Effects of dietary different

canthaxanthin levels on growth

performance, antioxidant capacity,
biochemical and immune-physiological
parameters of white shrimp (
Litopenaeus Vannamei ) Samia Fawzy
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Aquaculture
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Effects of dietary different canthaxanthin levels on growth performance,

antioxidant capacity, biochemical and immune-physiological parameters of
white shrimp (Litopenaeus Vannamei)
Samia Fawzy a, f, 1, Weilong Wang a, b, c, 1, Meiqin Wu d, Ganfeng Yi a, e, Xuxiong Huang a, b, c, *
a
Centre for Research on Environmental Ecology and Fish Nutrition of the Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, China
b
Shanghai Collaborative Innovation Center for Cultivating Elite Breeds and Green-culture of Aquaculture Animals, Shanghai Ocean University, China
c
National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, China
d
College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China
e
Beijing Dabeinong Technology Group Co., Ltd, Beijing 100080, China
f
Department of Animal Wealth Development, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: An 8-week feeding trial was conducted to evaluate the effects of different dietary canthaxanthin (CX) levels on
Canthaxanthin growth performance, pigmentation, antioxidant capacity, hemolymph biochemical parameters, immune
Growth performance response, and resistance to hypoxia stress of white shrimp (Litopenaeus vannamei). Juveniles (initial weight 1.15
Pigmentation
± 0.12 g) were fed with five iso‑nitrogenous and isolipidic diets supplemented with/without CX: 0 (control), 50,
Antioxidant
100, 200, and 400 mg kg− 1 diet. Results showed that the growth performance, survival, and feed conversion ratio
Litopenaeus vannamei
improved significantly in CX supplemented treatments compared to the control. The redness of cooked shrimp
tended to increase with increasing CX level; however, no significant difference was observed among the treat­
ments that received CX of more than 100 mg kg− 1. Further, the activities of digestive enzymes, total antioxidant
capacity, and peroxidase increased significantly (P < 0.05), while the activities of glutathione peroxidase,
catalase, and malondialdehyde decreased significantly (P < 0.05) in the hepatopancreas with increasing dietary
CX levels. The immune-related enzymes (alkaline phosphatase, lysozyme, alanine aminotransferase, and
aspartate aminotransferase) and hemolymph biochemical parameters (cholesterol, high-density, and low-density
lipoprotein) are significantly affected by different dietary CX levels. Total carotenoids and astaxanthin contents
in shrimp muscle, shell, head, and whole-body showed an upward trend with the increment of dietary CX. After
exposure to hypoxia stress, juveniles in 200 mg kg− 1 supplementation treatment exhibited the highest LT50 value
among all the treatments. Moreover, broken-line regression analysis indicated that a dose of 173.73 to 202.13
mg kg− 1 of CX was suitable in the diet of L. vannamei as a potential carotenoid source for substituting dietary
astaxanthin in the shrimp feed industry.

1. Introduction
Shrimp has tremendous importance among seafood products due to
the significant economic benefits it yields (Mialhe et al., 2013). White
shrimp (L. vannamei) is a major cultivated species which exhibits rapid
growth rate and tolerance to a wide range of environmental conditions
such as temperature and salinity (Landsman et al., 2019; Li et al., 2017).
Nowadays, intensive shrimp farming is widely practiced to meet the
market demand and generate more profit (Shinji et al., 2019). As a result
of this intensification, shrimp are losing their natural red color and
suffering from “blue disease” (Chien and Jeng, 1992). Unfortunately,
crustaceans do not have the pathway for de novo synthesis of the pig­
ments (Goodwin, 1984). Under such conditions, only the dietary inter­
vention through carotenoids supplementation can maintain the desired
color, improve the product quality and further raise their market price
(Kalinowski et al., 2005; Parisenti et al., 2011). The typical red color of

* Corresponding author at: Centre for Research on Environmental Ecology and Fish Nutrition of the Ministry of Agriculture and Rural Affairs, Shanghai Ocean
University, China.
E-mail address: xxhuang@shou.edu.cn (X. Huang).
1
These authors have contributed equally to this work and share first authorship.

https://doi.org/10.1016/j.aquaculture.2022.738276
Received 3 January 2022; Received in revised form 30 March 2022; Accepted 18 April 2022
Available online 21 April 2022
0044-8486/© 2022 Published by Elsevier B.V.
S. Fawzy et al. Aquaculture 556 (2022) 738276

crustaceans could be obtained by incorporating astaxanthin into their Table 1

diets (Niu et al., 2009; Wade et al., 2017a). Nevertheless, astaxanthin is Formulation and composition of the experimental diets (g kg− 1, dry matter
an expensive carotenoid supplement that leads to a significant increase basis).
in feed costs despite constituting a small portion from their diet (Hansen Ingredients Treatments
et al., 2016). In this regard, several trials have been done to find other Control CX50 CX100 CX200 CX400
carotenoid sources that can come up with results similar to those of
Caseina 425 425 425 425 425
astaxanthin but at lower prices. Ample evidence had proven that
Lysineb 15 15 15 15 15
canthaxanthin (CX) could be as effective as, if not better than, astax­ Methionineb 10 10 10 10 10
anthin in pigmentation of Atlantic salmon (Buttle et al., 2001) and red L-arginineb 20 20 20 20 20
porgy (Kalinowski et al., 2015). Additionally, it has been reported that Fish oila 40 40 40 40 40
shrimp could efficiently utilize dietary synthetic CX and deposit it as Soybean lecithina 30 30 30 30 30
PUFAc 8 8 8 8 8
astaxanthin in their tissues (Yamada et al., 1990). Consequently, CX Cholesterolb 5 5 5 5 5
would be a substrate for astaxanthin, the most abundant carotenoid in Vitamin mixtured 12 12 12 12 12
crustaceans. Mineral mixturee 34.4 34.4 34.4 34.4 34.4
CX (β,β-carotene-4,4′ -dione), also known as red ketocarotenoid, is a a-Starcha 50 50 50 50 50
Sucroseb 50 50 50 50 50
member of xanthophylls and is widely used as a feed additive in aqua­
Glucoseb 50 50 50 50 50
culture (Rebelo et al., 2020). Its market value was estimated at 75 Glucosamine-HClb 8 8 8 8 8
million dollars in 2017 and is expected to hit 85 million dollars by 2024, Sodium citrateb 3 3 3 3 3
with aquaculture and livestock sectors utilizing about 40% of the total Sodium succinateb 3 3 3 3 3
volume (Mussagy et al., 2021). Moreover, synthetic CX is considered the Wheat gluten mealb 55 55 55 55 55
Ca(H2PO4)2a 51.6 51.6 51.6 51.6 51.6
primary dietary source available in the market that could provide suf­ Cellulosea 130 129.5 129 128 126
ficient supply as the natural sources are limited (Pi et al., 2020; Sanchez Canthaxanthinf 0 0.5 1 2 4
et al., 2013). Due to its attractive color and pigmentation property, CX Total 1000 1000 1000 1000 1000
was added predominantly to the diets of Salmon, Rainbow trout (Baker a
Yuehai Feed Group Co., Ltd., Zhejiang, China.
et al., 2002; Buttle et al., 2001), and Penaeus monodon shrimp (Niu et al., b
Shanghai Macklin Biochemical Co., Ltd., Shanghai, China.
2012) for their flesh color enhancement. In addition to color improve­ c
PUFA: (eicosapentaenoic acid) EPA 4 g and (docosahexaenoic acid) DHA 4 g,
ment, previous studies have reported multiple benefits for CX, such as Shanghai Macklin Biochemical Co., Ltd., Shanghai, China.
growth promotion, antioxidant activities, and immune modulation d
Vitamin mixture (12 g kg− 1 diet) (vitamin A free): p-aminobenzoic acid
properties (Elia et al., 2019; Shahidi and Brown, 1998; Venugopalan 0.092 g; biotin 0.004 g; inositol 3.668 g; nicotinic acid 0.368 g, Ca-pantothenate
et al., 2013). The antioxidative role of carotenoids is closely associated 0.552 g; pyridoxine-HCl 0.112 g; riboflavin 0.072 g; thiamine-HCl 0.036 g;
with improving the resistance of aquatic animals to the stressors during menadione 0.036 g, α-tocopherol 0.184 g; cyanocobalamin 0.0008 g; cholecal­
the culture period (Niu et al., 2009). Oxygen depletion (hypoxia) ciferol 0.012 g; stay-C 1.36 g, folic acid 0.008 g; choline chloride 5.5 g.
e
Mineral mixture (34.38 g kg− 1 diet): C6H10CaO6⋅5H2O, 1.75; K2HPO4, 11.98;
generally results in oxidative stress and increased ROS release, which
MgSO4.7H2O, 12.28; NaH2PO4, 7.04; C6H5O7Fe⋅5H2O, 0.23; CuSO4.5H2O, 0.34;
negatively affects the survival and growth performance of shrimp
ZnSO4⋅7H2O, 0.48; CoCl2, 0.07; MnSO4⋅H2O, 0.21.
(Zhang et al., 2013). It had been reported that the increase in dietary and f
Canthaxanthin: CarophyllⓇ Yellow containing 10% canthaxanthin made by
body carotenoids could alleviate the destructive effects of hypoxia, so DSM Nutrition, Switzerland.
CX as a carotenoid may be effective in such cases (Chien and Shiau,
2005).
premixed and then added to the dry ingredients with continuous mixing.
Therefore, CX, with its numerous properties and relatively low
Subsequently, water (30% of dry ingredients mixture) was added and
market price compared with astaxanthin, may be a potential carotenoid
carefully mixed for another 10 min. Finally, the blend was transferred
source in the shrimp diet. As long as the utilization efficiency differs
into a single-screwed mincer to produce pellets (1.5 mm in diameter).
from one species to another, it was essential to study the effect of dietary
The pellets were then dried in a dryer mechanical convection oven at
CX on L. vannamei. Based on this background, the current study was
35 ◦ C until moisture content was reduced to about 10%. Once the
carried out to investigate the effect of different dietary CX levels on
experimental diets were dried, they were packed and stored at − 20 ◦ C
growth performance, pigmentation, antioxidant activity, immune
until use. The approximate analysis of the diets is shown in Table 2.
response, and resistance to hypoxia stress of L. vannamei. The findings of
the current study would contribute to achieving a cost-effective feed
formulation to control the blue disease of farmed shrimp. 2.2. Shrimp and culture conditions

2. Materials and methods Juvenile white shrimp (L. vannamei) were obtained from

2.1. Diet formulation and preparation

Table 2
Proximate composition (g kg− 1, dry matter basis) of the experimental diets.a
A chemically synthesized pigment (CarophyllⓇ yellow) containing
Parametersa Treatments
10% CX produced by DSM Nutrition, Switzerland, was used as a source
for CX in the current experiment. Five semi-purified pelleted diets were Control CX50 CX100 CX200 CX400
formulated to be iso‑nitrogenous and isolipidic as described in Table 1 Crude Protein 471.20 ± 475.80 ± 473.33 ± 475.33 ± 476.70 ±
and supplemented with different levels of CX (0, 50, 100, 200, 400 mg (CP) 1.45 1.81 0.50 0.98 2.55
kg− 1 of diet). Casein was used as the major protein source. Crystalline Crude Lipid 62.93 ± 61.87 ± 60.27 ± 61.80 ± 60.63 ±
(CL) 3.24 1.63 5.26 5.22 1.64
amino acids were added to meet the amino acid requirement of this 78.57 ± 79.43 ± 80.17 ± 81.00 ± 85.73 ±
species according to Zhou et al. (2012). Fish oil, soybean lecithin, Ash
2.72 0.55 0.63 0.55 2.04
cholesterol, and PUFA were used as the lipid source. The mineral and 49.31 ± 98.86 ± 199.85 ± 398.37 ±
CX (mg kg− 1) NDb
vitamin mixtures were added according to Xie et al. (2012) with slight 0.25 0.11 0.88 0.49
modifications. The diet-making process started with weighing all dry a
Data were expressed as mean ± S.E.M from triplicate groups. CX,
ingredients and mixing thoroughly with a mixer (B10, Rudong, China) canthaxanthin.
for 10–15 min. Lipid ingredients and fat-soluble components were also b
ND: not detected.

2
S. Fawzy et al.
Haixingnong Aquaculture Cooperatives (Shanghai, China). Juveniles
were maintained in a cement pool at 27 ± 0.5 ◦ C and fed the control diet
for two weeks to acclimate to water conditions. After the acclimation, six
hundred juveniles with mean initial weight (1.15 ± 0.12 g) were
selected and randomly distributed into 15 PVC net cages (Length = 1 m,
Width = 1 m, and Height = 1.2 m) fitted inside a cement pond (Length =
10 m, Width = 5 m, and Height = 1.5 m) corresponding to 40 shrimp/
cage. The cement tanks used in the study were provided with a drainage
system and water flow system. Each tank’s water was changed every two
days to two-thirds of its capacity. Each experimental diet was tested in
triplicate in a randomized design. The experiment was carried out in­
door and lasted for 56 days. During this period, juveniles were manually
fed four times per day (7:00, 12:00, 17:00, and 22:00). A fixed feeding
method was applied in which the shrimp were fed at 5–8% of their body
weight. In order to determine the amount of feed required, a weekly
random sampling was done to check the average body weight of shrimp.
The diet was introduced through disk-shaped feeders placed inside the
rearing nets. Therefore, the uneaten feed was kept inside the feeders and
could be easily collected. Three hours after feeding, the uneaten feed
was collected while the fecal matter was siphoned out of the cages. All
uneaten food was freeze-dried to calculate feed intake and feed effi­
ciency ratio. During the experiment, water conditions were measured
everyday: temperature, 29–32 ◦ C; dissolved oxygen concentration, > 5
mg L− 1; ammonia nitrogen, < 0.05 mg L− 1; pH, 8.0–8.5; salinity, 1–2‰.
2.3. Determination of growth performance parameters
At the end of 8 weeks feed trial, all the shrimp were fasted for 24 h,
and the survivors from each cage counted and weighed to evaluate the
growth performance based on the following formulae:
Body weight gain (BWG, %) = [(final weight-initial weight)/initial
weight] × 100.
Specific growth rate (SGR, % day− 1) = [(Ln final weight-Ln initial
weight)/duration] × 100.
Feed conversion ratio (FCR) = dry weight of feed consumed (g)/live
weight gain (g).
Survival (%) = (final number of shrimp/initial number of shrimp) ×
100.
2.4. Sampling techniques
After counting and weighing, 15 shrimp from each treatment (5
shrimp per cage × 3 replicates) were randomly sampled and used to
analyze whole-body composition and carotenoid content. Using a 1-mL
sterile syringe, hemolymph was collected from the ventral sinus of 10
shrimp per cage. Also, the hepatopancreas of 6 shrimp from each cage
were dissected out and collected separately in 5 mL tubes. Hemolymph
and hepatopancreas samples were frozen immediately in liquid nitro­
gen, while whole-body shrimp samples were chilled in ice. Finally, all
samples were transported to the laboratory and stored at − 80 ◦ C until
analysis.
2.5. Determination of biochemical parameters
According to the Association of Official Analytical Chemists, the
proximate composition (crude protein, crude lipid, and ash) of whole
shrimp and experimental diets were determined after freeze-drying and
grinding (AOAC, 2006). Briefly, crude protein was analyzed using the
Kjeldahl method (2300-Autoanalyzer, Foss Tecator, Sweden). Crude
lipid was determined by the ether extraction method using Soxtec Sys­
tem HT (Soxtec System HT6, Sweden). Ash content was determined by
pre-incineration on a hot plate followed by 6 h in a muffle furnace at
550 ◦ C.
For assaying enzymatic activities, serum was obtained from the su­
pernatant by centrifuging the hemolymph at 10000 rpm, 4 ◦ C for 10
min. Parameters including urea, cholesterol, triglycerides, high-density
3
Aquaculture 556 (2022) 738276
lipoprotein (HDL), and low-density lipoprotein (LDL) were measured by
the colorimetric method performed using a chemistry analyzer
(iChem340, China). Serum was also used for the determination of non-
specific immune parameters, including lysozymes (LZM), aspartate
aminotransferase (AST), alanine transaminase (ALT), and alkaline
phosphatase (AKP). All enzymes were assayed using Nanjing Kits
(Nanjing Jiancheng Bioengineering Institute, China) according to the
instructions of the manufacturer.
Furthermore, hepatopancreas was prepared by homogenization,
dilution with shrimp saline solution and centrifugation at 8000 rpm,
4 ◦ C for 15 min (Wang et al., 2019). Hepatopancreas samples were used
for assaying the activities of antioxidant enzymes, including total anti­
oxidant capacity (T-AOC), peroxidase (POD), glutathione peroxidase
(GSH-Px), Malondialdehyde (MDA), catalase (CAT), and digestive en­
zymes (protease, lipase, and amylase). Nanjing Kits (Nanjing Jiancheng
Bioengineering Institute, China) were used according to the instructions
of the manufacturer.
2.6. Color reading
The color was assessed using a colorimeter (Chroma Meter CR400,
Konica Minolta Sensing Inc., Osaka, Japan) after putting shrimp samples
into plastic bags and placed into a boiling water bath for 3 min and
cooling in tap water (about 5 ◦ C) (Ju et al., 2011). The color values were
expressed according to CIE L*a*b* color space (Nickell and Bromage,
1998), with L*, a*, and b* representing lightness, redness, and yellowness
of the cooked shrimp, respectively.
2.7. Determination of carotenoid content
Total carotenoid content in the whole-body shrimp, shell (including
carapace, telson, and uropod), muscle, and head tissues (without
hepatopancreas) was quantified spectrophotometrically. Dried and
crushed samples of the whole body (1.5 g), muscle (2 g), shell (1 g), and
head (1 g) were weighed and placed separately into 50 mL poly­
propylene tubes in triplicates. Chloroform was added to the samples,
thoroughly mixed for 10 min, centrifuged at 4 ◦ C at 10000 rpm for 5
min, and stored for 2 h in darkness. This step was repeated three times
until the extract became completely clear. The extracts were collected
and pooled into new tubes, then dried in a vacuum evaporator (Eyela SB
1100, Japan). After that, 8 mL acetone was added to each dried sample.
3 mL of the solution were used for measuring the carotenoid concen­
tration using a spectrophotometer (Puxi T6, China) at 475 nm and E1%
value of 2100 against a blank of acetone (Wang et al., 2021).
The remaining 5 mL were dried by N2 and delivered for Ultra Per­
formance Liquid Chromatography (UPLC, Waters ACQUITY, USA)
analysis after re-dissolving in 2 mL of mobile phase solution (Acetoni­
trile: Methanol, 70:30 v/v). UPLC was equipped with Waters ACQUITY
H-Class BEH C18 column (1.7 μm, 2.1 mm × 150 mm). The mobile
gradient phase consisted of A (100% dH20), B (Acetonitrile: Methanol,
70:30 v/v), and C (100% methyl tert-butyl ether). Initial ratio of 5% A:
95% B for 16 min, 5% A: 10% B: 85% B for 39 min, then returned to
initial ratio over 4 min at a flow rate of 0.2 mL/min. An ultraviolet-
visible detector set quantified relative amounts of carotenoids to 475
nm by calculating the area under the curve of each peak and it to a
known standard. The standards of astaxanthin, CX, echinenone, zeax­
anthin, and β-carotene were purchased from Sigma-Aldrich (USA), and
the β-cryptoxanthin standard was purchased from CaroteNature (Swit).
Since relatively significant differences were detected in the amount of
free and esterified carotenoids in shrimp tissues, only the free-type ca­
rotenoids were considered and presented in the current study.
2.8. Hypoxia stress resistance test
Fifteen shrimp from each treatment were randomly selected after the
feeding trial and distributed into 5 L white plastic buckets in triplicates
S. Fawzy et al. Aquaculture 556 (2022) 738276

with five shrimp per bucket. Each bucket was filled with the same water
(temperature 28 ◦ C, salinity 1‰, pH 8.2, and DO 1 mg L− 1). DO in the
test buckets was measured using an oxygen meter (HACH HQ30d, USA),
while low DO conditions were maintained by nitrogen gas bubbling.
Shrimp survival was recorded and expressed as LT50 for each hour
during the resistant test. LT50 (the time required for 50% mortality of
tested shrimp by a stressor) was calculated by a regression equation
according to Yokoyama et al. (2005). The equation is as the following:
3.2. Shrimp whole-body composition
The findings of the shrimp whole-body composition analysis are
presented in Table 4. CX50 and CX100 treatments differed significantly (P
< 0.05) from the control in crude protein content. In contrast, dietary CX
feeding did not significantly affect (P > 0.05) the crude lipid and ash
content of the shrimp body.

Y = a X+b 3.3. Body color reading

X = time to individual death of shrimp.
LT50 (X) was obtained when Y = log10 (50) =1.7.
The equation resulted from the plotting of values of log survival
against the time of mortality to determine LT50 for each treatment. The
values obtained from the equation were statistically compared, and the
higher value indicated a greater tolerance to hypoxia stress.
2.9. Statistical analysis
The statistical analyses were performed using SPSS 20.0. All data are
presented as means ± standard error of the mean (S.E.M., n = 3). One-
way analysis of variance was used to test the differences among the
treatments at p-value (P < 0.05), followed by Duncan’s multiple range
test to compare the treatments’ means. The broken-line analysis was
performed using OriginPro 2021. The relationships among parameters
were estimated by Pearson coefficient of correlation, thus visualized by
heatmap of correlation, using ggcorrplot package (ver. 0.1.2.999) and
heatmaply package (ver. 1.3.0) with R version 4.1.2.
The color reading values of cooked shrimp fed different dietary CX
levels are shown in Fig. 1. Shrimp fed with CX-containing diets exhibited
higher (P < 0.05) redness (a*) values than the control treatment, how­
ever, lightness (L*) showed an opposite trend. Further, CX100, CX200, and
CX400 treatments were similar in a* and L* values. Although yellowness
(b*) values were higher in CX fed treatments than in the control, no
significant difference (P > 0.05) was observed among treatments.
3.4. Antioxidant capacity
Dietary manipulation significantly affected the hepatopancreatic
antioxidant parameters of L. vannamei (Table 5). T-AOC showed a sig­
nificant increase (P < 0.05) in shrimp-fed CX-containing diets compared
to those fed the control diet. POD activity showed a similar trend, but
CX400 treatment did not exhibit a significant difference with the control
treatment. On the contrary, GSH-Px, MDA, and CAT levels decreased
significantly in CX supplemented treatments (P < 0.05) compared to the
control treatment.

2.10. Ethical statement 3.5. Hemolymph biochemical and hemato-immunological parameters

The present experimental procedures were carried out in strict The immunological parameters in the hemolymph of L. vannamei fed
accordance with the recommendations in the ethical guidelines of EU with different CX levels were represented in Table 6. The findings
Directive 2010/63/EU for animal experiments. cleared that the AKP and LZM activities tended to increase markedly (P
< 0.05) when dietary CX levels increased, while AST and ALT showed an
3. Results opposite trend. However, a higher dose of CX (400 mg kg− 1) caused
neither a further significant increase in AKP and LZM nor a significant
3.1. Growth performance parameters and feed utilization decrease in AST and ALT.
The results of hemolymph biochemical parameters of the studied
Growth performance (FBW, BWG, and SGR), FCR, and the survival shrimp are shown in Table 7. The findings showed that with increasing
rate of shrimp fed with experimental diets are given in Table 3. The dietary CX levels, cholesterol showed a significant decreasing trend
results indicated that all the parameters as mentioned above improved compared to the control treatment (P < 0.05). The lowest levels of HDL,
significantly in shrimp fed with CX supplemented diets compared to LDL, and TP were observed in the control treatment, which significantly
those fed the control diet (P < 0.05). CX400 treatment had the lowest differs (P < 0.05) from the higher CX supplemented treatments (CX200
FBW, BWG, and SGR among CX supplemented treatments, while there and CX400). In contrast, the parameters of triglycerides and urea in
was no significant difference (P > 0.05) in survival among them. experimental treatments did not show a significant difference (P > 0.05)
among all the treatments.
Table 3
Growth parameters and nutrient utilization of Litopenaeus vannamei fed the 3.6. Digestive enzymes activities
experimental diets for 56 days.
Parametersa Treatments The effect of dietary CX on digestive enzymes activities is shown in
Control CX50 CX100 CX200 CX400
Table 4
7.67 ± 9.21 ± 9.38 ± 9.62 ± 8.57 ±
FBW (g) Whole-body proximate analysis (g kg− 1, dry matter basis) of Litopenaeus van­
0.28a 0.11c 0.14c 0.14c 0.17b
SGR (% 3.64 ± 3.97 ± 3.99 ± 4.04 ± 3.84 ± namei fed with experimental diets for 56 days.
day− 1) 0.06a 0.02c 0.02c 0.02c 0.03b Parametersa Treatments
667.35 ± 821.33 ± 837.52 ± 861.96 ± 757.12 ±
BWG (%) Control CX50 CX100 CX200 CX400
28.94a 11.04c 14.35c 14.54c 17.12b
1.63 ± 1.36 ± 1.49 ± 1.44 ± 1.31 ± Crude Protein 767.60 ± 780.93 ± 792.73 ± 771.26 ± 767.63 ±
FCR
0.03c 0.03a 0.02b 0.03ab 0.05a (CP) 4.73a 4.87b 0.69c 1.93ab 2.70a
86.67 ± 95.83 ± 95.00 ± 97.50 ± 96.67 ± Crude Lipid 56.36 ± 54.17 ± 54.63 ± 53.17 ± 53.07 ±
Survival (%)
3.63a 2.20b 1.44b 1.44b 2.20b (CL) 4.78 0.46 2.33 3.26 0.68
a 122.27 ± 115.93 ± 124.87 ± 119.43 ± 125.47 ±
FBW, final body weight; FCR, feed conversion ratio; BWG, body weight gain; Ash
2.04 1.68 1.28 3.33 2.94
SGR, specific growth rate. Data were expressed as mean ± S.E.M from triplicate
a
groups. Mean values in the same row with different letters are significantly Data were expressed as mean ± S.E.M from triplicate groups. Mean values in
different (P < 0.05). the same row with different letters are significantly different (P < 0.05).

4
S. Fawzy et al. Aquaculture 556 (2022) 738276

Table 7
Effect of dietary different canthaxanthin levels on hemolymph biochemical pa­
rameters of Litopenaeus vannamei.
Parametersa Treatments

Control CX50 CX100 CX200 CX400

HDL (mmol 0.07 ± 0.10 ± 0.09 ± 0.12 ± 0.11 ±

L− 1) 0.01a 0.01abc 0.01ab 0.01c 0.01bc
LDL (mmol 0.25 ± 0.29 ± 0.29 ± 0.42 ± 0.37 ±
L− 1) 0.02a 0.01a 0.02a 0.02b 0.01b
CHO (mmol 2.51 ± 2.41 ± 2.01 ± 1.33 ± 0.78 ±
L− 1) 0.31c 0.36bc 0.29b 0.29ab 0.17a
TG (mmol 0.45 ± 0.51 ± 0.43 ± 0.48 ± 0.46 ±
L− 1) 0.01 0.02 0.02 0.03 0.01
46.90 ± 54.14 ± 59.20 ± 60.46 ± 52.25 ±
TP (g L− 1)
Fig. 1. Color parameters of cooked whole-body shrimp fed with different levels 0.45a 0.74b 1.34c 0.89c 0.41b
of canthaxanthin for 56 days. Data were expressed as mean ± S.E.M from Urea (mmol 7.86 ± 7.58 ± 7.31 ± 7.27 ± 7.07 ±
triplicate groups. Bars with different letters represented significant differences L− 1) 0.19 0.35 0.38 0.26 0.59
between various treatments (P < 0.05). a
Data were expressed as mean ± S.E.M from triplicate groups. Mean values in
the same row with different letters are significantly different (P < 0.05). HDL,
high-density lipoprotein; LDL, low-density lipoprotein; CHO, cholesterol; TG,
Table 5 triglycerides; TP, total protein.
Effect of dietary different canthaxanthin levels on hepatopancreatic antioxidant
statuses of Litopenaeus vannamei.
Parametersa Treatments

Control CX50 CX100 CX200 CX400

T-AOC (mmol 0.07 ± 0.20 ± 0.20 ± 0.17 ± 0.16 ±

mg− 1 protein) 0.01a 0.01c 0.01c 0.01b 0.01b
POD (U mg− 1 0.37 ± 0.49 ± 0.50 ± 0.43 ± 0.39 ±
protein) 0.01a 0.01c 0.01c 0.01b 0.02a
GSH-Px (U mg− 1 8.07 ± 6.85 ± 6.74 ± 5.12 ± 5.34 ±
protein) 0.33c 0.20b 0.35b 0.37a 0.40a
MDA (nmol mg− 1 0.28 ± 0.20 ± 0.17 ± 0.12 ± 0.18 ±
protein) 0.01d 0.01c 0.01b 0.01a 0.01b
CAT (U mg− 1 1.30 ± 1.03 ± 0.74 ± 0.69 ± 0.87 ±
protein) 0.02c 0.07b 0.06a 0.05a 0.07ab
a
Data were expressed as mean ± S.E.M from triplicate groups. Mean values in
the same row with different letters are significantly different (P < 0.05). T-AOC, Fig. 2. Effect of dietary different canthaxanthin levels on digestive enzymes
total antioxidant capacity; POD, peroxidase; GSH-Px, glutathione peroxidase; activities in hepatopancreas of Litopenaeus vannamei. Data were expressed as
MDA, malondialdehyde. CAT: catalase. mean ± S.E.M from triplicate groups. Bars with different letters represented
significant differences between various treatments (P < 0.05).

Table 6
Effect of dietary different canthaxanthin levels on hemato-immunological pa­ with experimental diets for 56 days are shown in Fig. 3. With increasing
rameters of Litopenaeus vannamei. the dietary CX levels, the total carotenoid content in different tissues
significantly increased (P < 0.05). The highest carotenoid content in the
Parametersa Treatments
muscle, head, shell, and whole-body was observed in shrimp fed with
Control CX50 CX100 CX200 CX400 400 mg kg− 1 CX. Meanwhile, the lowest content was found in the control
AKP (U 100 2.65 ± 3.64 ± 4.03 ± 4.79 ± 4.29 ± treatment.
mL− 1) 0.33a 0.33b 0.15bc 0.27c 0.16bc The profile of carotenoids in various tissues of L. vannamei (muscle,
30.70 ± 27.71 ± 24.88 ± 20.93 ± 23.07 ±
AST (U L− 1) shell, head, and whole-body) analyzed by UPLC was given in Table 8.
0.88d 0.83c 0.79b 0.72a 0.43a
103.25 ± 95.12 ± 88.92 ± 70.09 ± 81.74 ±
ALT (U L− 1)
3.73d 2.07c 1.32bc 2.46a 0.42b
LZM (U 83.33 ± 115.38 ± 147.44 ± 224.36 ± 160.26 ±
mL− 1) 6.41a 11.10ab 6.41bc 6.41d 16.95c
a
Data were expressed as mean ± S.E.M from triplicate groups. Mean values in
the same row with different letters are significantly different (P < 0.05). AKP,
alkaline phosphatase; AST, aspartate aminotransferase; ALT, Alanine amino­
transferase; LZM, lysozyme.

Fig. 2. It was observed that the dietary CX inclusion significantly (P <

0.05) affected the activities of digestive enzymes in shrimp hepatopan­
creas. The highest (P < 0.05) protease and lipase activities were detected
in CX200 treatment, while amylase was detected in the CX400 treatment.
The control treatment had the lowest levels (P < 0.05) of the three
enzymes.
Fig. 3. Effect of different dietary canthaxanthin levels on total carotenoid
content in different tissues of Litopenaeus vannamei. Data were expressed as
3.7. Carotenoid contents mean ± S.E.M from triplicate groups. Bars with different letters represented
significant differences between various treatments (P < 0.05). TC,
The total carotenoid contents of different tissues of L. vannamei fed total carotenoid.

5
S. Fawzy et al. Aquaculture 556 (2022) 738276

Table 8 (P > 0.05) from CX100 and CX200 treatments in the head. Relative to
Effect of dietary canthaxanthin levels on carotenoids composition in tissues (μg astaxanthin concentration, CX content in L. vannamei tissues was lower.
g− 1) of Litopenaeus vannamei.
Tissue CDa Treatments 3.8. Quantification of optimum dietary CX level for L. vannamei
Control CX50 CX100 CX200 CX400
Fig. 4 presented the findings obtained after performing the broken-
Muscle
line regression analysis to determine the optimum dietary CX levels.
0.12 ± 0.14 ± 0.26 ± 0.37 ± 0.42 ±
Astaxanthin
0.01a 0.02a 0.01b 0.01b 0.04c The analysis was based on SGR, survival, a* (redness), protease, MDA,
0.003 ± 0.004 ± 0.009 ± 0.011 ± 0.013 ± AST, lysozyme, and LT50. These parameters figured out that the optimal
Canthaxanthin
0.001a 0.001a 0.001b 0.001c 0.001c dietary CX levels were 177.42, 173.72, 180.40, 193.71, 188.14, 200.30,
0.005 ± 0.005 ± 0.008 ± 0.013 ± 0.012 ± 202.13, and 198.76 mg kg− 1 diet, respectively.
Zeaxanthin
0.001a 0.001a 0.001b 0.001c 0.001c
0.04 ± 0.04 ± 0.05 ± 0.08 ± 0.10 ±
β-cryptoxanthin
0.01a 0.01a 0.01a 0.01b 0.01c 3.9. Hypoxia stress resistance test
0.06 ± 0.07 ± 0.08 ± 0.15 ± 0.17 ±
Echinenone
0.01a 0.01a 0.01a 0.01b 0.01b After exposure to low DO stress, the time required for the death of
0.09 ± 0.15 ± 0.18 ± 0.23 ± 0.31 ±
β-Carotene 50% of the stressed shrimp (LT50) was calculated and presented in Fig. 5.
0.01a 0.01b 0.01b 0.02c 0.02d
LT50 values were significantly (P < 0.05) influenced by the dietary in­
clusion of CX. The maximum LT50 value was observed in the CX200
Head
2.10 ± 4.17 ± 5.46 ± 6.60 ± 6.88 ±
treatment with significant differences from the other treatments, while
Astaxanthin the minimum value was given by the control treatment (P < 0.05).
0.25a 0.04b 0.65bc 0.26c 0.90c
0.07 ± 0.08 ± 0.05 ± 0.06 ± Further, no significant difference was observed in LT50 between CX100
Canthaxanthin ND
0.01bc 0.01c 0.01b 0.01bc and CX400 treatments.
0.06 ± 0.08 ± 0.09 ± 0.12 ± 0.12 ±
Zeaxanthin
0.01a 0.02ab 0.01ab 0.03b 0.01b
0.62 ± 0.80 ± 1.05 ± 0.86 ± 0.66 ± 3.10. Person correlation analysis
β-Cryptoxanthin
0.12 0.20 0.43 0.22 0.06

Echinenone
0.86 ± 1.17 ± 1.48 ± 1.13 ± 1.50 ± A heat map visualization was used to present the correlation asso­
0.18 0.24 0.47 0.27 0.32 ciation between the carotenoid composition in shrimp whole body
0.87 ± 1.13 ± 0.81 ± 1.11 ± 1.83 ±
β-Carotene
0.08a 0.12ab 0.05a 0.04ab 0.19b
(Fig. 6) and whole-body carotenoids with biochemical and immune-
physiological parameters (Fig. 7). The results indicated significant and
positive associations between carotenoids in shrimp whole body and
Shell
2.56 ± 3.39 ± 3.35 ± 4.08 ± 5.48 ± amylase, a* (redness), AKP, LT50, LDL, and HDL. While, ALT, AST, L*, CL,
Astaxanthin
0.22a 0.42ab 0.43ab 0.20b 0.12c GSH-Px, Urea, and CHO showed an opposite pattern.
0.30 ± 0.54 ± 0.68 ± 0.78 ± 1.08 ±
Canthaxanthin
0.02a 0.05b 0.02c 0.03c 0.02d 4. Discussion
0.10 ± 0.18 ± 0.22 ± 0.23 ± 0.30 ±
Zeaxanthin
0.01a 0.02b 0.01b 0.02b 0.01c
1.26 ± 1.63 ± 1.77 ± 1.80 ± 2.74 ± Carotenoid pigments are multi-functional feed additives. They are
added to the diets of aquatic animals, essentially for improving their
β-Cryptoxanthin
0.10a 0.18ab 0.06b 0.12b 0.11c

Echinenone
1.65 ± 3.02 ± 3.06 ± 3.76 ± 5.03 ± organoleptic properties (De Carvalho and Caramujo, 2017; Pereira da
0.21a 0.18b 0.41b 0.23b 0.01c
Costa and Campos Miranda-Filho, 2020) and consequently providing
2.77 ± 3.88 ± 4.07 ± 4.31 ± 5.07 ±
β-Carotene
0.15a 0.36ab 0.34b 0.25b 0.54b better market prices (Parisenti et al., 2011). Moreover, carotenoids were
recognized to perform many physiological and metabolic functions with
evidence for their beneficial effects on physical performance (Man­
Whole body
0.54 ± 0.99 ± 1.39 ± 1.64 ± 2.13 ± ikandan et al., 2020; Wade et al., 2017b). Our results indicated that
Astaxanthin
0.05a 0.04b 0.10c 0.02d 0.02e dietary inclusion of CX dramatically improved growth performance,
Canthaxanthin
0.05 ± 0.17 ± 0.21 ± 0.30 ± 0.34 ± enhanced survival, and decreased the FCR of Litopenaeus vannamei. The
0.01a 0.01b 0.01b 0.01c 0.03c
results were consistent with the previous studies on giant tiger shrimp
0.04 ± 0.08 ± 0.10 ± 0.12 ± 0.15 ±
Zeaxanthin
0.01a 0.01b 0.01bc 0.01c 0.01d
(P. monodon) fed with CX supplemented diet (Niu et al., 2012), oriental
0.42 ± 0.47 ± 0.57 ± 0.81 ± 0.84 ± river prawn (Macrobrachium nipponense) fed with lutein supplemented
β-Cryptoxanthin
0.06a 0.03a 0.04a 0.07b 0.06b diet (Ettefaghdoost and Haghighi, 2021); and swamp crayfish, Pro­
0.66 ± 0.76 ± 0.94 ± 1.18 ± 1.34 ± cambarus clarkii (Cheng and Wu, 2019), L. vannamei (Flores et al., 2007;
Echinenone
0.10a 0.08a 0.16ab 0.10bc 0.12c
Liu et al., 2018; Wang et al., 2020), and kuruma shrimp, Marsupenaeus
0.58 ± 0.65 ± 0.84 ± 0.98 ± 1.22 ±
β-Carotene
0.02a 0.03ab 0.01b 0.01b 0.02c japonicas (Wang et al., 2018a) fed with astaxanthin supplemented diets.
a
Furthermore, the studies carried out on a variety of fish species showed
Data were expressed as mean ± S.E.M from triplicate groups. Mean values in
that dietary CX supplementation could boost the growth and survival of
the same row with different letters are significantly different (P < 0.05). CD,
Lake Kurumoi rainbowfish, Melanotaenia parva (Allen) (Meilisza et al.,
carotenoids.
2017), Atlantic salmon (Torrissen, 1984), and red porgy, Pagrus pagrus
(Kalinowski et al., 2015).
Astaxanthin, CX, zeaxanthin, β-cryptoxanthin, echinenone, and β-caro­
The growth-promoting role of carotenoids such as CX could be
tene were detected as the main carotenoids in the tissues of L. vannamei.
related to their capability on activating digestion and metabolism (Amar
The six carotenoids mentioned above were significantly (P < 0.05)
et al., 2001), shortening the molting cycles intervals (Petit et al., 1997),
affected by the dietary manipulation and showed increasing trends in
inhibition of nicotinamide adenine dinucleotide phosphoric acid
different tissues by increasing dietary CX supplementation. The most
(NADPH) reductase, decreasing energy consumption, and further pro­
abundant carotenoid accumulated in various tissues was astaxanthin.
moting the biosynthesis and growth (Ohno et al., 2011) in aquatic ani­
The highest astaxanthin content in all tissues was given by CX400
mals. Additionally, the higher survival rate of shrimp fed with CX
treatment, which was significantly different (P < 0.05) from other
supplemented diets could be associated with the superior antioxidant
treatments in muscle, shell, and whole-body but did not differ markedly
activity of carotenoids which ameliorates the effect of stressors and

6
S. Fawzy et al. Aquaculture 556 (2022) 738276

Fig. 4. Broken-line analysis of optimal dietary CX levels based on SGR, survival, a* (redness), protease, MDA, AST, lysozyme, and LT50.

7
S. Fawzy et al.
Fig. 5. Time to 50% mortality (h) after exposure to hypoxia stress tolerance of
Litopenaeus vannamei fed with different levels of canthaxanthin for 56 days.
Data were expressed as mean ± S.E.M. from triplicate groups. Bars with
different letters represented significant differences between various treatments
(P < 0.05).
Fig. 6. The correlation heatmap characterizes the Pearson correlation co­
efficients (r) between the carotenoid composition in shrimp whole body. The
red bar means the positive correlation, while the blue bar means the negative
correlation. Moderate correlation was defined following (0.5 < |r| < 0.8) and
high correlation (0.8 < |r| < 1), respectively. (For interpretation of the refer­
ences to color in this figure legend, the reader is referred to the web version of
this article.)
prolongs the shrimp life (Cheng and Wu, 2019; Niu et al., 2012). Wade
et al. (2017a) demonstrated that carotenoid supplementation could
improve only the weight gain but not the survival of P. monodon.
Moreover, other counterpart studies on kuruma shrimp Penaeus japoni­
cus (Yamada et al., 1990), P. monodon (Boonyaratpalin et al., 2001),
tropical spiny lobster Panulirus ornatus (Barclay et al., 2006), L. vannamei
(Ju et al., 2011) and Chinese mitten crab Eriocheir sinensis (Jiang et al.,
2020) indicated that the growth and survival were similar regardless of
addition or omission of dietary carotenoids. The dissimilarity of these
studies with ours could be attributed to the variations in diet composi­
tion, culture conditions, state of animal health, species-specific carot­
enoid requirements, source of carotenoid supplement, and the initial
carotenoid level in shrimp (Chien and Jeng, 1992; Wade et al., 2017b).
In the aquaculture industry, dietary carotenoid additives constitute
about 15–20% of the salmon feed cost (Forsberg and Guttormsen, 2006),
which dramatically increase the total production costs of some aquatic
animals (Hansen et al., 2016). Therefore, it was necessary to determine
8
Aquaculture 556 (2022) 738276
Fig. 7. The heatmap characterizes the Pearson correlation coefficients (r) be­
tween the whole-body carotenoids with other measured parameters in the
current study. The blue bar suggests that the whole-body carotenoids positively
correlate with the other parameters, while the orange bar means the negative
correlation. The asterisk (* and **) indicates P < 0.05 and P < 0.01, respectively.
Moderate correlation was defined following (0.5 < |r| < 0.8) and high corre­
lation (0.8 < |r| < 1), respectively. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
the optimum CX level for better physical performance. In such a way,
only the requirements of the species will be provided, wastage will be
avoided, and the feed cost will be maintained.
One of the most critical indicators for the nutritive value of the
cultured species is the proximate composition (Shao et al., 2014). Ac­
cording to Kawamura et al. (2020), carotenoids intake could stimulate
protein synthesis leading to an increase in protein content and muscle
mass. This agrees with our study where the protein content in the body
of shrimp fed CX-containing diets was significantly higher than that fed
the control diet, which was reflected by the growth performance. A
similar finding was observed for P. monodon (Niu et al., 2014) and
E. sinensis (Long et al., 2017). The authors suggested that the efficiency
of carotenoids in reducing oxidative stress and hence, energy expendi­
ture is possibly the cause for the elevation of protein concentration.
CX has been reported to have potent antioxidant and immune
modulation properties (Venugopalan et al., 2013). T-AOC is an essential
index for both enzymatic and non-enzymatic antioxidant activities, and
its higher value indicates an improved antioxidant capacity. The current
study revealed that CX feeding significantly increased T-AOC compared
to the control diet. This is in the line of findings on P. monodon (Niu
et al., 2012) and O. mykiss (Cui et al., 2009; Elia et al., 2019). The au­
thors concluded that the antioxidant activity was greatly enhanced after
feeding a diet supplemented with CX compared to CX-free diet. POD, a
pivotal antioxidant enzyme, also increased in the current study with
increasing dietary CX supplementation which is similar to the finding of
Long et al. (2017) on E. sinensis. Another major enzyme in the antioxi­
dant defense system, CAT, acts as a scavenger for hydrogen peroxide
(H2O2) and catalyzes its decomposition into molecular oxygen and
water to protect the cell from oxidative damage (Chien et al., 2003;
Ighodaro and Akinloye, 2018). In the current study, CAT activity
decreased significantly by increasing the dietary CX levels. Similar
findings were observed on astaxanthin fed L. vannamei (Zhang et al.,
2013), P. monodon (Niu et al., 2014), Oncorhynchus mykiss (Rahman
et al., 2016), and lutein fed M. nipponense (Ettefaghdoost and Haghighi,
2021). The GSH-Px enzyme also participates in the removal of H2O2. Its
decreasing activity in CX supplemented treatments compared to the
control treatment indicates that higher cell protection was provided by
CX as reported on yellow perch, Perca flavescens (El-Gawad et al., 2019),
and characins, Hyphessobrycon callistus (Wang et al., 2006). Addition­
ally, Pearson correlation analysis indicated that GSH-Px activity was
S. Fawzy et al.
significantly and positively reflected by carotenoid level in shrimp body
(Fig. 7). These authors suggested that carotenoid pigments, including
CX, have a vigorous O2 quenching activity. Therefore, they could
effectively neutralize and remove the free radicals and hence, lower the
need to produce endogenous antioxidant enzymes like CAT and the
GSH-Px, which is consistent with the findings of the current study.
Further, increased CX supplementation level resulted in a significant
decrease in MDA level in L. vannamei. According to Munoz et al. (2000),
MDA level reflects the degree of cellular damage and lipid peroxidation.
So, the decreased MDA level in our study indicates CX’s effective role in
the inhibition of lipid peroxidation and protection of the cell against free
radicals (Sahin et al., 2014). Combining these data, the efficient anti­
oxidant properties of CX can be concluded.
It is well-known that shrimp, as a crustacean, rely entirely on the
innate immune system to defend themselves against the invading
pathogens (Zhao et al., 2009). Innate immunity includes two main
components, the cellular and humoral immune responses. Hemocytes
are the critical element that mediates cellular immunity and are
involved in the secretion of various humoral components in the hemo­
lymph (Li and Xiang, 2013; Liu et al., 2020). In this respect, non-specific
immune biomarkers (AKP, AST, ALT, and lysozyme) in the serum were
used to evaluate the immune response of L. vannamei after feeding with
CX. AKP is a pivotal regulatory enzyme that participates in phosphory­
lation and dephosphorylation processes in organisms and its role in
improving the absorption and utilization of nutrients in shrimp. Lyso­
zyme is another important index of non-specific immunity in crusta­
ceans due to its function as a potent killer for Gram-positive bacteria
(Wang et al., 2018c). The activity of lysozyme points out an enhance­
ment of the immune response of aquatic animals. The results of the
current study showed that the activities of AKP and lysozyme signifi­
cantly increased in shrimp fed with CX supplemented diets compared to
those fed the control diet. Our findings were in agreement with the
studies carried out on P. clarkii (Cheng and Wu, 2019), E. sinensis (Jiang
et al., 2020), and crucian carp, Carassius auratus (Wu and Xu, 2021). The
similarity between the present and the previous studies clarifies that CX,
like other carotenoids, exerts a significant role in improving the non-
specific immunity of shrimp. Given the vital role of AST and ALT en­
zymes in protein metabolism, they are widely used to evaluate the
health status of aquatic animals (Haque et al., 2021). The activities of
these enzymes in serum reflect the physiological status of hep­
atopancreatic cells because their higher levels in hemolymph indicate
severe damage and increased leakage of hepatopancreatic cells (Ette­
faghdoost and Haghighi, 2021; Mohankumar and Ramasamy, 2006).
The current study demonstrated that shrimp fed with CX exhibited
reduced levels of AST and ALT in comparison to those fed the control
diet. Similarly, Ettefaghdoost and Haghighi (2021), Lim et al. (2017),
and Niu et al. (2012) demonstrated that carotenoids supplementation
significantly decreased the activities of AST and ALT in the serum of
shrimp. Combining the results, dietary CX could be a beneficial immu­
nostimulant for enhancing the health status of the studied shrimp.
Hepatopancreas is the primary digestive gland in shrimp. It combines
the functions of the intestine, liver, and pancreas, taking part in the
synthesis of digestive enzymes, absorption of nutrients, and their
metabolism (Vogt, 2019). Therefore, the hepatopancreatic digestive
enzymes were used as indicators for nutritional physiology in the
studied shrimp. Evaluation of digestive enzymes activities gives an
indication about the digestion processes and nutrient utilization effi­
ciency, which is reflected by the growth of aquatic animals (Abolfathi
et al., 2012; Lovett and Felder, 1990). Lutein, a carotenoid pigment, had
been reported to significantly increase the activities of the digestive
enzymes of M. nipponense (Ettefaghdoost and Haghighi, 2021). Likewise,
Wang et al. (2018b) noticed an improvement in the activities of diges­
tive enzymes in kuruma shrimp, M. japonicus after feeding astaxanthin
added diet. Similarly, our findings revealed that the dietary incorpora­
tion of CX markedly increased protease, amylase, and lipase activities
more than CX-free diet. Together with the previous reports, these
9
Aquaculture 556 (2022) 738276
findings support the positive role of carotenoids in enhancing the
digestion process and nutrient utilization, which probably explains the
better growth performance of shrimp in the treatments received CX
compared to those in the control treatment.
Due to the disability to synthesize carotenoids de novo, L. vannamei
and other crustaceans mainly depend on the dietary carotenoids to ac­
quire the attractive reddish coloration (Goodwin, 1984; Wade et al.,
2017b). Moreover, the color of crustaceans’ bodies is determined by
factors such as the concentration and composition of carotenoids, spe­
cies, and environmental conditions (Long et al., 2017; Niu et al., 2012).
Our results clearly indicated that the total carotenoids and astaxanthin
contents in the shell, muscle, head, and whole body of L. vannamei
increased significantly and positively with increasing dietary CX levels
(Fig. 6). Deposition of CX primarily in the form of astaxanthin supports
the findings on P. japonicus (Yamada et al., 1990) and P. monodon
(Boonyaratpalin et al., 2001), which implied that crustaceans could
transform or modify the pigments precursors like CX into astaxanthin
and accumulate them in different tissues. Jiang et al. (2020) also stated
that the red color is related to astaxanthin content in the exoskeleton of
E. sinensis. Consistent with this report, Pearson correlation analysis
indicated that the redness (a*) value of cooked shrimp in treatment
received CX was significantly and positively affected by astaxanthin
levels in shrimp body in the current study (Fig. 7). Additionally, CX had
been proved to be as effective as astaxanthin in the pigmentation of
Atlantic salmon (Buttle et al., 2001). On the contrary, the L* value of
cooked shrimp showed a decreasing trend with increasing dietary CX,
and it was also found that carotenoids included in diets decreased the
lightness of red king crab, Paralithodes camtschaticus (Daly et al., 2013),
and red porgy, P. pagrus (Kalinowski et al., 2005). The results confer that
L. vannamei could utilize the dietary CX effectively to improve their red
appeal and other biological functions. One more observation is that the
lowest total carotenoids and astaxanthin contents were observed in
muscle tissue which is consistent with the findings reported earlier (Niu
et al., 2012). Nevertheless, the carotenoid deposition in muscle is
beneficial for the consumer’s health since carotenoids are effective an­
tioxidants besides having other properties that make them an essential
human food supplement (De Carvalho and Caramujo, 2017).
Shrimp culture under an intensive system is constantly subjected to
different environmental stressors such as hypoxia (Chien and Shiau,
2005) that can arise from temperature fluctuations or the formation of
algal blooms due to the presence of organic pollutants (Belão et al.,
2011). Hypoxia causes significant economic losses and is considered a
threat to productivity due to its negative impact on shrimp survival
(Diaz and Rosenberg, 2011). Previous studies showed that the
enhancement of resistance in penaeid shrimp to hypoxia stress is closely
related to dietary carotenoids uptake (Niu et al., 2012; Niu et al., 2009).
Moreover, a positive correlation was detected between the improvement
of shrimp resistance to stressors and pigment concentration in the diet
and tissue (Chien et al., 2003). Similarly, in the current study, LT50
values increased with increasing dietary CX levels, with the maximum
values observed for the shrimp-fed diets containing the higher CX levels
(200 and 400 mg kg− 1). Therefore, it can be concluded that dietary CX
pigment could enhance the resistance of the studied shrimp against
hypoxia stress and prolong its life, as reported by Chien et al. (1999) and
Niu et al. (2012) on P. monodon, and Zhang et al. (2013) on L. vannamei.
This is probably associated with the antioxidant property of carotenoids
(Table 5), which is closely linked to stress resistance (Niu et al., 2014).
They play a vital role in protecting sensitive structures of the cell from
oxidative damage, which is reflected by an increase in shrimp survival.
5. Conclusion
Overall, the findings of the current study revealed that supplemen­
tation of the diet with CX in the range of 173.73 to 202.13 mg kg− 1 is of
primary importance to shrimp growth performance and health status.
Therefore, with its numerous properties and relatively low market price,
S. Fawzy et al.
antioxidant and metabolic conversion characters, CX could be a poten­
tial astaxanthin precursor in the diet of L. vannamei.
Credit author statement
Weilong Wang finished the data analysis and worked on paper
revision; Fawzy Samia finished the biochemical analysis and drafted the
manuscript; Meiqin Wu put forward relevant experimental guidance;
Ganfeng Yi and Xuxiong Huang designed the research. All authors read
and gave final approval of the manuscript.
Declaration of Competing Interest
The authors have no conflict of interest to declare.
Acknowledgments
The work was supported by the Shanghai Agriculture Applied
Technology Development Program, China (No.
202102080012F00761); National Science Foundation of China
(31902385); Chinese Postdoctoral Science Foundation (219724);
Shanghai Collaborative Innovation Center for Cultivating Elite Breeds
and Green-culture of Aquaculture Animals, Key Laboratory of Aquatic
Functional Feed and Environmental Regulation of Fujian Province Open
Project (FACE20200001); and China Scholarship Council (CSC) sup­
ported this research through a Ph.D scholarship (No.2018 GBJ008472).
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