86 
Fishery Bulletin 109(1 ) 
considered and if a constant estimate of duration is 
used. In the case of Japanese flounder, the duration 
of the HD stage decreased from 19.1 to 5.3 h between 
9.2° and 19.7°C. If an HD-stage oocyte is used as a 
spawning marker and 2 out of 10 sampled females 
have HD-stage oocytes in their ovaries, the estimated 
spawning frequency by Equation 1 will be very differ- 
ent, i.e., 0.25 at 9.2°C and 0.91 at 19.7°C, leading to 
answers that differ by a factor of 3.6 ( = [0.2x24/5.3]/ 
[0.2x24/19.1]). 
Each spawning marker has advantages and disad- 
vantages for estimating spawning frequency. The merit 
of using POFs as spawning markers is that females 
with day-1 or day-2 old POFs are usually found away 
from spawning aggregations and estimates of spawn- 
ing frequency are therefore less biased (Hunter and 
Goldberg, 1980; Picquelle and Stauffer, 1985; Ganias 
et ah, 2003). However, aging or staging is difficult be- 
cause the process of degeneration is continuous (Ganias 
et ah, 2003). On the other hand, the merit of using 
oocytes at the MN and HD stages as spawning mark- 
ers is that aging or staging is easy. However, in some 
cases estimates of spawning frequency with the MN- 
or HD-stage oocytes provide biased estimates because 
of the nonuniformity of their distribution, i.e., during 
spawning aggregations. Therefore, in cases where fish 
populations spawn within a restricted period of a few 
hours, as occurs with some clupeid fish populations 
(Stratoudakis et al., 2006) and sampling is also con- 
ducted within a restricted time period, the POF is an 
appropriate spawning marker because staging is easier 
as the age of POF appears intermittently (Hunter and 
Goldberg, 1980; Hunter and Macewicz, 1985). But when 
fish spawn over a prolonged period (over many hours in 
a day) at the population level, or when sampling time 
is not restricted to a short period, oocyte at the MN 
or HD stage is appropriate spawning marker because 
aging or staging is much easier than POFs, and the 
use of Equation 1 is recommended. Even in species that 
form spawning aggregations around spawning events, 
a MN-stage oocyte may be a useful spawning marker 
1000 
| 900 
<D 
| 800 
03 
g 700 
CD 
O 
oj 600 
> 
O 
o 
° 500 
5 10 15 20 25 
Temperature (°C) 
Figure 6 
Changes in the range (from the mini- 
mum to the maximum) of sizes of oocytes 
at the hydrated stage (HD) as a function 
of temperature ( T ) for Japanese flounder 
( Paralichthys olivaceus). The minimum 
and maximum sizes of oocytes at the 
HD stage were replaced with the maxi- 
mum size of oocytes at the late migra- 
tory nucleus stage (A), and with the 
average diameter of ovulated eggs (□), 
respectively, in a 48-h experiment at 
each temperature. The broken line shows 
the average diameter of the maximum 
sizes of oocytes at the late migratory 
nucleus stage (645 pm) and the solid 
line shows the regression line for the 
diameter of ovulated eggs. See Equation 
3 in the main text. 
because oocytes at the MN stage appear several hours 
before spawning and the duration of the MN stage is 
relatively long. In any case, precise evaluation of the 
duration of a marker and its changes due to ambient 
temperature is essential, especially for species that 
spawn over long time periods within a day. 
Table 3 
Parameters of equations used to determine stage duration (D) at temperature (T); D=axe~ bT) for Japanese flounder ( Paralichthys 
olivaceus) with statistics (n=number of data points; /^coefficient of determination; P=probability of error in the regressions). 
The reduction rate of D for an increase in temperature of 10°C (D lT+10) /D T ) is also shown. MN(E)=at early migratory nucleus 
stage; MN(L)=at late migratory nucleus stage; HD=at hydrated stage; POF(new)=new postovulatory follicle. 
Stages 
a 
b 
D(T+10)/ D T 
n 
r 2 
P 
MNlE) 
186 
0.186 
0.16 
6 
0.88 
0.006 
MN(L) 
42.4 
0.103 
0.36 
7 
0.90 
0.001 
HD(calculated) 1 
59.4 
0.122 
0.30 
6 
1.00 
<0.001 
POF(new) 
62.9 
0.118 
0.31 
6 
0.74 
0.028 
MN(L) + HD 
98.4 
0.111 
0.33 
6 
0.96 
<0.001 
MN(E) + MN(L) + HD 
332 
0.159 
0.20 
5 
0.98 
0.001 
1 Stage duration at each temperature was calculated with the size range and the growth rate of HD-stage oocytes (Eq. 2). 
I 
