Kurita et al.: The effect of temperature on the duration of spawning markers in Parahchthys olivaceus 
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Table 1 
Criteria for each developmental stage of oocyte, ovulated egg, and postovulatory follicle of Japanese flounder (Paralichthys 
olivaceus). 
Description 
Developmental stage 
Abbreviation 
Histology 
Whole mount appearance 
with transmitted light 
Yolk granule 
YG 
Yolk granules present. Many small oil 
droplets are distributed around the 
nucleus which is located in the center 
(Fig. 1A). 
Oocytes are slightly dark and oil droplets, 
which are located around the nucleus, 
look like a dark shadow (Fig. IB). 
Early migratory 
nucleus 
MN(E) 
Yolk granules are larger. Oil droplets 
fuse and are distributed unevenly to 
one side (within 180° from the center 
of nucleus) beside the nucleus (Fig. 1C). 
Uneven distribution of oil droplets can be 
recognized as dark shadow. Difficult to 
distinguish from YG (Fig. ID). 
Late migratory 
nucleus 
MN(L) 
Oil droplets fuse into 1-3 big droplets 
(Fig. IE). 
Yellowish oil droplets can be easily recog- 
nized (Fig. IF) and usually distinguish- 
able from MN ( E ) and YG. 
Hydrated 
HD 
In the earlier phase, yolk granules 
fuse progressively and start to become 
irregularly shaped (Fig. 1G). In the 
later phase, all yolk glanules fuse into 
a uniform yolk mass that occupies the 
inside of the oocyte (Fig. 11). Oocyte is 
still surrounded by the follicle layer. 
In the earlier phase, the whole mount 
appearance turns opaque and dark (Fig. 
1H). In the later phase the whole mount 
appearance turns translucent and a big 
yellowish oil droplet is prominent (Fig. 
1J). Oocytes at this stage are easily 
distinguished from other stages of oocytes. 
New ovulated egg 
OV 
A uniform yolk mass occupies the inside 
of the egg. Egg is free from follicle layer. 
Egg coexists with POF(new). 
Appearance is similar to the oocyte at 
the late HD stage. 
New postovulatory 
follicle 
POF(new) 
Granulosa cells are clearly recognized. 
Cell membrane and nucleus of granu- 
losa cells are intact (Fig. IK). 
HD-stage oocytes were monitored for seven other trials 
(four females; 484-710 mm TL). The former seven trials 
where the duration was monitored for more than two 
spawning markers, conducted between 9.2° and 22.6°C, 
were used for analyses of the approximate evaluation of 
the duration of spawning markers and the size range 
of hydrated oocytes, the latter of which is the range 
between the maximum and the minimum diameters of 
hydrated oocytes through the final maturation process 
at each temperature of the experiment). In total, 20 
events of hydration from 13 trials conducted between 
9.2° and 19.7°C were used for analyses of growth rate 
and the duration of HD-stage oocytes. 
Histological and whole mount examination 
for evaluation of the duration of the spawning marker 
Approximate evaluation of the duration for spawning 
markers was conducted as follows. A part of each can- 
nulated sample was dehydrated and embedded in resin 
(Historesin), sectioned at 4 pm thickness, and stained 
with 2 % toluidine blue and 1% borax. The occurrence 
of oocytes at the yolk granule (YG; Table 1), MN(E), 
MN(L), and HD stages, and POF(new) were recorded 
by a combination of histological examination under a 
light microscope and whole mount examination under 
a binocular microscope (Fig. 1). The start time of the 
duration of each stage was estimated as the mid-point 
of the two consecutive sampling times when the devel- 
opmental stage was not observed and then observed. 
Similarly, the end time was the mid-point of the two 
consecutive sampling times when the developmental 
stage was observed and not observed. The duration of 
oocytes at the MN(E) and MN(L) stages, and POF(new) 
were estimated at each trial, and the relationships with 
temperature were analyzed as the exponential formulae. 
Measurement of oocyte diameter for evaluation 
of duration of the HD stage 
The duration of HD stage was estimated more precisely 
as the range of hydrated oocyte diameter divided by the 
