Aquaculture 568 (2023) 739299
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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture
Comparative assessment of plasma cortisol and fecal corticoid metabolites
(FCM) of Atlantic salmon (Salmo salar L.) subjected to acute- and
long-term stress
Jingwen Ding a, *, Bengt Finstad c, Lars Christian Gansel a, Ann-Kristin Tveten a,
Steffen Hageselle Blindheim b, Yanran Cao a
a
Department of Biological Sciences Aalesund, Faculty of Natural Sciences, Norwegian University of Science and Technology, Larsgardsvegen 2, 6009 Aalesund, Norway
The Industrial and Aquatic Laboratory (ILAB), Thormøhlensgate 55, N-5006 Bergen, Norway
c
Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Sampling stress
Long-term stress
Plasma cortisol
Fecal corticoid metabolites (FCM)
Atlantic salmon
Farmed Atlantic salmon (Salmo salar L.) are subject to a variety of stressors throughout production. Elevations of
cortisol level in the blood are one of the major endocrine primary stress responses in vertebrates and are widely
used as stress indicator. However, blood sampling is invasive and stressful procedures. Cortisol instantly released
into the blood at sampling can easily interfere with the initial stress response of interest. Fecal corticoid me­
tabolites (FCM) have been suggested for a less invasive assessment of stress in fish. In the present study, we
evaluated stress responses by using plasma cortisol and FCM as stress indicators in Atlantic salmon. Atlantic
salmon (with average weight 42 g) were exposed to 3 different stressors: 1) parr-smolt transformation; 2)
infestation with salmon lice (Lepeophtheirus salmonis) for two-weeks; 3) infection with infectious salmon anaemia
virus (ISAV) for four weeks. The results demonstrated that FCM levels correlated well with the plasma cortisol
levels at single-point sampling during long-term stress (p < 0.05). Significant increases of both plasma cortisol
and FCM were found two weeks after initiated 24 h light. Each tank was sampled twice, with around 40-min
interval. The chasing during the first sampling was used as an acute stressor. The effects of sampling proced­
ures on plasma cortisol and FCM levels are compared. Blood and feces were analyzed by an Enzyme-Linked
Immunosorbent Assay (ELISA). Plasma cortisol and FCM levels increased within 40 min after fish perceived
an acute stressor when the average weight of fish was below 100 g. Attenuated cortisol stress responses to acute
stress were found after fish experiencing long-term stress. Collectively, FCM reflects changes in plasma cortisol
and can be an alternative to analysis of plasma cortisol in Atlantic salmon, complementing evidence of using FCM
as stress indicators for monitoring and improving fish welfare. The results highlight that standard sampling
procedures and good experimental designs should be established to ensure robust and repeatable results in the
study of stress based on the FCM measurement. Future study will investigate representative timepoints for FCM
sampling, with particular attention to the types of stress and sizes of the fish.
1. Introduction
Atlantic salmon is the most productive product in the Norwegian
aquaculture industry. Norwegian salmon production was almost 1.5
million metric tons for human consumption in 2021, with a first-hand
value of 76 billion NOK (Directorate of fisheries, 2022). Traditionally,
smolts stay in open net-pens in the sea for grow-out until they are
slaughtered. Fish in net cages are in direct interaction with the sur­
rounding environment, and different environmental conditions and
production factors can disturb the homeostasis of fish. The well-being of
fish can deteriorate and lead to compromised health and performance,
when disturbances are overly severe, prolonged or repeated (Barton,
2002; Moberg, 2000). Several studies demonstrate that poor welfare can
result in low growth rate, immunosuppression, and high mortality in
salmon aquaculture (Calabrese et al., 2017; Huntingford et al., 2006;
Oliveira et al., 2021). To achieve sustainable growth in aquaculture
industry, the well-being of fish should be considered inevitably. Mean­
while, it is important to assess the severity of stressors and prevent
* Corresponding author.
E-mail address: Jingwen.ding@ntnu.no (J. Ding).
https://doi.org/10.1016/j.aquaculture.2023.739299
Received 11 July 2022; Received in revised form 19 November 2022; Accepted 22 January 2023
Available online 24 January 2023
0044-8486/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
J. Ding et al.
Aquaculture 568 (2023) 739299
stressing situations from happening.
Several types of indicators are used for quantifying stress in fish,
based on measurements of pre- and post-stressor levels of the indicators.
The levels of stress hormones in blood become reliable and direct indi­
cator to evaluate stress level of the fish, since they are the products of the
primary stress responses. When fish perceive a stressor, catecholamines
from chromaffin tissue are released immediately, and the hypothalamicpituitary-interrenal (HPI) axis becomes activated. The activated hypo­
thalamus secretes corticotropin-releasing hormone (CRH) and other
secretagogues. CRH stimulates anterior pituitary gland into releasing
cortisol secretagogues in blood, primarily adrenocorticotrophic hor­
mone (ACTH). ACTH further activates the releases of glucocorticoid
hormone cortisol from interrenal cells within the head kidney into the
blood circulation (Barton, 2002; Gamperl et al., 1994; Mommsen et al.,
1999). Unlike for catecholamines, there is a time lag from synthesizing
cortisol to it entering the blood circulation system. Therefore, it is
feasible to measure cortisol baseline levels in fish. Stress hormones bind
to their receptors in the cell of target tissues, evoking the secondary
stress responses that enable fish to cope with stress. For example,
cortisol mobilizes the energy reserves, by increasing gluconeogenesis,
which releases more energy through substrate-level phosphorylation
and oxidation reactions to fish (Kuo et al., 2015; Mommsen et al., 1999).
Circulating cortisol level increases significantly as response to different
physical, chemical, and biological stressors, and it is widely used as an
acute stress indicator (Sundh et al., 2010). However, circulating cortisol
level starts to increase within minutes after a stressful situation (Marco,
2001), and sampling fish from commercial production cages can be
time-consuming. Fish are captured often by using large dip nets or nets
with weighted bottom ropes. Capture effects are hardly avoided, since
fish are crowded and exposed to the air before they can be anesthetized
and blood samples taken. Therefore, sampling procedures themselves
can influence the plasma cortisol level when sample size is too large, or/
and long handling time before fish can be anesthetized and blood can be
sampled. Meanwhile, blood sampling itself is an invasive procedure and
it will affect fish welfare.
Other methods have been developed for measuring cortisol, to
minimize interference from sampling procedures themselves and extend
valid sampling time when using cortisol level as stress indicator in
aquaculture industry. Cortisol will be metabolized to corticoids metab­
olites (glucuronidated and sulphated cortisol in fish), via a series of
metabolism and inactive processes, diffusing out of fish mainly through
the hepato-biliary-fecal/urine route (Mommsen et al., 1999; Sadoul and
Geffroy, 2019). Unbound cortisol can be also excreted from fish by
passive diffusion though gills (Scott and Ellis, 2007). So far, fish scales,
feces, urine, and mucus are validated non-invasive matrices for
measuring cortisol level in fish (Aerts et al., 2015; Cao et al., 2017;
Fernández-Alacid et al., 2019; Sadoul and Geffroy, 2019). Feces can be
collected from individual fish by using simple, low- or non-invasive
methods (Cao et al., 2017). The level of fecal corticoids metabolites
(FCM) seems to be relatively stable, as it does not degrade significantly
at room temperature during a 16-h test, which could lower requirements
of transporting and storing before analysis (Reimers et al., 1982; Turner
Jr et al., 2003). It is also proven that FCM keep strong positive corre­
lation with cortisol level in plasma, which indicates FCM can represent
the stress situation of fish (Cao et al., 2017). FCM develops through the
actions of bile and intestinal mixing, representing the integrated average
of a short-term stress levels in the blood, rather than estimates stress
level at blood sampling point. Feces turn out to be the most promising
matrix for long-term monitoring of stress in fish farms, after comparing
storage requirements and invasiveness of sampling procedures of
different matrices on measuring cortisol level. However, to the best of
our knowledge, no study so far demonstrates whether FCM keeps stable
during sampling, and the measurements of FCM eliminate potential
misleading acute cortisol spike from the sampling itself.
Plasma cortisol has limitations on demonstrating whether fish is
stressed under long-term stress situations (Madaro et al., 2015;
Pickering and Pottinger, 1989). Several studies demonstrate that when
fish experience stress over long time periods, plasma cortisol no longer
represents stress level of fish since there is no significant differences
from their baseline level, and plasma cortisol level is less sensitive to the
acute stress events after fish experienced long-term stress (Madaro et al.,
2015). Although there is no a consensus endocrine profile of FCM for
chronically stressed animals, most researchers expect increased levels of
FCM (Dickens and Romero, 2013; Palme, 2019). The potential role of
FCM in evaluating fish stress level under long-term stress is unknown
yet. The present study aims to compare plasma cortisol and FCM levels
in Atlantic salmon related to sampling procedure and three long-term
stress situations. We hypothesize that there are no significant differ­
ences between long-term stress groups and pre-stress groups in the
plasma cortisol levels. FCM levels are significantly correlated with
plasma cortisol levels in the groups without long-term stressors and
sampling itself has less effect on FCM level than on plasma cortisol level.
2. Materials and methods
2.1. Animal husbandry
A total of 270 Atlantic salmon (strain Stofnfiskur, Benchmark Ge­
netics, Iceland) were used in this study. The experiment was conducted
at the Industrial and Aquatic Laboratory (ILAB) in Bergen, Norway, and
it lasted ~15 weeks from the beginning of August until the end of
November in 2020. The fish arrived at the facility weighing approxi­
mately 42 g, and they were assigned at random to three replicate 450 L
tanks. Fish stayed in their original tanks throughout the experiment,
except for 60 individuals (20 fish from each triplicated tank) that were
moved to a new tank for challenge with salmon lice copepodids.
Stocking density was between 7.3 kg/m3 to 13.6 kg/m3 during the
experiment. All environmental parameters were standardized and
optimized for all tanks. The water flow was kept at 800–1000 L/h and
water temperature was 12–14 ◦ C over the course of the experiment. In
the lice-infested tank, the water flow was reduced to 600–700 L/h about
30 min during lice challenge, then the flow was increased back to
normal. Water temperature and other environmental parameters were
the same as among tanks. The fish were fed excessively with 24 h
automatic feeding and were kept under 24 h continuous light, after a
14–days acclimatization period with 12:12 light.
The study was part of a commercial experiment, which was approved
by the Norwegian Food Safety Authority (FOTS ID: 24246). The exper­
iment was in accordance with guidelines of Directive 2010/63/EU and
the Norwegian laws and regulations on animal experimentation in sci­
entific research. All animal procedures were approved by the Norwegian
Food Safety Authority based on the guidelines of the Norwegian Animal
Welfare Act.
2.2. Experimental design
This study consisted of three stress-events as shown in Fig. 1: parrsmolt transformation, sea lice (Lepeophtheirus salmonis) challenge and
infectious salmon anaemia virus (ISAV) infection. After a two–weeks
acclimation period, all fish went through parr-smolt transformation
together. Parr-smolt transformation was induced artificially by using 24
h continuous light for four weeks. Then fish were transferred to 34 ‰
seawater and stayed in seawater until the trial ended. Five weeks after
launching seawater transfer, the remaining fish were divided into two
groups for bath challenge with either salmon lice (strain LsGulen, pro­
duced by ILAB) or ISAV (produced by the Norwegian Veterinary Insti­
tute). For the salmon lice infestation group, 20 fish from each of the
three replicate tanks were transferred to a new tank, where they were
exposed to salmon lice copepodids around 30 min at reduced water level
(one-third of normal), with water flow-through but no extra oxygenation
supply. 30 copepodids per fish were used to challenge fish (average
weight of fish was 84.5 g) as described by Hamre et al. (2009). 14 days
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Aquaculture 568 (2023) 739299
Fig. 1. Schematic diagram of the experimental setup and sampling groups. The above part demonstrates the timeline of three stress-challenges and other operations.
The red markers indicate the time point of conducting stress events. One black scale unit represents one week period. The bottoms black arrows point out the
sampling groups in the timeline, including two control groups (C1 and C2) and four stress groups (S1, S2, S3 and S4). Except for stress group 3, sampling procedures
demonstrate that 3 fish were sampled at each time and 6 fish were sampled from each triplicate tanks for five sampling groups. For the stress group 3, 18 fish were
sampled 6 times from the same tank and 3 fish were sampled at each time. The blue arrow in the sampling procedures present the sampling order. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
post infestation (dpi) with salmon lice, fish were sampled for the stress
level measurement. For the ISAV infection group, the remaining fish
were kept in their original tanks and were bath-infected with 15 mL of
106.3 TCID50 /mL ISAV in 150 L seawater. Fish were sampled to monitor
their stress level on the 28 days after ISAV infection.
were collected from the caudal vein using EDTA (ethyl­
enediaminetetraacetic acid) vacutainers (VACUETTE® TUBE, Greiner
bio-one, Austria), centrifuged for 10 min at 3040 ×g, and plasma was
removed and stored at − 80 ◦ C until cortisol analysis. Gut contents were
used as feces sample in this experiment to avoid affecting gut and in­
testinal samples for histology and other analysis in the experiment. Gut
contents from each fish were collected by using a lab spatula to gently
press from beginning of middle intestine to posterior after fish dissec­
tion. Feces samples were stored at − 80 ◦ C prior to analysis.
2.3. Design of sampling point
Samplings were performed at 6 sampling groups, and 18 fish were
sampled for blood and feces per sampling group (Table 1): one week
before initiating parr-smolt transformation(Control group 1; C1), two
weeks after initiating 24 h light (Stress group 1; S1), one week after
salmon launching seawater period (Stress group 2; S2), one day before
infection challenge with salmon lice or ISAV (Control group 2; C2), two
weeks after salmon lice infestation (Stress group 3; S3) and four weeks
days after infection with ISAV (Stress group 4; S4). For the group S3, 18
fish were netted from one tank 6 times consecutively and 3 fish were
sampled each time. For the other five sampling groups, 18 fish were
sampled from three replicated tanks (6 fish per tank), each tank had
been sampled two times consecutively with 3 fish at each sampling
point. Therefore, each sampling groups had two sampling points, except
for the salmon lice infestation tank. Fish from the first sampling point
were without disturbing from sampling effects and fish from the second
sampling point were with disturbing from sampling effects (Fig. 1). For
all 6 sampling groups, the time interval between each netting out was
kept around 40 min. Fish were euthanized immediately with an over­
dose of Finquel vet. (Tricaine mesylate, 0.18 g/L, MSD Animal Health).
Fork length and wet weight of each fish were recorded. Blood samples
2.4. Cortisol measurement
Analysis of cortisol concentration in plasma and fecal samples was
performed by using a commercial ELISA kit (Cortisol ELISA KIT; Neo­
gen® Corporation, KY, USA). All samples were prepared according to
the manufacturer’s instructions. Plasma cortisol was extracted by add­
ing 1 mL diethyl ether (AnalaR NORMAPUR® ACS, VWR) to 100 μL
plasma in a 2 mL microcentrifuge tube. The sample and solvent were
vortex mixed for two minutes, and centrifuged (16,900 ×g for five mi­
nutes) to allow the phases to separate. The bottom aqueous phase was
removed, the organic phase was left in the original tube and evaporated
at room temperature under a stream of nitrogen. The residue was dis­
solved in 300 μL of diluted extraction buffer (extraction buffer from the
commercial ELISA kit was 5-fold diluted with deionized water) before
plasma cortisol was measured. The FCMs were extracted based on the
method developed by Cao et al. (2017). One ml 100% methanol (purity
≥99.9%, hypergrade for LC-MS, LiChrosolv®) was mixed with 100 mg
solid feces in a 2 mL microcentrifuge tube, the sample and solvent were
vortex mixed for two minutes, and then centrifuged (16,900 ×g for 5
min) to allow the phases to separate. The organic upper phase was
transferred into a new tube, and the solvent was evaporated with a
stream of nitrogen at room temperature. A 300 μL diluted extraction
buffer (extraction buffer from the commercial ELISA kit was 5-fold
diluted with deionized water) were added and vortexed for 2 min for
dissolving all residue in the tube. The extracts were kept at 4 ◦ C for
further measurement. Cortisol measurement was performed according
to the manufacturer’s protocol. Plasma cortisol and FCM extracts were
Table 1
Sampling groups with description of the timeline.
Control group 1 (C1)
Stress group 1 (S1)
Stress group 2 (S2)
Control group 2 (C2)
Stress group 3 (S3)
Stress group 4 (S4)
one week before initiating parr-smolt transformation.
two weeks after initiating 24 h light.
one week after salmon launching seawater period.
one day before infection challenge with salmon lice or ISAV.
two weeks post salmon lice infestation.
four weeks post ISAV infection.
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Aquaculture 568 (2023) 739299
further diluted until the cortisol concentration was within the analytical
range, by adding extracts into diluted extraction buffer in the microtiter
plates and mixing with pipettes. Both plasma and feces samples were
analyzed in duplicate. To stop the reaction, 50 μL of 1 N HCl were
applied to each well and the absorbance was measured at 450 nm using a
Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scienti­
fic). Cortisol concentration was calculated based on the standard curve
on each plate and the dilution factors for each sample.
course of the experiment (p < 0.01). Resting cortisol level of pre-smolts
(C1) and post-smolts (C2) samples were compared, no statistically sig­
nificant difference was found either in plasma or in feces matrix (p >
0.05). There was a significant increase on both plasma cortisol and FCM
during parr-smolt transformation, with their peak values at S1, with
64.3 ng/mL and 334 ng/g, respectively. One week after salmon
completed parr-smolt transformation (S2), plasma cortisol dropped to
41.1 ng/mL, and there was a statistically significant difference between
C1 and S2. Although FCM level at S2 was higher than two control groups
(C1 and C2), statistically significant differences were only found be­
tween S2 and C1 sampling groups for FCM. Both plasma cortisol and
FCM levels of salmon lice infested group (S3) were statistically signifi­
cant different from their respective control groups C1 and C2 (p < 0.01).
For the ISAV infection group (S4), there was a modest elevation on
plasma cortisol concentration (fold increase of 1.53 compared to C2),
while FCM level did not increase (from 39.5 ng/g at C2 to 35.2 ng/g at
S4). There were no statistically significant differences on either plasma
cortisol or FCM level compared with both baseline level (p > 0.05).
2.5. Statistical analysis
All statistical analyses were performed in R software™ version 4.0.4
(R Development Core Team, 2021). A p-value below 0.05 was used for
all tests for statistically significant difference. Firstly, outliers of data
and normality of data at group level were assessed by using Tukey’s
3.0*IQR method and Shapiro-Wilk test, respectively. If log trans­
formation proved to have insufficient power to achieve data normally
distributed, corresponding non-parametric test was used. Spearman’s
rank correlation coefficient was used to assess linear relationships be­
tween cortisol in feces and plasma, evaluating whether FCM was
correlated with cortisol in plasma. Kruskal-Walli’s test was used to
compare variance. Where applicable, dunn’s test of multiple compari­
sons were used to assess significant differences between groups. Data
with all samples from five sampling groups (C1, S1, S2, C2 and S4) were
analyzed by two-way ANOVA, to examine the influence of two inde­
pendent variables (sampling groups and sampling procedures) on
plasma cortisol and FCM, respectively. Differences between sampling
groups (C1, S1, S2, C2 and S4) demonstrated that the effect of stress
events on cortisol levels. All five sampling groups were sampled two
times with 40 min interval in between (two sampling points), differences
between two sampling points were used to demonstrate the effects of
sampling procedures on plasma cortisol and FCM levels.
3.2. Correlation between plasma cortisol and FCM
Plasma cortisol and FCM were analyzed for correlation in four cat­
egories. C1 and C2 were combined as one category for the control levels.
S1 and S2 were combined as one category for the parr-smolt trans­
formation levels. S3 and S4 were as two dependent categories for 14days sea lice infestation and 28-days ISAV infection levels, respec­
tively. Statistically significant positive correlations were identified be­
tween plasma cortisol and FCM (Spearman’s p < 0.05) in all four
categories (Table 2).
Table 2
Correlations (R) between plasma cortisol and FCM were analyzed in the control
levels (C1 and C2), the parr-smolt transformation levels (S1 and S2), the 14-days
sea lice infestation (S3) and in the 28-days ISAV infection (S4) levels.
3. Results
3.1. Effect of stress events on cortisol level
FCM
Plasma cortisol and FCM levels were analyzed from samples
collected from all six sampling groups, and the mean values ± standard
error of mean (SEM) for plasma cortisol and FCM are shown in Fig. 2.
Both plasma cortisol and FCM level varied significantly during the
Plasma cortisol
C1 & C2
S1 & S2
S3
S4
R = 0.64
R = 0.59
R = 0.84
R = 0.68
p = 0.6E-05
p = 0.00041
p = 1.3E-06
p = 0.0037
Fig. 2. Plasma cortisol (A.) and FCM (B.) level (mean
± SEM) of Atlantic salmon sampled under control
conditions, or after exposure to three different
stressors. Fish were sampled at six timepoints; C1: one
week before initiating parr-smolt transformation, S1:
two weeks after initiating 24 h light, S2: one week
after salmon launching seawater period, C2: one day
before infection challenge, S3: two weeks post salmon
lice infestation, S4: four weeks post ISAV infection.
Bar diagrams represent the mean cortisol values
(+/− ) standard error of mean (SEM). Lower-case
different letters indicate statistically significant dif­
ferences (P < 0.05), which was assessed by Wilcoxon
test.
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Aquaculture 568 (2023) 739299
and fourth sampling points and original fifth and sixth sampling points,
respectively. There were statistically significant differences only be­
tween the first and the third sampling point, to both plasma cortisol and
FCM levels (Fig. 4).
Only first three fish from each triplicate tank were used for evalu­
ating the effects of long-term stressors on plasma cortisol and FCM
levels, to eliminate the effects of sampling procedures on results (Fig. 5).
Statistically significant increase under long-term stress situations was
only found in the fish from S1 (p < 0.05). There was no statistically
significant difference between S1 and S2 on plasma cortisol levels,
although statistically significant decreases from S1 to S2 were found on
FCM levels. There were no statistically significant differences among
other sampling groups (C1, C2, S2, S3 and S4).
3.3. The impact of sampling on plasma cortisol and FCM level
Plasma cortisol and FCM level were compared between sampling
points to investigate the impact of sampling on plasma cortisol and FCM
levels. The impact of sampling to group S3 was analyzed separately from
other five groups, since sampling from the salmon lice infestation group
was different from other sampling groups. In the salmon lice infestation
group, 18 fish were collected from the same tank. In the other five
sampling groups, 18 fish were sampled from triplicated tanks. In all five
sampling groups, the median values of both plasma cortisol and FCM at
the second sampling point (D2, with dark grey boxplot in the Fig. 3)
were higher than their levels at the first sampling point (D1, with light
grey boxplot in the Fig. 3). Plasma cortisol levels of the second sampling
points were significantly higher than the first sampling points in C2 and
S4 (Wilcoxon test’s p < 0.01). The increase-rate of plasma cortisol levels
between two sampling points were 61% in group C1 and were lower
than 46% in groups S1 and S2. Plasma cortisol levels of the second
sampling point increased 640% and 270%, compared to the levels of the
first sampling points in C2 and S4, respectively. Statistically significant
differences between two sampling points were found in control groups
(C1 and C2) and S2 for FCM (Wilcoxon test’s p < 0.05). The increaserates of FCM between two sampling point were 56% and 177% in C1
and C2, respectively. The increase-rates of FCM between two sampling
point was 54% for S2. FCM levels increased lower than 26% at the
second sampling points, compared to their first sampling point in the
stress groups (S1 and S3). Two-way ANOVA indicated that stress events
and sampling procedures both affect plasma cortisol level significantly
(p < 0.001), and there were statistically significant differences between
sampling point 1 and sampling point 2 on plasma cortisol level (p <
0.001). Stress events had statistically significant effects on FCM levels (p
< 0.001), sampling procedures significantly affected FCM levels (p <
0.05).
In S3, both plasma cortisol and FCM level increased continuously
between each sampling point until the fifth sampling point, where the
peak levels of both plasma cortisol and FCM were found. To demonstrate
the effect of continuous sampling on plasma cortisol and FCM level,
sampling points were regrouped as three sampling points: original first
and second sampling points were combined as the first sampling point,
the second and third sampling points were composed of original third
4. Discussion
Atlantic salmon parr and smolts had cortisol baseline levels at 10.8
and 4 ng/mL, respectively. These values agree with the baseline levels
for smolts in other studies (Culbert et al., 2022; McCormick et al., 2007;
Pankhurst et al., 2008; van Rijn et al., 2020). Plasma cortisol levels in
parr were slightly elevated and might be explained by one week accli­
mation period too short for the fish fully recover. In the present study,
the baseline levels of FCM for both parrs and smolts were 22 ng/g.
Baseline levels of FCM from unextracted feces supernatant has been
reported to be 14.4 ng/g in Atlantic salmon (Cao et al., 2017). Further,
baseline levels of cortisol and corticosterone averaged 3.4 and 14.8 ng/g
in parrotfishes (Scarus sp. and Sparisoma sp.) (Turner Jr et al., 2003).
Basal plasma cortisol levels can vary with life stages, reproductive states,
the sex, and the different species of fishes (Sadoul and Geffroy, 2019). It
is unclear what factors that can modulate baseline levels of FCM in fish.
In present study, the results demonstrated that smaller differences be­
tween two baseline levels for FCM (both are 22 ng/g) than for plasma
cortisol, confirming that it is also feasible to measure basal FCM levels
and further apply into stress evaluation.
Parr-smolts transformation and increases in cortisol levels in plasma
are well established (McCormick, 2012; McCormick et al., 2007; Nilsen
et al., 2008). However, only few studies have so far addressed the
question of how this change is demonstrated on other matrices. After
being exposed to 24-h light for two weeks in the present study, there was
Fig. 3. Plasma cortisol (A.) and FCM (B.) level for 5 different sampling groups with two sampling points for each sampling group. Light grey (D1) is for first sampling
point and dark grey (D2) is for the second sampling point. Asterisks indicate the degree of significance (*: p ≤ 0.05, **: p ≤ 0.01) as assessed by Wilcoxon test.
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Fig. 4. Plasma cortisol (A.) and FCM (B.) level in salmon lice infection group with three sampling points. 6 fish were in each sampling points. The first sampling point
was composed of the original first and second sampling points, the second and third sampling points were composed of original third and fourth sampling points and
original fifth and sixth sampling points, respectively. Asterisks indicate the degree of significance (*: p ≤ 0.05, **: p ≤ 0.01) as assessed by Wilcoxon test.
Fig. 5. Plasma cortisol (A.) and FCM (B.) levels (mean ± SEM) of Atlantic salmon sampled with first three fish from each triplicate tanks under pre-stressed con­
ditions (C1 and C2) and three different long-term stress: after exposured to parr-smolt transformation (S1 and S2), salmon lice infestation (S3) and ISAV infection
(S4). Lower-case different letters indicate statistically significant differences (p < 0.05), which was assessed by Wilcoxon test.
a 5-fold increase in plasma cortisol levels, and cortisol levels in circu­
lation remained high up to 5 weeks after being exposed to 24 h light.
Plasma cortisol levels were followed by a gradual decrease after sea
water transfer. This observation of changes in plasma cortisol during
parr-smolt transformation is accordance with regular pattern of cortisol
responses in Atlantic salmon, with plasma cortisol level staying elevated
during the parr-smolt transformation and declining to baseline levels
after about one month in sea water (McCormick, 2012; McCormick
et al., 2007; Nilsen et al., 2008). Elevated cortisol levels play an
important role of changing the osmoregulatory capacity, developing of
seawater tolerance in osmoregulatory tissues (Björnsson et al., 2011;
Wendelaar Bonga, 1997). FCM presented a similar time course response
to plasma cortisol during the parr-smolt transformation period. The peak
values of FCM were found at the same time point as the peak plasma
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cortisol values, but with a 10-fold increase. This may be explained by
increased intestinal fluid transport during the parr-smolt trans­
formation, resulting in faster FCM accumulation and by the intestine
switching its role from preventing water inflow in freshwater environ­
ment to actively absorb ions and water in seawater environment (Sun­
dell et al., 2003). It should be noted that statistically significant
differences were found in FCM values between S1 and S2, but not in
plasma cortisol values. This may be explained by decreased cortisol
secretion and stimulated cortisol clearance, resulting in different accu­
mulation rates of FCM during parr-smolt transformation (Sundell et al.,
2003). To the best of our knowledge, present study demonstrated for the
first time that FCM levels correlate with plasma cortisol levels during the
parr-smolt transformation period, suggesting FCM levels are good sub­
stitutes for plasma cortisol levels to monitor fish welfare during the parrsmolt transformation period.
For long-term stress situations, several studies indicate that the
absence of clearly elevated levels of plasma cortisol limits the use of
plasma cortisol as a stress indicator (Fast et al., 2008; Madaro et al.,
2015; Pickering and Pottinger, 1989). Plasma cortisol levels of S3 (14
dpi with salmon lice (Lepeophtheirus salmonis)) and S4 (28 dpi with ISAV)
groups were 5.86 ng/mL and 11 ng/mL, respectively. There were no
significant elevations in plasma cortisol level in S3 and S4, compared to
pre-stressed groups (C1 and C2). Finstad et al. (2000) demonstrated that
cortisol levels were significantly higher in lice infested fish than control
fish throughout the 40-days sampling period. However, there were no
significant differences in our experiment. This may be explained by
lower number of lice per fish in our experiment (110 copepodids per fish
in Finstad et al. (2000)). As findings by Ross et al. (2000), increased
plasma cortisol was related with the number of louse on fish. Low
plasma cortisol level during ISAV infection may be explained by the long
infection period, resulting in elevated cortisol levels below peak levels
(Carbajal et al., 2019; Fast et al., 2008; Ross et al., 2000). These findings
are consistent with the results from several long-term stress studies in
fish, showing down-regulation of the HPI axis during chronic stress and
stress adaptation in fish (Carbajal et al., 2019; Fast et al., 2008; Madaro
et al., 2015). Increased cortisol reallocates energy distribution, helping
fish to adapt to stressful situations and maintain homeostasis (Mommsen
et al., 1999). Whereas long-term stress may induce a potential habitu­
ation from exhaustion or desensitization, easing up the function of
cortisol in stress responses. Therefore, single-point plasma cortisol may
not be a good assessment of the long-term stress responses in fish when it
is used as stress indicator. Further studies should consider that how long
the elevated plasma cortisol can stay during the long-term stress, and
when it will induce irreversible damage to fish. Meanwhile, other
matrices should be developed and validated when cortisol level is used
as single stress indicator for long-term stress events.
Furthermore, FCM levels of long-term stressed groups (S3 and S4)
were compared to those of pre-stress groups (C1 and C2), to check
whether FCM interprets the past HPI axis activity, based on the results of
Fig. 5. Chronically stressed fish presented higher FCM levels than prestressed levels (22 ng/g) in both 14 days after salmon lice infestation
(30 ng/g) and 28 days after ISAV infection groups (31.8 ng/g), although
there was no significant difference among these groups. Based on the
routes of metabolism and excretion of circulating cortisol, inactive
cortisol is principally excreted as conjugates via the kidneys into the
urine or via the bile into the feces (Touma and Palme, 2005). The time
lag between cortisol in circulation and in feces is related to gut passage
time (Palme et al., 1996), which indicates that feces may not be a good
matrix for accumulating corticoids metabolisms for the long term.
However, the FCM levels should be related to a two-stages process, se­
cretions cortisol into circulation system and the clearance rate. Faster
clearance of corticosteroids was found in the seawater period than in the
freshwater period in coho salmon (Oncorhynchus kisutch). The same
study also demonstrated that increased clearance rate of corticosteroids
from the blood when fish were under chronic stress situations (Redding
et al., 1984). In the present study, the plasma cortisol levels were twice
as high in S4 as in S3, while these two groups shared similar FCM levels.
It may be explained by long-term stressors changing the clearance rate
through the hepato-biliary-fecal route. More time points should be
addressed in the further study, to develop and validate FCM as stress
indictor in long-term stress situations. Meanwhile, long-term stress can
change gut physiology in humans, including negative effects on intes­
tinal microbiota (Konturek et al., 2011). Similar results were found in
Atlantic salmon showing that confinement long-term stress can change
the structure of gut microbiome (Uren Webster et al., 2020). Additional
studies should investigate whether specific corticoid metabolisms are
significantly changed by the microbiota and corelated with long-term
stress, instead of the total amount of corticoid metabolites.
Plasma cortisol and FCM values with and without sampling effect
were compared, to demonstrate how sampling procedure affect the re­
sults of stress levels. Sampling procedures biased the results of S2 and
S3. There were continuous increases in both plasma cortisol and FCM
values in 1.5-h sampling procedures. S2 and S3 demonstrated significant
difference from pre-stressed group, when sampling effect was included
(6 fish with two sampling points from each tank were used). No signif­
icant differences were found between long-term stressed group (S2 and
S3) and pre-stressed groups (C1 and C2), when sampling effect was
excluded (only first 3 fish were used). The results highlight the impor­
tance of sampling procedures, and repeated sampling should be avoided
to assure robust and repeatable results, as sampling can affect results.
Plasma cortisol and FCM value were higher in the fish groups
sampled secondly in relation to those in the fish groups sampled firstly.
With data from all sampling groups and two sampling points, both
sampling procedures (two different sampling points) and stress events
affected plasma cortisol levels significantly (p < 0.001). There were
significant differences of FCM values among the individual samples from
the first sampling point and the second one (p < 0.05). Stress events
themselves seems to be the main reason for the significant differences in
FCM between different sampling groups (p < 0.001). The time interval
between two sampling points was about 40 min, which resulted in sig­
nificant differences on plasma cortisol between two sampling points.
Increased plasma cortisol levels are in line with results of previous
studies that plasma cortisol reached peak levels around 30 min after
Atlantic salmon experienced acute confinement stress (Einarsdóttir and
Nilssen, 1996; Espelid et al., 1996; Fast et al., 2008; Gamperl et al.,
1994). We expected delayed increases in FCM levels in the second
sampling point, since the time lag between cortisol in circulation and in
feces is related to gut passage time (Palme et al., 1996). Feces should
extend the time window in which FCM levels are unaffected by the
sampling procedure and eliminate potentially misleading acute cortisol
spike caused by the sampling itself (Bosson et al., 2009; Palme et al.,
2005). One study demonstrated that content in the small intestine
incased 2 h post feeding and peaked 12 h post feeding in Atlantic salmon
(Aas et al., 2017). However, FCM levels elevated 40 min after the first
sampling, which was much earlier than we had expected. The gut pas­
sage time in fish varies with temperature, season, osmotic stress, activ­
ity, body size and metabolic rate (Aas et al., 2017; Jobling et al., 1977;
Tseitlin, 1980). Lower temperature and larger fish require longer gut
passage time in fish (Bromley, 1987; Jobling et al., 1977). The average
weight of fish was 82 g in the present experiment, while the average
weight of fish was 1131 g in Aas et al. (2017). The suggested digestibe
duration for fish has a postitive correlation with the fish weight based on
the equation in Tseitlin (1980). Inceased FCM levels between two
sampling points may also be explained by the sampling method. Instead
of stripping feces from the pelvic fins to the anus, feces samples were
collected from the middle intestine and posterior intestine segment. The
time lag between handling provoked stress responses and the reflection
on corticoid metabolisms in intestinal contents should be shorter (Aas
et al., 2017; Storebakken et al., 1999). Our results combined with other
studies which indicate intrinsic and external factors can modulate FCM
levels. Based on the result of this study, we will propose further study of
the time course and dynamics of FCM after fish perceive stress and
7
J. Ding et al.
Aquaculture 568 (2023) 739299
precise the adequate time window for FCM sample collection. The fish
size and the stress type could be the major factors that decide the time
window for sampling feces, further obtaining the representative levels of
FCM.
In the present study, plasma cortisol levels of the second sampling
points were higher than the first ones in all groups, but statistically
significant differences were only found between two sampling points
after fish completed parr-smolt transformation. This result is in line with
the study by Carey and McCormick (1998), which demonstrates that
Atlantic salmon smolts are more responsive to stress than parr. For FCM,
significantly higher values in the second sampling point groups were
detected in two pre-stress groups and S2. This may be explained by more
sensitive response to acute stressors when fish is under resting state.
Meanwhile, for pre-smolts and smolts respectively, their control groups
had higher increase-rates of both plasma cortisol and FCM between two
sampling points than their stressed groups. These results combine with
other recent studies that an acute stress challenge to the fish differen­
tiates whether fish is chronically stressed or not (Basrur et al., 2010; Fast
et al., 2008; Madaro et al., 2015; Samaras et al., 2021).
As expected, we identified a positive correlation between plasma
cortisol and FCM in all samples, confirming that FCM fluctuations reflect
cortisol levels in the circulation system. Feces can be collected by
manually stripping fish from the pelvic fins to the anus of fish (Cao et al.,
2017), and/or discharged feces can be collected from tanks directly
(Sundell et al., 2003). Both methods are much less problematic for the
fish than blood sampling. Meanwhile, it is feasible to sample feces from
euthanized fish. Large sampling amount or the limited working envi­
ronment usually require long sampling time, and it is difficult to collect
blood samples long after fish have been euthanized, due to blood
coagulation. Our results are consistent with other recent studies, sug­
gesting the FCM can be used as a non-invasive substitute of measuring
plasma cortisol in Atlantic salmon (Cao et al., 2017; Sadoul and Geffroy,
2019; Uren Webster et al., 2020).
As this study was part of a commercial experiment, we had limited
access to the experimental designs and sampling procedures. Besides
blood and feces sample, different types of tissue samples were collected
from individual fish. Therefore, the fish were sampled twice with around
40 min interval from each tank. To the best of our knowledge, this is for
the first time noticed that FCM levels had already increased in 40 min
after fish (average weight <100 g) perceived an acute stressor. Further
study will focus on the time course and dynamics of FCM when fish
perceive an acute stressor. The factors affect the clearance rate of FCM in
fish, with particular attention to the size of the fish, will be investigated.
salar L.) subjected to acute- and long-term stress”, contribute to the work
and agree with the author contribution in the cover letter.
Jingwen Ding: Conceptualization, Software, Validation, Formal
analysis, Investigation, Data Curation, Writing - Original Draft, Writing Review & Editing, Visualization, Project administration.
Yanran Cao: Conceptualization, Methodology, Validation, Investi­
gation, Resources, Writing - Review & Editing, Supervision, Project
administration, Funding acquisition
Lars Christian Gansel: Validation, Resources, Writing - Review &
Editing
Ann-Kristin Tveten: Conceptualization, Writing - Review & Editing,
Funding acquisition.
Bengt Finstad: Writing - Review & Editing.
Steffen Hageselle Blindheim: Investigation, Project administration,
Writing - Review & Editing.
Funding
This study was funded by Regional Research Fund Møre og Romsdal
(Grant numbers: 317833), China Scholarship Council (CSC) and Nor­
wegian University of Science and Technology (NTNU).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
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The current results demonstrated the feasibility of using FCM as the
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Authors statement
All authors, in the article of “Comparative assessment of plasma
cortisol and fecal corticoid metabolites (FCM) of Atlantic salmon (Salmo
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