Aquaculture 388–391 (2013) 30–34

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Using osmotic pumps to deliver hormones to induce sexual maturation

of female Japanese eels, Anguilla japonica
Hirohiko Kagawa a,⁎, Nobuhiro Fujie a, Hitoshi Imaizumi b, Yoshitsugu Masuda b, Kentaro Oda c,
Junichi Adachi d, Akefumi Nishi e, Hiroshi Hashimoto f, Kazuhisa Teruya g, Shunji Kaji h
a
Department of Marine Biology and Environmental Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
b
Shibushi Laboratory, National Research Institute of Aquaculture, Fisheries Research Agency, Shibushi, Kagoshima 899-7101, Japan
c
Marine Fisheries Research and Development Center, Fisheries Research Agency, Minatomirai, Yokohama, Kanagawa 220-6115, Japan
d
Headquarters, Incorporated Administrative Agency, Fisheries Research Agency, Minatomirai, Yokohama, Kanagawa 220-6115, Japan
e
Amami Station, National Center for Stock Enhancement Fisheries Research Agency, Hyo Setouchi Oshima, Kagoshima 894-2414, Japan
f
Seikai National Fisheries Research Institute, Fisheries Research Agency, Ishigaki, Okinawa 907-0451, Japan
g
Research Center for Subtropical fisheries, Seikai National Fisheries Research Institute, Fisheries Research Agency, Ishigaki, Okinawa 907-0451, Japan
h
Kamiura Branch, National Research Institute of Aquaculture, Fisheries Research Agency, Saiki, Oita 876-2602, Japan

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

Article history: This study examines how the continuous administration of various hormones using an osmotic pump, a
Received 24 April 2012 long-term sustained hormone release system, influences the induction of sexual maturation in females of
Received in revised form 12 January 2013 the Japanese eel, Anguilla japonica. Human chorionic gonadotropin (hCG), salmon pituitary extract (SPE),
Accepted 21 January 2013
and gonadotropin-releasing hormone analogue (GnRHa) were tested, and the consequent egg quality was
Available online 28 January 2013
evaluated. The implantation of osmotic pumps loaded with SPE induced vitellogenesis and increased the
Keywords:
gonadosomatic index (GSI) at 39–110 days. In comparison, pumps loaded with hCG inconsistently induced
Female Japanese eels early vitellogenesis, while those loaded with GnRHa did not exhibit any stimulatory effect. Fewer eels attained
Sexual maturation full maturity when using the osmotic pump system compared with SPE-injected female eels. However, after
Human chorionic gonadotropin the final treatment, the number of eels that ovulated and the time required for ovulation were similar for both
Salmon pituitary extract the osmotic pump and injection groups. Moreover, more eggs were spawned in the SPE-loaded osmotic pump
GnRH group than in the SPE-injected group. Egg quality was similar for both experimental groups. Therefore, the implan-
Osmotic pump tation of an osmotic pump loaded with SPE represents a reliable method for inducing vitellogenesis and obtaining
ovulated eggs from sexually immature Japanese eels.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Cultivated female eels tend to remain sexually immature under
captive rearing conditions (Yamamoto et al., 1972). However, studies
have shown that repeated weekly injections of salmon pituitary extract
(SPE) over a 10-week period effectively induce vitellogenesis, resulting
in female eels fully maturing to produce fully grown oocytes at the
migratory nucleus stage (Kagawa, 2003; Kagawa et al., 2005; Ohta
et al., 1996a). In addition, human chorionic gonadotropin (hCG) induces
spermatogenesis and spermiation in male eels (Ohta et al., 1996b).
However, the weekly injection of hormones requires repetitive handling
of the broodstock, which incurs substantial labor, time, and monitoring
costs, as well as causing stress and increased mortality of the fish. Over
⁎ Corresponding author. Tel./fax: +81 985 58 7223.
E-mail address: kagawa@cc.miyazaki-u.ac.jp (H. Kagawa).
the years, a variety of methods have been developed, such as cholesterol
pellets and copolymer pellets, for the sustained release of gonadotropin-
releasing hormone analogue (GnRHa), which effectively induce oocyte
maturation and ovulation in female cultivated fish (Mylonas and Zohar,
2001; Zohar and Mylonas, 2001). Recently, our research group developed
a long-term hormone delivery system using an osmotic pump (Kagawa et
al., 2009). The implantation of a single hCG-loaded osmotic pump
successfully induced spermatogenesis, and increased the gonadosomatic
index (GSI) at 35–42 days post-implantation. However, the use and effi-
cacy of osmotic pumps for inducing sexual maturation have not been
studied in female eels.
Therefore, we examined the effects of administering hCG, SPE, and
GnRHa via an osmotic pump (which has the potential of long-term
sustained hormone-release) to induce sexual maturation in females of
the Japanese eel, Anguilla japonica. Furthermore, we compared the egg
quality of eels implanted using the SPE-loaded osmotic pump with
those induced to sexually mature using the standard SPE-injection
method.

0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.01.025
 H. Kagawa et al. / Aquaculture 388–391 (2013) 30–34
2. Materials and methods
2.1. Fish and hormonal treatments
Cultured female Japanese eels were either obtained from a fish farm or
from the National Research Institute of Aquaculture, Fisheries Research
Agency, Japan. After the eels were acclimated to seawater, they were
maintained in indoor 400-L circulating tanks under a natural photoperiod
at a water temperature of 20 °C, without food. After anesthetization with
2-phenoxyethanol (Nacalai Tesque, Tokyo, Japan), the fish were weighed
and then treated with various hormones.
An osmotic pump (Osmotic Pump Type 2002; Alzet Osmotic
Pumps Co., Cupertino, CA; diameter=7 mm, length=30 mm, reservoir
volume=approximately 200 μL) that releases a constant amount of
hormones over a long period (Kagawa et al., 2009) was loaded with
GnRHa, hCG or SPE. According to the manufacturer's instruction manual,
the osmotic pump releases a maximum of 5 μL of a given solution per day
for approximately 45–50 days when fish are maintained at a water tem-
perature of 20 °C. We validated this specification of the expected volume
of hormone solution in the osmotic pump reservoir at the end of the
experiment by measuring the remaining solution volume. Moreover,
using ELISA kit (Wako Pure Chemical Industry, Osaka) we confirmed
that the levels of hCG in the serum remained constant throughout the
experiment (Kagawa et al., 2009). An osmotic pump was implanted
into the peritoneal cavity of each eel after cutting an approximately
8-mm opening in the abdomen with a fine scalpel. The wound was not
sutured, but healed naturally within 2 weeks.
hCG (Teikoku Zhoki Co. Ltd., Tokyo, Japan) was dissolved in 0.9%
sterilized sodium chloride solution. GnRHa, des-Gly10-[D-Ala 6]-LH-RH
ethylamide (Sigma) was dissolved in 0.9% sodium chloride, containing
0.1% bovine serum albumin (BSA). The addition of BSA prevents the ad-
hesion of GnRHa to the reservoir wall of the osmotic pump. SPE was
prepared by homogenizing dried salmon (Oncorhynchus keta) pituitary
powder with 0.9% sodium chloride solution, followed by centrifugation
at 9700 ×g (Kagawa et al., 1995, 1997). SPE was concentrated with a con-
centrator (Vivapore 10/20; Sartorius Stedim Lab, Ltd., Gloucestershire,
UK), before loading it into the osmotic pumps. The pituitaries used in
the present study were obtained from ovulated or spermiated fish
migrating to the natal river for spawning. Therefore, higher volumes of
LH and FSH were expected to be present in the pituitary, as suggested
by a previous study (Swanson et al., 2003).
2.2. Experimental design
In Experiment 1 (Table 1), 20 fish were randomly divided into 3
groups (n = 6–7 eels/group): the saline control group, a single osmotic
pump loaded with hCG (120 IU/day/fish) group, and an osmotic pump
loaded with SPE (3 mg/day/fish) group. The fish were implanted with a
single osmotic pump on the first experimental day.
In Experiment 2, 23 fish were randomly divided into 4 groups (n=
5–6 eels/group). The first group was the control group, while the other
Table 1
The body weight of fish, hormone treatments, and experimental period of eels in each of the 3 experiments.
Experiments Body weight
b
Experiment 1 725 g (250–1201 g)
Experiment 2 490 g (405–66 5 g)
Experiment 3 552 g (350–765 g)
a
Hormones were administrated with an osmotic pump.
b
Mean body weight (min.–max.)
c
The osmotic pump was implanted twice during Experiment 3.
d
SPE was administrated viainjection. SPE: salmon pituitary extract; HCG: human chorionic gonadotropin; and GnRHa: gonadotropin-releasing hormone analogue.
31
3 groups were represented by eels implanted with a single osmotic
pump loaded with SPE (1.5 mg/day), hCG (75 IU/day/fish), or GnRHa
(5.25 μg/day/fish). The saline control group received a single osmotic
pump containing just 0.9% sodium chloride.
In Experiment 3, 32 fish were randomly divided into 2 groups
(n = 16 eels/group). In the first group, eels were first implanted
with a single osmotic pump loaded with SPE (4 mg/day/fish), with
the same osmotic pump being implanted again at 6 weeks after the
start of the experiment. In the second group, eels were injected
weekly with SPE (28 mg/week/fish).
2.3. Sampling
At 38 days post-implantation in Experiment 1, and at 51 days
post-implantation in Experiment 2, the eels were terminally anesthe-
tized with 2-phenoxyethanol and weighed. Their gonads were dissect-
ed and removed to calculate the gonadosomatic index (GSI). In brief,
sections of the ovaries obtained at the end of the experiments were
fixed in Bouin's solution. Serial 7-μm-thick paraffin sections were
stained with Mayer's hematoxylin and eosin. The developmental stages
of oocytes were assessed using the criteria presented in a previous
paper (Yamamoto et al., 1974).
2.4. Artificial insemination
In Experiment 3, after the completion of vitellogenesis (full-grown),
all fish received a final treatment to induce oocyte maturation and ovu-
lation according to a previously reported method (Kagawa, 2003; Ohta
et al., 1996a). In brief, female eels that contained oocytes of more than
850 μm in diameter at the migratory nucleus stage were injected with
SPE (30 mg/kg) as a priming dose, followed by an intra-peritoneal
injection of 17,20β-dihydroxy-4-pregnen-3-one (DHP; 2 mg/kg) 24 h
later. After the final injection of DHP, each female eel was maintained
separately in a spawning tank at 22 °C and under a natural photoperiod.
From 10 h after the DHP injection, the fish were checked for ovulation
at 3-h intervals by applying gentle pressure to the abdomen in an
anterior to posterior direction. Artificial fertilization was performed
according to a previously reported method (Ohta et al., 1996a). In
brief, 2 g of ovulated eggs (about 3300 eggs) obtained from 1 female
were artificially fertilized with 1 mL of pre-diluted milt.
2.5. Assessment of egg quality
Egg quality (fertility, hatchability, and survival of the larvae) until
7 days post hatch was assessed using an individual rearing method,
which is described in Unuma et al. (2004). In brief, fertilized eggs
were stocked in a 96-well plate, with 1 egg per well (wells were filled
with filtered seawater; pore size 0.2 μm), in addition to penicillin
G potassium (Banyu Pharmaceutical, Tokyo) at 100,000 IU/L and
streptomycin sulfate (Meiji Seika, Tokyo) at 0.1 g/L. The wells were
maintained at 23 °C. Dead larvae were not removed, the water was
Hormone treatmentsa Experiment period
SPE (3 mg/day/fish) 2005/12/21–2006/01/28
HCG (120 IU/day/fish)
SPE (1.5 mg/day/fish) 2006/08/04–2006/09/22
HCG (75 IU/day/fish)
GnRHa (5.25 μg/day/fish)
SPE (4 mg/day/fishc) 2008/06/09–2008/09/26
SPE (28 mg/week/fishd)
32 H. Kagawa et al. / Aquaculture 388–391 (2013) 30–34

not changed, and food was not provided. Larvae at 7 days post hatch
were fixed with 10% formalin, and observed for abnormalities under a
A
stereoscopic microscope (Kurokawa et al., 2008). The parameters used
in this study were calculated with the following formulas:

Fertility ð% Þ ¼ 100  ðthe number of eggs displaying cleavage at 4 h post fertilization=

the number of eggs used for assessmentÞ:

Hatchability ð% Þ ¼ 100  ðthe number of hatched larvae=

the number of eggs used for assessmentÞ:

Survival of larvae at 7 days post hatch ð% Þ

¼ 100  ðthe number of larvae that survived at 7 days post hatch=
the number of eggs used for assessmentÞ:

B
2.6. Statistics

All data are represented as the mean±SEM (Standard Error of

Mean). The differences were compared by one-way ANOVA and the
Newman–Keul's multiple comparison test. A result of P b 0.05 was con-
sidered statistically significant. Fish that died during the experiment
were excluded from the evaluation.

3. Results

3.1. Experiment 1

The mean GSI of the control group was 2.2 ±0.2 (Fig. 1), with fish
containing very small ovaries (Fig. 2A), in which the oocytes were at
the oil droplet and at the primary yolk globule stages (Table 2). The
implantation of a single hCG-loaded osmotic pump did not cause any
significant increase in the GSI (4.8 ± 0.5) of female eels (Fig. 1); howev-
er, their ovaries contained oocytes at the primary (5 of 7 fish) and
secondary yolk globule (2 of 7 fish) stages (Table 2). The mean GSI of
the SPE-loaded group (27.5 ± 4.1) was significantly higher compared
to the other 2 groups, with fish containing large ovaries with oocytes
at the migratory nucleus stage (Fig. 2B, Table 2).
Fig. 2. Photographs of (A) the ovary of a female Japanese eel implanted with a single
osmotic pump loaded with 0.1% bovine serum albumin (BSA) in 0.9% sodium chloride
(control) and (B) the ovary of a female Japanese eel implanted with a single osmotic
pump loaded with salmon pituitary extract (SPE, 3 mg/day/fish). The arrows indicate
the ovary and the arrowhead indicates the osmotic pump.
nucleus or secondary yolk globule stage, respectively (Table 3), while
the remaining fish had oocytes at oil droplet or primary yolk globule
stage.

3.2. Experiment 2 3.3. Experiment 3

In both the saline control and GnRHa-loaded osmotic pump groups
(Fig. 3), the mean GSI was very low (1.3±0.1 and 1.5 ± 0.2, respective-
ly), and most fish contained ovaries with oocytes at the oil droplet stage
(Table 3). Implantation of an osmotic pump loaded with hCG did not re-
sult in any significant increase in GSI (3.2± 0.5) (Fig. 3); however, 2 of
the 6 females contained ovaries with oocytes at the primary yolk glob-
ule stage (Table 3). SPE implantation significantly increased the GSI,
with 2 out of 6 fish containing ovaries with oocytes at the migratory
35
30
25
20
GSI
In the osmotic pump implanted group, 10 out of the 16 female eels
had attained full maturity by the end of experiment (Table 4). These
fish contained ovaries with oocytes at the migratory nucleus stage
that were approximately 850 μm in diameter. The remaining 5 fish
contained immature ovaries. One of the 16 female eels died during
the final treatment.
In the osmotic pump injected group, 8 out of the 10 fully mature
female eels ovulated after receiving the final treatment, 1 eel did
not ovulate, and 1 eel died (Table 5). In the SPE injection group, 13
out of the 15 females ovulated, with just 2 not ovulating by the final
b treatment.
Table 2

Oocyte developmental stages of Japanese eels at the initial and the final stages of Ex-
15 periment 1.

10 Treatments Initial stage Final stage

a
5 a Oil PYG Oil PYG SYG TYG MN
0 Cont – – 6 4 0 0 0
Control HCG SPE
HCG 3a 4 0 5 2 0 0
SPE 3 4 0 0 0 0 7
Fig. 1. Effects of human chorionic gonadotropin (hCG, 120 IU/day/fish) and salmon pitu-
itary extract (SPE, 3 mg/day/fish) on the gonadosomatic index (GSI) of female Japanese Cont: Control; HCG: human chorionic gonadotropin; SPE: salmon pituitary extract; Oil:
eels, administrated via osmotic pump. The control fish (control) were implanted with a oil droplet stage; PYG: primary yolk globule stage; SYG: secondary yolk globule stage;
single osmotic pump loaded with 0.1% bovine serum albumin (BSA) in 0.9% sodium TYG: tertiary yolk globule stage; and MN: migrate nucleus stage.
a
chloride. Different letters indicate statistically significant differences (Pb 0.05). Number of fish.
 H. Kagawa et al. / Aquaculture 388–391 (2013) 30–34 33

24 Table 4
Effects of different salmon pituitary extract administration methods on Japanese eel
20
vitellogenesis.
16 b Methods Number of fish
GSI

12 Fullly grown Immature Dead

8 a
a Injection
4 a a a
0 b
Control GnRHa HCG SPE
Treatments
Fig. 3. Effects of the single implantation of osmotic pumps loaded with gonadotropin-
releasing hormone analogue (GnRHa, 5.25 μg/day/fish), salmon pituitary extract (SPE,
1.5 mg/day), or human chorionic gonadotropin (hCG, 75 IU/day) on the gonadosomatic
index (GSI) of female Japanese eels. The control fish (control) were implanted with a single
osmotic pump loaded with 0.1% bovine serum albumin (BSA) in 0.9% sodium chloride. Differ-
ent letters indicate statistically significant differences (Pb 0.05).
The ovulated egg volume and number of eggs were significantly
higher in the osmotic pump implanted group than in the injection
group (Table 6). The egg quality of both groups is shown in Fig. 4.
There was no significant difference in fertility and normal cleavage
rate, between the osmotic implanted and injection groups; however,
other factors, such as hatchability, survival rates at 3 and 7 days post
hatch, and normal larvae rates at 3 and 7 days post hatch, were signif-
icantly higher in the osmotic pump implanted group compared to the
injection group.
4. Discussion
This study confirms the effectiveness of using osmotic pumps to
induce the maturation of captive female eels, complementing a previ-
ous study on the maturation of captive male Japanese eels (Kagawa et
al., 2009). Specifically, SPE was found to be a more effective hormone
preparation in this process compared with hCG and GnRHa, which
had minimal or no effect.
The dose of SPE used in the present study varied among the 3
experiments (from 1.5 mg to 4 mg/day/fish). The total amount of
SPE released from the osmotic pump within a week (21.7–
50.4 mg/kg/week, calculated from the data of mean body weight and
daily release rates of SPE) were similar to or higher than the quantity
used for the injection technique (20–40 mg/kg body weight/week) in
previous experiments (Ijiri et al., 1998; Kagawa, 2003). Ijiri et al. (1998)
indicated that eels treated with high-dose injections (40 mg SPE/kg
body weight/week) reached full maturity sooner and with greater
efficiency compared to those treated with low-dose injections (20 mg
SPE/kg body weight). Similar results were obtained in the present
study. However, other factors might also induce and complete vitellogen-
esis in female eels. For instance, the developmental stage of the ovary
before SPE injection directly influences the success rate of artificial matu-
ration and the time required to reach the final maturation stage, as indi-
cated in a previous study (Ijiri et al., 1998).
Table 3
Oocyte developmental stages of Japanese eels at the initial and the final stages of Ex-
periment 2.
b
Osmotic pump 16 10 (63.4 ) 5 (31.3) 1 (6.3)
16 15 (93.7) 1 (6.3) 0 (0)
Number of fish used.
Percentage.
The present study demonstrates that hCG can stimulate vitellogene-
sis in the female eels. However, from the data of GSI, hCG showed a
lower potency than SPE for inducing vitellogenesis. Since SPE contains
not only gonadotropins (LH and FSH) but also many other hormones,
such as GH and TSH, which are also implicated in vitellogenesis in fish
(Moussavi et al., 2009; Rocha et al., 2007), in cooperation with
gonadotropin.
The implantation of an osmotic pump loaded with GnRHa did not
stimulate vitellogenesis, with GSI and histological observations remaining
similar to those of the control group. Similar results were obtained for
male eels (Kagawa et al., 2009). GnRH treatment alone (Dufour et al.,
1991) has been generally unsuccessful in inducing vitellogenesis of
sexually immature female silver eels, because luteinizing hormone syn-
thesis and release are not stimulated (Dufour et al., 1991; Yaron et al.,
1995). In comparison, combined treatments with GnRHa and a dopamine
antagonist (blocker of dopamine D2-subtype receptor or blocker of
catecholamine biosynthesis) trigger luteinizing hormone release and
vitellogenesis in female European eels pretreated with estradiol-17β or
testosterone (Dufour et al., 2003). Therefore, it is possible that an osmotic
pump loaded with both GnRHa and a dopamine antagonist might pro-
gressively induce vitellogenesis in female Japanese eels.
Final treatments (SPE priming dose followed by DHP injection) in-
duced similar levels of oocyte maturation and ovulation in both the
osmotic pump and injection groups. Therefore, the SPE-loaded osmotic
pump could be used to obtain fully grown female eels with ovulated
eggs. Egg quality in both the injection and osmotic pump groups was
similar with respect to fertility and normal cleavage rates. Moreover,
the volume and number of ovulated eggs was significantly higher in
the osmotic pump implanted group compared to the injection group.
These results indicate that fully grown female eels induced by the
implantation of the SPE loaded osmotic pump produce an abundance
of good quality eggs. However, the fertility of eggs was low in the cur-
rent study (approximately 25% in the injection group) and hatchability
(approximately 10%) when compared to the results of previous studies
(40% hatchability) (Kagawa, 2003; Kagawa et al., 2005). Therefore, it is
important to determine whether higher quality eggs are obtained from
female eels implanted with SPE-loaded osmotic pumps in future
studies.
The present study confirmed that long-term SPE treatment via an
osmotic pump presents an effective method for inducing vitellogene-
sis in female Japanese eels. The SPE implantation dose used in the
present study (1.5–7.2 mg/kg/day) significantly increased the GSI of
female eels. Fully grown female eels obtained from implantation of
the osmotic pump were successfully induced for oocyte maturation

Treatments Initial stage Final stage Table 5

Oil PYG Oil PYG SYG TYG MN Effects of final treatments on the induction of oocyte maturation and ovulation in Jap-
anese eels.
Cont 5a 0 4 1 0 0 0
GnRHa 6 0 6 0 0 0 0 Methods Number of fish
HCG 6 0 2 4 0 0 0
Ovulation Non-ovulation Dead
SPE 6 0 1 1 2 0 2
Osmotic pump 10a 8 (80.0)b 1(10.0) 1(10.0)
Cont: Control, HCG: human chorionic gonadotropin; SPE: salmon pituitary extract; Oil:
Injection 15 13 (86.7) 2 (13.3) 0 (0)
oil droplet stage; PYG: primary yolk globule stage; SYG: secondary yolk globule stage;
a
TYG: tertiary yolk globule stage; MN: migrate nucleus stage. Number of fish used.
a b
Number of fish. Percentage.
34 H. Kagawa et al. / Aquaculture 388–391 (2013) 30–34

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