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Aquaculture Research. 2022;00:1–14. wileyonlinelibrary.com/journal/are
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1© 2022 John Wiley & Sons Ltd.
Received: 23 May 2022
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Revised: 16 August 2022
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Accepted: 19 August 2022
DOI: 10.1111/are.16084
ORIGINAL ARTICLE
The effect of dietary nanocurcumin on the growth
performance, body composition, haemato- biochemical
parameters and histopathological scores of the Nile tilapia
(Oreochromis niloticus) challenged with Aspergillus flavus
El- Sayed Hemdan Eissa1 | Omaima H. Ezzo2 | Hala Saber Khalil3 |
Wesam Ahmed Tawfik4,5 | Ashraf A. El- Badawi6,7 | Nadia A. Abd Elghany8 |
Monga I. Mossa9 | Montaser M. Hassan10 | Mohamed M. Hassan10 |
Moaheda E. H. Eissa11 | Manal E. Shafi12 | Awatef Hamed Hamouda13
1Fish Rese arch Centre, Ar ish Universit y, Arish, Egypt
2Depar tment of Animal Reprodu ction and Ar tificial Inseminatio n, Natio nal Rese arch Centre, Do kki, Egypt
3Nationa l Institute of Oce anography and F isheries, (NIOF), Cairo, Eg ypt
4Holding Company for Biological P roduc ts and Vaccines, Giza, Egypt
5Naqaa Nanotechnolog y Netwo rk, Eg ypt
6Centra l Labo rator y for Aquacultu re Resea rch, Ab bassa , Abo- Hammad, Egypt
7Biolog y Depar tment, Univer sity Col lege, Umm- Al Qura University, Makkah, Saudi Ar abia
8Fish Diseases De part ment, Animal H ealth Re search Institute, Agriculture Research Ce ntre (A RC), Dok ki, Eg ypt
9Botany and Microbiolog y Departme nt, Facul ty of Science, Arish Universit y, Arish, E gypt
10Depa rtme nt of Biology, Colle ge of Scien ce, Taif Univer sity, Taif, Saudi A rabia
11Aquaculture Depar tment, Facult y of Fish and Fishe ries Technology, As wan Universit y, Aswan , Egypt
12Depar tment of Biologi cal Sci ences Zoology, Faculty of Sciences , King Abdulaziz Univer sity, Jed dah, Sa udi Arabia
13Fish Health and Diseases Departme nt, Facul ty of Fis h and Fisheries Technolog y, Aswan Un iversity, Aswa n, Egypt
Correspondence
El- Sayed Hem dan Eiss a, Fish Researc h
Centre, Arish Un iversi ty, Arish , Egypt.
Email: sayed_hemd@hotmail.com-
Hala Sab er Khal il, National Ins titute of
Oceanography and Fisheries, (NIOF),
Cairo, E gypt .
Email: halasaber2011@yahoo.com
Funding information
Taif University, Grant/Award Numb er:
TURSP- 2020/119
Abstract
The study aims to investigate the potential effect of nanocurcumin as feed additive
in the diet of Oreochromis niloticus to improve its growth performance, health status
and resistance against Aspergillus flavus. The control group was fed on a basal diet
without nanocurcumin, and four diets T1, T2, T3 and T4 were supplemented with 10,
25, 40 and 55 mg/kg of nanocurcumin, respectively, in triplicate (20 fish/replicate).
The duration of the feeding trial was 60 days. The final body weight, weight gain, spe-
cific growth rate and survival rate showed significantly (p < 0.05) increased values in
the nanocurcumin groups than the control. Fish fed with nanocurcumin supplementa-
tion showed improvement in RBCs, haemoglobin, total protein, albumin and globulin
while there was a decrease in the liver enzymes (AST and ALT), glucose and alkaline
phosphatase. The creatinine was also decreased in fish fed nanocurcumin. The di-
gestive enzymes amylase and lipase increased in the nanocurcumin- treated groups,
and the triglycerides values showed non- significant increase, whereas the cholesterol
values showed non- significant decrease in T1 and T4. Meanwhile, the cortisol was
2
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EISSA et a l.
1 | INTRODUC TION
Curcumin [(E, E)- 1, 7- bis (4- hydroxy- 3- methoxy- phenyl)- 1,
6- heptadiene- 3, 5- dione], of the ginger family, is a bis- α, β-
unsaturated β- diketone and is an active ingredient of Curcuma longa
(turmeric). For centuries in traditional medicine, the powder of
C. longa was considered as an antiinfective agent especially in the
countries of Southeast Asia (Zheng et al., 2018). However, there are
some drawbacks related to it s solubility, bioavailability and metabo-
lism that hinder its therapeutic uses (Karthikeyan et al., 2020).
Nowadays, the application of nanotechnology is a very promising
advancement in the aquaculture industry (Khalil, Maulu, et al., 2022). To
ove rcome the drawba ck s of the trad it ion al cur cu min powde r, na no tech-
nology facilitated the nanoencapsulation of curcumin into nanocurcumin
(Karthikeyan et al., 2020). Nanocurcumin has powerful immunomodula-
ti o n effec ts on bo t h an ima l s and hu mans . It has go o d bio l ogic al an d phar-
macological properties, such as being an anti- inflammatory, antioxidant
and antimicrobial agent (Jayaprakasha et al., 2006; Kohli et al., 2005;
Mohamed et al., 2020). The solubility, absorption rate and bioavailability
of nanocurcumin are higher than curcumin (Hani & Shivakumar, 2014;
Kurita & Makino, 2013; Ma et al., 20 07; Song et al., 2 011). Therefore,
lower doses of nanocurcumin can be used instead of large ones to be
more cost- effective (Alagawany et al., 2021).
There are lots of studies dealing with curcumin as feed ad-
ditive in diets of dif ferent fish species (Baldissera et al., 2018; El-
Barbary, 2018; Mahmoud et al., 2017). On the other hand, El Basuini
et al. (2022) reported that incorporating nanocurcumin at a level of
≥100 mg/kg diet (particularly at 150 mg/kg) improved non- specific
immune responses (Lysozyme and bactericidal activities), antioxidant
enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathi-
one peroxidase [GPX]), and maintained healthier gastrointestinal mi-
crobiota in Nile tilapia under chronic low- temperature stress.
Oreochromis niloticus is one of the topmost cultured fish spe-
cies in Egypt and around the world (Khalil et al., 2021; Kord, Maulu,
et al., 2021). There is a great interest among aquaculturist s about
this species because of its high market demands, adaptability
to various environmental conditions and resistance to diseases
(Allam et al., 2020; Maulu et al., 2021). Tilapia culture is one of the
world's fastest growing aquaculture food industries (Khalil, Momoh,
et al., 2022). However, farming fish under oppor tunistic pathogens
or stressful circumst ances has serious consequences on their health,
metabolism and physiological reac tions (Xavier et al., 2020).
Fungi are op portuni stic pathogens When infecting freshwater fish,
they may cause high mor talities, hinder the growth rate and reduce
hatchability especially in chronic infection, including mycotoxins pro-
duction when fish are subjected to stress of any type such as poor
water quality (Abd El Ghany & Elias, 2014; Abd El- Tawab et al., 2020;
Noor El- Deen et al., 2018). One of the feed contaminants is the
Aspergillus species, which produces aflatoxins (Ferreira et al., 2013).
Aspergillus flavus produces aflatoxin B1, which causes hepatotoxic and
carcinogenic effects (Williams et al., 200 4). The superfluous utilization
of antimicrobials in fish environment causes a lot of problems, in partic-
ular, the risk of drug resistance, which is considered as a public health
hazard. Therefore, the utilization of phytogenics like nanocurcumin as
antimicrobials substitution has become of interest in fish industr y.
The present study aims to assess the effect of nanocurcumin-
treated diet on the growth performance, response to feed intake,
survival rate, body composition and blood biochemical parameters
of O. niloticus. Furthermore, to determine the antifungal efficacy of
nanocurcumin on A. flavus challenged O. niloticus.
2 | MATERIALS AND METHODS
2.1 | Preparation of curcumin nanoparticles
Curcumin (Chemajet Comp., Egypt), the bioactive compound of tur-
meric and Dichloromethane (Elgomhoreya pharmaceutical company,
Egypt) were used in the preparation of the curcumin nanoparticles.
Briefly, the solvent- antisolvent method was used for the preparation
nearly the same in all groups. At the end of the trial, the fish were challenged with
Aspergillus flavus for 15 days. Aspergillus flavus resu lted in the mor t ality of 10 0% of the
control group and the groups with lower doses of nanocurcumin (T1 and T2) within
the first week and second week post challenge respectively. In the treatments with
high doses (T3 and T4), higher survival rates were recorded in a dose- dependent man-
ner. The pathogenicity of Aspergillus flavus was confirmed histopathologically. It was
concluded that the dietary supplementation of nanocurcumin enhanced the health
status of O. niloticus by improving the haemato- immunological response and body
composition parameters of the fish, and protected it from the Aspergillus flavus infec-
tion with optimum inclusion levels of 25– 40 mg/kg diet.
KEY WORDS
Aspergillus flavus, fish gills, fish skin and muscles, growth per formance, haemato- immunological
response, nanocurcumin, Oreochromis niloticus, survival
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EISSA et al.
of curcumin nanoparticles according to Kakran et al. (2012) with
modifications in the antisolvation technique, according to El- Sayed
et al . (2018). Curcumin extracts were prepared in dichloromethane as
an organic solvent. The syringe was filled with 10 ml of the prepared
solution and was quickly injected at a fixed flow rate (10 ml/min) into
the deionized water (antisolvent) of a definite volume at a ratio of 1:9
under magnetic stirring (1000 rpm). Stirring was allowed for 2 h. Then
the formed curcumin nanopar ticles were filtered and dried.
2.1.1 | Characterization of nanocurcumin
Characterization was done in the Electron Microscope Unit at the
Regional Center of Mycology, Al- Azhar University, Cairo, Egypt.
Transmission electron microscopy (TEM) micrograph shows the na-
noparticles of curcumin extract of particle size 10– 50 nm with uni-
form size distribution (Figure 1). The particles are nearly spherical in
shape. The purity of the curcumin source was 99%.
2.2 | Experimental diet and fish samples
This experiment was carried out in a private fish farm in Abbassa,
Abo- Hammad, Egypt. A total number of 400 O. niloticus with av-
erage initial body weight of 5.00 ± 0.30 g was maintained into a
concrete pond for acclimation. To exclude any external lesions or
abnormalities, 20 fish were examined externally. Then, 300 fish (20
fish/pond, three triplicate per treatment), were stocked into 15 sep-
arate concrete ponds (1000 L each). The water quality parameters
in ponds were as follows; water temperature 26.21 ± 3.20°C , pH
7.50 ± 0.26, dissolved oxygen 7.21 ± 0.61 mg L−1 and total ammonia-
nitrogen 0.10 ± 0.06 mg L−1.
After acclimatization, the nanocurcumin was added to the fish
feed in the four treatment groups as follows: 10 mg of nanocur-
cumin/kg (T1), 25 mg of nanocurcumin/kg (T2), 40 mg of nanocur-
cumin/kg ( T3), 55 mg of nanocurcumin/kg (T4), and the control (no
additives). The fish were fed the experimental diet s twice daily for
60 days, at a rate of 3% of their body weight (Table 1).
2.2.1 | Sampling
From each pond, three fish were anaesthetized with tricaine meth-
ane sulfonate (MS- 222) with the dosage of 100 mg L−1 and wer e used
for the collection of blood and serum samples via the caudal blood
vessel using heparinized tubes as anticoagulant and without heparin
respectively. The serum was obtained by centrifugation of the blood
at 3500 g for 15 min at 4°C, then kept at −20°C until further analysis.
From the same fish, samples from gills, liver and ovary were
collected and preserved in 10% formalin for histopathological
examinations.
2.3 | Growth parameters
After the end of the feeding trial and calculation of survival rate (100
* [Final number of fish/initial number of fish]), fish were weighed
individually to calculate the following parameters:
FIGURE 1 Characterization
of nanocurcumin turmeric under
transmission electron microscopy.
4
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EISSA et a l.
Where FW: final fish weight (g); IW: initial fish weight (g).
2.4 | Blood haematology and
biochemical parameters
Red blood cells (RBCs) and white blood cells (WBCs) were counted
using a haemocy tometer with Natt & Herrick's solution, accord-
ing to Houston (1990) and Stoskopf (1993) respectively. The hae-
moglobin was measured according to the method of Blaxhall and
Daisley (1973 ) after the addition of Drabkin's solution. From the ob-
tained serum, the total protein, albumin and globulins contents were
determined, according to Peters Jr. (1970) , Doumas an d Bi ggs (1972),
and Peters Jr. et al. (1982). Aspartate aminotransferase (AST), ala-
nine aminotransferase (ALT) and alkaline phosphatase (ALP) were
determined, according to Reitman and Frankel (1957).
Creatinine was determined according to Heinegard and
Tiderstrom (1973). Serum glucose was determined by using a blood
suga r determination kit, ‘Boehringer’, as described by Trinder (1969).
Activities of amylase and lipase were measured by the methods of
Bernfeld (1955) and Furne et al. (2005) respectively. The serum cho-
lesterol and triglycerides were estimated by the methods described
by Thomas (1992) and Friedewald et al. (1972) respec tively. The cor-
tisol was determined by the Automated Chemiluminescence System
(ACS: 180).
2.5 | Chemical analysis
Diet and fish body composition analyses were performed using
the standard methods of Horwitz (2010) to determine crude pro-
tein, crude lipid, moisture and ash levels. The fish and feed moisture
levels were determined by oven drying to a constant weight at 70
and 105°C, respectively, according to AOAC (2016). The content
of protein for both fish and diets was calculated using the Kjeldahl
method. The fish and diets ash levels were calculated using a muffle
furnace at 550°C for 8– 10 h. The fish and diets lipid content were
Weight gain (WG, g)=FW −IW.
Specific growth rate (SGR)=100∗(Ln (FW)−Ln (IW)) ∕experimental days.
Feed conversion ratio based on dry matter (DM):(FCR)
=
feed intake
(
g
)
as dry weight
∕
weight gain
(
g
)
.
TAB LE 1 The diet ingredients and proximate chemical composition (% on dry matter basis) of the experimental diets used in the present
study
Ingredient Control T1 (Nanocur10) T2 (Nanocur25) T3 (Nanocur40)
T4
(Nanocur55)
Fish meal (CP 72%) 11.0 0 11.0 0 11.0 0 11.00 11 .0 0
Soybean meal (CP 48%) 36.00 36.00 36.00 36.00 36.00
Rice bran 20.00 20.00 20.00 20.00 20.00
Wheat bran 20.00 20.00 20.00 20.00 20.00
Yellow corn 6.00 5.99 5.97 5.96 5 .94
Nanocurcumin – 0.01 0.02 0.04 0.05
Fish oil 1.50 1.50 1.50 1.50 1.50
Soybean oil 1.50 1.50 1.50 1.50 1.50
Molasses 2.00 2.00 2.00 2.00 2.00
Dicalcium phosphate 1.00 1.00 1.00 1.00 1.00
Vitamin and mineral premixa1.00 1.00 1.00 1.00 1.0 0
Chemical composition
Dry matter (DM) 91.50 91.48 91.42 91.50 91.50
Crude protein (CP) 30.28 30.28 30.28 30.28 30.28
Crude lipids (CL) 8.17 8.16 8.17 8.16 8.16
Crude fibre (CF) 6.62 6. 61 6.61 6. 62 6.62
Nitrogen- free extract (NFE)b4 7.70 47.71 47. 70 47. 70 47. 71
Ash 7. 23 7.21 7. 23 7. 2 3 7. 2 3
Growth Energy (GE)c3.74 3.73 3.73 3.73 3 .73
aVitamin mixture (mg/k g premix): vitamin A (3200 IU), vitamin B1 (129 mg), vitamin B2 (570 mg), vitamin E (2550 mg ), vitamin B6 (520 mg), vitamin
B12 (40 mg), vitamin D3 (390 IU), biotin (8550 mg), inositol (33 0 mg), choline chloride (3 000 mg), par a- amino benzoic acid (8990 mg), niacin (25. 50 mg),
vitamin C (2500 mg), pantothenic acid (3000 mg), copper (20 mg), iodine, cobalt (6 mg), manganese (280 mg), and iron (150 mg).
b
NFE
=100 −
[
%Ash +%CL +%CP +%CF
]
.
cGE was calculated as 5.65, 9.45, and 4.11 kcaL/g for protein, lipid, and carbohydrates respec tively.
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EISSA et al.
determined using the chloroform/methanol extraction method, ac-
cording to AOAC (2016).
2.6 | Aspergillus flavus infection and sampling
The A . flavus isolated from O. niloticus was provided by the
Microbiological Unit of Fish Diseases Department, Animal
Health Institute, Dokki, Giza, Egypt. For the preparation of
spores' suspension, A. flavus was cultured on Sabourauds dex-
trose agar (SDA, Difco) with penicillin (100 UI/ml) and strepto-
mycin (100 μg/ml), then incubated at 25°C for 7 days according to
Willoughby (1994). To co llect co ni dial ma ss, ster il e d is tille d water
(20 ml) was added to each plate and the suspension was collected
in s te ri le tube s. Two laye rs of s te rile ga uze we re used to f il ter the
suspension.
An erythrocyte counting chamber of a haemocytometer
was used to calculate and adjust the conidial suspension to be
4 × 103 conidia/ml in sterile distilled water (Willoughby, 1994).
Then, 10 of the remaining fish from each group were injected I/P
(intraperitoneal injection with a sterile needle) with 0.2 ml of A. fla-
vus (4 × 103 conidia/ml), and were observed daily for 15 days. At
the end of the exp erimental tri al , samples from gills, liver and ovary
were collected from all fish groups and preserved in 10% formalin
for histopathological examinations. Specimens from liver, gills and
muscle at the injection site from each living or dead fish were mi-
croscopically examined and inoculated into PDA (Potato Dextrose
Agar) plates to assure re- isolation and identification of the fungus
(Willoughby, 1994).
2.7 | Histopathological examination
Tissue specimens from suspected organs were fixed in buffered for-
malin (10%). Then they were embedded in paraffin, sectioned and
stained with Haematox ylin and Eosin (H & E) as well as the Periodic
Acid Schif f method (PAS) (Rober ts, 1978).
2.8 | Statistical analysis
Data were statistically analysed with a one- way analysis of variance
(ANOVA) test using spss version 22, spss, IL, USA . Differences were
considered significant at p < 0.05. Polynomial regression analysis
was done for growth per formance parameters and feed conversion
ratio to determine the linear impacts of nanocurcumin supplemen-
tation on the tested variables (Yossa & Verdegem, 2015). Survival
rate of O. niloticus- fed nanocurcumin diets and post challenged with
A. flavus for 15 days was assessed by the Kaplan– Meier method with
survival curves, Log- rank (Mantel- Cox) test.
3 | RESULTS
3.1 | Growth and response to feed intake
Th e fi nal body weigh t, weig ht gain and specif ic grow th ra te were sig-
nificantly (p < 0.05) higher in all fish groups fed with nanocurcumin
compared with the control (Table 2). The nanocurcumin supplemen-
tation linearly increased significantly in the final weight (g), specific
growth rate (SGR% g/day), weight gain (g), and feed conversion
ratio values of O. niloticus compared with those fed the control diet
(p < 0.05). The highest FW, WG, SGR and FCR values were noticed in
the T3 treatment as shown in Figure 2. The second- order polynomial
regression between FW, WG, SGR and FCR and nanocurcumin sup-
plementation levels showed that the optimal nanocurcumin levels
for maximum growth and feed utilization was 40 mg/kg diet. On the
other hand, the survival rate of O. niloticus was not influenced by
nanocurcumin- treated diets (p > 0.05) (Table 2).
3.2 | Blood haematology and
biochemical parameters
The RBCs and haemoglobin showed significantly (p < 0.05) increased
values in fish fed nanocurcumin, while the values of WBCs showed no
TAB LE 2 Growth performance and feed efficiency of Oreochromis niloticus fed diets supplemented with varying levels of nanocurcumin
for 60 days
Item
Tes t di et s
Control T1 (Nanocur- 10) T2 (Nanocur- 25) T3 (Nanocur- 40)
T4
(Nanocur- 55)
Initial weight (g) 5.08 ± 0.02 5.12 ± 0.04 5.15 ± 0.02 5.24 ± 0.02 5.16 ± 0.05
Final weight (g) 19.2 3 ± 1.19b26.70 ± 0.99a28.66 ± 0.81a28.30 ± 0.62a28 .13 ± 0.53a
Weight gain (g) 14.15 ± 1 .18b21.65 ± 0.95a23 . 51 ± 0.84a23.05 ± 0.60a22.97 ± 0.49a
SGR (%/day) 2.21 ± 0.09b2.75 ± 0.05a2.86 ± 0.05a2.81 ± 0.03a2.82 ± 0.02a
FCR 1.22 ± 0.09a1.21 ± 0.03a1.16 ± 0.03a1.06 ± 0.03a1.08 ± 0.061a
Survival (%) 91.00 ± 1.60 93.00 ± 2. 20 100 ± 0.00 95.0 0 ± 3. 20 95.0 ± 3.20
Note: Values expressed as means ± S.E.M. (n = 3). Different superscript letters indicate significant differences for each pairwise comparison between
treatments. The mean difference is significant at 5% level.
Abbreviations: FCR, Feeding Conversion Ratio; SGR , Specific Growth Rate.
6
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EISSA et a l.
difference from the control (p > 0.05) (Table 3). The ALT and glucose
activities significantly (p < 0.05) decreased in T1 and T4 groups and in-
significantly decreased in T2 and T3 compared with the control group.
Meanwhile, the AST and alkaline phosphatase insignificantly decreased
in the different treated groups than in the control. The supplementa-
tion of nanocurcumin significantly (p < 0.05) increased the serum total
protein and albumin levels than in the control group after 60 days. On
the other hand, globulin insignificantly increased in the different groups
than in the control. The lowest significant levels of creatinine were ob-
served in T1, T3 and T4 (p < 0.05) and were non- signific ant in T2.
The amylase activity significantly (p < 0.05) increased in all
groups fed nanocurcumin than in the control, meanwhile lipase in-
significantly (p > 0.05) increased in all groups of fish fed nanocur-
cumin in comparison with the control. The triglycerides showed
insignificant (p > 0.05) decreased values in the control group than in
the other groups. Moreover, the cholesterol insignificantly (p > 0.05)
decreased in T1 and T4. While, cortisol showed no differences in all
treated groups (Table 3).
3.3 | Chemical composition of the whole fish body
The whole- body composition of the juvenile tilapia was improved by
the nanocurcumin (p > 0.05) in terms of the protein content, DM and
ash content in comparison with the control group (Table 4).
3.4 | The survival rate after Aspergillus
flavus challenge
The fish began to die on the 3rd day post challenge. The infected fish
showed signs of haemorrhages on different parts of the external body
surface with increased mucus secretions and detachment of scales. On
postmortem examination, the liver appeared pale, enlarged with white
nodules scattered on its surface and a distended gallbladder.
Aspergillus flavus was re- isolated from the internal organs of
these infected fish. After the first and second weeks of the A. fla-
vus challenge, O. niloticus showed survival rates with significant
differences among all treated groups and the control (Table 5 and
Figure 6). The fish fed on nanocurcumin exhibited higher survival
rates than the control in a dose- dependent manner.
3.5 | Histopathology
The transver se sections of fish gills (Figure 3) revealed that in the T1
grou p, the gi ll s sho we d epith eli al prol if era tion, lead ing to la me lla r fu-
sion, and fungal spores appeared on the gill racker. Ovaries showed
the presence of fungal spores between the ovarian follicles. The liver
of the control group revealed the presence of A. flavus spores in the
hepatocytes (Figure 4). Liver showed congestion in hepatopancreatic
blood vessels and hepatic sinusoids with mononuclear inflammatory
FIGURE 2 The relationship between
final weight (g), specific growth rate
(SGR; %g/day), weight gain (g), and feed
conversion ratio of Oreochromis niloticus
fed diets supplemented with varying
levels of nanocurcumin for 60 days.
y = -1.0643x2 + 8.2797x + 11.984
R2 = 0.9389
15
17
19
21
23
25
27
29
31
Control T1 T2 T3 T4
Final weight (g)
y = -1.2707x2 + 9.3613x + 6.462
R2 = 0.9499
15
17
19
21
23
25
27
29
Control T1 T2 T3 T4
Weight gain
y = -0.0721x
2
+ 0.5719x + 1.672
R
2
= 0.8866
0.
6
1.
1
1.
6
2.
1
2.
6
3.
1
3.
6
Control T1 T2 T3 T4
SGR (%/day)
y = 0.0007x
2
- 0.0473x + 1.28
R
2
= 0.8595
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
Control T1 T2 T3 T4
Feed conversion ratio
slevelruc-onaNslevelruc-onaN
|
7
EISSA et al.
cells infiltrations, as well as the presence of many fungal spores
around the hepatopancreas with melanin pigment deposition. Some
hepatocytes showed necrotic changes. Liver revealed congestion of
hepatoportal blood vessels and the presence of many fungal spores
in the lumen of the hepatoportal blood vessels (Figure 4).
Many fungal spores (dark pink colour) were observed scat-
tered around the hepatoportal, hepatopancreatic blood vessels and
around hepatic sinusoids (Figure 5). In T3, ovar y showed haemor-
rhage and mononuclear inflammatory cells infiltration with melanin
pigment deposition between ovarian follicles.
4 | DISCUSSION
Dietar y nanocurcumin has been acknowledged as an anti-
inflammatory compound (Calder, 2013; Jacob et al., 2007), exerting
protec tive ef fec t s ag ainst inf lam ma to r y di sease s along wit h the inh i-
bition of the NF- κB protein complex that is involved in the synthesis
of Interleukin- 1 (IL−1) and tumour necrosis factor- alpha (TNFα) (Zhao
et al., 2005). There are a lot of studies dealing with curcumin as feed
additive in diets of different fish species (Baldissera et al., 2018; El-
Barbary, 2018; Mahmoud et al., 2017). However, to the best of the
TAB LE 3 Haematological and serum biochemical parameters of Oreochromis niloticus- fed diets supplemented with varying levels of
nanocurcumin for 60 days
Item
Tes t di et
Control T1 (Nanocur- 10) T2 (Nanocur- 25) T3 (Nanocur- 40)
T4
(Nanocur- 55)
RBCs (×10/m m3) 0.83 ± 0.31b1.27 ± 0.03a1.23 ± 0.03a1.33 ± 0.03a1.23 ± 0.03a
Haemoglobin (g /100 ml) 4.81 ± 0.06b5.19 ± 0.13a5.28 ± 0.08a5 .17 ± 0.03a5.23 ± 0.06a
WBCs (×103/mm3)31.36 ± 0.65a30.54 ± 0.29a30.36 ± 0.50a31.21 ± 0.54a31.37 ± 0. 67a
ALT (U/L) 19.3 1 ± 1.47a9. 54 ± 0.72b16.52 ± 2. 76a13.27 ± 3.12a9.26 ± 0. 47c
AST (U/L) 18.54 ± 3.34a15. 59 ± 2.33a14. 37 ± 2.99a13.47 ± 3.07a14.52 ± 3.27a
ALP (U/L) 115.91 ± 14.0 4a78.68 ± 8.23a109.10 ± 6.56a95.15 ± 14. 84a109.75 ± 4.54a
Total protein (g/dl) 3.91 ± 0.03e5.16 ± 0.29d5.66 ± 0.10b6.18 ± 0.06a5.56 ± 0.06c
Albumin (g/dl) 2.29 ± 0.08e3 . 51 ± 0.34c3.72 ± 0.25b4.38 ± 0.05a3.26 ± 0 .10d
Globulin (g/dl) 1. 61 ± 0.06a1.65 ± 0.32a1.94 ± 0.34 a1.79 ± 0.10a2. 31 ± 0.14a
Creatinine (mg/dl) 0.63 ± 0.04a0.17 ± 0.06d0 .61 ± 0.1 2a0.4 0 ± 0.02b0.27 ± 0.02c
Glucose (mg/dl) 135. 84 ± 8.02a8 9.85 ± 9. 90c133.89 ± 7.86a116.37 ± 2.48a111.17 ± 5.20 b
Cortisol (mg/dl) 99. 30 ± 0. 27a98.77 ± 0.38a98.44 ± 0.26a99.14 ± 0.35a99. 0 3 ± 0.36a
Amylase (U/L) 6.92 ± 0. 34b19.72 ± 4.73a44.70 ± 24.53a23.66 ± 5.93a14.9 1 ± 1.83a
Lipase (U/L) 29. 23 ± 0.21a33.44 ± 1.87a30.13 ± 3.63a34.01 ± 2.01a31.55 ± 1.0 4a
Cholesterol (mg/dl) 179. 44 ± 1.27a175.01 ± 2.60a19 3.51 ± 2.40 c195. 45 ± 2.44b174. 62 ± 10.78a
Triglycerides (mg/dl) 95.57 ± 47.29 a116 .29 ± 12 .49a148.14 ± 7. 4 9 a111. 57 ± 3 .69a152 .70 ± 4.50a
Note: Values expressed as means ± S.E.M. (n = 3). Different superscript letters indicate significant differences for each pairwise comparison between
treatments. The mean difference is significant at the level 5%.
Abbreviations: ALP, Alkaline phosphat ase; ALT, Alanine aminotransfer ase; AS T, Aspartate aminotransferase; RBCs , Red blood cells.
TAB LE 4 Body composition of Oreochromis niloticus- fed diets supplemented with varying levels of nanocurcumin for 60 days
Final
Parameter Initial Control
T1
(Nanocur- 10)
T2
(Nanocur- 25)
T3
(Nanocur- 40)
T4
(Nanocur- 55)
DM (%) 24.01 ± 0.08d25.23 ± 0.52c26.07 ± 0. 41bc 26 .24 ± 0.52bc 27. 51 ± 0.36a27.05 ± 0.26ab
Protein (%) 53.31 ± 0.23c56.03 ± 0.38ab 56.26 ± 0.32ab 55.08 ± 0.18 b56 .61 ± 0.32a56.06 ± 0. 51ab
Ether Ex trac t (%) 20.84 ± 0.26b25.48 ± 0.19a24.91 ± 0.11a24.71 ± 0.09a25.08 ± 0.41 a24. 85 ± 0.35a
Ash (%) 18.58 ± 0 .12a14 .49 ± 0.08b14. 59 ± 0.07b14. 54 ± 0.09b14.21 ± 0 .19b14.55 ± 0.07b
Gross Energy (kc al/100 g) 496.3 0 ± 32.11b563.21 ± 1 .76 a564.38 ± 1.98a55 9.01 ± 4.26a560.42 ± 3.40a561 .8 4 ± 2.74a
Note: Values expressed as means ± S.E.M. (n = 3). Different superscript letters indicate significant differences for each pairwise comparison between
treatments. The mean difference is significant at the level 5%.
Abbreviation: DM, Dry Matter.
8
|
EISSA et a l.
author's knowledge very few studies were conducted on the nano-
curcumin as feed additive (Alagawany et al., 2021; Moniruzzaman
& Min, 2020). The current results revealed that nanocurcumin im-
proved the health status and haemato- immunological parameters
of O. niloticus, which may enable the fish to combat the A. flavus
infection.
4.1 | Growth and response to feed intake
In the present study, the supplementation of nanocurcumin for
60 days resulted in the improvement of FBW, WG, SGR and sur-
vival rate of O. niloticus. This was in line with earlier studies con-
ducted on curcumin (Cui et al., 2017; Mahmoud et al., 2017; Sruthi
TAB LE 5 Survival rate of Oreochromis niloticus- fed diets supplemented with varying levels of nanocurcumin for 60 days and post
challenged with Aspergillus flavus for 15 days
Fish number Control T1 T2 T3 T4
Initial number 10.00 10.00 10.00 10.00 10.00
After the first week of A. flavus infec tion (%) 0.00 30.00 40.00 70.00 80.00
After the second week of A. flavus infec tion (%) 0.00 0.00 0.00 30.00 40.00
FIGURE 3 Effect of dietary
nanocurcumin on the histopathological
scores of the Nile tilapia (Oreochromis
niloticus) challenged with Aspergillus flavus.
Transverse sections of fish gills H & E
100×, n = 10. Control group (ca, Cb), T 1
group (T1a, T1b), T 2 group (T2a, T2b), T 3
group (T3a, T3b), T 4 (T4a, T4b).
|
9
EISSA et al.
et al., 2018). The improved growth per formance may be attributed
to the curcumin enhancing the activities of the digestive enz ymes
such as lipase and amylase, which improved the feed digestion, as
well as its consumption and utilization (Prasad & Aggarwal, 2011;
Yitbarek, 2015). When compared with the control, fish fed
nanocurcumin- supplemented diet had greater levels of digestive
enzymes activity in their plasma or intestines (Jiang et al., 2016;
Midhun et al., 2016; Sruthi et al., 2018). Similar research found that
feeding Nile tilapia diets with different concentrations of cinnamon
nanoparticles improved the growth performance by enhancing the
activity of the digestive enzymes (Abdel- Tawwab et al., 2018).
Additionally, nanocurcumin may increase the palatability of
feed leading to increased feed conversion ratio, as obser ved in
our study. The nanocurcumin derived from turmeric is considered
as a polyphenolic subst ance that works as a growth- stimulator
for good intestinal bacteria (functioning similarly to a prebiotic),
thus, may prevent the growth of microbial pathogens (Gessner
et al., 2 017).
Kaur et al. (2020) reported enhanced growth indices in Labeo
rohita- fed turmeric supplemented diet due to the increase in the in-
testinal villi heights and goblet cell numbers, which in turn increase
the surface area for nutrients’ absorption.
4.2 | Blood haematology and
biochemical parameters
Haemato- biochemical indicators are significant instruments fre-
quently used to assess the state of health of the fish, nutritional intake
and capacity to adapt to their environment (Abdel- Tawwab, 2016;
FIGURE 4 Effect of dietary
nanocurcumin on the histopathological
scores of the Nile tilapia (Oreochromis
niloticus) challenged with Aspergillus flavus.
Vertical sections in hepatopancreatic
tissue H & E 400×, n = 10. Control group
(ca, Cb), T 1 group (T1a, T1b), T 2 group
(T2a, T2b), T 3 group (T3a, T3b), T 4 (T4a,
T4b ).
10
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EISSA et a l.
Kord, Srour, et al., 2021). Diet supplementation with nanocurcumin
improved haematological parameters (RBCs and haemoglobin con-
tents) in comparison with the control group.
The WBCs were the same between all groups indicating the
healthy condition of fish. It is well known that the values of the total
serum proteins, albumin and globulin are effective indicators of the
humoral immunit y. In the present study, these values were elevated
in fish fed nanocurcumin suggesting its immunomodulatory effect
on O. niloticus, which may be attributed to the effect of curcumin
as an antimicrobial and immunomostimulant agent (Abdelrazek
et al., 2017; Eissa et al., 2019; Kord, Maulu, et al., 2021), as well as
its effect on the antigen presentation, lymphoid cell populations and
cytokine production (Sankar et al., 2010; Varalakshmi et al., 2008).
The liver enzymes, ALT, AST and alkaline phosphatase, are in-
dications of the liver health status and are elevated in case of liver
dysfunction (Javed et al., 2016; Kord, Srour, et al., 2021). In this
study, these enzymes showed lower concentrations in the fish fed
nanocurcumin in comparison with the control group as a result of the
FIGURE 5 Effect of dietary nanocurcumin on the histopathological scores of the Nile tilapia (Oreochromis niloticus) challenged with
Aspergillus flavus. Vertical sections in fish skin and muscles H & E 4 00×, n = 10. Control group (C), fungal spores (SP) between hepatocytes
of control group, PAS, T 1 group, T1a: Fungal spores (SP) in gill racker, PAS, T1b: Fungal spores (SP) in the gills, and T1c: Fungal spores (SP)
between hepatoc ytes, T2 group, T2a, Haemorrhage (he) and mononuclear inflammatory cells infiltration (Mn) with pigment deposition
melanin (me) between ovarian follicles; T2b, Congestion of hepatoportal blood vessels and presence of fungual spores; T2c, Fungual spores
in lumen of hepatopor tal blood vessels.
|
11
EISSA et al.
antioxidant and hepatoprotective properties of curcumin (Elgendy
et al., 2016).
The level of serum creatinine is a good indication of kidney
health status because it is excreted via the kidney and it is elevated
in the case of nephrotoxicity (Cengiz, 2006; Walker et al., 1990). The
fish fed nanocurcumin showed lower levels of creatinine compared
with the control group, and this may be attributed to the nephro-
protective effects of curcumin (Kaur et al., 2020).
Cor tisol is the major stre ss horm one in fish and is used for evalu-
ating the animal's welfare in aquaculture (Bastien & Benjamin, 2019;
Khalil et al., 2 019). In our study, the cortisol was stable in all tested
groups including the control. Mommsen et al. (1999) repor ted that
the fish blood glucose was elevated in response to stress. However,
in the present study, blood glucose was significantly decreased in
fish fed nanocurcumin with 10 and 55 mg concentrations/kg for
60 days and insignificantly decreased in the other groups compared
with the control. This indicated that nanocurcumin improved the
glucose level in O. niloticus.
Likewise, cholesterol insignificantly decreased in fish fed nano-
curcumin with 10 and 55 mg concentrations/kg for 60 days than in
the other groups, while the triglycerides insignificantly increased in
all groups than in the control due to the content of fat in the fish diet
(Van der Boon et al., 19 91).
4.3 | The survival rate after the A. flavus challenge
The clinical signs, postmortem and histopathologial signs that
appeared on O. niloticus post challenged with A. flavus were in
accordance with Abd El Ghany and Elias (20 14). The experimental
inoculation of O. niloticus with A. flavus indicated that it is highly
pathogenic as the cumulative mortality was 100% in the challenged
control group within the first 7 days post inoculation. The survival
rate of the infected fish fed nanocurcumin indicated the protective
role of nanocurcumin against A. flavus infection, thus, confirming the
antioxidant, anti- inflammatory and antimicrobial effects of curcumin
(Jayaprakasha et al., 2006; Kohli et al., 2005; Mohamed et al., 2020).
5 | CONCLUSIONS
The present investigation used nanocurcumin in the diet formula-
tion of Nile tilapia. Dietary supplementation with nanocurcumin en-
hanced the growth parameters, haemato- immunological response
and body composition of O. niloticus, and protected it from A. fla-
vus infection. Furthermore, nanocurcumin dietar y supplementation
enhanced the survival rate following the fungal infection, hence, it
can be used as an alternative treatment to the traditional chemical
antifungal drugs. These results indicate that nanocurcumin can be
considered as a beneficial dietary supplement for Nile tilapia with
optimum inclusion levels of 25– 40 mg/kg diet. Finally, nanocurcumin
used in small doses is inexpensive, non- toxic and gives good results
that are equivalent to the use of turmeric or curcumin powder.
ACKNOWLEDGEMENTS
The authors extend their appreciation to Taif University for funding
the current work by Taif University Researchers Supporting Project
number (TURSP- 2020/119), Taif University, Taif, Saudi Arabia.
FIGURE 6 Kaplan– Meier method with survival curves, log- rank (mantel- cox) test of Oreochromis niloticus fed diets supplemented with
varying levels of nanocurcumin for 60 days and post challenged with Aspergillus flavus for 15 days.
Treatment01234RRR
Survivalrate
0
1
2
3
4
5
6
7
8
9
10
day
123456789101112131
41
5
RR
RR
RRRRR
R
R
RR
RR
12
|
EISSA et a l.
FUNDING INFORMATION
The current work was funded by Taif University Researchers
Suppor ting Projec t number (TURSP- 2020/119), Taif university, Taif,
Saudi Arabia.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVA ILAB ILITY STATE MEN T
The authors declare that all data can be provided upon reasonable
request.
ETHICS STATEMENT
The research was carried out in accordance with the international
standards for treating and utilizing animals in scientific experiments,
and the Institutional Animal Treatment and Use Committee (IACUC)
at Alexandria University (AU/2020/053127), Egypt.
ORCID
El- Sayed Hemdan Eissa https://orcid.org/0000-0002-7229-2175
Montaser M. Hassan https://orcid.org/0000-0002-7990-6969
Moaheda E. H. Eissa https://orcid.org/0000-0002-1733-6892
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How to cite this article: Eissa, E.-S., Ezzo, O. H., Khalil, H. S.,
Tawfik, W. A., El- Badawi, A. A ., Abd Elghany, N. A., Mossa, M.
I., Hassan, M. M., Hassan, M. M., Eissa, M. E. H., Shafi, M. E.,
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composition, haemato- biochemical parameters and
histopathologic al scores of the Nile tilapia (Oreochromis
niloticus) challenged with Aspergillus flavus. Aquaculture
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