82 
Fishery Bulletin 109(1 ) 
Table 2 
Experimental data obtained from ovarian tissue from Japanese flounder (Paralichthys olivaceus). Ovarian tissue was examined 
every two to four hours over a 27- or 48-hour sampling period. MN(E)=oocyte at early migratory nucleus stage; MN(L) = at late 
migratory nucleus stage; HD = at hydrated stage; POF(new)=new postovulatory follicles. 
Fish 
number 
Total length 
(mm) 
Experiment 
period 
Duration of 
experiment (hour) 
Temperature 
(°C) 
Spawning markers used for evaluating 
marker duration or estimating 
hydrated oocyte growth rate 1 
MN(E) 
MN(L) 
HD 2 
POF(new) 
1 
605 
7-9 June 2004 
48 
14.1 
Y 
Y 
Y(2) 
Y 
1 
12-14 July 2004 
48 
16.7 
Y 
Y 
Y(l) 
Y 
1 
9-11 Aug 2004 
48 
22.6 
Y 
Y 
Y 
2 
710 
30 May-1 June 2005 
48 
11. 3(11.9) 3 
Y 
Y 
Y(2) 
Y 
2 
30-31 June 2005 
27 
13.7 
Y(2) 
2 
20-22 July 2005 
48 
16.7 
Y(2) 
3 
707 
30 May-1 June 2005 
48 
11.2G1.9) 3 
Y(2) 
3 
30-31 June 2005 
27 
14.1 
Y(2) 
3 
20-22 July 2005 
48 
16.9 
Y 
Y 
Y(2) 
Y 
3 
11-13 Aug 2005 
48 
19.7 
Y 
Y 
Y(l) 
Y 
4 
484 
12-14 July 2004 
48 
16.8 
Y(l) 
5 
545 
30 May-1 June 2005 
48 
11.2 
Y(l) 
5 
30-31 June 2005 
27 
13.7 
Y(l) 
6 
730 
23-24 May 2006 
27 
9.2 
Y 
Y(l) 
Total 
6 
7 
20 
6 
1 “Y” indicates that data were obtained. 
2 Figures in parentheses show the number of hydration events used for calculation of the growth rate of hydrated oocytes. 
3 Figures in parentheses show the temperature at the second hydration event. 
growth rate of hydrated oocytes. Changes in those two 
variables were evaluated in relation to temperature as 
follows. For the measurements of oocyte diameter, the 
width of follicle layer was not included. 
First, the growth rate of hydrated oocytes was ex- 
amined. For each cannulated sample, the diameters 
of 30 hydrated oocytes were measured manually in 
a whole mount (dispersed sample observed under a 
binocular microscope) with image-analysis software 
(Image Pro PLUS, Media Cybernetics, Inc., Bethesda, 
MD). Growth rate of hydrated oocytes (pm/h) was 
calculated as the slope of the regression line between 
the average hydrated oocyte diameter and sampling 
time. The relationship between the growth rate of 
hydrated oocytes and temperature was established as 
the exponential formula. 
Next, to estimate the size range of diameters of hy- 
drated oocytes at each temperature unit of the experi- 
ment, ovulated egg diameter and the maximum MN(L)- 
stage oocyte diameter through 48 hours of sampling 
were measured as the substitution for the maximum 
and the minimum hydrated oocyte diameters, respec- 
tively. 
The diameter of 30 newly ovulated eggs (Table 1) that 
remained in the ovarian cavity from each cannulated 
sample were measured manually with image-analysis 
software and averaged. Ovulated eggs were distin- 
guished from large hydrated oocytes by histological 
examination; i.e., ovulated eggs did not have follicle lay- 
ers around them and coexisted with new postovulatory 
follicles. Diameters of ovulated eggs were measured for 
the two consecutive samples after the samples in which 
the largest hydrated oocytes in each hydration process 
were observed. 
The diameters of an additional 200-300 fixed, 
whole-mount oocytes at the YG, MN(E), and MN(L) 
stages from each cannulated sample were also meas- 
ured with image analysis software. Average diam- 
eters of oocytes at the MN(L) stage were calculated 
for each sample. When the MN(L) stage oocytes were 
distinguishable in whole-mount samples (Table 1), 
the diameters of these oocytes were measured and 
averaged. However, in some cases, the MN(L)-stage 
oocytes were difficult to distinguish from the MN(E)- 
stage oocytes in the whole-mount samples by diameter 
and appearance. In those cases, the proportion of the 
number of MN(L)-stage oocytes to the number of the 
developing oocytes (oocytes at the YG, MN(E), MN(L), 
and HD stages) was set as the same proportion of the 
number of HD-stage oocytes to the developing oocytes 
(Fig. 2). For the example in Figure 2, the proportion 
of MN(L)-stage oocytes to the developing oocytes was 
