Journal of Fish Biology (1997) 50, 1088–1093

Sperm motility and fertilization time span in Atlantic

salmon and brown trout—the effect of water temperature
T. V́*  T. J̈*†
*Department of Zoology, University of Stockholm, S-106 95 Stockholm, Sweden and
†Institute of Freshwater Research, S-179 93 Drottningholm, Sweden

(Received 19 October 1996, Accepted 6 December 1996 )

The effect of water temperature on the duration of sperm motility, the time lapse after
activation by fresh water and the fertility of eggs was studied in Atlantic salmon and brown
trout. Eggs of both species were fully fertile in fresh water after 512 s. No interspecific
differences were noted in egg fertility at the lower water temperatures, but the brown trout eggs
showed a higher resistance to high temperatures, indicating a better physiological thermotoler-
ance. A highly significant effect of temperature on the overall duration of sperm motility was
found, with a marked peak at 3–4) C for salmon and a weaker one for trout. After freshwater
activation the eggs of both species remained fertile for a longer time than the sperm were
mobile. ? 1997 The Fisheries Society of the British Isles

Key words: fertilization time span; sperm motility; temperature ; Salmo salar; Salmo trutta.

INTRODUCTION

In order to maximize reproductive success, gametes of both sexes must be

adapted to their physical environment. For stream-spawning salmonids, the
factors influencing the time span for successful fertilization are: limited ATP
storage in the sperm (Billard & Cosson, 1992); hypoosmotic effects on the egg
(Szöllösi & Billard, 1974) and sperm membranes (Stoss, 1983); water tempera-
ture (Wootton, 1990); and a rapid dispersal of sperm from the ejaculate by the
water flow (Ginzburg, 1972; Billard, 1986).
Atlantic salmon Salmo salar L., and brown trout S. trutta L., are cold water
stenotherms. The egg is the critical stage with respect to thermal stress and it is
assumed that salmonid eggs can tolerate only a temperature range of 0–16) C
(Peterson et al., 1977; Elliott, 1981). Furthermore, Lindroth (1947) suggested
that the duration of swimming time for salmonid spermatozoa is related
exponentially to the water temperature. Generally, sperm motility is considered
to be a good parameter that reflects sperm viability and fertilization ability
(Ginzburg, 1972; Aas et al., 1991).
Atlantic salmon and brown trout are phylogenetically closely related species,
with a similar reproductive ecology. Hence, it is reasonable to assume that
physiological properties of the gametes of both species should be similar, and
that they should possess a similar fertilization time span and duration of sperm
motility in response to water temperature.
The aim of this study was to investigate how the fertilization time span and the
duration of sperm motility of these two species was related to temperature.
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0022–1112/97/051088+06 $25.00/0/jb960373 ? 1997 The Fisheries Society of the British Isles
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MATERIALS AND METHODS

The fish used originated from the River Dalälven, where salmon and trout live
sympatrically (for more details about the River Dalälven stocks see Petersson et al.,
1996). During October and November 1995, two sets of experiments were carried out at
the Laboratory of Stream Water Ecology at Älvkarleby. The first set of experiments
studied the effect of water temperature on the fertilization time span of anadromous
Atlantic salmon and brown trout females. The second set of experiments was a study of
the effect of water temperature on the duration of sperm motility of mature parr of
salmon and trout.

FERTILIZATION TIME SPAN

The eggs were collected from five individually tagged anadromous salmon and five
female brown trout. Each batch was placed in a separate basket (90 ml), containing
15 ml physiological saline solution (0·9% NaCl) at ambient temperature (2–4) C) for no
longer than 8 h until artificial fertilization had occurred. Prior to the experiment the
saline solution was rinsed off, and the experiment started by putting the eggs in the water
at the temperature to be tested.
Anadromous male salmon (n=15) and trout (n=15), chosen at random, were stripped
for milt under tricaine methane sulphonate, MS-222 (Thomson and Joseph Ltd, U.K.)
anaesthetic. During stripping, care was taken to avoid contamination of the milt with
water, blood, faeces and urine. A mixture of pooled milt from three randomly chosen
individuals was stored on ice at 4) C for no longer than 8 h before use. Five different milt
mixtures were used for the fertilization of batches of eggs from five females of each
species.
The eggs from each individual female were divided into 25 batches, with 20 eggs in each
batch, and then activated by water at 2, 4, 8, 16 or 32) C, for a duration of 2, 8, 32, 128
or 512 s. Then 0·3 ml of the pooled milt samples were added. Different combinations of
individual males were used for each individual female. The amount of sperm used did not
differ significantly between the two species (spermatocrit: x̄(trout)=32·4&13·2 (..);
x̄(salmon)=24·6&5·4; t=1·2, d.f.=8, P>0·05). Ten minutes after the insemination, the
eggs were transferred into twin-row incubation holders containing river water at its
ambient temperature. During development, the eggs were treated weekly with malachite
green to avoid fungal infection. The numbers of eggs fertilized were counted at the late
gastrula stage in March 1996. Unfertilized eggs were recognizable easily because they
were opaque and almost white in colour.

DURATION OF SPERM MOTILITY

Milt was collected as above from six salmon parr and six brown trout parr and stored
on ice for no longer than 4 h in clean plastic beakers until the observations were made.
The effect of temperature on sperm motility was assessed using a Bürker chamber
placed on a Nikon dark-field contrast light microscope. Water temperature was held
constant by a Peltier element connected to the microscope. The intention was to use the
same geometrical temperature scale as for the fertilization time span, but due to technical
limitations of the Peltier element the highest temperature (32) C) could not be reached
accurately. Hence, the temperature of 28) C was used instead. A droplet of water was
placed in the chamber. When the temperature was 2, 4, 8, 16 or 28) C, a small droplet of
semen was placed in the chamber. Sperm motility was recorded by a video camera, at a
magnification of 100#. Both visual and video inspection showed that all sperm began to
move simultaneously in the water droplet. Duration of sperm motility was assessed using
a stopwatch that was started simultaneously with suspension of the sperm into the water.
Only forward movements by the spermatozoa were assessed as motility, whereas simply
vibrating sperm were assessed as immobile.

DATA ANALYSIS
Results were evaluated by ANOVA. Comparisons were made using the post hoc
Scheffé test. The level of significance was set at P=0·05.
1090 . ́  . ̈

100

80
% Fertilized eggs

60

40

20

0
2 8 32 128 512 2 8 32 128 512 2 8 32 128 512 2 8 32 128 512 2 8 32 128 512
Time (s)
F. 1. A three-way interaction plot of the mean percentages of fertilized eggs for brown trout (-) and
Atlantic salmon (.). The factors are species, temperature and time. Graphs represent, from left
to right, 2, 4, 8, 16 and 32) C.

RESULTS
FERTILIZATION TIME SPAN
Although fertilization was not affected significantly by time, there was a
significant overall difference between the salmon and the trout (F=27·6,
d.f.=1 : 200, P<0·0001), and between the water temperatures (F=177·3,
d.f.=4 : 200, P<0·0001) in regard to the frequency of fertilized eggs. At 32) C the
frequency of fertilized eggs of both species declined, but the decrease was
significantly greater for salmon (F=30·5, d.f.=4 : 200, P<0·0001; Fig. 1).

DURATION OF SPERM MOTILITY

There was a significant interspecific difference (F=38·2, d.f.=1 : 50,
P<0·0001), as well as significant differences with water temperature (F=16·5,
d.f.=4 : 50, P=0·0001; Fig. 2). Furthermore, there was a significant interaction
between these two factors (F=7·3, d.f.=4 : 50, P=0·0001). A post hoc Scheffe’s
test revealed that the interaction was due to significant differences in the sperm
motility duration of the salmon parr compared to the trout parr at the
temperatures of 2 (P<0·05) and 4) C (P<0·0001). In addition, the duration of
salmon sperm motility at temperatures of 2 (P<0·05) and 4) C (P<0·01) was
significantly longer than at 16) C, and the duration of sperm motility at
temperatures of 2 (P<0·05), 4 (P<0·0001) and 8) C (P<0·05) was significantly
longer than at 28) C.

DISCUSSION
There was no significant difference between the fertilization time spans of
Atlantic salmon and brown trout. There was, however, a difference between
the two species in regard to the percentage of eggs fertilized at 32) C and to the
duration of sperm motility in respect to water temperature, showing that the
gametes of Atlantic salmon and brown trout had different physiological response
to equivalent water temperatures.
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350

300

250

Motility (s)
200

150

100

50

0
2 4 8 16 28
Temperature (° C)
F. 2. A fourth-order polynomial function plot of the mean durations of sperm motility with
temperature for Atlantic salmon (r=0·83) and brown trout (r=0·63). -, Brown trout; ., Atlantic
salmon.

The trout had a significantly better fertilization rate than the salmon at 32) C.
Such a discrepancy may be caused by the trout sperm decreasing less in viability
or the trout eggs tolerating heat better, or both. A heat shock after fertilization
has been used for the induction of triploidy and the production of sterile
offspring (e.g. Benfey & Sutterlin, 1984; Crozier & Moffett, 1989; Teskeredžić
et al., 1993). Since both salmon and trout spawn at water temperatures between
0 and 10) C (Peterson et al., 1977), it seems unlikely that the physiological
properties of the gametes making them viable at such high temperatures would
evolve as an adaptation to spawning under natural conditions.
The results of this study show that the duration of sperm motility is
temperature dependent with an increase in duration of motility at low water
temperatures. This is in accordance with the previous studies on brown trout
(Lindroth, 1947; Billard & Cosson, 1992) and Atlantic salmon (Lindroth, 1947).
In addition, our observations indicate that the overall motility time of salmon
spermatozoa is significantly longer at low temperatures than those of the brown
trout. Lindroth (1947) reported similar sperm motility patterns for these two
species, with that of the salmon being longer than that of the trout. However, in
our study, the interspecific difference disappeared at temperatures at and above
8) C reaching eventually previously reported short motility times (Ginzburg,
1972; Stoss, 1983; Billard, 1986). An interesting result in our study was the
marked peak in the duration of motility of salmon sperm at 3–4) C and a
less-marked one for the trout (Fig. 2). Since the natural spawning temperature
in the River Dalälven corresponds with this value, adaptation of the thermal
optimum of the sperm allozymes to the local environment is indicated (see
Reynolds & Casterlin, 1980). The interspecific differences in the duration of
sperm motility may be accounted for by a slight difference in some of the
characteristic life-history traits of the two species. In the River Dalälven, the two
species live sympatrically, and the salmon appears to be more semelparous than
the brown trout (pers. obs.). Hence, the male salmon may invest more in sperm
quality (reflected as sperm motility) than the trout.
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The time span available for fertilization was longer than the duration of sperm
motility, which implies that the egg is capable of being fertilized for a longer time
than the sperm is mobile. A proximate mechanism underlying this intersexual
difference may be due to the lower energetic costs incurred by an ATP-generated
Na + /K + efflux in the activated immobile egg (Girard & Gatti-Favereau, 1993),
compared to a K + /Ca2+ efflux-influx (Morisawa, 1985; Cosson et al., 1989) and
the rapid depletion of ATP reserves that occur during the mobile phase of the
sperm (Billard & Cosson, 1992).
To summarize, no difference in the fertilization time spans of the two salmonid
species exists, even when water temperature is taken into account. However, the
freshwater-activated egg of the brown trout tolerates a heat shock better than the
egg of the salmon. The nature of the observed discrepancy in thermotolerance is
probably non-adaptive. The duration of motility of salmon sperm is longer at 2
and 4) C than that of the trout. The salmon is regarded as being more
semelparous, wherefore it is reasonable to assume that the male salmon invests
more in energetic terms in each gamete, thus enabling it to be mobile for a longer
time.

We thank the staff of the Fisheries Research Station at Älvkarleby for their help during
the experimental work; B. Afzelius, G. Svärdson and two anonymous referees for critical
reading of previous drafts of the paper; and P. Tallantire for improving the English of an
earlier version. This work was supported financially by The National Board of Fisheries
(TJ) and The Hierta Retzius Foundation by The Royal Swedish Academy of Sciences
(TV).

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