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RESEARCH
Impact ofavaried combinatorial mixture ofβ‑1, 3 glucan
andfructooligosaccharides ongrowth performance,
metabolism, intestinal morphometry, expression
ofantioxidant‑related genes, immunity, andprotection
againstVibrio alginolyticus inRed tilapia (Oreochromis
niloticus x O. mossambicus)
El‑SayedHemdanEissa1 · RagaaA.Ahmed2 · YasminM.AbdEl‑Aziz3 ·
BasmaM.Hendam4 · MoahedaE.H.Eissa5 · NohaI.ElBanna6
Received: 1 January 2024 / Accepted: 20 March 2024
© The Author(s) 2024
Abstract
Immunosaccharides such as β-glucans and fructooligosaccharide (FOS) strengthen the
host’s immune responses. This study examined the influences of three levels of the β-1,
3 glucan-FOS combination on Red tilapia performance. Four diets were prepared: T0 as
a control, while T0.5, T1, and T1.5 indicate the addition of β-1, 3 glucan-FOS mixture at
0.5%, 1%, and 1.5%, respectively. Then, 240 red Tilapia (Oreochromis niloticus x O. mos-
sambicus) (weight 6.1 ± 0.07gm) were allocated into four groups, and every group was fed
tested diets for 56 days. After the feeding trial, growth parameters, erythrogram profile,
liver and kidney function testes, glucose, histopathological analysis, and gene expres-
sions for antioxidants, catalase (CAT) and glutathione peroxidase (GPX) besides growth
hormone (GH) and insulin-like growth factor 1 IGF1-related markers were assessed. Red
tilapia fed T0.5, T1, and T1.5 exhibited consistent growth, survival rate, and homeostasis
compared with the control group. Different supplement concentrations displayed varying
levels of responses. The hepatorenal biomarkers (alanine transaminase (ALT = SGPT) and
aspartate transaminase (AST = SGOT), alkaline phosphatase (ALP = ALK), urea, and cre-
atinine) and glucose showed a significant reduction in the supplemented groups compared
with the control, especially in the T1 and T1.5 groups. The intestinal morphometric study
revealed that fish group fed on T1 represented the best result, whereas group T1.5, followed
by group T0.5, was moderately treated, compared to the control. The real-time quantita-
tive reverse transcription PCR (qRT-PCR) analysis displayed up-regulated expression of
antioxidant and growth-correlated genes in the T1.5 groups. After 56 days, the β-1,3 glucan-
FOS fed groups also exhibited an increase in survival rates compared to the control when
challenged with a pathogenic Vibrio alginolyticus. Current findings suggest that inclusion
of β-1,3 glucan-FOS in diets could enhance red tilapia biochemical parameters, growth,
and protection against pathogenic V. alginolyticus infection.
Handling editor: Amany Abbass
Extended author information available on the last page of the article
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Keywords Vibriosis· Immunosaccharides· Antioxidants· Immunostimulants·
Histopathology· Gene expression
Introduction
Aquaculture is a significant producer of animal protein and food globally, so it is necessary
to continue growing in response to the fastest-growing human population (Edwards etal.
2019; FAO, 2020; Eissa etal. 2023b). Particularly, marine aquaculture that has expanded
and predictable to boost sea food production in national and international levels (Chand
2022; Food Nations A U, 2020).
Red tilapia ‘a hybrid species of O. niloticus and O. mossambicus,’ is gaining more
acceptance for fish farming (Wang etal. 2020a, b), owing to its delightful flavor, easily
farming, fast growing, adaptability, and high resistance to disease, besides its omnivorous
feeding behavior (Haque etal. 2016).
Recently, the sustainability of finfish mariculture has faced challenges, including limited
aquafeed availability, climate-related issues, and disease outbreaks such as vibriosis (Vez-
zulli etal. 2010; Baker-Austin etal. 2018; Aly etal. 2019; Okon etal. 2023).
Prebiotics are known as immunosaccharides such as β-glucans, inulin, fructooligo-
saccharide (FOS), and mannanooligosaccharide (Akhter etal. 2015; Iswarya etal. 2018;
Dawood etal. 2020). Immunosaccharides strengthen the host defense mechanisms through
promoting the development and activity of beneficial gut flora, which changes the colonic
microbiota (Gibson and Roberfroid 1995), likewise, having various biological activities, as
antibacterial, anti-inflammatory, and antioxidant properties (Gibson and Roberfroid 1995).
β-Glucans are the most important prebiotics that applied in aquaculture (Iswarya etal.
2018; Jami etal. 2019; Abdelhamid etal. 2020; Eissa etal. 2023a). It is a linear polysac-
charides derived from cell walls of yeast, filamentous fungi, certain bacteria, and plants
(Dalmo and Bøgwald 2008; Meena etal. 2013), which has possess significant immuno-
logical impacts due to triggering the innate and humeral defense mechanisms (Abbas etal.
2014; Akhter etal. 2015). For instance, the utilization of β-glucans extracted from yeast
cell walls has been shown to improve disease resistance in tilapia against bacterial infec-
tions such as Aeromonas hydrophila (Barros etal. 2014; Iswarya etal. 2018) and Strep-
tococcus agalactiae infections (Pilarski et al. 2017). Meanwhile, no studies were found
to study its role against marine bacterial pathogens. In gastrointestinal tract, Song etal.
(2014) elucidated that the absorption of β-glucans by intestinal and gut lymphoid tissue
cells stimulates molecular and humoral immune responses, enhancing resistance to infec-
tions through increased cytokine production in immune cells (RingØ etal. 2010).
Likewise, FOS are considered the most promising prebiotics in aquaculture (Guerreiro
etal. 2015, 2016; Hu etal. 2019). Fructooligosaccharide is recognized to support growth
and survival of autochthonous bacteria of the gastrointestinal tract, such as Lactobacillus,
which possess b-fructosidase activity and therefore can hydrolyze FOS β-(2–1) glycosidic
bonds (Akhter etal. 2015; Wee etal. 2022). Furthermore, it considered an immunosac-
charide (Song etal. 2014) as it has a direct signaling capacity on human’s immune cells, by
activating toll-like receptors, mainly TLR2 and, to a lesser extent, TLR4 (Vogt etal. 2013)
that regulate the dendritic cell function.
Numerous studies have explored the impact of immunosaccharides on the immune sta-
tus of aquatic organisms (Guerreiro etal. 2016; Jami etal. 2019; Dawood etal. 2020).
However, to the best of our knowledge, this is the first study to assess the incorporation
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of β-glucans and FOS in red tilapia diets and test their effectiveness against marine patho-
genic bacteria. Furthermore, there is a lack of research on the combined composition of
prebiotics and their potential interactive effects on the performance and immunological
condition of red tilapia.
Hence, this study aimed to investigate the effects of dietary β-glucans, FOS combination
on growth performance, metabolism, antioxidant status, immunity, and protection against
V. alginolyticus infection of Red tilapia.
Materials andmethods
Diets
Four experimental foodstuffs were formulated to fulfill the nutritional needs of Red tilapia
(NRC2011). The control diet (T0) was prepared by mixing all the feed ingredients without
any supplementations. While the other three diets were prepared by mixing the feedstuffs
with the β-glucan and FOS mixture (Aquastem™ V) that purchased from Kemin Indus-
tries, Inc., USA, at 0.5 (T0.5), 1 (T1), and 1.5 (T1.5) g/kg diet, respectively. The constituents
and chemical composition of each prepared diets were illustrated in (Table1).
Table 1 Components and
chemical constituents of the
Experimenteddiets (g/kg on dry
matter basis)
*Β-1,3 G-FOS: β-1, 3 glucan and fructooligosaccharides. Analysis
was made according to Official Methods of Analysis, Association of
Official Analytical Chemists, Arlington (Horwitzand Latimer 2000).
Vitamin & Mineral premix Vitamin and mineral premix (1&2); each
100g had vitamins (Bi, 100mg; Vit A, 7,500,000 Iu; B3, 500mg; E,
100mg; B6, 150mg; B12, 2.5mg; K, 100 mg; folic acid, 100 mg;
pantothenic acid, 275mg; vit. D3, 7500 Iu). Minerals (Zn, 2.50mg;
Fe, 31.50mg; Mn, 16.00mg; Cu, 5.50; I, 0.55mg; P, 450mg; and Ca,
1.15 gm). NFE = 100 − [%ash + %protein + % lipid]
Feed ingredients T0T0.5 T1T1.5
Fish meal (70% CP) 210 210 210 210
Soybean meal (44% CP) 210 210 210 210
Yellow corn 220 220 220 220
Wheat bran 100 100 100 100
Rice bran 200 199.5 199 198.5
*β-1,3 G-FOS 0 0.5 1.0 1.5
Linseed oil 20 20 20 20
Vitamins premix (1) 20 20 20 20
Minerals premix (2) 20 20 20 20
Total 1000 1000 1000 1000
Chemical analysis (%)
Dry matter (DM) 91.50 91.48 91.42 91.50
Crude protein (CP) 30.32 30.28 30.41 30.32
Ether extract (EE) 5.62 5.60 5.61 5.61
Ash 5.61 5.63 5.61 5.61
Crude fiber (CF) 5.32 5.30 5.34 5.33
Nitrogen-free extract (NFE) 53.13 53.21 53.29 53.12
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All the grinded feed constituents were blended well, then 300 ml/kg of water were
added to form dough that was passed through a meat grinder mesh (3 mm), and the
obtained sticks were dried over night against a fan and then dried in a hot-air oven at 50
°C for 1 h. The dried sticks were squashed to form pellets (3 mm) (Awad etal. 2022).
Afterword, all prepared diets were kept in plastic containers and preserved in the refrig-
erator (4 °C) until use.
Fish husbandry andfeeding protocol
Two hundred and forty Red tilapia fingerlings with an average body weight of 6.1 ± 0.07g
were stocked into twelve concrete tanks (1 × 1 × 1.2-m diameter) and assigned to four
treatments in triplicate (20 fish/m3/tank), at a private fish farm in Damietta Governorate,
Egypt. During the two-week acclimatization period, fish were fed a basal diet (30%
protein) twice daily. Throughout 56 days of the trial, the first group was fed a basal
diet (T0) that assisted as a control, while the second (T0.5), third (T1), and fourth (T1.5)
groups were fed on β-1, 3 glucan-FOS supplemented diets (0.5, 1.0, and 1.5 g/kg diet),
respectively. Throughout the study, the tanks’ water was renewed by half each day, and
the typical parameters were maintained as follows: water temperature was 26 ± 0.29 (°C),
salinity was 2.5 ± 0.00 (ppt), pH was 7.8 ± 0.1, D.O was 6.93 ± 0.05 (mg/l), NH3 was
0.01 ± 0.00 (mg/l), NH4 was 0.36 ± 0.02, and NO2 was 0.039 ± 0.00. Fish were adapted to
a photoperiod of a 12:12 h L/D. The experimented fish were fed 6% of their body weight
twice daily (5 days/week) and suspended for a day before sampling and challenging
infection.
Growth performance
After 56 days of feeding trial, entirely fish groups were weighed; then, the growth param-
eters were calculated with the equations (Mohammadi etal. 2020):
where weight gain (WG) %, fish initial and final weights (IW and FW) in gram, and d was
days of raising.
Sampling procedures
When the feeding trial terminates, five fish from each group were randomly netted and
anaesthetized by bath immersion in 50 μl−1 clove oil solution for 5 min. Following anes-
thesia, blood samples were taken by the caudal vein puncture using heparinized syringes
(1600 UI/ml), for hematological assessment, while serum samples were taken without an
anticoagulant that centrifugated at 3500 rpm for 15 min at 4 °C. All serum samples were
kept at − 20 °C until used for biochemical analysis.
Weight gain,WG(g)= FW(g)− IW(g),
Specific growth rate(
SGR, %per day) = [(Ln(FW)− Ln(IW)∕d] ×
100,
FCR
= consumed feed(g)∕weight gain(g),
Survival(%) = (final number of fish∕initial number of fish) × 100.
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Body composition analysis
The nutrient composition analysis was done on experimental fish groups (3 fish/repli-
cate/group). Fish were initially weighted then dried at 105 °C in oven for 24 h after that
finally weighted to detect the moisture content. The Kjeldahl method was applied for
crude protein (N × 6.25) analysis. Additionally, the raw ash was evaluated via a muffle
furnace at 550 °C for 16 h and the total lipid level by chloroform–methanol extraction
method (Bligh and Dyer 1959). All the above analyses were done according to the Asso-
ciation of Analytical Communities; (Horwitzand Latimer 2000) procedure.
Hematological parameters
The total erythrocyte (RBCs) was counted by diluting the freshly collected blood sam-
ples with Natt-Herrick’s solution and counted by hemocytometer. Hemoglobin (Hb) lev-
els were determined by cyanomethemoglobin method using a spectrophotometer at 540
nm (Drabkin 1946). Packed cell volume (PCV) and red blood cell indices (MCV (fl),
MCH (pg), and MCHC (%)) were calculated (Jain 1986).
Serum bioindicators
Serum samples were analyzed spectrophotometrically to determine various parameters,
including total protein, albumin levels (Doumas etal. 1981), albumin and globulin ratio
(A/G ratio), and alanine and aspartate aminotransferases, alkaline phosphatase (ALT,
AST, and ALP) (Reitman and Frankel 1957), creatinine, urea, and uric acid concentra-
tions (Coulombe and Favreau 1963; Heinegård and Tiderström 1973). These analyses
were conducted following the protocols outlined by previous studies, and commercially
available Bio-Diagnostics kits from Bio-Diagnostics Co, Giza, Egypt, were used accord-
ing to the manufacturer’s instructions. Additionally, the glucose levels were measured
based on glucose oxidase method (Teixeira etal. 2017).
Histomorphology
The formalin preserved and fixed fish’s hepatic and intestinal specimens were dehy-
drated in a gradient series ethanolic concentrations (70%, 90%, and 100%) from three
fish per each group (n = 3). Following dehydration, the tissues were cleared with xylene,
then impregnated and blocked with molten paraffin wax, and were sectioned to 5-μm
thickness via a rotary microtome. Afterward, the tissue sections were prepared for
hematoxylin and eosin (H&E) staining (Suvarna et al. 2018). The histopathological
features and the intestinal-morphometrical parameters of the experimented fish groups
were analyzed under a light microscope (Leica DM 1750, US) (Pirarat etal. 2011).
Gene expression
Total RNA was obtained from 50mg of liver tissues (n = 5 samples per each group)
using TRIzol reagent (iNtRON Biotechnology, Inc., South Korea) following the compa-
ny’s protocol. The intensity of the extracted RNA was completed by Nanodrop (Uv–Vis
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spectrophotometer Q5000/Quawell, Quawell Technology, Inc., San Jose, CA, USA).
The complementary DNA (cDNA) was reversed using the Fast Hisenscript ™ RH ( −)
RT PreMix cDNA synthesis kit (iNtRON Biotechnology, Inc., South Korea) according
to the producer’s instructions. cDNA samples were then reserved at − 20°C until use.
Quantitative real-time PCR (qRT-PCR) was carried out to assess the expression of
hepatic antioxidant genes: catalase (CAT ) and glutathione peroxidase (GPX), and growth-
related genes: growth hormone (GH) and insulin-like growth factor (IGF-1) in tilapias. In
addition, beta-actin (β-actin) was used as reference (housekeeping) gene for normalizing
mRNA expressions of these genes. Used primer sequences and GenBank accession num-
bers were listed in Table2.
The qPCR of the β-actin (a housekeeping gene) and studied genes was implemented
by SYBR Green PCR Master Mix for the mRNA expression folds’ quantification of the
selected genes (SensiFast™ SYBR Lo-Rox kit, Bioline). The thermocycling settings were
95°C for 10min, after those 40 cycles at 94°C for 15s, 60°C for 1min, and to finish
72°C for 20s. Every target gene’s mRNA expression fold was standardized and normal-
ized to β-actin mRNA transcripts by the 2−ΔΔCT method (Schmittgen and Livak 2001).
Challenge infection
The pathogenicity test for settling the lethal dose fifty (LD50) of a pathogenic strain of V.
alginolyticus was done as described previously (Aly etal. 2021) that was obtained from
the Aquaculture Diseases Control Department, Fish Farming and Technology Institute,
Suez Canal University, Egypt. After the eighth week, three replicates from each fish group
(10 fish/replicate) were established and then were intraperitoneally injected with 0.1 ml of
pathogenic V. alginolyticus at a concentration of 1 × 109 CFU/ml. The challenged fish were
observed for 15 days for cumulative mortalities recording, where the other non-challenged
fish from all replicates were used for evaluating the hematological and immunological
parameters.
Statistical analysis
Data are displayed as means ± standard error (S. E). Normality and equal variance of data
were patterned using Shapiro–Wilk test and Kolmogorov–Smirnov tests. All statistical dif-
ferences were assessed by one-way ANOVA tests (SPSS version 22, SPSS Inc., Il, USA)
Table 2 Sequences of used primers for q-PCR analysis
Gene Primer’s sequence (5′-3′) GenBank accessation no Reference
CAT F: CCC AGC TCT TCA TCC AGA AAC
R: GCC TCC GCA TTG TAC TTC TT
XM_019361816.2 (Goes etal. 2019)
GPx F: CCA AGA GAA CTG CAA GAA CGA
R: CAG GAC ACG TCA TTC CTA CAC
NM_001279711.1 (Neamat-Allah etal. 2019)
GH F: ACA TCA TCA GCC CGA TCG AC
R: TCA GCA GCA AGA TTC CCG TT
XM_003442542.5 (Lian etal. 2017)
IGF-1 F: GCA GAT TGC TGA TGG CAT GG
R: TCA TTC CGA AGT CGC CGA T
KC506777.1 -
β-Actin F: CAG CAA GCA GGA GTA CGA TGAG
R: TGT GTG GTG TGT GGT TGT TTTG
XM_003455949.2 (Pang etal. 2013)
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according to the method described by Snedecor and Cochran (1989) with Duncan post-hoc
test (Duncan 1955) where differences in experimental groups occurred. Data were consid-
ered statistically significant at p < 0.05.
Fish mortalities result from the invivo experimental infection were compared with Chi
square subsequently a Kaplan–Meier survival analysis and post hoc analysis via Holm-
Sidak method. Differences between groups were measured significant when p < 0.05.
Ethical approval
Fish were maintained and managed following the guidelines for experimental trials in
animal research of the Ethics and Animal Welfare Committee of the Suez Canal Univer-
sity, Egypt. The ethical review board from the faculty of veterinary medicine approved the
experiment (ID: 2,023,032).
Results
Growth performance
All Red tilapia groups grown-up with the diet supplementation showed significant (p < 0.05)
increase in FW, WG, and SGR in a dose-dependent manner (Table3). Additionally, diet
supplementation yielded a higher survival in fish fed 1 and 1.5% β-glucan-FOS diets with
survival percent values of 97 ± 2.00% and 97 ± 1.22%, respectively, than groups fed on
0.5% β-glucan-FOS and basal diet (control group).
Body composition
By the end of the feeding period, analyzed fish groups that were fed 1 and 1.5% β-glucan-
FOS diets showed a significant rise in crude protein content when compared to tilapia fish
from the other groups (p < 0.05). Additionally, fish groups receiving β-glucan-FOS feeds
had significant higher raw ash content, while the total content of fat in the fish flesh was
lower than that of the control group’s fish (p < 0.05) (Table4).
Table 3 Growth performance and survival percentage of Red tilapia fed diets with 0%, 0.5%, 1%, and 1.5%
of the β-glucan-FOS post 56 days of feeding trial (means ± S. E)
Means ± S. E (n = 5 fish) with different superscripts in the same row are significantly different at p < 0.05
IW initial weight, FW final weight, WG weight gain, SGR specific growth rate
Parameters T0T0.5 T1T1.5
IW (g/fish) 6.10 ± 0.03 5.99 ± 0.05 5.96 ± 0.04 6.07 ± 0.05
FW (g/fish) 26.42 ± 0.41d28.19 ± 0.27c30.38 ± 0.34b32.38 ± 0.14a
WG (g) 20.32 ± 0.38d22.20 ± 0.22c24.42 ± 0.31b36.31 ± 0.18a
SGR (% per day) 1.14 ± 0.01d1.20 ± 0.00c1.26 ± 0.00b1.29 ± 0.01a
FCR 1.50 ± 0.02a1.41 ± 0.01b1.32 ± 0.01c1.28 ± 0.09c
Survival (%) 89 ± 1.00b89 ± 1.00b97 ± 1.22a97 ± 2.00a
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Hematological evaluation
The inclusion of β-glucan-FOS in the diets of Red tilapia had a significant effect on both
measured blood parameters and indices Table 5. Significant differences were detected
between the high β-glucan-FOS incorporation groups (T1 and T1.5) and the control-fish
group (T0), with no significant differences between thus supplemented groups (T1 and T1.5)
(p > 0.05).
Serum bioindicators
Serum biochemical indices, containing total protein, albumin, globulin, A/G ratio, liver
markers (ALT, AST, and ALP), renal function markers (creatinine, urea, and uric acid),
and glucose, were significantly changed by the inclusion of various levels of β-glucan-FOS
in the diets of Red tilapia (Table6). A significant upregulation in total protein, albumin,
globulin, and the A/G ratio (p < 0.05), besides significant decline was observed in the liver,
kidney’s indices, and glucose between β-glucan-FOS fed groups and the control group
(p < 0.05).
Histomorphology andintestinal‑morphometrical evaluation
Histopathologically, the hepatic specimens of control group showed normal lobular
arrangement and portal area structures, with intra-hepatic pancreases. Pancreatic acini
were organized and well-formed, with hepatocytes, sinusoids, and reticulio-endothelial
system appeared normal. Likewise, treatment groups (T0.5, T1, and T1.5) exhibited normal
Table 4 The whole-body composition (%) of red tilapia fed diets with 0%, 0.5%, 1%, and 1.5% of the
β-glucan-FOS post 56 days of feeding trial (means ± S. E)
Means ± S. E (n = 3 fish) with different superscripts in the same row are significantly different at p < 0.05
Parameters T0T0.5 T1T1.5
Moisture 78.36 ± 0.03d78.48 ± 0.02c78.58 ± 0.01b78.71 ± 0.01a
Protein 13.34 ± 0.00d13.37 ± 0.01c13.54 ± 0.00b13.68 ± 0.00a
Ash 6.97 ± 0.00b7.10 ± 0.00a7.10 ± 0.00a7.09 ± 0.00a
Lipid 11.60 ± 0.00a11.54 ± 0.00b11.52 ± 0.00c11.53 ± 0.00bc
Table 5 Blood parameters of red tilapia fed diets with 0%, 0.5%, 1%, and 1.5% of the β-glucan-FOS post 56
days of feeding trial (means ± S. E)
Means ± S. E (n = 5 fish) with different superscripts in the same row are significantly different at p < 0.05
Parameters T0T0.5 T1T1.5
RBCs (106/µl) 1.19 ± 0.01d1.31 ± 0.01c1.37 ± 0.01b1.42 ± 0.01a
HB (g/dl) 6.33 ± 0.05c7.46 ± 0.06b7.87 ± 0.03a7.89 ± 0.04a
PCV (%) 27.86 ± 0.05c28.39 ± 0.03b28.77 ± 0.03a28.84 ± 0.01a
MVC (µm3) 232.51 ± 1.92a218.13 ± 2.41b209.81 ± 1.47c202.49 ± 1.42d
MCH (pg) 52.81 ± 0.31c55.44 ± 0.29b57.40 ± 0.29a57.74 ± 0.52a
MCHC (%) 22.71 ± 0.13c26.26 ± 0.07b27.38 ± 0.05a27.38 ± 0.06a
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hepatic parenchyma and melanomacrophage populations. Active pancreatic acini with
large number of zymogenic granules were seen in T1, with fat globules partially or totally
replaced hepatocytes cytoplasm. The largest amount was seen in group T0.5 (55–60% of the
hepatocytes were affected), with mild dilatation of hepatic sinusoids was also seen in this
group (Fig.1).
The control group’s anterior intestine exhibited normal lining epithelium and the
mucosa with microvilli. A variable number of goblet cells and a few lymphocytes were
seen besides, the mucosa was tangled and folded, while the supplemented groups (T0.5, T1,
and T1.5) pointed out a comparative variability regarding the villous length, villous width,
crypts of Lieberkühn length, muscle coat thickness, and the number of villous and crypts
goblet cells (Fig.2).
Table 6 Serum biochemical parameters of Red tilapia fed diets with 0%, 0.5%, 1%, and 1.5% of the
β-glucan-FOS post 56 days of feeding trial (means ± S. E)
Means ± S. E (n = 5 fish) with different superscripts in the same row are significantly different at p < 0.05
Parameters T0T0.5 T1T1.5
Total protein (g/dl) 3.17 ± 0.01d3.46 ± 0.03c3.65 ± 0.02b3.96 ± 0.02a
Albumin (g/dl) 1.34 ± 0.01c1.64 ± 0.02b1.67 ± 0.01b1.94 ± 0.03a
Globulin (g/dl) 1.83 ± 0.00b1.84 ± 0.01b1.98 ± 0.02a2.03 ± 0.02a
A/G ratio 0.73 ± 0.01d0.89 ± 0.00b0.84 ± 0.01c0.96 ± 0.02a
ALT (U/l) 47.49 ± 0.11a46.33 ± 0.12b45.95 ± 0.05c45.42 ± 0.10c
AST(U/l) 130.33 ± 2.56a121.79 ± 1.89b118.25 ± 0.81b116.59 ± 0.51b
ALP (U/l) 26.16 ± 0.34a23.99 ± 0.14b22.01 ± 0.13c21.22 ± 0.14c
Creatinine(mg/dl) 60.76 ± 0.22a56.57 ± 0.27b50.87 ± 0.33c48.12 ± 0.26d
Urea (mg/dl) 22.78 ± 0.34a21.29 ± 0.13b19.46 ± 0.22c18.73 ± 0.1d
Uric acid (mg/dl) 1.67 ± 0.04a1.44 ± 0.04b1.32 ± 0.07b1.11 ± 0.04c
Glucose (mg/dl) 132.06 ± 0.8a125.73 ± 0.6b125.74 ± 0.6b122.03 ± 0.7c
Fig. 1 Representative micrographs of livers of Red tilapia fed diets with 0%, 0.5%, 1%, and 1.5% of the
β-glucan-FOS post 56 days of feeding trial (H&E staining). All fish groups show normal hepatic appear-
ance with intra-hepatic pancreases and active populations of zymogenic granules (red arrow). Supple-
mented groups (T0.5, T1, and T1.5) show a proportional hepatocellular cytoplasmic basophilia and active
acinar arrangement with fat globules (yellow arrow) with the greatest amount in T0.5, with mild sinusoidal
dilatation is also seen (red star). Scale bar = 30 µm and 50 µm
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The intestinal morphometry showed that the best results were seen in T1 followed by
T1.5; meanwhile, the comparative least values were recorded in the control fish followed by
group T0.5. The goblet cell populations estimation least counted number was seen in control
group T0 (0–1 cell/HPF) (Fig.2 A, B), but 6–8 cells/HPF were seen in T1 (Figs.2E, F).
T1.5, and T0.5 showed 3–5, 1–3 cells/HPF, respectively (Fig.2G, H, C, D). The proportions
of the intestinal villi, crypts of Lieberkühn length, and thickness of the muscle layer in
addition to villous width were recorded and graphically illustrated in Fig.3.
Fig. 2 Representative micrographs of intestines of Red tilapia fed diets with 0%, 0.5%, 1%, and 1.5% of the
β-1,3 G-FOS post 56 days of feeding trial (H&E staining). Variable dimensional changes regarding villous
length (light blue arrows), villous width (brown arrows), crypt of Lieberkühn length (green arrows), and
muscular coat thickness (black stars). Mucosal folds of distal intestine (red stars) and lymphocytic aggrega-
tions (white arrows) are seen. Scale bar = 20 µm and 50 µm
Fig. 3 Chart showing dimensions of the intestinal villi length (VIL.L), villi width (VIL.W), crypts of
Lieberkühn length (CRY.L), and thickness of the muscle layer (MU.CO.TH) in different experimental
groups. Means ± S. E (n = 3 fish) with different superscripts in the same row are significantly different at
p < 0.05)
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Furthermore, the distal intestine of control group revealed normal structure with broad
and short villi. Whereas among the treated groups (T0.5, T1, and T1.5) were exhibited normal
appearance, however a mild to moderate number of lymphocytes were seen infiltrating the
lamina epithelial particularly in T0.5 and T1.5 (Fig.2E, F).
Expression ofantioxidant andgrowth associated genes
Antioxidant genes expressions for CAT and GPX were studied in liver tissues (Fig.4A,
B). A significant (p < 0.05) up-regulation of relative mRNA were detected in the T1 and
T1.5 groups. However, T0.5 fish showed a significantly higher up-regulation than the control
group. Moreover, growth-related genes expression for GH and IGF1 were investigated in
liver tissues (Fig.4 C, D). A significant (p < 0.05) up-regulation of relative mRNA were
detected in β-glucan-FOS supplemented groups compared to the control group.
Fig. 4 Genes expression of catalase CAT (A) and glutathione peroxidase GPX (B), growth-related genes
(growth hormone GH (C) and insulin-like growth factor IGF1 (D) in liver of red tilapia fed diets with 0%,
0.5%, 1%, and 1.5% of the β-glucan-FOS post 56 days of feeding trial. Means ± S. E (n = 5 fish) with differ-
ent superscripts in the same row are significantly different at p < 0.05
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Challenge infection andcommutative mortality percentage
The cumulative mortality percentage of the four treatments 15 days post-challenge against
V. alginolyticus is presented in Fig.5. Dead fish exhibited typical signs of V. alginolyticus
septicemia, when compared to the control group, T0. The cumulative mortality values for
the fish groups fed diets with 0.5%, 1%, and 1.5% of the β-glucan-FOS (T0.5, T1, and T1.5)
were considerably lower. The T1 and T1.5 fish groups showed the highest resistance with the
low mortality 35 ± 0.19% and 45 ± 0.19%).
Discussion
New dietary supplement strategies for health- and growth-promoting components, such as
probiotics, prebiotics, synbiotics, and phytobiotics, were studied. When maximizing the
addition range of specific immunostimulants, growth parameters, physiological aspects
such as metabolism, antioxidant status, and disease resistance should ought to be given due
regard.
In the present study,1% and 1.5% β-glucan and FOS inclusion in the food of Red tilapia
significantly improved in FBW, WG, and SGR, as well as in survival rate. Concurrently,
these prebiotics were shown to improve the growth performance of Chu’s croaker, Nibea
coibor (Li etal. 2019), FBW, WG, and SCR on Nile tilapia (Abdelhamid etal. 2020), tur-
bot (Scophthalmus maximus) larvae (Miest et al. 2016), and common carp (Hoseinifar
etal. 2014). These findings could be correlated to the fact that immunosaccharides not only
strengthen gut-beneficial bacteria communities but also promote their growth (Hoseinifar
etal. 2011) and their survival. Furthermore, they boost the activity of digestive enzymes
(Miest etal. 2016). Thus, β-glucan and FOS can be used in aquaculture to promote growth,
as well as improve the health and survival of aquatic species (Rohani etal. 2022; Wee etal.
2022).
Among the proximate body compositions, crude protein and ash contents were signif-
icantly higher in dietary β-glucan-FOS groups than the control, while lipid content was
lowered. The findings are comparable with those of Ebrahimi etal. (2012), who found that
Fig. 5 The mortality rate per-
centage of red tilapia fed diets
with 0%, 0.5%, 1%, and 1.5% of
the β-glucan-FOS post 15 days of
the challenge infection against V.
alginolyticus
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increased body protein, lipid content, and improved body composition in common carp
flesh were associated with increased dietary prebiotic levels. Furthermore, Nile tilapia fed
1.5% β-glucan had a significantly raised ash content (Lu etal. 2019). Moreover, oligosac-
charides have been shown to increase both body composition and flesh quality in Grass
carp (Ctenopharyngodon idella) (Lu et al. 2023). Similar results were obtained in Nile
tilapia-fed prebiotics and cultivated at various salinities (Hersi etal. 2023). While, on the
contrary, previous studies assumed that dietary prebiotic levels not affected neither the ash
(Ebrahimi etal. 2012) nor lipid contents (Lu etal. 2019). The better body composition is
most likely because of enhancing feed utilization and promoting weight gain caused by
these prebiotics.
Hematological assessment is reliable indicators of the physiological and health status
of fish that revealed significant elevation in erythrogram among Red tilapia fed β-glucan-
FOS incorporation diets. Meanwhile, no-significant variations between the two highest
doses (T1 and T1.5) in the most parameters were detected. In line with that, Novak and Vet-
vicka (2008) reviewed that, in the mid-1980s, previous studies discovered one of the most
vital properties of β-glucans is its potential to activate hematopoiesis in a manner simi-
lar to granulocyte monocyte-colony stimulating factor. The significant elevation in hema-
tocrit values considered as a macro-analysis of the fish immune state, since the number
of immune cells (WBC) in the immune activation rises erythrogram (Akhter etal. 2015).
Khani etal. (2017) observed a large increase in WBC populations in koi carp given Chlo-
rella vulgaris (CV), which increased the overall leukocytic count; thus, he proposed that
this increase had been triggered by the glucans in the C. vulgaris cell wall. Also, Aly etal.
(2022) obtained comparable results in Nile tilapia.
The current study elucidated that globulin levels specifically elevated in 1% and 1.5%
β-glucan-FOS fed fish. In the same context, Abdelhamid etal. (2020) noticed in Nile tila-
pia intoxicated with diazinon and treated with β-glucan supplemented feed, the total pro-
tein and globulin levels were significantly up-raised compared to control (DZN) group.
Likewise, Soleimani etal. (2012) recorded significant upgraded in the total immunoglobu-
lin levels in Caspian roach (Rutilus rutilus) fry fed diets supplemented with 2 and 3% FOS.
These evidence indicates that both β-glucans and FOS can modulate the immune responses
that may be attributed to the positive effect of prebiotics such as β-glucans and immunosac-
charides on the gut-associated lymphoid tissue (GALT) (Hoseinifar etal. 2015), as well as
the resemblance between these prebiotics and the polysaccharide layers in the bacterial cell
wall, stimulate immunological responses and immunoglobulins production.
Song etal. (2014) declared that immunosaccharides primarily increase phagocytic cell
function and the immunoglobulins response in the host. FOS, either alone or in combi-
nation, dramatically increased lysozyme, and phagocytic activity, which aided in the
enhancement of innate and adaptive immune responses. On the contrary, no enhancement
in total or specific immunoglobulin production were noticed in Nile tilapia (Whittington
etal. 2005) and Gilthead sea bream (Guzmán-Villanueva etal. 2014) when fed diets with
β-glucans.
The hepatorenal proficiency biomarkers, among fish groups fed β-glucan-FOS showed
significant reduction in the serum levels of ALT, AST, creatinine, urea, and uric acid. Con-
sistently, in diazinon-intoxicated Nile tilapia previous study reported low levels of ALT
and AST when treated with β-glucan (Abdelhamid etal. 2020). The hepato-renal protec-
tion enhanced by the dietary supplementation can be accredited to the antioxidant activity
of β-glucan, that protects against oxidative injury as well as leukocyte apoptosis (Şener
etal. 2006). In accordance with our suggestions, El-Murr etal. (2016) detected that β-1,3-
glucan scavenges the free radicals and inhibits lipid peroxidation in the hepatic tissue
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(El-Murr etal. 2016). In addition to protect tissue damage by inhibiting early activation of
tissue nuclear factor-КB (NF-B) and NF-IL-6. It has been proposed that β-glucan bind to
scavenger receptors and block monocyte membrane interactions with traditional scavenger
ligands.
Meanwhile, adding β-glucan to the diet considerably lowered glucose levels. Shalaby
etal. (2006) elucidated that glucose quantification in fish blood is a key biomarker of phys-
iological stress caused by a variety of internal and external variables. In contrast, Guerreiro
etal. (2015) noticed unaffected plasma glucose values of European sea bass (Dicentrar-
chus labrax) fed FOS supplementation in diets.
Histopathological analysis of the prebiotic-supplemented fish’s livers revealed acti-
vation of pancreatic acinar cells, thus indicating that prebiotics as FOS improved diges-
tive enzyme secretions and activities in fish (Wu etal. 2013). Furthermore, the intestine
demonstrated that dietary supplementation with a β-glucan-FOS mixture could upturn
the intestinal morphometry and lymphocyte populations. In the same context, it has been
reported that dietary inclusion of FOS in the red drum (Sciaenops ocellatus) diet enhanced
the height of microvilli (Zhou etal. 2010); also, Wu etal. (2013) recorded a significant
increase in microvilli length of the mid-intestine with dietary supplementation of FOS in
Blunt snout bream fingerlings. The improvement in intestinal microvillus morphometry,
particularly enriching apical brush border integrity, led to an increase in the surface area
for absorption of the available nutrients, which in turn enhanced intestinal function (Rohani
etal. 2022) and consequently improve feed utilization, besides growth performance (Abdel
Rahman et al. 2022; Amenyogbe et al. 2020). Moreover, rising inhabited lymphocytes
reflect the boosted immune response provoked by the administered compounds, thus
advancing the overall performance of the examined fish (Ringø etal. 2014). Additionally,
Volman etal. (2008) explained the protective effect of 1,3 β-glucans orally administered as
receptor-mediated interactions with specialized epithelial cells transport macromolecules
in the Peyer’s patches (microfold cells), which induced cytokine production and heightened
resistance to infection.
For the quantitative assay of antioxidant and growth genes, β-glucan-FOS (containing
0.5, 1 and 1.5%) groups displayed a similar cytokine and growth profiles. The mixture
of high doses of β-glucan and FOS was found to significantly increase the expression of
the CAT and GPX genes, which are the genes for the major first-line antioxidant enzymes
(Ahmed etal. 2022) that support cellular self-mechanisms by suppressing and preventing
the formation of reactive oxygen species in host cells (Ighodaro and Akinloye 2018). These
enzymes aid in the removal of reactive oxygen species, protecting cells from harmful free
radicals.
Furthermore, a significant up-regulated expression in the GH and IGF1 profiles were
detected in supplemented groups especially T1.5 group. The inclusion of β-glucans in the
tilapia diet has led to an promote growth which is supported by Yan et al. (2013) who
found that the IGF1 is the marker gene of miR-206, which is a key regulator of tilapia
growth.
The increased IGF1 mRNA expression level in the hepatic tissue of red tilapia signi-
fying elevated serum IGF, which functions similarly to insulin in promoting fish growth
(Wang etal. 2020a). Moreover, IGF1 is one of the essential components of IGF signaling,
which controls the growth as well as development of skeletal muscle in a variety of verte-
brates (Ibrahim etal. 2022).
The existing study stated that the dietary inclusion of β-glucan-FOS significantly
decreased the cumulative mortality of Red tilapia after being challenged by V. alginolyti-
cus. Similar findings were detected in various fish species fed β-glucan inclusion diets and
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following challenges with Aeromonas veronii infection in koi (Cyprinus carpio) (Lin etal.
2011) and A. hydrophila infection in Nile tilapia (Barros etal. 2014). In addition to protect-
ing fish against bacterial infections, immunosaccharides also, for example, FOS increase
the resistance of Caspian roach’s (Rutilus rutilus) resilience to saline stress (Soleimani
etal. 2012). The protection of red tilapia against infection may be attributed the fact that
immunosaccharides stimulate the immune system directly though enhancing both innate
humoral and cellular defense mechanisms. Novak and Vetvicka (2008) reviewed that
β-glucan binding to dectin-1 (β-glucan major receptor on Macrophages) and, as a result,
activates macrophages that considered the basic effector cells in host defense against
microbes as bacterial pathogens. β-Glucan binding activates macrophages through various
processes, including increased chemokinesis, chemotaxis, migration to phagocyted parti-
cles, degranulation, adhesive molecules, adhesion to endothelium, and tissue migration. It
triggers intracellular processes, including respiratory burst, increased hydrolytic enzyme
activity, and signaling processes that lead to activating professional phagocytes and boost-
ing phagocytosis.
Conclusion
The present study provided evidence that a dietary inclusion of 1% β-glucans-FOS mix-
ture could significantly enhance the growth, metabolism, and non-specific immunity of
Red tilapia through improving gut microbiota, intestinal absorption, feed utilization, and
antioxidant enzyme activity. The benefit of this was demonstrated by a higher resistance to
V. alginolyticus infection. Increased consumption of antioxidant-rich foods or antioxidant
supplements will improve the body’s potential to minimize the risk of disease incidence.
Thus, these environmentally and consumer-friendly immunosaccharides can assist aqua-
culture production in moving steadily in the direction of sustainability by improving fish
health.
Abbreviations FOS: Fructooligosaccharides; CAT : Catalase; GPX: Glutathione peroxidase; GH: Growth
hormone; IGF-1: Insulin-like growth factor; ALT:Alanine transaminase; AST:Aspartate transaminase;
ALP:Alkaline phosphatase; qRT-PCR: Real-time quantitative reverse transcription PCR; TLR2:Toll-like
receptors 2; LD50:The lethal dose fifty
Acknowledgements The authors would like to thank workers in fish farmers for their help during the exper-
iment and in samples collection.
Author contributions E. E: Conceptualization; investigation; methodology; data curation; validation; formal
analysis; writing–review and editing. R. A: conceptualization; writing –review and editing; resources. Y. A:
Methodology; data curation; investigation; writing –original draft. B.H: Methodology; investigation; data
curation; writing –original draft. M. E: methodology; data curation; investigation; writing–original draft.
N. E: Conceptualization; methodology; investigation; validation; data curation; formal analysis; writing –
review and editing. All authors have read and approved the final version of the submitted manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority
(STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Data availability All data generated and analyzed during this study are included in this published article.
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Declarations
Ethics approval All experiments and sampling procedures were conducted according to the Guidelines of
the Animal Care of the Faculty of Veterinary Medicine, Suez Canal University, Egypt’s Scientific Research
Ethics Committee, coded: (2023032).
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-
mons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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Authors and Aliations
El‑SayedHemdanEissa1 · RagaaA.Ahmed2 · YasminM.AbdEl‑Aziz3 ·
BasmaM.Hendam4 · MoahedaE.H.Eissa5 · NohaI.ElBanna6
* El-Sayed Hemdan Eissa
Sayed.Hemdan@env.aru.edu.eg; sayedhemd@gmail.com
* Noha I. ElBanna
Noha.Elbana.fish@suez.edu.eg; Dr_noha_elbanna@hotmail.com
Ragaa A. Ahmed
ragaa10@yahoo.com
Yasmin M. Abd El-Aziz
yasminabdelaziz2012@yahoo.com
Basma M. Hendam
Basmahendam@mans.edu.eg
Moaheda E. H. Eissa
moahedaelsayed@gmail.com
1 Fish Research Centre, Faculty ofAgricultural Environmental Sciences, Arish University, El-Arish,
Egypt
2 Aquaculture Department, Faculty ofFish andFisheries Technology, Aswan University, Aswan,
Egypt
3 Zoology Department, Faculty ofScience, Port Said University, PortSaid42526, Egypt
4 Department ofAnimal Wealth Development, Faculty ofVeterinary Medicine, Mansoura
University, Mansoura35516, DakahliaGovernorate, Egypt
5 Biotechnology Department, Fish Farming & Technology Institute, Suez Canal University,
Ismailia41522, Egypt
6 Aquaculture Diseases Control Department, Fish Farming & Technology Institute, Suez Canal
University, Ismailia41522, Egypt
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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