Aquaculture

Aquaculture, 120 (1994) 151-169

Preliminary evidence that chronic confinement stress

reduces the quality of gametes produced by brown
and rainbow trout
P.M. Campbell”,*, T.G. Pottingerb, J.P. Sumptef
“Department of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex UBS 3PH, UK
bInstituteof Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside,
Cumbria LA22 OLP, UK

(Accepted 5 October 1993 )

Abstract

This study presents preliminary evidence that periods of chronic confinement stress ex-
perienced during the final stages of reproductive development not only disrupt the repro-
ductive endocrinology of trout, but also result in reduced egg size in rainbow trout (On-
corhynchus mykiss) and, most importantly, significantly lower survival rates for progeny
from both stressed brown (Salmo trutta) and rainbow trout compared to progeny from
unstressed controls. Groups of male and female brown and rainbow trout were subjected
to one or two episodes of confinement stress in the months immediately prior to spawning.
Plasma levels of cortisol, testosterone, 17P-estradiol and vitellogenin were determined fol-
lowing the 2-week period of confinement. Plasma levels of cortisol were significantly ele-
vated in all groups of stressed fish, whereas plasma levels of testosterone were significantly
reduced in some, but not all, groups of trout. Plasma levels of 17gestradiol were unaf-
fected by confinement stress, whereas the plasma vitellogenin levels of the stressed female
rainbow trout were significantly reduced. Female rainbow trout subjected to two episodes
of confinement stress, experienced 1 and 3 months prior to spawning, produced eggs which
were signiftcantly smaller than eggs from control fish. Eggs from ovulated brown and rain-
bow trout females were fertilised with milt from males subjected to the corresponding
treatment regime. Subsequent development of the fertilised eggs was then monitored
through the stages of eying, hatch and swim-up. The significantly reduced survival rates
observed in the progeny from the stressed crosses compared to the controls indicates that
stress reduces the quality of gametes produced by trout.

*Corresponding author.

0044-8486/94/%07.00 Q 1994 Elsevier Science B.V. All rights reserved

SSDI0044-8486(93)E0237-4
152 P.M. Campbell et al. /Aquaculture 120 (1994) X51-169

1. Introduction

Stress is a ubiquitous feature of life and reproduction is one physiological pro-

cess which is particularly sensitive to its disruptive effects. Acute and chronic
stress have been shown to adversely affect a range of reproductive indices, includ-
ing a suppressive effect on reproductive endocrinology in humans (Rabin et al.,
1988), other mammals (Sapolsky, 1985; Moberg, 1987), birds (Petitte and
Etches, 1991), reptiles (Moore et al., 199 1 ), amphibians (Zerani et al., 199 1)
and fish (Billard and Gillet, 198 1; Pickering et al., 1987; Carragher et al., 1989;
Pottinger and Pickering, 1990). There is considerable evidence that corticoste-
roids, acting at the level of the hypothalamus (Moore and Zoeller, 1985), pitui-
tary gland (Pi-Hseuh, 1987; Brann et al., 1990) and the gonads (Danisova et al.,
1987; Carragher and Sumpter, 1990) mediate the suppressive effects of stress on
reproduction. A great deal of work has been directed at elucidating the mecha-
nisms believed to mediate stress-induced inhibition of reproductive functions and
the anatomical sites at which these effects take place (for review, see Rivier and
Rivest, 199 1). The ultimate consequences of stress in terms of overall reproduc-
tive success have, however, received much less attention. It is possible to measure
the concentrations of a wide variety of hormones and neurotransmitters involved
in coordinating the reproductive process, and other reproductive parameters such
as sperm counts and motility, egg size and chemical composition, etc., in an at-
tempt to assess the inhibitory influence of acute and chronic stress on reproduc-
tive function. However, it is the successful production of viable offspring which
attests to any animal’s full reproductive capability and it is the effect of stress on
this ultimate reproductive parameter which is most important.
In the fish farming industry it has long been recognised that batches of eggs
collected from different fish can have survival rates which vary widely. Egg losses
to the eyed stage of development average N 18% in UK hatcheries; however, con-
siderable individual variation is observed, since some batches of eggs have sur-
vival rates, up to Cmonth-old fry, in excess of 85%, whilst others experience 100%
mortality at fertilisation (Bromage et al., 1992). As a consequence, the factors
which determine gamete quality, particularly egg quality, have been subjected to
much research on behalf of the fish cultivation industry, yet, apart from overri-
pening (a change which is determined primarily by the time of stripping), no
specific factors have been identified that directly correlate with gamete quality
and progeny survival. The chemical composition and physical dimensions of the
egg, the density and motility of the sperm, the nutrition of the parent fish and
husbandry practices used for the maintenance of the eggs and the broodstock are
all factors implicated in determining progeny survival (Springate et al., 1984;
Bromage and Cumaranatunga, 1988); however, there is little hard evidence link-
ing any of these parameters with fertility or subsequent offspring survival.
Previous work, using brown (Salmu trutta) and rainbow trout (Oncorhynchus
mykiss), has shown that chronic stress, and consequent elevation of plasma cor-
tisol concentration, has a suppressive effect on reproductive endocrinology, re-
sulting in significantly reduced plasma testosterone levels in males and signifi-
 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169 153

cantly reduced plasma sex steroids and vitellogenin levels in females (Carragher
et al., 1989). The aim of this study was to establish the consequences of these
stress-induced endocrine dysfunctions on gamete quality. We have already shown,
in a previous study, that rainbow trout subjected to frequently repeated acute
stress during the 9 months prior to spawning, a period when the gonads are rap-
idly developing, produce gametes of poorer quality than unstressed control fish
(Campbell et al., 1992). This complementary study was designed to examine the
effects of a single (or double) period of chronic stress during the final stages of
maturation on the quality of gametes produced by trout.

2. Methods and materials

The fish used in these studies were 3-year-old rainbow (Annan strain) and
brown (Hungerford strain) trout that had been maintained at densities of 30 and
16 fish per 1500-litre tank, respectively, prior to the experiments. To determine
the effect of periods of prolonged stress (2 weeks duration) on a variety of repro-
ductive parameters in brown and rainbow trout, groups of maturing trout were
taken from their rearing tanks ( 1500 litres, supplied with lake water at 35 l/min,
overall temperature range 2-l 8°C) and confined, in groups of 4, in small cov-
ered troughs (45 x 30 x 15 cm) supplied with a constant flow of Windermere lake
water ( 15 l/min) sufficient to ensure no deleterious change in water quality. Fish
were rapidly netted (3-4 fish at a time) and anaesthetised, before being carefully
transferred to the confinement troughs. A similar number of control fish were
maintained in large rearing tanks and left undisturbed for the duration of the
experiment. The fish, in both the stressed and control groups, were not fed during
the 2 weeks that the stressed group were confined. Outside the period of conline-
ment, the fish were fed, once daily, with commercial trout pellets at a rate of 1%
body weight per day. In each experiment the fish were divided into 2 groups: one
group was subjected to a 2-week period of confinement stress whilst the other,
control group, was left undisturbed for the duration of the experiment. In each
experiment there were equal numbers of fish in the stressed and control groups.
In each of the experiments, the first using brown trout (Salvo trutta) and the
second using rainbow trout ( Oncorhynchus mykiss), two complementary studies
were run concurrently. Hence, each experiment consisted of two parts, A and B.
Part A of each experiment determined the effects of the 2-week confinement stress
on the reproductive endocrinology of the stressed fish. Part B of each experiment
determined the effect of the 2-week confinement stress on the quality of gametes
produced by the affected fish. In Part B, the tanks used to hold the “stressed”
fish, following their period of confinement, were identical to the tanks used to
hold the control fish. Following confinement the “stressed” fish were held under
identical rearing conditions to those of the unstressed control fish.
154 P.M. CampbeN et al. /Aquaculture 120 (1994) 151-169

The brown trout study (Experiment I)

The brown trout (mean weight= 732 2 32.1 g, n= 40) were divided into 4
groups: one group of stressed and one group of control fish were used in each of
the two parts, A and B, of Experiment 1. Part A of Experiment 1 was designed to
assess the effects of a 2-week confinement stress on brown trout reproductive
endocrinology. In this part of the experiment, 12 male and 8 female brown trout
were subjected to a 2-week confinement stress (beginning in early October). The
control fish for part A of Experiment 1 ( 12 males and 8 females) were main-
tained in the large rearing tanks and left undisturbed for the duration of the ex-
periment. In Part B, 8 male and 8 female brown trout were used to study the
effects of one period of chronic confinement stress (beginning early October) on
the quality of gametes produced by brown trout (Experiment 1B). The control
fish for Part B (8 males and 8 females) were maintained in the large rearing tanks
and left undisturbed throughout the experiment. At the end of the 2-week period
of confinement stress, the fish in Experiment 1A were anaesthetised (2-phenox-
yethanol, 1: 2000) and blood samples were taken from the caudal vessels using
heparinised syringes. The fish were then weighed and measured, before being killed
by cervical severance. The blood samples were stored on ice in precooled tubes
for < 30 min and centrifuged at 4°C. The resultant plasma was stored in l-ml
aliquots at - 20” C until assayed for steroids. The brown trout in Experiment 1B
were returned to their stock tanks following the 2-week confinement, where they
were observed to rapidly resume normal feeding behaviour, and subsequently
ovulated in early November. Eggs were stripped under anaethesia, within 2 days
of ovulation, from the stressed and control females into separate, dry, plastic
bowls, and in each case were fertilised with a pooled sample of milt collected from
between 2 and 4 males subjected to the corresponding regime. Hence, within the
two treatment groups, gametes were collected from each fish and pooled for fer-
tilisation and rearing. Milt from all 8 control males and all 8 stressed males was
used at the fertilisation stage. The pooled fertilised eggs were divided equally be-
tween 4 egg trays, producing 2 trays of eggs from stressed parents and 2 trays of
eggs from control parents. The subsequent survival of the progeny was monitored
through egg development, hatching, and up to the swim-up stage of development.

The rainbow trout study (Experiment 2)

The rainbow trout (mean weight = 1462 5 55.8 g, n=24) were divided into 4
groups: one group of stressed and one group of control fish were used in each of
the two parts, A and B, of Experiment 2. Part A of Experiment 2 was designed to
assess the effects of a 2-week period of confinement stress on rainbow trout re-
productive endocrinology. In this first part of Experiment 2,6 male and 6 female
rainbow trout were subjected to 2 weeks confinement stress (beginning in mid-
November). An equal number of control fish were maintained undisturbed in
the large rearing tanks. Part B of Experiment 2 was designed to assess the effects
of two separate periods of chronic confinement stress (beginning in mid-Novem-
ber and early December), each of 2 weeks, on the quality of gametes produced by
stressed rainbow trout. In Part B, 8 male and 8 female rainbow trout were sub-
 PM. Campbellet al. /Aquaculture 120 (1994) 151-169 155

jetted to two periods of confinement stress. As in Part A, an equal number of

control fish were maintained undisturbed in the large rearing tanks for Part B of
Experiment 2. Final blood samples were taken from the fish in Experiment 2A at
the end of the 2 week-confinement period and treated in the same way as de-
scribed previously for the brown trout study. In Experiment 2B, the fish were
returned to their tanks after the chronic stress, and reached full sexual maturity
approximately 1 month later, when fecundity and egg size were recorded in ma-
ture females and sperm counts were estimated for mature males from both the
stress and control groups. The females were checked every l-2 days for signs of
ovulation. Following ovulation, eggs were stripped, the coelomic fluid drained
and discarded, the total number of eggs weighed and a subsample of eggs taken
for fertilisation. A dissecting microscope fitted with an eyepiece micrometer was
used to measure egg diameters; the diameters of 10 eggs from each female were
measured on a random orientation basis (to the nearest 0.1 mm) and individual
egg volumes calculated. Ten eggs from each female were also weighed (to the
nearest 0.001 g). The fecundity of each female was estimated by dividing the
total weight of eggs collected by the mean individual egg weight.
When required for the fertilisation stage of the experiment, mature males were
rapidly netted from the appropriate stress or control groups and anaesthetised.
Milt was then stripped from the males by gentle abdominal massage and collected
into dry plastic tubes. An aliquot of milt from each male was diluted 1: 1000 in
0.5% NaCl (Sigma Chemical Company Ltd.) and used for estimating sperm
counts using a haemocytometer (chamber volume 0.00025 mm3) under a phase-
contrast microscope at 250x magnification. Ten squares were counted per fish.
The following formula was used:
sperm -- DXN
mm3 milt- SXK
where D= dilution ( 1000)) N= total number of sperm counted, S= number of
squares counted, and K= cubic capacity of one square in mm3.
The gametes, collected separately from each rainbow trout, were held sepa-
rately for fertilisation and rearing. The eggs from stressed females were fertilised
with milt from stressed males and the eggs from control females were fertilised
with milt from control males. The males were used only once, to fertilise a batch
of 300 eggs from a single female. Hence, each cross was a single pair mating and
each fish participated in only one mating. Five experimental crosses were set up
using the stressed group (i.e., 5 stressed males were crossed with 5 stressed fe-
males); only 5, instead of the expected 8, crosses could be established because
two of the original 8 confined females died before ovulation and one was showing
early signs of bacterial infection and consequently was removed from the experi-
ment. Fifteen experimental crosses were set up using unstressed control fish (i.e.,
15 control males were crossed with 15 control females).
It was considered important not to use a large excess of sperm at the fertilisa-
tion stage, because doing so might mask any reduction in sperm quality. Previous
156 P.M. Campbellet al. /Aquaculture 120 (1994) 151-169

work (reviewed by Billard, 1992) has shown that 10 ~1 of milt, once appropri-
ately diluted, is sufficient to fertilise a few hundred eggs. Thus the 300 eggs from
each female were fertilised with 10 pl of milt. Each sperm sample was first diluted
1: 10 in a non-activating solution of 40 mmol KC1 (Sigma Chemical Company
Ltd.). The inhibitory effect of this diluent on trout sperm motility was checked
before any sperm dilutions were carried out; it was found to completely inhibit
motility. This was then further diluted 1: 100 in 125 mmol NaCl buffered to pH
9 with Tris (Billard, 1992). This solution activates the sperm, which are then
motile for about 30 s. The 1: 1000 dilution of milt, final volume 10 ml, was added
immediately to a batch of 300 eggs which were then gently stirred before being
left undisturbed for 10 min. The fertilised eggs were placed in separate compart-
ments within egg trays in incubators supplied with a constant flow of filtered lake
water (temperature range during embryo development 2-6 ‘C ) . Subsequent sur-
vival of the progeny was monitored through egg development, hatching and up
to 28 days post-hatch.

Radioimmunoassays
Plasma steroid levels were measured using established, previously validated
radioimmunoassays: Cortisol levels were determined according to Pickering et
al. (1987), testosterone levels were measured according to Pottinger and Pick-
ering ( 1985 ) and 17#I-estradiol levels were measured according to Carragher et
al. ( 1989). Rainbow trout vitellogenin was measured as described in Sumpter
( 1985) and Copeland et al. (1986), and brown trout vitellogenin was measured
using the homologous assay described in Norberg and Haux ( 1988 ) .

Data analysis
Student’s two-tailed t-test for unpaired comparisons was used to determine the
significance of the observed effects. A statistically significant difference was in-
dicated when the P-value was less than 0.05. The results of replicate treatments
were tested for similarity, shown to be similar, and then combined.The progeny
survival data was transformed, by conversion to proportions and by arcsin, be-
fore statistical analysis was performed. Because fertilised brown trout eggs, within
treatment groups, were placed in two large pools for fertilisation and rearing, iz= 2
only and thus the brown trout progeny survival data was analysed using chi-square
analysis (2 x 2 contingency tables),

3. Results

Weights and lengths

There were no significant differences in either weights or lengths between the
stressed and control brown trout, of both sexes, at the end of Experiment 1. Sim-
ilarly, there were no significant differences in either weights or lengths between
the stressed and control female rainbow trout at the end of Experiment 2. How-
ever, the male rainbow trout subjected to 2 weeks confinement stress in Experi-
 P.M. Campbell et al. /Aquaculture 120(1994) 151-169 157

ment 2 had, on average, significantly lower mean weights than the males in the
control group (P~0.05). Hence, exposure to chronic confinement stress for 2
weeks did not, in the majority of cases, cause significant alterations in body weight,
the exception being the male rainbow trout.

Plasma cortisol
All groups of trout subjected to 2 weeks chronic confinement stress in Experi-
ments 1 and 2 had significantly higher plasma cortisol levels compared to control
fish (stressed female rainbow trout = 37.5 2 7.5 ng cortisol/ml compared to con-
trols = 7.2 ? 1.5 ng cortisol/ml; stressed male rainbow trout= 18.0 + 5.7 ng corti-
sol/ml compared to controls= 1.8 +-0.2 ng cortisol/ml; stressed female brown
trout = 6.9 t 1.2 ng cortisol/ml compared to controls= 2.6 + 0.8 ng cortisol/ml;
stressed male brown trout= 12.2 + 3.4 ng cortisol/ml compared to controls =
2.8 ? 0.4 ng cortisol/ml; Fig. 1A: PC 0.05).

Plasma testosterone
The plasma testosterone (T) levels of the stressed male rainbow trout and fe-
male brown trout were significantly reduced compared to controls (stressed male
rainbow trout = 67.2 + 4.7 ng T/ml compared to controls = 144.15 14.1 ng T/ml;
stressed female brown trout=38.7 If:5.6 ng T/ml compared to controls =
57.3 ? 6.1 ng T/ml; Fig. 1B: P~0.05). However, the plasma testosterone levels
of the stressed female rainbow trout and male brown trout were unaffected by the
2 weeks of confinement stress (stressed female rainbow trout = 130.12 11.4 ng
T/ml compared to controls= 136.02 16.8 ng T/ml; stressed male brown
trout= 18.12 2.4 ng T/ml compared to controls= 11.7 t 1.7 ng T/ml).

Plasma 1 ?b-estradiol
The stress regime employed had no effect on plasma 17pestradiol levels in
female brown or rainbow trout in Experiments 1 and 2, since the stressed females
had plasma 17pestradiol levels which were not significantly different from con-
trol values (stressed female rainbow trout = 37.9 i 6.1 ng E,/ml compared to
controls= 37.0+- 3.6 ng E,/ml; stressed female brown trout= lO.O? 1.2 ng E/ml
compared to controls= 12.8 22.6 ng E,/ml) (Fig. lc).

Plasma vitellogenin
The plasma vitellogenin (VTG) levels of the female rainbow trout were signif-
icantly reduced (stressed=23.1 t 4.4 mg VTG/ml compared to controls =
42.8 2 4.9 mg VTG/ml: P-z 0.05). There was no significant difference in plasma
vitellogenin levels between the female brown trout subjected to a 2-week period
of confinement stress and the controls (stressedz23.2 + 3.2 mg VTG/ml com-
pared to controls= 35.6& 8.7mg VTG/ml) (Fig. Id).

Egg parameters
Eggs obtained from female rainbow trout subjected to episodes of chronic con-
finement stress in Experiment 2 were significantly smaller than eggs obtained
from control females (Fig. 2). Ten eggs from each female rainbow trout were
158 P.M. Campbellet al. /Aquaculture 120 (1994) 151-169

weighed and measured; this is a suffkiently large sample size because eggs from
an individual are of a very similar size (Tyler et al., 1990a). The weight was
significantly lower (P< 0.05 ) for eggs from stressed female rainbow trout when
compared to eggs from control females (0.622 5 0.04 g for eggs from stressed fe-
male rainbow trout compared to 0.7 12 + 0.02 g for eggs from controls) (Fig. 2A).
The mean volume of eggs obtained from stressed female rainbow trout was also
significantly less (P-=0.05) than that of eggs from controls (96.05 5.0 mm3 for
eggs from stressed females compared to 115.1+ 3.4 mm3 for eggs from controls
(Fig. 2B). The fecundity of the female rainbow trout was unaffected by the re-
gime of two separate episodes of prolonged stress (Fig. 2~). Egg parameters were
not recorded for the brown trout females in Experiment 1.

Sperm counts
The sperm counts of the male rainbow trout subjected to periods of chronic
confinement stress were not significantly different from those of the controls
( 16.0 +-1.0~ 1O9sperm/ml of milt for stressed males compared to 13.6 + 1.2~ 1O9

n STRESS

Ia CONTROL

*p<o.os
 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169 159

0
Female FNll*k

Rambw Trout 1 BmwnTmut ]

Fig 1. Mean plasma steroid levels in chronically stressed and control maturing brown and rainbow
trout of both sexes; mean plasma cortisol (a) and testosterone (b ) levels in male and female trout,
and mean plasma 17/?-estradiol (c) and vitellogenin (d) levels in the female trout. Shaded columns
represent stressed fish, open columns represent controls. Values are means 2 s.e.m.

sperm/ml of milt for control males (Fig. 3 ). Sperm counts were not carried out
on the brown trout males.

Progeny survival
There was a significant difference in survival rates between the eggs from the
chronically stressed and control brown trout females up to the eyed stage of de-
velopment in Experiment 1 (% survival of progeny from stressed brown trout =
68.3% compared to progeny of controls= 95.7%, PC 0.01) (see Fig. 4a). In this
experiment, fertilised eggs, within a treatment group, were pooled and placed in
2 separate egg trays. It should be noted that the results obtained for the two egg
trays (containing eggs from different individuals stripped on successive days)
were very consistent (egg trays 1 and 2: 96survival of progeny from stressed brown
trout=66.0% compared to progeny of unstressed control=95.1%, P-=0.01; egg
160 P.M. Campbell et al. /Aquaculture I20 (1994) 151-169

q CONTROL

(b)
150

I
q CONTROL

q CONTROL

Fig 2. Effect of two periods of chronic confinement stress, experienced close to final maturation, on
mean weight of 10 eggs (a), egg volume (b) and fecundity (c ) of the female rainbow trout. Eggs were
obtained from 5 stressed and 15 control females. Ten eggs from each female were measured. Values
are means+ s.e.m.

trays 3 and 4: % survival of progeny from stressed fish = 70.6% compared to prog-
eny of controls = 96.2%, P< 0.01). Over 90% of the eggs from the control crosses
became eyed, whereas only around 70% of the eggs from the stressed crosses be-
came eyed, indicating a very good fertilisation rate in the control crosses and
 P.M. Campbelletal. /Aquaculture120(1994)151-169 161

Fig. 3. Effect of two periods of chronic confinement stress, experienced close to final maturation, on
sperm density in milt of male rainbow trout. Values are means I! s.e.m.

so

60
q Chronic Stress
fa Controls
40

**p<o.o1
20

0
MD HATCH SWIM-UP

q Chronic Stress
ca Controls

“p<O.Ol

MB) HATCH SWIM-UP

Fig. 4. Cumulative survival rates (a) and mortality rates at each developmental stage (b) of eggs and
embryos from chronically stressed and control brown trout. The rate of survival (and hence mortal-
ity) was assessed at three developmental stages: the eyed stage, hatch and swim-up.
162 P.M. Campbell et al. /Aquaculture 120 (1994) 141-169

W Chronic Stress
la Controls

EYED HAm SWIM-UP 2%DAYS

Fosr-HATal

Devebpnreotalstaee

m,

EYED HAlCH SWE+UP ZEDAYS

POST-HATCH

-a-lP

Fig. 5. Cumulative survival rates (a) and mortality rates at each developmental stage (b) of eggs and
embryos from chronically stressed and control rainbow trout. The rate of survival (and hence mor-
tality) was assessed at four developmental stages: the eyed stage, hatch, swim-up and 28 days post-
hatch.

possibly a reduced rate of fertilisation in the stressed crosses. This difference in

survival was maintained through the stages of hatch and swim-up, so that at the
end of the monitoring period 62.7% of the progeny from the stressed crosses were
still alive, compared to 9 1.O%of the progeny from the controls (PC 0.0 1) (see
Fig. 4a). The differences in survival between the two groups at any stage of de-
velopment can be attributed to more mortalities in the progeny of the stressed
fish before the eyed stage (Fig. 4b).
Very similar results were obtained from the rainbow trout in Experiment 2.
There was a significant difference in the survival rates between the eggs from the
chronically stressed and control rainbow trout at the end of the monitoring pe-
riod in Experiment 2 (Fig. 5a). A significant difference in survival rates between
the eggs from the stressed and the control rainbow trout was evident early on, at
the eyed stage of development (016survival of progeny from stressed crosses =
 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169 163

53.6Ir22.1%compared to 96.2 5 1.3% survival of progeny from unstressed con-

trol crosses; P-c 0.05) (Fig. 5a). This difference in survivorship was maintained
throughout development, up to the end of the monitoring period, when
44.1? 18.6% of the progeny from the stressed crosses were still alive compared to
84.8 If 2.5% of the progeny of the control crosses, PC 0.05)(Fig. Sa). The differ-
ences in survivorship between the two groups at any stage of development can be
attributed to more mortalities in the progeny of the stressed rainbow trout before
the eyed stage (Fig. 5b); it is not known whether this was due to a failure at the
fertilisation stage or to deaths at an early stage of embryonic development.

4. Discussion

This study shows that exposure of brown and rainbow trout to episodes of
chronic stress during the later stages of reproductive development results in re-
duced egg size (at least in rainbow trout) and, most importantly, significantly
lower survival rates for progeny from stressed fish, of both species, compared to
progeny of unstressed controls. In addition, exposure to chronic stress in the
months immediately prior to spawning can result in reduced plasma sex steroid
levels, although this effect was variable between sexes and species.
It is well established that chronic crowding stress and elevated plasma cortisol
levels are associated with reduced growth of trout (Pickering and Stewart, 1984))
most probably as a result of reduced food intake and decreased efficiency of food
utilisation (Fagerlund et al., 198 1; Trzebiatowski et al., 198 1). Hence, food was
withheld from both the stressed and control fish during the period of confine-
ment, in an attempt to avoid a difference in nutritional status between the two
groups of fish influencing the results (see later). Excluding the male rainbow trout
in Experiment 2, when the control males were significantly heavier than the
stressed males, there were no significant differences in weights or lengths between
the chronically stressed and control fish in any experiment. It is not known why
the control male rainbow trout were heavier than the stressed fish; it is possible
that the difference was apparent at the beginning of the experiment, but unfor-
tunately the fish were not weighed at this time, so we do not know. Alternatively,
it is possible that the control male rainbow trout in Experiment 2 mistakenly
received food during the 2 weeks of the experiment, leading to the differences in
final weight between the stressed and control males. However, our results on ga-
mete quality cannot be explained by this size difference in one group of trout (out
of 4), because pronounced effects of stress on plasma steroid and vitellogenin
(VTG) levels were observed in both sexes of brown trout and also in female
rainbow trout exposed to chronic confinement stress, in which no significant dif-
ferences in weights and lengths occurred between the stressed and control fish,
indicating that stress and not nutritional factors was responsible for the observed
effects.
Prolonged confinement of trout in spatially restrictive holding tanks, a method
of mimicking the chronic overcrowding that can occur on fish farms, is a reliable
164 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169

method of invoking a chronic stress response (Pottinger et al., 1992; Pickering et

al., 1987). In this study, confinement of brown and rainbow trout elicited signif-
icant elevation of plasma cortisol levels in both male and female fish. relative to
unconfined controls. Confinement also significantly reduced testosterone levels
in male rainbow trout and female brown trout, but not in female rainbow trout
or male brown trout. It is not clear why some, but not all, of the groups of trout
showed reduced testosterone levels when subjected to chronic stress.
Similar to the results of Carragher et al. ( 1989) obtained when fish were im-
planted with cortisol to mimic the effects of stress, plasma sex steroid levels of
the female rainbow trout in this study were unaffected by confinement stress and
plasma cortisol elevation, whereas the plasma VTG levels of the females in both
studies were significantly reduced by the treatment. Hence, the mechanism me-
diating this disruption of vitellogenesis in this species is more complex than sim-
ply a stress-induced reduction in plasma 1‘i/3-estradiol levels directly resulting in
a decline in VTG production by the liver. The variable effect of stress and plasma
glucocorticoid elevation on plasma steroid levels, particularly in females, has been
noted in a number of studies using a variety of species. Studies using amphibians
and reptiles, including bullfrogs, turtles and alligators, show a consistent negative
effect of stress and concomitant plasma corticosterone elevation on androgen lev-
els in males (Licht et al., 1983; Mahmoud et al., 1989; Elsey et al., 199 1 ), whereas
the effect of stress on plasma estradiol levels in females was found to be much
more variable. However, despite the much less dramatic effect of stress on plasma
levels of gonadal steroids in female alligators compared to males, a profound ef-
fect of stress on ovulation and nesting success in females was observed (Elsey et
al., 1990). These results indicate that the degree of stress-induced aberrations in
plasma steroid levels does not necessarily reflect the extent to which reproductive
success is eventually impaired. This hypothesis is supported by this study, in which
levels of gonadal steroids were not significantly affected by stress in some of the
stressed groups of fish, whereas reproductive success of these fish was markedly
reduced.
Once ovulation had commenced, the female fish were checked every l-2 days
and the eggs removed from the body cavity by manual stripping when individuals
were found to have ovulated. This regular checking for ovulation was important
since ovulated eggs of oviparous teleosts become over-ripe if retained in the body
cavity and show a progressive reduction in viability (reviewed in Bromage and
Cumaranatunga, 1988). This process of post-ovulatory aging is poorly under-
stood, but it is known that it has a profound effect on the ability of the eggs to be
fertilised and to develop further through the stages of eying, hatch and swim-up
(Statova et al., 1982; McEvoy-Barton, 1984). The eggs from control females in
this study had fertilisation, eying and hatching rates of over 9096, which is high,
comparing very favourably with the maximum survival rates obtained by Sprin-
gate et al. ( 1984) and other authors.
The effects of stress on oogenesis and spermatogenesis have been investigated
in only a relatively small number of studies and in these cases the fish were ex-
posed, not to crowding stress, but to sublethally low pH (acid stress). A few of
 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169 165

these studies have examined the consequences of exposure in terms of fertility,

gamete quality and larval growth and development. Exposure of trout to suble-
thally low pH resulted in elevated cortisol levels, reduced growth and hence re-
duced fecundity, lower plasma sex steroid levels and delayed ovulation (Tam and
Payson, 1986; Tam et al., 1990). In addition, adult rainbow trout exposed to acid
stress in the 6 weeks prior to spawning produced gametes which demonstrated
reduced embryonic survival when incubation was conducted at ambient pH
(Weiner et al., 1986). The study by Weiner et al. ( 1986) showed that progeny of
acid-exposed female rainbow trout and male controls had reduced survival, in-
dicating that oogenesis was adversely affected by stress. A similar, but less pro-
nounced, reduction in the survival of progeny from acid-exposed males and con-
trol females was also noted, suggesting that oogenesis was more sensitive to acid
stress than spermatogenesis. It is possible that the mechanisms resulting in the
reduction in gamete quality in fish exposed to low pH are different from the
mechanisms causing reduced progeny survival in fish exposed to crowding stress.
Although some of these studies employing acid stress suggest that it is already
established that stress adversely affects gamete quality in fish, in fact interpreta-
tion of the results in these studies is complicated by the fact that fish chronically
exposed to sublethally low pH had reduced calorific intake and hence did not
grow as well as the control fish (Tam et al., 1990). In addition, it might be spec-
ulated that the energetic costs of maintaining homeostasis under acid conditions
would divert resources from gonadal growth. Since the number of eggs produced
by a female is proportional to body weight, it is not surprising that the smaller,
acid-stressed fish had reduced absolute fecundity. A similar argument applies to
egg size, which is related to body size (Bromage and Cumaranatunga, 1988).
Thus, the deleterious effects of acid stress on oogenesis and spermatogenesis ob-
served in many of these studies cannot be distinguished from the effects of poor
nutrition. It is well known that reduced food intake in mammals, including hu-
mans, is associated with hypothalamic dysfunction and reversible sterility. Sim-
ilarly, in fish decreased food availability has been reported to suppress gameto-
genesis (Love, 1980) and reduce fecundity (Billard and Gillet, 198 1). This
problem of distinguishing between the direct consequences of stress on reproduc-
tion and the indirect consequences due to nutritional factors is discussed compre-
hensively by Tyler et al. ( 1990) in relation to oocyte atresia (and hence reduced
fecundity) in rainbow trout. In the study reported here, food was withheld from
both the stressed and control groups during the period of stress to ensure that
both groups received and consumed equal amounts of food during the course of
the experiment.
In this study, chronically stressed female rainbow trout produced significantly
smaller eggs than unstressed control tish. However, there was no significant cor-
relation between egg size and subsequent egg and fry survival rates (r~0.31;
P= 0.19). These results indicate that egg size had no direct implications as far as
overall egg quality and fry survival are concerned. The importance of egg size in
determining egg quality has been difficult to ascertain because of conflicting re-
sults from various studies and because of problems in separating the effects of egg
166 P.M. Campbell et al. /Aquaculture 120 (1994) 151-169

size on egg/fry survival rates from the effects of other factors such as age, strain
and nutritional status of the parent fish. Springate and Bromage ( 1985) and
Campbell et al. ( 1992), using rainbow trout, found that egg size had no direct
effect on egg quality or fry survival, a finding supported by this study. At the
present time the balance of evidence indicates that egg size has no direct impli-
cations as far as egg quality is concerned. The female trout exposed to periods of
chronic stress in Experiment 2 did not show any change in fecundity (Fig. 2~).
In female rainbow trout the reproductive cycle starts at least 12 months prior to
spawning (Sumpter et al., 1984), and the number of eggs recruited for develop-
ment that year appears to be decided at a very early stage (Tyler et al., 1990).
Since the fish were not exposed to stress until 1 and 3 months prior to spawning,
this was probably too late in the cycle to affect fecundity.
Presently, there are no truly dependable criteria for estimating sperm quality;
the length of time and intensity of spermatozoon motility (Terner, 1986; Moccia
and Munkittrick, 1987; Billard and Cosson, 1992 ), the percentage of motile sper-
matozoa (Levanduski and Cloud, 1988), sperm density (Moccia and Munkit-
trick, 1987; Scott and Baynes, 1980) and the chemical composition of the sem-
inal plasma (Hwang and Idler, 1969; Morisawa and Morisawa, 1988) are all
parameters that have been measured in an attempt to assess sperm quality. How-
ever, there is little evidence directly linking any of these parameters with fertility.
Hence, despite the development of many tests to evaluate sperm quality, it is still
true that the ability of sperm to successfully fertilise eggs and produce viable off-
spring remains the only truly dependable test of sperm quality. In this study, sperm
counts, and hence sperm densities, were not significantly different in male rain-
bow trout exposed to episodes of chronic stress from those in controls. In a re-
lated study, male rainbow trout subjected to repeated acute stress during the 9
months prior to spawning did have significantly lower sperm counts than the un-
stressed control fish (Campbell et al., 1992)) although this difference in sperm
density appeared to have no effect on the fertilisation rate. It should be noted that
only a relatively few males were used in the preliminary studies reported here,
and that the stress regime employed in the two studies differed considerably.
The lower progeny survival of the stressed crosses does indicate a stress-in-
duced reduction in gamete quality, which could be attributed to deleterious ef-
fects on sperm, or eggs, or both. The fact that the study using brown trout, which
used a large excess of milt from a number of different males at the fertilisation
stage, also showed a significant reduction in survival of progeny from chronically
stressed fish, suggests that egg quality is susceptible to the deleterious effects of
stress. The reduced survival of the progeny from the stressed fish compared to
the controls attests to the fact that exposure to periods of chronic stress experi-
enced close to the time of spawning reduces the quality of the gametes produced.
These results are of interest, not only from a fundamental point of view, but also
because at a very practical level they have direct implications in the field of aqua-
culture. These results show that the conditions to which fish farm broodstock are
subjected during the final phase of sexual maturation will be an important factor
in determining the quality of gametes produced, and that this will be reflected in
 P.M. Campbeltetal. /Aquaculture 120(1994/ 151-169 167

the subsequent progeny survival rate. The regime of one or more episodes of
chronic stress used in this experiment may mimic conditions on some fish farms,
where trout can be subjected to periods of chronic overcrowding or confinement
stress during various farming procedures. Future research should expand this ex-
perimental design by fertilising eggs from stressed females with milt from control
males, and vice versa, in an attempt to determine which of the sexes has the ga-
metes more susceptible to the deleterious affects of stress.
The increased mortality, previously reported by Campbell et al. ( 1992), in
progeny from acutely stressed rainbow trout, occurred primarily between eying
and hatch. Progeny of brook trout exposed to acid stress also show reduced sur-
vival to hatch and lower hatching success than progeny from unstressed control
groups (Mount et al., 1988; Weiner et al., 1986 ). In contrast, the increased mor-
tality observed in this study in the progeny from the chronically stressed brown
and rainbow trout occurred primarily before the eyed stage of development. It is
not known whether this was due to lower fertilisation rates in the stressed crosses
or to deaths at an early stage of embryonic development. The difference in the
timing of the deaths of the progeny in our two studies (Campbell et al., 1992 and
this study) may reflect the severity or the timing of the stress experienced, since
the trout in this study were subjected to chronic stress at a time when the fish
were nearly fully mature, whereas the rainbow trout in the previously reported
study (Campbell et al., 1992) were subjected to repeated acute stress during the
9 months preceding spawning. Although this difference in the timing of mortality
in the two studies appears real, it will require further work, especially more exten-
sive studies on the effects of chronic stress, before it can be accepted unequivocally.
In conclusion, we have presented preliminary evidence that periods of chronic
stress during the final stages of reproductive development not only disrupt the
reproductive endocrinology of trout, but can also result in reduced egg size and
in significantly lower survival rates for progeny from stressed trout compared to
progeny from unstressed control fish.

Acknowledgements

This work was supported by the Natural Environment Research Council

(NERC).

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