Aquaculture 246 (2005) 173 179

www.elsevier.com/locate/aqua-online

Effect of temperature on incubation period and hatching success of

obscure puffer Takifugu obscurus (Abe) eggs
Zhou Yanga,b,c,*, Yafen Chenb
a

Jiangsu Key Laboratory of Bioresource Technology, School of Biological Sciences, Nanjing Normal University, 122 Ninghai Road,
Nanjing 210097, Peoples Republic of China
b
Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing 210008,
Peoples Republic of China
c
Graduate School, Chinese Academy of Sciences, Beijing 100039, Peoples Republic of China
Received 14 September 2004; received in revised form 14 December 2004; accepted 17 December 2004

Abstract
Artificially fertilized eggs of obscure puffer Takifugu obscurus were obtained by induced spawning of cultured broodstock
and incubated at temperatures of 15, 19, 23, and 27 8C. The results showed that the optimal temperature for obscure puffer
embryonic development ranged from 19 to 23 8C, based on total hatch rate, viability of newly hatched larvae 24 h post-hatch,
and total mortality rate of eggs. At the given temperature range, the times taken for 50% embryos to hatch were 11.3, 6.6, 5.0,
and 4.2 days, respectively. There was significant difference in time to 50% hatch among the temperatures used in this
experiment. The power law model, quadratic equation, exponential equation, and effective degree-day model all provided good
fits for the relationship between incubation temperature and time to 50% hatch, with r 2 values greater than 0.90. The formulae
for these were y=1031.7T 1.6885, y=44.721 3.1574T+0.0615T 2, y=34.663e 0.0813T and y=78.905/(T 7.6033), respectively,
where y is time to 50% hatch in days, and T is incubation temperature in degrees Celsius. The effective degree-day model was
determined to be the best model because of efficient computation, good fit to the experimental data, and most importantly, the
derived parameters, k (the sum of effective degree-days) and t 0 (the temperature of biological zero), have important biological
meaning. Based on the effective degree-day model, the t 0 and k values were calculated as 7.6033 8C and 78.905 degree-days,
respectively.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Obscure puffer; Takifugu obscurus; Temperature; Embryonic development; Time to 50% hatch; Hatching success

1. Introduction
* Corresponding author. Jiangsu Key Laboratory of Bioresource
Technology, School of Biological Sciences, Nanjing Normal
University, 122 Ninghai Road, Nanjing 210097, Peoples Republic
of China. Tel.: +86 25 83598916; fax: +86 25 57714759.
E-mail address: yangzhouff@vip.sina.com (Z. Yang).
0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.12.030

There are approximately 100 different species of

puffer fish in the world. Some species, such as
bullseye puffer Sphoeroides annulatus (Duncan et

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Z. Yang, Y. Chen / Aquaculture 246 (2005) 173179

al., 2003; Komar et al., 2004), tiger puffer Takifugu

rubripes (Miyaki et al., 1992; Chuda et al., 1997;
Matsuyama et al., 1997), purple puffer Takifugu
porphyreus (Fujita and Abe, 1992), brown puffer
Takifugu exascurus (Fujita and Honma, 1991), and
obscure puffer Takifugu obscurus (Yang and Chen,
2004, in press; Yang and Yang, 2004), have been
studied with regard to seeding production because
they appear to be promising aquaculture species.
Most species inhabit marine or coastal waters
throughout their life cycle, and only a few species,
such as the obscure puffer T. obscurus, are
anadromous. They migrate to freshwater rivers for
reproduction during the spawning season of February to May. The newly hatched larvae remain in
freshwater for several months before they emigrate
to sea. Most feeding and growth of subadults take
place at sea over several years. Approaching
maturity, the adults return to freshwaters to spawn
(Yuan and Xie, 1986). Obscure puffer is a fish with
considerable commercial importance in China owning to its high-quality meat, but overexploitation and
environmental degradation are diminishing its natural populations (Yang and Chen, 2003). Although
we have successfully developed the technique of
induction ovulation in both wild and cultured
obscure puffer in order to protect and increase
natural populations and meet the increasing demand
for consumption (Yang and Chen, 2004, in press),
little is known about the biological and ecological
requirements of this species as a basis for fishery
management, commercial cultivation, and replenishing natural populations.
An essential step in the successful culture of any
species is to understand the optimal environmental
conditions for egg incubation. Temperature is one of
the most decisive environmental variables affecting
embryonic development in fish eggs (Brannas, 1987;
Beacham and Murray, 1990; Baynes and Howell,
1996; Bermudes and Ritar, 1999; Kamler, 2002).
Therefore, determination of the optimal temperature
for obscure puffer egg incubation is necessary to
maximize the seeding production. The present
investigation is a part of a larger study of effects
of extrinsic factors on the hatching success and
survival of obscure puffer eggs. The result of this
study will be useful in improving the production in
hatcheries.

2. Materials and methods

2.1. Fertilized eggs collection
Artificially fertilized eggs of obscure puffer were
obtained by induced spawning of cultured broodstock maintained in 20 m2 concrete tanks. Twenty
individuals of 3-year-old females (above 0.65 kg
body weight) were injected intraperitoneally with
[d-Ala6-Pro 9-Net]-luteinising hormone releasing
hormone analogue (LHRH-a) at a dose of 30 Ag
kg 1 body weight (Yang and Chen, in press).
Multiple injections were given and the interval
between injections was 36 h. Beginning 2 days
after initiation of hormonal treatment, abdominal
palpation was performed every day to check
expansion and hardening of the abdomen due to
the hydration of oocytes which indicates completion
of final oocyte maturation. Most females ovulated
after the fourth LHRH-a injections (Yang and
Chen, in press). The eggs were stripped manually
and artificially fertilized. At 15 h post-fertilization,
when fertilized eggs were at the stage of middle
blastula, dead and physically damaged eggs were
removed using a wide-mouth pipette and only
developing fertilized eggs were placed into experimental units.
2.2. Conditions of incubation
Experiments were conducted in water baths equipped with thermoregulators and immersion heaters or
coolers. Experimental temperatures of incubation
were 15, 19, 23, and 27 8C. There were three
replicates for each of the 4 treatments. Positions of
temperature treatment replicates were randomized
within the water baths. Eggs were transferred and
counted using a wide-mouth pipette. Experimental
incubation units consisted of 100-ml glass beakers
filled with 50 ml sterilized freshwater. Eggs were
stocked at a number of 50 eggs per beaker. All
temperature gradients were adjusted at an appropriate
rate from initial temperature of 19 8C to their final
temperatures within 3 hours. Eggs were incubated
statically in the beakers under natural light and
photoperiod. Fifty percent of the incubation water in
each beaker was replaced daily with new sterilized
freshwater.

Z. Yang, Y. Chen / Aquaculture 246 (2005) 173179

For all replicates, mortalities were removed and

counted each day, until all the larvae were hatched.
At the same time, three eggs were sampled every
day from each replicate using a wide-mouthed
pipette and were observed quickly through a
dissecting microscope for determination of the
developmental stage. Sampled eggs were returned
to their respective incubation units. Total hatch rate
was determined as the percentage of stocked
embryos that hatched, regardless of viability. Newly
hatched larvae were removed and held in static
containers for a further 24 h to observe their
continued normal development and immediate
post-hatch viability. After 24 h, deformed and
moribund larvae were counted. Viability of newly
hatched larvae 24 h post-hatch was determined as
the percentage of total hatch that was alive and
normally developed at 24 h post-hatch. Total
mortality rate represented the combined percentage
of dead eggs, abnormal fry, and dead and moribund
larvae.
Frequency of observation must be increased when
eggs were at the stage of pre-hatching. To determine
the effect of temperature on the rate of embryonic
development, we adopted time to 50% hatch as an
index. Time to 50% hatch is defined as the time
interval from egg activation until 50% of the
fertilized eggs have hatched (Kamler, 2002). To
determine the time to 50% hatch (time in day in this
experiment), the percentages of eggs that have
hatched were monitored every few hours and the
times of these observations are recorded once
hatching began.
2.4. Statistical analysis
All data (total hatch rate, viability of newly
hatched larvae 24 h post-hatch, total mortality rate,
time to 50% hatch) were presented as meanF
standard error of mean (S.E.M.). The influence of
temperature on above indices was analyzed by
analysis of variance (ANOVA). Statistical significance was established at a=0.05. Total hatch rate
was described using a second-order polynomial.
Several models are available to describe the relationship between the hatching time and incubation

temperature (Hamel et al., 1997b; Kamler, 2002),

four of which were used in this study. These models
were as follows:
Power law model: y=aT b
Quadratic equation: y=a+bT+cT 2
Exponential equation: y=ae T
Effective degree-day model: y=k/(T t 0)
where y is time to 50% hatch in days, T is incubation
temperature in degrees Celsius, k is the sum of
effective degree-days, t 0 is the temperature at developmental zero, and a, b, and c are constants.

3. Results
3.1. Total hatch rate
Total hatch rates at 15, 19, 23, and 27 8C were
77.0%, 96.0%, 95.3%, and 92.7%, respectively.
Hatching success was significantly lower ( Pb0.05)
at 15 8C than at higher temperatures, but not
significantly different ( PN0.05) in hatching success
at 19, 23, and 27 8C. When total hatch rates were
assessed across the range of temperature (1527 8C)
and fitted with a second-order polynomial, 1923 8C
was optimal for hatching (Fig. 1).

100
90

Total hatch rate (%)

2.3. Data collection

175

80
70
y = -0.3385x2 + 15.377x - 76.601
R2 = 0.7764

60
50
40
10

15

20

25

30

Temperature (C)
Fig. 1. Total hatch rate for eggs incubated at different temperatures,
calculated as a percentage of eggs fertilized. The curve was a
second-order polynomial.

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Z. Yang, Y. Chen / Aquaculture 246 (2005) 173179

14

Time to 50% hatch (day)

Viability of newly hatched

larvae 24 h post-hatch (%)

ab

100
80

60
40

20

12
10
8
6
4
2
0

15

19

23

10

27

15

20

25

30

Temperature (C)

Temperature (C)
Fig. 2. Viability of newly hatched larvae incubated at different
temperatures. Vertical bars represent one standard error. Bars with
different superscripts denote significant difference at Pb0.05
(ANOVA).

Fig. 4. Relationship between incubation temperature and time to

50% hatch for obscure puffer embryos. The curves fitted according
to the power law model (thinner line), quadratic equation (thicker
line), and exponential equation (broken line), respectively.

3.2. Viability of newly hatched larvae 24 h post-hatch

3.3. Total mortality rate

Viability of newly hatched larvae 24 h post-hatch

at 15, 19, 23, and 27 8C were 95.7%, 98.6%, 99.3%,
and 99.3%, respectively. Viability of newly hatched
larvae 24 h post-hatch was significantly lower
( Pb0.05) at 15 8C than at 23 and 27 8C, while there
was no significant difference ( PN0.05) among 19, 23,
and 27 8C treatments (Fig. 2).

Total mortality rates at 15, 19, 23, and 27 8C were

35.3%, 4.6%, 4.0%, and 8.0%, respectively. Total
mortality rate was significantly higher ( Pb0.05) at 15
8C than at the other three temperatures, but not
significantly different ( PN0.05) at 19, 23, and 27 8C
(Fig. 3).

45

0.3

35
30
25
20
15

10

19

23

1/(Time to 50% hatch)

(day-1)

Total mortality rate (%)

40

0.2

0.1

5
0
15

27

Temperature (C)
Fig. 3. Total mortality rate of obscure puffer embryos incubated at
different temperatures. Vertical bars represent one standard error.
Bars with different superscripts denote significant difference at
Pb0.05 (ANOVA).

0.0
10

15

20

25

30

Temperature (C)
Fig. 5. Relationship between incubation temperature and rate of
embryonic development. The fitted line was based on the effective
degree-day model.

Z. Yang, Y. Chen / Aquaculture 246 (2005) 173179

3.4. Rate of embryonic development

Rate of embryonic development to hatch was
accelerated with increase in incubation temperature.
The times taken for 50% of the embryos to hatch at 15,
19, 23, and 27 8C were 11.3, 6.6, 5.0, and 4.2 days,
respectively. There were significant differences in time
to 50% hatch among all temperatures of this experiment. The power law model, quadratic equation,
exponential equation, and effective degree-day model
also provided accurate fits for the relationship between
temperature and developmental time to the hatch, with
r 2 values greater than 0.90 (Figs. 4 and 5). The
formulae for these were y=1031.7T 1.6885 , y=
44.721 3.1574T+0.0615T 2, y=34.663e 0.0813T and
y=78.905/(T 7.6033), respectively.

4. Discussion
Lower survival of obscure puffer eggs during early
development at 15 8C suggested that egg incubation at
or below this temperature is not suitable for this
species. Although there was no significant difference
in total hatch rate and total mortality rate among the
three higher temperatures, trend towards lower total
hatch rate and higher total mortality rate existed at the
warmer temperature of 27 8C. Therefore, the optimal
temperature for incubating obscure puffer eggs is
about 19 to 23 8C according to the results of total
hatch rate, viability of newly hatched larvae 24 h posthatch and total mortality rate of eggs incubated at 15,
19, 23, and 27 8C.
Within a viable range, incubation temperature
strongly affects the rate of embryonic development
of fish. Generally, lower temperature retards the rate
of embryonic development and higher temperature
accelerates it (Marangos et al., 1986; Pepin, 1991;
Blaxter, 1992; Mihelakakis and Kitajima, 1994; Hart
and Purser, 1995; Hart et al., 1996; Hamel et al.,
1997b; Mihelakakis and Yoshimatsu, 1998; Hansen
and Falk-Petersen, 2001; Kamler, 2002). In the
present study, time from fertilization to 50% hatch
of obscure puffer eggs decreased from 11.3 days at
15 8C to 4.2 days at 27 8C and was consistent with
the widely observed phenomena in many other fishes
(Marangos et al., 1986; Pepin, 1991; Blaxter, 1992;
Mihelakakis and Kitajima, 1994; Hart and Purser,

177

1995; Hart et al., 1996; Hamel et al., 1997b;

Mihelakakis and Yoshimatsu, 1998; Hansen and
Falk-Petersen, 2001; Kamler, 2002). Furthermore,
the effect of increasing incubation temperature from
15 8C to 27 8C on reducing the incubation time is
accurately described by the power law model,
quadratic equation, exponential equation, and effective degree-day model, respectively. Like in some
other fish species (Hamel et al., 1997b; Kamler,
2002), more than one model can be used to describe
the relationship between water temperature and
incubation time of obscure puffer eggs. From the
results of the statistical analysis only, it is impossible
to identify which of the power law model, quadratic
equation, exponential equation, or effective degreeday model should be the best because the regression
coefficients for all models were only slightly different. However, the common weakness of the power
law model, quadratic equation, and exponential
equation is that the derived parameters (a, b and
c) have no biological meaning (Hamel et al., 1997b;
Kamler, 2002). In addition, a comparison of temperature requirements between species is not possible
with those models (Kamler, 2002). Although the
effective degree-day model has often been criticized
because of its lack of accuracy at extreme temperatures (Wagner et al., 1984; Highley et al., 1986), it
is still used by many researchers (Haylor and
Mollah, 1995; Hamel et al., 1997a,b; Weltzien et
al., 1999; Kamler, 2002) because the derived
parameters (k and t 0) have important biological
meaning and the model is easy to compute and
often gives a very good fit to the experimental data.
We also approved of this model on the basis of our
results. In addition, the effective degree-day model is
temperature-independent and has been applied and
confirmed in many other fish species (Haylor and
Mollah, 1995; Weltzien et al., 1999; Kamler, 2002).
The t 0 value is the threshold temperature at which
ontogeny is theoretically arrested (i.e. temperature of
biological zero), and the k value is the number of
degree-days above the threshold temperature (i.e. the
sum of effective degree-days). These parameters
were shown to be useful for interspecific comparison. However, their suitability for intraspecific
comparisons (e.g. comparing populations from different latitudes or altitudes) remains to be demonstrated
(Kamler, 2002).

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Z. Yang, Y. Chen / Aquaculture 246 (2005) 173179

In summary, our results showed that the optimal

temperature for incubating obscure puffer eggs ranged
from 19 to 23 8C. The effective degree-days for the
obscure puffer eggs were constant in this experiment.
The t 0 value and k value are 7.6033 8C and 78.905
degree-days, respectively.

Acknowledgements
We would like to thank Xinke Zhu for technical
help in spawning and maintaining the obscure
puffer. Thanks are directed to Xi Chen and Jie
Hua for their assistance in observing the embryonic
development. Our sincere thanks are also due to
Dr Binhe Gu for polishing the English. This
investigation was partially funded by Science Foundation for Youths of NJNU (2001XQ22) and bThreeProjectQ of Aquaculture in Jiangsu Province of China
(PJ2002-31).

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