AN ABSTRACT OF THE THESIS OF
Reynaldo Patina
for the degree of
Department of Fisheries and Wildlife
Title:
Master of Science
presented on
in the
May 4, 1983
CLEARANCE OF PLASMA CORTISOL IN COHO SALMON, ONCORHYNCHUS
KISUTCH, AT VARIOUS STAGES OF SMOLTIFICATION
Signature redacted for privacy.
Abstract approved:
Carl B. Schreci
The metabolic clearance rate (MCR) of plasma radioactivity after a
single intracardial injection of 3H-cortisol was elevated during the
Spring in yearling coho salmon, Oncorhynchus kisutch.
The reasons for
this increase in MCR are not clear, but graphical analysis suggested
that there may be a seasonal correlation between MCR and gill
Na/K-ATPase activity.
The main plasma metabolite of 311-cortisol was
3H-cortisone, and it appeared very rapidly following injection of the
original radiotracer.
The stress of handling as used in the injection
protocol and various anesthetic treatments did not affect clearance of
cortisol from plasma.
CLEARANCE OF PLASMA CORTISOL IN COHO SALMON, ONCORHYNCHUS KISUTCH,
AT VARIOUS STAGES OF SMOLTIFICATION
by
Reynaldo Patin()
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements of the
degree of
Master of Science
Completed May 4, 1983
Commencement June 1984
Approved:
Signature redacted for privacy.
Associate Pkofessor of Fisheries in charge of major
Signature redacted for privacy.
Head of Department of Fisheries and Wildlife
Signature redacted for privacy.
Dean of Gradua
'school
r)
J
Date thesis is presented
May 4, 1983
Typed by Adrian Hunter and LaVon Mauer for
Reynaldo Patiflo
ACKNOWLEDGEMENTS
I thank my major professor, Carl B. Schreck, for his guidance and
encouragement throughout this study.
J. Michael Redding provided
valuable criticism and assisted in the experiments of chapter I, and
for this he was made co-author of that chapter.
I appreciate the tech-
nical help received from Cynthia K. Pring and Amy Crook.
The fish for this study were provided by the Oregon Department of
Fish and Wildlife and the U.S. Fish and Wildlife Service.
Finally, this study would not have been possible without the
generous fellowship given to me by the Oregon State University Sea
Grant College Program.
TABLE OF CONTENTS
Page
CLEARANCE OF PLASMA CORTISOL IN COHO SALMON, ONCORHYNCHUS
KISUTCH, AT VARIOUS STAGES OF SMOLTIFICATION
Introduction
Materials and Methods
Experimental Design and Results
Discussion
21
Bibliography
26
Appendix
30
1
2
5
TABLE OF FIGURES
Figure
Page
Metabolic clearance rate of corticosteroids and gill
Na/K-ATPase activity in yearling coho salmon
9
Weight, condition factor, plasma thyroxine, and plasma
cortisol in yearling Big Creek-Sol Duc coho salmon.
13
Weight, condition factor, plasma thyroxine, and plasma
cortisol in yearling Eagle Creek coho salmon.
15
Plasma thyroxine and gill Na/K-ATPase activity in
yearling coho salmon reared at different densities
37
LIST OF TABLES
Table
Page
Percent composition of total plasma radioactivity
4h after 3H-cortisol injection in yearling coho
salmon.
10
Plasma radioactivity and half-life in yearling coho
salmon following a 5 min exposure to anesthetic and
intraperitoneal administration of 3H-cortisol.
18
Plasma radioactivity in unhandled and handled
coho salmon juveniles after oral administration of
3H-cortisol.
20
Density index, total weight of fish, fork length,
weight, and condition factor in yearling coho
salmon.
35
Plasma cortisol, interrenal nuclear diameter, relative metabolic clearance rate, and half-life of
plasma tritium after 3H-cortisol administration in
yearling coho salmon reared at different densities.
38
Mortality, plasma sodium, condition factor, and
behaviour of yearling coho salmon reared at different densities and following a 24 h exposure to
seawater.
40
CLEARANCE OF PLASMA CORTISOL IN COHO SALMON, ONCORHYNCHUS KISUTCH,
AT VARIOUS STAGES OF SMOLTIFICATION1
Reynaldo Patiffo
Carl B. Schreck2
J. Michael Redding
Oregon Cooperative Fishery Research Unit3
Oregon State University
Corvallis, OR 97331
Running Title:
Cortisol Clearance During Salmon Smoltification
lOregon State University Agricultural Experiment Station Technical
Paper No.
2To whom reprint request should be sent
3Cooperators are Oregon State University, Oregon Department of Fish and
Wildlife, and U.S. Fish and Wildlife Service
CLEARANCE OF PLASMA CORTISOL IN COHO SALMON, ONCORHYNCHUS KISUTCH,
AT VARIOUS STAGES OF SMOLTIFICATION
The parr-smolt transformation (smoltification) of anadromous
salmonids consists of a series of morphological, physiological, and
behavioral changes that culminate in the migration of the fish from
freshwater streams to the ocean (Hoar 1963, 1976).
Physiological
changes commonly studied during smoltification are those observed in
gill Na/K-ATPase, thyroid and, to a lesser extent, interrenal activity.
However, the biological significance of these changes has not yet been
clearly established.
Histological (Fontaine and Olivereau 1957, 1959; Olivereau 1962,
1975; McLeay 1975; Komourdjian et al. 1976; Nishioka et al. 1982) as
well as plasma hormone concentration (Fontaine and Hatey 1954;
Langhorne and Simpson 1981; Specker and Schreck 1982) studies of
Atlantic (Salmo salar) and coho (Oncorhynchus kisutch) salmon indicate
higher interrenal activity at the time of smoltification, suggesting an
involvement of corticosteroid hormones in this process (Specker 1982).
Basal circulating levels of corticosteroids are difficult to ascertain
because they are extremely responsive to stress in salmonids (Schreck
1981).
Since hormone concentrations are a function of secretion and
clearance rates, we investigated the metabolic (plasma) clearance rate
(MCR) of radioactivity following injection of 3H-cortisol in coho
salmon.
The MCR of a hormone has been established as a useful estimate
of its overall metabolism (Tait and Burstein 1964; DiSteffano 1982)
and, as suggested by the results of the present study, corticosteroid
2
MCR may not be as sensitive to acute handling stress as plasma
corticosteroid concentrations.
The MCR of 3H-cortisol and its metabolites was determined at
various times during smoltification in two populations of coho salmon.
In addition to measurements of endogenous plasma cortisol, we monitored
changes in other putative smoltification "markers" (e.g., gill
Na/K-ATPase activity and plasma thyroxine).
MATERIALS AND METHODS
Experimental animals.
Juvenile coho salmon from the Fall Creek
stock and from a cross of Big Creek and Soleduc stocks were obtained
through the Oregon Department of Fish and Wildlife in 1980 and 1981,
respectively.
For the 1982 experiments, fish were obtained from Eagle
Creek National Fish Hatchery (U.S. Fish and Wildlife Service).
All
fish were held in freshwater (10-12° C) and under natural photoperiod
at Smith Farm Hatchery, Oregon State University, and fed daily with
Oregon Moist Pellets.
Fall Creek fish were reared in a circular
flow-through tank (diameter, 1.4 m; volume, 0.7 m3); Big Creek-Soleduc
fish were reared in a large circular tank (diameter, 1.8 m; volume 1.7
m3) from January to mid-April 1981, and were then transferred to a 0.7
m3 tank until the end of the experiments; and Eagle Creek fish were
reared in two 0.7 m3 tanks from late December 1981 through the end of
their rearing period.
For the smoltification studies, fish density was not deliberately
adjusted for growth of fish.
Due to the periodic sampling, however,
the total weight of fish in 1982, as determined by total fish number
3
and mean weight of 15-20 fish, did not vary appreciably over the course
of the study (10.8-15.0 kg per tank).
In 1981, fish were also sampled
for purposes other than for the present experiment, and estimates of
the rearing density at any one time could not be made.
However, the
rearing density toward the end of their rearing period may have been
slightly lower than at the start of the experiment.
General Methods.
For the cortisol clearance experiments, fish were
administered 3H-cortisol and then sampled at determined times to
measure plasma radioactivity levels.
The purity of 311-cortisol was
confirmed by thin layer chromatography (TLC) followed by analysis of
radioactivity in the chromatogram.
Fish were killed by a blow to the
head, and their blood was taken from the severed caudal peduncle into
ammonium-heparinized capillary tubes.
Blood samples were centrifuged
within 30 minutes and 20 Ml of plasma were placed in glass
scintillation vials, treated with 200 ML of Protosole, and dissolved in
10 ml of scintillation fluid.
Plasma radioactivity was determined with
a liquid scintillation spectrophotometer (Packard 2425) and multiplied
by body weight to correct for variation due to weight differences
(except when 3H-cortisol was given orally).
Only small quantities of plasma could be obtained from each fish,
and therefore separation of authentic radioactive cortisol by use of
chromatographic techniques could not be adequately performed on
individual plasma samples.
In the 1982 MCR experiments, however, an
estimate of the mean percentage of plasma radioactivity accounted for
by cortisol was obtained by pooling plasma samples taken at 4h
following 3H-cortisol injection in each experiment.
4
The pooled plasma samples, 20 41 per sample from 6 fish, were
placed in test tubes where an ethanol solution containing 10 pg of
unlabelled cortisol, cortisone, corticosterone, 11-deoxycortisol and
11-deoxycorticosterone had been previously evaporated to dryness.
samples were thoroughly mixed and a 20
ul
determine total plasma radioactivity.
A known amount of
The
aliquot was then taken to
4_14 C-cortisol (Research Products International) was added to the
remaining plasma, the plasma extracted once with 1 ml cold
dichloromethane, and the extract washed successively with 100 41 of
cold 0.05 M NaOH and 100 41 of cold distilled water.
About 500 41 of
the extract was then transferred to a clean test tube, evaporated,
spotted 4 times with 2 drops of methanol (I); dichloromethane (9) on a
TLC sheet (Baker-fle" silica gel 1B2-F) and subjected to a
2-dimensional development.
The solvent systems used were chloroform
(19): ethanol (1) and dichloromethane (150): methanol (9): water (0.5)
(Quesenberry et al. 1965) for the first and second developments,
respectively.
The areas containing the unlabelled marker steroids were
located under ultraviolet light and marked.
The chromatograms were
then treated with EN3HANCE0 (New England Nuclear) and fluorographed
using Kodak X-OMAT AR films (Eastman Kodak Co.) as described by the
manufacturers.
The only radioactive areas which could be detected
after 24-88h fluorography were those corresponding to authentic
cortisol and cortisone.
Therefore, only the cortisol and cortisone
areas were cut out of the TLC sheets and placed in glass scintillation
vials.
To avoid correcting for differential quench effects, all
scintillation vials contained 20 41 of plasma (radioactive or
5
non-radioactive), 200 pl of Protosol®, 1 cm2 of TLC sheet (with or
without radioactive steroids) and 10 ml of scintillation fluid.
The
recoveries of cortisol and cortisone from fish plasma after extraction
with dichloromethane and chromatographic development are similar
(Leloup-Hatey 1976).
Therefore, the recovery of 14C-cortisol was used
as estimate of the recoveries of both 3H-cortisol and 3H-cortisone.
Using the preceding procedure, the 3H-cortisol:
3H-cortisone ratios
at 10 min, 0.5, 1, and 4h following 3H-cortisol injection were
calculated for the first (February) 1982 MCR experiment.
EXPERIMENTAL DESIGN AND RESULTS
Clearance during smoltification.
To determine the MCR of plasma
radioactivity following injection of 3H-cortisol, groups of 10 Big
CreekSoleduc fish (1981) were acclimated to small circular
flow,-through tanks (diameter 0.6 m; volume 0.1 m3) for about 1 week
before the experiment.
In 1982, Eagle Creek fish were taken directly
from the rearing tanks at the time of the experiment.
The fish were
anesthesized with 50 mg/liter tricaine methanesulfonate and injected
intracardially with 1,2,6,7-3H-cortisol (1981:
ng, New England Nuclear; 1982:
International).
2.5-3.6
uCi, 9.5-13.7
3-5 pCi, 10.2-17 rig, Research Products
The quantity of 3H-cortisol injected was consistent
on any given experiment, and in the 1981 experiments was mixed with
unlabelled cortisol (approx. 110 rig/dose) and administered in 50 01 of
ethanol:Cortland's saline without glucose (5:95), but in 1982 was
administered alone in 25 pl of carrier solution.
Omission of
unlabelled cortisol from the carrier solution was shown not to affect
6
clearance of radioactivity from plasma following the injection of
3H-cortisol (Redding 1982).
Six to 10 individuals were sampled at 10
min, 0.5, 1, 4, 12, and 24h after the injection to determine plasma
radioactivity levels.
A previous study at Smith Farm Hatchery with
additional sampling times (Redding 1982) suggested that the present
sampling times are appropriate to determine the MCR.
Weight-corrected
plasma radioactivity values were expressed as percentage of injected
dose per ml of plasma.
The disappearance curves of radioactivity showed a fast and a slow
component intercepting at 1-1.5h in a semilogarithmic plot, suggesting
that cortisol may be approximately distributed in two "compartments."
However, the initial portion of the curve was somewhat variable.
This
variability most likely reflects the inaccuracy of our blind delivery
of tracer into the heart (some tracer may leak out into the
pericardial cavity), and makes compartmental analysis of the
disappearance curves meaningless.
Alternatively, a numerical method
of integrating the area under the curve avoids assumptions of
compartmental distributions (Normand and Fortier 1970).
Moreover, the
rate of dose delivery does not affect results obtained by stochastic
(non-compartmental) analysis as it does in compartmental analysis
(Shipley and Clark 1972), and hence the leakage of 3H-cortisol into
the pericardial cavity is not of concern since it is
likely
that most
of the radioactive steroid would be absorbed into the circulation
within the sampling period (24h).
We therefore used the numerical
technique described by Normand and Fortier (1970) to calculate the
MCR.
7
The MCR of plasma radioactivity following injection of 3H-cortisol
was determined 8 times in 1981 and 4 times in 1982 (Fig. 1).
For the
statistical analysis, the first 4 estimates of MCR in 1981 were
averaged, and this average value and the first (February) estimate of
MCR in 1982 were designated as the control or pre-smolt value for each
year respectively.
Subsequent MCR estimates were compared to their
respective control with Student ti-tests for means with unequal
variances (Snedecor and Cochran 1980) and with confidence levels of a
divided by the number of comparisons (Bonferroni technique; Miller
1981).
There was a distinctive peak in the MCR of cortisol and its
metabolites in yearling salmon during the spring of both years (Fig.
1).
In 1981, the MCR increased and reached a maximum level in late
April (P< 0.01), and returned to pre-smolt values in late June (F>
0.05).
A similar pattern of MCR changes was observed in 1982, with
the MCR higher in April (P < 0.01) but lower in May and early July (P
< 0.01) than in February.
The only steroid metabolite of 3H-cortisol identified in plasma
was 311-cortisone.
311-cortiso1:3H-cortisone ratios in the February
1982 experiment were 3.95, 1.00, 0.76, and 0.39 at 10 min, 0.5, 1, and
4h following 3H-cortisol injection, respectively, indicating that
plasma 3H-cortisol was rapidly converted into 3H-cortisone in the
peripheral tissues.
At 4h after the injection, most of the plasma
radioactivity could be accounted for by 3H-cortisol and 3H-cortisone
in all four 1982 experiments (Table 1).
8
FIGURE 1.
Metabolic clearance rate (MCR) of plasma tritium following
intracardial injection of 3H-cortisol, and gill Na/K-ATPase
activity in Big Creek-Soleduc (BC-SD) and Eagle Creek (EC)
yearling coho salmon.
BD-SD values were obtained by E. K.
Birks and R. D. Ewing (unpublished results) from the
Oregon Department of Fish and Wildlife.
standard errors around mean values.
Bars represent
9
-6
-3 0
3
5
EC (1982)
/
.08-"
CA
S.
.07--
S.
.06-
-3
JAN
F IGURE 1.
FEB MAR APR MAY JUN
JUL
10
TABLE 1.
Percent composition of total plasma radioactivity 4h after
3H-cortisol injection in yearling coho salmon.
Date
3H-cortisol
3H-cortisone
Unidentified
Metabolites
February 24
24
61
15
April 22
34
52
14
May 24
46
47
7
July 2
36
56
8
11
Physiological "markers" of smoltification.
To monitor the
progress in smoltification, 15-20 fish were taken from the rearing
tanks at the time of every MCR determination (1981) or within a day of
every new and full moon (1982).
Fish were netted from the tanks in
groups of 2 or 3 until the total desired number for each sampling date
was obtained.
Blood was taken from the severed caudal peduncle into
heparinized tubes and kept on ice for 30-60 min until centrifuged.
Plasma samples were stored at -20° C until cortisol (Redding 1982) and
thyroxine (Specker and Schreck 1982) levels were determined by
radioimmunoassay.
Gill tissue samples were also taken in 1982 to
determine Na/K-ATPase activity as described by Johnson et al. (1977),
and modified by E. K. Birks, R. D. Ewing, and G. L. Peterson, Oregon
Department of Fish and Wildlife, and Department of Biochemistry and
Biophysics, Oregon State University; the protein content in tissue
homogenates was determined according to Bradford (1976).
Plasma levels of cortisol in 1981 yearling salmon decreased from
January throughout the experimental period (Fig. 2).
In 1982, the
general pattern of plasma cortisol changes was similar to that of
1981, except for the relatively high values observed in early March
and late July (Fig. 3).
Plasma thyroxine was higher during the spring and early summer
than during the winter in both years (Figs. 2, 3), and gill
Na/K-ATPase activity in 1982 was elevated from March to early May
(Fig. 1).
Gill Na/K-ATPase activity and plasma thyroxine in 1982 were
significantly correlated (Pearson's product moment test; r = 0.36; P <
0.01) when data obtained at all sampling dates were analyzed
1 2
FIGURE 2.
Weight, condition factor, plasma thyroxine, and plasma cortisol in yearling Big Creek-Soleduc coho salmon.
represent standard errors around mean values.
Bars
a
C-
>
c-
I
;In
I
0
I
a
41b.
I
I
I
0
(ng/ml)
(100.g/cm3)
(g)
Ca
THYROXINE
CONDITION FACTOR
WEIGHT
(ng/ml)
CORTISOL
14
FIGURE 3.
Weight, condition factors, plasma thyroxine, and plasma
cortisol in yearling Eagle Creek coho salmon.
represent standard errors around mean values.
Bars
C
r
c-
Z
C
C-
-,C
>
C
33
'0
>
XI
>
E
CO
m
-n
z
>
c_
-
f
(9)
WEIGHT
I
0
0
.4
7.
i i
:,
T
_.
_.
(100-g/cm3)
7
I
s.
(ng/ml)
CONDITION FACTOR THYROXINE
°
hi
°
16
?
?
a
(ng/ml)
CORTISOL
?
8
16
simultaneously, but not when data of any given date were considered
individually (P> 0.05).
In both years, the condition factor declined during the Spring and
reached the lowest values in late April or early May (Figs. 2,3).
The
vernal nadir in condition factor has been used as criterion of
smoltification in some salmonids (see Wedemeyer et al. 1980).
If this
were true also for coho salmon, it would appear that our fish
"smolted" between April and May.
Validation of experimental protocol.
We evaluated the effects of
anesthetics on plasma clearance of radioactivity following 3H-cortisol
administration.
On 13 December 1980, Fall Creek fish (mean wt. 28.1
g) were anesthesized for 5 min with 50 mg/liter tricaine
methanesulfonate (unbuffered or buffered with 600 mg/liter sodium
bicarbonate or 17 mg/liter imidazole) or 300 ppm 2-phenoxyethanol.
Unanesthesized fish served as controls.
(Addition of buffer to the
anesthetic solution does not have a considerable effect on the pH of
the solution due to the already high degree of hardness of the water
used [100 mg/liter as CaCO3; Strange and Schreck 1978].
We included
buffered solutions in these experiments since a preliminary report by
Fabacher (1980) suggested that 600 mg/liter sodium bicarbonate could
affect liver function in fish.)
The fish were then injected
intraperitoneally with 2.5 uCi 1,2,6,7-3H-cortisol (9.5 ng, New
England Nuclear) and 110 ng of unlabelled cortisol given in 50 Ill of
injecting solution.
The fish were returned to fresh water, and blood
samples were taken at 3 and 7h after the injection.
In a similar
experiment on 18 February 1981, Big Creek-Soleduc salmon (mean wt.
17
14.0 g) were anesthesized for 5 min with 100 mg/liter tricaine
methanesulfonate (unbuffered or buffered with sodium bicarbonate) or
2-phenoxyethanol.
The fish were then injected with 3H-cortisol and
sampled at 1, 3, and 7h after the injection.
Mean plasma
radioactivity levels at each sampling time were compared using ANOVA,
and the half-life of plasma radioactivity was also calculated for
groups with 3 sampling times as described by Normand and Fortier
(1970) for a mono-exponentially decreasing curve, and compared among
treatments using the student t'-test described previously.
Although,
as previously stated, the disappearance curves contain more than one
exponential component, measurements taken 1-1.5h and later following
3H-cortisol injection can be approximately described by a single
exponential term.
Therefore, the half-lives of radioactivity after
1-1.5h may be used to estimate the relative metabolism of cortisol
among treatments.
None of the above anesthetic treatments affected clearance of
plasma radioactivity following 3H-cortisol administration in coho
salmon (Table 2).
However, these results did not rule out the
possibility that the overall handling procedure may have affected
corticosteroid clearance since the control fish were also physically
handled.
We conducted the following experiment in order to
investigate this possibility.
Five groups of 8 Eagle Creek fish (mean
wt. 47.4 g at end of experiment) were acclimated to 0.1 m3 circular
tanks for 6 weeks before the experiment.
3H-cortisol (Research
Products International) was dissolved in ethanol and mixed with Oregon
Moist Pellets at a ratio of 6 pCi (20.4 ng) per gram of feed.
The
TABLE 2.
Plasma radioactivity and half-life in yearling coho salmon following a 5 min exposure to anesthetic
and intraperitoneal injection of 311-cortisol.
TKEATMENT
Time after
injection(h)
Control
MS222
50 mg/L
_
se
se
x
se
x
MS222-imidazole
50-17 mg/L
x
se
2-phenoxyethanol
300 ppm
x
se
3
50.8
4.2a
63.0
5.0b
63.7
11.4b
61.3
38b
65.5
6.0b
7
16.9
3.5
21.1
3.9b
26.6
3.0b
28.5
3.2b
24.3
31b
100 mg/L
100-600 g/L
300 ppm
1
115.5
9.1
86.8
22.3b
105.6
11.6b
94.9
21.5b
3
51.7
15.2
72.7
7.2b
44.2
7.2b
58.7
7.76
7
19.8
4.2
22.3
3.7b
26.1
5.3b
20.9
29b
2.7
0.9
2.9
0.8c
3.0
07c
2.8
0.6c
Half-life
aN
MS222-NaHCO3
50-600 mg/L
5-6; units
counts per minute
weight (g)
10-3
bNot significantly different from corresponding control (ANOVA; P ) 0.05)
cNot significantly different from control (C-test; P > 0.05)
19
pellets were carefully mixed with the ethanol solution until the
latter evaporated, and the treated diet was stored frozen for 24-48h
until used.
Each group of fish received 3.5 g of the prepared feed 3
times on 20 August 1982 and once in the morning of the following day.
One group was sampled 3h after last feeding to determine plasma
radioactivity levels in the fish.
Two groups of fish were taken from
their tanks 4.5h after last feeding, anesthetized with 50 mg/liter
tricaine methanesulfonate, injected intracardially with 25
ul
of
Cortland's saline, and subsequently returned to their respective tanks
to simulate the treatment protocol used in determining MCR.
were not observed to regurgitate food while being handled.
remaining 2 groups were not disturbed.
The fish
The
One disturbed and 1
undisturbed group were simultaneously sampled at 10 min and at 4h
after handling to determine their plasma radioactivity levels.
Plasma
radioactivity values were not individually corrected for body weight
since it is unlikely that all fish consumed the same amount of treated
feed.
However, each group received equal rations, and therefore the
mean plasma radioactivity levels were corrected for the total fish
weight in the respective group.
The slightly lower levels of plasma radioactivity observed in the
handled fish (Table 3) may have been caused by water gain following
stress in fresh water (see Mazeaud et al. 1977).
However, the
difference between treatments was not statistically significant at
either 10 min or the 4h post-handling.
20
TABLE 3.
Plasma radioactivity in unhandled and handled (netting,
anesthesia, and injection) coho salmon juveniles after
oral administration of 3H-cortisol.
Treatment
Time
(min)a
Unhandled
Handled
-x
se
-90
24.8
36b
10
33.3
3.0
24.8
2.9c
240
28.7
3.5
23.3
2.9c
-x
se
aTime of injection = 0
bN = 8; units = counts per minute
total group weight (g)
10-4
cNot significantly different from unhandled (t-test; P> 0.05)
__
__
21
DISCUSSION
The MCR of plasma tritium after injection of 3H-cortisol increased
in yearling coho salmon during the spring.
However, graphical
comparison of the pattern of changes in MCR and endogenous cortisol
concentration shows no apparent correlation (positive or negative)
between these variables.
Therefore, assuming that changes in MCR of
radioactivity in plasma reflect similar changes in the MCR of
authentic cortisol, no correlation would seem to exist between the
rate of overall cortisol metabolism and endogenous plasma cortisol
levels.
Under this assumption, it would appear that endogenous plasma
cortisol levels may give an adequate relative estimate of the cortisol
secretion rate since changes in plasma levels were several-fold
greater than changes in cortisol MCR.
It has been suggested that long-term (chronic) changes in plasma
levels of some steroids can influence their rate of degradation
(Southren et al. 1968; Redding 1982).
Therefore, it may be possible
that seasonal changes in other plasma corticosteroids, e.g.,
cortisone, could be a factor influencing the changes observed in the
MCR of cortisol and its metabolites during smoltification.
Alternatively, like in adult salmon (Donaldson and Fagerlund 1969,
1970; Fagerlund and Donaldson 1969), sex steroids may influence the
MCR of corticosteroids in juvenile coho salmon.
Preliminary evidence
suggests that plasma levels of sex steroids may increase during
smoltification of coho salmon (S. Sower, personal communication).
the other hand, it is also possible that the rate of corticosteroid
On
2 2
metabolism during smoltification is a secondary effect of events
occurring independently of plasma steroid concentrations.
Our finding that cortisone is the major plasma metabolite of
cortisol is in accordance with previous studies on adult sockeye
salmon, Oncorhynchus nerka, (Idler and Tuscott 1963; Donaldson and
Fagerlund 1968, 1972) and eels, Anguilla anguilla, (Leloup-Hatey 1974,
1976, 1979).
The rapidity of the conversion of 3H-cortisol into
311-cortisone in our studies suggests that, like in adult sockeye
salmon (Donaldson and Fagerlund 1972), plasma cortisone is not
appreciably converted into plasma cortisol in juvenile coho salmon.
In eels, on the other hand, there is a significant interconversion
between plasma cortisol and plasma cortisone (Leloup-Hatey 1976,
1979), and consequently the rate of appearance of radioactive
cortisone following a single injection of radioactive cortisol
(Leloup-Hatey 1976) is relatively reduced as compared with salmon.
Neither the anesthetic treatment by itself nor the overall
handling procedure (netting, anesthesia, and injection) significantly
affected the rate at which plasma tritium disappeared from the plasma
following 3H-cortisol administration.
Tricaine methanesulfonate, the
anesthetic used in our MCR measurements, has been shown to bind to
cytochrome P450 and inhibit microsomal mixed function oxidase activity
in the liver of some fish species including brook trout, Salvelinus
fontinalis, (Fabacher 1982a,b).
Microsomal mixed function oxidase is
presumably involved in the hepatic hydroxylation of some steroids, but
hydroxylated hepatic metabolites of cortisol have been found only in
23
relatively small amounts in mammals (Cope 1972), while in fish no such
metabolites have been detected at this time (Truscott 1979).
Handling of salmonids rapidly increases their plasma cortisol
levels (Schreck 1981).
In mammals, acute changes (Southren et al.
1968) or diurnal fluctuations (de Lacerda et al. 1973) in plasma
levels of steroids which show high-affinity and low-capacity binding
to specific plasma proteins are known to affect their own MCR, perhaps
due to changes in the fraction of "available" hormone (Tait and
Burstein 1964).
However, the function of specific plasma proteins in
protecting corticosteroids from being metabolized is limited since
corticosteroids which bind to transcortin with 40% or less affinity
than cortisol behave as if only bound to albumin (Daniel et al. 1982),
and therefore may be regarded as being essentially all "available" for
metabolism at any given plasma concentration.
If plasma
corticosteroid extraction by tissues in salmon behaves as in mammals,
then cortisol would be little protected from metabolism in salmon
since its binding affinity to salmon plasma proteins is 100-fold less
than to mammalian transcortin (Freeman and Idler 1971).
The present
and previous (Redding 1982) results from our laboratory suggest that
acute changes in plasma cortisol levels within the physiological range
do not influence its MCR in juvenile coho salmon.
Previous studies in juvenile salmonids have suggested an increase
in plasma cortisol during the Spring (Langhorne and Simpson 1981;
Specker and Schreck 1982).
In the present study, plasma cortisol in
Eagle Creek salmon increased in July.
In Big Creek-Soleduc salmon,
plasma cortisol decreased with time, although no early summer samples
2 4
were taken and it is conceivable that plasma cortisol could have
increased at this time.
The differences in results between previous
studies and the present one could be due to differences in the timing
of plasma cortisol changes between stocks or to differences in rearing
conditions.
On the other hand, it may be possible that despite the
care taken not to disturb the fish for endogenous cortisol
measurements, the resting levels of cortisol in our fish may have been
altered by some stress imposed on the fish.
No consistent trend for
plasma cortisol to increase with sampling order was apparent within
samples taken at any given sampling date.
However, a wide variation
was observed when mean plasma cortisol values were relatively high.
One possible explanation for these wide variations is that some fish
may have been inadvertently stressed before or during sampling.
In 1982, fish for endogenous hormone determinations were sampled
within a day of the new and full moons since previous reports have
suggested that changes in plasma thyroxine during smoltification may
be related to the phase of the moon (Grau et al. 1981).
Although the
late June increase in plasma thyroxine observed in 1981 and 1982
occurred close to a new moon, no clear correlation between plasma
thyroxine changes and the lunar cycle was apparent at other times.
Visual analysis of the graphs for MCR of 3H-cortisol and its
metabolites and gill Na/K-ATPase activity suggests that the timing and
direction of their changes closely coincided.
Measurements of gill
Na/K-ATPase activity on the Big Creek7.Soleduc stock were performed by
E. K. Birks and R. D. Ewing (unpublished results) on fish reared at
the Corvallis Laboratory of the Oregon Department of Fish and Wildlife,
25
only 0.5 km from our hatchery, with the same water supply, and in the
same year when we conducted our experiments.
The significance of the
apparent relationship between MCR and gill Na/K-ATPase, however, is
unknown.
Our finding that plasma thyroxine titers and gill
Na/K-ATPase activity were correlated among, but not within, groups of
fish sampled at different dates corroborates the conclusions of Folmar
and Dickhoff (1981) who suggested that these physiological variables
change independently, although simultaneously, following changes in
the environment.
Unpublished results from studies at our laboratory
on the effects of rearing density on coho salmon smoltification
provide further evidence for this notion.
In conclusion, the results of our study show that the plasma
clearance rate of corticosteroids changes during smoltification of
coho salmon, although it is unclear whether these changes are
regulated by seasonal changes in plasma levels of corticosteroids or
by some other factor.
26
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Donaldson, E. M., and Fagerlund, U. H. M. (1972).
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27
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17, 233-250.
Grau, E. G., Dickhoff, W. W., Nishioka, R. S., Bern, H. A., and
Folmar, L. C. (1981).
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Hoar, W. S. (1963).
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Hoar, W. S. (1976).
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physiology. J. Fish. Res. Board Can. 33, 1234-1252.
Idler, D. R., and Truscott, B., with the collaboration of Freeman, H. C.,
Chang, Y., Schmidt, P. J., and Ronald, A. P.
(1963).
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(B)
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199, 345-354.
28
Komourdjian, M. P., Saunders, R. L., and Fenwick, J. C.
(1976).
Evidence for the role of growth hormone as part of a
"light-pituitary axis" in growth and smoltification of Atlantic
salmon (Salmo salar).
Can. J. Zool. 54, 544-551.
Langhorne, P., and Simpson, T. H. (1981). Natural changes in serum
cortisol in Atlantic salmon (Salmo salar L.) during parr-smolt
transformation.
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p. 349. Academic Press, London.
Leloup-Hatey, J.
(1974).
Influence de l'adaptation a l'eau de mer sur
la fonction interrenalienne de l'Anguille (Anguilla anguilla L.).
Gen. Comp. Endocrinol. 24, 28-37.
Leloup-Hatey, J.
(1976). Method de mesure des vitesses d'epuration
metabolique et de secretion du cortisol chez l'anguille
(Anguilla anguilla L.). Can. J. Physiol. Pharmacol. 54, 262-276.
Leloup-Hatey, J.
(1979).
Interrenal secretion and peripheral
production of cortisone in eel (Anguilla anguilla L.).
Proc.
Indian Natn. Sci. Acad. B, 45, 436-442.
Mazeaud, M. M., Mazeaud, F., and Donaldson, E. M.
(1977). Primary and
secondary effects of stress in fish: Some new data with a genral
review. Trans. Am. Fish. Soc. 106, 201-212.
McLeay, D. S. (1975). Variations in the pituitary-interrenal axis
and the abundance of circulating blood-cell types in juvenile coho
salmon, Oncorhynchus kisutch, during stream residence.
Can. J.
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Miller, R. G. (1981).
Simultaneous statistical inferences.
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Nishioka, R. S., Bern, H. A., Lai, K. V., Nagahama, Y., and Grau, E. G.
(1982).
Changes in the endocrine organs of coho salmon during
normal and abnormal smoltification. An electron microscope study.
Aquaculture 28, 21-38.
Normand, M., and Fortier, C.
(1970). Numerical versus analytical
integration of hormonal disappearance data.
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Olivereau, M. (1962). Modifications de l'interrenal du smolt (Salmo
salar L.) au cours du passage d'eau douce en eau de mer.
Gen. Comp. Endocrinol. 2, 565-573.
Olivereau, M. (1975). Histophysiologie de l'axe hypophysiocorticosurrenalien chez la saumon de l'Atlantique (cycle en eau douce vie
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29
Quesenberry, R. O., Donaldson, E. M., and Ungar, F. (1965).
Descending and ascending chromatography of steroids using thin
layer chromatography sheets. Steroids 6, 167-175.
Redding, J. M. (1982). "Stress, osmoregulation, and the hormone
cortisol in yearling coho salmon, Oncorhynchus kisutch."
Ph.D.
Thesis. Oregon State University, Corvallis.
Schreck, C. B. (1981). Stress and compensation in teleostean fishes:
Response to social and physical factors.
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(A. D. Pickering, ed.), pp. 295-321.
Academic Press, London.
Shipley, R. A., and Clark, R. E.
(1972). Tracer methods for in vivo
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Theory and applications. Academic Press, New York and
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Snedecor, G. W., and Cochran, W. G. (1980).
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Statistical methods.
Southren, A. L., Gordon, G. G., and Tochimoto, S.
(1968). Further
study of factors affecting the metabolic clearance rate of
testosterone in man. J. Clin. Endocrinol. 28, 1105-1112.
Specker, J. L. (1982).
Interrenal function and smoltification.
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Specker, J. L., and Schreck, C. B. (1982). Changes in plasma
corticosteroids during smoltification of coho salmon,
Oncorhynchus kisutch. Gen. Comp. Endocrinol. 46, 53-58.
Strange, R. J., and Schreck, C. B. (1978). Anesthetic and handling
stress in survival and cortisol concentration in yearling chinook
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35, 345-349.
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(1964).
In vivo studies of steroid
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Truscott, B. (1979).
Steroid metabolism in fish: Identification of
steroid moieties of hydrolyzable conjugates of cortisol in the
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Wedemeyer, G. A., Saunders, R. L., and Clarke, W. C.
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Environmental factors effecting smoltification and early marine
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APPENDIX
30
EFFECTS OF REARING DENSITY ON YEARLING COHO
SALMON, ONCORHYNCHUS KISUTCH
The parr-smolt transformation (smoltification) of anadromous
salmonids is a critical process by which these fish prepare for
residence in the ocean (Hoar 1963, 1976).
Migration, sea water
adaptation, and other activities of the smolting salmon may be
affected by stress (Schreck 1981, 1982).
Therefore, in the case of
hatchery-reared salmon, certain stressful culture practices which are
known to affect the physiology of juvenile salmon such as handling
(Wedemeyer 1976), medication treatment (Bouck and Johnson 1979), and
rearing density (Fagerlund et al. 1981) may have short and/or long
term effects on the process of smoltification.
Indeed, Sandercock and
Stone (1982) have shown that the rate of adult return is inversely
proportional to rearing density in coho salmon.
The rate of adult return may be an effective estimate of the
quality of hatchery practices during rearing of juveniles.
However,
in order to make the operation economically worthwhile, it may be
necessary for hatcheries to meet a minimum requirement in terms of
absolute numbers of adults returning to the hatchery and commercial
fisheries.
Consequently, a trade-off between rearing density and
adult rate of return must be sought in order to determine the minimum
rearing density that will yield the desired returns.
The present investigation was performed at Smith Farm Hatchery,
Oregon State University, as part of a study conducted by the Oregon
Cooperative Fishery Research Unit and Eagle Creek National Fish
31
Hatchery to establish an appropriate rearing density for Eagle Creek
coho salmon.
The experiments at Smith Farm included replicates of the
experiments performed at Eagle Creek such as sea water challenges,
gill Na/K-ATPase activity, interrenal nuclear diameter, plasma
cortisol and plasma thyroxine measurements, as well as corticosteroid
metabolic clearance rate determinations which could not be performed
at Eagle Creek.
MATERIALS AND METHODS
On 12 January 1982, fish from each experimental raceway at Eagle
Creek were transported to Smith Farm and placed in flow-through
circular tanks (diameter, 0.9 m; volume, 0.3 m3) at about their former
densities (low, medium and high, in duplicates), and a combination of
fish from medium and low denisites was used to create a single very
high density treatment.
The water flow was set at 15 liters/min.
Fish were fed Oregon Moist Pellets twice daily until satiation.
A
plastic mesh was used to cover the water outlets in an attempt to
avoid gas supersaturation which commonly occurs in the water source of
this area during the Spring.
However, high mortalities due to this
problem occurred in March in one replicate of each treatment including
the very high density.
Therefore, except for the very high density
group where the fish seemed to recover quickly, these affected groups
were dropped from the experiment.
signs of disturbance.
The remaining groups did not show
During this period, the water flow was
increased as high as possible since earlier experience has shown that
32
this measure effectively reduces supersaturation.
The flow was
restored to 15 liters/min on April 21.
The high mortality which occurred in the very high density group
brought its density down between the medium and the high density
groups and from hereon it will be referred to as the medium-high
density.
Occasional, albeit few, mortalities were also observed in
the other groups, but there was no clear correlation between mortality
rate and rearing density.
The density indices1 at the end of the
rearing periods were 0.11, 0.29, 0.43, and 0.55, for the low, medium,
medium-high and high density groups respectively.
The experiments were conducted the same week during which the fish
at Eagle Creek were released (May 4-8).
Ten fish from each of the 4
densities were sampled to determine plasma levels of cortisol and
thyroxine, and gill Na/K-ATPase activity, as described in the main
manuscript.
To determine interrenal nuclear diameter in the low and
high density treatments, the head kidneys were dissected out, placed
in Bouin-Hollande solution for 40 h, washed with tap water and
transferred to 657. ethanol until embeeding in paraffin.
cut at 5 4m and stained with hematoxylin eosin.
Sections were
The mean nuclear
index (long + short axis/2) of 40 interrenal nuclei, 10 selected at
random from each of 4 different clusters, was used to estimate
interrenal nuclear diameter.
1
Density index: weight (lbs)/volume (ft3)/length (in). The English
system is used to express density since it is still widely used by
hatcheries in the United States. For a metric description of
density see Material and Methods and Table 1.
33
To determine the kinetics of cortisol and its metabolites, 3.6 pCi
(12.2 ng) of 1, 2, 6, 7-3H7cortisol (Research Products International)
without unlabelled cortisol was injected intracardially into each
fish, and 7-10 individuals were sampled at 1, 4, and 8 h after the
injection (injecting procedures and determination of plasma
radioactivity were as described in the main manuscript).
The ability of the fish to withstand a seawater challenge (Clarke
and Blackbourn 1977) was tested by placing 10 individuals per
treatment in 5 gal plastic buckets containing aerated artificial
seawater for 24 h (Instant Ocean:
30-31 ppt; 12-13° C).
Mortalities
and behaviour were recorded and the survivors were sampled to
determine total plasma Na levels with a flame photometer (Coleman o 51
Ca).
Mean length, weight and condition factor for each group were
estimated from measurements of the fish used in the preceeding
experiments, except those of the seawater challenge.
Total weight of
fish per group was calculated by adding the weight of the fish sampled
in all experiments to the weight of the remaining fish.
Linear regression analysis showed that the decline of radiactivity
in plasma after 3H-cortisol injection followed a straight line in a
semilogarithmic plot (R2: 0.80-0.91) between 1 and 8 h.
Therefore,
the slopes and intercepts of the decline curves were compared using
the general linear approach to multiple regression with indicator
variables (Neter and Wassterman 1974), testing equality of slopes
first and then of intercepts.
In addition, an estimate of the
34
metabolic clearance rate was obtained by calculating the inverse of
the area under the clearance curve (see main manuscript), and
expressing the values in units relative to the value for the low
density.
The final values thus obtained were referred to as relative
metabolic clearance rates (RMCR).
The other results were analyzed using ANOVA, and if significant
differences were found, the means were subsequently compared using
Duncan's multiple range test.
The correlation between plasma
thyroxine and gill Na/K-ATPase activity was analyzed with Pearson's
product moment test.
RESULTS
Rearing density affected growth of fish (Table 4).
Mean length
and weight were lowest and highest in the high and low density groups,
respectively.
However, the condition factor did not seem to vary
significantly among groups.
Higher densities depressed plasma thyroxine titers (P < 0.05;
Fig.
4).
Gill Na/K-ATPase activity tended to decrease with
increasing density, but this trend was not significantly (P> 0.05;
Fig. 4).
Correlation analysis showed a significant covariance (P<
0.05) between plasma thyroxine levels and gill Na/K-ATPase activity
when the results from all treatments were analyzed simultaneously, but
not when single groups were considered individually (P> 0.05).
Interrenal nuclear diameters were not significiantly different
between the low and high density treatments (P> 0.05; Table 5), even
if the groups were divided into 2 subgroups (5 fish per subgroup) of
Table 4.
Final density index (DI), total weight of fish, fork length, weight, and condition factor
of yearling coho salmon reared at different densities. Values on the same column followed
by a common letter are not significantly different (ANOVA and Duncan's multiple range
test; P> 0.05; N = 35-40).
DI
(lbs/ft3/in)
Total
weight (kg)
x
Length
Weight
(cm)
(g)
se
x
Condition factor
(100
g/cm3)
--
se
se
0.55 (high)
15.1
14.1
0.2a
28.3
1.2a
0.99
0.01a
0.45 (medium-high)
12.5
14.7
0.2a,b
32.9
1.3a
0.98
0.01a
0.29 (medium)
8.0
14.3
0.2b,c
28.9
1.3a,b
0.97
0.01a
0.11 (low)
3.1
15.0
0.2c
34.7
1.3b
1.01
0.01a
3 6
FIGURE 4.
Plasma thyroxine (dark bars) and gill Na/K-ATPase activity
(light bars) in coho salmon reared at different densities
(see text).
values.
Bars represent standard errors around mean
I
Co
I
os
.'.:-
......
::::::::"::; :::::::::::: i :: :: :::::::::::
es
':
. ::::: - ::::; :::::; ::. : :.: .: - : .:;:;::.::: .:::::: :: ::: ::: :
i
GILL Na/K-ATPase
(pmole P1/mg protein/h)
I
v
]:::::: :::::; :::::::: :::::::: :: :: ::: ::::: .: ..
'11117:::::::: ::: : : : : : : .:..
t
(Iw/6u)
3NIXOEIA1-1.1.
a
z
2
z
a
a
38
Table 5.
Plasma cortisol, interrenal nuclear diameter (IND), relative
metabolic clearance rate (RMCR), and half-life of plasma
tritium after intracardial injection of 3H-cortisol in
yearling coho salmon reared at different densities
(s:significantly different from low density group; t'-test;
P < 0.05).
Cortisol
(ng/m1)1
Density
x
se
High
58.8
29.5
Medium-High
41.5
Medium
Low
1
x
5.78
IND
RMCR
relative
Half-life
(am)
units)2
(h)2,3
se
0.07
x
se
--
x
se
1.24
0.08
2.58
0.19
15.8
1.14
0.05
2.61
0.17
20.2
7.6
1.22
0.06
2.96
0.21
41.1
20.8
1.00
0.07s
2.96
0.33
5.75
0.04
N = 9-10
2 N = 35-41
3 Calculated as described by Normand and Fortier (1970).
39
largest and smallest fish and comparisons among all 4 subgroups were
made.
In addition, plasma levels of cortisol were similar among
treatments (P) 0.05; Table 5).
The RMCR of cortisol and its metabolites seemed to increase with
density, and a t'-test for means with unequal variances (Sendecor and
Cochran 1980) between the low and the high density showed a
significant difference (Table 5).
The slopes of the clearance curves
did not vary with density (P> 0.05), but the intercepts showed a
trend to decrease with increasing density and were significantly lower
for the medium and high density groups (P < 0.05)
This finding
indicates that the area under the clearance curves decreased with
density, and consequently that the metabolic clearance rate may have
increased since the area under the curve and the metabolic clearance
rate are inversely related.
The osmoregulatory performance of juvenile coho salmon was
markedly affected by rearing density as shown by the results of the
seawater challenge (Table 6).
Mortality rates ranged from 0% for the
low density to 50% for the high density treatments.
plasma Na was increased at the higher densities.
Besides, total
Finally, behavioural
observations showed that the most and least healthy fish after the
seawater challenge were those from the low and high densities,
respectively.
DISCUSSION
It has been previously reported that growth of salmonids can be
affected by rearing density (Reftie and Kittelsen 1976; Reftie 1977;
Table 6.
Mortality, plasma sodium, condition factor, and behaviour of yearling coho salmon reared
at different densities and following a 24 h exposure to artifical seawater.
Initial
number of fish placed in seawater was 10 fish per treatment.
Mortality
Density
(%)
Plasma
sodium
(meq/liter)
Condition factor
(100
g/cm3)
se
Behaviour2
se
High
50
240
9
0.83
0.02a
lethargic/moribund
Medium-High
10
205
10a
0.89
0.02a
lethargic
Medium
111
184
7a,b
0.90
0.03a
lethargic
0
175
8b
0.90
0.02a
active
Low
1
One fish escaped; 1 out of 9 fish died.
2 Basis of estimation was avoidance behaviour observed at sampling.
41
Fagerlund et al. 1981).
In the present study, length and weight of
the fish were depressed at the higher densities, but in contrast to
the previous reports, no differences in condition factor were evident
at the end of the rearing period.
I must be noted that depression of
growth by high rearing densities may not always occur.
Joe Banks
(U.S. Fish and Wildlife Service) found no differences in growth of
fish reared under various densities in a prelimiary experiment at
Willard Fish Hatchery, Willard, WA (personal communication).
Food deprivation or certain diet compositions have a depressing
effect on thyroid activity in fish (e.g., Higgs and Bales 1979 and
references therein).
It is possible that the depression in plasma
thyroxine observed at the higher densities in this study may have been
caused by a reduced energy intake since food conversion efficiency of
coho salmon may be reduced by increasing the population density
(Fagerlund et al. 1981).
In any case, the process of smoltification
could be adversely affected by the reduced thyroxine levels since
thyroxine may play an important role in the regulation of this process
(Hoar 1976; Folmar and Dickhoff 1980; Wedemeyer et al. 1980).
Statistical analysis showed no significant differences in gill
Na/K-ATPase activity among the groups, but there was a consistent
trend toward decreasing activity with increasing density.
A
depression of gill Na/K-ATPase activity at high rearing densities has
also been observed by Dr. Wally Zaugg (National Marine Fisheries
Service, Cook, WA) at Eagle Creek Hatchery (personal communication).
Interestingly, correlation analysis showed a siginificant
covariance between thyroxine levels and gill Na/K-ATPase activity when
42
all groups were included in the analysis, but not when each group was
considered individually.
As has been suggested before (Folmar and
Dickhoff 1981; see also main manuscript), it appears that there is no
cause-effect relationship between thyroxine and gill Na/K-ATPase
activity, but rather that they may be under a common control (e.g.,
photoperiod, energy intake, and so forth).
Plasma cortisol titers and interrenal nuclear diameter appeared to
be unaffected by rearing density.
However, the results of this study
suggest that the metabolic clearance rate of cortisol may have
slightly increased with density.
This would reflect a higher
secretion of cortisol at the higher densities.
The lack of difference
in interrenal nuclear diameter between the high and low density fish
may be explained by the fact that light microscopical observations of
fish interrenal tissue may not always reflect its state of activity
(Nishioka et al. 1982).
This study has shown that rearing density can affect seawater
adaptability of coho salmon juveniles.
know whether the incidence
of
It would be interesting to
"stunting" in coho salmon juveniles upon
transfer of the fish to seawater (Folmar et al. 1982; Nishioka et al.
1982) is related to their rearing density in freshwater and/or
seawater.
For example, the depression of plasma thyroxine in
freshwater fish at high rearing densities may be further accentuated
upon transfer to seawater resulting in the characteristically
low-plasma-thyroxine and slow-growing stunt.
In conclusion, the results of this study show that the physiology
and seawater adaptability of juvenile coho salmon can be markedly
43
affected by rearing density.
It is stil too early to suggest an
appropriate rearing density for Eagle Creek coho salmon, but it
appears that rearing densities above 0.29 (density index) may prove to
be highly detrimental to the fish.
44
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