The influence of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii

Cryobiology 46 (2003) 190–193
www.elsevier.com/locate/ycryo
Brief communication
The in?uence of cooling rate, developmental stage, and the
addition of sugar on the cryopreservation of larvae
of the pearl oyster Pinctada fucata martensiiq
Youn Hee Choi and Young Jin Chang*
Department of Aquaculture, Pukyong National University, 599-1 Daeyeon-3-dong, Nam-gu, Busan 608-737, Republic of Korea
Received 6 September 2002; accepted 21 January 2003
Abstract
This paper examines the e?ects of cooling rate, developmental stage, and the addition of sugar on the cryopreser-
vation of the larvae of the pearl oyster, Pinctada fucata martensii. The survival rates of frozen–thawed trochophores was
43.1% at a cooling rate of 1.0 °C/min. The survival rate of frozen–thawed larvae increased with developmental stage,
except for umbo stage larvae, and the late D-shaped larvae showed a survival rate as high as 91%. The addition of sugar
(0.2 M glucose or sucrose) improved the survival rate of larvae. These results indicate that the preferred cooling rate,
developmental stage, and sugar for the cryopreservation of pearl oyster larvae are 1 °C/min, late D-shaped larvae and
0.2 M glucose or sucrose.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Pearl oyster; Pinctada fucata martensii; Cooling rate; Developmental stage; Sugar
Cells are, in general, damaged severely during
Mytilus edulis [10] and the Paci?c oyster, Cras-
freezing and thawing. An appropriate cooling rate
sostrea gigas [2]. The optimal cooling rate for the
is necessary to avoid intracellular freezing: Mazur
cryopreservation of bivalve embryos and larvae
[5] proved that survival rate depends on the cool-
depends on the species as well as the use of seed-
ing rate, subject to the type of cell. For many
ing. In particular, the survival rate of thawed
mammalian cells the appropriate cooling rate has
embryos depends on the developmental stage; in
been de?ned, and in bivalve studies the preferred
general, late bivalve embryos show a higher sur-
cooling rate has been reported for the blue mussel,
vival rate than early embryos [2,10]. There are few
studies of cryopreservation in larvae, especially of
late larval stages, such as the D-shaped larvae and
q This work was supported by Grant No. R01-2000-000-
umbo stage larvae, which resemble the mother
00214-0 from the Basic Research Program of the Korea Science
bivalve. The addition of sugar is known to be one
and Engineering Foundation.
*
of factor that could improve the survival of
Corresponding author. Fax: +82-51-628-7430.
E-mail address: yjchang@pknu.ac.kr (Y.J. Chang).
cryopreserved embryos, and there are reports of
0011-2240/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0011-2240(03)00016-6

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Y.H. Choi, Y.J. Chang / Cryobiology 46 (2003) 190–193
191
the e?ects of glucose, sucrose, and trehalose in
)12 °C. Seeding was induced by dipping the straws
bivalves [1,2,8,9]. Most of these studies in bivalves
into liquid nitrogen. The straws were then cooled at
concerned Paci?c oyster embryos. A study is nee-
a rate of 1 °C/min from )12 to )35 °C at which
ded to investigate the e?ect of adding sugars to the
temperature they were quickly immersed in liquid
solutions used for the preservation of the embryos
nitrogen and held there for one hour. The straws
and larvae of various bivalve species. The aim of
were thawed in a water bath at 25 °C for 10 s and
the present paper is to describe the in?uence of
after thawing, the larvae were transferred to a glass
cooling rate, developmental stage, and the addi-
tube to which fresh arti?cial seawater (ASW) was
tion of sugar for the cryopreservation of larvae of
gradually added to avoid osmotic shock. ASW
the pearl oyster, Pinctada fucata martensii.
contained NaCl 2.7 g, KCl 0.07 g, NaHCO3 0.05 g,
Broodstock of arti?cial sex-matured pearl oys-
CaCl2 0.12 g, MgCl 0.46 g, and distilled water
2
ters were obtained in March and April from the
100 ml. The larvae were fed mixed diets of I. gal-
Nam-Hae Marine Hatchery, Korea and were
bana, C. calcitrans, and P. lutheri.
cleaned and placed in a 1000 L tank containing
To investigate e?ect of developmental stage, the
gently aerated, ?ltered seawater until spawning
following seven developmental stages were used;
induction. The oysters were checked before trans-
early and late
trochophore, early
and late
fer into the tank and their shell length (SL), shell
D-shaped larvae, and early, middle, and late umbo
height (SH), and shell width (SW) were measured
larvae. Larvae were obtained at 13 h, 17 h, 1 day, 3
by a vernier caliper: total wet weight (TW) was
days, 5 days, 7 days, and 12 days after fertilization,
measured by electrical balance. The data were as
respectively. The cryoprotectant was 2.0 M Me2SO
follows:
SL ¼ 64:6 Æ 6:0 mm,
SH ¼ 70:5 Æ 5:8
mixed with 0.2 M sucrose. Larvae were cryopre-
mm, SW ¼ 23:8 Æ 1:9 mm, and TW ¼ 42:7 Æ 9:3
served using the freezing and thawing protocol
g. The broodstock were placed in the shade for 2 h
described above. To study the e?ect of di?erent
and were then subjected to thermal stimulation by
sugars, sucrose was replaced by fructose or glucose
heating the seawater from 23 to 29 °C for 1 h in a
in some experiments and the cryoprotectant con-
50 L tank. The fertilized eggs were incubated in a
centrations were 1.0, 2.0, 2.5, or 3.0 M Me2SO.
60,000 L tank at 26 °C until the cryopreservation
The sugar concentrations were 0.2 or 0.5 M. D-
experiment was carried out. The rearing of larvae
shaped larvae (early stage: SL 78:0 Æ 1:6 lm and
was carried out at the hatchery and the larvae were
SH 68:8 Æ 3:4 lm, late stage: SL 83:8 Æ 4:6 lm and
then transported to the laboratory for all the
SH 72:6 Æ 5:1 lm) were used in this experiment
cryopreservation experiments. Larvae and post-
and were cryopreserved using the freezing and
thawed larvae were fed a mixture of Isochrysis
thawing protocol described above. In each exper-
galbana, Chaetoceros calcitrans, and Pavlova lu-
iment, the survival rate of frozen–thawed trocho-
theri. SL and SH of larvae were calculated using a
phore and D-shaped larva or umbo stage larva
computer picture analysis program (Matrox Elec-
was determined by light microscopy after 1 h of
tronic Systems, Canada).
supplying food. The trochophore larvae were es-
At 13–17 h after fertilization, trochophore larvae
timated by counting the larva showing rotary
(60:5 Æ 1:6 lm) were equilibrated with a freezing
motion with cilia, while the D-shaped and umbo
medium composed of 0.2 M sucrose and 2.0 M di-
stage larvae were calculated by counting those
methyl sulfoxide (Me2SO) for 15 min at room
showing a beating heart and active cilia. The sur-
temperature (20–22 °C) and transferred into 0.5 ml
vival rates of larvae in cryopreservation experi-
straws (FHK, Japan) at room temperature. The
ments were expressed as means. The signi?cance of
straws were immediately placed in a programmed
di?erences between mean survival rates for each
freezer (Samwon Freezing Engineering, Korea)
factor were tested by DuncanÕs multiple range
that was set to an initial temperature of 0 °C. The
post-hoc test. Di?erences with a probability value
larvae were cooled at rates of 0.25, 0.5, 0.75, and
(P ) of 0.05 or less were considered signi?cant.
1 °C/min from 0 °C to )12 °C and the larvae were
No survival was observed with 1.0 M Me2SO
kept at )12 °C for a period of 10 min after seeding at
with cooling rates of 0.25, 0.5, 0.75, and 1 °C/min,

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192
Y.H. Choi, Y.J. Chang / Cryobiology 46 (2003) 190–193
whereas with 2.0 M Me
Table 1
2SO survival was observed
at all cooling rates, 1 °C/min giving the highest
E?ect of sugars on the cryopreservation of early D-shaped
larvae (SL 78:0 Æ 1:6 lm and SH 68:8 Æ 3:4 lm)
survival (43%). The other cooling rates gave sur-
vivals between 12:6 Æ 3:0% and 21:4 Æ 4:6%. With
Sugar
Concentration
Survival rate
the Paci?c oyster, the published optimal cooling
of Me2SO (M)
(Means Æ
Kind
Concentration
SEM, %)
rate and seeding temperature were di?erent from
(M)
our results, that is 2 and )12 °C [2], 0.3 and )6 °C
Fructose
0.2
1.0
6:7 Æ 2:2d
[8], 1.5 and )7 °C [4], and 2 and )7 °C [7]. With the
2.0
40:2 Æ 4:5a
blue mussel the optima were 0.5 and )5 °C [10].
0.5
1.0
3:7 Æ 0:4d
The survival of frozen–thawed larvae was depen-
2.0
20:5 Æ 7:4c
dent on the developmental stage and increased
Glucose
0.2
1.0
0d
signi?cantly with the stage of development up to
2.0
44:3 Æ 5:4a
D-shaped larvae but decreased during develop-
0.5
1.0
0d
2.0
38:0 Æ 6:3a
ment to the next and the umbo stage (P < 0:05).
The highest survival rate was obtained with late
Sucrose
0.2
1.0
0d
D-shaped larvae (87% survival) (Fig. 1). Chao et al.
2.0
29:3 Æ 8:6b
0.5
1.0
0d
[2] emphasized that the most important factor for
2.0
39:2 Æ 8:8a
successful cryopreservation is the quality of the
Values with di?erent superscripts within a column di?er
embryo. The e?ect of including sugars di?ered
(P < 0:05).
with the concentration of the cryoprotectant (Ta-
ble 1) and the survival rate was signi?cantly higher
with 2.0 M Me2SO than with 1.0 M Me2SO
(P < 0:05). The e?ect of the sugar depended on the
larval stage; glucose and sucrose had extremely
positive e?ects with late D-shaped larvae. Survival
Fig. 1. Survival rate of frozen–thawed larvae of pearl oyster
(Pinctada fucata martensii) at each developmental stage,
cryopreserved with 2.0 M Me2SO and 0.2 M sucrose. ET, early
trochophore; LT, late trochophore; ED, early D-shape larva
(SL 78:0 Æ 1:6 lm and SH 68:8 Æ 3:4 lm); LD, late D-shaped
arva (SL 83:8 Æ 4:6 lm and SH 72:6 Æ 5:1 lm); EU, early umbo
stage larva (SL 95:1 Æ 8:3 lm and SH 88:3 Æ 8:9 lm); MU,
middle
umbo
stage
larva
(SL
112:3 Æ 8:7 lm and SH
Fig. 2. Survival rates of frozen–thawed late D-shaped larvae of
103:5 Æ 9:6 lm);
and
LU,
late
umbo
stage
larva
(SL
pearl oyster (Pinctada fucata martensii) cryopreserved with
185:6 Æ 21:1 lm and SH 174:8 Æ 16:6 lm). Di?erent alphabetic
Me2SO and sugar. Di?erent alphabetic letters on the bars are
letters on the bars are signi?cantly di?erent (P < 0:05).
signi?cantly di?erent (P < 0:05).

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Y.H. Choi, Y.J. Chang / Cryobiology 46 (2003) 190–193
193
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Document Outline

• The influence of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii

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