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03 - Kagawa 2013 - Using Osmotic Pumps To Deliver Hormones To Induce Sexual Maturation
03 - Kagawa 2013 - Using Osmotic Pumps To Deliver Hormones To Induce Sexual Maturation
Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online
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 the years, a variety of methods have been developed, such as cholesterol
pellets and copolymer pellets, for the sustained release of gonadotropin-
Cultivated female eels tend to remain sexually immature under releasing hormone analogue (GnRHa), which effectively induce oocyte
captive rearing conditions (Yamamoto et al., 1972). However, studies maturation and ovulation in female cultivated fish (Mylonas and Zohar,
have shown that repeated weekly injections of salmon pituitary extract 2001; Zohar and Mylonas, 2001). Recently, our research group developed
(SPE) over a 10-week period effectively induce vitellogenesis, resulting a long-term hormone delivery system using an osmotic pump (Kagawa et
in female eels fully maturing to produce fully grown oocytes at the al., 2009). The implantation of a single hCG-loaded osmotic pump
migratory nucleus stage (Kagawa, 2003; Kagawa et al., 2005; Ohta successfully induced spermatogenesis, and increased the gonadosomatic
et al., 1996a). In addition, human chorionic gonadotropin (hCG) induces index (GSI) at 35–42 days post-implantation. However, the use and effi-
spermatogenesis and spermiation in male eels (Ohta et al., 1996b). cacy of osmotic pumps for inducing sexual maturation have not been
However, the weekly injection of hormones requires repetitive handling studied in female eels.
of the broodstock, which incurs substantial labor, time, and monitoring Therefore, we examined the effects of administering hCG, SPE, and
costs, as well as causing stress and increased mortality of the fish. Over 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
⁎ Corresponding author. Tel./fax: +81 985 58 7223. those induced to sexually mature using the standard SPE-injection
E-mail address: kagawa@cc.miyazaki-u.ac.jp (H. Kagawa). 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 31
2. Materials and methods 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
2.1. Fish and hormonal treatments (5.25 μg/day/fish). The saline control group received a single osmotic
pump containing just 0.9% sodium chloride.
Cultured female Japanese eels were either obtained from a fish farm or In Experiment 3, 32 fish were randomly divided into 2 groups
from the National Research Institute of Aquaculture, Fisheries Research (n = 16 eels/group). In the first group, eels were first implanted
Agency, Japan. After the eels were acclimated to seawater, they were with a single osmotic pump loaded with SPE (4 mg/day/fish), with
maintained in indoor 400-L circulating tanks under a natural photoperiod the same osmotic pump being implanted again at 6 weeks after the
at a water temperature of 20 °C, without food. After anesthetization with start of the experiment. In the second group, eels were injected
2-phenoxyethanol (Nacalai Tesque, Tokyo, Japan), the fish were weighed weekly with SPE (28 mg/week/fish).
and then treated with various hormones.
An osmotic pump (Osmotic Pump Type 2002; Alzet Osmotic 2.3. Sampling
Pumps Co., Cupertino, CA; diameter=7 mm, length=30 mm, reservoir
volume=approximately 200 μL) that releases a constant amount of At 38 days post-implantation in Experiment 1, and at 51 days
hormones over a long period (Kagawa et al., 2009) was loaded with post-implantation in Experiment 2, the eels were terminally anesthe-
GnRHa, hCG or SPE. According to the manufacturer's instruction manual, tized with 2-phenoxyethanol and weighed. Their gonads were dissect-
the osmotic pump releases a maximum of 5 μL of a given solution per day ed and removed to calculate the gonadosomatic index (GSI). In brief,
for approximately 45–50 days when fish are maintained at a water tem- sections of the ovaries obtained at the end of the experiments were
perature of 20 °C. We validated this specification of the expected volume fixed in Bouin's solution. Serial 7-μm-thick paraffin sections were
of hormone solution in the osmotic pump reservoir at the end of the stained with Mayer's hematoxylin and eosin. The developmental stages
experiment by measuring the remaining solution volume. Moreover, of oocytes were assessed using the criteria presented in a previous
using ELISA kit (Wako Pure Chemical Industry, Osaka) we confirmed paper (Yamamoto et al., 1974).
that the levels of hCG in the serum remained constant throughout the
experiment (Kagawa et al., 2009). An osmotic pump was implanted 2.4. Artificial insemination
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 In Experiment 3, after the completion of vitellogenesis (full-grown),
sutured, but healed naturally within 2 weeks. all fish received a final treatment to induce oocyte maturation and ovu-
hCG (Teikoku Zhoki Co. Ltd., Tokyo, Japan) was dissolved in 0.9% lation according to a previously reported method (Kagawa, 2003; Ohta
sterilized sodium chloride solution. GnRHa, des-Gly10-[D-Ala 6]-LH-RH et al., 1996a). In brief, female eels that contained oocytes of more than
ethylamide (Sigma) was dissolved in 0.9% sodium chloride, containing 850 μm in diameter at the migratory nucleus stage were injected with
0.1% bovine serum albumin (BSA). The addition of BSA prevents the ad- SPE (30 mg/kg) as a priming dose, followed by an intra-peritoneal
hesion of GnRHa to the reservoir wall of the osmotic pump. SPE was injection of 17,20β-dihydroxy-4-pregnen-3-one (DHP; 2 mg/kg) 24 h
prepared by homogenizing dried salmon (Oncorhynchus keta) pituitary later. After the final injection of DHP, each female eel was maintained
powder with 0.9% sodium chloride solution, followed by centrifugation separately in a spawning tank at 22 °C and under a natural photoperiod.
at 9700 ×g (Kagawa et al., 1995, 1997). SPE was concentrated with a con- From 10 h after the DHP injection, the fish were checked for ovulation
centrator (Vivapore 10/20; Sartorius Stedim Lab, Ltd., Gloucestershire, at 3-h intervals by applying gentle pressure to the abdomen in an
UK), before loading it into the osmotic pumps. The pituitaries used in anterior to posterior direction. Artificial fertilization was performed
the present study were obtained from ovulated or spermiated fish according to a previously reported method (Ohta et al., 1996a). In
migrating to the natal river for spawning. Therefore, higher volumes of brief, 2 g of ovulated eggs (about 3300 eggs) obtained from 1 female
LH and FSH were expected to be present in the pituitary, as suggested were artificially fertilized with 1 mL of pre-diluted milt.
by a previous study (Swanson et al., 2003).
2.5. Assessment of egg quality
2.2. Experimental design
Egg quality (fertility, hatchability, and survival of the larvae) until
In Experiment 1 (Table 1), 20 fish were randomly divided into 3 7 days post hatch was assessed using an individual rearing method,
groups (n = 6–7 eels/group): the saline control group, a single osmotic which is described in Unuma et al. (2004). In brief, fertilized eggs
pump loaded with hCG (120 IU/day/fish) group, and an osmotic pump were stocked in a 96-well plate, with 1 egg per well (wells were filled
loaded with SPE (3 mg/day/fish) group. The fish were implanted with a with filtered seawater; pore size 0.2 μm), in addition to penicillin
single osmotic pump on the first experimental day. G potassium (Banyu Pharmaceutical, Tokyo) at 100,000 IU/L and
In Experiment 2, 23 fish were randomly divided into 4 groups (n= streptomycin sulfate (Meiji Seika, Tokyo) at 0.1 g/L. The wells were
5–6 eels/group). The first group was the control group, while the other maintained at 23 °C. Dead larvae were not removed, the water was
Table 1
The body weight of fish, hormone treatments, and experimental period of eels in each of the 3 experiments.
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:
B
2.6. Statistics
3. Results
3.1. Experiment 1
The mean GSI of the control group was 2.2 ±0.2 (Fig. 1), with fish
Fig. 2. Photographs of (A) the ovary of a female Japanese eel implanted with a single
containing very small ovaries (Fig. 2A), in which the oocytes were at
osmotic pump loaded with 0.1% bovine serum albumin (BSA) in 0.9% sodium chloride
the oil droplet and at the primary yolk globule stages (Table 2). The (control) and (B) the ovary of a female Japanese eel implanted with a single osmotic
implantation of a single hCG-loaded osmotic pump did not cause any pump loaded with salmon pituitary extract (SPE, 3 mg/day/fish). The arrows indicate
significant increase in the GSI (4.8 ± 0.5) of female eels (Fig. 1); howev- the ovary and the arrowhead indicates the osmotic pump.
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 nucleus or secondary yolk globule stage, respectively (Table 3), while
the SPE-loaded group (27.5 ± 4.1) was significantly higher compared the remaining fish had oocytes at oil droplet or primary yolk globule
to the other 2 groups, with fish containing large ovaries with oocytes stage.
at the migratory nucleus stage (Fig. 2B, Table 2).
In both the saline control and GnRHa-loaded osmotic pump groups In the osmotic pump implanted group, 10 out of the 16 female eels
(Fig. 3), the mean GSI was very low (1.3±0.1 and 1.5 ± 0.2, respective- had attained full maturity by the end of experiment (Table 4). These
ly), and most fish contained ovaries with oocytes at the oil droplet stage fish contained ovaries with oocytes at the migratory nucleus stage
(Table 3). Implantation of an osmotic pump loaded with hCG did not re- that were approximately 850 μm in diameter. The remaining 5 fish
sult in any significant increase in GSI (3.2± 0.5) (Fig. 3); however, 2 of contained immature ovaries. One of the 16 female eels died during
the 6 females contained ovaries with oocytes at the primary yolk glob- the final treatment.
ule stage (Table 3). SPE implantation significantly increased the GSI, In the osmotic pump injected group, 8 out of the 10 fully mature
with 2 out of 6 fish containing ovaries with oocytes at the migratory 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
35 out of the 15 females ovulated, with just 2 not ovulating by the final
30 b treatment.
25
Table 2
20
GSI
Oocyte developmental stages of Japanese eels at the initial and the final stages of Ex-
15 periment 1.
24 Table 4
Effects of different salmon pituitary extract administration methods on Japanese eel
20
vitellogenesis.
16 b Methods Number of fish
GSI
Table 6 References
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4
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a
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35
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