Kurita et al.: The effect of temperature on the duration of spawning markers in Paralichthys olivaceus 
87 
To our knowledge, this is the first report to clearly il- 
lustrate the duration and growth rate of oocytes during 
final maturation, on an individual-fish basis, in relation 
to ambient water temperature. Final maturation, in 
this study, is considered to have progressed normally 
for the following three reasons, although successive 
sampling in general would produce strong stress on 
animals. First, past in vitro incubation experiments of 
Japanese flounder at 15°C have shown that the dura- 
tion of the HD stage is 12 hours and the duration of the 
final maturation process is about 33 hours (Matsubara 
et ah, 1995). These results are comparable to ours, 
namely that the estimated duration of the HD stage is 
10 hours and the duration of final maturation (MN[E], 
MN[L], and HD stages) is 30 hours at 15°C. Second, 
other studies also have reported the negative relation- 
ship between the duration of final maturation (from 
injection of pituitary extracts or gonadotropin-releas- 
ing hormone [GnRH] to ovulation) and ambient water 
temperature (common carp [Cyprinus carpio], Drori 
et ah, 1994; streaked prochilod [Prochilodus platen- 
sis], Fortuny et al., 1988) as seen in this study. Third, 
hydration is driven by the osmotic gradient caused 
by protein hydrolysis of yolk and ion accumulation 
through many enzyme catalyses (Cerda et al., 2007). 
The rate of enzyme catalysis increases as temperature 
increases, within a range of appropriate temperatures. 
Therefore, it is reasonable that the growth rate of hy- 
drated oocytes increases, or in other words, duration 
of this stage decreases as ambient water temperature 
increases. 
In some trials, females did not produce a new batch 
of oocytes that entered the final maturation process 
during the 48-h experiment period, probably because 
of the stress experienced during the sampling proce- 
dure. However, once a batch of oocytes entered the MN 
stage, those oocytes completed final maturation and 
proceeded to ovulation, even when under the stressful 
conditions of the experiments. This finding indicates 
that GnRH or gonadotropin secretion, which induce 
a batch of oocytes to proceed to final maturation, is 
likely susceptible to stress, but the process of the fi- 
nal maturation of oocytes is less susceptible, at least 
for Japanese flounder. In addition, fish could ovulate 
oocytes but could not spawn them during the 48-h ex- 
periment period. Spawning behaviour or the endocrine 
control of spawning, or both, are also likely susceptible 
to stress. 
Successive sampling of oocytes from individual fe- 
males with a catheter is a useful method for clarifying 
the diurnal rhythm of the final maturation of fish spe- 
cies, especially those with a long spawning time distri- 
bution within a day at the population level. However, 
careful consideration should be paid for reducing stress 
to fish during the sampling procedure. Anesthetizing 
and handling seem to be the main sources of stress. 
In this study, we tried to reduce stress by weakly an- 
esthetizing fish for a short duration. We also did not 
use a scoop net to pick up fish because the fish would 
move around in the tank to escape the net and strug- 
gle in the net, both of which would cause undue stress 
and physical injury to the fish, e.g., injuries to their 
body surface and internal bleeding. Instead, we lowered 
the water level of the tank, gently picked up the fish, 
placing both hands under its body, put it into a crate 
made of styrene foam, which floated on the surface of 
water, carried the crate with the fish and placed the 
fish in a bath filled with 0.08% 2-phenoxyethanol sea 
water without touching the fish. During this procedure, 
the fish did not struggle much and seemed to be less 
stressed. We also experimented with taking ovary sam- 
ples by cannulation without anaesthesia; i.e., we picked 
up the fish with both hands placed under its body, 
put it on a board floating on the surface of water, and 
then took samples of oocytes. These operations took 
10-20 seconds and females stayed still on the board. 
The latter method seemed to be a better procedure to 
reduce stress. 
Spawning frequency is, in general, estimated by us- 
ing the fraction of fish with a spawning marker and 
the duration of the marker as seen in Equation 1 
(Priede and Watson, 1993; Murua et al., 2003). Dura- 
tion of oocytes at the MN and HD stages, in addition 
to POFs (Fitzhugh and Hettler, 1995; Ganias et al., 
2007), changes as temperature changes. Thus, accu- 
rate estimates of the duration of a marker in relation 
to temperature are essential for estimating spawning 
frequency accurately, especially for those fish species 
that have long spawning-time distribution within a day 
at the population level and experience a wide range of 
ambient water temperatures during their long spawn- 
ing season. 
Acknowledgments 
We thank the staff at Miyako National Center for Stock 
Enhancement for their help with the experiments. We 
are grateful to K. X. Pham for his assistance in the 
experiments. We are also grateful to T. Matsubara and 
M. Matsuyama for providing valuable information and 
to four anonymous referees for their valuable reviews 
of an earlier version of the manuscript. This work was 
supported by a grant-in-aid from the Fisheries Research 
Agency of Japan. This contribution is catalogued as B127 
by the Tohoku National Fisheries Research Institute. 
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