Fish Physiol Biochem (2020) 46:1679–1698
https://doi.org/10.1007/s10695-020-00821-9
Integrated biomarker parameters response to the toxic
effects of high stocking density, CuSO4, and trichlorfon
on fish and protective role mediated by Angelica sinensis
extract
HuaTao Li & YuTing Ma & Ying Liu & Min Wu &
Jiao Long & XiaoQin Jing & SiShun Zhou & Ping Yuan &
Jun Jiang
Received: 6 November 2019 / Accepted: 14 May 2020 / Published online: 15 June 2020
# Springer Nature B.V. 2020
Abstract The present study explored the protective role
of dietary the extract of Angelica sinensis (EAs) on high
density, CuSO4, or trichlorfon-treated Crucian carp
(Carassius auratus auratus). Firstly, the study showed
that the optimum density for growth and growth inhibi-
tion was 0.49 and 0.98 fish L−1 water, respectively.
Dietary EAs relieved the high density–induced growth
inhibition in Crucian carp. The appropriate concentra-
tion of EAs for recovery of growth was estimated to be
4.30 g kg−1 diet in high-density fish. Moreover, high
density decreased both digestive and absorptive enzyme
activities and increased lipid oxidation in digestive or-
gans, suggesting the ability of high density to induce
oxidative damage. However, dietary EAs inhibited the
oxidative damage through elevating ROS scavenging
ability and enzymatic antioxidant activity in digestive
organs. Secondly, our data demonstrated that the appro-
priate concentration of CuSO4 to induce the decrease in
H. Li (*) : Y. Ma : Y. Liu : J. Long : X. Jing : S. Zhou :
P. Yuan
Key Laboratory of Sichuan Province for Conservation and
Utilization of Fishes Resources in the Upper Reaches of the
Yangtze River, College of Life Sciences, Neijiang Normal
University, Neijiang 641100 Sichuan, China
e-mail: huataoli217@126.com
M. Wu
Archives, Neijiang Normal University, Neijiang 641100 Sichuan,
China
J. Jiang
College of Animal Science and Technology, Sichuan Agricultural
University, Chengdu 611130 Sichuan, China
feed intake (FI) was 0.8 mg Cu L−1 water. Dietary EAs
returned to FI of Crucian carp treated with CuSO4. The
appropriate concentration of EAs for recovery of FI was
estimated to be 4.25 g kg−1 diet. Moreover, dietary EAs
suppressed the CuSO4-induced decrease in digestion
and absorption capacity and increase in protein metab-
olism in digestive organs of Crucian carp. Finally, the
present results suggested that dietary EAs inhibited the
trichlorfon-induced rollover (loss of equilibrium) in
Crucian carp. The appropriate concentration of EAs
for inhibition of rollover was estimated to be
4.18 g kg−1 diet. Moreover, trichlorfon stimulated not
only the decrease in energy metabolism but also lipid
and protein oxidation, suggesting that trichlorfon caused
loss of function and oxidative damage in muscles of
fish. However, dietary EAs improved muscular function
and inhibited oxidative damage via quenching ROS and
elevating non-enzymatic and enzymatic antioxidant ac-
tivity in muscles of trichlorfon-induced fish. So, EAs
could be used as an inhibitor of high density, CuSO4,
and trichlorfon stress in fish.
Keywords Angelica sinensis . Stocking density .
CuSO4 . Trichlorfon . Crucian carp
Introduction
Fish farming at a high stocking density is actually to
maximize the utilization of production infrastructure of
fish, so as to improve farm profitability (Wei 2011).
However, as reported in some studies, it seems that high
1680
stocking density can inhibit the growth of fish consid-
ering the reductions in feed consumption (Li et al.
2012). Feed consumption is closely associated with the
digestion and absorption of fish (Gisbert et al. 2009). So,
it is possible that high stocking density caused the
decrease in digestive and absorptive capacities that sup-
presses the feed consumption as well as growth in fish.
The effects of high stocking density on digestion and
absorption might be related to oxidative-antioxidant
status in the digestive organs of fish. According to some
reports, high stocking density leads to crowding stress in
fish (Li et al. 2012). One common feature from stresses
is the production of reactive oxygen species (ROS)
(Chen and Dickman 2005), which is capable of reaction
with the most biologically important molecules at a high
rate, thereby leading to cellular oxidative damage in
aquatic animals (Lushchak 2011). Therefore, high
stocking density might induce oxidative damage to di-
gestive organs that led to decreased digestion and ab-
sorption capacity as well as feed usage in fish. Even so,
few studies have analyzed how high stocking density
affects fish digestive organs. So, it is important to ex-
pand our knowledge about the effect of high stocking
density on digestive organs and how to protect against
the high stocking density–induced reductions in feed
consumption and growth in fish.
As demonstrated by studies, high stocking density can
cause the low efficiency of immunological responses
(Marco et al. 2008) and increase the number of opportu-
nistic pathogens (Ellis 2002), thus resulting in the overuse
of disinfectants and pesticides in aquaculture systems
(Harikrishnan et al. 2011). Copper sulfate (CuSO4) is often
applied to contain pathogen infections (Sun et al. 2014).
The hepatopancreas and intestine serve as the most impor-
tant digestive organs for fish to digest and absorb nutrient
from feed (Hong et al. 2015). Nevertheless, according to
related reports, Cu performs the function of inducing ROS
to be overproduced and can be mainly accumulated in
hepatopancreas and intestine of fish (Bopp et al. 2008).
Moreover, the exposure to CuSO4 for 4 days induces
oxidative damage in the abovementioned two organs
(Jiang et al. 2011), and the exposure to CuSO4 for 7 days
reduces the feed consumption and weakens the growth
performance in fish (Boeck et al. 1997). Thus, it is possible
that the digestion and absorption capacity is reduced by the
CuSO4-mediated oxidative damage in the digestive organs,
as a result of which the feed consumption is lowered and
the growth is weakened in fish. In spite of this, there are
only limited information with regard to how CuSO4 affects
Fish Physiol Biochem (2020) 46:1679–1698
the digestion and absorption of fish. So, we shall improve
our understanding about the effect of CuSO4 on the diges-
tion and absorption capacity and the way to prevent the
CuSO4-induced reductions in feed consumption in fish.
In the aquaculture system, as a type of the organophos-
phorus pesticides (OPPs), trichlorfon has been met with a
wide range of applications in the control of parasitic
organisms (Chang et al. 2006). The skeletal muscles play
an essential role in body movement for animals (Johnston
et al. 1977). Nevertheless, as reported, trichlorfon is con-
centrated in the muscle (Shen et al. 2009) and is capable to
cause balance loss in fish (Li et al. 2019c). The effects of
trichlorfon on the balance of fish might be related to
oxidative damage in muscles of fish. Based on the relevant
studies, due to trichlorfon, ROS is overproduced (Xu et al.
2009) and oxidative damage is caused in the hepatopan-
creas of fish (Li et al. 2019c). Therefore, trichlorfon might
induce the oxidative damage to muscles, which weakens
the balance capacity of fish. Even so, only little informa-
tion focuses on studying how trichlorfon affects the
oxidative-antioxidant status in the muscle of fish. There-
fore, we shall make effort to improve our understanding of
how trichlorfon causes the oxidative damage in muscles
and find the way to prevent trichlorfon from negatively
affecting muscular function in fish.
Angelica sinensis (As), the Chinese angelica, serves as
a famous medicinal herb and a kind of food material which
has various pharmacological activities (Yeh et al. 2014).
Based on our previous study, dietary the extract of As
(EAs) is beneficial to promote the capacity of the growth,
digestion, and absorption in fish (Li et al. 2019b). On that
account, it is reasonable to speculate that EAs have the
function of mitigating the high stocking density–induced
changes in fish growth. However, there are few studies
focusing on analyzing the effect of EAs on fish at high
density. It has been reported that dietary EAs inhibits
CuSO4-mediated oxidative damage in digestive organs
of fish (Li et al. 2019a). Therefore, it is possible that EAs
suppress CuSO4-induced reductions in feed consumption
in fish. However, the information on the effects of EAs on
digestion and absorption in CuSO4-treated fish is limited.
Besides, studies have revealed that dietary EAs quench the
trichlorfon-induced functional damage by improving the
antioxidative capacity in the digestive organs of fish (Li
et al. 2019c). Thus, it is speculated that EAs have the
function of scavenging trichlorfon from the negatively
affected muscular function in fish. Nevertheless, little in-
formation can be found that focus on the effects exerted by
EAs on the muscles of trichlorfon-treated fish.
Fish Physiol Biochem (2020) 46:1679–1698
How EAs affect the growth, digestion, and absorp-
tion or the antioxidant status in high density, CuSO4, or
trichlorfon-treated fish was explored in the present
study. The study is aimed at evaluating the anti-stress
effects of EAs on fish. It seems that the study results can
contribute to the use of EAs to inhibit the stress of fish.
Materials and methods
Chemicals
Cyclohexane, ethyl ether, and acetone were of analytical
grade and provided by the Chengdu Kelong Chemical
Reagent Factory (Chengdu, Sichuan, China). Copper
sulfate pentahydrate (CuSO4·5H2O, analytical grade)
was provided by the Shanghai Chemical Reagent Fac-
tory (Shanghai, China). Folin-ciocalteu reagent was ob-
tained from Hefei Bomei Biotech. Co., LTD (Hefei,
Anhui, China). Gallic acid (≥ 99%) was purchased from
Shanghai Ekear Biotech. Co., LTD (Shanghai, China).
Trichlorfon (≥ 90%) was bought from the Shanghai
Biochemical Reagent Co., LTD (Shanghai, China).
Quercetin, kaempferol, and ferulic acid (≥ 99%) were
bought from the Sigma-Aldrich Co., LLC (St. Louis,
MO, USA). Other chemicals were of analytical grade.
Preparation of EAs
The roots of As were sourced from the Chengdu Pharma-
ceuticals market of China (Chengdu, Sichuan, China). The
identification of botany was performed in the Neijiang
Normal University, where researchers assigned the vouch-
er samples a reference number and deposited them subse-
quently. The dried herb was ground to a powder using a
Chinese medicine mill (Ronghao RHP-2000A, Zhejiang,
China). And then, 50 g of the powder was sequentially
extracted with 450 ml of cyclohexane, ethyl ether, acetone,
and water at 20 °C for 8 h using an Agitator (Dalong
OS40-S, Beijing, China), in accordance with the recom-
mendation of Dalar et al. (2014) and Li et al. (2016). The
extraction was repeated three times under the same condi-
tions. After filtration, the solutions were concentrated and
dried using a rotary evaporator (Jinye RE-52CS,
Shanghai, China) until constant mass was achieved. Fol-
lowing extraction, the dry cyclohexane extract (CHE),
ethyl ether extract (EEE), acetone extract (AE), and aque-
ous extract (AQE) were kept in dark place in sealed bottles
and preserved at − 80 °C before use.
1681
Determination of total phenolic content
The phenol content of EAs was quantified according to
the method of Yuan et al. (2005). The phenol content of
EAs was expressed as gallic acid equivalents. Three
replicates were prepared for each sample. The yield rate
and phenolic content of EAs are showed in Table 1. The
EEE presented the maximum content of phenolic com-
pounds among the extracts examined (P < 0.05).
The composition analyses of EAs
A saturated solution was freshly prepared by dissolving
EEE in methanol (chromatographic grade). The saturated
solution was injected to high-performance liquid chroma-
tography (HPLC) for quantitative analysis of ferulic acid,
quercetin, and kaempferol before being filtrated by germ
filter with 0.22-μm hole as described by Zhao et al.
(2003), with slight modifications. Standards of ferulic
acid, quercetin, or kaempferol (1 mg) were weighed and
dissolved in 1 mL of methanol to give serial concentra-
tions, and three injections were performed for each dilu-
tion. The standard curves were calibrated by using the
linear least-squares regression equation derived from the
peak area. The concentrations of ferulic acid, quercetin,
and kaempferol in the sample were calculated according
to the regression parameters derived from the standard
curves. The HPLC (Agilent 1100 HPLC, Wilmington,
DE, USA) was installed with an Agilent Zorbax SB C18
column (250 mm × 4.6 mm i.d., particle size = 5 μm).
Solvents were (A) 0.5% formic acid in water and (B)
100% methanol. The gradient of A:B was 0–45 min, 95:5
gradients up to 5:95. The flow rate of mobile phase was
kept constant at 1.0 mL min−1. Peaks were detected at
254 nm (Fig. 1, Table 2).
Table 1 Yield rate and phenolic content of cyclohexane extract
(CHE), ethyl ether extract (EEE), acetone extract (AE), and aque-
ous extract (AQE) of Angelica sinensis
Extracts Yield (g kg dry herb−1) Phenols (mg g dry extract−1)
CHE 54.05 ± 3.12b 26.50 ± 1.76c
c
EEE 62.18 ± 3.66 48.28 ± 2.81d
a
AE 43.54 ± 2.47 17.76 ± 1.21a
d
AQE 106.50 ± 6.07 21.09 ± 1.34b
Values are means ± S.D. of 3 replicates. Values in the same
column with different superscripts are significantly different
(P < 0.05)
1682 Fish Physiol Biochem (2020) 46:1679–1698
DAD1 A, Sig=254,10 Ref=off (SSS\20200117000009.D)

40.255
mAU

1600

37.734
1400

1200

1000

800

40.575
600

22.352 - ferulic acid

44.488
400

33.472 - kaempferol
30.858 - quercetin

44.005
200

39.504

44.250
36.066

41.986
42.233
26.641

38.120

43.292
41.327
41.698
37.045

38.777

42.710
35.772
17.135

35.451
10.821

11.880

17.834

23.354

35.113
14.984

16.789

19.410
19.889

24.797
25.318
25.676

27.882

29.295
30.231
31.219
32.206
32.825

34.388
22.906

28.390
29.018
0

0 5 10 15 20 25 30 35 40 min

Fig. 1 The high-performance liquid chromatography (HPLC) of ethyl ether extract (EEE) of Angelica sinensis. This experiment was
repeated three times with similar results achieved

Experimental fish and diets Li et al. (2019b) was applied for feed production
(Table 3). Table 1 lists the formulation of basal diet.
Crucian carps (Carassius auratus auratus) were provid-
ed by a fish farm in Neijiang (Sichuan, China) and have
adapted to the conditions of lab (22.0 ± 1 °C, natural Stocking density trial
photoperiod). Water has an index sign that meets the
Standards for Drinking Water Quality (GB5749-2006). Density assays
Water was flowing and constant aerated by YQJ-700G
pumps (Shanghai, China). We formulated 7 experimen- The trial was conducted based on the procedures proposed
tal diets based on previous study (Li et al. 2017). The by Papoutsoglou et al. (2006), which were modified slight-
basal diet was comprised of 34.82% crude protein to- ly. A total of 720 Crucian carp (the initial weight averaged
gether with 5.52% crude lipid. In EEE diets, the basal 8.0 ± 0.2 g) that were obtained from the acclimatization
diet was added with EEE premix for providing 0.0, 1.0, aquariums were divided into 8 treatment groups randomly,
2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 g EEE kg−1, with reduced each of which was equipped with 4 replicate aquariums
amount of cellulose as a compensation. The method of (plastics, rectangle, and 31-L capacity). Rearing densities
in 8 treatment groups were 5, 10, 15, 20, 25, 30, 35, and 40
Table 2 The composition analyses of ethyl ether extract (EEE) of fish aquarium−1 (the equivalent of 161.3, 322.6, 483.9,
Angelica sinensis by high-performance liquid chromatography 645.2, 806.5, 967.7, 1129.0, and 1290.3 fish m−3 or 1.29,
(HPLC) 2.58, 3.87, 5.16, 6.45, 7.74, 9.03, and 10.32 kg m−3),
Compound Retention Peak area (mAU × s) Amount (%) respectively. Fish in each treatment group were fed the
name time (min) basal diet for 56 days and for six times on a daily basis
(08:00, 10:00, 12:00, 14:00, 16:00, and 20:00 h) and were
Ferulic acid 22.352 1275.622 0.355 observed carefully to ensure the apparent satiation without
Quercetin 30.858 326.733 0.040 overfeeding. The collection of uneaten pellets was per-
Kaempferol 33.472 47.587 0.008 formed after 30 min of manual feeding, followed by
This experiment was repeated three times with similar results drying and weighting (Li et al. 2012). Feed intake (FI),
achieved as well as mortality, was recorded daily. Dead fish were
Fish Physiol Biochem (2020) 46:1679–1698 1683

Table 3 Composition and nutrients content of the basal diet

Proximate analysis3


Ingredients % % SGR % day−1
Fish meal 25.0 Dry matter 93.25 ln final weight − ln initial weight
¼ 100 
Soybean meal 32.0 Crude protein 34.12 experimental duration ðdayÞ
Wheat flour 36.6 Crude lipid 5.24
DL-methionine 0.70 Crude ash 8.37



Threonine 0.40 FE ð%Þ ¼ 100  WG g fish−1 =FI g fish−1
Fish oil 1.50
Soybean oil 1.80
Vitamin mixture1 1.00
Protection assays
2
Mineral mixture 1.00
A total of 1020 Crucian carp (initial weight averaged
1
Per kilogram of vitamin mix: retinyl acetate (500,000 IU g−1 ), 8.8 ± 0.3 g) that were obtained from the acclimatization
0.80 g; cholecalciferol (500,000 IU g−1 ), 0.48 g; DL-α-tocopherol
aquariums were divided into 9 groups at random, each
acetate (50%), 20.00 g; menadione (23%), 0.43 g; thiamin nitrate
(90%), 0.11 g; riboflavine (80%), 0.63 g; pyridoxine HCl (81%), of which was equipped with 4 replicate aquariums.
0.92 g; cyanocobalamin (1%), 0.10 g; ascorhyl acetate (93%), Rearing densities in 9 treatment groups were 15, 30,
7.16 g; D-calcium pantothenate (90%), 2.73 g; niacin (99%), 30, 30, 30, 30, 30, 30, and 30 fish aquarium−1, respec-
2.82 g; D-biotin (2%), 5.00 g; meso-inositol (99%), 52.33 g; folic
tively. The experimental conditions and procedures ex-
acid (96%), 0.52 g
2 hibited a similarity to those in density assays. Fish in 9
Per kilogram of mineral mix: FeSO4·7H2O (20% Fe), 69.70 g;
CuSO4·5H2O (25% Cu), 1.20 g; ZnSO4·7H2O (23% Zn), 21.64 g; treatment groups were fed the diets containing 0 (con-
MnSO4·H2O (32% Mn), 4.09 g; Na2SeO3·5H2O (1% Se), 2.50 g; trol), 0, 1, 2, 3, 4, 5, 6, and 7 g EEE kg−1 diet for 56 days,
KI (4% I), 2.90 g; CaCO3, 897.98 g respectively. At the end of feeding trial, FI, WG, SGR,
3
Proximate analysis was carried out by the procedures by Zhou and FE were calculated. After a quick removal, hepato-
et al. (2008) pancreas and intestine were stored at − 80 °C to deter-
mine the activity of lipase, trypsin, alpha-amylase (am-
not replaced. The experiment was performed under similar ylase), Na+,K+-ATPase, and alkaline phosphatase
condition as that of acclimation. Counting and weighting (AKP) as well as the content of protein. The anti-
of fish in every aquarium were performed before the trail hydroxyl radical (AHR) and anti-superoxide anion
was ended. The survival rate (SR), weight gain (WG), (ASA) capacity, the malonaldehyde (MDA) level and
specific growth rate (SGR), and feed efficiency (FE) were the glutathione peroxidase (GPx), superoxide dismutase
calculated based on the data of the initial and final number (SOD), and catalase (CAT) activity in hepatopancreas
and the initial and final weight as well as the FI of fish and intestine were also measured at the same time.
(Chen et al. 2019; Cui et al. 2017; Ai et al. 2006; Zhou
et al. 2008; Ai et al. 2007; Lin and Shiau 2009).
CuSO4 trial


FI g fish−1
Stress assays
¼ feed consumed ðgÞ=final number of fish
CuSO4 exposure was carried out based on the guidelines
for fish acute bioassays of the general Organization for
SR ð%Þ ¼ 100 Economic Co-operation and Development (OECD)
(OECD 1993) following the report of Jiang et al.
 ðfinal number=initial number of fishÞ
(2011). CuSO4·5H2O was dissolved into distilled water
to obtain a final concentration of water (1.0 g Cu L−1),

for preparation of the stock solution. A total of 210 fish
WG g fish−1 from the acclimatization aquariums were assigned to 7


groups on a random basis (each group was equipped
¼ final weight g fish−1 –initial weight g fish−1
with 3 experimental aquariums and each aquarium had
10 fish). The stock solution was diluted with water to
1684
attain the desired concentrations of 0 (control), 0.5, 0.6,
0.7, 0.8, 0.9, and 1.0 mg Cu L−1, respectively, so as to
prepare the test solutions in the experimental aquariums
of 7 groups. The experiment was implemented under the
similar conditions to acclimation conditions. Fish in
each concentration group were maintained under the
experimental conditions for 4 days. During the test, the
basal diet was provided, the test solutions were used to
renew the water, with the fish mortality, and FI being
monitored on a daily basis. We determined the fish
mortality and FI in each concentration group at the end
of the CuSO4 exposure.
Protection assays
The implementation of feeding trial followed the proce-
dures proposed by Li et al. (2019a), which were modi-
fied slightly. A total of 420 fish (initial weight averaged
9.3 ± 0.1 g) from the acclimatization aquariums were
divided into 7 groups at random (each group had 4
experimental aquariums and each aquarium had 15
fish). Experimenters fed fish in 7 treatment groups, the
diets that contained 0 (control), 1, 2, 3, 4, 5, and 6 g EEE
kg−1 diet for 30 days, respectively. They fed the fish in
each group 6 times each day and observed these fish
carefully for ensuring satiation without overfeeding for
30 days. The experiment was conducted under the sim-
ilar conditions to acclimation conditions.
After feeding trial, 30 fish from the control group, 1,
2, 3, 4, 5, or 6 g EEE kg−1 diet group, experienced 4 days
of exposure to the water at the concentration of 0.8 mg
Cu L−1, respectively (Li et al. 2019a). Additional 30 fish
in the control group experienced exposure to the clean
water. Each treatment group was equipped with 3 repli-
cate aquariums, each of which had 10 fishes. The ex-
periment was conducted under the same conditions and
by the same procedures as those in the CuSO4 exposure.
At the end of CuSO4 stress, FI in each treatment group
was confirmed. Ethyl carbamate was used to anesthetize
fish in water, which were then collected and kept in ice.
Experimenters drew the blood from the caudal vein of
15 collected fish in each treatment group in the heparin-
ized syringes. The blood samples received 15 min of
centrifugation. The plasma was collected to determine
the plasma ammonia content (PAC). Subsequently, the
hepatopancreas and intestine were quickly removed and
stored at − 80 °C to determine the activity of lipase,
trypsin, amylase, Na+,K+-ATPase, AKP, glutamate-
Fish Physiol Biochem (2020) 46:1679–1698
pyruvate transaminase (GPT), and glutamate-
oxaloacetate transaminase (GOT).
Trichlorfon trial
Stress assays
Trichlorfon exposure was implemented according to the
bioassays by Li et al. (2019c).
Protection assays
The feeding trial was the same as that in Protection
assays of CuSO4 trial.
After feeding trial, 30 fishes from the control group,
1, 2, 3, 4, 5 or 6 g EEE kg−1 diet group, experienced
4 days of exposure to 2.4 mg trichlorfon L−1 water,
respectively as described by Li et al. (2019c). Additional
30 fishes in the control group were exposed to the clean
water. Each treatment group was equipped with 3 repli-
cate aquariums, each of which had 10 fishes. The ex-
periment was conducted under the same condition and
by the same procedures as those in the Protection assays
of CuSO4 trial. At the end of trichlorfon stress, the rate
of rollover (loss of equilibrium, % of total) in each
treatment group was confirmed. Ethyl carbamate was
used to anesthetize fish in water, which were then col-
lected and kept in ice. After a quick removal from the
base of dorsal fin, muscles were stored at − 80 °C to
determine the capacity of ASA and AHR, MDA, protein
carbonyl (PC), reduced glutathione (GSH), and protein
level and the activity of Na+,K+-ATPase, lactate dehy-
drogenase (LDH), GOT, GPT, glutathione reductase
(GR), and glutathione S-transferase (GST).
All the abovementioned procedures have been
granted the approval from the Institutional Animal Care
and Use Committee of Neijiang Normal University
following the guidelines of the Institutional Ethics Com-
mittee of the Chinese Institute of Chemical Biology.
Biochemical analysis
The methods of Liu et al. (2017) were adopted to deter-
mine the trypsin, lipase, and amylase activity. The
method of Jiang et al. (2015) helped measure the
Na + ,K + -ATPase, AKP, and LDH activity. The
methods of Li et al. (2019a) were adopted to assay the
GOT and GPT activity and PAC. The method of
Bradford (1976) was adopted to measure the
Fish Physiol Biochem (2020) 46:1679–1698
concentration of protein. ASA and AHR capacity and
PC level as well as GR and GPx activity were deter-
mined using the detection kits (Nanjing Jiancheng Bio-
engineer Institute, Nanjing, China) as described by Jiang
et al. (2015). Superoxide anion (O2-) and hydroxyl
radical (·OH) were generated by the action of xanthine
and xanthine oxidase and Fenton reaction, with the
electron acceptor added, respectively. A coloration re-
action is developed using the Griess reagent. The color-
ation degree is directly proportional to the quantity of
O2- or ·OH in the reaction. The color intensity was read
at 550 nm. ASA and AHR capacity was expressed as U
of activity per gram and milligram of protein, respec-
tively. The content of PC was evaluated by derivatiza-
tion with 2,4-dinitrophenylhydrazine and precipitating
with trichloroacetic acid. The amount of carbonyls was
measured at 370 nm and expressed as nanomole of
carbonyl per milligram of protein. GR activity was
assayed by measuring the GSH formed when the oxi-
dized glutathione (GSSG) reduced by reduced nicotin-
amide adenine dinucleotide phosphate. The color inten-
sity was read at 340 nm. GR activity was expressed as U
of activity per gram of protein. GPx activity was mea-
sured by monitoring the GSH consumption rate of hy-
drogen peroxide (H2O2) using dithionitro benzoic acid
(DTNB). The color intensity was measured at 412 nm.
GPx activity was expressed as U of activity per milli-
gram of protein. The MDA and GSH level as well as the
GST activity was determined using those proposed by
Yuan et al. (2014). Lipid peroxidation levels were de-
termined based on the MDA level generated by oxidiz-
ing fatty acids. In the presence of thiobarbituric acid,
MDA started producing colored thiobarbituric acid-
reacting substances (TBARS) that were measured at
532 nm and expressed as nanomole of TBARS per
milligram of protein. The method of GSH assay is based
on the formation of yellow color when DTNB reacts
with compounds containing sulfhydryl groups. The col-
or intensity was read at 420 nm. The amount of GSH
was expressed as milligrams of GSH per gram of pro-
tein. Activity of GST was measured by monitoring the
formation between GSH and 1-chloro-2,4-
dinitrobrnzene adduct that was measured at 412 nm
and expressed as U of activity per milligram of
protein. The method of Wang et al. (2015) was
employed to measure the CAT and SOD activity. CAT
activity was determined by measuring the decrease in
H2O2 concentration at 405 nm. The CAT activity was
expressed as U of activity per milligram of protein. SOD
1685
activity was determined following the ration of auto-
oxidation caused by O2- in samples. O2- generated
through the xanthine/xanthine oxidase system oxidized
hydroxylamine into nitrite and then presented color
under the action of chromogenic reagent, which could
be determined at 550 nm. SOD activity was expressed
as U of activity per milligram of protein.
Statistical analysis
The data was indicated as the mean ± standard deviation
(SD). The one- or two-way analysis of variance
(ANOVA) was performed on the data. The significant
difference was confirmed via Duncan’s multiple range
tests. The significance level was determined as 95%.
SPSS 13.0 for Windows software (SPSS, USA) assisted
with the statistical analysis.
Results
How the stocking density affects the growth
performance in fish
Table 4 demonstrates a gradual increase in the FBW,
WG, SGR, and FI while a gradual decrease in the FE
with increasing stocking density below the density of
0.48 fish L−1 (P < 0.05). However, FBW, WG, SGR,
and FE exhibited no differences at the density above
(Table 4). It is worthwhile to note that the FBW, WG,
SGR, FI, and FE above the densities of 0.48 fish L−1
gradually decreased with increasing stocking densi-
ties to 0.97 fish L−1 (Table 4) (P < 0.05). A further
increase in the stocking densities failed to induce the
difference in parameters above (Table 4), however,
significantly increased mortality at the density of
1.13 and 1.29 fish L−1 in Crucian carp (Table 4)
(P < 0.05). Based on the broken-line regression meth-
od with WG considered (Fig. 2), the optimum density
for the growth was estimated to be 0.49 fish L−1
(P < 0.05). The maximum density for the minimum
growth performance is estimated to be 0.98 fish L−1
(Fig. 2) (P < 0.05), which failed to cause the death of
Crucian carp. Thus, 0.98 fish L−1 was selected as an
appropriate high density for inducing growth inhibi-
tion in fish, and the stocking density also served for
the later experiments.
1686 Fish Physiol Biochem (2020) 46:1679–1698

Table 4 Initial body weight (IBW), final body weight (FBW), weight gain (WG), specific growth rate (SGR), feed intake (FI), feed efficiency (FE), and survival ratio (SR) of Crucian carp

1.29 (fish L−1)

7.98 ± 0.16a
20.27 ± 0.61a
12.29 ± 0.51a
1.66 ± 0.04a
27.65 ± 1.12a
44.54 ± 3.37a
93.75 ± 1.44a
1.13 (fish L−1)

b
8.09 ± 0.14a
a

28.16 ± 1.37a
a
20.82 ± 0.89
12.73 ± 0.98
1.69 ± 0.10

45.19 ± 2.55
96.43 ± 1.43
28.75 ± 1.79ab
8.04 ± 0.18a
a

c
0.97 (fish L−1)

21.06 ± 0.82
13.02 ± 0.95
1.72 ± 0.10

45.38 ± 3.64
100.00 ± 0.00 Fig. 2 Broken-line analysis of weight gain (WG) for Crucian carp
fed the basal diet for 56 days at different stocking densities. Values
Values are mean ± SD of 4 replicates. Values in the same row with the different superscripts are significantly different (P < 0.05) are mean ± SD of 4 replicates

How dietary EEE affects the growth performance

29.82 ± 1.56bc
b

b
7.92 ± 0.16a

c
0.81 (fish L−1)

24.05 ± 1.00
16.13 ± 1.00
1.98 ± 0.08

54.07 ± 1.22
100.00 ± 0.00

in high-density fish

Compared to the control group, high-density induced

the decrease in FBW, WG, SGR, FI, and FE in Crucian
carp. However, dietary EEE significantly increased pa-
30.90 ± 1.26bc

rameters above in Crucian carp (Table 5) (P < 0.05).

8.07 ± 0.15a
c

c
0.65 (fish L−1)

27.08 ± 1.41
19.01 ± 1.44
2.16 ± 0.10

61.45 ± 2.89
100.00 ± 0.00

Besides, FBW, WG, SGR, FI, and FE presented a slow

uptrend as the dietary EEE level increased (Table 5)
(P < 0.05). After above parameters increased to the max-
imum, no difference occurred as the EEE level was
further increased (Table 5). The FBW, WG, SGR, and
d

d
7.99 ± 0.17a

31.65 ± 1.61c

c
0.48 (fish L−1)

29.39 ± 1.24
21.40 ± 1.27
2.32 ± 0.09

67.66 ± 3.80
100.00 ± 0.00

FI reached the maximum for fish fed diets that contained

4.0 g EEE kg−1 diet (Table 5) (P < 0.05). The FE
reached the maximum for fish fed diets that contained
3.0 g EEE kg−1 diet (Table 5) (P < 0.05). In spite of this,
relying on the broken-line regression method consider-
ing recovery rate of WG (RWG) (Fig. 3), the proper
fed the basal diet for 56 days at different stocking densities

d
8.05 ± 0.16a

31.16 ± 1.65c

c
0.32 (fish L−1)

29.22 ± 1.16
21.16 ± 1.19
2.30 ± 0.08

68.05 ± 5.23
100.00 ± 0.00

concentration for growth was 4.30 g EEE kg−1 diet as

estimated (P < 0.05).

How dietary EEE affects the activity of digestive

cd

and absorptive enzymes in high-density fish

d

29.77 ± 1.30b
d
8.00 ± 0.17a

c
0.16 (fish L−1)

28.71 ± 1.18
20.71 ± 1.07
2.28 ± 0.05

69.74 ± 5.54
100.00 ± 0.00

Compared to the control, high-density caused decreas-

ing activity of lipase and amylase in hepatopancreas and
of trypsin, amylase, Na+,K+-ATPase, and AKP in intes-
tine of Crucian carp (Table 6) (P < 0.05). In spite of this,
FBW (g fish )

dietary EEE at 4.0 g kg−1 diet contributed to an effective

IBW (g fish−1)
−1

−1
WG (g fish )
−1
SGR (% d )
FI (g fish−1)

improvement of the activities exhibited by these en-

SR (%)
FE (%)

zymes in the digestive organs of Crucian carp at high

Item

density (Table 6) (P < 0.05). There was no difference in

Fish Physiol Biochem (2020) 46:1679–1698 1687

Table 5 Initial body weight (IBW), final body weight (FBW), weight gain (WG), specific growth rate (SGR), feed intake (FI), feed efficiency (FE), and survival ratio (SR) of Crucian carp at

2.23 ± 0.11de
30.50 ± 1.45d
21.76 ± 1.47d
8.74 ± 0.34a

32.12 ± 0.54c
67.80 ± 5.61c
100.00 ± 0.00a
0.98 + 7 (E7)

30.10 ± 1.62d
d

d
8.76 ± 0.33a

100.00 ± 0.00a
21.34 ± 1.63
2.20 ± 0.11
32.17 ± 0.74
66.44 ± 5.47
0.98 + 6 (E6)

cd
29.99 ± 1.49d
d
8.83 ± 0.29a

100.00 ± 0.00a
21.16 ± 1.72
2.18 ± 0.14
32.03 ± 1.22
66.04 ± 4.65
0.98 + 5 (E5)

Fig. 3 Broken-line analysis of recovery rate of weight gain

(RWG) for Crucian carp at high stocking densities by feeding
diets containing different levels of ethyl ether extract (EEE) of
Angelica sinensis for 56 days. Values are mean ± SD of 4 repli-
cates. RWG (%) = (E − Y)/ (D − Y) × 100 (in Table 5)
Values are mean ± SD of 4 replicates. Values in the same row with the different superscripts are significantly different (P < 0.05)
28.52 ± 1.15cd
cd

cd
8.77 ± 0.29a

100.00 ± 0.00a
19.76 ± 1.01
2.11 ± 0.06
31.97 ± 1.03
61.82 ± 2.98
high stocking density by feeding diets containing different levels of ethyl ether extract (EEE) of Angelica sinensis for 56 days

0.98 + 4 (E4)

the activity exhibited by trypsin in the hepatopancreas of

Crucian carp (Table 6) (P < 0.05).
b
8.75 ± 0.26a
27.46 ± 1.01c
c

100.00 ± 0.00a

How dietary EEE affects the antioxidant capacity

18.71 ± 1.02
2.04 ± 0.07
29.58 ± 1.35
63.23 ± 1.62
0.98 + 3 (E3)

in high-density fish

Compared to the control, high-density caused the AHR

and CAT activity to decrease in the hepatopancreas and
24.62 ± 1.29b
b

b
8.80 ± 0.32a

100.00 ± 0.00a

intestine of Crucian carp (Table 7) (P < 0.05), while

15.83 ± 1.16
1.84 ± 0.08
29.08 ± 0.92
54.46 ± 4.19
0.98 + 2 (E2)

dietary EEE led to an effective improvement on the

AHR and GPx activity in the hepatopancreas and the
AHR and CAT activity in the intestine of Crucian carp
at high density (Table 7) (P < 0.05). Moreover, high
ab
22.95 ± 0.99b
b

b
8.87 ± 0.30a

100.00 ± 0.00a

density significantly caused the increase in the MDA

14.08 ± 0.90
1.70 ± 0.07
28.31 ± 0.83
49.77 ± 3.76
0.98 + 1 (E1)

level in the intestine and hepatopancreas of Crucian

carp. However, dietary EEE inhibited the indicator
above in high-density fish (Table 7) (P < 0.05). The
Densities (fish L−1) + EEE (g kg−1 diet)

activity exhibited by ASA and SOD increased in intes-

8.81 ± 0.29a
20.88 ± 0.93a
a

100.00 ± 0.00a
12.07 ± 0.81
1.54 ± 0.07
25.83 ± 1.81
46.76 ± 1.57

tine of Crucian carp at high density (Table 7) (P < 0.05).

0.98 + 0 (Y)

Moreover, relying on dietary EEE, the indicator as

mentioned above further increased in intestine of
Crucian carp at high density (Table 7) (P < 0.05). Nev-
ertheless, the difference in these parameters is not sig-
d
8.77 ± 0.33a
32.99 ± 1.23e
e

100.00 ± 0.00a
24.22 ± 1.54
2.37 ± 0.13
35.15 ± 1.29
68.89 ± 3.23

nificant. No difference existed with regard to the ASA

0.49 + 0 (D)

activity in hepatopancreas and the GPx activity in intes-

tine in Crucian carp (Table 7) (P < 0.05).

How CuSO4 exposure affects the FI in fish

FBW (g fish−1)
IBW (g fish−1)

−1
WG (g fish )
−1
SGR (% d )
−1
FI (g fish )

Relative to the control, after being exposed to CuSO4,

SR (%)
FE (%)

the FI in Crucian carp decreased (Fig. 4A) (P < 0.05).

Item

Besides, the FI presented a slow-paced decrease with

1688 Fish Physiol Biochem (2020) 46:1679–1698

Table 6 The activities of trypsin, lipase, amylase, Na+,K+- diet and 0.98 fish L−1 feeding the diet containing 4 g kg−1 ethyl
ATPase, and alkaline phosphatase (AKP) in hepatopancreas or ether extract of Angelica sinensis for 56 days
intestine of Crucian carp at 0.49 and 0.98 fish L−1 feeding the basal

0.49 + 0 0.98 + 0 0.98 + 4

Hepatopancreas
Trypsin (U mg−1 protein) 957.58 ± 61.34a 930.79 ± 68.77a 945.21 ± 61.16a
−1 b a
Lipase (U mg protein) 35.57 ± 2.42 31.76 ± 1.55 42.45 ± 2.48c
Amylase (U mg−1 protein) 0.85 ± 0.03c 0.71 ± 0.02a 0.78 ± 0.02b
Intestine
Trypsin (U mg−1 protein) 1246.42 ± 68.04b 1037.92 ± 59.90a 1166.75 ± 82.92b
−1 ab a
Lipase (U mg protein) 26.42 ± 1.79 24.79 ± 1.60 29.07 ± 3.01b
−1 c a
Amylase (U mg protein) 0.86 ± 0.04 0.63 ± 0.02 0.76 ± 0.04b
Na+,K+-ATPase (U mg−1 protein) 4.03 ± 0.20b 3.31 ± 0.22a 5.07 ± 0.26c
−1 b a
AKP (U g protein) 421.04 ± 17.74 375.11 ± 18.08 428.34 ± 25.56b

Values are mean ± SD of 4 replicates, with 5 fish in each replicate. Values with the different superscripts in the same row are significantly
different (P < 0.05)

the increase in the Cu level (Fig. 4A) (P < 0.05). The FI
in Crucian carp under the treatment of 0.8 mg Cu L−1
water reached the minimum value as estimated (Fig.
4A) (P < 0.05). Further increasing the Cu concentration
failed to cause the difference in the FI of Crucian carp
(Fig. 4 A) (P < 0.05), however, significantly increased
mortality when the concentrations of Cu were 0.9 and
1.0 mg L−1 in Crucian carp (Fig. 4 B) (P < 0.05).
Hence, the study confirmed 0.8 mg L−1 as a proper
concentration of Cu in water to induce the FI change of
fish, and the concentration also served for later
experiments.

Table 7 The content of malondialdehyde (MDA) and the activ- at 0.49 and 0.98 fish L−1 feeding the basal diet and 0.98 fish L−1
ities of anti-superoxide anion (ASA), anti-hydroxy radical (AHR), feeding the diet containing 4 g ethyl ether extract of Angelica
superoxide dismutase (SOD), catalase (CAT), and glutathione sinensis kg−1 for 56 days
peroxidase (GPx) in hepatopancreas and intestine of Crucian carp

0.49 + 0 0.98 + 0 0.98 + 4

Hepatopancreas
ASA (U g−1 protein) 60.74 ± 2.24a 63.76 ± 4.08a 65.56 ± 3.95a
−1 c a
AHR (U mg protein) 273.05 ± 7.14 223.98 ± 11.17 249.66 ± 15.78b
−1 a c
MDA (nmol mg protein) 10.02 ± 0.67 13.30 ± 0.91 11.69 ± 0.83b
−1 a a
SOD (U mg protein) 117.21 ± 4.81 123.70 ± 4.87 126.47 ± 5.67a
−1 b a
CAT (U mg protein) 30.09 ± 1.40 23.25 ± 1.65 24.42 ± 1.85a
GPx (U mg−1 protein) 421.34 ± 22.53a 428.81 ± 30.91a 479.19 ± 29.13b
Intestine
ASA (U g−1 protein) 51.33 ± 2.88a 56.29 ± 4.20ab 59.86 ± 2.55b
−1 b a
AHR (U mg protein) 279.58 ± 17.53 235.56 ± 9.99 293.79 ± 19.12b
−1 a b
MDA (nmol mg protein) 11.23 ± 0.58 14.09 ± 0.88 11.73 ± 0.61a
SOD (U mg−1 protein) 115.35 ± 4.28a 121.38 ± 5.76ab 129.29 ± 5.01b
−1 c a
CAT (U mg protein) 20.21 ± 1.58 12.11 ± 0.84 16.78 ± 0.76b
−1 a a
GPx (U mg protein) 384.67 ± 23.63 393.18 ± 14.15 406.02 ± 24.04a

Values are mean ± SD of 4 replicates, with 5 fish in each replicate. Values with the different superscripts in the same row are significantly
different (P < 0.05)
Fish Physiol Biochem (2020) 46:1679–1698
Fig. 4 Feed intake (FI; A) and mortality (B) of Crucian carp
followed by exposure to graded levels of Cu for 4 days. Values
are mean ± SD of three replicates, with 10 fish in each replicate.
Bars with different superscripts are significantly different
(P < 0.05)
How dietary EEE affects the FI change of fish induced
by CuSO4
Table 8 explains how dietary EEE affects the FI of
fish after exposure to CuSO4. Relative to the con-
trol, being exposed to CuSO4 for 4 days signifi-
cantly decreased FI of Crucian carp (Table 8) (P
< 0.05). Nevertheless, dietary EEE supplements
administered for 30 days effectively increased FI
of Crucian carp treated with CuSO4 (Table 8) (P
< 0.05). Also, the FI presented a slow increase as
the dietary EEE level increased (Table 8) (P
< 0.05). With the increase in the EEE concentra-
tion to 4.0 g kg −1 diet, FI of CuSO 4 -treated
Crucian carp reached the maximum for all EEE
groups as estimated (Table 8) (P < 0.05). Howev-
er, according to the broken-line regression method
based on the recovery rate of FI (RFI) (Fig. 5), the
proper concentration for FI was 4.25 g EEE kg−1
diet as estimated.
1689
How dietary EEE affects the change in the digestive,
absorptive, and metabolic parameters in fish induced
by CuSO4
Relative to the control, being exposed to CuSO4 led
to an obvious decrease in the trypsin and amylase
activity in the hepatopancreas of Crucian carp and
the lipase, amylase, and AKP activity in the intes-
tine (Table 9) (P < 0.05). Even so, dietary EEE
contributed to an effective improvement on the
lipase activity in the hepatopancreas of Crucian
carp and the lipase, amylase, Na+,K+-ATPase, and
AKP activity in the intestine (Table 9) (P < 0.05).
Nevertheless, CuSO4 and dietary EEE could not
cause any difference in the trypsin activity in the
intestine of Crucian carp relative to the control
(Table 9) (P < 0.05). Moreover, the exposure to
CuSO4 markedly enhanced the GOT and GPT ac-
tivity in hepatopancreas and PAC in Crucian carp
relative to the control (Table 9) (P < 0.05). How-
ever, dietary EEE markedly reduced PAC in
Crucian carp (Table 9) (P < 0.05). Although dietary
EEE induce the decrease in the GOT and GPT
activity in the hepatopancreas of CuSO4-treated
Crucian carp, the difference is not significant
(Table 9) (P < 0.05).
How dietary EEE affects the change in rollover of fish
induced by trichlorfon
Table 8 explains how dietary EEE affects the
rollover of fish after being exposed to trichlorfon.
Relative to the control, being exposed to trichlor-
fon for 4 days considerably increased the rollover
of Crucian carp (Table 8) (P < 0.05). Nevertheless,
dietary EEE supplements administered for 30 days
effectively decreased the rollover of trichlorfon-
treated Crucian carp (Table 8) (P < 0.05). In ad-
dition, the increase in the dietary EEE level led to
a gradual decrease in the rollover of fish (Table 8)
(P < 0.05). When increasing EEE concentration to
4.0 g kg−1 diet, rollover of Crucian carp treated
with trichlorfon reached the minimum value spe-
cific to all EEE groups as estimated (Table 8) (P
< 0.05). However, according to the broken-line
regression method based on inhibitory rate of roll-
over (IR) (Fig. 5), the proper concentration for
inhibitory rollover was 4.18 g EEE kg−1 diet as
estimated.
1690 Fish Physiol Biochem (2020) 46:1679–1698

Table 8 Feed intake (FI) and rollover of Crucian carp fed diets containing different levels of ethyl ether extract of Angelica sinensis (EEE)
for 30 days, following Cu or trichlorfon exposure for 4 days

EEE (g kg−1 diet) FI Rollover

Cu (mg L−1) % of body weight Trichlorfon (mg L−1) % of total

0 + 0.0 (K) 4.10 ± 0.24f + 0.0 (K) 0.00 ± 0.00a

a
0 + 0.8 (Y) 0.09 ± 0.00 + 2.4 (Y) 100.00 ± 0.00f
1 + 0.8 (E1) 0.72 ± 0.04b + 2.4 (E1) 77.50 ± 1.27e
c
2 + 0.8 (E2) 1.37 ± 0.04 + 2.4 (E2) 69.17 ± 5.29d
d
3 + 0.8 (E3) 2.00 ± 0.08 + 2.4 (E3) 59.17 ± 4.20c
e
4 + 0.8 (E4) 3.15 ± 0.15 + 2.4 (E4) 44.16 ± 1.44b
e
5 + 0.8 (E5) 3.23 ± 0.14 + 2.4 (E5) 43.33 ± 2.43b
6 + 0.8 (E6) 3.20 ± 0.11e + 2.4 (E6) 42.50 ± 2.55b

Values are mean ± SD of three replicates, with 10 fish in each replicate. Values with the different superscripts in the same column are
significantly different (P < 0.05)

How dietary EEE affects the change in metabolic

parameters in the muscle of fish induced by trichlorfon

In comparison with the control, being exposed to trichlor-

fon markedly decreased the Na+,K+-ATPase, LDH, and
GPT activity in muscles of Crucian carp (Table 10) (P
< 0.05). Even so, no differences in the activities of GOT
in the muscle were found in Crucian carp (Table 10)
(P < 0.05). Interestingly, dietary EEE contributed to an
effective improvement to the parameters above in the
muscle of Crucian carp (Table 10) (P < 0.05).

How dietary EEE affects the change in antioxidant

status in the muscle of fish induced by trichlorfon

Being exposed to trichlorfon obviously improved the

Fig. 5 Broken-line analysis of recovery rate of feed intake (RFI,
A) and inhibitory rate of rollover (IR, B) for Crucian carp fed diets
containing different levels of ethyl ether extract of Angelica
sinensis (EEE) for 30 days, following Cu (A) or trichlorfon (B)
exposure for 4 days. RFI (%) = (E − Y)/ (K − Y) × 100 (in Table 8);
IR = Y − E (in Table 8). Values are mean ± SD of three replicates,
with 10 fish in each replicate
MDA and PC level in muscles of Crucian carp com-
pared to the control (Table 11) (P < 0.05). Nevertheless,
dietary EEE at the concentrations of 4.0 g kg−1 diet
prevented the trichlorfon-induced increase in above pa-
rameters in muscles of Crucian carp in an effective
manner (Table 11) (P < 0.05). Moreover, in comparison
with the control, being exposed to trichlorfon caused the
GSH level and the AHR and GR activity to decrease in
muscles of Crucian carp (Table 11) (P < 0.05). Trichlor-
fon did not cause any difference in the ASA and GST
activity in muscles of Crucian carp compared with the
control (Table 11) (P < 0.05). While dietary EEE led to
an obvious improvement of the GSH level and the
AHR, GR, and GST activity in muscles of Crucian carp
treated with trichlorfon (Table 11) (P < 0.05).
Fish Physiol Biochem (2020) 46:1679–1698 1691

Table 9 The activities of trypsin, lipase, amylase, glutamate- intestine and plasma ammonia content (PAC) of Crucian carp
oxaloacetate transaminase (GOT), and glutamate-pyruvate trans- fed diet containing 4.0 g ethyl ether extract of Angelica sinensis
aminase (GPT) in hepatopancreas; the activities of trypsin, lipase, (EEE) kg−1 for 30 days, followed by exposure to 0.8 mg Cu L−1
amylase, Na+,K+-ATPase, and alkaline phosphatase (AKP) in water for 4 days

Control Control + Cu EEE + Cu

Hepatopancreas
Trypsin (U mg−1 protein) 1375.03 ± 107.27b 1106.42 ± 86.04a 1227.03 ± 80.84a
−1 a ab
Lipase (U mg protein) 40.91 ± 2.21 41.33 ± 2.36 45.84 ± 3.74b
−1 b a
Amylase (U mg protein) 1.40 ± 0.07 1.21 ± 0.06 1.29 ± 0.07a
−1 a b
GOT (U g protein) 28.18 ± 2.11 32.31 ± 1.74 29.04 ± 1.87ab
−1 a b
GPT (U g protein) 18.52 ± 1.42 23.66 ± 1.26 21.91 ± 1.54b
Intestine
Trypsin (U mg−1 protein) 1753.47 ± 106.69a 1707.12 ± 114.25a 1809.82 ± 96.72a
−1 b a
Lipase (U mg protein) 32.04 ± 2.37 27.60 ± 2.10 36.19 ± 2.79b
−1 c a
Amylase (U mg protein) 1.42 ± 0.05 1.10 ± 0.04 1.30 ± 0.04b
+ + −1 a a
Na ,K -ATPase (U mg protein) 1.71 ± 0.07 1.73 ± 0.12 2.06 ± 0.10b
AKP (U g−1 protein) 192.72 ± 9.55b 159.54 ± 6.93a 211.76 ± 11.44c
Plasma
PAC (μmol mL−1) 310.37 ± 12.37a 339.72 ± 17.29b 305.99 ± 22.32a

Values are mean ± SD of 4 replicates, with 5 fish in each replicate. Values with the different superscripts in the same row are significantly
different (P < 0.05)

Discussion density for growth was estimated to be 0.49 fish L−1 in

Dietary EAs recovered growth performance
in high-density fish
People recognize the stocking density as an essential hus-
bandry factor in the intensive aquaculture (Marco et al.
2008). As illustrated by reports, it seems that improper
stocking densities can negatively affect the growth of fish
as well as lower their immune competence (Lupatsch et al.
2010). However, the optimum stocking densities vary with
species, size of fish, water quality, and length of growing
season, etc. (Rowland et al. 2006). Here, the optimum
Crucian carp. The FBW, WG, SGR, FI, and FE above the
densities of 0.49 fish L−1 gradually decreased with in-
crease in stocking densities. These indexes suggested that
high stocking density suppressed growth in Crucian carp.
This result is in excellent agreement with the report in
Amur sturgeon (Li et al. 2012). The appropriate density for
inducing growth inhibition was estimated to be 0.98 fish
L−1 in Crucian carp. There is little information to date
regarding the proper density for inducing growth inhibi-
tion in fish. It is possible to use these parameters as an
experimental model for assessing how drugs and
chemicals affect the growth in high-density fish.

Table 10 The activities of Na+,K+-ATPase, lactate dehydroge- fed diet containing 4.0 g ethyl ether extract of Angelica sinensis
nase (LDH), glutamate-oxaloacetate transaminase (GOT), and (EEE) kg−1 for 30 days, followed by exposure to 2.4 mg trichlor-
glutamate-pyruvate transaminase (GPT) in muscle of Crucian carp fon L−1 water for 4 days

Control Control + trichlorfon EEE + trichlorfon

Na+,K+-ATPase (U mg−1 protein) 1.26 ± 0.08b 0.96 ± 0.06a 1.30 ± 0.07b

LDH (U g−1 protein) 374.12 ± 22.60c 313.34 ± 13.36a 344.76 ± 14.06b
−1 ab a
GOT (U g protein) 18.20 ± 1.19 16.37 ± 0.85 18.83 ± 1.46b
−1 c a
GPT (U g protein) 13.05 ± 1.06 9.36 ± 0.54 11.09 ± 0.83b

Values are mean ± SD of 4 replicates, with 5 fish in each replicate. Values with the different superscripts in the same row are significantly
different (P < 0.05)
1692
Table 11 The levels of malondialdehyde (MDA), protein carbon-
yl (PC), and reduced glutathione (GSH) and the activities of anti-
superoxide anion (ASA), anti-hydroxy radical (AHR), glutathione
reductase (GR), and glutathione S-transferase (GST) in the muscle
Control
ASA (U g−1 protein) 188.13 ± 14.16a
AHR (U mg−1 protein) 227.64 ± 14.08c
−1 a c
MDA (nmol mg protein) 3.56 ± 0.26
−1 a b
PC (nmol mg protein) 2.87 ± 0.21
−1 b a
GSH (mg g protein) 12.83 ± 0.48
−1 b a
GR (U g protein) 40.03 ± 2.76
GST (U mg−1 protein) 136.92 ± 6.34a
Values are mean ± SD of 4 replicates, with 5 fish in each replicate. Values with the different superscripts in the same row are significantly
different (P < 0.05)
Several studies showed that high densities can de-
crease the performance of growth in fish (Santos et al.
2010; Tolussi et al. 2010). In harmony with studies
above, in this study, high density (0.98 fish L−1) induced
the decline in FBW, SGR, WG, FI, and FE in Crucian
carp. However, these parameters exhibited an obvious
increase due to dietary EEE for 56 days in high-density
fish. The proper concentration of EEE for RWG was
4.30 g kg−1 diet as estimated in high-density fish. This is
in accordance with the report that dietary EAs could
enhance the performance of growth in carp (Li et al.
2019b; Li et al. 2019d). As suggested by these results,
dietary EEE can recover the growth in high-density fish.
There are no official reports discussing how EAs affect
the growth performance in high-density fish.
Dietary EAs suppressed the decrease in the digestion
and absorption capacity in fish induced by high density
The digestion and absorption capacities of fish deter-
mine their growth (Zhou et al. 2007). The amylase,
trypsin, and lipase in the intestine and hepatopancreas
remarkably help fish to digest nutrients, and their activ-
ity is capable to reflect the digestion ability of fish
directly (Ling et al. 2010). The study found high density
stimulated the activity exhibited by above enzymes to
decrease in the digestive organs of Crucian carp. How-
ever, dietary EEE enhanced the activity exhibited by
these enzymes in high-density Crucian carp, which well
supported the report that dietary EAs increased the
activities of trypsin, lipase, and amylase in the digestive
organs of carp (Li et al. 2019b). There are no formal
reports that talked about how high density affects the
Fish Physiol Biochem (2020) 46:1679–1698
of Crucian carp fed diet containing 4.0 g ethyl ether extract of
Angelica sinensis (EEE) kg−1 for 30 days, followed by exposure to
2.4 mg trichlorfon L−1 water for 4 days
Control + trichlorfon EEE + trichlorfon
174.18 ± 11.85a 192.22 ± 9.64a
181.44 ± 10.22a 206.35 ± 10.44b
5.77 ± 0.37 4.22 ± 0.42b
3.30 ± 0.20 3.05 ± 0.18ab
9.41 ± 0.64 11.95 ± 0.68b
33.59 ± 2.92 41.39 ± 2.85b
134.19 ± 4.25a 147.91 ± 8.35b
digestive enzyme activity in fish. These results con-
firmed that high density could decrease the digestive
capacity of fish. However, dietary EAs could improve
the digestive capacity in high-density fish. Na+,K+-
ATPase and AKP in intestine contribute to nutrient
absorption, and their activity represents the absorption
ability exhibited by fish (Zhou et al. 2007). According to
the current study, high density significantly decreased
Na+,K+-ATPase and AKP activities in intestine of
Crucian carp. However, dietary EEE inhibited the high
density–induced decreases in above parameters in intes-
tine of Crucian carp. This is in line with the report in
carp treated with trichlorfon (Li et al. 2019c). Few
papers explained how the high density affects the activ-
ity exhibited by absorptive enzyme in the intestine of
fish. The data as described above suggested that high
density could depress the absorptive capacity in fish.
However, dietary EAs could improve the absorptive
capacity in high-density fish.
Dietary EAs inhibited the oxidative damage in digestive
organs of fish induced by high density
There is a correlation between the normal function of the
cells in digestive organs and their oxidative/
antioxidative state (Shoveller et al. 2005). MDA results
from lipid oxidation, and its content is capable of
reflecting the oxidative/antioxidative state in cells (Li
et al. 2013). Here, dietary EEE suppressed the increase
in the MDA content induced by high density in the
hepatopancreas and intestine in Crucian carp, which
conforms the report in the Cu-induced carp (Li et al.
2019a). As suggested by these results, high-density
Fish Physiol Biochem (2020) 46:1679–1698
results in the increase in oxidative status in fish’s diges-
tive organs. The improved oxidative status is reported to
initiate the function and structure change of essential
biomolecules, thus leading to cell injury (Pisoschi and
Pop 2015). On that account, the high density–induced
loss of partial function in digestion and absorption may
be ascribed to oxidative damage in fish’s digestive
organs. Even so, dietary EEE inhibited the oxidative
damage to the digestive organs of fish caused by high
density. Based on studies, ROS (O2- and ·OH) showed a
strong involvement in the lipid oxidation (Lushchak
2011). The current study found that the AHR capacity
was restored by the dietary EEE, which was decreased
in the intestine and hepatopancreas in high-density
Crucian carp. ASA capacity was improved by dietary
EEE in the intestine of high-density Crucian carp. The
result well met the report that dietary EAs raised the
ASA and AHR activity in the intestine and hepatopan-
creas of carp (Li et al. 2019b). By now, no information
once focused on the association between the high den-
sity and the ROS in fish’s digestive organs.
Aquatic organisms possess antioxidants capable of
quenching ROS and suppressing the lipid oxidation in
cells (Wu et al. 2011). SOD enables O2- to enzymatical-
ly degrade to H2O2 (Lin et al. 2011). CAT helps detox-
ify H2O2 to water and O2 (Jannuzzi et al. 2018). GPx
degrades lipid hydroperoxides taking GSH as the sub-
strate (Lin and Shiau 2009). The current study found the
ability of dietary EEE to recover the CAT activity in the
digestive organs, which was reduced in high-density
Crucian carp. The SOD and GPx activity was enhanced
due to the dietary EEE in the digestive organs of high-
density Crucian carp. These results support the report
that dietary EAs raised the activity exhibited by CAT,
SOD, and GPx in the intestine and hepatopancreas of
carp (Li et al. 2019b). Nevertheless, scarce information
can be found about how the high density affects the
antioxidant enzymes in the digestive organs of fish.
Hence, dietary EAs improved the scavenging ability of
ROS as well as maintained the enzymatic antioxidant
activity, thereby cutting down the oxidative damage
induced by high density in fish’s digestive organs.
Dietary EAs inhibited the decrease in the digestion
and absorption capacity and protein metabolism in fish
induced by CuSO4
Xenobiotic-mediated environmental stress is an impor-
tant determining factor in the maintenance of fish health
1693
as fish are frequently exposed to such components (Jena
et al. 2009). In this study, FI was significantly sup-
pressed in Crucian carp treated with CuSO4 in water,
which is in agreement with a report in Mystus vittatus
(Subathra and Karuppasamy 2007). CuSO4 at the con-
centrations of 0.8 mg L−1 induced the minimum value of
FI, which failed to cause the death of Crucian carp.
Thus, the study selected 0.8 mg L−1 as the proper
concentration of CuSO4 for inducing the decrease in
FI of fish.
The FI of fish can reflect their digestion and absorp-
tion capacity (Hong et al. 2015). In this study, dietary
EEE suppressed the decrease in the FI of Crucian carp
induced by CuSO4. The proper concentration of EEE for
RFI was 4.25 g kg−1 diet as estimated. The FI of fish is
closely associated with the activity of digestive and
absorptive enzymes in the hepatopancreas and intestine
(Ling et al. 2010). As discovered in the study, due to the
exposure to CuSO4, the activity of trypsin, amylase,
lipase, and AKP decreased in the digestive organs of
Crucian carp. However, dietary EEE improved these
parameters while enhancing Na+,K+-ATPase activity
in Crucian carp treated with CuSO4. This is consistent
with the previous reports that dietary EAs had the func-
tion of enhancing the trypsin, amylase, and lipase activ-
ity (Li et al. 2019a) as well as suppressing the decreases
in the activity of Na+,K+-ATPase and AKP induced by
trichlorfon in hepatopancreas and intestine of carp (Li
et al. 2019c). The data as described above suggested that
EAs could strengthen the digestion and absorption ca-
pacity exhibited by CuSO4-treated fish. GOT and GPT
can help to utilize amino acids into tricarboxylic acid
cycle as a source of energy by deamination (Boyko et al.
2012; Gottlieb et al. 2003). Ammonia acts as the major
end product of the protein catabolism in teleosts (Zhou
et al. 2007). The application of protein in fish can be
evaluated considering the GOT and GPT activity and
PAC (Liu et al. 2009). As revealed by these results,
relying on CuSO4, PAC, and GOT and GPT activity
increased in hepatopancreas of Crucian carp. These
results demonstrated that CuSO4 activated the increase
in protein decomposition for energy supply in fish.
There is scarce information focusing on how CuSO4
affects the protein metabolism in fish. However, it has
been reported that the Cu-exposed fish need to use more
energy to sustain their normal metabolism (Subathra and
Karuppasamy 2007). The effects of CuSO4 on protein
metabolism may present a close relation to the cortisol
in fish. Studies have demonstrated that Cu elevates
1694
plasma cortisol level in fish (Dethloff et al. 1999). With
the increase in cortisol secretion, the catabolic processes
to create an energetic sink would increase (Li et al.
2012). Protein is the main energy source of fish (Cui
et al. 2017). So, it may provide with a possible mecha-
nism for the enhancement of protein metabolism in fish
treated with CuSO4 that increase cortisol secretion for
energy. This hypothesis shall be clarified in further
research. In this study, the CuSO4-induced changes in
GOT and GPT activity as well as PAC were significant-
ly recovered by dietary EEE supplementation in Crucian
carp. Based on previous studies in our lab, dietary EAs
raised the GOT and GPT activity in hepatopancreas and
reduced PAC in carp (Li et al. 2019a; Li et al. 2019b).
All these proved that EAs were capable of improving
the protein metabolism of fish treated with CuSO4.
Dietary EAs suppressed the partial function loss
in muscles of fish induced by trichlorfon
Trichlorfon inhibited the acetylcholinesterase at the neu-
romuscular junction and synapses of the skeletal mus-
cle, thereby playing its toxic action, which resulted in
the balance loss in fish (Guimara et al. 2007). The study
found out about an obvious increase in the rate of
rollover in Crucian carp treated with trichlorfon, which
conforms to our previous report (Li et al. 2019c). As
suggested in the study result, trichlorfon induced the
loss of balance in fish. However, dietary EEE sup-
pressed the rollover in Crucian carp under the induction
of trichlorfon. The proper concentration of EEE for IR
was 4.18 g kg−1 diet as estimated. Accordingly, EAs
could maintain the balance of trichlorfon-treated fish.
The balance of fish exhibited a close relation to the
energy metabolism in muscles (Johnston et al. 1977).
LDH acts as an essential enzyme in energy production
anaerobic pathway, which takes charge of catalyzing the
inter-conversion between pyruvate and lactate in the
glycolysis in fish (Coelho et al. 2011). Herein, trichlor-
fon decreased LDH activity in muscles of Crucian carp,
which conforms to the report in euryhaline fish (Rao
2006). However, dietary EEE suppressed the
trichlorfon-induced decrease in LDH activity in muscles
of Crucian carp. GPT and GOT serve as the most
important protein metabolism enzymes (Hong et al.
2015). As found by the study, trichlorfon decreased
the GPT and GOT activity in muscles of Crucian carp.
Nevertheless, dietary EEE recovered the GPT and GOT
activity in muscles of Crucian carp induced by
Fish Physiol Biochem (2020) 46:1679–1698
trichlorfon, which well conforms to the report that die-
tary EAs raised the GOT and GPT activity in muscles of
carp (Li et al. 2019a; Li et al. 2019b). A significant
portion of ATP has been applied to preserving the
Na+,K+-ATPase activity required for maintaining the
cytoplasmic ionic milieu so as to prevent the colloidal
osmotic lysis (Cimen 2008). The study found the ability
of trichlorfon to decrease the Na+,K+-ATPase activity in
muscles of Crucian carp, which well meets the report in
the gills of fish (Baldissera et al. 2019). However, die-
tary EEE protected against the trichlorfon-induced re-
duction in Na+,K+-ATPase activity in muscles of
Crucian carp. The results well support that in the intes-
tine of carp (Li et al. 2019c). Only few papers pay
attention to how EAs affects LDH, GOT, GPT, and
Na+,K+-ATPase in muscles of Crucian carp. According
to the present results, trichlorfon stimulated not only
imbalance of fish but also suppression of energy metab-
olism, suggesting the loss of partial function in muscles
of fish. However, dietary EAs inhibited the function
impairment in muscles of fish induced by trichlorfon.
There may be a close association between the function
and oxidative-antioxidative status in muscles of fish.
Dietary EAs suppressed the decrease in the antioxidant
capacity in muscles of fish induced by trichlorfon
PC and MDA are the products of cellular component
oxidation (Li et al. 2013). Herein, the exposure to tri-
chlorfon improved the MDA and PC contents in mus-
cles of Crucian carp, which indicated that the lipid and
protein oxidation increased in muscles of fish. However,
dietary EEE helped to decrease the MDA and PC con-
tents in muscles of trichlorfon-treated Crucian carp,
which meant that the lipid and protein oxidation were
depressed in fish. These results well meet the report in
the digestive organs of carp (Li et al. 2019c). By now,
no studies have focused on how EAs affects the lipid
and protein oxidation in muscles of trichlorfon-treated
fish. In line with the present results, trichlorfon stimu-
lated not only loss of function but also the cellular
component oxidation, suggesting that trichlorfon caused
oxidative damage in muscles of fish. Even so, dietary
EAs suppressed the oxidative damage induced by tri-
chlorfon in muscles of fish.
There is a close association between cellular compo-
nent oxidation and ROS in fish (Li et al. 2013). Here,
trichlorfon in water depressed the AHR and ASA activ-
ity in muscles of Crucian carp. Nevertheless, dietary
Fish Physiol Biochem (2020) 46:1679–1698
EEE elevated their activities in muscles of Crucian carp
induced by trichlorfon. These results confirmed that
dietary EAs suppressed the decrease in abilities of scav-
enging ROS in muscles of fish induced by trichlorfon,
satisfying the report in the digestive organs of carp (Li
et al. 2019c). There may be an association between the
effect mechanism of EAs on ROS and the GSH content
in muscles of fish. GSH acts a major non-enzymatic
antioxidant which is able to scavenge intracellular ROS
directly (Masella et al. 2005). As found out in the
present study, trichlorfon decreased the GSH level in
muscles of Crucian carp. However, dietary EEE restored
the GSH level in muscles of Crucian carp induced by
trichlorfon. The intracellular GSH reduced GSSG to
GSH by virtue of GR, thereby remaining at a high level
(Chen et al. 2009). In the study, dietary EEE recovered
the activity of GR in muscles of Crucian carp, which
decreased after trichlorfon exposure. GST is an enzyme
which helps to detoxify harmful electrophilic endoge-
nous compounds and exogenous compounds via the
GSH conjugated to the target molecules (Modén and
Mannervik 2014). Herein, the activity of GST did not
decrease obviously in muscles of Crucian carp follow-
ing trichlorfon exposure. However, dietary EEE in-
creased the activity of GST in muscles of trichlorfon-
treated Crucian carp. These results well meet the report
in the hepatopancreas and intestines of carp (Li et al.
2019c). There are no formal reports that paying attention
to how EAs affects the GSH, GR, and GST in the
muscles of fish. The abovementioned results demon-
strated the ability of dietary EAs to mitigate oxidative
damage induced by trichlorfon via quenching ROS and
elevating the enzymatic and non-enzymatic antioxidant
activity in muscles of fish.
The effect of dietary EAs on oxidative damage in-
duced by trichlorfon in muscles of fish may be ascribed
to the active ingredients of EAs. As reported in studies,
ferulic acid acts as an active ingredient in As (Wu and
Hsieh 2011). In this study, the content of ferulic acid in
EEE is 3.55 mg g−1, which is 8 times higher than that
(0.40–0.44 mg g−1) in As from Sichuan of China (Zhao
et al. 2003). Studies have illustrated the good absorption
of ferulic acid in the intestinal mucosa in rats (Adam
et al. 2002) and its capability to reduce the level of ROS
in rats’ aortas (Suzuki et al. 2007). As reported in our
study, ligustilide is the most abundant active ingredient
in Angelica sinensis (Li et al. 2020). It has been reported
that ligustilide has the function of decreasing the intra-
cellular ROS level in both the murine macrophages (Su
1695
et al. 2011) and the human endothelial cells (Choi et al.
2018). Besides, according to the relevant studies, orally
administered ligustilide reduced the level of MDA and
PC in neuronal of mice and increased the activity of
SOD and CAT in brain (Feng et al. 2012; Kuang et al.
2014). All these reports demonstrated that dietary EAs
can improve the antioxidant capacity in fish.
Conclusion
To sum up, the study firstly found that the optimum
density for growth was 0.49 fish L−1 water in Crucian
carp. High stocking densities caused the decrease in
growth performance. The appropriate density for induc-
ing growth inhibition was 0.98 fish L−1 water in Crucian
carp. Dietary EAs restored the growth in the high-
density Crucian carp. The proper concentration of EAs
for RWG was 4.30 g kg−1 diet in high-density fish.
Moreover, this study indicated that due to the high
density, the MDA content was increased and the AHR,
CAT, trypsin, lipase, amylase, Na+,K+-ATPase, and
AKP activity was reduced in digestive organs of
Crucian carp. However, dietary EAs restored these pa-
rameters and increased the ASA, SOD, and GPx activity
in high-density Crucian carp. Hence, dietary EAs
inhibited the high density–induced decrease in digestion
and absorption as well as antioxidant capacity in diges-
tive organs of Crucian carp. Secondly, our data demon-
strated that CuSO4 exposure decreased FI of Crucian
carp. The proper concentration of CuSO4 to induce the
decrease in FI was 0.8 mg Cu L−1 water. Moreover,
CuSO4 increased PAC and GOT and GPT activities in
hepatopancreas and decreased trypsin, amylase, lipase,
and AKP activity in the digestive organs of Crucian
carp. However, dietary EAs returned to FI of Crucian
carp treated with CuSO4. The proper concentration of
EAs for RFI was 4.25 g kg−1 diet. Moreover, dietary
EAs suppressed the CuSO4-mediated change in the
parameters as mentioned above and enhanced Na+,K+-
ATPase activity in the digestive organs of Crucian carp.
As confirmed by these results, dietary EAs helped to
mitigate the CuSO4-induced decrease in digestion and
absorption capacity and increase in protein metabolism
in the digestive organs of fish. Finally, the present
results suggested that trichlorfon induced the rollover
in Crucian carp. Dietary EAs suppressed the rollover in
Crucian carp induced by trichlorfon. The proper con-
centration of EAs for IR was 4.18 g kg−1 diet as
1696
estimated. Moreover, dietary EAs suppressed the in-
creases in the MDA and PC level and the decrease in
the GSH level and the LDH, GOT, GPT, Na+,K+-
ATPase, AHR, and GR activity induced by trichlorfon
while enhanced the GST activity in the muscle of
Crucian carp. These results revealed that trichlorfon
stimulated not only imbalance of fish and decrease in
energy metabolism but also cellular component oxida-
tion, suggesting that trichlorfon caused oxidative dam-
age in fish’s muscles. However, dietary EAs inhibited
the oxidative damage induced by trichlorfon via
quenching ROS and elevating the enzymatic and non-
enzymatic antioxidant activity in muscles of fish. So,
EAs could be used as an inhibitor of high density,
CuSO4, and trichlorfon stress in fish.
Funding information This research was financially supported
by Sichuan Science and Technology Program (2018JY0214), the
Scientific Research Fund of Sichuan Provincial Education Depart-
ment (16ZB0302), the Doctoral Research Fund (14B07), and the
Patent Project (Z2019027) of Neijiang Normal University. The
authors wish to thank the personnel of these teams for their kind
assistance.
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