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Fishery Bulletin 109(1) 
a restricted period of a few hours in the early night, 
and sampling time is restricted to just after spawning 
(Hunter and Goldberg, 1980; Hunter and Macewicz, 
1985; Funamoto and Aoki, 2002). However, for many 
other species, spawning time is not synchronized at the 
population level (i.e., egg release by the population is 
not restricted to a few hours of the day) or the sampling 
period for these species is broader. In this case, spawn- 
ing frequency ( S : 1/day) is corrected by the duration (£•) 
of a spawning marker i as follows: 
S = F x (24 / t t ) (1) 
(Priede and Watson, 1993; Murua et al., 2003). 
Many multiple-batch spawning fish have a long 
spawning season, and as a consequence, they spawn 
at a wide range of water temperatures. Therefore, the 
information on the duration of spawning markers in 
relation to ambient water temperature is critical for 
estimating spawning frequency accurately. So far, the 
duration of a stage of POF has been reported to vary 
depending on the ambient temperature (Fitzhugh and 
Hettler, 1995; Ganias et al., 2007). However, to our 
knowledge, temperature effects on the duration of oo- 
cytes at MN and HD stages have not been reported. 
In many previous studies, the duration of spawning 
markers at a given temperature were evaluated by the 
observation of ovaries which were dissected out of peri- 
odically sampled females taken from a spawning popu- 
lation in the field or laboratory (Hunter and Goldberg, 
1980; Hunter and Macewicz, 1985; Matsuyama et al., 
1988, 2002; Shiraishi et al., 2005). This type of sam- 
pling schedule is valid when spawning occurs synchro- 
nously or over a short period (h) in the day. However, 
many fish species, e.g., European plaice ( Pleuronectes 
platessa) and Atlantic mackerel ( Scotnber scombrus ) 
have a relatively long spawning-time distribution (in 
hours per day) at the population level (Ferraro, 1980; 
Nichols, 1989; Walsh and Johnstone, 1992; Nichols and 
Warnes, 1993; Scott et al., 1993), and consequently, 
it is not possible to accurately estimate the duration 
of spawning markers by fish-group-based studies. For 
these fish, periodical observations of the presence of 
spawning markers should be taken from the same fish 
for a period of 24 hours. Periodic sampling of oocytes 
and POFs from individual captive fish by cannulation 
(e.g., Kjesbu et al., 1996a; Kennedy et al., 2008; Wit- 
thames et al., 2010), is a promising method for estimat- 
ing the duration of spawning markers. 
In this study, we used Japanese flounder (Paralichthys 
oliuaceus) (also called the “bastard halibut”) as a model 
species. The flounder is a commercially important bot- 
tom fish that inhabits coastal areas <150 m in depth. 
This species is a multiple-batch spawner and the most 
active fish spawn every day (Hirano and Yamamoto, 
1992). A batch of advanced oocytes among vitellogenic 
oocytes (ca. 300-600 pm) enters the final oocyte matu- 
ration process and is ovulated (Kurita and Kjesbu, 
2009). This species is also a typical indeterminate 
spawner that continues to produce vitellogenic oocytes 
during the spawning season (Murua and Saborido- 
Rey, 2003). Each individual female typically spawns 
over a period of three months (Hirano and Yamamoto, 
1992), and the spawning season of the population lasts 
4-5 months (Takeno et al., 1999). Japanese flounder 
experience temperatures of 7-19°C during their spawn- 
ing season (Y. Kurita, unpubl. data). Spawning occurs 
throughout 24-h periods at the population level (Y. 
Kurita, unpubl. data). Thus, the duration of spawning 
markers during final maturation in relation to tempera- 
ture is crucial information for accurately estimating 
spawning frequency. 
The objective of this study is to evaluate the effects 
of temperature on the duration of spawning markers 
(i.e., oocytes at the MN and HD stages, and POFs) of 
Japanese flounder by successive sampling of ovarian 
tissue from individuals with a catheter over a range of 
ambient temperatures typically encountered during the 
spawning season. 
Materials and methods 
Experiments 
To evaluate the duration of spawning markers over a 
range of temperatures, oocyte developmental stages 
relating to final maturation, and as well as POF degen- 
eration stages (Table 1) of individual captive females 
were documented periodically. Two kinds of analyses 
were conducted: 1) an approximate evaluation of the 
duration for those markers; and 2) a fine-scale evalua- 
tion of the duration of hydrated oocytes. 
Spawning females were held separately with two 
males each in cylindrical tanks with a diameter of 2.5 
m and a water depth of 1 m (ca. 5 m 3 in volume) during 
the period covering the spawning season. A constant 
flow of seawater at ambient temperature was provided. 
Ovary samples of 1-1.5 mL were taken from each in- 
dividual every 2-4 hours through 27-48 h with a thin 
(inner diameter; 2 mm) soft catheter (Pipelle de Cornier, 
Laboratoire C.C.D., France). At each catheterization, 
fish were weakly anesthetized with 0.08% 2-phenoxy- 
ethanol sea water for 30-120 seconds until fish did 
not beat their tails when they were turned over in the 
water. Samples were fixed in 3.6% phosphate buffered 
formaldehyde and subjected to the following analyses. 
Samplings were conducted for 27-48 hours for a total 
of 14 trials (four trials for 27 hours and 10 trials for 
48 hours) for six females (total length [TL] 484-730 
mm) and with ambient temperature ranging from 9.2° 
to 22.6°C (Table 2). Out of 14 trials, the duration of all 
stages of oocytes during final maturation, i.e., early 
migratory nucleus (MN[E]), late migratory nucleus 
(MN[L|), and HD stages, and new postovulatory fol- 
licles (POF[new]) (Table 1) were monitored for five trials 
(three females; TL 605-710 mm); MN(E), MN(L), and 
POF(new) for one trial (one female; TL 605 mm); and 
MN(L) and HD for another trial (one female; TL 730 
mm). Moreover, the duration and growth rate of only 
