Aquaculture 217 (2003) 647 – 662

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Feed intake, growth and ionoregulation in Atlantic

salmon (Salmo salar L.) smolts in relation to dietary
addition of a feeding stimulant and time of
seawater transfer
Hilde Toften *, Arne M. Arnesen, Malcolm Jobling
Norwegian Institute of Fisheries and Aquaculture Research, N-9291 Tromsø, Norway

Received 2 May 2002; received in revised form 18 July 2002; accepted 7 August 2002

Abstract

Feed intake, growth and ionoregulation of Atlantic salmon (Salmo salar L.) were studied in fish
fed diets either with or without squid extract supplementation in combination with transfer from fresh
water to seawater (SW) at different times during parr – smolt transformation (10 April, 3 May and 19
June). Water temperature was held at approximately 8 jC. All groups of fish showed a temporary
depression in feeding and growth after SW transfer. The depression was most severe in fish
transferred in April, intermediate in fish transferred in May and shortest in fish transferred in June.
Nevertheless, squid extract resulted in increased feeding and growth, especially in fish transferred to
SW in June, providing evidence that squid extract contains substances that serve as feeding
stimulants. The results indicate that there may be a potential to improve SW performance of salmon
by adding squid extract to feed. The results also indicate that Atlantic salmon may be transferred to
SW over a relatively long period during spring, but to minimise the duration of appetite depression,
late transfer may be preferable. Individual feeding rates following short-time SW exposure were not
correlated with plasma chloride concentrations in any of the groups, indicating that appetite
suppression is not a direct result of osmo-ionoregulatory failure.
D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Salmo salar; Feeding stimulant; Seawater transfer; Feeding; Growth; Ionoregulation

*
Corresponding author. Present address: Fiskeriforskning, N-9291 Tromsø, Norway. Tel.: +47-77-62-90-00;
fax: +47-77-62-91-00.
E-mail address: hilde.toften@fiskforsk.norut.no (H. Toften).

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 4 0 4 - 0
648 H. Toften et al. / Aquaculture 217 (2003) 647–662

1. Introduction

A critical phase in the life cycle of Atlantic salmon (Salmo salar L.), and many other
salmonids, is the period following the transfer to seawater (SW), a period often associated
with depressions in appetite and growth (MacLeod, 1977; McKay and Gjerde, 1985;
Usher et al., 1991; Arnesen et al., 1993a,b; Jørgensen and Jobling, 1994; Stradmeyer,
1994; McCarthy et al., 1996). The causal factors involved in the appetite depression are
not known, but it is possible that feed manipulations could influence appetite and growth
in this phase and enhance the performance of newly released smolts.
The application of small quantities of feeding stimulants can improve the palatability of
formulated feeds resulting in improved feeding and growth rates (Mackie and Mitchell,
1985; Hara, 1992; Toften et al., 1995; Toften and Jobling, 1997). Thus, it might be possible
to add feeding stimulants to feed to enhance appetite return in salmon smolts in the period
immediately following transfer to SW. Feeding stimulants are often found in extracts
prepared from the tissues of common prey organisms (Carr and Darby, 1986; Carr et al.,
1996), and squid extracts may have such properties (Mackie and Mitchell, 1985; Fukuda
et al., 1989; Johnstone and Mackie, 1990; Toften et al., 1995; Toften and Jobling, 1997).
In Atlantic salmon, the appetite suppression following SW transfer is transient and may
vary in duration. Some authors have found that appetite suppression may last for 5– 7
weeks (Usher et al., 1991; Stradmeyer, 1994; McCarthy et al., 1996), whereas others have
found an earlier return of appetite (Jørgensen and Jobling, 1994). Usher et al. (1991)
suggested that variations in the timing of the return of appetite may be related to
differences in SW adaptability, such that Atlantic salmon smolts transferred to SW at
various times during the spring period of parr –smolt transformation may show differences
in feeding and growth performance.
To test whether the dietary addition of a putative feeding stimulant, squid extract,
would result in faster appetite recovery we examined feed intake, growth and ionoregu-
lation in groups of Atlantic salmon transferred to SW at different times during the
smoltification process (10 April, 3 May and 19 June) and then fed diets either with or
without squid extract.

2. Materials and methods

2.1. Experimental animals

The experiment was conducted at Kårvika Aquaculture Research Station, Tromsø,

using Atlantic salmon (Salmo salar L.) of the AquaGen strain (AquaGen, Sunndalsøra,
Norway). The fish hatched in January and were first fed in mid-March 1994. From March
to September 1994 the fish were reared under continuous light (LD 24:0) in fresh water
(FW) at 10 –12 jC. From October to December 1994 the photoperiod was gradually
reduced to LD 6:18, and from December to March 1995 the fish were subjected to
simulated winter conditions (LD 6:18, 4– 6 jC). On 20 –21 March 1995, 840 fish were
marked individually (Fingerling tag, Floy Tag, Seattle, USA) and divided randomly
amongst 12 tanks (70 fish per tank). An additional 200 untagged fish were transferred to
 H. Toften et al. / Aquaculture 217 (2003) 647–662 649

similar tanks, these fish being used for the assessment of smolt status in SW challenge
tests. The fish were held in circular, centrally drained, fibreglass tanks (300 l) supplied
with FW at a rate of 9 l min
1, and a circumferential water current of about 0.11 m s
1
was created by directing the water inflow through vertical, perforated inlet pipes, as
described by Christiansen and Jobling (1990). Tanks were illuminated using 36-W
daylight fluorescent tubes (Osram, Germany) providing 450– 800 lx approx. 10 cm above
the water surface. To synchronise parr – smolt transformation, the photoperiod was
switched from short day (LD 6:18) to continuous light (LD 24:0) and the temperature
was raised to 8 jC on 28 March 1995. No changes in photoperiod and water temperature
were made thereafter. The square-wave photoperiod regime used is known to induce parr –
smolt transformation (Duston and Saunders, 1995; Sigholt et al., 1995; Björnsson et al.,
2000; Handeland and Stefansson, 2001).

2.2. SW challenge tests

The SW challenge test, a commonly used indicator of seawater adaptation, examines

the ability of the fish to regulate the ionic composition of body fluids following SW
transfer (Clarke and Blackburn, 1977; Clarke et al., 1996; Clarke, 2000). The SW
challenge tests (n = 10) were conducted regularly from 28 March to 16 June 1995. Fifteen
fish were transferred directly from FW to a circular tank (water volume 300 l) supplied
with running SW (9 l min
1, 34 g l
1, 8 jC). After 24 h of exposure to SW, fish were
netted and killed by a blow to the head, and blood samples were immediately collected
from the caudal vessels using a syringe-vacuum tube system (Venoject tubes with Li-
Heparin; Terumo, Leuven, Belgium). Blood was stored on ice (max 15 min) and then
centrifuged (8 min, 2780  g). Thereafter, plasma was removed and stored at
80 jC
until analysed for chloride concentrations (Corning 925, Ciba-Corning Diagnostics, Essex,
UK). No mortality occurred during SW exposure.

2.3. Experimental protocol

Groups of fish were transferred to SW (34 g l
1) at three times during the spring: 10
April, 3 May and 19 June. The timing of the transfers was decided based on the results of
the SW challenge tests (Fig. 1). The April group was transferred to SW when fish in the
SW challenge test had relatively high plasma chloride concentrations (significantly higher
( p < 0.01, Tukey – Kramer post hoc test) than those registered later) (Fig. 1). The second
group was transferred in early May when fish in the SW challenge tests showed the lowest
plasma chloride concentrations (Fig. 1). The last group (June group) was not transferred to
SW until SW challenge tests revealed elevated plasma chloride concentrations (signifi-
cantly higher ( p < 0.01, Tukey – Kramer post hoc test) than those registered in early May),
which indicated that the hypoosmoregulatory ability of the fish was declining. For transfer
of the groups from FW to SW the salinity change was achieved by rapidly switching the
water supply in each tank from FW to SW. Saltwater concentration was measured by a
salinity refractometer (Atago).
During SW rearing, the water current in the tanks was kept at about 0.11 m s
1. A water
inflow rate of 9 l min
1 was maintained for fish transferred to SW in April and May, but was
650 H. Toften et al. / Aquaculture 217 (2003) 647–662

Fig. 1. Plasma chloride concentrations in Atlantic salmon (n = 15) after 24 h of exposure to seawater (34x ,8
jC) during parr – smolt transformation. The photoperiod was switched from short (LD 6:18) to long day (LD
24:0) on 28 March. The dates selected for transfer of fish from fresh water to seawater were 10 April, 3 May and
19 June.

adjusted to 13 l min
1 for fish transferred in June. The mean water temperatures ( F SE)
during the 30 days of rearing were 7.9 ( F 0.3), 8.1 ( F 0.3) and 8.2 ( F 0.3) jC in the April,
May and June groups, respectively. Information about the characteristics and stocking
densities of the fish in the different treatments is given in Table 1.

Table 1
Number of fish (N), weight (W), fork length (L), condition factor (CF) and stocking density (D) at day 2 (4) for
groups of Atlantic salmon transferred directly from fresh water to seawater at different times during the parr –
smolt transformation (10 April, 3 May and 19 June)
Group R N W (g) L (cm) CF D (kg m
3)
10 April
B 1 59 47.0 ( F 0.7) 15.8 ( F 0.1) 1.19 ( F 0.01) 9.2
B 2 59 46.7 ( F 0.7) 15.6 ( F 0.1) 1.22 ( F 0.01) 9.2
BS 1 63 47.6 ( F 0.7) 15.8 ( F 0.1) 1.19 ( F 0.01) 9.4
BS 2 55 46.8 ( F 0.7) 15.7 ( F 0.1) 1.20 ( F 0.01) 8.9
3 May
B 1 60 58.1 ( F 0.8) 16.9 ( F 0.1) 1.20 ( F 0.01) 11.6
B 2 63 59.9 ( F 0.9) 17.1 ( F 0.1) 1.19 ( F 0.01) 12.6
BS 1 61 56.7 ( F 0.9) 16.9 ( F 0.1) 1.18 ( F 0.01) 11.5
BS 2 62 59.1 ( F 0.9) 17.1 ( F 0.1) 1.18 ( F 0.01) 12.2
19 June
B 1 58 86.8 ( F 1.5) 19.9 ( F 0.1) 1.09 ( F 0.01) 16.8
B 2 56 91.1 ( F 1.3) 20.4 ( F 0.1) 1.08 ( F 0.01) 17.0
BS 1 57 86.5 ( F 1.3) 19.9 ( F 0.1) 1.10 ( F 0.01) 16.5
BS 2 59 88.3 ( F 1.6) 20.1 ( F 0.1) 1.08 ( F 0.01) 17.4
Groups were fed either basic feed (B) or squid supplemented feed (BS) (two replicates (R) for each diet). The
photoperiod was increased to long day (LD 24:0) 2, 5 and 12 weeks before seawater transfer for the April, May
and June groups, respectively. Values for W, L and CF are means ( F SEM).
 H. Toften et al. / Aquaculture 217 (2003) 647–662 651

2.4. Diets

In FW the fish were fed in excess with 3-mm NUTRA pellets (T. Skretting, Stavanger,
Norway) using automatic disc feeders timed to distribute feed for 6 h each day. On 28
March 1995, when the photoperiod was switched to continuous light, the time during
which fish were fed was increased to 8 h per day. Following transfer to SW, the NUTRA
pellets were replaced with test feeds, and two dietary treatments (with two replicates each)
were applied. The fish were fed either basic feed (B) or basic feed plus 5 g kg
1 squid
extract (BS). The feeds were offered in excess, and were distributed for 8 h per day
1 by
automatic disc feeders. The basic feed (Table 2) was formulated to be similar to a
commercial feed (FK-VEKST, Felleskjøpet, Sandnes, Norway). A pellet size of 4 mm was
used, based on the recommendations of Wankowski and Thorpe (1979). Samples of feed
were marked with ballotini glass beads (Jencon’s, Leighton Buzzard, Bedfordshire,
England) for feed intake measurements: ballotini of two sizes (490 – 700 and 230 –320
Am) and concentrations (30 and 7 g kg
1) were included in different batches of feed. The
BS feed was prepared by coating the basic feed with squid extract. The squid extract was
prepared by mixing squid powder (Rieber & Son, Bergen, Norway) with distilled water,
and thereafter, the mixture was stirred for 30 min following heating to 70 jC. The
homogenate was then centrifuged (8275  g, 25 min), the upper aqueous phase siphoned
off and vacuum evaporated (>
70 jC; 36– 48 h) to dryness. The squid extract was then
stored frozen (
70 jC) until needed. For coating the feed, squid extract powder was
redissolved in distilled water and sprayed onto the surface of the feed pellets. To avoid any
differences in texture and water content between the two test feeds, the B-feed was coated
with distilled water. After coating, the feeds were dried at room temperature to remove
surplus water. All diets were stored at
20 jC until used.

2.5. Measurements

Feed intake, body weight (nearest 0.1 g) and fork length (nearest mm) were measured
on all fish in each tank on three occasions. For fish transferred to SW in April and May, the

Table 2
Ingredients and chemical composition of the test feed (basic feed: B)
Ingredients (g kg
1)
Fish meal (Norse-LT 94) 220
Fish meal (Norse-Mink) 210
Fish oil (capelin) 140
Soya protein concentrate 90
Fish ensilage concentrate 40
Mineral- and vitamin premix

Chemical analysis (g kg
1)
Dry matter 910
Protein 456
Lipid 193
Carbohydrate 181
Ash 80
652 H. Toften et al. / Aquaculture 217 (2003) 647–662

measurements were conducted on days 2, 16 and 30. For fish transferred to SW in June,
there were some technical problems, so the first sampling was carried out on day 4 after
transfer to SW. The other two samplings were conducted on days 16 and 30. On days 2 (4)
and 30, 10 fish from each tank were killed and blood samples were taken for analysis of
plasma chloride concentration.
Feed intake was determined by the radiographic method originally described by Talbot
and Higgins (1983), but with some minor modifications (Jobling et al., 1993). Calibration
curves were determined to describe the relationship between the weight of marked feed
and the number of glass beads. On days that feed intake was to be measured, the fish were
fed as usual, except that ordinary feed was replaced with labelled feed. Following feeding,
the fish were anaesthetised with benzocaine (50 mg l
1), weighed, length measured and
X-radiographed. After the measurements, the fish were returned to their rearing tanks and
fed unlabelled feed until the next sampling. After the X-ray plates had been developed, the
number of ballotinis present in the gastrointestinal tract of individual fish was counted,
thereby allowing an estimate to be made of the amount of feed consumed.
The feed intake measurements were conducted at relatively frequent intervals (14 days),
giving the possibility that not all of the glass beads would have been evacuated from the
gut before the next sampling (McCarthy et al., 1993; Jobling et al., 1995). To avoid
problems of overestimation of feed intake, we used feeds that were labelled with ballotinis
of different sizes.
The fish used for blood sampling were the first fish netted from each tank for X-raying.
After netting, the fish were killed by a blow to the head, and blood was collected and
treated as previously described. After the blood sampling was completed, the fish were X-
radiographed, weighed and length measured.
Specific growth rates (SGR) of each fish were calculated according to the formula:

SGR ¼ ððlnWT
lnWt Þ=ðT
tÞÞ  100 ð1Þ

where WT and Wt are weights in grams at times T and t, respectively, and (T
t) is the time
in days between weighings. Corrected values of WT and Wt were used, i.e. weights of feed
ingested were subtracted from total weights recorded.
Condition factor (CF) was calculated for each fish as:

CF ¼ W  100=L3 ð2Þ

where W is weight in grams and L is fork-length in centimeters.

2.6. Statistics

Data on feed intake, SGR and plasma chloride values were normally distributed
(Lilliefors test), so these (replicates pooled) are reported as arithmetic means ( F SEM).
For feed intake and growth data, a two-way repeated measures ANOVA was used to
investigate for possible differences between treatments (transfer time and feed type) over
time. For feed intake, growth data, plasma chloride values and condition factor, one-way
ANOVA (with body weight as a co-factor) was used to test for significant differences
between treatments, and the Tukey – Kramer test was used for post hoc pairwise
 H. Toften et al. / Aquaculture 217 (2003) 647–662 653

comparisons between groups. No significant differences in plasma chloride concentrations

were found between fish fed B or BS feed, so within each smolt group, chloride data for
both feed types were pooled. A chi-square test of independence was used to investigate for
possible differences in the proportions of non-feeding fish between treatments (transfer
time and feed type). Correlation analyses (Pearson) were used to examine for relationships

Fig. 2. Feed intake in Atlantic salmon measured 2 (4), 16 and 30 days after direct transfer from fresh water to
seawater at different times during parr – smolt transformation (10 April, 3 May and 19 June). Within each transfer
time, filled stars indicate significant differences between fish fed B (basic feed) and BS (basic plus 5 g kg
1
squid extract). Uppercase letters (A, B) indicate significant differences between feed intakes of fish transferred to
seawater at different times and fed the B-feed, whereas lowercase letters (a, b) indicate significant differences
between feed intakes of fish transferred to seawater at different times and fed the BS-feed. For fish transferred to
seawater in June, the first feed intake measurement was conducted on day 4.
654 H. Toften et al. / Aquaculture 217 (2003) 647–662

between measured parameters. In all tests, differences with a probability level of < 0.05
were considered significant. Statistical computations were performed with SYSTAT 5.1
(Wilkinson, 1992) and SYSTAT 9.0 (SPSS, 1999).

3. Results

During the 30 days of SW rearing, mortality was low ( < 1.3%) in all groups of fish.
Feeding and growth were low in all treatment groups in the period immediately following
transfer to SW (Figs. 2 –4). The proportions of non-feeding fish decreased as time
progressed (Fig. 4), and feed intake and growth increased (Figs. 2 and 3).
The addition of squid extract to the basic feed tended to give increased feeding and
growth, and the effect was most pronounced for fish transferred to SW in June. In fish
transferred to SW in June both feed intake and growth of fish fed the BS feed were

Fig. 3. Specific growth rates of Atlantic salmon during two periods after direct transfer from fresh water to
seawater at different times during parr – smolt transformation (10 April, 3 May and 19 June). Within each transfer
time, filled stars indicate significant differences between fish fed B (basic feed) and BS (basic plus 5 g kg
1
squid extract). Uppercase letters (A, B) indicate significant differences between growth rates of fish transferred to
seawater at different times and fed the B-feed, whereas lowercase letters (a, b) indicate significant differences
between growth rates of fish transferred to seawater at different times and fed the BS-feed. For fish transferred to
seawater in June, the first time interval used for calculation of SGR was days 4 to 16.
 H. Toften et al. / Aquaculture 217 (2003) 647–662 655

significantly higher than in fish given the B feed (Figs. 2 and 3). In the other groups, the
feed intake of fish fed the squid supplemented diet (BS) was significantly higher than that
of fish fed the basic feed (B) on day 2 for fish in the April group and on day 30 for fish in
the May group (Fig. 2). In the latter, the proportion of fish with empty stomachs on day 30
was significantly lower in fish fed the BS feed (Fig. 4).
There were significant differences in proportions of non-feeding fish, feed intake and
growth rates between fish transferred to SW at different times (Figs. 2– 4). The two-way

Fig. 4. Proportions of fish (%) with empty stomachs in groups of Atlantic salmon monitored on days 2 (4), 16 and
30 following direct transfer from fresh water to seawater at different times (10 April, 3 May and 19 June). Within
each transfer time, filled stars indicate significant differences between fish fed B (basic feed) and BS (basic plus 5
g kg
1 squid extract). For fish transferred to seawater in June, the first measurements were conducted on day 4.
656 H. Toften et al. / Aquaculture 217 (2003) 647–662

repeated measures ANOVA revealed that the differences were linked to transfer time rather
than feed type. Proportions of non-feeding fish were significantly lower and feed intake
and growth rates were significantly higher in fish transferred to SW in June than in fish
transferred to SW in either April or May (Figs. 2– 4). During the latter part of the
experiment, rates of feed intake and growth were higher in fish transferred to SW in May
than in those transferred in April (Figs. 2 and 3).
There were significant differences in plasma chloride concentrations between fish
transferred to SW at different times (Fig. 5). In fish transferred to SW in April, plasma
chloride concentrations at first sampling were significantly higher than those of fish
transferred to SW in May and June (Fig. 5). After 30 days in SW, however, plasma
chloride concentration in the April group had decreased significantly, and was similar
to that of fish transferred to SW in May (Fig. 5). Fish transferred to SW in May
showed the lowest chloride concentrations of all groups on the first sampling date, and
levels were similar on day 30 (Fig. 5). On day 30, the plasma chloride concentration in
the fish transferred in June was higher than that of fish transferred earlier (Fig. 5). The
condition factor on day 2 (4) was significantly lower in fish transferred to SW in June
than in those transferred in either April or May (Table 1). There were no correlations
between individual feed intakes and plasma chloride concentrations in fish within any
of the groups. When body weight was included as a co-variate in the ANOVA tests it
was not found to have a significant effect on plasma chloride level, feed intake or
growth.

Fig. 5. Plasma chloride concentrations of Atlantic salmon (n = 10), 2 (4) and 30 days after direct transfer from
fresh water to seawater at different times (10 April, 3 May and 19 June) (feed groups pooled). Filled stars indicate
significant differences between sampling times for a given treatment. Uppercase letters (A, B) indicate significant
differences between chloride concentrations on the first sampling date, whereas lowercase letters (a, b) indicate
significant differences between chloride concentrations on day 30. Values that are significantly different are
marked with the same letter.
 H. Toften et al. / Aquaculture 217 (2003) 647–662 657

4. Discussion

The present study was undertaken to study the effects of dietary supplementation of a
putative feeding stimulant, squid extract, on SW performance of Atlantic salmon.
The addition of squid extract to the basic feed tended to enhance feeding and growth,
and this was especially pronounced in fish transferred to SW in June. This indicates that
the squid extract acted as a feeding stimulant, in agreement with previous findings (Mackie
and Mitchell, 1985; Fukuda et al., 1989; Johnstone and Mackie, 1990; Toften et al., 1995;
Toften and Jobling, 1997). The implication is that the addition of feeding stimulants to
feed may have a potential for improving the performance of salmon smolts in the period
immediately following the transfer to SW.
Feeding and growth of all groups were depressed in the early phase of SW rearing,
but during the course of the experiment, the proportion of non-feeders decreased (Fig.
4) and the rates of feed intake and growth increased (Figs. 2 and 3). These findings
generally corroborate the results of previous studies carried out on Atlantic salmon
smolts (Usher et al., 1991; Jørgensen and Jobling, 1994; Stradmeyer, 1994; McCarthy
et al., 1996; Arnesen et al., 1998). There were, however, pronounced differences in
feeding and growth between the groups of fish transferred to SW at different times,
with late transfer to SW leading to the quickest return of appetite. By day 16, over
90% of the fish transferred to SW in June were feeding, and high rates of ingestion
were restored within 30 days. A similar early return of appetite was reported by
Jørgensen and Jobling (1994), but other authors have observed that 35 –50 days may
elapse before most fish display normal appetite (Usher et al., 1991; Stradmeyer, 1994;
McCarthy et al., 1996). The latter results seem to be more in line with the findings for
fish transferred to SW in May in the present work. Although feeding and growth were
low in fish transferred to SW in April, our results indicate that the appetite of these
fish was not permanently depressed; almost 80% of the fish had resumed feeding
within 30 days and their feed intake was increasing by the end of the experiment.
Thus, under the conditions of our study, it was possible to transfer Atlantic salmon to
SW over a relatively protracted time (about 2 months) during the spring, but late
transfer seemed to be most appropriate to minimise the period with appetite depression.
It has been suggested that the variation in timing of the return of appetite may be related
to differences in SW adaptability (Usher et al., 1991). In the present study, plasma chloride
concentrations after 48 h of SW exposure were highest in fish transferred to SW in April,
and were lowest in fish transferred in May. This, along with the results of the SW
challenge tests, indicates that the fish transferred to SW in May had high hypoosmor-
egulatory capacity. However, the condition factor of these fish was still high (Table 1),
indicating that the fish may not have completed the parr –smolt transformation. Reduced
condition factor is considered a reasonably reliable indication of smoltification in Atlantic
salmon (Farmer et al., 1978; Saunders et al., 1985; Hoar, 1988; Stefansson et al., 1991;
Clarke et al., 1996; Clarke, 2000), even though the ability to regulate plasma ions may not
be directly coupled to changes in condition factor (Solbakken et al., 1994; Berge et al.,
1995; McCormick et al., 1995). Sigholt et al. (1998) reported that parr –smolt trans-
formation was completed approximately 400 jC-days following exposure of the fish to a
long day photoperiod, and this would translate to a transfer time in mid-may in our
658 H. Toften et al. / Aquaculture 217 (2003) 647–662

experiment, i.e. 2 weeks after the date (3 May) we used. In fish transferred to SW in April,
the elevated chloride concentrations after 48 h in SW (Fig. 5), relatively poor hypoosmor-
egulatory ability (as indicted by the SW challenge test; Fig. 1) and a high condition factor
(Table 1) indicate that these fish had not completed the parr – smolt transformation. Thus,
problems related to incomplete parr – smolt transformation may explain why fish trans-
ferred to SW in April did not resume feeding as quickly as fish in the other groups. After
30 days in SW, however, fish transferred to SW in April seemed to be well acclimated to
SW because plasma chloride concentrations were similar to those of fish transferred to SW
in May (Fig. 5).
Several authors have reported that the salinity tolerance and hypoosmoregulatory
ability of salmonids decrease if the fish are held in FW water beyond parr – smolt
transformation (‘‘desmoltification’’) (Evropeytseva, 1962; McCormick and Saunders,
1987; Hoar, 1988; Soivio et al., 1988; Duston et al., 1991; Clarke et al., 1996; Clarke,
2000). Results of the SW challenge tests provided evidence that the fish had started to
‘‘desmoltify’’ by mid-June (Fig. 1), and after 30 days in SW, fish transferred in June had
significantly elevated plasma chloride concentrations compared to fish transferred in both
April and May (Fig. 5). Nevertheless, it was the fish transferred to SW in June that had the
most rapid recovery of appetite and grew best during the early phase of SW rearing (Figs.
2 and 3). These findings suggest that the appetite suppression observed in Atlantic salmon
smolts after SW transfer is caused by mechanisms other than those directly linked to
osmo- and ionoregulatory imbalance. Further, Usher et al. (1991) observed that the
appetite of smolts was depressed for up to 30 –40 days following transfer to SW, despite
the fact that the fish appeared to be SW-acclimated within 10 days. In addition, there were
no indications of a relationship between low rates of feed intake and interference with
drinking or any suppression of digestive activity (Usher et al., 1988, 1990). Further, the
feeding and growth patterns did not appear to be linked to size differences at the time of
transfer. Although the fish transferred in June were larger than those transferred in April
and May, we found no evidence of an effect of size on plasma chloride, feed intake or
growth. Thus, the general positive relationship between body size and hypoosmoregula-
tory capacity found in young salmonids (McCormick and Saunders, 1987; Duston and
Saunders, 1990; Arnesen et al., 1992) did not seem to apply in the present experiment.
This may have been because all fish were above a critical threshold size for SW transfer.
The differences in feeding and growth between fish transferred to SW in April, May
and June observed in our study might reflect other developmental changes. During the
parr – smolt transformation, there are many changes in metabolism, body composition and
endocrinology (Hoar, 1988; Boeuf, 1993; Clarke et al., 1996; Clarke, 2000). For example,
plasma concentrations of growth hormone (GH) increase during smoltification in Atlantic
salmon (Björnsson et al., 1989, 1995, 2000; Boeuf and Prunet, 1989; Stefansson et al.,
1991; McCormick et al., 1995), and GH is known to have a seawater-adapting,
hypoosmoregulatory role (Björnsson, 1997). A growth-promoting effect of GH has also
been documented in salmonids (Boeuf, 1993; Björnsson et al., 1995; De Pedro and
Björnsson, 2001) and there is increasing evidence that GH influences feeding behaviour
(Johnsen and Björnsson, 1994; Jönsson et al., 1996; De Pedro and Björnsson, 2001). In the
light of this it is tempting to speculate that the fish transferred to SW in June in the present
study might have had a more favourable GH status than those transferred earlier, resulting
 H. Toften et al. / Aquaculture 217 (2003) 647–662 659

in increased appetite and growth in SW. Additional studies would be needed to elucidate
this possibility.
Several authors have reported that sensory information may have different effects
according to the internal physiological state of the animal (Cabanac et al., 1968;
Wyrwicka, 1969; Booth, 1977, 1980). Given this, the differences in responses to dietary
addition of squid extract between fish transferred to SW at different times (Fig. 2) may
have been related to differences in physiological state: fish transferred to SW in June were
both most ready to feed and were most influenced by the feeding stimulant.

5. Conclusions

The results of this study provide further evidence that squid extract is a feeding
stimulant for salmonids, and the implication is that it can be added to feed to improve the
performance of salmon in the period immediately following transfer to SW. It was possible
to transfer fish to SW over a relatively protracted period (about 2 months from mid-April
to mid-June) during the spring, but late transfer seemed most appropriate to minimise the
length of time with appetite depression after SW transfer. The differences in feeding and
growth performance between treatment groups (transfer time and feed type) seem to be
related to differences in internal physiological state that are not directly linked to seawater
adaptability or osmoregulatory failure.

Acknowledgements

We thank the staff of Kårvik Aquaculture Research Station for technical assistance.
This work was supported by the Norwegian Research Council (project nos. 105606/130
and 108350/120).

References

Arnesen, A.M., Halvorsen, M., Nilssen, K.J., 1992. Development of hypoosmoregulatory capacity in Arctic charr
(Salvelinus alpinus) reared under either continuous light or natural photoperiod. Can. J. Fish. Aquat. Sci. 49,
229 – 237.
Arnesen, A.M., Jørgensen, E.H., Jobling, M., 1993a. Feed intake, growth and osmoregulation in Arctic charr,
Salvelinus alpinus (L.), following abrupt transfer from freshwater to more saline water. Aquaculture 114,
327 – 338.
Arnesen, A.M., Jørgensen, E.H., Jobling, M., 1993b. Feed intake, growth and osmoregulation in Arctic charr,
Salvelinus alpinus (L.), transferred from freshwater to saltwater at 8 jC during summer and winter. Fish
Physiol. Biochem. 12, 281 – 292.
Arnesen, A.M., Johnsen, H.K., Mortensen, A., Jobling, M., 1998. Acclimation of Atlantic salmon (Salmo salar
L.) smolts to ‘‘cold’’ sea water following direct transfer from fresh water. Aquaculture 168, 351 – 367.
Berge, Å.I., Berg, A., Fyhn, H.J., Barnung, T., Hansen, T., Stefansson, S.O., 1995. Development of salinity
tolerance in underyearling smolts of Atlantic salmon (Salmo salar) reared under different photoperiods. Can.
J. Fish. Aquat. Sci. 52, 243 – 251.
660 H. Toften et al. / Aquaculture 217 (2003) 647–662

Björnsson, B.Th., 1997. The biology of salmon growth hormone: from daylight to dominance. Fish Physiol.
Biochem. 17, 9 – 24.
Björnsson, B.Th., Thorarensen, H., Hirano, T., Ogasawara, T., Kristinsson, J.B., 1989. Photoperiod and temper-
ature affect plasma growth hormone levels, growth, condition factor and hypoosmoregulatory ability in
juvenile Atlantic salmon (Salmo salar) during parr – smolt transformation. Aquaculture 82, 77 – 91.
Björnsson, B.T., Stefansson, S.O., Hansen, T., 1995. Photoperiod regulation of plasma growth hormone levels
during parr – smolt transformation of Atlantic salmon: implications for hypoosmoregulatory ability and
growth. Gen. Comp. Endocrinol. 100, 73 – 82.
Björnsson, B.T., Hemre, G.I., Bjørnevik, M., Hansen, T., 2000. Photoperiod regulation of plasma growth hor-
mone levels during induced smoltification of underyearling Atlantic salmon. Gen. Comp. Endocrinol. 119,
17 – 25.
Boeuf, G., 1993. Salmonid smolting: a pre-adaptation to the oceanic environment. In: Rankin, J.C., Jensen, F.B.
(Eds.), Fish Ecophysiology. Chapman & Hall, London, pp. 105 – 135.
Boeuf, G., Prunet, P., 1989. Growth hormone and thyroid hormones during Atlantic salmon, Salmo salar L.,
smolting, and after transfer to seawater. Aquaculture 82, 257 – 268.
Booth, D.A., 1977. Appetite and satiety as metabolic expectancies. In: Katsuki, Y., Sato, M., Takagi, S.F.,
Oomura, Y. (Eds.), Food Intake and Chemical Senses. University of Tokyo Press, Tokyo, pp. 317 – 330.
Booth, D.A., 1980. Conditioned reactions in motivation. In: Toates, F.M., Holliday, R.T. (Eds.), Analysis of
Motivational Processes. Academic Press, London, pp. 77 – 102.
Cabanac, M., Minaire, Y., Adair, E.R., 1968. Influence of internal factors on the pleasantness of a gustative sweet
sensation. Commun. Behav. Biol. 1, 77 – 82.
Carr, W.E.S., Darby, C.D., 1986. Chemically stimulated feeding behaviour in marine animals. J. Chem. Ecol. 12,
989 – 1011.
Carr, W.E.S., Netherton, J.C., Gleeson, R.A., Derby, C.D., 1996. Stimulants of feeding behavior in fish: analysis
of tissues of diverse marine organisms. Biol. Bull. 190, 149 – 160.
Christiansen, J.S., Jobling, M., 1990. The behaviour and the relationship between food intake and growth of
juvenile Arctic charr, Salvelinus alpinus L., subjected to sustained exercise. Can. J. Zool. 68, 2185 – 2191.
Clarke, W.C., 2000. Smolting. In: Stickney, R.R. (Ed.), Encyclopedia of Aquaculture. Wiley, New York,
pp. 879 – 884.
Clarke, W.C., Blackburn, J., 1977. A seawater challenge test to measure smolting of juvenile salmon. Tech. Rep.-
Fish. Mar. Serv. 705 (11 pp.).
Clarke, W.C., Saunders, R.L., McCormick, S.D., 1996. Smolt production. In: Pennell, W., Barton, B.A. (Eds.),
Principles of Salmonid Culture. Elsevier, Amsterdam, pp. 517 – 567.
De Pedro, N., Björnsson, B.Th., 2001. Regulation of food intake by neuropeptides and hormones. In: Houlihan, D.,
Boujard, T., Jobling, M. (Eds.), Food Intake in Fish. Blackwell Science, Oxford, pp. 269 – 296.
Duston, J., Saunders, R.L., 1990. The entrainment role of photoperiod on hypoosmoregulatory and growth related
aspects of smolting in Atlantic salmon (Salmo salar). Can. J. Zool. 68, 707 – 715.
Duston, J., Saunders, R.L., 1995. Advancing smolting to autumn in age 0+ Atlantic salmon by photoperiod and
long-term performance in sea water. Aquaculture 135, 295 – 309.
Duston, J., Saunders, R.L., Knox, D.E., 1991. Effects of increases in freshwater temperatures on loss of smolt
characteristics in Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. 48, 164 – 169.
Evropeytseva, N.V., 1962. Comparative analysis of the smoltification process among the young of different
ecological forms of Atlantic salmon. Uchenye Zap. Leningr. Gos. Univ. 311, 46 – 76 (Translated from
Russian: Fish. Res. Board Can. Transl. Ser., 431, Ottawa, Canada).
Farmer, G.J., Ritter, J.A., Ashfield, D., 1978. Seawater adaption and parr – smolt transformation of juvenile
Atlantic salmon, Salmo salar. J. Fish. Res. Board Can. 35, 93 – 100.
Fukuda, K., Kohbara, J., Zeng, C., Hidaka, I., 1989. The feeding-stimulatory effects of squid muscle extracts on
the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 55, 791 – 797.
Handeland, S.O., Stefansson, S.O., 2001. Photoperiod control and influence of body size on off-season parr –
smolt transformation and post-smolt growth. Aquaculture 192, 291 – 307.
Hara, T.J., 1992. Fish Chemoreception. Chapman & Hall, London. 373 pp.
Hoar, W.S., 1988. The physiology of smolting salmonids. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology,
vol. 11B. Academic Press, New York, pp. 275 – 343.
 H. Toften et al. / Aquaculture 217 (2003) 647–662 661

Jobling, M., Christiansen, J.S., Jørgensen, E.H., Arnesen, A.M., 1993. The application of X-radiography in
feeding and growth studies with fish: a summary of experiments conducted on Arctic charr. Rev. Fish.
Sci. 1, 223 – 237.
Jobling, M., Arnesen, A.M., Baardvik, B.M., Christiansen, J.S., Jørgensen, E.H., 1995. Monitoring feeding
behaviour and food intake: methods and applications. Aquacult. Nutr. 1, 131 – 143.
Johnsen, J.I., Björnsson, B.Th., 1994. Growth hormone increases growth rate, appetite and dominance in juvenile
rainbow trout, Oncorhynchus mykiss. Anim. Behav. 48, 177 – 186.
Johnstone, A.D.F., Mackie, A.M., 1990. Laboratory investigations of bait acceptance by the cod, Gadus morhua
L.: identification of feeding stimulants. Fish. Res. 9, 219 – 230.
Jönsson, E., Johnsson, J.I., Björnsson, B.T., 1996. Growth hormone increases predation exposure of rainbow
trout. Proc. R. Soc. Lond., B 263, 647 – 651.
Jørgensen, E.H., Jobling, M., 1994. Feeding and growth of exercised and unexercised juvenile Atlantic salmon
(Salmo salar) in fresh water, and performance after transfer to sea water. Aquacult. Int. 2, 154 – 164.
Mackie, A.M., Mitchell, A.I., 1985. Identification of gustatory feeding stimulants for fish—Application in
aquaculture. In: Cowey, C.B., Mackie, A.M., Bell, J.G. (Eds.), Nutrition and Feeding in Fishes. Academic
Press, London, pp. 177 – 189.
MacLeod, M.G., 1977. Effects of salinity of food intake, absorption and conversion in rainbow trout (Salmo
gairdneri Richardson). Mar. Biol. 43, 93 – 102.
McCarthy, I.D., Houlihan, D.F., Carter, C.G., Moutou, K.A., 1993. Variation in individual food consumption rates
of fish and its implication for the study of fish nutrition and physiology. Proc. Nutr. Soc. 52, 411 – 420.
McCarthy, I.D., Carter, C.G., Houlihan, D.F., Johnstone, R., Mitchell, A.I., 1996. The performance of all-female
diploid and triploid Atlantic salmon smolts on transfer together to sea water. J. Fish Biol. 48, 545 – 548.
McCormick, S.D., Saunders, R.L., 1987. Preparatory physiological adaptations for marine life of salmonids:
osmoregulation, growth and metabolism. Am. Fish. Soc. Symp. 1, 211 – 229.
McCormick, S.D., Björnsson, B.T., Sheridan, M., Eilertson, C., Carey, J.B., O’Dea, M., 1995. Increased day-
length stimulates plasma growth hormone and gill Na + , K + -ATPase in Atlantic salmon (Salmo salar). J.
Comp. Phys. B 165, 245 – 254.
McKay, L.R., Gjerde, B., 1985. The effect of salinity on growth of rainbow trout. Aquaculture 49, 325 – 331.
Saunders, R.L., Henderson, E.B., Harmon, P.R., 1985. Effects of photoperiod on juvenile growth and smolting of
Atlantic salmon and subsequent survival and growth in sea cages. Aquaculture 45, 55 – 66.
Sigholt, T., Staurnes, M., Jakobsen, H.J., Åsgård, T., 1995. Effects of continuous light and short-day photo-
period on smolting, seawater survival and growth in Atlantic salmon (Salmo salar). Aquaculture 130,
373 – 388.
Sigholt, T., Åsgård, T., Staurnes, M., 1998. Timing of parr – smolt transformation in Atlantic salmon (Salmo
salar): effects of changes in temperature and photoperiod. Aquaculture 160, 129 – 144.
Soivio, A., Virtanen, E., Muona, M., 1988. Desmoltification of heat-accelerated Baltic salmon (Salmo salar) in
brackish water. Aquaculture 71, 89 – 97.
Solbakken, V.A., Hansen, T., Stefansson, S.O., 1994. Effects of photoperiod and temperature on growth and
parr – smolt transformation in Atlantic salmon (Salmo salar L.) and subsequent performance in seawater.
Aquaculture 121, 13 – 27.
SPSS, 1999. Systat 9: Statistics. SPSS, Chicago, USA.
Stefansson, S.O., Björnsson, B.T., Hansen, T., Haux, C., Taranger, G.L., Saunders, R.L., 1991. Growth, parr –
smolt transformation, and changes in growth hormone of Atlantic salmon (Salmo salar) reared under different
photoperiods. Can. J. Fish. Aquat. Sci. 48, 2100 – 2108.
Stradmeyer, L., 1994. Survival, growth and feeding of Atlantic salmon, Salmo salar L., smolts after transfer to sea
water in relation to the failed smolt syndrome. Aquacult. Fish. Manage. 25, 103 – 112.
Talbot, C., Higgins, P.J., 1983. A radiographic method for feeding studies on fish using metallic iron powder as a
marker. J. Fish Biol. 23, 211 – 220.
Toften, H., Jobling, M., 1997. Feed intake and growth of Atlantic salmon, Salmo salar L., fed diets supplemented
with oxytetracycline and squid. Aquacult. Nutr. 3, 145 – 151.
Toften, H., Jørgensen, E.H., Jobling, M., 1995. The study of feeding preferences using radiography: oxytetracy-
cline as a feeding deterrent and squid extract as a stimulant in diets for Atlantic salmon. Aquacult. Nutr. 1,
145 – 149.
662 H. Toften et al. / Aquaculture 217 (2003) 647–662

Usher, M.L., Talbot, C., Eddy, F.B., 1988. Drinking in Atlantic salmon smolts transferred to seawater and
relationship between drinking and feeding. Aquaculture 73, 237 – 246.
Usher, M.L., Talbot, C., Eddy, F.B., 1990. Effects of transfer to SW on digestion and gut function in Atlantic
salmon smolts (Salmo salar L.). Aquaculture 90, 85 – 96.
Usher, M.L., Talbot, C., Eddy, F.B., 1991. Effects of transfer to SW on growth and feeding in Atlantic salmon
smolts (Salmo salar L.). Aquaculture 94, 309 – 326.
Wankowski, J.W.J., Thorpe, J.E., 1979. The role of food particle size in the growth of juvenile Atlantic salmon
(Salmo salar L.). J. Fish Biol. 14, 351 – 370.
Wilkinson, L., 1992. SYSTAT for Windows: Statistics, Version 5 Edition. SYSTAT, Evanston, IL. 750 pp.
Wyrwicka, W., 1969. Sensory regulation of food intake. Physiol. Behav. 4, 853 – 858.