreover, for the four measurements, the di¡erence
between 1 and 3 individuals L À 1 was also signi¢cant
(Po0.05) (Table 1). Survival of juvenile seahorses
among the treatments was signi¢cantly di¡erent
(Kruskal^Wallis ANOVA, H 5 4.153, Po0.05) after 40
days (Table 1).

Discussion
It is well known that stocking density can a¡ect
growth and survival in ¢sh aquaculture; generally,
higher densities result in a lower ¢sh growth rate
(e.g. Wallace et al. 1988; Haylor 1991; Christianssen
et al.1992; Bj˛rnsson1994; Hossain et al.1998; Feldlite
& Milstein 1999; Irwin et al. 1999; Salas-Leiton et al.
2008). The results presented here indicate the same
pattern of the negative e¡ect of increasing stocking
density on the growth of early-stage juvenile H. erectus, i.e. the growth parameters (HT, WG and SRG)
of the seahorses at the highest density (5 individuals L À 1) were signi¢cantly reduced (Table 1).
A similar e¡ect has also been observed in late-stage

juvenile H. abdominalis (Woods 2003). Another
e¡ect frequently associated with the stocking
density is the high size variations within the reared
group of ¢sh (Irwin et al. 1999; Lambert & Dutil
2001). Perhaps due to the short experimental time,
size heterogeneity was not signi¢cant in this study
(Table 1).

Table 1 Final height (HT, mm), wet weight (WW, mg), weight gain (WG, %), condition factor (CF), speci¢c growth rate (SGR,
%day À 1) and survival (%) of juvenile Hippocampus erectus at stocking densities of 1, 3 and 5 individuals L À 1 over the 25 and
40 days (mean Æ SD)
Time
Day 25

Day 40

Density (L À 1)

Final HT

1
3
5
1
3
5

2.79 Æ 0.22Ã
2.82 Æ 0.28Ã
2.64

3.58
3.31
3.16

Æ
Æ
Æ
Æ

0.28
0.30Ãa
0.33Ã
0.38

Final WW
63.9 Æ 21.3Ã
65.9 Æ 21.3Ã
53.7
127.4
103.1
82.4

Æ
Æ
Æ
Æ

17.3
21.6Ãa
20.4Ã

19.7

WG
2646 Æ 257Ã
2614 Æ 234Ã
2139
104
86
72

Æ
Æ
Æ
Æ

175
8Ãa
7Ã
11

CF

SGR

Survival

0.30
0.29
0.28
0.29

0.28
0.27

13.24 Æ 0.37Ã
13.19 Æ 0.31Ã

90.3
86.7
86.2
87.8
69.6
52.9

Æ
Æ
Æ
Æ
Æ
Æ

0.03
0.05
0.04
0.03
0.04
0.04

12.43
4.80
4.07

3.76

Æ
Æ
Æ
Æ

0.13
0.49Ãa
0.21Ã
0.24

Æ
Æ
Æ
Æ
Æ
Æ

4.5
4.2
3.8
3.9Ãa
4.2Ã
2.8

ÃFinal HT, WW, WG and SGR at 1, 3 individuals L À 1 were signi¢cantly higher than that at 5 individuals L À 1; a indicates ¢nal HT, WW,
WG and SGR at 1individuals L À 1 was signi¢cantly higher than that at 3 individuals L À 1.

r 2010 The Authors

Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 9^13

11


Growth of seahorse at di¡erent densities D Zhang et al.

An inverse relationship between survivorship
and stocking density has been reported for many ¢sh
species (e.g. Wallace et al. 1988; Haylor 1991; Christianssen et al. 1992; Bj˛rnsson 1994; Hossain et al.
1998; Feldlite & Milstein 1999; Irwin et al. 1999;
Salas-Leiton et al. 2008). The same e¡ect was also
found in the seahorse, H. abdominalis (Woods 2003).
Probably due to the short experimental time of this
study, di¡erences in survival between the stocking
densities were not signi¢cant, although survival at
higher densities tended to be lower (Table 1). In seahorses, conspeci¢c grasping and wrestling with each
other is considered to a¡ect juvenile growth and survival at higher stocking densities where the incidence of such behaviour increased (Woods 2003).
Similar behavioural interactions were also observed
in the early-stage juveniles in this study. Although
we did not quantify this behaviour accurately, the incidence of the behaviour was the highest at the highest stocking density.
Compared with other ¢sh species, seahorses appear not to be suitable for culture in high density.
This may be a consequence of their habit of using
their tails to anchor themselves, which can cause interference among conspeci¢cs at higher densities. To
date, instances of all the high survival in cultured
seahorses have been found at low stocking densities
of around or o1individual L À 1 (e.g.Woods 2000a, b;
Job et al. 2006; Lin et al. 2008). Five individuals per litre for an early-stage seahorse may be the high-density limit. For example, low survival of 20^40% over
21 days at 5 juveniles L À 1 was found for
H. reidi (Olivotto et al. 2008). Survival of the H. erectus

at a stocking density of 6 juveniles L À 1 over 35 days
was only 50.67% (Correa et al. 1989), similar to what
we found over the 40 days for the 5 juveniles L À 1
treatment in the present study.
The strategy of commercial ¢sh aquaculture is to
maximize the economic return in relation to culture investment. High stocking density is a common strategy
to reduce the overall production cost and to improve
¢sh health, growth and survival. A trade-o¡ between
growth/survival and stocking density is essential for
the successful mass culture of seahorses, especially for
medicinal purposes. Based on the results of this study,
we recommend a stocking density of 3 juveniles L À 1 for
the early-stage H. erectus. Because of signi¢cant growth
reduction and lower survival of juvenile seahorses after
25 days at higher densities (i.e. 3, 5 juveniles L À 1), the
density should be reduced to 1^2 juveniles L À 1 (D.
Zhang & J. Lin unpubl. obs.) to avoid reduced and variable growth and high mortality after 25 days.

12

Aquaculture Research, 2010, 42, 9^13

Acknowledgments
This study was supported by a special research fund
for the national nonpro¢t institutes (East China Sea
Fisheries Research Institute).

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13


Aquaculture Research, 2010, 42, 14^21

doi:10.1111/j.1365-2109.2010.02483.x

Sedimentation and sediment characteristics in
sea cucumber Apostichopus japonicus (Selenka)
culture ponds
Yichao Ren, Shuanglin Dong, Fang Wang, Qinfeng Gao, Xiangli Tian & Feng Liu
Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China
Correspondence: S Dong, Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266100, China.
E-mail:

Abstract
Annual sedimentation, re-suspension rates and contents of particulate organic carbon (POC), particulate
organic nitrogen (PON) and total phosphorus (TP) in
the sediment were investigated in two sea cucumber
culture ponds at Rongcheng, Shandong Province,

China. The results showed that the average £ux of total particulate matter in the ponds was 22.1g m2 d À 1.
The average re-suspension rate of the sediment in the
ponds was 81.7%. The re-suspension rates in spring
and autumn were higher than those in summer and
winter. The mean contents of POC, PON and TP in the
sediment of the ponds were 4.4, 0.5 and 0.6 (mg g À 1),
respectively, and the mean contents of Chlorophyll a
(Chl a) and pheophytin in the sediment were 8.1 and
12.1 mg g À 1 respectively. The POC, PON and TP contents in the sediment of the ponds increased during
the period of sea cucumber aestivation (summer)
and hibernation (winter), while they decreased during the feeding periods. The organic matter accumulation rate and the contents of POC, PON, TP, Chl a
and pheophytin in the sediment were even lower
than those in the pond without sea cucumber
(Po0.05). The results demonstrated that sea cucumber culture can e¡ectively stop nutrient accumulation at the bottom of the cucumber culture ponds.

Keywords: sea cucumber, ponds, sedimentation,
sediment, chlorophyll, pheophytin, POC, PON,TP

Introduction
Particular organic matter (POM) sedimentation and
re-suspension play an important role in the material

14

and energy transformation from primary producers
to benthic consumers in aquatic ecosystems, and the
process of pelagic^benthic coupling has a considerable in£uence on the biotic community in inter-tidal
zones and shallow seas (Danovaro, Croce, Dell’Anno,
Fabiano, Marrale & Martorano 2000). Re-suspension
nutrients restores to the overlying water and has a

feedback e¡ect on the vertical £ux and the ecosystem
structure in shallow sea (Sun & Zhan 2002).
The quality of settling particulate matter depends
on the seasonal variations in phytoplankton biomass
and community composition (Josefson & Rasmussen
2000) as well as the re-suspension process, and it can
in£uence, in turn, the growth of aquatic invertebrates.
The £ux and characteristic of POM are di¡erent in
di¡erent waters due to various bio-productivity and
hydrological conditions. There have been several reports on the £ux and sources of total particulate material (TPM) in the ocean (Honjo, Manganini & Wefer
1988; Sasaki, Hattori & Nishizawa 1988; Lohrenz,
Knauer, Asper & Tuel 1992; Wassmann 1993; Kawahata 2002), and a few studies have dealt with biodeposition of scallop culture (Zhou, Yang, Liu, Yuan,
Mao, Liu, Xu & Zhang 2006) and ¢sh cage culture
(Sutherland, Martin & Levings 2001; Cromey, Nickell
& Black 2002) in coastal areas.
The sea cucumber Apostichopus japonicus (Selenka)
is a commercial species in the coasts of Asia (Liao
1997) and is cultured in coastal ponds in north China.
Sea cucumber, being a deposit feeder, ingests large
amounts of sedimentary material, absorbing nutrition
from it (Yingst 1976). Particular organic matter in the
water column is a potentially available food source for
sea cucumber after sedimentation. However, quantitative studies of its sedimentation and re-suspension

r 2010 The Authors
Aquaculture Research r 2010 Blackwell Publishing Ltd


Aquaculture Research, 2010, 42, 14^21

rates are scarce in ponds. The sea cucumber culture
pond is a shallow, semi-closed water body, with large
deposit feeder (sea cucumber) in it. Therefore, its POM
sedimentation and re-suspension characteristics
should be quite di¡erent from those of other waters.
Particular organic matter in the water column is
not only the potentially available food source for sea
cucumber but it can also a¡ect sediment quality and
bottom environment; therefore, for sea cucumber
culture it is very important to know sedimentation,
re-suspension and its e¡ect on sediment. The present
study was conducted to investigate mainly the sedimentation £ux and the sediment characteristics of
the sea cucumber culture ponds in order to gain an
insight into the pool of organic matter (OM) and the
e¡ect of sea cucumber culture on the sediment.

Materials and methods
Study ponds
The two ponds investigated (Pond 1 and Pond 2), approximately 2 ha (100 Â 200 Â 2 m) each, are located at Rongcheng, Shandong Province, China. The
seawater in the ponds was routinely exchanged during the spring tide, while the sluices were closed during the neap tide. Juvenile sea cucumbers of about
5.0 g ind À 1 were stocked at a density of 10 ind m À 2
in April 2007. No supplemental food was provided
during the sea cucumber culture period.

Sampling and measurement
Straight-sided cylindrical traps with an aspect ratio of
4 (White 1990) were used for collecting sinking particles. A net, approximately 0.8 cm in mesh, was used to
cover the upper end of the trap to prevent large nektons
from entering. Ten sides were selected to set up the
traps evenly in the pond. At each site, vertical sediment

traps with three replicates were installed on the sediment. The traps were deployed for 10 days and taken
back to a lab for analysis. Overlying water of the traps
was carefully removed using a siphon after standing
for 12 h. Distilled water was used to rinse the salt of
the samples. The dry weight of the total sediment deposition was obtained after drying the samples at
60 1C to a constant weight. The trapped material was
analysed for nutrient contents. Particulate organic
carbon (POC) and particulate organic nitrogen (PON)
were analysed using a PE-24 CHN analyzer (Heraecus,
Banau, Germany) after acidi¢cation with 0.1N HCl to

Sedimentation in sea cucumber culture ponds Y Ren et al.

remove carbonate (Fabiano, Danovaro & Fraschetti
1995). The total phosphorous (TP) content was measured according to Mudroch, Azcue and Mudroch
(1997). Samples for Chlorophyll a (Chl a) and pheophytin were analysed according to Lorenzen and Jeffrey (1980).
Surface samples of sediment (0^1cm) were collected
at monthly intervals using aWuttke standard box corer
beside the sediment traps (three replicates in each site).
A series of sediment samples for comparison were also
collected from an adjacent pond where no organism
was cultured. The samples were processed within 3 h
of collection (Gerino, Stora, Poydenot & Bourcier1995).
The methods to determine POC, PON, TP, Chl a and
pheophytin of the sediment samples were the same as
those for the settling particles. Sediment samples were
stored at À 20 1C before analytical treatment.
Water samples were collected above every sediment
trap using 2.5 L Van Doorn bottles for analysis of salinity, suspended particle content, Chl a, pheophytin
and phytoplankton of the water column. Temperature

and salinity were measured in situ. A one-litre water
sample was ¢ltered through a Whatman GF/C
(0.45 mm pore size, 25 mm diameter, weight), rinsed
with a small volume of Milli-Q water and dried at
60 1C for 24 h, and then weighed to determine the
suspended particle content. Chlorophyll a and pheophytin were determined according to Lorenzen and
Je¡rey (1980). Samples were stored at À 20 1C before
analytical treatment. The abundance of phytoplankton species was determined by counting the cells
in a phytoplankton-counting chamber (Popovicha &
Marcovecchio 2007) using an Olympus microscope
(Tokyo, Japan).
The re-suspension rates were estimated using the
method proposed by Sun and Zhan (2002).

Statistical analysis
Data were analysed using SPSS 13.0 for Windows statistical software. Di¡erences in the parameters were
analysed using one-way analysis of variance, followed by SNK tests. Di¡erences were considered to
be signi¢cant if Po0.05.

Results
Water quality of sea cucumber culture ponds
There were no signi¢cant di¡erences in the water
quality (temperature, salinity, dissolved oxygen

r 2010 The Authors
Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21

15

Aquaculture Research, 2010, 42, 14–21

Sedimentation in sea cucumber culture ponds Y Ren et al.

10

Chl a content, ugL–1

(DO), pH, Chl a and content of suspended particles)
between the two investigated ponds. The annual
water temperature ranged from 0.0 Æ 1.5 to
28.7 Æ 1.4 1C, and the highest water temperature occurred in August and the lowest occurred in January.
Water salinity of the ponds ranged from 27.6 to
31.1g L À 1, and pH ranged from 7.9 to 8.2. Annual
DO contents were above 5.0 mg L À 1. The contents of
suspended particulate matter in the water column
ranged from 9.5 to 18.1mg L À 1; it was less in summer
due to weak wind and in winter due to ice covering
and less phytoplankton (Fig. 1).

8

6

4

2

0

Time

Phytoplankton in the ponds
The average Chl a content in the water ranged from
2.5 to 5.5 mg L À 1, with an average of 3.7 mg L À 1, and
the maximum Chl a content occurred in July (Fig. 2).
The mean number of phytoplankton was 18.5 Â
104 cell L À 1 in the two sea cucumber culture ponds,
and no signi¢cant di¡erence was detected between
the two ponds (P40.05).
Diatoms were the predominant species, constituting
85.1% of the total phytoplankton species. Dino£agellates accounted for 6.1%; the others, including Chlorophyta, Cryptophyta, Euglenophyta, Chrysophyta and
Cyanophyta, only accounted for 8.8% (Fig. 3).

Phytoplankton group composition, %

Figure 2 Annual Chl a contents in the water column of
sea cucumber culture ponds. Values have been averaged
per month. The values were given as means Æ SD (n 5 5).

100

80

60

40

20

0

Time

Vertical £uxes of particulate matters and
re-suspension
There were no signi¢cant di¡erences between the
two sea cucumber culture ponds in the £uxes of
26
SPM

24
22
20
18
16
14
12
10
8
6

Time

Figure 1 Contents of suspended particulate matter in the
water column of sea cucumber culture ponds. The values
have been averaged per month. Values were given as
means Æ SD (n 5 5).

16

Figure 3 Compositions of di¡erent phytoplankton
groups in sea cucumber culture ponds. The values have
been averaged per month.

TPM, POC, PON, Chl a and pheophytin according to
the monthly analysis (P40.05). Total particulate material £ux ranged from 10.2 to 38.3 g m À 2 day À 1,
with an average of 22.1g m À 2 day À 1, and it was
higher during spring or autumn than that during
summer or winter. The maximum TPM £ux in Pond
1 reached 43.5 g m À 2 day À 1 and it reached
40.2 g m À 2 day À 1 in Pond 2, both occurring in May.
However, the minimum TPM £ux in Pond 1 was
8.2 g m À 2 day À 1 and 8.0 g m À 2 day À 1 in Pond 2,
both occurring in January. The POC content in the
settling particles collected by traps in the two sea cucumber culture ponds in October reached
44.1mg g À 1, which was higher than that in the other
months, and no signi¢cant di¡erences were observed
among other months (P40.05). Particulate organic
carbon £ux in the two ponds ranged from 377.0 to

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Aquaculture Research, 2010, 42, 14^21

Sedimentation in sea cucumber culture ponds Y Ren et al.

1201.0 mg m À 2 day À 1, with an average of 756.1

mg m À 2 day À 1, and it had a signi¢cant correlation
with phytoplankton biomass (r 5 0.822, P 5 0.001).
The PON contents in the settling particles in the two
ponds were higher in October and November; however,
there were no signi¢cant di¡erences among other
months (Po0.05). Particulate organic nitrogen £ux
ranged from 45.5 to 174.8 mg m À 2 day À 1, with an
average of 92.1mg m À 2 day À 1. Two peaks occurred
in the TP contents of settling particles collected from
the two sea cucumber culture ponds in August and
January respectively, and the maximum TP contents
in the settling particles occurred in August, which
reached 1.0 mg g À 1. Total phosphorus £ux in the
two sea cucumber culture ponds ranged from 9.2 to
22.7 mg m À 2 day À 1, with an average of 15.6 mg
m À 2 day À 1. Chlorophyll a £ux ranged from 1.0 to
4.4 mg m À 2 day À 1, with an average of 2.5 mg
m À 2 day À 1. The Chl a £ux was higher in spring and
autumn than in summer and winter. Pheophytin £ux
in the two sea cucumber culture ponds was higher in
August, September and October than in the other
months. The pheophytin £ux of the two ponds ranged
from 2.3 to 22.7 mg m À 2 day À 1, with an average of
8.4 mg m À 2 day À 1. The pheophytin £ux was higher
than the Chl a £ux in the ponds. Re-suspension rates
of the sediment ranged from 59.6% to 95.5%, and they
were lower in summer and winter compared with
those in spring and autumn (Table 1).

Annual C/N of the settling particles in the traps ranged from 9.6 to 12.7, and it was lower in winter and

higher in autumn (Po0.05). The Chl a/pheophytin ratio ranged from 0.2 to 0.5, and the POC/Chl a ratio ranged from 131.0 to 829.9, with an average of 312.1. The
maximum POC/Chl a ratio occurred in July (Table 2).

Table 2 Ratio of C/N, Chl a/pheophytin and POC/Chl a of
the settling particles in the pondsÃ

Time

C/N
(a/a)

POC/Chl
a (w/w)

Chl a/
pheophytin (w/w)

April 2007
May 2007
June 2007
July 2007
August 2007
September 2007
October 2007
November 2007
December 2007
January 2008
February 2008
March 2008
April 2008

Average

10.8
11.6
11.4
10.0
10.2
12.1
12.7
12.1
9.6
9.7
9.8
11.0
11.1
10.9

312.0
239.8
427.4
829.9
131.0
301.1
294.1
439.2
387.5
377.0
299.1
262.5
312.1

354.8

0.5
0.4
0.4
0.4
0.2
0.2
0.2
0.4
0.3
0.3
0.4
0.5
0.5
0.4

ÃValues have been averaged per month (n 510).
POC, particulate organic carbon; Chl a, chlorophyll a; a/a, atom/
atom; w/w, weight/weight.

Table 1 Annual £uxes of TPM, POC, PON,TP, Chl a and pheophytin in the sea cucumber culture pondsÃ

Time
April 2007
May 2007
June 2007
July 2007
August 2007
September

2007
October 2007
November
2007
December
2007
January 2008
February 2008
March 2008
April 2008
Average

TPM
POC
PON
TP
Chl a
Pheophytin
Resuspension
(g m À 2 day À 1) (mg m À 2 day À 1) (mg m À 2 day À 1) (mg m À 2 day À 1) (mg m À 2 day À 1) (mg m À 2 day À 1) rate (%)
24.8
38.3
14.6
18.0
23.6
29.3

Æ
Æ
Æ

Æ
Æ
Æ

1.2
2.7
1.8
2.6
3.8
3.2

782.5
1104.3
512.9
626.7
484.8
1084.5

Æ
Æ
Æ
Æ
Æ
Æ

72.6
22.1
71.6
79.8
28.1

73.9

87.0
113.4
58.9
57.7
74.2
146.7

Æ
Æ
Æ
Æ
Æ
Æ

5.8
13.4
7.0
19.4
7.4
12.7

17.3
21.6
11.6
19.4
16.6
22.7

Æ
Æ
Æ
Æ
Æ
Æ

1.4
2.1
0.6
2.3
3.4
2.5

2.5
4.4
1.2
0.9
3.7
3.6

Æ
Æ
Æ
Æ
Æ
Æ

0.1
0.4

0.3
0.2
0.9
0.5

5.0
9.9
2.9
2.3
15.5
20.5

Æ
Æ
Æ
Æ
Æ
Æ

0.6
0.4
0.5
0.3
2.8
3.3

89.6
95.5
89.6
66.5

67.1
83.8

28.8 Æ 0.7
31.8 Æ 1.8

1200.6 Æ 75.9
1142.4 Æ 88.0

155.5 Æ 1.4
174.8 Æ 12.3

16.6 Æ 0.5
16.2 Æ 1.6

4.0 Æ 0.8
2.6 Æ 0.3

22.7 Æ 0.9
6.8 Æ 0.6

90.5
92.1

14.5 Æ 2.5

465.3 Æ 65.2

52.2 Æ 6.6

10.8 Æ 1.6

1.2 Æ 0.2

4.0 Æ 0.5

79.3

Æ
Æ
Æ
Æ

59.6
69.8
87.9
91.0
81.7

10.2
11.9
19.1
22.1
22.1

Æ
Æ
Æ
Æ

1.2
0.5
1.5
1.3

377.0
419.8
754.6
874.3
756.1

Æ
Æ
Æ
Æ

38.9
26.2
86.5
244.0

45.5
50.8
80.6
99.9
92.1

Æ
Æ
Æ

Æ

5.7
1.5
14.4
22.2

9.4
9.2
16.6
15.1
15.6

Æ
Æ
Æ
Æ

1.1
0.4
0.3
1.1

1.0
1.4
2.8
2.8
2.5

Æ

Æ
Æ
Æ

0.1
0.4
0.5
0.2

3.3
3.9
5.7
6.0
8.3

0.5
0.8
0.6
0.6

ÃValues have been averaged and were given as means Æ SD (n 510).

TPM, total particulate matter; POC, particulate organic carbon; PON, particulate organic nitrogen; TP, total phosphorus; Chl a, chlorophyll a.

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17

Aquaculture Research, 2010, 42, 14–21

Sedimentation in sea cucumber culture ponds Y Ren et al.

1.2

POC contents in the sediment, mgg–1

10

PON
TP

1.0
.8
.6
.4
.2
0.0

Time

Figure 5 Annual particulate organic nitrogen (PON)
and total phosphorus (TP) contents in the sediment of
sea cucumber culture ponds. The values have been averaged per month. Values were given as means Æ SD
(n 510).
Chl a and pheophytin contents, ugg–1

The ponds investigated had the same sediment quality before the sea cucumber stocking. In April 2007,
the sediment contents of POC, PON and TP in Pond 1

were 2.5 Æ 0.2, 0.2 Æ 0.0 and 0.7 Æ 0.0 mg g À 1, respectively, and the POC, PON and TP contents in the
sediment of Pond 2 were 2.5 Æ 0.1, 0.2 Æ 0.0 and
0.7 Æ 0.0 mg g À 1 respectively. In the pond without
sea cucumber, the sediment contents of POC, PON
and TP were 2.7 Æ 0.1, 0.2 Æ 0.1 and 0.6 Æ 0.0
mg g À 1 respectively. During 1 year of culture, no signi¢cant di¡erences were observed in the contents of
POC, PON and TP in the sediment between Pond1and
Pond 2 by monthly analysis (P40.05). In April 2008,
when the experiment was completed, the sediment
contents of POC, PON and TP in Pond 1 were
3.4 Æ 0.2, 0.4 Æ 0.0 and 0.7 Æ 0.0 mg g À 1, respectively, and in Pond 2 they were 3.4 Æ 0.1, 0.4 Æ 0.0
and 0.6 Æ 0.1mg g À 1 respectively. Nevertheless, the
sediment contents of POC, PON and TP in the pond
without sea cucumber were 10.3 Æ 0.5, 1.2 Æ 0.0
and 0.9 Æ 0.1mg g À 1, respectively, which were signi¢cantly higher than those in sea cucumber culture
ponds (Po0.05). The mean POC contents in the sediment of the sea cucumber culture ponds ranged from
2.4 to 7.7 mg g À 1 (Fig. 4), the PON contents ranged
from 0.2 to 0.9 mg g À 1 and the TP contents ranged
from 0.3 to 1.0 mg g À 1 (Fig. 5). Two peaks occurred
in summer and winter for all above matters in the
two sea cucumber culture ponds. The pheophytin
contents in the sediment of the sea cucumber culture
ponds were signi¢cantly higher than that of Chl a
(Po0.05) (Fig. 6). Chlorophyll a and pheophytin con-

PON and TP contents, mgg–1

Sediment characteristics of the ponds

100

Sea cucumber
Without sea cucumber

80

60

40

20

0

Chl a

Pheophytin

Figure 6 Mean Chl a and pheophytin contents in the sediment of sea cucumber culture ponds and the pond without sea cucumber during the investigated period. Values
were given as means Æ SD (n 5 60).

tents in the sediment of sea cucumber culture ponds
were signi¢cantly lower than those in the pond without sea cucumber (Fig.6). The annual mean sediment
contents of POC, PON and TP of the two sea cucumber culture ponds were 3.9, 0.4 and 0.6 mg g À 1, respectively, however they were 8.0, 0.9 and
0.8 mg g À 1, respectively, in the pond without sea cucumber. Thus, the annual mean POC, PON and TP
contents in the sediment of the sea cucumber culture
ponds were signi¢cantly lower than those in the
pond without sea cucumber (Po0.05) (Fig.7).

8

6

4

2

0

Time

Figure 4 Annual particulate organic carbon (POC) contents in the sediment of sea cucumber culture ponds. Values have been averaged per month. The values were
given as means Æ SD (n 510).

18

Discussion
Previous studies have shown that OM deposition
is a key process in the open sea (Takahashi 1986;

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POC, PON and TP contents, mgg–1

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Sedimentation in sea cucumber culture ponds Y Ren et al.

Sea cucumber
Without sea cucuber

8

6

4

2

0

POC

PON

TP

Figure 7 Mean particulate organic carbon (POC), particulate organic nitrogen (PON) and total phosphorus (TP)
contents in the sediment of sea cucumber culture ponds
and the pond without sea cucumber during the investigated period.Values were given as means Æ SD (n 5 60).

Danovaro et al. 2000), and the quality and quantity of
OM input to the sediment from the water column
is very important for macrobenthic production
(Gre’mare, Amoroux, Charles, Dinet, Riaux-Gobin,
Baudart, edernach, Bodiou, Ve’tion, Colomines &

Albert 1997). The present study showed that in sea
cucumber culture ponds, the TPM sedimentation
rates were high, which ranged from 10.2 to
38.3 g m2 day À 1. There was a signi¢cant correlation
between the POC contents in TPM and the biomass
of phytoplankton (r 5 0.822, P 5 0.001). In the sea
cucumber culture ponds, the Chl a and pheophytin
sedimentation rates reached 2.5 and 8.4 mg m À 2
day À 1, respectively, which implied that phytoplankton took a certain proportion of the settling particulate matter. Diatoms in the water column occupied
490% of the phytoplankton during spring and
autumn that could provide good-quality food to sea
cucumber after settling to the bottom. The Chl a
£ux in the two sea cucumber culture ponds was
4.0 Æ 0.8 mg m À 2 day À 1 in October and it was
4.4 Æ 0.4 mg m À 2 day À 1 in May, which indicated
that there were many diatoms settling to the bottom
from the water layer.
The sea cucumber culture ponds were shallow;
therefore, the re-suspension of the sediment was not
a negligible process. In the present study, the re-suspension rates of settling particulate matter ranged
from 60.0% to 95.5%, which was similar to the values
found in Erie Lake (55^95%), the Peel-Harvey Estuary (69^92%) and the coastal areas of East China
Sea (72.75^96.96%) respectively (Gabrielson & Lukatelich 1985; Sun & Zhan 2002). The wave and tide driven by wind are the main causes of re-suspension of

sedimentation matter (Rhoads 1974). In this study,
the re-suspension rates were lower in summer and
winter than those in spring and autumn due to weak
wind in summer and ice covering and less phytoplankton in winter (Table 1).
Research on shallow areas of water showed that
the particles collected by traps were complex (Lundagaard & Olesen 1997), which contained both newly

born particles and re-suspension ones. Generally, a
lower C/N of the particles implies that there is a high
portion of unstable OM in the settling particles (Nickell, Black, Hughes, Overnell, Brand, Nickell, Breuer &
Harvey 2003). In this study, the mean C/N of the settling particulate matter in the trap samples ranged
between 9.6 and 12.7, and the values in autumn
(12.1^12.7) were signi¢cantly higher than those in
winter (9.6^9.8). The settling particles in autumn
had undergone more cycles of sedimentation and resuspension processes, leaving more stable OM in the
settling particles, and might be less nutritional for
sea cucumber.
In this study, the average TPM £ux in the ponds
was 22.1g m2 day À 1, comprising 756.1mg C, 92.1mg
N and 15.6 mg P, and it was 10 times lower than that
in Sungo Bay, China, where ¢lter-feeder scallop was
cultured (Cai, Fang & Liang 2003). Sedimentation
plays an important role in the energy transformation
from primary producers to benthic consumers (Lesser 2006). In a high density of sea cucumber culture
ponds, only natural sedimentation might not supply
su⁄cient food for sea cucumber. In practice, polyculture of sea cucumber with scallop or jelly¢sh is an
e⁄cient way to accelerate the sedimentation of organic particles and improve the productivity of sea
cucumber (Zhou et al. 2006; Zheng, Dong,Tian,Wang,
Gao & Bai 2009). The polyculture of sea cucumber
with ¢lter feeders can not only accelerate sedimentation but also alleviate nutrient loadings of nearby
coasts e¡ectively.
The sea cucumber grew roughly from April to
early June, after aestivation from July to September,
grew again from October to December and then they
went into hibernation from January to March. In this
study, the POC, PON and TP contents in the sediment
of sea cucumber culture ponds increased during the

period of sea cucumber aestivation and hibernation,
while in contrast, they decreased during the feeding
periods. In shrimp and ¢sh culture ponds, OM accumulation is frequent at the bottom (Allan, Moriarty &
Maguire 1995); however, OM accumulation and the
contents of POC, PON, TP, Chl a and pheophytin in
the sediment of sea cucumber culture ponds were

r 2010 The Authors
Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21

19


Sedimentation in sea cucumber culture ponds Y Ren et al.

even lower than those in the pond without sea cucumber (Po0.05) (Fig. 7). The results demonstrated
that sea cucumber culture can e¡ectively stop nutrient accumulation at the bottom of the culture ponds.

Acknowledgments
This work was supported by National Projects
of Scienti¢c and Technical Supporting Programs of
China (No. 2006BAD09A01), Hi-Tech Research and
Development Program of China (No. 2006AA
10Z409) and Natural Science Fundation of China
(No. 30871931). We thank Fisheries Research Institute, Enterprise Homey Group, for helping with the
experiments.

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21


Aquaculture Research, 2010, 42, 22^30

doi:10.1111/j.1365-2109.2010.02488.x

Cryopreservation of sperm from natural and
sex-reversed orange-spotted grouper
(Epinephelus coioides)
Taweesin Peatpisut & Amrit N Bart
Aquaculture and Aquatic Resources Management, Asian Institute of Technology, Pathumthani,Thailand
Correspondence: A N Bart, Aquaculture and Aquatic Resources Management, Asian Institute of Technology, PO Box 4, Klong Luang,
Pathumthani 12120,Thailand. E-mail:

Abstract

Introduction

The shortage of males and/or sperm has been an impediment to the aquaculture of orange-spotted
grouper (Epinephelus coioides). This study reversed orange-spotted grouper females into males using hormone implants. A cryopreservation protocol for
sperm was developed using normal males, and then
using similar procedures the cryopreservation of
sperm from sex-reversed males was compared. Immature, young and mature female ¢sh were injected
with 4 mg kg À 1 BW 17a methyltestosterone as implants and the gonad development stage was monitored over a 120-day period. All treated females
converted into functional males within 120 days of
the experimental period. Younger females (2Y) were
all males within 30 days, although not all were capable of fertilizing fresh ova until day 60. The time
after injection to sex reversal in immature ¢sh was
50% shorter than in older females. Postthaw fertilization (81%, 82%) and hatching (45%, 47%) of cryopreserved sperm from natural males were the highest
in trehalose (15^20%) with 150 mmol NaCl treatment; however, it was less than the control (89% fertilization and 69% hatch). There was no di¡erence in
the postthaw fertilization and the hatch percentages
between sex-reversed male sperm (64% and 46% respectively) compared with natural male sperm (59%
and 49%). The ¢ndings of this study suggest the potential use of sex-reversed males and cryopreserved
sperm for commercial production of orange-spotted
grouper seed for aquaculture.

Orange-spotted grouper (Epinephelus coioides) is an
important commercial aquaculture species in Southeast Asia (Boonyaratpalin 1997; Millamena 2002).
However, its culture is constrained by an inconsistent
supply of seed to meet the increasing aquaculture
demand (Liao & Leanìo 2008). Because of the protogynous hermaphroditic nature of this species, obtaining an adequate number of mature males from the
wild has been problematic (Quinitio, Caberoy & Reyes
1997). Maintaining ¢sh in a hatchery for breeding
would increase the number of males but the time

and cost of maintaining ¢sh for 6^8 years before sex
inversion would naturally make it economically prohibitive (Quinitio et al.1997). Moreover, the volume of
expressible milt is low in many species during the o¡
peak spawning period. Some authors have successfully increased the volume of milt by inducing males
using luteinizing hormone releasing hormone analogue (LHRHa) (Barry, Castanos & Fast 1991).
One of the solutions to produce more spermiating
males is hormonal therapy. Several studies have
reported a successful induction of sex reversal in protogynous hermaphrodites by treatment with androgens (Chen, Chow, Chao & Lim 1977; Chao & Chow
1990; Tan-Fermin, Garcia & Castillo 1994). Sex reversal by hormonal treatment have been successful in
orange-spotted grouper and dusky grouper (Epinephelus marginatus; Yeh, Kuo, Ting & Chang 2003c;
Sarter, Papadaki, Zanuy & Mylonas 2006). Although,
a successful sex change in 2-year-old orange-spotted
grouper was possible using a17a methyl-testosterone
implant (1000 mg kg À 1 BW), the quality of the sperm
was not assessed (Yeh, Kuo, Ting & Chang 2003b).
Sex reversal of potato grouper, Epinephelus tukula, in

Keywords: sperm cryopreservation, sex-reversed,
orange-spotted grouper, sperm quality, Epinephelus
coioides

22

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Aquaculture Research, 2010, 42, 22^30

Cryopreservation of sperm from Epinephelus coioides T Peatpisut & A N Bart

vitellogenic females was also reported (Yeh, Dai, Chu,
Kuo, Ting & Chang 2003a). While sex reversal using
androgen treatment has been successful in a number
of protogynous hermaphroditic species, these studies
examined only a single age group (i.e. Yeh et al.
2003b, c; Sarter et al. 2006). There has not been a
study to examine reversal e⁄ciency with varying age.
Cryopreserved sperm would be another solution to
the timely availability of functional sperm for fertilization and fry/¢ngerling production. Cryopreserved
sperm of orange-spotted grouper would allow yearround seed production, reduce the number of males
needed in the hatchery and facilitate arti¢cial propagation. Although cryopreservation techniques have
been well established for many ¢sh species, only a limited number of studies have been carried out on
groupers. Spermatozoa cryopreservation has been
achieved in greasy grouper (Epinephelus tauvina;
Withler & Lim1982), malabar grouper (Epinephelus malabaricus; Chao,Tsai & Liao 1992; Gwo 1993) and k elp
grouper (Epinephelus moara; Miyaki, Nakano, Ohta &
Kurokura 2005).These studies were carried out under
laboratory conditions and motility was used as the
only indicator of success. The postthaw fertilization,
hatch and survival rates of larvae from frozen-thawed
spermatozoa are often lacking. Moreover, only one
study on dusky grouper reported the successful cryopreservation of sperm from sex-reversed males (Cabrita, Engrola, Conceic°aìo, Pousaìo-Ferreira & Dinis 2009).
Selection of appropriate cryoprotectants is important to successful cryopreservation of spermatozoa.
There are many cryoprotectants successfully used in
freezing protocols including dimethyl sulphoxide
(DMSO) and propylene glycol (PG; Billard, Cosson &
Crim 1993; Gwo 1993, 1994; Richardson, Crim, Yao &
Short 1995; Cabrita et al. 2009). Trehalose, a novel
cryoprotectant, has been used in studying only a limited number of freshwater species (Miyaki et al. 2005),

but has not been tested in marine species.
Year-round availability of spermiating functional
males and/or sperm of orange-spotted grouper are
critical to the commercial production of this species.
The aim of this study was to: (1) better understand
the age-related sex reversal e⁄ciency and (2) to develop a simple protocol for sperm cryopreservation of
sex-reversed orange-spotted grouper.

spawning season based on their age and health. The
experimental ¢sh were maintained and prepared for
reproduction in a 30 m3 circular concrete tank
(+5 m and 1.6 m deep) connected to a recirculating
system in the Aquaculture and Aquatic Resources
Management (AARM) hatchery at the Asian Institute
of Technology (AIT),Thailand. Fish were maintained
in saline water (30%) at 26^30 1C and fed to satiation
every other day with frozen whole yellow striped scad
(Selaroides leptolepis). Moreover, once a week a vitamin E (400 IU) capsule (Mega, Samutprakarn, Thailand) was inserted in the frozen ¢sh before feeding.
The experiment comparing an age group e¡ect on
sex-reversal was initiated at the end of August 2006,
while cryopreservation experiments were initiated in
early January 2007. Fish were selected from the pool
of 30 for sex reversal and cryopreservation studies.
Sampled ¢sh were anaesthetized with 50 mg L À 1 of
benzocaine for 3 min or until the opercular movement was signi¢cantly reduced. Fish were canulated
using a £exible polyethylene tube through the genital
pore and examined microscopically to determine
their sex stage (Fig. 1). The age of the ¢sh was determined based on hatchery data. Fifteen 1.5^4.5-yearold females (2.41^8.73 kg) at the perinucleolus oocyte
stage (transparent oocyte, 30^130 mm diameter) were
chosen for the experiment. The selected ¢sh were

divided into three age groups of ¢ve individuals
each of 1.5^2.5 years (2.56 Æ 0.21kg), 2.6^3.5 years
5.05 Æ 1.04 kg) and 3.6^4.5 years (7.26 Æ 0.95) and
identi¢ed as group 2, 3 and 4Y respectively. Experimental females were individually weighed and tagged
(Fish eagle PIT tags, Biomark, ID, USA). In each group,
three females were implanted with methyltestosterone (MT), and two ¢sh served as controls.

Materials and methods
Experimental ¢sh and design
Thirty orange-spotted groupers cultured in cages
were purchased from several farms during the post-

Figure 1 Gonadal tissue was sampled by passing a catheter through the genital pore and gently vacuuming the
gonadal lining. The tissue was examined microscopically
to determine the sex stage.

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23


Cryopreservation of sperm from Epinephelus coioides T Peatpisut & A N Bart

Induced sex change
The hormone pellet for implantation was prepared by
mixing 350 mg of 17a MT, 1mL of 80% ethanol,
190 mg of cholesterol powder (mixture of 95^100%
cholesterol and 0^5% animal lard) and 10 mg of cocoa butter (modi¢ed from Lee, Tamaru & Kelly 1986).
The mixture was passed through a press pelletizer

and the pellets were cut to achieve 10 mg per pellet
with 35 total pellets.While the smallest ¢sh (2.41kg)
received a single pellet, the largest ¢sh (8.73 kg) were
implanted with 3.5 pellets. The MT implant containing 4.0 mg kg À 1 BW was injected intramuscularly
below the base of dorsal ¢n. The control ¢sh were implanted with blank implants.

The examination of functional males
On days 30, 60, 90 and 120 after implantation with
MT, the gonadal stages of the treated ¢sh were determined. During the early morning period, gonadal tissue was collected using a plastic canulation catheter
(Feeding Tube CH 6, City Medical Supply, Bangkok,
Thailand). The catheter was passed through the genital pore to remove the gonadal lining. The biopsy was
examined under a microscope and photographed
using a digital camera. The gonadal stage was classi¢ed into F1, F2, F3 and I stages in females. Males were
classi¢ed into M1 and M2 stages (Fig. 2).

Aquaculture Research, 2010, 42, 22–30

F1, presence of transparent oocytes (30^130 mm
oocyte diameters).
F2, presence of some dark oocytes (130^250 mm
oocyte diameters).
F3, presence of oocytes with transparent circle surrounding the yolk (4400 mm diameter).
I, presence of a few sperm in the biopsy tissue.
M1, presence of sperm cells but not motile upon
activation.
M2, presence of motility competent sperm cells.

Sperm cryopreservation
Collection and cryopreservation of sperm from natural
and sex-reversed males

Spermiation was induced 72 h before stripping males
s
by implanting pellets containing LHRHa (Suprefact ,
Hoechst AG, Maine, Germany) at 10 mg kg À 1. The pellets were prepared using a similar procedure as
described for MT. Pellets were implanted intramuscularly behind the dorsal ¢n. From the pool of remaining 15 ¢sh, three males of 8 years old (10.2^12.5 kg)
were selected as donors of sperm for the cryopreservation experiment. The sperm from three males was
collected by applying gentle pressure to the abdominal area along the midline and aspirated with a micropipette (Pipetman, Gilson, France). Caution was
exercised to prevent contamination of the sperm with

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2 The ovary of a female orange-spotted grouper: (a) F1 stage: presence of transparent oocytes (Â10), (b) F2 stage;
presence of dark oocytes (Â10) and (c) F3 stage; presence of oocytes with transparent circle surround the yolk plate (Â10),
(d) I stage: presence of some recognizable sperm cells in the biopsy tissue (Â10), (e) M1 stage: presence of non-motile sperm
cells (Â10) and (f) M2 stage: presence of motility competent sperm cells (Â40).

24

r 2010 Blackwell Munksgaard
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