さけ・ます資源管理センター
研究報告 第 5 号 27-31頁
2002年12月

Bulletin of the National Salmon
Resources Center, No. 5: 27-31
December 2002

RESEARCH NOTE
Effects of Cortisol and Growth Hormone on the Seawater
Tolerance of Sockeye Salmon (Oncorhynchus nerka)
Masatoshi Ban
Research Division, National Salmon Resources Center,
2-2 Nakanoshima, Toyohira-ku, Sapporo 062-0922, Japan(dukeban @salmon.affrc.go.jp)
Abstract. − In order to determine hormonal effects on the seawater tolerance of sockeye salmon(Oncorhynchus nerka), cortisol or growth hormone (GH) were administered via intraperitoneal
injection at 1 μg per body weight for two weeks in March and May in the laboratory. In March, thegill Na+, K+-ATPase activity of the cortisol group was 1.5-fold higher than that of the control groupinjected only with saline. Plasma sodium concentrations 24 h after transfer to seawater for the cortisol and GH groups declined to 164.3 mM and 153.5 mM, respectively, while the control group had
191.1 mM. In May, however, the gill Na+,K+-ATPase activity in all these treatments elevated to
16.8 - 17.1 μmols Pi/mg pro/h and the plasma sodium concentrations decreased to 148.0 - 154.5
mM. Administration of cortisol and GH had no effect on seawater tolerance in May. These results
indicate that cortisol and GH are important hormones promoting seawater tolerance in sockeye
salmon, while effects of these hormones may vary with administration time.
Key words : sockeye salmon, seawater tolerance, gill Na+, K+-ATPase, cortisol, growth hormone

Introduction
Juvenile salmonids generally show parr-smolt
transformation (smoltification) as a pre-adaptation to
ocean life. Smoltification is a complex phenomenon
composed of morphological, physiological andbehavioral changes (Wedemeyer 1980). The major
physiological processes of smoltification include anincrease in hypo osmoregulatory ability (seawater
tolerance) and activation of gill Na +, K +-ATPase
activity (Hoar 1988). Gill Na+, K+-ATPase has animportant role for ion transport in seawater. Thesechanges during smoltification are controlled by theendocrine system.
McLeay (1975) found activation of the pituitaryinterrenal axis during smoltification in Atlantic
salmon (Salmo salar). Specker and Schreck (1982)
and McLeese et al. (1994) observed changes in theplasma corticosteroids with smoltification of coho

salmon (Oncorhynchus kisutch) and steelhead trout(O. mykiss). Elevation of corticosteroids duringsmoltification has also been shown to correlate withan increase in gill Na+, K+-ATPase activity in salmon(Specker et al. 1982; Shrimpton et al. 1994).
Besides cortisol, evidence of the involvement of
pituitary growth hormone (GH) in the smolting
process was observed in both physiological and histological studies (Parry 1958; Saunders and Henderson 1970; Komourdjian et al. 1976a; McCormick1996). These results indicate that cortisol and GH
are important endocrinological factors for seawater
tolerance in salmonids.
However, the roles of cortisol and GH involved
with seawater tolerance in the sockeye salmon (O.
nerka) are still unclear. In this paper, effects of cortisol and GH on seawater tolerance of sockeye salmon
were investigated as a preliminary study to elucidate
the hormonal mechanisms of seawater tolerance.

Materials and Methods
Contribution A No. 31 from the National Salmon
Resources Center.
© 2002 National Salmon Resources Center

Fish
Sockeye salmon used for this experiment were28

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 5, 2002

from brood-year 1994 stocks, which had been rearedin an outdoor concrete pond at the Chitose Hatchery,
National Salmon Resources Center in Hokkaido.
Yearling fish collected from the pond were divided
into three groups composed of 30 individuals (each)on March 12, and May 7, 1996. Fish were maintained in 25 l plastic tanks with flow-through fresh
water from a groundwater source, and held at 10゜Cfor one week in order to acclimate them to new conditions prior to hormone treatment.
At each sampling time, all fish were anesthetizedwith MS-222 (100 mg/l), weighed, and their fork
lengths were recorded.
Hormone treatment
Hormone administrations were performed for
two weeks from March 19 and May 13, respectively.
Fish in both months were allocated to one of threetreatment groups: control (saline, n=30), salmon
growth hormone (GH, n=30) or cortisol (n=30). Fishfrom their respective groups received an intraperitoneal injection of saline, GH (Kyowa Hakko Kogyo
Co. ltd, dissolved in distilled water), and cortisol(Sigma, dissolved in ethanol) on six alternating days.
Seawater tolerance
To determine the seawater tolerance of treatedfish, seawater challenge tests were conducted at theend of treatment administrations on March 31 andMay 25. Twenty fish from each of three treatment
tanks were directly transferred into a 60 l tank filledwith aerated artificial seawater (Aqua-Ocean, JapanPet Drugs co., ltd.) at a salinity of 33 ppt and 10゜C.
After 24 h, fish were anesthetized and blood was taken from their caudal veins using a micro tube.
Serum was obtained by centrifugation at 1,000 g for
15 min at 4゜
C and stored at -40゜
C until assays forplasma sodium were performed. Ten fish from eachtreatment group were placed in fresh water for 24 h.
The gill filaments from each fish were removed tomeasure the gill Na +, K +-ATPase activity. The
excised gill filaments were placed in ice-cold
homogenizing solution (250 mM sucrose, 6 mM
EDTA 2Na, 20 mM imidazole, pH 6.8) and stored at-40 ゜
C until assays for enzyme activity were performed.
+

according to the method of Ban and Yamauchi(1991). Defrosted gill tissues were homogenized ina homogenizing solution and centrifuged at 1,000 g
for 5 min at 4゜
C. The supernatant was incubated for
20 min at 37゜
C. Phosphorus (Pi) produced from the
incubation was measured according to the method ofGoldenberg and Fernandez (1966). Protein determination was made according to the method of Lowry
et al. (1951). Na + , K + -ATPase activity was
expressed as micromoles Pi per milligram protein
per hour (μmols Pi/mg pro/h).
Plasma sodium concentrations (mM) were measured using an Atomic Absorption and Flame Emission Spectrometer (Shimadzu, AA-640-13). Defrosted plasma was diluted 1,000 x with distilled water
for analysis.
Statistical analysis
The data were subjected to a one-way analysis ofvariance (ANOVA) followed by a Student's t-test to
determine significant differences in plasma sodium
concentrations and gill Na +, K +-ATPase activity
among the three groups in each collection time. A
probability level of less than 0.05 was consideredsignificant.

Results
Fork length and body weight of all three groups
increased during the experimental period (Table 1).
There was no significant difference in body size
among the three groups in each month.
The gill Na+, K+-ATPase activities of GH andcortisol groups were higher than that of the control
group in March. In particular, the enzyme activity of
the cortisol group significantly differed from thecontrol group and reached 6.0 μmols Pi/mg pro/h
Table 1. The mean fork length and body weight of sockeye
salmon injected saline (control), growth hormone (GH) or
cortisol in March and May 1996.
Treatment

March 31

Control10.4±0.1*

11.1±0.5GH

10.6±0.2

12.1±0.7

Cortisol10.4±0.3

11.5±0.9

Control11.9±0.2

16.9±1.5GH

12.5±0.417.9±1.8

Cortisol12.4±0.2

17.8±2.0

May 25+Gill Na , K -ATPase activity and plasma sodium
concentrations
Gill Na +, K +-ATPase activity was determined

Fork length (cm) Body weight (g)Date* Mean±SE

Ban − Effects of cortisol and growth hormone on seawater tolerance

Fig. 1. Gill Na +, K +-ATPase activity of sockeye salmon
injected with saline (Con), cortisol (Cor) or growth hormone(GH) in March and May. Bars indicate SE. Asterisks above
columns indicate a significant difference (p<0.05) amongexperimental groups in March and May.

Fig. 2. Serum sodium concentrations of sockeye salmon
injected with saline (Con), cortisol (Cor), and growth hormone (GH) in March and May. Bars indicate SE. Asterisks
above columns indicate a significant difference (p<0.05)
among experimental groups in March and May.

(Fig. 1). The plasma sodium concentrations of GH
and cortisol groups in March were 153.5 mM and164.3 mM, respectively (Fig. 2). These levels weresignificantly lower than that of the control group(191.1 mM).

Discussion
There are many reports concerned with effects ofcortisol and/or GH on seawater tolerance and gill
Na+, K+-ATPase activity. In coho salmon, plasma
sodium levels after transfer to seawater are lowered29

by cortisol treatment (Richman and Zaugg 1987).
Also, oral administration of cortisol has a positiveeffect on the salinity tolerance of masu salmon (O.
masou) parr (Ouchi 1985) and cortisol implants activate gill Na+, K+-ATPase in coho salmon (Specker etal. 1994). Furthermore, the gill Na+, K+-ATPase
activity of Atlantic salmon, amago salmon (O. rhodurus) and coho salmon are activated by GH treatment (Komourdjian et al. 1976b; Miwa and Inui1985; Richman et al. 1987). Bolton et al. (1987) also
demonstrated that juvenile rainbow trout (O. mykiss)given injections of chum salmon (O. keta) GH or
ovine GH have lower increases in plasma sodium
concentrations than control groups injected withsaline only. These results show that cortisol and GH
are important hormones promoting an adaptation toseawater in salmonid fishes. In the present study, theexperimental groups treated with cortisol or GH had
decreased plasma sodium concentrations after transfer to seawater and increased gill Na+, K+-ATPase
activity. These results indicate that cortisol and GH
also have an important function in improving theseawater tolerance of sockeye salmon by stimulating
their gill Na+, K+-ATPase activity.
Effects of cortisol and GH on seawater tolerance
were not the same in March fish. The plasma sodium concentration of the GH group was lower thanthat of the cortisol group, while the gill Na+, K+ATPase activity of the cortisol group was higher
than that of the GH group. Richman et al. (1987)
reported the similar result that implants of bovine
growth hormone in hypophysectomized coho salmon
did not increase the gill Na+, K+-ATPase activity, and48 hrs after fish transfer to seawater these implants
reduced plasma sodium concentrations. An explanation for these results may be due to differences in the
optimal dosage of each hormone or the administration period. However, McCormick (1996) and Pelisand McCormick (2001) observed a more positiveeffect on the development of seawater tolerance
using a combination of cortisol and GH, compared toseparate cortisol and GH treatments. These findings
suggest that cortisol and GH have different roles inthe developmental process of gill Na+, K+-ATPase or
sodium excretion ability in seawater, though both
hormones cooperate to develop seawater tolerance
during smoltification. We need more research tounderstand the interaction between cortisol and GH.
In May fish, the GH and cortisol groups showed
dramatic increases in the gill Na+, K+-ATPase activi-

30

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 5, 2002

ty and it reached 16.8 - 17.1 μmols Pi/mg pro/h(Fig. 1). Their plasma sodium concentrations after
transfer to seawater synchronously decreased to148.0 - 152.5 mM (Fig. 2). These values were equal
to levels observed in sockeye salmon smolts rearedat the Chitose Hatchery (Ban and Yamauchi 1991).
However, the gill Na+, Ki+-ATPase activity and plasma sodium concentrations of the control group wereat the same levels as in the GH and cortisol groups
and there was no significant difference among thethree groups. Exogenous cortisol and GH had noeffect on seawater adaptation in May as comparedwith the results in March. A reason for this result
may be due to endogenous cortisol and GH. In thepresent experiment, endogenous concentrations ofcortisol and GH were not measured. Under normal
rearing conditions, yearling sockeye salmon show
temporal surges in endogenous cortisol and GH during March and April (Ban, unpublished data). Following these surges, the fish have a peak of seawater
tolerance in May. Therefore, it seems that sockeye
salmon used in this experiment in May had alreadydeveloped seawater tolerance via their endogenous
cortisol and GH prior to the hormonal treatments.
The present study suggests that cortisol and GH
are involved with the development of seawater tolerance in sockeye salmon. More detailed research isnecessary to understand the respective roles of eachhormone in seawater tolerance.

Acknowledgements
I wish to express appreciation to Dr. G. Curt
Fiedler of University of Ryukyus, Tropical Biosphere Research Center, for revision of this manuscript.

References
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Hirano. 1987. Osmoregulatory actions of growth
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コーチゾルと成長ホルモンがベニザケの海水適応能に与える影響伴真俊

コーチゾルと成長ホルモン(GH)が，ベニザケの海水適応能に与える影響を調べるため，体重当り1μgの濃度に調整した各ホルモンを，腹腔内へ2週間に亘って注射した．同様の実験を3月と5月の2回行った．3月のコーチゾル群における鰓のNa + ，K + ATPase活性は，生理食塩水を投与した対照群に比べて約1.5倍高い値を示した．海水移行24時間後の血中ナトリウム (Na) 濃度は，対照群が191.1 mMだ
ったのに対して，コーチゾル群とGH群は各々164.3 mMと153.5 mMを示した．しかし，5月には3群とも共通して鰓のNa + ，K + -ATPase活性が16.817.1μmols Pi/mg pro/hまで上昇するとともに，海水移行後の血中Na濃度は148.0-154.4 mMまで低下し，ホルモンの投与効果が認められなかった．これらの結果は，コーチゾルとGHが，ベニザケの海水適応能獲得過程に重要な役割を果たすことを示している．しかし，ホルモンの効果は投与時期により異なることが予想される．