Fisheries Research 174 (2016) 30–39

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Fisheries Research
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Reproductive biology of female Pacific bluefin tuna, Thunnus orientalis,

in the Sea of Japan
Yumi Okochi a,b,∗ , Osamu Abe a , Sho Tanaka c , Yukio Ishihara d , Akio Shimizu b,e
a
National Research Institute of Far Seas Fisheries, Fisheries Research Agency, 5-7-1, Orido, Shimizu, Shizuoka 424-8633, Japan
b
Tokyo University of Marine Science and Technology, The Graduate School of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477,
Japan
c
Tokai University, School of Marine Science and Technology, 3-20-1, Orido, Shimizu, Shizuoka 424-8610, Japan
d
Tottori Prefecture Fisheries Experimental Station, 107, Takenouchidanchi, Sakaiminato, Tottori 684-0046, Japan
e
National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fukuura, Yokohama, Kanagawa 236–8648, Japan

a r t i c l e i n f o a b s t r a c t

Article history: A histological analysis of ovaries was conducted to examine reproductive characteristics of female Pacific
Received 7 April 2015 bluefin tuna, Thunnus orientalis (Temminck and Schlegel, 1844), in the known spawning ground in the Sea
Received in revised form 4 August 2015 of Japan. A total of 1040 females caught with purse seines were sampled from late May to early August
Accepted 22 August 2015
in 2011 and 2012. The spawning season began in mid-June and continued until early August. The sex
ratio for the catch in 2012 was nearly 1:1. The estimated spawning time was 1700–2200 h based on the
Keywords:
occurrence of 0 h postovulatory follicles. The estimated mean spawning frequency and mean spawning
Spawning
interval were 0.91 per day and 1.10 days, respectively, indicating that mature females spawn nearly every
Maturity
Fecundity
day in the Sea of Japan. The fork lengths at 50% and 95% mature were 114.4 cm and 133.6 cm, respectively.
Sea of Japan Batch fecundity was positively correlated with fork length, gilled and gutted body weight, and gonad
weight. The mean batch fecundity was 6.4 million oocytes, and the mean relative batch fecundity per
gilled and gutted body weight was 122 oocytes. This study provides some comprehensive information
on reproductive biology of female Pacific bluefin tuna in the Sea of Japan.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction mation on the reproductive parameters of Pacific bluefin tuna is

Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel),
is one of the largest tuna species and is found throughout the
Pacific Ocean. This species is an international fishery resource with
high commercial value and is fished in many countries with vari-
ous fishing equipment (ISC, 2014). The stock assessment for Pacific
bluefin tuna is conducted by the International Scientific Commit-
tee for Tuna and Tuna-like Species in the North Pacific Ocean (ISC).
The latest assessment, in 2014, concluded that the spawning stock
biomass is near its historically lowest level (ISC, 2014). Biological
parameters such as age, maturity schedule, fecundity, spawning
frequency, and duration of the spawning period are essential for
assessing the stock status as fishery resources. However, infor-
∗ Corresponding author at: National Research Institute of Far Seas Fisheries,
Fisheries Research Agency, 5-7-1, Orido, Shimizu, Shizuoka 424-8633, Japan.
Fax: +81 54 335 9642.
E-mail addresses: okochi@affrc.go.jp (Y. Okochi), turtlea@affrc.go.jp (O. Abe),
sho@scc.u-tokai.ac.jp (S. Tanaka), ishiharay@pref.tottori.jp (Y. Ishihara),
aneko@affrc.go.jp (A. Shimizu).
scarce (ISC, 2013). Pacific bluefin tuna have two spawning grounds,
one in the northwestern Pacific Ocean during May and June and
one in the Sea of Japan during July and August. Larvae have been
collected in May and June from waters off Taiwan to areas near
the Ryukyu Islands in the northwestern Pacific Ocean (Yabe et al.,
1966; Ueyanagi, 1969; Tan and Chen, 1975; Tanaka et al., 2006;
Abe et al., 2014). Histological observations of ovaries indicate that
the spawning activities in the northwestern Pacific Ocean start in
May and peak in late May and early June (Chen et al., 2006). In
the Sea of Japan, information on spawning grounds and spawn-
ing seasons has mainly been derived from observations of the
occurrence of larvae and juveniles. Larvae have been collected in
July and August (Okiyama, 1974; Okiyama and Yamamoto, 1979;
Kitagawa et al., 1995; Abe et al., 2014), and spawning date esti-
mates based on counting daily increments in otoliths have revealed
that juveniles collected in the Sea of Japan hatch during July and
August (Tanaka et al., 2007; Itoh, 2009). Nonetheless, few histologi-
cal investigations of gonads, which is the best method to investigate
fish reproductive biology, have been conducted in the Sea of Japan.
The objective of this study is to better understand the reproductive

http://dx.doi.org/10.1016/j.fishres.2015.08.020
0165-7836/© 2015 Elsevier B.V. All rights reserved.
 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39
40˚N
Sea of Japan
36˚N
Sakai Fishing Port
32˚N
132˚E 136˚E 140˚E
Fig. 1. Map indicating fishing locations in the Sea of Japan for Pacific bluefin tuna
sampled in this study and the location of Sakai Port. Samples were collected in 2011
and 2012.
biology of female Pacific bluefin tuna caught in the Sea of Japan
using histological analyses.
2. Materials and methods
2.1. Sample collection and sex ratio
A total of 1040 fresh ovaries from landed female Pacific bluefin
tuna caught with commercial purse seines in the Sea of Japan were
sampled in 2011 and 2012 at Sakai Port in Tottori Prefecture (Fig. 1).
The landing period at Sakai Port was late May to early August. The
proportion of port sampling times to landing times were 84.4% in
2011 and 85.7% in 2012. Port samplings were conducted around
0800 h and the mean time between catch (ending time of opera-
tion) and sampling was 29 h 24 min (SD 14 h 56 min). Ovaries were
randomly collected, and sections of the ovaries were preserved in
10% buffered neutral formalin about 1–2 h after port sampling. Fork
length (FL, cm), gilled and gutted body weight (BW, kg), and gonad
weight (GW, g) were recorded for each female.
The mean FL (±SD) of the sampled females was 139.6 (±20.5) cm
in 2011 and 151.6 (±16.8) cm in 2012. The FL frequency distribution
varied with seasons (Fig. 2). In 2011, females with a FL smaller than
130 cm were collected in late May, early June, and July. Beginning
in June, females between 130 and 160 cm were frequently sampled.
In 2012, the FL of females collected in late May was between 120
and 140 cm FL. In June and July, females between 140 and 150 cm
FL were observed. Females collected in early August had a FL of 120
to 150 cm.
The sex ratio was examined for the catch in 2012. Sex was iden-
tified by examining gonads when fishermen gilled and gutted the
fish at Sakai Port. Sex identification was performed for 14 of 27
landings in 2012. The numbers of males and females were counted,
and a chi-square test was used to examine the sex ratios.
2.2. Histological observation and ovarian developmental
classification
Fixed ovarian pieces were embedded in paraffin wax, and sec-
tions with a thickness of 6 ␮m were mounted on microscopic slides
and stained with Mayer’s haematoxylin-eosin (HE). The develop-
mental stages of the oocytes were classified into 6 stages: the
perinucleolus stage (PNS), yolk vesicle stage (YVS), early yolked
stage (EYS), advanced yolked stage (AYS), migratory nucleus stage
(MNS), and hydrated stage (HS) (Chen et al., 2006). The most
advanced group of oocytes (MAGO) was recorded, and atretic
31
yolked oocytes were classified into ␣ and ␤ atresia according to
the criteria of Hunter et al. (1986).
The post-ovulatory follicle (POF) is an important structure that
provides evidence of past spawning. In previous reports for ayu
(Plecoglossus altivelis) (Shimizu et al., 2007) and Atlantic cod (Gadus
morhua) (Witthames et al., 2010), the basement membrane of
the POF showed a PAS (periodic acid-Schiff)-positive reaction and
became distinct as the POF regressed. For the staging of POFs of
Pacific bluefin tuna, parts of the sections were stained with both
PAS-haematoxylin and HE. POFs were classified into 0 h, <12 h, and
>12 h old stages (Fig. 3). In 0 h old POF, granulosa cells are dis-
tinct, and the apertures between granulosa cells and theca cells
are visible in both HE and PAS staining (Fig. 3g and j). The base-
ment membrane was positively stained with PAS. In <12 h old POF,
the granulosa cells are still distinct, but apertures between granu-
losa cells and theca cells became narrow and granulosa cells formed
irregular lines (Fig. 3h and k). In >12 h POF, regressed granulosa cells
are still visible but not clear (Fig. 3i and l). The basement membrane
stained with PAS becomes more distinct as it regresses. PAS staining
is therefore a useful method for investigating time course changes
in POFs in tunas. After confirmation of the morphological appear-
ances of POFs stained with both HE and PAS staining, all POFs are
classified into 0 h, <12 h, or >12 h old stages using sections stained
with HE.
Based on the criteria of Schaefer (1998), ovaries which contained
MAGOs of AYS and no or minor (<50%) atresia were classified as
active (Table 1, Fig. 3). Ovaries which contained MAGOs of unyolked
(PNS, YVS) or EYS, or MAGOS of EYS or AYS and more than 50%
atresia were classified as inactive. Then each ovary were classed
into immature, developing, spawning capable, spawning, regress-
ing or regressed classes based on observations of MAGOS, POFs, and
atresia according to the criteria of Farley et al. (2013). As for the
criteria developing class, ovaries which contained MAGOs of EYS
and no or minor atresia of yolked oocytes are classed into devel-
oping in this study, because atresia at the stage of early yolked
oocytes would occur. Females whose ovaries classed as immature
or developing were considered immature. Females with ovaries in
spawning capable, spawning, regressing, or regressed classes were
considered mature.
2.3. Fish body indices
The gonad index (GI) was calculated as GI = GW/FL3 × 104 , where
GW is the gonad weight in grams and FL is the fork length in
centimeters. To evaluate the body condition of each fish, Fulton’s
condition factor (CF) was calculated as CF = BW/FL3 × 105 where BW
is the gilled and gutted body weight in kilograms and FL is the fork
length in centimeters.
2.4. Size at maturity
The Richards (1959) function was used to describe the maturity
schedule. The following equation, reported by Schaefer (1998) was
fitted to the proportion of mature females by 5-cm FL interval:
P FL = [1 − (1 − m)e−K(FL−c )]1/(1−m)
where PFL is proportion mature at a fork length (FL) in centimeters,
and m, K, c are the parameters. The parameters were estimated
using nonlinear least squares function in R software (ver. 3.1.2).
2.5. Spawning frequency
Spawning frequency was estimated by the POF method in accor-
dance with Hunter and Goldberg (1980) and Hunter and Macewicz
(1985). The ratio of the females with <24 h POFs to mature females
32 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39

Fig. 2. Fork length (FL) frequency distributions by month of sampled female Pacific bluefin tuna caught in 2011 and 2012. Each month was divided into early period (1st to
10th), middle period (11th to 20th), and late period (21st to 31st).

Table 1
Classification of maturity status, reproductive activity, and ovarian developmental classes of Pacific bluefin tuna.

Maturity status Reproductive activity Ovarian developmental class MAGOs and POF Atresia of yolked oocytes

Immature Inactive Immature PNS or YVS, no POF All atresia absent

Immature Inactive Developing EYS, no POF No or some atresia may be present
Mature Active Spawning capable AYS, no POF <50% ␣ atresia, ␤ atresia may be present
Mature Active Spawning MNS or HS, and/or POF <50% ␣ atresia, ␤ atresia may be present
Mature Inactive Regressing – potentially reproductive AYS, no POF ␣ atresia common (≥50%), ␤ atresia may be present
Mature Inactive Regressed PNS or YVS, no POF 100% ␣ atresia, ␤ atresia may be present

POF, post-ovulatory follicles; PNS, perinucleolus stage, YVS, yolk vesicle stage; EYS, early yolked stage; AYS, advanced yolked stage; MNS, migratory nucleus stage; HS,
hydrated stage.

was evaluated as the spawning frequency. The inverse of the
spawning frequency was considered the spawning interval.
2.6. Batch fecundity
Batch fecundities were estimated from the ovaries with MAGO
of HS and without new POFs (Hunter et al., 1985). A subsam-
ple of 0.5 g was removed from the interior side in the middle
part of a formalin-fixed ovary, and hydrated oocytes were sepa-
rated from the tissue under a stereomicroscope. The number of
hydrated oocytes was counted for each subsample. Subsamples
were taken from the ovary at least twice, and the mean number
of hydrated oocytes between subsamples was converted into the
batch fecundity based on the total gonad weight. When the coef-
 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39 33

Fig. 3. Micrographs of oocyte developmental stages of Pacific bluefin tuna. (a) Unyolked stages (perinucleolus stage, PNS; yolk vesicle stage, YVS), (b) early yolked stage,
(c) advanced yolked stage, (d) migratory nucleus stage, (e) hydrated stage. (f) ␣ atresia and ␤ atresia. Sections were stained with haematoxylin-eosin. (g)–(l): Comparison
of post-ovulatory follicle (POF) images by staining method. POF classified as (g) and (j) 0 h old, (h) and (k) <12 h old, and (i) and (l) >12 h old. (g)–(i): haematoxylin-eosin
staining. (j)–(l): PAS-haematoxylin staining. The basement membrane shows a PAS-positive reaction. Scale bars represent 200 ␮m.

ficient of variation (CV) between two subsamples was higher than tests indicated no differences in the numbers of males and females
10%, extractions of the subsample were conducted until the CV was in each period (Table 2; P > 0.1). The difference in the numbers
less than 10%. of males and females through the total sampling period was not
significant (P = 0.5983), indicating nearly equal sex ratios.
2.7. Data

Information about each set by purse-seine vessels from which
fish were sampled, including date, start and end times, and sea sur-
face water temperature (SST), were copied from vessel log books.
Each catch month was divided into an early, middle, and late period.
Early period was set from 1st to 10th, middle period was set from
11th to 20th, and late period was set from 21st to 31st. SSTs associ-
ated with catches were used to report spawning temperatures. The
catch times were considered the end times of operation and were
used to estimate the spawning rhythm.
3. Results
3.1. Sex ratio
The numbers of males and females landed in 2012 were aggre-
gated by month and classified as early, middle and late. Chi-square
3.2. Occurrence of ovarian developmental classes
Of the 1040 ovaries, 139 ovaries were immature class, 20 ovaries
were developing class, 88 ovaries were spawning capable class, 806
ovaries were spawning class, 2 ovaries were regressing class, and
5 ovaries were regressed class. The spawning class exhibited the
highest proportion in both years, with values of 70.2% in 2011 and
93.8% in 2012. The proportion of the immature class was 15.3% in
2011 and only 2.8% in 2012.
GIs varied with maturity status and ovarian developmental class
(Fig. 4). The GIs of immature females were less than 3.5. The GIs of
reproductively active mature females differed by individual, rang-
ing from 0.67 to 21.91. The GIs for inactive mature females ranged
from 0.67 to 2.98. The mean GIs (±SD) of each ovarian develop-
ing class were 0.72 (±0.44) for immature class, 1.67 (±0.51) for
developing class, 4.39 (±3.10) for spawning capable class, 7.81
34 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39

Table 2
Numbers of male and female Pacific bluefin tuna caught and sampled in the Sea of Japan and the results of Chi-square tests.

Month Male (n) Female (n) Male fraction Chi-square

Late May 18 11 0.62 1.69

Mid-June 27 20 0.57 1.04
Late June 98 111 0.47 0.81
Early July 56 50 0.53 0.34
Mid-July 79 72 0.52 0.32
Late July 33 34 0.49 0.01
Total 311 298 0.51 0.28

Sex identification was performed for the landings in 2012.

Fig. 5. Seasonal changes in frequencies of ovarian developmental classes in 2011

and 2012. Numbers above the bars are sample sizes. Each month was divided into
early period (1st to 10th), middle period (11th to 20th), and late period (21st to
31st).

Fig. 4. Relationships between fork length (FL) and gonad index (GI) by ovarian devel-
opmental class for (a) immature females, (b) reproductively active mature females,
and (c) reproductively inactive mature females. Data are pooled for 2011 and 2012.
(±2.97) for spawning class, 2.04 (±1.34) for regressing class, and
1.62 (±0.69) for regressed class.
3.3. Spawning season
The immature class was mainly observed in late May in both
2011 and 2012, with frequencies higher than 80% (Fig. 5). The fre-
quency of developing class was 10.2% in late May and early June
in 2011 and less than 1.5% in other periods. Ovaries classified in
spawning capable class were observed from late May to late July
in 2011 and from late May to early July in 2012. The frequency of
spawning capable class was relatively higher in early and mid-June
in 2011. From mid-June, spawning class dominated, with frequen-
cies higher than 70%. Spawning class females were caught in same
area as the fishing area (Fig. 1). Ovaries classified inregressing class
were observed in late June in 2011 and 2012. Ovaries of regressed
class occurred in early June, early July, and late July with frequencies
of less than 1.5%.
The values of the mean GI and mean CF of mature females varied
throughout the sampling periods (Fig. 6). The mean GIs were less
than 2.2 from late May to early June in both years, greater than 7.5
from mid-June to mid-July, and decreased in late July. The mean CFs
of mature females were highest in late May and decreased through-
out the sampling period. The decrease in the mean CF from late May
to early August was 14.0% in 2011 and 12.6% in 2012.
The SSTs at which reproductively active spawning class ovaries
were collected after mid-June in 2011 ranged from 19.3 to 27.7 ◦ C
(mean = 23.2), and in 2012 ranged from 20.0 to 27.7 ◦ C (mean = 23.4)
(Fig. 7). The percentages of observations for spawning class ovaries
at SSTs equal or greater than 22 ◦ C was 77.9% in 2011, and 61.6% in
2012.
 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39
Fig. 6. Seasonal changes in gonad index (GI) and condition factor (CF) of mature
female Pacific bluefin tuna in 2011 and 2012. Bars show the standard deviations.
Fig. 7. Frequencies of sea-surface temperature (SST) at which the ovaries classified
as the spawning class collected after mid-June in 2011 and 2012.
3.4. Size at maturity
Immature females had a FL of between 85 and 151 cm, and the
smallest mature female was 107 cm (Fig. 8). The proportion mature
at FL (PFL ) was expressed by the following Richards function:
P FL = [1 − (1 − 1.149)e−0.138(FL−112.110) ]1/(1−1.149)
35
Fig. 8. Maturity schedule for female Pacific bluefin tuna. The dotted arrow indicates
the FL at 50% mature. Data are pooled for 2011 and 2012.
Fig. 9. Numbers of ovaries with migratory nucleus stage oocytes (MNS), hydrated
stage oocytes (HS), and 0 h old postovulatory follicles (0 h POF) by catch time.
FL at 50% mature was 114.4 cm and FL at 95% mature was
133.6 cm, corresponding to approximately 3 years old and 4 years
old, respectively, based on the fitted growth curve of Shimose et al.
(2009).
3.5. Daily spawning rhythm
During the spawning period, MAGOs of MNS were observed
between 0800 and 1800 h, and 95.6% of these were observed
between 0800 and 1300 h (Fig. 9). MAGOs of HS were observed
between 1000 and 2100 h, and 97.8% were observed between 1200
and 1800 h. The difference in the end of catch times for MNS fish and
HS fish was 3 h, being similar to that of 4 h for bigeye tuna (Nikaido
et al., 1991). Females with 0 h POFs were caught between 1200
and 2200 h, and 99.4% of these females were caught between 1700
and 2200 h. The mean catch times of MNS, HS, and 0 h POFs were
1103, 1503, and 1928 h, respectively. MAGOs of MNS were first
observed at 0800 h, and 0 h POFs were mainly observed after 1700 h,
indicating that it took approximately 9 h from the commencement
of oocyte maturation to spawning. Therefore, the main spawning
activities likely occurred between 1700 and 2200 h, based on the
appearance of 0 h POF.
3.6. Spawning frequency
Spawning frequency was estimated using the females sampled
from mid-June to early August, the peak of the observed spawn-
ing class (Fig. 5). Eight females that were caught during 1700 and
2200 h with >12 h old POFs and MAGOs of AYS were considered
36 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39

Table 3
Numbers of mature females, females with <24 h old POFs, spawning frequency, spawning interval, and percentages of daily spawners of mature females for Pacific bluefin
tuna in the Sea of Japan.

Year Month Mature female (n) Female with <24 h old POFs (n) Spawning frequency Spawning interval Daily spawners/mature females (%)

2011 Mid-June 45 32 0.71 1.41 53.3

Late June 140 112 0.80 1.25 70.0
Early July 155 148 0.95 1.05 81.3
Mid-July 95 91 0.96 1.04 91.6
Late July 68 63 0.93 1.08 83.8
Early August 41 41 1.00 1.00 97.6
2012 Mid-June 19 18 0.95 1.06 94.7
Late June 101 91 0.90 1.11 79.2
Early July 63 56 0.89 1.13 77.8
Mid-July 45 45 1.00 1.00 95.6
Late July 66 64 0.97 1.03 87.9
Early August 18 18 1.00 1.00 72.2
Total 856 779 0.91 1.10 81.0

>24 h post-spawning and were excluded from the fish with <24 h
POF in the estimation of the spawning frequency. The spawning fre-
quency and the spawning interval in mature females are shown in
Table 3. The spawning frequency in mature females in each period
was higher than 0.9 except in mid-June and late June in 2011. Only
in reproductively active mature females, the spawning frequency
was 0.92, and the spawning interval was 1.09 days. These results
indicate that mature females spawn nearly every day in the Sea of
Japan.
Females with both early and late POFs and late POFs and MAGOs
being MNS or HS are evidence of daily spawning activity (Schaefer,
1996), assuming the POFs are not detectable for greater than 24 h.
We considered that females having both <12 h POFs and >12 h POFs,
and MNS or HS and >12 h POFs as daily spawners in this study. The
proportion of daily spawners to mature females peaked in early
August in 2011 and in mid-July in 2012. The proportion of the two-
year total also indicated that most mature females spawned daily.

3.7. Batch fecundity

Batch fecundities were estimated from 67 females (116–170 cm

FL). The batch fecundities increased with FL, BW, and GW (Fig. 10),
and the relationships between the batch fecundity (BF) and each
size parameter were expressed by the following equations:

BF = 1.898 × 105 × FL − 199.473 × 105 (r 2 = 0609)

BF = 1.720 × 105 × BW − 23.843 × 105 (r 2 = 0.648)

BF = 0.016 × 105 × GW + 12.820 × 105 (r 2 = 0.819)

The highest correlation was with GW. The batch fecundities

ranged from 0.78 to 13.89 million oocytes, and the mean (±SD)
value was 6.41 (±2.68) million oocytes. Relative batch fecundities
per gram BW ranged from 27 to 213 oocytes, and the mean value
(±SD) was 122 (±36).

4. Discussion

4.1. Sex ratio

The nearly equal sex ratios observed in the present study are
consistent with results for Pacific bluefin tuna in the waters off
Taiwan, in the spawning ground of the northwestern Pacific Ocean
(Chen et al., 2006). For Atlantic bluefin tuna (T. thynnus), sex ratios
differ by sizes for the eastern stock around the Balearic Islands
spawning ground (Aranda et al., 2013b) and for larger fish in spawn-
ing schools of the western stock (Baglin, 1982). The possibility of
such differences in size classes or seasons should also be examined
for Pacific bluefin tuna in the future.
Fig. 10. Relationships between batch fecundities and fork length (FL), gilled and
gutted body weight (BW), and gonad weight (GW).
4.2. Spawning season and area
Among ovarian developmental classes, the spawning class was
in the highest proportion in 2011 and 2012. The occurrence of
reproductively active females indicates that the spawning of this
 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39
species starts in mid-June and continues until early August in the
Sea of Japan. The proportions of the reproductively active stage and
spawning frequencies were higher than 90% from mid-June in both
2011 and 2012. Higher mean GI values were observed from mid-
June to mid-July. The ratios of daily spawners to mature females
were higher than 90% in mid-July in both years. These results indi-
cate that spawning activity peaks during July in the Sea of Japan.
The end of the spawning season was not determined in the present
study because all females had spawning class ovaries, even in early
August at the end of the fishing season. However, the mean GI
of mature females decreased after mid-July, and the mean CF of
mature females was lowest in early August, suggesting that spawn-
ing activities were declining and that the end of the spawning
season in the Sea of Japan likely occurs in middle or late August.
Spawning class females were caught in the same area as the
fishing area. Okiyama (1974) reported the collection of a few post-
larvae of Pacific bluefin tuna in August from the central area of
Sea of Japan (38◦ –39◦ N, 138◦ –139◦ E). Nishikawa et al. (1985) also
reported that many larvae were collected in the western Sea of
Japan in early August. Besides the above areas, although collected
larvae were few, they were also collected in the northern part of the
Sea of Japan (39◦ –41◦ N, 139◦ –140◦ N) in recent research cruise (Abe
et al., 2014). The area of spawning found in this study was similar
to that described from larval surveys. However Abe et al. (2014)
reported that the hot spots of Pacific bluefin tuna spawning ground
were in the central-western Sea of Japan (36◦ –39◦ N, 132◦ –137◦ E)
based on the ocean numerical models. Therefore, considering the
result of Nishikawa et al. (1985), main spawning area in the Sea of
Japan may be the central-western part.
4.3. Spawning temperature
Tunas are generally known to spawn in waters with a SST of
≥24 ◦ C, based on the occurrence of larvae and spawning activity
of adults (Schaefer, 2001b). For yellowfin tuna (T. albacares), the
SST where spawning females were sampled in the eastern Pacific
Ocean (EPO) ranged from 21.5 to 30.5 ◦ C, but 85.3% were collected
between 26.0 and 30.0 ◦ C (Schaefer, 1998). Spawning skipjack tuna
(Katsuwonus pelamis) were sampled in the EPO at SSTs of ≥24 ◦ C
(Schaefer, 2001a). In the present study, the spawning class ovaries
were collected at SSTs between 19.3 and 27.7 ◦ C. Thus the spawning
of Pacific bluefin tuna in the Sea of Japan occurs at lower temper-
atures than generally reported. Such spawning activities in lower
temperatures have also been reported for a related species, Atlantic
bluefin tuna. Active spawning females of Atlantic bluefin tuna have
been collected in waters with a SST of 22.5–24.9 ◦ C (Karakulak
et al., 2004). Around the Balearic Islands, Atlantic bluefin tuna start
to spawn in waters >20.5 ◦ C, and their preferred temperature has
been estimated as 21.5–26.5 ◦ C based on larval collection (Alemany
et al., 2010). For cultured Pacific bluefin tuna, spawning occurred
with water temperatures from 21.6 to 29.2 ◦ C (Miyashita et al.,
2000a). However, Pacific bluefin tuna larvae have been collected
at higher temperatures, in waters with a temperature of approx-
imately 27 ◦ C (Tan and Chen, 1975) or 25.0–28.8 ◦ C (Tanaka et al.,
2006) in the northwestern Pacific Ocean and ≥26 ◦ C in the Sea of
Japan (Kitagawa et al., 1995). A recent study collected larvae at SSTs
of 23.9 to 29.5 ◦ C between May and August in the northwestern
Pacific Ocean and in the Sea of Japan (Abe et al., 2014). Miyashita
et al. (2000b) reported that the eggs of Pacific bluefin tuna hatched
at temperatures between 19.9 and 31.5, with an optimum temper-
ature of 25 ◦ C. Miyashita et al. (2000a) indicated that the beginning
factors of spawning were rise of water temperature and long-day
photoperiod. We therefore conclude that low levels of spawning
by Pacific bluefin tuna can occur in SSTs <24 ◦ C, as observed in June
in the Sea of Japan. However, such environments may not be opti-
mal for hatching eggs or larval survival. Reproductive strategies of
37
Pacific bluefin tuna involving spawning in colder water may require
additional consideration.
4.4. Size at maturity
We estimated that the FL at 50% mature was 114.4 cm FL and
that the FL at 95% mature was 133.6 cm. Chen et al. (2006) reported
that almost all Pacific bluefin tuna caught by the Taiwanese longline
are greater than 180 cm FL and sexually mature. Difference in size
distributions between spawning areas has been also reported by
Itoh (2006). Therefore our result is the first information on FL at
50% mature of this species and our estimates are similar to reports
for eastern Atlantic bluefin tuna of an L50 of 103.6 cm FL and a 100%
maturity rate at sizes above 135 cm, corresponding to 3 years of age
and 4–5 years of age, respectively (determined from the spiniform
ray) (Corriero et al., 2005). Size frequencies showed that in late May
and early June, the fish were mostly smaller than 130 cm FL, and
the females collected in this period had ovaries in the immature or
developing class. From mid-June, when most fish of larger FL classes
were collected, nearly all ovaries were classified into the mature or
spawning class. Purse seine fishing in the Sea of Japan tends to catch
immature and relatively small fish early in the fishing season during
May and early June, and catch larger reproductively active fish as
the season progresses. To accurately estimate a maturity schedule
for the stock in the Sea of Japan larger sample sizes throughout the
length range and spawning season need to be collected from both
the purse seine fishery and from other fisheries.
4.5. Spawning rhythms
Based on the appearance of 0 h POFs, the estimated spawning
time of Pacific bluefin tuna was between 1700 and 2200 h. This
estimate is similar to a report that spawning occurs between 1731
and 2336 h in reared Pacific bluefin tuna (Masuma et al., 2006). In
Atlantic bluefin tuna in transport cages, however, nocturnal spawn-
ing between 0300 and 0500 h has been reported (Gordoa et al.,
2009). Compared to the mean apparent time of migratory nucleus
oocytes (1933 h) and hydrated oocytes (20:56 h) for South Pacific
albacore (Farley et al., 2013), the mean appearance times of those
oocytes stages in this study were much earlier. Additionally big-
eye tuna (Nikaido et al., 1991) and yellowfin tuna (Schaefer, 1996,
1998; Itano, 2000) are also reported as nocturnal spawners. Due
to a lack of samples, spawning activities at night between 2200
and 0800 h could not be examined in this study. Although there
is no clear consideration which explains the reasons for spawn-
ing time differences from other tuna species, it might be due to
spawning strategy. Given the required time for hatching of Pacific
bluefin tuna, 32 h at 24 ◦ C (Miyashita et al., 2000b), spawning during
evening hours would result in a nocturnal hatch.
4.6. Spawning frequency
The ratio of daily spawners to mature females indicates that
most of the mature females spawned every day during the spawn-
ing season. The spawning frequency of 0.91 is similar to the
frequency of 0.87 for Atlantic bluefin tuna (Aranda et al., 2013b),
but much higher than the frequency of 0.3 for Pacific bluefin tuna
caught by longline in the northwestern Pacific Ocean (Chen et al.,
2006). There are two possible explanations for this difference. One
is a true difference in the reproductive parameter itself by area
or size, and the other is the sampling bias caused by fishing gear.
Sampling gear can influence the estimates of reproductive param-
eters of Atlantic bluefin tuna, with spawning frequencies of 0.84
for purse seine samples and 0.33 for longline samples (Medina
et al., 2007). Davis and Farley (2001) demonstrated that larger and
spawning southern bluefin tuna (T. maccoyii) are more frequently
38 Y. Okochi et al. / Fisheries Research 174 (2016) 30–39
found in shallower water, whereas non-spawning fish were caught
in deeper water. In light of these results, the estimates of spawning
frequency based on females caught with purse seiners, one of the
surface fisheries would be higher than those from fish caught with
longlines. Although there is a possibility that mature non-spawning
females were not caught because of fishing gear selectivity, spawn-
ing females appear to spawn daily in the Sea of Japan according
to another parameter, “proportion of daily spawners” (the ratio
of females having <12 h and >12 h POFs and females having >12 h
POFs and MAGOs of MNS or HS to mature females). The presence of
<12 h and >12 h POFs is the consequence of spawning on successive
days, and the presence of the >12 h POFs and MAGOs of MNS or HS
indicates that successive spawning will occur in the near future. If
considerable effects of fishing gear bias were present, the “propor-
tions of daily spawners” should be even lower than the spawning
frequency (i.e., if the fish were to spawn every two or more days
and only spawning fish were caught because of fishing gear selec-
tivity, the value of the “proportion of daily spawners” should be
zero because >12 h POFs would disappear during the non-spawning
days). The quantity of available food may vary in area and also affect
reproduction. Spawning intervals in Japanese anchovy (Engraulis
japonica) vary with feed quantities (Tsuruta, 1992). Itano (2000)
reported that mature yellowfin tuna in actively feeding schools
were in daily or near-daily spawning condition. Research on feed-
ing activities during the breeding season in each spawning ground
might be one of the factors to elucidate the reason for differences
in spawning frequencies among spawning grounds.
4.7. Batch fecundity
The batch fecundities were positively correlated with body size,
as reported for Pacific bluefin tuna in the northwestern Pacific
Ocean (Chen et al., 2006). Using the formula obtained by Chen et al.
(2006), batch fecundities in the Sea of Japan were estimated to be
larger than those in the northwestern Pacific Ocean for a given size.
For example, the batch fecundities for the 170-cm-FL size class were
estimated to be 12 million eggs in the Sea of Japan and 3 million
eggs in the northwestern Pacific Ocean. The sampled females in this
study were smaller than that of females caught in the northwest-
ern Pacific Ocean, where the females caught were generally larger
than 180 cm (Chen et al., 2006). To obtain a more precise com-
parison, additional samples of larger females in the Sea of Japan
need to be analyzed. For bigeye tuna (T. obesus), the fecundity of
females caught by longlines differs by areas, and the difference is
also correlated with the values of the gonadosomatic index (Nikaido
et al., 1991). Food quantity influences fecundity in captive Japanese
anchovy (Tsuruta, 1992), and Margulies et al. (2007) also reported a
positive correlation between daily food intake and batch fecundity
in yellowfin tuna. Further research on feeding activity is needed
to evaluate the spatial variability in fecundity and spawning fre-
quency.
Annual fecundity is estimated from the multiplication of the
batch fecundity, spawning frequency, and spawning period. We
observed that the spawning season lasted at least 2 months. If
the spawning period of individuals lasts for 2 months, the aver-
age annual fecundity, calculated from the mean batch fecundity of
females between 116 and 170 cm FL and the mean spawning fre-
quency of 0.91, is estimated to be 349 million oocytes. By contrast,
average annual fecundities were estimated to be 70–80 million eggs
for Atlantic bluefin tuna in the Balearic Sea (Medina et al., 2007;
Aranda et al., 2013b), using two weeks spawning period based on
an archival tag study in the Gulf of Mexico (Gunn and Block, 2001).
The study of satellite archival tags showed that the mean spawn-
ing period of Atlantic bluefin tuna was 23.9 days around Balearic
Islands (Aranda et al., 2013a). If the values of 14 and 24 days were
applied to our result as the individual spawning period, the aver-
age annual fecundity would be estimated at comparable values of
82 and 140 million oocytes, respectively. Farley et al. (2013) cal-
culated the potential annual fecundity of albacore (T. alalunga)
by age-related estimation from monthly batch fecundities. Fur-
ther information on the variation in batch fecundity based on age,
season, and the individual spawning period will enable precise esti-
mations of the annual fecundity of stock.
5. Conclusion
This investigation describes some reproductive parameters for
smaller and younger female Pacific bluefin tuna, especially from
spawning schools in the Sea of Japan. Investigations on matu-
rity and reproductive activity of fish caught with other fishing
gear throughout the Sea of Japan are also needed to clarify their
reproductive biology. Spawning frequency and batch fecundity
evaluated in this study were higher than those of larger and older
Pacific bluefin tuna in the northwestern Pacific Ocean, another
important spawning ground. There are many differences between
the two areas in environmental factors such as food availability
and water temperature, the size distributions of the fish, and fish-
ing gear. To clarify the source of the differences in reproductive
parameters, both environmental factors and fishing gear selec-
tivity should be evaluated. Improved estimates of Pacific bluefin
tuna reproductive parameters including maturity and fecundity are
required for use in stock assessments, and further investigations
should be undertaken.
Acknowledgements
This research was implemented as part of the Research Project
on Japanese Bluefin Tuna conducted by the Fisheries Agency of
Japan. We sincerely appreciate the support of the Tottori Prefectural
Fisheries Experiment Station, the Fisheries Cooperative Associa-
tion of Sakaiminato, and the fishermen who cooperated in sample
collection.
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