さ け・ま す 資 源 管 理 セ ン タ ー
研究報告第4号
1-5 頁
2001 年 12 月

Bulletin of the National Salmon
Resources Center, No. 4: 1-5
December 2001

RESEARCH NOTE
Effects of Handling Stress on Osmoregulation of JuvenileSockeye Salmon (Oncorhynchus nerka) in Seawater
Masatoshi Ban
Research Division, National Salmon Resources Center,
2-2 Nakanoshima, Toyohira-ku, Sapporo 062-0922, Japan(dukeban @salmon.affrc.go.jp)

Abstract. − Effects of handling stress on osmoregulatory ability of juvenile sockeye salmon(Oncorhynchus nerka) were investigated to assess responses of fish sampled for physiologicalstudies. Yearling juveniles were initially reared in either fresh water (May) or natural seawater(August), and subsequently separated into three treatment groups. For each treatment, fish wereplaced in artificial seawater (33 ppt) using one of three handling methods. Fish were either carefullytransferred to the seawater (Group 1), hung on a fishing hook and suspended in the seawater (Group2), or confined in a small net and dipped into the seawater (Group 3). Serum sodium concentrationand gill Na+ ,K+-ATPase activity were measured 4 hrs after transfer of fish. In May and August, theserum sodium concentrations of Groups 2 and 3 fish were significantly higher than that of Group 1
fish, whereas there was no significant difference in the gill Na+ ,K+ -ATPase activity among threegroups. These results indicate that handling stress may disturb osmoregulatory ability of fish inseawater without activating enzyme in the gills and that the serum sodium concentration increases
in a short period.
Key words: sockeye salmon, handling stress, osmoregulatory ability

Introduction
It has been reported that salmonids respondphysiologically to exogenous stresses. Chronicstresses cause stunt, disease or ultimately death(Fagerlund et al. 1995), while acute stresses drastically increase the plasma levels of catecholamines
and cortisol, and stress-related hormones, as well asheart rate (Wedemeyer 1969; Pickering et al. 1982;
Sumpter et al. 1986). Stresses also affect reproductive activity and metabolic changes (Strange et al.
1979; Nikinmaa et al. 1983; Redding and Schreck
1983; Kubokawa et al. 1999). It has been noted that
confinement stress decreases plasma levels of sex
steroid hormones during the breeding season in rainbow trout Oncorhynchus mykiss (Kubokawa et al.
1999) and gill Na+,K+-ATPase activity, a key enContribution A No. 22 from the National Salmon
Resources Center.
賛 2001 National Salmon Resources Center

zyme of osmoregulation in seawater, in rainbow
trout (Strange et al. 1979). Hauling stress also induces metabolic changes in an osmoregulatory compensation (Nikinmaa et al. 1983; Redding andSchreck 1983).
When physiological studies are carried out using
wild salmonids, collection of samples is always accompanied with stresses. Fishing methods such asthe long-line fishing and the set net are consideredto impose acute and extreme stresses to the caught
fish. To precisely estimate data from fish, it is indispensable that affects of stress are taken into consideration. However little information is known
regarding effects of fishing stress on the osmoregulatory ability of wild salmonids. The aim of the present study is to investigate effects of handling stressupon the osmoregulatory ability of yearling sockeye
salmon (Oncorhynchus nerka) in seawater, in order
to assess responses of fish caught for physiologicalstudies to fishing implements. Here, long-line andset net fishing conditions were simulated to examine
potential stresses.

2

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 4, 2001

ity level of less than 0.05 was considered significant.

Materials and Methods
Results and Discussion
Thirty yearling sockeye salmon smolts were randomly collected from a freshwater rearing pond at
the Chitose Hatchery, National Salmon Resources
Center on May 5, 1996. Smolts were divided into
three treatment groups of ten fish. Each group experienced a different transfer method into a 60 l tankfilled with artificial seawater (33 ppt). Smolts wereeither carefully transferred to the tank (Group 1),
hung on a fishing hook suspended in the tank(Group 2) or confined in a small net (30 cm × 30cm) and dipped in the tank 10 cm below the water
surface (Group 3). Groups 2 and 3 were designed as
experimental simulations of long-line fishing and set
net, respectively. Four hours after each treatment,
blood samples were collected in microtubes fromcut caudal fins and centrifuged at 3,000 g for 15 min
to separate serum. The resultant sera were stored at-40oC until assay. The gill filaments were also sampled and immersed in homogenizing solution (250mM sucrose, 6 mM EDTA 2Na, 20 mM imidazole,pH 6.8), and frozen at -80oC. Serum sodium concentration (mEq/l) was measured using an Atomic Absorption and Flame Emission Spectrometer(Shimadzu, AA-640-13). Gill Na+,K+-ATPase activity was measured using the method of Ban and Yamauchi (1991).
On May 7, 1996, one hundred of yearling sockeye salmon smolts were transferred from a fresh water pond into a fiberglass tank (3 tons). They wereacclimated gradually from a salinity of 20 to 33 ppt
for two weeks and kept in full-strength seawater (33ppt) until early August. In a preliminary study (unpublished data), I found that the osmoregulatory
function of yearling sockeye salmon reared in seawater became stable in August. The aerated and circulated seawater was controlled at 8oC. Fish were fed
at 3% body weight per day with a commercial dry
diet (Oriental Yeast, Co, Ld.). On August 4, 30 sockeye salmon were randomly collected from the tank.
These fish were subsequently divided into threegroups and subjected to the same transfer treatments
as indicated above.
The data were subjected to a one-way analysis ofvariance (ANOVA) followed by Student t-test to determine significant differences in serum sodium concentration and gill Na+,K+-ATPase activity among
the three groups in each collection time. A probabil-

For both sampling dates (May 5 and August 4),
there were no significant differences in the fork
length of yearling sockeye salmon among three treatment groups (Table 1). The mean serum sodium concentration of Group 1 fish was 154.4 mEq/l in Mayand 165.6 mEq/l in August (Fig. 1). Wedemeyer etal. (1980) reported that a serum sodium concentration of less than 170.0 mEq/l in seawater was optimal range for smolt. Therefore, Group 1 fish musthave attained a high seawater tolerance in bothmonths. However, serum sodium concentration ofGroups 2 and 3 fish were 177.8 and 176.7 mEq/l(May), and 216.9 and 218.1 mEq/l (August), respectively. These values were significantly (p<0.05)
higher than that of Group 1 fish. Waring et al.
(1996) reported a similar elevation in serum sodiumTable 1. Mean fork length (cm) ± S. E. of yearling sockeye
salmon in Groups 1, 2, and 3 sampled on May 5 and August
4, 1996.
Date sampledGroup 1

Group 2

Group 3May 5

12.6 ± 0.3

13.1 ± 0.4

12.5 ± 0.3

Aug. 419.5 ± 0.8

18.2 ± 0.8

18.0 ± 1.4

Fig. 1. Mean serum sodium concentration of yearling sockeye salmon 4 hrs after transfer from fresh water to artificial
seawater in May (white column) and from natural seawaterto artificial seawater in August (black column) for different
treatment groups. Group 1 fish were transferred carefully andslowly as control. Group 2 fish were hung on a fishing hookand suspended in seawater. Group 3 fish were confined in asmall net and dipped in seawater. Bars indicate SE. Asterisks
above columns show significant difference (p<0.05) between
Group 1 and Groups 2 and 3 in the same period.

Ban − Handling stress on osmoregulation of sockeye salmon

3

concentration in accordance with confinement stressin turbot (Scophthalmus maximus). These results indicate that acute stresses such as hanging or confinement hinder the normal sodium regulatory functionsof yearling sockeye salmon in seawater.
The mean gill Na+,K +-ATPase activity of thethree group fish ranged from 18.0 to 19.4 µmols
Pi/mg pro./h in May and from 13.2 to 14.1 µmols
Pi/mg pro./h in August (Fig. 2). These enzyme activities are similar to those of yearling sockeye
salmon smolt reported by Ban and Yamauchi(1991). Reductions in the mean enzyme activity observed during May and August may be due to a seasonal change, but not an effect of body size. This isindicated by lack of correlation between body sizeand enzyme activity in both months. Furthermore, I
similarly found a slight reduction in the enzyme activity of normal yearling sockeye salmon reared inseawater during spring and summer seasons in a preliminary study (unpublished data). For both months,
a significant difference in this activity was not observed between the control and stressed groups. It is
well known that gill Na+,K +-ATPase plays a valuablerole in ionoregulation of marine animals (Towle1981). Specifically, this enzyme is activated during
excretion of superfluous sodium ions by salmonids
in seawater (Wedemeyer et al. 1980; Ban andYamauchi 1991). Since little change in gill Na+,K+-

ATPase activity occurred with the handling stresses,
it appears that elevations of serum sodium concentration in the stressed groups are not due to decreased
sodium excretion ability of gill Na+,K +-ATPase.
Fletcher (1992), however, noted that handling stressincreased net water efflux via the gill filaments anddisturbed the serum ion balance of a marine flatfish(Pleuronectes platessa). Therefore, it seems that aserum change due to increased water efflux via thegill filaments and subsequent serum water loss in accordance with handling stress could explain the elevated serum sodium concentrations of stressed
yearling sockeye salmon in the present study.
There are several reports on physiological responses to stresses in salmonids reared in fresh water (Utida et al. 1972; Strange and Schreck 1978;
Graham et al. 1982; Sumpter et al. 1986). After
strenuous exercise, the plasma sodium levels increased in rainbow trout (Graham et al. 1982). Thisphenomenon is explained in that increases in intracellular lactate accompanied with exercise stress activate an inflow of water and sodium into the body.
Plasma cortisol levels also increase under the influence of acute stress in chinook salmon (O. tshawytscha) and brown trout (Salmo trutta) (Strange andSchreck 1978; Sumpter et al. 1986). Utida et al.
(1972) reported that the injection of cortisol increased the amount of water and sodium transported
through the intestine of Japanese eel (Anguilla japonica) in fresh water. In this study, changes in theinternal water movement or plasma cortisol concentrations after acute stresses were not measured. However, I might expect that an acute stress induces
elevation in the plasma cortisol levels of yearlingsockeye salmon in a short period, and that increased
cortisol stimulates sodium inflow passage from theintestine, even in seawater. It is possible that increases of serum sodium concentration (Fig. 1)
cause an extra inflow of sodium ion from organs
other than the gills. This inflow may exceed the excretion capacity of sodium ion via Na+,K +-ATPase in
the gills.
A drastic change in the salinity of ambient water
also imposes stress on fish (Wedemeyer et al. 1990).
The experimental fish in May were transferred from
fresh water to seawater, whereas those in August
were moved from natural seawater to artificial seawater. It is possible that the fish in May, but not inAugust, received an intensive stress. However, serum sodium concentrations of the stressed fish inFig. 2. The mean gill Na+,K+-ATPase activity of yearlingsockeye salmon 4 hrs after transfer from fresh water to artificial seawater in May (white column) and from natural seawater to artificial seawa ter in August (black column) for
different treatment groups. Group 1 fish were transferredcarefully and slowly a s control. Group 2 fish were hung on afishing hook and suspended in seawater. Group 3 fish wereconfined in a small net and dipped in seawater. Bars indicate
SE. There was no significant difference among groups in thesame period.

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 4, 2001

4

May were lower than those in August. One reason
for this is that gill Na+,K+-ATPase activity of fish in
May was higher than in August, whose fish were already acclimated to seawater. This phenomenon
may be an excessive preparation by smolt in advance of the drastic changes in ambient salinity. Furthermore, Barton et al. (1985) reported that cohosalmon (O. kisutch) smolt responded more distinctly
to acute handling stress than pre-smolt fish. The present results show that sockeye salmon smolt in fresh
water acquires tolerance not only for extreme environmental changes in salinity that accompany seaward migration, but also for some unpredictablestresses.
The present study warns investigators to consider the effects of handling stress on physiologicalcondition when examining osmoregulation of wild
salmonids.

Acknowledgements
I wish to express appreciation to Dr. Akihiro
Takemura and Dr. G. Curt Fiedler for revision ofthis manuscript.

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5Clarke. 1980. Environmental factors affecting
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べた．淡水飼育されたスモルトを 5 月に，また天然海水飼育された幼魚を8 月に，10 尾ずつの3群に分けて，塩分 33の人工海水へ移行した．人工海水へ安静に移した群を G-1，釣針に掛けて海水中に吊した群を G-2，小さな網袋に入れて海水中に漬けた群を G3 とした．各処置後 4 時間目の血中ナトリウム (Na)濃度と鰓の Na+ ,K+-ATPase 活性を測定した．両月とも，G-2 と G-3 の血中 Na 濃度は G-1 に比べて有意に高まったが，鰓の Na +,K+ -ATPase 活性には有意差が認められなかった．この結果は，ハンドリングによるストレスが，海水中の魚における鰓以外の浸透圧調節機能を阻害し，比較的短時間に血中ナトリウム濃度を上昇させることを示している．

ハンドリングがベニザケ幼魚の海水中における浸透圧調節機能に与える影響
伴真俊

生理学的研究に供試する魚が漁具により受ける影響を把握するため，種々のハンドリングストレスがベニザケ 1 年魚の浸透圧調節機能に与える影響を調