Aquaculture Research, 2016, 113

doi:10.1111/are.13066

Effects of body weight on the growth and physiology

of Salmo salar L. during smoltification
Zhang Mo1, Gao Xiaolong1,2, Li Xian1 & Liu Ying3
1

Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China

University of Chinese Academy of Sciences, Beijing, China

Dalian Ocean University, Dalian, China

Correspondence: L Ying, College of Marine Technology and Environment, Dalian Ocean University, 116023, Dalian, China.
E-mail: yingliuaquaculture@gmail.com

Abstract
To study the effects of body weight on growth
and physiological response mechanism of S. salar,
fish of three average body weights were selected:
30.25
2.12 g (SW1), 24.27
1.59 g (SW2)
and 18.05
2.46 g (SW3). Condition factor,
daily weight gain and feed conversion efficiency
were significantly higher in the SW2 group compared with the other treatment groups
(P < 0.05). Food conversion ratio decreased with
increasing body weight, and weight and specific
growth rate of body length were highest in the
SW2 group (P < 0.05). Larger fish (SW1 and
SW2) had higher haemoglobin concentration
and number of red blood cells. The number of
white blood cells, lymphocytes and neutrophils
were greatest in the SW3 group (P < 0.05), suggesting that these fish had strong resistance to
disease. The content of total protein, albumin
and urea were significantly higher in the SW2
group compared with other groups (P < 0.05).
The concentration of total cholesterol and
triglyceride increased with increasing body size
(P < 0.05). Hormone (T3 and T4) levels first
increased significantly and then decreased in fish
of various sizes (P < 0.05), and the concentration of cortisol rose, fell and then rose again in
SW1 and SW2 groups. Light affected the endocrine activity of S. salar by influencing the
growth performance of the fish, and smaller
sized fish exhibited decreased smoltification rate
and a reduced success rate of smoltification.
Based on these findings, S. salar weighing about
24 g were chosen to begin the process of
smoltification.

2016 John Wiley & Sons Ltd

Keywords: Salmo salar L., smoltification, body

weight, growth index, physiological and biochemical index, hormone indexes
Introduction
Salmo salar L. is an anadromous fish. As juveniles
swim from fresh water to seawater, they undergo
smoltification (Handeland, Imsland, Bj
ornsson, Stefansson & Porter 2013), during which their physiology, morphology and behaviour change in order
to adapt to the marine environment (Sundh, Nilsen, Lindstr
om, Hasselberg-Frank, Stefansson,
McCormick & Sundell 2014). The endocrine system of S. salar regulates the physiological changes
that occur during smoltification. In addition to
establishing an effective osmotic regulation mechanism, the fish undergoes physiological changes
that better adapt it to the catadromous lifestyle.
For example, the slender body shape improves the
fishs ability to swim in seawater and the ability of
haemoglobin to carry oxygen (Nilsen, Ebbesson,
Madsen, McCormick, Bj
ornsson, Prunet, Andersson & Stefansson 2007).
Fish size, growth rate and energy storage in the
body all affect smoltification and sexual maturity
(Porter, Duncan, Mitchell & Bromagea 1999;
Whalen & Parrish 2011). Bagliniere and Maisse
(1985) and Whalen and Parrish (2011) asserted
that the youngest individuals grow most rapidly in
a population and that body growth inhibits the
development of sex glands. Thorpe, Talbot, Miles
and Keay (1990) reported that development of the
sex gland is inhibited after smoltification and that
body size before smoltification has an obvious clear
effect on body size after smoltification, as sexual

Physiology of Salmo salar L. during smoltification Z Mo et al.

maturity is delayed (Thorpe & Metcalfe 1998).

Bagliniere and Maisse (1985) and Kristinsson,
Saunders and Wiggs (1985) also found that the
sex glands gradually matured during the first
spring after smoltification. Thus, the initial size of
the fish body during the early stage of smoltification is of great significance to the growth of the
fish body and development of sex glands after
smoltification.
Light is an important ecological factor that
affects food intake, metabolism, reproduction and
other physiological functions (Fyhn, Clarke &
Withler 1991). With the development of semiconductor technology, packaged chips containing
light emitting diodes (LEDs) at various wavelengths and powers have been developed and
extensively applied in all walks of life (Song, Qiu,
Wang, Yu & Liu 2013). In ecological studies,
they have been used to assess the effects of light
on many organisms. The effects of light cycle on
growth of fish may depend on the light-hypophysis axis, which stimulates the hypophysis and
influences the synthesis and secretion of growth
hormones (Bjornsson, Taranger, Hansen, Stefansson & Haux 1994). The growth of some fish
can be promoted under continuous lighting or
under an extended light cycle. Hansen, Karlsen,
Taranger, Hemre, Holm and Kjesbu (2001) examined the effects of two light cycles on the growth
performance of Gadus morhua in net-cage culture.
They found that compared with the natural light
cycle, continuous lighting increased the growth
rate of G. morhua. Endal, Taranger, Stefansson
and Hansen (2000) studied the effects of light on
the growth and sexual maturity of S. salar in
net-cage culture and reported that an extended
natural light cycle during both winter and spring
could promote the growth of S. salar. Other
reports suggest that a short light cycle will inhibit the growth and development of some fish
(Taranger, Haux, Hansen, Stefansson, Bj
ornsson,
Walther & Kryvi 1999). Duncan, Mitchell and
Bromage (1999) examined the effects of different
light cycles on the growth and sexual maturity of
S. salar and found that the group exposed to continuous light and the one under L:D 9:15 had
the maximum and minimum body weight gain
respectively. Therefore, determining the light conditions that lead to successful smoltification and
inhibition of the development of sex glands of
S. salar is necessary for optimal culture of this
species.

Aquaculture Research, 2016, 113

Recirculating aquaculture systems (RASs) are

economical, sustainable and environmentally
friendly aquaculture systems that have been used
extensively around the world. In a RAS, the living
environment of aquatic organisms is controllable
in that conditions such as light, temperature,
salinity and nutrient and waste concentrations
can be optimized (Dalsgaard, Lund, Thorarinsdottir, Drengstig, Arvonen & Pedersen 2013). The
operation of RASs requires limited water replenishment because wastes are removed, and wastewater
discharge is reduced, both of which lessen the cost
of aquaculture. The goal of this study was to evaluate the different growth characteristics and physiological activities of S. salar of various sizes in a
RAS in order to identify the optimal body size at
which fish should begin smoltification. The data
will be useful for developing environmentally
friendly, sustainable and healthy cultures of
S. salar.
Materials and methods
Source and acclimation of S. salar
Specimens of S. salar were obtained from Shandong Oriental Ocean Sci-Tech, Yantai, Shandong,
and this experiment was carried out at the Mouping Subsidiary of Shandong Oriental Ocean SciTech. The experimental fish were of three average
body weights, with a healthy body and no signs of
wounds or disease. They were fed in the airtight
RAS. Water from an unpolluted local underground
well was aerated and cooled and introduced into
the RAS.
The RAS was provided by Shandong Oriental
Ocean Sci-Tech. The culture pond was a quadrangle with floor area of 16 m2, height of 2 m and
effective depth of 1.5 m. The water volume of each
culture pond was 24 000 L. Water inflow was
through an inclined jet along the walls that was
controlled by a fluid flow metre and a centralized
computer system. The dissolved oxygen concentration of the water was monitored and maintained
at >6 mg L1. Water was drained through the
drainage pipe in the middle of the pond. Before
stocking the pond, water was added to 0.5 cm
depth, and all over the pond should be washed
out and sterilized with 15 ppm hypochlorous solution. The LEDs used in the experiment were
designed at the Institute of Semiconductors,
Chinese Academy of Sciences, and manufactured
2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Aquaculture Research, 2016, 113

by HZH Photoelectric Technology, Wuxi, Jiangsu.

A SP-10 spectrograph (Hangzhou Everfine Photoelectrical Information, Hangzhou, China) was used
to measure the light intensity of each experimental
group at 5 cm above the water surface in the middle of the pond, and the height of the projector
lamp was adjusted to keep the light intensity
aligned.
After experimental fish were placed in a pond,
no feed was provided until after the first 3 days
had elapsed. Norway Skretting pellet feed (crude
protein >48.0%, crude fat >18.0%, crude fibre
<1.0%, crude ash <12.0%; Stavanger, Norway)
then was given at 7:00 hours and 15:00 hours on
a daily basis. The daily feed amounted to 1% of
the fish body weight. The majority of fish first
rushed for food and then dispersed after being fully
fed. After 30 min of feeding, food residue and faeces were collected and the quantity of residual
food was recorded.
The airtight RAS equipped with biological,
chemical, electronic and engineering technologies
performed mechanical and biological filtration to
remove food residues, faeces, NH3-N, NO2-N and
other hazardous substances; treated the culture
water through sterilization, aeration, CO2 removal
and temperature conditioning; and then returned
it to the culture pond. This system recycles the
culture water, saves water resources, maintains a
high dissolved oxygen content in the water, maintains good water quality and increases the yield of
fish.
Experimental design
The following three body sizes of S. salar were
used in this experiment: 30.25
2.12 g (SW1),
24.27
1.59 g (SW2) and 18.05
2.46 g
(SW3). Three replicates of each experimental
group were tested. The culture density was
20 kg m3, and the experiment lasted for 94 days.
After fish stocking, they were allowed to acclimate
to the environment for 10 days, with the following
conditions: water temperature 13C; pH 7.5; dissolved oxygen concentration >6 mg L1; and a
natural light:dark photoperiod (12:12). During the
period of acclimation, the fish were provided with
Norway Skretting pellet feed as a food source daily
at 7:00 hours and 15:00 hours, with the feeding
quantity equivalent to 1% of the body weight, to
keep them fully fed.

2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Physiology of Salmo salar L. during smoltification Z Mo et al.

For the first 142 days (6 weeks) of the experiment, fish were exposed to a 12L:12D light cycle;
for days 4384 days (second 6 weeks), they were
exposed to full light (24L:0D) to induce smoltification. The light intensity of each treatment group
was 8.60
0.34 W m2, and the light cycle was
controlled by a clock controller. Samples were
taken once every 14 days (six times in total). At
each time point, 100 fish were selected and their
body length and weight were measured. They then
were returned to the pond.
A portable multiparameter water quality measurer (YSI556MPS; YSI, Yellow Springs, OH, USA)
was used to determine the dissolved oxygen content, pH value and temperature of the water on a
daily basis. The ammonia and nitrite concentrations of the water were measured every 3 days
using Nesslers reagent colorimetry and N(1naphty1)-ethylenediaminedihydrochloride
spectrophotometry respectively.
Blood was sampled after starving the experimental fish for 12 h. Every 14 days, 10 fish were
randomly selected from each treatment group
and quickly placed in a MS-222 (200 mg L1)
bucket to be anaesthetized in order to minimize
the stress reaction caused by their being caught.
After 2 min, a 1-mL sterilized disposable syringe
was used to sample blood from the tail vein of
each fish. The blood sample was kept at ambient
temperature, allowed to separate into layers and
then centrifuged at 4C for 10 min. The serum
was transferred to a centrifuge tube using a pipette, and the tube was sealed and stored at
80C. A BC-1800 full-automatic blood cell analyser was used to determine the haematological
indexes of blood, and a BS-180 full-automatic
biochemical analyser was used to determine the
biochemical indexes of serum (both devices manufactured by Shenzhen Mindray Bio-Medical Electronics, Shenzhen, China). Concentrations of total
protein (TP), albumin (ALB), total bilirubin (TBil), uric acid, carbamide, glucose, total cholesterol, triglyceride and creatine kinase (CK) were
measured.
Kits purchased from Tianjin Jiuding Bio-Medical
Engineering were used to determine the concentrations of cortisol, triiodothyronine (T3) and thyroxine
(T4). Liquid balance competition radioimmunoassays (RIAs) were performed, and measurements
were taken using a c-RIA counter (SN-695B,
Shanghai, China). The competitive immune reac-

Physiology of Salmo salar L. during smoltification Z Mo et al.

Aquaculture Research, 2016, 113

tion was produced by adding a certain amount of

specific antibody to the blood sample and then
adding 125I-Cortisol following the instructions provided by the manufacturer.

data obtained were expressed graphically using

Sigmaplot.

Formulas used to calculate parameter values

Water quality in the RAS

Condition factor, CF W=L3  100

Daily weight gain, DWG W2  W1 =t2  t1
Specific growth rate, SGR 100  ln W2
 ln W1 =t2  t1
Food intake, FI 100  F=0:5  W2 W1
 t2  t1 
Food conversion rate, FCR F=nW2  W1 
Food conversion efficiency, FCE
100  W2  W1 =F
Coefficient of body weight variation; SVW
SD=XW  100
Coefficient of body length variation; SVL
SD=XL  100
where t1 and t2 were the days at the beginning
and end of the experiment, respectively, W1 and
W2 were the wet weight (g) of S. salar at the
beginning and end of the experiment, respectively,
L1 and L2 were the S. salar body length (cm) at
the beginning and end of the experiment, respectively, n was the number of fish and F was the
weight of food intake during the experimental process (dry weight, g). XW and XL were the average
body weight (g) or body length (cm) of S. salar
in different size groups, SD was the standard
deviation.
Statistical analysis
Data were expressed as the mean
standard error
(mean
SE), and logarithmic transformation was
done to satisfy the homogeneity test of variances
and standard normal distribution. SPSS16.0 was
used to do the statistical analysis, and the difference between different treatment groups was
examined by one-way analysis of variance (ANOVA)
and Duncan multiple comparison analysis; differences were significant at the P < 0.05 level. The
4

Results

As S. salar is a cold-water fish, the water temperature, concentration of dissolved oxygen and pH
value were appropriately maintained for the culture of specimens. Ammonia and nitrite concentration did not differ significantly among treatment
groups (F = 1.47, P = 0.144). Table 1 shows that
the water quality parameters were maintained
within the safe range for the growth of S. salar.
Growth characteristics of S. salar
CF ranged from 0.64 to 0.85 for fish of different
sizes and differed significantly among size groups
(Table 2). The maximum CF (0.85) occurred in
group SW2. DWG, FCE, FI and FCR also differed
significantly from group to group (PDWG <0.001,
PFCE = 0.001, PFCR = 0.002, PFI = 0.004), and the
first four were highest in group SW2. However,
FCR was significantly higher in SW3 than in any
other group (F = 25.57, P = 0.001).
Figure 1 shows that body length SGR in group
SW3 was always significantly lower than that in
any other groups (F = 30.91, P < 0.001). This
result indicates that induction of smoltification in
the small group occurred too early, that fish
(SW3) were too young and small to undergo the
growth that normally would be expected to occur
during smoltification. When the first 42 days light
cycle (12L:12D) shifted to the subsequent 6 weeks
of full light, the body length of S. salar in the three
groups significantly increased, and the increase
was significantly greater in group SW2 than in
the other two groups (F = 13.62, P = 0.011).
Body weight SGR in SW3 group was always
slow (Fig. 2) and it dropped at day 56, indicating
that the attempt to induce smoltification occurred
too early in this group of small sized fish. When
the condition was changed to full light, the fish
body was not able to adapt to the new condition.
However, the SGR of the SW1 and SW2 fish
increased significantly when they were exposed to
full light, suggesting that smoltification occurred
at that time. The body weight of the fish in the
three groups significantly increased by the end of
the experiment, and this was more significant in
2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Physiology of Salmo salar L. during smoltification Z Mo et al.

Aquaculture Research, 2016, 113

Table 1 Water quality parameters

Parameters

Acclimation

Temperature (C)
Dissolved oxygen (mg L1)
pH
Ammonia (mg L1)
Nitrite (mg L1)

13.72
8.58
7.58
0.014
0.005

0.03a
0.51a
0.50a
0.001a
0.001a

142 days
13.15
8.62
7.57
0.015
0.004

0.04a
0.91a
0.41a
0.002a
0.001a

4284 days
13.55
8.55
7.49
0.014
0.005

0.06a
0.45a
0.23a
0.001a
0.001a

Values are expressed as mean
SE (n = 4). The different letters on the parameters in one row mean significant differences (P < 0.05); the same ones mean no
significant differences (P > 0.05).

Table 2 Growth indexes of Salmo salar L. in different size

groups
Experimental groups
Growth
indexes
DWG (g d1)
CF
FCE (%)
FCR
FI (%)
NY
(g m2 d1)

SW1
0.46
0.73
70.38
1.23
0.63
4.36

SW2

0.07b
0.12b
6.67b
0.34b
0.11b
0.03b

0.62
0.85
84.17
1.29
0.75
5.36

SW3

0.09a
0.36a
5.76a
0.53b
0.16a
0.05a

0.32
0.64
65.14
1.54
0.37
4.75

0.05c
0.73c
4.75b
0.35a
0.07b
0.02b

Values are expressed as mean
SE (n = 5). The different letters on the parameters in one row mean significant differences
(P < 0.05); the same ones mean no significant differences
(P > 0.05).

group SW1 compared with the other groups

(F = 29.53, P < 0.001). The value for group SW3
significantly decreased (F = 10.17, P = 0.022).
The SV of body weight of S. salar in group
SW3 varied greatly over the course of the experiment (Fig. 4, F = 75.37, P < 0.001). Within the
first 6-week light cycle (12L:12D), the SV of
body weight in groups SW1 and SW2 did not
vary significantly (F = 2.57, P = 0.101), but
after the condition changed to full light, it varied
narrowly in all three groups. At 84 days, the
value in the SW3 group was significantly higher
than that in any other groups (F = 70.59,
P < 0.001).
Haematological indexes

group SW2 than in the other two groups

(F = 44.57, P < 0.001).
The SV of body length of S. salar in the different
groups did not significantly change in the first
6 weeks of the study (F = 1.53, P = 0.141)
(Fig. 3), but when the condition was changed to
full light, it significantly increased in the SW1 and
SW2 groups. The change was more significant in

After smoltification, the numbers of white blood

cells (WBCs), lymphocytes and neutrophils were
highest in the SW3 group, although the values did
not differ significantly among the three groups (see
Table 3, F = 1.39, P = 0.155). The concentration
of haemoglobin (HGB) was highest in group SW1,
but this also fell with the decrease of culture size,
and significant differences were identified among

Figure 1 Body
lengthspecific
growth rate of Salmo salar L. in different size groups. Values are
expressed as mean
SE (n = 5).
The same superscript means that
the differences are not significant,
while the different superscript
means that the differences are significant (P < 0.05).
2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Physiology of Salmo salar L. during smoltification Z Mo et al.

Aquaculture Research, 2016, 113

Figure 2 Body
weightspecific
growth rate of Salmo salar L. in different size groups. Values are
expressed as mean
SE (n = 5).
The same superscript means that
the differences are not significant,
while the different superscript
means that the differences are significant (P < 0.05).

Figure 3 Coefficient
of
body
length variation in Salmo salar L.
in different size groups. Values are
expressed as mean
SE (n = 5).
The same superscript means that
the differences are not significant,
while the different superscript
means that the differences are significant (P < 0.05).

Figure 4 Coefficient
of
body
weight variation in Salmo salar L.
in different size groups. Values are
expressed as mean
SE (n = 5).
The same superscript means that
the differences are not significant,
while the different superscript
means that the differences are significant (P < 0.05).

the three groups (F = 24.11, P = 0.001). The number of red blood cells (RBCs) did not differ significantly between groups SW1 and SW2 (F = 1.25,
P = 0.176), but the value was significantly lower in
group SW3 (F = 15.84, P = 0.008). The number of
6

thrombocytes fell with the increase in culture size,

and the three groups differed significantly
(F = 19.66, P = 0.002). The number of neutrophil
was close among these groups, and no significant
difference was identified (F = 1.04, P = 0.221).
2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Physiology of Salmo salar L. during smoltification Z Mo et al.

Aquaculture Research, 2016, 113

Table 3 The change in haematological indices of different size Salmo salar L. in smoltification
Haematological indices

SW1
9

1

White blood cell (WBC, 10 L )

Lymphocyte (LYMP, 109 L1)
Haemoglobin (HB, g L1)
Red blood cell (RBC, 1012 L1)
Thrombocyte (109 L1)
Neutrophile granulocyte (GRAN, 109 L1)

318.62
104.63
119.46
0.57
311.13
95.02

SW2

8.24
2.18a
0.77a
0.04a
5.77c
2.67a

323.75
108.37
108.41
0.54
337.86
97.48

SW3

6.15
2.54a
1.43b
0.08a
2.45b
4.56a

329.03
110.04
98.41
0.45
349.27
98.84

8.39a
4.36a
0.89c
0.07b
4.47a
5.33a

Values are expressed as mean
SE (n = 5). The different letters on the parameters in one row mean significant differences
(P < 0.05); the same ones mean no significant differences (P > 0.05).

Biochemical indexes

Hormonal indexes

The concentrations of TP and ALB in the serum of

S. salar L. were highest in group SW2 because the
smoltification process ended during the experiment,
and significant differences in concentrations of the
compounds were detected among the experimental
groups (see Table 4, PTP = 0.005, PALB = 0.002).
The concentration of total cholesterol and triglyceride in the serum increased with increasing fish
body size, and significant differences were identified
among these groups (SW1 > SW2 > SW3;
PTC = 0.007, PTG = 0.004). However, no significant
difference in glucose concentration was found
among the groups (F = 1.40, P = 0.139). The concentration of uric acid and carbamide in metabolites
was highest in group SW2. Their concentrations in
groups SW1 and SW2 did not differ significantly
(PUA = 0.127, PUREA = 0.209), but they were
significantly higher than those in group SW3
(PUA = 0.002, PUREA = 0.009). The concentration
of T-Bil in group SW3 was significantly higher
than that in the other two groups (F = 16.22, PT-Bil
= 0.005). The concentration of CK was highest in
group SW3, but no significant difference was identified among the three groups (F = 1.34, P = 0.169).

Within the first 42 days, the concentration of T3

in S. salar in each size group increased, but no
significant difference was detected among the
three groups (F = 2.35, P = 0.106). The concentration of T3 in groups SW1 and SW2 was highest on day 56, whereas it began to decrease in
group SW3 on day 42 and was always lower than
that in any other groups. The concentration of T3
in group SW1 was significantly higher than that
in groups SW2 and SW3 (F = 22.69, P = 0.001),
but no significant difference was identified
between groups SW2 and SW3 (Fig. 5, F = 1.85,
P = 0.132).
The concentration of T4 of S. salar in groups
SW1 and SW2 decreased significantly on day 28
but reached the highest value on day 56 (Fig. 6,
F = 38.26, P < 0.001). The concentration of T4
in group SW3 significantly increased on day 28
(F = 24.36, P = 0.001), reached the highest value
on day 70 and then decreased. The concentration
of T4 in group SW1 and SW2 began to decline on
day 56. At this time point (56 days), the maximum T4 value (2.36 lg dL1) was found in group
SW1. At the end of the experiment, the concentra-

Table 4 The change in plasma biochemical indices of different size Salmo salar L. in smoltification
Plasma biochemical indices

SW1

Total protein (TP, g L1)

Albumin (ALB, g L1)
Total bilirubin (T-Bil, lmol L1)
Uric acid (UA, mmol L1)
Carbamide (UREA, mmol L1)
Glucose (Glu, mmol L1)
Total cholesterol (TC, mmol L1)
Triglyceride (TG, mmol L1)
Creatine kinase (CK, mmol L1)

49.15
6.97
10.42
47.63
5.34
4.65
3.57
3.05
537.84

SW2

2.67b
0.18b
0.67b
0.33a
0.37a
0.19a
0.58a
0.47a
9.36a

56.23
7.78
10.55
50.45
5.56
4.92
3.02
2.44
532.73

SW3

1.38a
0.22a
0.57b
0.75a
0.58a
0.74a
0.97b
0.34b
6.48a

40.12
6.02
11.87
43.26
5.11
4.88
2.64
1.89
540.37

2.55b
0.36c
0.97a
0.98b
0.83b
0.83a
0.34c
0.85c
9.94a

Values are expressed as mean
SE (n = 5). The different letters on the parameters in one row mean significant differences
(P < 0.05); the same ones mean no significant differences (P > 0.05).

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Aquaculture Research, 2016, 113

Figure 5 The change in T3 concentration of different size Salmo

salar L. in smoltification. Values
are expressed as mean
SE
(n = 5). The same superscript
means that the differences are not
significant, while the different
superscript means that the differences are significant (P < 0.05).

Figure 6 The change in T4 concentration of different size Salmo

salar L. in smoltification. Values
are expressed as mean
SE
(n = 5). The same superscript
means that the differences are not
significant, while the different
superscript means that the differences are significant (P < 0.05).

Figure 7 The change in cortisol

concentration of different size
Salmo salar L. in smoltification.
Values
are
expressed
as
mean
SE (n = 5). The same
superscript means that the differences are not significant, while the
different superscript means that
the differences are significant
(P < 0.05).

tion of T4 did not differ significantly among the

three groups (F = 1.14, P = 0.209).
During smoltification, the concentration of cortisol in S. salar in groups SW1 and SW2 first

increased then decreased and increased again

(Fig. 7). In the early stage of this experiment, the
concentrations of cortisol in each group were similar, suggesting that the culture environment was
2016 John Wiley & Sons Ltd, Aquaculture Research, 113

Aquaculture Research, 2016, 113

steady and the fish exhibited no stress reaction.

The concentration of cortisol in groups SW1 and
SW2 reached the highest value on day 42, and no
significant difference was detected between these
two groups (F = 1.28, P = 0.172). The value in
group SW3 was highest on day 56, but it was significantly lower than that in groups SW1 and
SW2 (F = 32.64, P < 0.001). After day 56, the
cortisol concentration in each group began to
decrease, although it rose slightly in groups SW1
and SW2 at 84 days.
Discussion
In this study, physiological and biochemical
indexes were used to evaluate the relationship
between S. salar and its environment. Changes in
these indexes influence the body structure and
external morphology of S. salar during smoltification so that it can adapt to changes in the external
environment. Fish require a certain amount of
time to adapt, and during this time, metabolism
and the intake and utilization of nutrients change
(Heras, M-Sitcha, Y
uferaa, Mancera & MRodrgueza 2015). Generally, haematological
indexes are used to evaluate the health status of
S. salar under toxicological, physiological and
pathological conditions (Oppedal, Berg, Olsen, Taranger & Hansen 2006), and they serve as indexes
for evaluation of the bodys adaptability to the
environment (Chatzifotis, Papadaki, Despoti, Roufidou & Antonopoulou 2011).
Effects of smoltification size on the haematological
indexes of S. salar
The size at which S. salar enter the early stage of
smoltification affects their haematological and
physiological indexes. At the end of the experiment, the number of WBCs, lymphocytes and neutrophils varied only slightly from group to group,
indicating that fish of all sizes were healthy. In the
treatment group with the largest fish (SW1), the
number of RBCs and the HGB concentration were
highest. When the light condition changed after
6 weeks to induce smoltification, the appearance
of the fish changed, and the largest quantity of
RBCs and the highest HGB concentration were
found in the blood of the most rapidly growing
S. salar, indicating that these SW1 fish had
quicker adaptability to the environment, stronger
swimming ability and higher efficiency of oxygen
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Physiology of Salmo salar L. during smoltification Z Mo et al.

transport via blood, which maintained the basic

physiological activities of these fish (Almeida,
Meletti & Martinez 2005). Previous studies suggested that aquaculture size and HGB concentration are positively correlated (Mazur & Iwama
1993; Trenzado, Morales & Higuera 2006). When
HGB concentration rises, the number of RBCs also
increases, and HGB in RBCs is likely to be bound
to oxygen, thereby increasing the content of oxygen transported to each tissue and satisfying the
demand for energy (Fivelstad, Waagb, Stefansson
& Olsen 2007). When the light condition was
changed to full light, it strengthened the effect of
smoltification and changed the physiological
rhythm of the fish. S. salar with greater body
weight had greater demand for hematopoietic raw
materials, larger intake of nutrients, quickened respiration and higher growth rate, and greater numbers of SW1 fish underwent smoltification than in
the other two treatment groups.
Effects of smoltification size on the biochemical
indexes of S. salar
Total protein, which mainly keeps the plasma balanced, provides energy, transports nutrients and
promotes blood coagulation (Hung, Liu, Li, Stroebakken & Cui 1997), is a complex mixture composed of ALB, globulin, fibrinogen, prothrombin
and other proteins. TP content is generally used to
detect any disease that fish may have and to
assess intake of nutrients and potential environmental pathogenic factors (Tocher, Bendiksen,
Campbell & Bell 2008). ALB is the most important
specific protein; it mainly keeps the plasma colloid
osmotic pressure in balance, transports the slightly
soluble small organic molecules and inorganic ions
and supplies nutrients (Wuenschel, Jugovich &
Hare 2005).
In this experiment, significant differences in
levels of TP and ALB in the blood of S. salar were
found among the size groups. The results indicate
that the resistance to disease of fish in group SW2
was strong and that consumption of proteins was
low. When reared under the same external conditions, the dynamic equilibrium between decomposition and anabolism of organic ingredients differs
among fish of different sizes, and thus a significant
difference in protein content among the treatment
groups was detected. In blood, the concentration
of CK in myocytes rises during smoltification,
which indicates that muscular damage has

Physiology of Salmo salar L. during smoltification Z Mo et al.


occurred or is occurring in the body (Rehulka,

Minark & Rehulkov
a 2004). In this experiment,
the concentration of CK in group SW3 was significantly higher than that in any other group, indicating that small S. salar are vulnerable to
muscular damage under the lighting conditions
used and that this size group is not suitable for
starting the smoltification process.
When fish are in an unfavourable living condition or affected by pathological changes, T-Bil content in the plasma increases, which leads to liver
damage (Skov, Larsen, Frisk & Jokumsen 2011).
In this experiment, the T-Bil content in group
SW3 differed significantly relative to that in groups
SW1 and SW2, indicating that small fish may
result experience liver damage during the process
of smoltification. Urea and uric acid mainly constitute the nitrogenous end products of protein catabolism in fish, and levels of these compounds are
used to evaluate the health of the fish kidney
(Wilkie 2002). The concentration of urea in group
SW2 was the highest during smoltification, and
sources include excretion by fish and urea from
food solubilized in the blood.
During smoltification, external morphological
changes take place along with the metabolic
changes (Machado, Garofaloj, Roselino, Kettelhut
& Migliorini 1988). During metabolism, fish use
stored energy to maintain normal physiological
activities (Rotllant & Tort 1997). The total content
of protein, fat, sugar and other organic matter
decreases and the content of water and inorganic
matter increases, which means that the relative
content of each component in the fish body always
changes within a period of time (Shearer, Parkins,
Gadberry, Beckman & Swanson 2006; Auperin &
Geslin 2008). Changes of one specific environmental factor (e.g., temperature, salinity, light) can
result in massive energy consumption, decreased
content of protein and other organic matter and
increased relative content of water (McConnachiea, Cook, Patterson, Gilmour, Hinch, Farrell
& Cooke 2012). Through catabolism, key forms of
energy such as protein, fat and sugar are transported via blood cells to each tissue, thereby contributing to the inter-tissue cycle of matter and
energy flow (Begg & Pankhurst 2004). In this
study, no significant difference in the concentration of glucose was detected in the serum of the
different sized S. salar, which suggests that utilization rate of glucose by S. salar is low and that glucose is not the primary substrate of energy

10

Aquaculture Research, 2016, 113

metabolism. The concentration of protein in the

fish with greater body weight was higher than
that in smaller fish, and a significant difference
was identified among groups. This suggests that a
sufficient amount of protein is accumulated in larger fish to maintain normal physiological activity
and that protein might become the primary substrate to fuel basic metabolism activity. Fat is
another important energy storage substance in
fish. However, fat must first accumulate, so protein
will be consumed during this process. Metabolic
activity is intense at this time, but it gradually stabilizes as fish begin to adapt to their surroundings
(Rios, Moraes, Oba, Fernandes, Donatti, Kalinin &
Rantin 2006).
Effects of smoltification size on the hormone
indexes of S. salar
The hypothalamuspituitarythyroid (HPT) plays
a significant role in the regulation of the neuroendocrine system (K
uhn, Geris, Geyten, Mol & Darras
1998). Thyrotropin-releasing hormone (TRH) is
transported to the adenohypophysis through the
hypophyseal portal system where it is applied to
the thyrotropic cells, thereby releasing thyroid
stimulating hormone (TSH). Under the effect of
TSH, a small amount of T3 and a lot of T4 are
generated. As soon as T3 and T4 are released into
the blood, they affect target cells, where they promote sugar, fat and protein metabolism (McKenzie,
Cataldi, Marco, Mandich, Romano, Ansferri,
Bronzi & Cataudella 1999). Results of this experiment suggest that T3 and T4 in the blood of
S. salar reached the highest level on day 56 before
falling since the first 6 weeks of smoltification and
that the activity of the HPT axis decreased as
amount of light increased. Similar findings were
reported for of Salvelinus fontinalis (Vijayan &
Leatherland 1990). The content of T4 decreases as
a result of its conversion to T3, which is triggered
by the reduction in food intake by S. salar under
changing light conditions or increasing concentration of cortisol in the blood. During smoltification,
energy transport and metabolism of S. salar might
result from self-regulation and self-adaptation in
response to the extended duration of lighting
(Pickerng & Duston 1983; Fast, Hosoya, Johnson
& Afonso 2008).
Cortisol is closely related to the stress reaction
in teleosts (Sumpter, Dye & Benfey 1986). Cortisol
is secreted when the fish body is under stress,
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Aquaculture Research, 2016, 113

which explains why the cortisol level is widely

accepted as the index that reflects the stress reaction of fish (Pelis & McCormick 2001). In this
work, the concentration of cortisol in groups SW1
and SW3 began to fall after 56 days because
S. salar gradually adapted to the extended duration of lighting, although the value slightly rose
on day 70. Barton, Peter and Paulencu (1980)
demonstrated that the concentration of plasma
cortisol in fish will significantly rise under external
environmental stimuli but that the level will
decline and approach the previous level over time
increase as the fish begin to adapt to the surrounding environment. Cortisol keeps energy
metabolism primarily operating via sugar metabolism, maintains a continuous supply of glucose to
vital organs and promotes the degradation of fat,
thereby compensating for energy consumption in
the early stage of the stress reaction and enhancing the defence capability of organisms (Thomas &
Robertson 1991).
In conclusion, the smaller S. salar in the SW3
group were not at a suitable size to begin smoltification under the conditions used in this experiment. Once smoltification began, growth and the
increase in physiological indexes of fish in group
SW2 were relatively fast, but the growth and some
blood biochemistry indexes in SW1 were not better
than those in SW2. So S. salar weighing 24 g
should be selected for the smoltification, thereby
optimizing the culture conditions and increasing
the culture yield per water body at lower cost.
Acknowledgments
This research was supported by the National Natural Science Fund of China (No. 31472312,
41306152, 31402283), the Qingdao Innovation
Talents Program (13-CX-16), the National Key
Technologies R&D Program (2014BAD08B09)
and the earmarked fund for the Modern Agroindustry Technology Research System (CARS-48).
We are also indebted to Tian Huiqin for assistance in measuring fish and Wang Chaoxi and
Dr. Chi Liang for their kind comments about our
experiments.
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