BIOLOGY
OF
REPRODUCTION
47,
Stress
1140-1150
Reduces
the Quality
of Biology
Institute
of Gametes
CAMPBELL,2’3 T.G.
P.M.
Department
(1992)
Produced
POTFINGER,4
and
by Rainbow
J.P.
Trout1
SUMPTER3
and Biochemistry,3
Brunel
University,
Uxbridge,
of Freshwater
Ecology,4
Windermere
Laboratory,
Cumbria
LA22 OLP United
Kingdom
Middlesex
UB8 3PH,
Far Sawrey,
Ambleside
United
Kingdom
ABSTRACT
In this
gamete
study
9 mo
the
we
quality
carried
prior
out
with
mill
from
in somatic
acute
nificantly
fish
Time
milt
the
males
weight
stress
from
subjected
compared
of ovulation,
male
the
from
regime.
groups
two
at the
perhaps
control
Hence,
fish.
to adversely
affect a range
of reproductive
ing a suppressive
effect on reproductive
[1, 2], mammals
phibians
[12-14],
evidence
pothalamus
that
fish
the
the
deal
suppressive
of work
mechanisms
success
believed
have,
There
includin
[9-11],
effects
has been
amhygo-
of stress
directed
to mediate
received
less
parameters
such
as sperm
counts
and
motility,
at-
Accepted
August
Received
This
May
work
‘Correspondence.
was
18,
10,
by the
parents.
In humans,
female
egg
to repeated
in females,
for progeny
advancing
from
and
trout.
it has
thus
of the
study
been
reproductive
failure
rather
endometrial
failure
[25]. The parameters
sperm
quality
are still not well
the
sig-
stressed
by rainbow
age
factors
that
a
affect
estab-
results
from
inadequacy
that deterdefined
and
gamete
quality
are
and
in vitro
fertilization).
parameters
that
However,
correlate
the
best
identification
with
fertility
and
gamete
quality
in these
number
of tiny
can be obtained,
be removed
thermore,
from
a number
there
are subsequent
embryonic
survival
takes
in utero
place
and
large
species
have used the rainbow
is egg
eggs,
often
and these
of different
difficulties
development,
or within
scale
studies
reThe limiting
factor
individuals.
Furin monitoring
because
an opaque
this
egg.
trout as a model
animal
of stress in terms
quality
its
quantity,
because
mode
usually
In this study
the biological consequences
and
produc-
at different
must often
to study
of gamete
of reproduction
of-
fers
many advantages.
Female
rainbow
trout
undergo
a very pronounced
annual reproductive
cycle, during
which
two large ovaries
develop.
Immediately
prior
to spawning,
the ovaries
together
fer-
comprise
approximately
20% of the body
weight.
At ovulation
2000-3000
eggs per kg body weight,
each measuring
approximately
5 mm, are synchronously
released
into the
body cavity [26]. Following ovulation, the eggs can be man-
by NERC.
(0895)
trout
produced
with
differences
and implantation
mine
egg and
we
1992.
FAX:
of gametes
age-related
oocyte
quality
tion; only a small
stages of maturation,
1992.
funded
rates
were
fertilized
no
size
egg
during
fertilized
were
of rainbow
reduced
survival
with
were
There
exposure
were
of
terms
counts
sperm
females
female
this
for studying
tion 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.
The successful
fertilization
of eggs and subsequent
development
of offspring
will depend
greatly
on the quality
produced
each
monitored.
and
and
ovulated
little possibility
of performing
the
quired
to establish
these
relationships.
size, and chemical
composition,
etc., in an attempt
to assess
the inhibitory
influence
of acute
and chronic
stress
on reproductive
function.
However,
it is the
successful
produc-
of gametes
quality
stress
offspring
survivorship
and the factors
that affect
gamete
quality
are difficult
to achieve
in most
animals,
especially
in species
such
as mammals
and humans
where
there
is
tention.
It is possible
to measure
the concentrations
of a
wide
variety
of hormones
and neurotransmitters
involved
in coordinating
the reproductive
process,
and other
reproductive
lower
declines
of gamete
stress-in-
much
the
lished
that
diminished
nation
on
at
functions
and the anatomtake place
[for review,
see
of stress
in terms
of overall
however,
but
from
in
acute
of considerable
interest,
not only from a fundamental
point
of view, but also because
a better
understanding
would
undoubtedly
improve
the success
of the many
different
assisted
reproduction
techniques
(such
as artificial
insemi-
is considerable
acting
at the level of the
gland
[20, 211, and the
duced
inhibition
of reproductive
ical sites at which
these
effects
24]. The ultimate
consequences
reproductive
indices,
endocrinology,
[8], reptiles
[15-18].
corticosteroids,
[3, 13, 19], pituitary
nads [5, 22, 23] mediate
reproduction.
A great
elucidating
birds
13-7],
and
reduces
tility
then
in ovulation
significantly
stress
Stress is a ubiquitous feature of life and reproduction
is
one physiological
process
that is particularly
sensitive
to its
disruptive
effects. Acute and chronic
stress have been shown
eggs
was
experiment,
delay
importantly,
INTRODUCTiON
humans
of the
eggs
females,
from
Eggs
of stress
to repeated
in mature
300
fertilized
consequences
subjected
groups.
Approximately
end
biological
were
recorded
control
in a significant
most
the
trout
were
and
of the
resulted
to study
rainbow
egg size
stressed
development
and,
unstressed
female
and
the
both
development
in males,
and
treatment
same
animal
as a model
male
fecundity,
from
Subsequent
between
Counts
to progeny
fish,
to the
reproductive
sperm
trout
of 30 mature
in diluent.
or length
during
lower
Groups
to spawning.
of iO
rainbow
the
used
quantity.
Ott the
dilution
sperm
have
and
274348.
1140
STRESS
ually
dures
recovered
from
females
and fertilized
externally
from
and
it
the males.
is possible
by routine
with milt
REDUCES
stripping
similarly
Eggs are then
placed
to monitor
fertilization
THE
ternal
sequent
be
fertilization
development
from
each
10
eye. Thus
system
for
the
8
ti
since
stage
female,
is readily
manipulated,
of the eggs can be very
C,)
C,)
C,)
I-
ex-
and the subeasily
mon-
6-
I-
c)
itored.
Previous
work
shown
acute
that
evation
nology,
stage
obtained
12
in incubation
trays,
success
and sub-
the study of the factors
that determine
gamete
quality,
a very large number
of eggs-all
at exactly
the same
using
and
this
animal
chronic
stress
have a suppressive
effect
resulting
in significantly
one levels
in males
and
steroids
and vitellogenin
this study
induced
model,
and
the
plasma
has
cortisol
el-
endocritestoster-
significantly
reduced
levels
in females
[17].
was to establish
the
endocrine
dysfunction
trout,
on reproductive
reduced
plasma
consequences
on gamete
1141
GAMETES
OF
proceremoved
sequent
development
of the eggs by the naked
the rainbow
trout constitutes
an excellent
model
of maturation-can
QUALITY
4.
C
2’
0
z
plasma
sex
The aim of
0
of this stressquality
and
5
10
15
20
25
WEEK
30
35
40
No.
quantity.
MATERIALS
Animals
and
AND
FIG. 1. Experimental
design.
The experiment
began in mid-April
and
ended in January
1991. The Stress was applied
randomly
throughout
the
38 wk. At the beginning,
the stress was applied,
on average,
once a week;
later in the experiment,
the fish were stressed more frequently.
The interval
between
each stress and the time of day the stress was applied varied ran-
METHODS
Treatment
domly.
Three-year-old
rainbow
trout
(Annan
strain)
were
maintained
at a density
of 30 fish (mean
weight
1462 ± 55.8 g,
n = 90)/tank
in 1500-L outdoor
fiberglass
tanks, each supplied
fish
with
were
a constant
fed
once
flow
daily
of lake
with
1% body
weight
per day. To
peated
acute stress
on a variety
in both
maturing
male and
females,
water
(35
commercial
L/min).
feed
determine
the
of reproductive
female
rainbow
all of which
had
The
at a rate
of
effects
of reparameters
trout,
two groups
of 30
spawned
in the previous
year, and two groups
of 30 maturing
males
were
subjected
to repeated acute stress
during
the 9 mo prior to spawning.
A similar number
of control fish were maintained
in identical tanks
but were
not subjected
to the stress.
The fish
were
stressed
by exposure
to a brief period
of emersion;
this involved
allowing
the water
to drain
completely
from
the
mm
have
tanks,
before
been
stressful
[30], and
leaving
the
experiment,
fish were
exposed
to the
stimulus
[27] as have
sheep
birds
[31]; hence
the emersion
at random
intervals
To assess
whether
procedure,
and blood
fish
air
the tanks
were
slowly
allowed
shown
to acclimate
to repeated
to prevent
emersion
a subsample
was sampled
4 mo
removed
prior
with
about
3
Trout
to a
to spawning.
During
as little disturbance
Collection
each
ice
the
to
for
fecundity,
females,
males from
female
and
and
both
ovulated
on
resultant
blood
for cortisol,
[32].
using
from
ovulation. The
ovulated
female
into
egg
sperm
stress
size
1-2
a dry
<
re-
1991, which
days the
anesthesia,
to the
eggs
stripped
bowl,
the
fitted
egg
measured
0.1 mm)
exfor
by this method,
eggs were
plastic
was
fish were
to check
gentle
pressure
direction;
mature
the genital pore
mature
were
counts
were
estiand control
groups.
15 January
This was done
by applying
in an anterior-to-posterior
eggs from
each
female
were
entation
basis (to the nearest
on
and
from
coelomic
of eggs
taken
for
with
an
diameters;
a random
individual
eye10
oriegg
volumes
were
calculated.
Ten eggs from each female
were
also weighed
(to the nearest
0.001 g). The fecundity
of each
ment,
on
at 4#{176}C.
The
termed
Day 0. Every
under
2-phenoxyethanol
following
osus by means
kept
ovulation,
are easily extruded
female
syringes,
first
thereafter
amined,
the fish remaining
in the tanks, and anesthetized
in 2-phenoxyethanol
(1:2000; Sigma Chemical
Company
Ltd., Poole,
Dorset, UK). Blood samples were taken from the sinus yenof heparinized
of
in mature
for mature
The
centrifuged
of Gametes
Time
corded
mated
then
stored
at -20#{176}Cuntil assayed
validated
radioimmunoassay
fertilization.
A dissecting
microscope
piece
micrometer
was used to measure
tank
the
sampling,
as possible
and
fluid was drained and discarded, the total number
was weighed,
and a subsample
of 300 eggs was
(see Fig. 1).
to be a stressful
taken
from
each
halfway
through
mm,
plasma
was
a previously
ovulation.
abdomen
[28], rats [29], cattle
stress
was applied
acclimation
continued
of fish was
approximately
for
to refill.
exposures
30
collected
When
was
estimated
by the
required
mature
mean
for
males
by dividing
the
individual
egg
the fertilization
were
rapidly
total
weight
weight.
stage
netted
of eggs
of the
from
the
experiappro-
1142
CAMPBELL
priate
+
stress
or control
seminal
groups
anesthetised.
and
fluid) was then stripped
massage
and collected
abdominal
Data
(sperm
Milt
from the males
into dry plastic
by gentle
tubes.
An
aliquot
of milt from each male was diluted
1:1000
in 0.5%
NaCI (Sigma
Chemical
Company
Ltd.) and used
for estimating
sperm
counts
using
a hemocytometer
(chamber
volume
0.00025
at 250
The
mm3)
under
magnification.
X
following
a phase-contrast
Ten
squares
was
used:
formula
sperm
were
per
fish.
total number
number
cubic
mm3)
Weights
and
capacity
lengths
t-test for unpaired
comparisons
the significance
of the observed
counted
of one
square
mean
number
tween
the stressed
of days
of all fish
were
for
to ovulation
and control
unpaired
(0.00025
recorded
groups
were
combined
following
The
conversion
analysis
(1) egg
to
progeny
progeny
data
survival
were
from
the
using
be-
the Student’s
survival
The
out
compared
using
to proportions
was performed.
size and progeny
to generate
calculations
were
carried
State College,
PA).
were
females
comparisons.
and (2) sperm
counts
and
simple
regression
analysis.
in mm3
used
females
began to ovulate on 15 January 1991, designated
Day 0, and all ovulated over the following 65 days. The
were
transformed,
by
arcsin,
before
statistical
relationships
between
counted
was
effects.
Statistical difference
was indicated
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
t-test
of sperm
of squares
Student’s
determine
S x K
D = dilution (1000)
S=
Analysis
DXN
mm3milt
where
microscope
counted
ET AL.
both
tested
by
treatment
regression
MINITAB
data
and by
Possible
survival,
line.
(Minitab
All
Inc.,
stripping.
RESULTS
Fertilization
and
Progeny
An in vitro fertilization
of the experiment.
The
Suwival
each fish, were
held separately
The eggs obtained
from stressed
milt
from
stressed
fertilized
were
selected
males
with
randomly
Plasma
technique
was used for this stage
gametes,
collected
separately
from
for fertilization
females were
the eggs
and
milt from
control
and
male
each
from
control
males.
was
and rearing.
fertilized
with
The
used
females
males
only
to
ng cortisol/mI
to ovulate in both the stress and control groups.
It was important
not to use a large excess of sperm,
These
stress
doing
quality.
Previous
appropriately
once
so might
hundred
eggs.
fertilized
with
diluted
1:10
ent
on
trout
dilutions
were
hibit
motility.
Thus
1:1000
the
sperm
Ltd.).
motility
in overall
has shown
is sufficient
eggs
300
Each
in a nonactivating
from
milt
solution
The
was
that
to
each
sample
female
was
of 40 mM
inhibitory
checked
sperm
10 p.1 of milt,
fertilize
a few
were
first
pre-
KC1 (Sigma
effect
of this
before
any
dilusperm
carried
out; it was found
to completely
inThis was then
further
diluted
1:100
in 125
mM NaCI buffered
tivates
the sperm,
mediately
any reduction
10 p.1 of milt.
Company
Chemical
mask
work
[33]
diluted,
to pH
which
9 with Tris [33].
are then
motile
This
for
solution
30 sec.
acThe
dilution of milt, final volume
10 ml, was added
imto a batch of 300 eggs that were then gently stirred
before
being
left undisturbed
for 10 mm. The fertilized
eggs
were placed
in separate
compartments
within egg trays in
incubators
supplied
with a constant
flow of filtered
lake
water. Subsequent
survival
of the progeny
was monitored
through
egg development,
hatching,
and up to 28 days after
hatching.
significantly higher
in the unstressed
cortisol/ml;
compared
cause
half way through
the experiment,
1 h afstress, plasma cortisol levels in the stressed
and control fish were compared
(Fig. 2). The results showed
that plasma cortisol levels in the fish subjected to the acute
fertilize
a batch
of 300 eggs from a single
female.
Thirty
experimental
crosses were set up using the first15 females
be-
Levels
Approximately
ter an emersion
stress were
were
once,
Cortisol
that
compared
stressed
to control
results show
was effective
the
Weights
than plasma
cortisol levels
control fish (stressed males
fish
had
not
and
Lengths
to control
males
=
=
13.5
26.4
±
±
3.2
4.7 ng
5.9 ng cortisol/ml
1.5 ng cortisol/mI).
that the regimen
of repeated
emersion
in provoking
a stress response
and thus
females
females
acclimated
=
=
33.8
10.8
±
±
to this
form
of stress.
There were
no significant
differences
in either
somatic
weights
or lengths between
the stressed
and control
fish at
the end of the experiment
(Fig. 3). The condition
factors
((weight
(g) #{247}
(length3
(cm)))
x 100) of these fish were
also calculated
and found not to differ significantly
between
the two groups
(stressed
males
= 1.56 ± 0.03 compared
to control
males = 1.62 ± 0.04; stressed
females
= 1.46 ±
0.03 compared
to control
females
= 1.41 ± 0.03). The fish
exposed
to the regimen
of repeated
acute stress were observed to recover
rapidly from the stress and were fed normally throughout
the course
of the experiment.
Ovulation
and
Egg Parameters
The first reproductive
parameter
observed
to be affected
by stress was timing
of ovulation.
The females
began
to
ovulate
on 15 January
1991, designated
Day 0, and the majority ovulated
over the following
65 days. Those females
STRESS REDUCES
THE
QUALITY
1143
OF GAMETES
(a)
(a)
WEIGHT
FEMALES
2000
**
40’
#{149}STRESS
U
30’
o
1000
-
CONrROL
C,)
1.
**p=o.009
20
0
0
10
SEX
LENGTH
(b)
0
CONTROL
MALES
(b)
*
30
C,
z
20
*p=o.037
0
SEX
1
0
FIG. 3. Mean weight
(a) and
rainbow
trout at the end of the
(n = 15).
10
mean length
experiment.
(b) of stressed
and
Values
are means
control
SEM
±
0
STRESS
CONTROL
FIG. 2. Plasma cortisol concentrations
in female
(a) and male (b) rainbow trout 1 h after an emersion
stress performed
mid-way
through
the 9
mo of the experiment.
Values are mean ± SEM In = 10).
0
51
0
that did not ovulate
during
this period,
which was an equal
number
of fish in both the stressed
and control
groups,
were found to be immature
at the end of the experiment.
The mean number
of days to ovulation
were compared
between
the stressed
and control
females
and ovulation
was
found to be delayed
significantly
(p = 0.031) in the females
exposed
to the regimen
of repeated
acute stress (Fig. 4).
Eggs obtained
from females
subjected
to repeated
acute
stress were significantly
smaller
than eggs obtained
from
control
females
(Fig. 5). Ten eggs from each female
were
weighed
and measured
(this is a sufficiently
large sample
size because
eggs from an individual
female
are of a very
similar
size [26]). The weight
was significantly
lower (p <
0.05)
for
eggs
from
stressed
females
compared
to eggs
from
C,)
5.
5.
O
#{149}
STRESSED
GROUP
‘
CONTROL
GROUP
z
0
20
DAY
40
OP
60
80
OVULATION
FIG. 4. Effect
of repeated
acute stress on the timing
of ovulation
in
female
rainbow
trout. The day on which the first female
ovulated
(15 January 1991) was designated
Day 0. The last mature
fish ovulated
65 days
later (on 21 March 1991).
The difference
in numbers
between
the two groups
is due to the fact that 5 more stressed
than control
females
died during
the course of the experiment.
1144
CAMPBELL
(a)
0.8
EGG
ET AL.
compared
of the fish
WEIGHT
to 115.1 ± 3.4 mm3 for controls).
The fecundity
was unaffected
by the regimen
of repeated
acute
stress
(Fig.
5).
Sperm
Counts
C,)
0.7
The
z
51
sperm
varied
I-’
‘a.
*tJ
015
0
sperm
were
0.6
of the
counts
between
counts
males
used
3.4 and 24.0 X io
of the males subjected
significantly lower
(p
0.05)
<
in this
experiment
sperm/mi
to repeated
than
the
of milt. The
acute stress
sperm
of the control males (10.4 ± 0.7 X i09 sperm/mi
for stressed males compared
to 13.6 ± 1.2 X
ml for control
males;
Fig. 6).
51
counts
of milt
sperm/
io
0.5
STRESS
CONTROL
Progeny
Survival
There
(b)
EGG
125
tween
to the
VOLUME
was
developing
the chorion
E
no significant
embryo
(Fig.
crosses
became
rate. A significant
vival
progeny
rates
hatch
between
swim-up
CONTROL
the
±
5.6%
stage-a
from
compared
critical
67.0 ± 6.1% of the
still alive,
compared
(c)
the
NSD
3000’
eyed,
indicating
a
difference
in surthe
to 94.3
stressed
crosses
time
stressed
and
con-
±
1.41%
for prog-
survival
of the progeny
very
slightly
more
pro(Fig. 7a). Thus,
at the
when
progeny
to 86.6
the controls
(p < 0.01).
nod,
28 days post-hatch,
FECUNDITY
4000’
be-
was,
detected from by stressed
the time crosses
the eggsto
survival however,
of progeny
76.5
=
rates
crosses
up
eyes of the
are visible
to the naked
eye through
7a). Over
90% of eggs
from
both
the
eny from
controls).
This reduced
from
stressed
fish became
only
nounced
as development
continued
STRESS
in survival
and control
when
the
stressed
and the control
very good
fertilization
trol
hatchedcrosses (%
*
difference
the eggs from
the stressed
eyed
stage
of development,
fry start
the
to feed-
from
the stressed
fish
± 2.4%
of the progeny
At the end of the monitoring
63.4 ± 7.3% of the progeny
were
still
alive,
compared
from the control
crosses.
deaths
of the progeny
during
opment
more
clearly
illustrated
pefrom
to 84.8
2.5% of the progeny
The timing
of the
is probably
were
from
by
±
devel-
observing
IZ
2000’
NSD
20
1000’
15
STRESS
00
CONTROL
I-
Z
FIG. 5. Effect
(b), and fecundity
10
o
of repeated
acute stress on egg weight
(a), egg volume
Ic) of female
rainbow
trout. Values are means
± SEM
*p=:0.033
U
(n=15).
515
51
CI)
control
males
The
was
control
females
compared
mean
volume
also significantly
females
(0.638
± 0.02 g for eggs
from stressed
feto 0.712 ± 0.02 g for eggs from controls).
of eggs obtained
less (p < 0.05)
(101.9
±
3.6
mm3
from
than
for
0
stressed
females
that of eggs from
sperm
stressed
(n
females
FIG.
=
6.
density
15).
Effect
of
in milt
STRESS
repeated
acute
of
male
CONTROL
stress
during
rainbow
trout.
the
Values
preceding
are
means
9 mo on
±
SEM
STRESS
REDUCES
THE
(a)
QUALITY
OF GAMETES
progeny
stressed
100
1145
from stressed
control
fish.
Following
fish
exposure
compared
to the
to progeny
repeated
acute
from
stress
un-
regime
for a period
of 20 wk, blood
samples
were
taken
from
a
subsample
of fish 1 h after an emersion
stress.
The significantly
higher
cortisol
levels
in the stressed
fish compared
80
51
60
00
#{149}STRESS
51
CONTROL
40
I-
z51
#{149}p<
o.oi
U
51
20
51
0.
counted
sensitive
that the
0
E’YHI
HATCH
SWIM-UP
28 DAYS
the
POSThATCH
DEVELOPMENTAL
for by the fact that brown
trout are a species
more
to stress
than rainbow
trout,
but may also indicate
fish in this experiment
had begun
to acclimate
to
emersion
maturing
STAGE
(b)
25
51
*
20
stress
trout
also
to stress
compared
this
became
case
proceeded,
attenuated
C,
to some
show
extent.
However,
a substantially
to immature
gradually
fish
more
mature
reduced
[35].
mature
this effect could also have
stress
response
observed.
Since
as the
and
response
the
fish
in
experiment
been
involved
The important
in the
point,
though, is that the fish subjected
to emersion
stress
had
significantly
higher
plasma
cortisol levels than the Unstressed control fish. After 20 wk of exposure
to repeated,
U
15
‘a
to the controls
indicated
that acclimation
had not occurred
at this stage. Brown
trout subjected
to acute
handling
stress
show
a much
more
pronounced
peak of cortisol
(over
100
ng/ml)
1-2 h post-stress
[34]. This difference
may be ac-
intermittent emersion
stress some
degree of acclimation
would be expected
to have taken place [36], although this
would have been prevented to some degree by the random
_____
timing of the stress; this slow but steady acclimation could
have continued until spawning, possibly resulting in the acute
stresses becoming
EYED
HATCH
28 DAYS
SWIM-UP
STAGE
the
FIG. 7. Survival
(a) and mortality
(b) rates of eggs and embryos
obtained from stressed
and control
rainbow
trout. The rate of survival
land
hence mortality)
was assessed
at four developmental
stages: the eyed-egg
stage, hatch, swim-up,
and 28 days post-hatch.
the
degree
of mortality
ment
we
mortality
assessed
observed
occurred
difference
was not
primarily
in the
significant
hatch
there
was
between
the
sequently,
mortality
there
of the
was
eny
of the
stages
around
mortality
(p =
the time of hatch.
There
was a
rate at the eyed
stage,
but this
0.069).
However,
at the time of
a significant
difference
of the
was no
progeny
stressed
significant
from the
correlation
8).
in the
and
between
mortality
control
difference
two groups
egg
fish.
Sub-
in the rate of
of fish. There
size
and
sensitive
phases
concentrations
crease
in both
approaches.
in the
of
male
Hence,
reproductive
and
female
rainbow
the
number
y-O.446x
+
25.637,
tOO
cycle,
im-
trout
OA)81,
in-
as spawning
of stresses
R.equareth
when
hormones
0
to which
the
pO.13
o8
80
..
60
.
U
40
IU
I
StTOS5ed
20
0
Controls
prog70
80
100
90
EGG
This study shows that exposure
of rainbow
trout
to incidences of acute stress during reproductive
development
results in delayed ovulation and reduced
egg size in fecounts
in males,
lower
survival
reproductive
the
rate
DISCUSSION
males,
significantly
lower
sperm
haps most importantly,
significantly
most
blood
of develop-
(Fig. 7b). This shows
that the increased
in the progeny
from
the stressed
fish
progeny
no significant
survival
(Fig.
at each
less effective. It was
portant
to maintain
the efficacy
of the stresses
during
the
final half of the experiment
since this is probably one of
POST-HATCH
DEVELOPMENTAL
progressively
and, perrates for
VOLUME
110
120
(mm
130
140
3)
FIG. 8. Simple
regression
analysis
of egg size (volume)
and progeny
survival
revealed
no significant
correlation
between
these two parameters.
When the data from the two treatment
groups
is analyzed
separately
or
combined
to generate
the regression
line (as above),
no significant
correlation between
egg size and egg and fry survival
is noted. Regression
equation for stressed
group: y = 0.487x
+ 13.989,
r = 0.237.
p = 0.395;
Regression
equation
for controls:
y = -0.226x
+ 110.696.
r = 0.316, p = 0.251.
1146
fish
were
subjected
increased
during
the
between
Weeks
to December),
September
9 mo
prior
CAMPBELL
El
was
The
to spawning
22 and 32 of the experiment
at which time plasma
(early
levels of
AL.
reproductive
have
been
disruptions
attributed
secretion
[53],
to low
resulting
pH
exposed
suppressing
in delayed
stress
gonadotropin
ovulation,
ology
reducing
drates
and
vitellogenin
synthesize
vitellogenin
by the liver is stimulated
[54]. The synthesis
of
by increasing
levels
of estradiol
released
However,
stress was then
reduced
January when
spawning
ber,
the
females
ovulation.
reduced
fish
were
frequency
and eventually
was imminent.
checked
every
handling
due
stress
both groups of fish were
handling stress during the
days
emersion
stress.
fish
different
the former
the latter
The
that
ovulated
from
that
of the
signs
stress
had
deterioration
of emersion
of
to be
in
collection
subjected to equal
stripping
procedure,
amounts
of
the effects
viability
late
for
the
[39]. Since
of stress on reproduction
observed
to have
been
caused
by the earlier
trol
few
the additive
effects
associated
with gamete
acute
stopped
in early
From late Decem-
The frequency
of the emersion
at this time to prevent
a possible
health
of the
in this study were likely
repeated
exposure
to
of the
in the
first
fish
and
progeny
from
spawning
the
con-
season
was
thus,
although
to ovulate;
no
fish were subjected
to more
handling
stress than
fish immediately
prior to ovulation, this had no
obvious effect on the quality of the eggs produced.
Once
ovulation had commenced,
the female fish were checked
every 1-2 days and the eggs were
removed
the body
from
cavity by manual stripping when individuals were found to
have ovulated. This was important since ovulated eggs of
oviparous
teleosts become
cavity
these
and
eggs
overripe
show
reduction
ability [40,41].
This process
of post-ovulatory
understood,
but it is known
that it has
on the ability
of the eggs to be fertilized
further
through
the stages of eying, hatch,
46]. The
tilization,
eggs from
control
females
eying,
and hatching
rates
compare
very
obtained
The
have
favorably
by Springate
effects
of stress
been
lethally
investigated
low
pH
(acid
with
in studies
stress)
stress
have
than control
fish
duced
fecundity
commersoni)
of bleach
been
maximum
survival
rates
fish
exposed
to sub-
A few
of these
pollutants.
to ovulate
held at neutral
pH
[48,49].
Fish (white
exposed
to environmental
kraft mill effluent (BKME)
significantly
and also
sucker,
in
deto
later
to show
re-
Catostomus
stress
in the form
also show increased
age to maturity, lower fecundity, and reduced
egg size [50].
Stress has also been shown
to delay or completely
inhibit
ovulation
Rainbow
levels
stituent
deposition
in mammals
[3,4] including
humans
trout exposed
to acid stress
have
of vitellogenin,
the
[51], and sublethal
by suppressing
precursor
exposure
uptake
[1, 2].
lower
plasma
of the major
yolk
to cyanide
inhibits
at the
ovarian
level
conyolk
[52].
from
ability
the
in brook
and
rainbow
had
reduced
rainbow
physi-
carbohy-
the
reduced
constituto lower
Tam et al. [48] and Weiner
change
in estradiol
levels
respectively,
by Weiner
survival,
metabolize
of the major
yolk
simply
be attributed
survival
of progeny
been
demonstrated
[49, 55, 56]. A study
eny of acid-exposed
fected
duction
trout,
to
ovary.
levels of estradiol,
since
reported
no significant
posure.
Reduced
acid stress
has
following
from fish
in a number
acid
et al. [55] showed
that
trout females
and control
indicating
oogenesis
ex-
exposed
to
of studies
progmales
is adversely
by acid stress. A similar, but less pronounced,
in the survival
of progeny
from acid-exposed
afremales
and control
females
was also noted,
suggesting
that oogenesis is more sensitive to exposure
to acid stress than spermatogenesis. Fathead minnows
and brook trout exposed to
low pH exhibited ovarian histological changes, particularly
an increase in oocyte
atresia and depression
of reproductive success,
which
were
directly
of environmental
stress
experienced
Although
lished
these
that
stress
studies
adversely
associated
with
the
level
[53, 57].
suggest
affects
that it is already
estabgamete
quality in fish, in
fact interpretation
of the results
of the studies
employing
acid stress is complicated
by the fact that the fish chronically exposed
to sublethally
low pH had reduced
calorific
intake and hence
did not grow
as well as the control
fish
[53]. In addition,
it might
be speculated
that the energetic
costs
of maintaining
homeostasis
under
acid
conditions
would
divert
resources
from
gonadal
growth.
Since
the
body
the consequences
of exposure
quality,
and larval growth
and
female
brook
trout
exposed
shown
circulating
et al. [55]
number
other
authors.
and spermatogenesis
using
or
aging is poorly
a profound
effect
and to develop
and swim-up
[42-
in this study had ferof over
90%,
which
et al. [47] and
on oogenesis
studies
have examined
terms
of fertility,
gamete
velopment.
For example,
acid
the
in vi-
liver’s
plasma
levels
of this precursor
ent in acid-stressed
fish cannot
if retained in the body
a progressive
the
liver
ad-
creasing
The
and
on
affects
[38].
metabolism
and
verse
males
carbohydrate
to acid
sex steroids and vitellogenin are rapidly increasing in the
maturing females [37] and plasma
androgen
levels
are inin maturing
on
in fish
fish
of eggs
weight,
had
produced
by a female
it is not surprising
reduced
egg size, which
eterious
effects
fecundity.
is proportional
that the smaller,
A similar
to
acid-stressed
argument
applies
to
is related to body size [40]. Thus the delof acid stress
on oogenesis
and spermato-
genesis
observed
in many
of these
studies
cannot
be distinguished
from the effects of poor nutrition.
It is well known
that reduced
food
intake
in mammals
and humans
is associated
with hypothalamic
dysfunction and reversible sterility. Similarly,
in fish decreased
food availability
has been
reported
to suppress
gametogenesis
[58] and reduce
fecundity
[15]. This problem
of distinguishing
between
the
direct consequences
of stress on reproduction
and the indirect
consequences
due to nutritional
factors
is discussed
comprehensively
by Tyler
et al. [26] in relation
to oocyte
atresia
(and hence
the study
reported
reduced
here
fecundity)
there
were
ences
in either
somatic
weight
exposed
to repeated
acute stress
in rainbow
no signfficant
or length
and the
between
unstressed
trout.
In
differthe fish
control
REDUCES
STRESS
fish. The regimen
chosen
not only
of random,
repeated,
because
in life stress
multiple
events
in a series,
but
because
exposure
also
effect
on overall
[34] using
brown
separated
acute stresses was
usually
occurs
as
by varying
of fish
to acute
food intake.
For
trout,
a species
THE QUALITY
OF GAMETES
in an
attempt
little
hard
intervals,
with
fertility.
has
spermatozoon
lated with
stress
little
example,
Pickering
et al.
more
sensitive
to stress
do
not
to
1147
assess
evidence
sperm
directly
reside
in the
length
of time
same
portion
sible
eggs
Rainbow
resumed
bow
tween
trout,
showed
fertilization
rapidly
early
in the
follow-
ever, this correlation
was lost;
between
initial sperm
density
ilarly,
in this
and
ing
study
resumed
the
cantly
rainbow
normal
an emersion
In this study,
smaller
to acute
behavior
trout
feeding
stress.
stressed
eggs
stress
the
recovered
behavior
female
than
by Wedemeyer
following
day.
very
immediately
trout
unstressed
produced
control
[59]
Sim-
sperm:
egg
ratio,
Moccia
[68]. In the present
density
was evident,
indicate
overall
that egg
egg quality
tion
portance
of egg
size
has
of conflicting
results
problems
in separating
survival
rates
strain,
studies,
from
mortality
difficult
from
various
the effects
the
and nutritional
smaller
eggs
creased
been
effects
as far as
The im-
to ascertain
studies
of egg
of other
and
size
because
of
on egg/fry
factors
such
as age,
status
of the parent
fish. In
have
been
shown
to experience
[60,61].
However,
Springate
and
some
inBrom-
age [62], using
rainbow
trout,
found
that egg size had no
direct
effect
on egg quality
or fry survival,
a finding
supported
by this study.
Similarly,
in studies
using brook
trout
and orangethroat
darters,
egg volume
was positively
correlated
with juvenile
size at hatching
and size at yolk sac
resorption,
but had no significant
effect on embryonic
survival,
developmental
hatchlings
time,
[63, 64]. The
or
present
subsequent
growth
uncertainty
surrounding
rate
of
the
characteristics
implication
as far as egg
trout
exposed
did not show
rainbow
trout,
quality
egg
size
has
is concerned.
no direct
The
female
to repeated
acute
stress
in this experiment
any change
in fecundity
(Fig. 5). In female
the reproductive
cycle starts
approximately
12 mo prior
to spawning,
and the number
of eggs recruited
for development
that year is decided
at a very early stage.
The fish in the present
study
were
not exposed
to stress
until 9 mo prior
to spawning,
probably
too late in the cycle
to affect fecundity.
At present,
there
are
timating
sperm
quality.
no truly dependable
In mammals,
humans,
length
of time and intensity
of spermatozoon
70], the percentage
of motile
spermatozoa
sity [68, 72], and the chemical
composition
plasma
[73-75]
are
all parameters
that
have
criteria
and
for esfish, the
motility
[66[71], sperm
denof the seminal
been
measured
in the
the
no
seasonal
our study
of
corremotility
it is pos-
using
rain-
season,
how-
correlation
rate was ev-
for fertilization
motility
per
change
covered
has
sperm,
which
resulted
tests of sperm
quality
probably
means
of assessing
likely fertility
By reproducing
in the laboratory
up to fertilization,
some
aspects
dif-
in the
techniques
of movement
assess
but also the quality
widely
recognized
se
in sperm
only a por-
males
were
ripe.
of sperm
function
for many
techniques
of human
not
only
to
the
of that movethat functional
provide
a more
valuable
and quality
of sperm
[79].
the natural
events
leading
of human
and mammalian
sperm
quality
can be evaluated,
for example,
by monitoring
progress
through
cervical
mucus
or gels and assessing
the
ability
of the sperm
to penetrate
the membrane
of hamster
eggs [78]. Despite
the
sessing
sperm
quality,
are
that
study,
although
number
of motile
sperm
ment
[66,78].
It is now
the
indicates
Later
instead,
a positive
and fertilization
development
of increasingly
sophisticated
evaluate
sperm
quality,
including
the analysis
to successfully
remains
the
Not enough
of evidence
[68],
the critical
factor
sperm,
not the
assisted-reproductive
effects
of egg size on progeny
survival
has occurred
because
of differences
in age and size of parental
fish, varying
culture
conditions,
and,
most
importantly,
uncontrolled
variation
in the ripeness
of eggs [41]. At the present
time
balance
intensity
sperm,
Munkittrick
season.
of the period
when
The need for assessment
ferent
because
and
reproductive
there
was no significant
correlation
between
egg size and
egg and fry survival
rates (p = 0.13; Fig. 8). These
results
size has no direct
implication
and fry survival
are concerned.
and
of the
is
a significant
positive
correlation
berate and subjective
motility
assessments
They concluded
that
the number
of motile
was
However,
there
parameters
to obtain
highly
motile
sperm
incapable
of fertilizing
[77]. Using low sperm
concentrations,
near the critical
ident.
signifi-
fish.
of these
not invariably
positively
[76]. Since
fertility
and
than
rainbow
trout
[17], showed
a temporary
cessation
of
feeding
following
an acute
handling
stress;
however,
this
was not sufficient
to impair
growth
during
the experiment.
trout subjected
normal
feeding
However,
any
In salmonids,
motility
are
fertilizing
ability
quality.
linking
dividual
fertilize
eggs and produce
only
truly dependable
test
is known
about
the specific
attributes
needed
ponents
and fish
to
be placed
and
to be
the
function,
most
on various
motility
useful
In humans
of sperm
identify
viable
of sperm
importance
and
valuable
remain
the
estimates
of sperm
[66] and
a useful
fish
assessment
[68],
offspring
quality.
of in-
further
studies
diagnostic
of semen
quality.
Evidence
from
studies
is contradictory
regarding
that should
density
sonably
value
of these
functional
tests in asis still true that the ability
of sperm
it
com-
human,
mammal,
the importance
parameters,
although
sperm
most widely
used and reaquality
at the present
time.
sperm
of sperm
cumstances;
for example,
it is generally
density
in the human
ejaculate
falls
density
has
quality
been
shown
in certain
cir-
accepted
that if sperm
below
10-20
million!
ml then men tend to be infertile
[80]. In this study,
sperm
counts-and
hence
sperm
densities-were
significantly
reduced
in male fish exposed
to the regimen
of repeated
acute
stress
compared
to unstressed
control
fish. The differences
in sperm
density
between
the stressed
and control
groups
had
no
effect
on
the
fertilization
rate,
possibly
indicating
1148
no
CAMPBELL
effect
of stress
relatively
high
been
a sufficient
quality.
Ideally,
this would
too many
the
sperm
crosses
does
quality
Little
work
sperm,
(each
been
although
stripping
despite
there
may
very large
The lower
indicate
only
carried
it has
been
involving
the
still
have
to mask any reduction
to titre out the sperm,
numbers
progeny
a stress-induced
after
out
the
reduc-
effects
of stress
that frequent
stress)
results
exposure
to repeated
acute
stress
reduces
on
stripping
in a de-
of the gametes
produced.
These
results
only from a fundamental
point
of view,
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