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Potential Symbiotic Effects of β-1,3 Glucan, and Fructooligosaccharides on the Growth Performance, Immune Response, Redox Status, and Resistance of Pacific White Shrimp, Litopenaeus vannamei to Fusarium solani Infection

El-Sayed Hemdan Eissa
Ragaa A. Ahmed
Nadia A. Abd Elghany
Amal Elfeky
Saadea Saadony
Norhan H. Ahmed
Salah El-Sayed Sakr
Geraldine B. Dayrit
Charlene Princess S. Tolenada
Adlene Anne C. Atienza
Mahmoud Mabrok
12 and
Hala F. Ayoub
Fish Research Centre, Arish University, Arish 45511, Egypt
Department of Aquaculture Nutrition, Faculty of Fish & Fisheries Technology, Aswan University, Aswan 81528, Egypt
Department of Fish Diseases, Animal Health Research Institute, ARC Dokki Giza, Cairo 12618, Egypt
Department of Animal Resources, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt
Department of Animal Production, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt
Department of Animal Production, Faculty of Agriculture, Zagazig University, AlSharqia 44519, Egypt
Fish Resources Department, Faculty of Agricultural and Environmental Sciences, Arish University, Arish 45511, Egypt
The Graduate School, University of Santo Tomas, Manila 1008, Philippines
Department of Medical Microbiology, College of Public Health, University of the Philippines Manila, Manila 1000, Philippines
Department of Medical Technology, Far Eastern University, Manila 1015, Philippines
School of Medical Laboratory Science, Far Eastern University-Dr. Nicanor Reyes Medical Foundation, West Fairview, Quezon City 1118, Philippines
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt
Department of Fish Health and Management, Central Laboratory for Aquaculture Research (CLAR), Agricultural Research Center (ARC), Abbassa 44662, Sharqia, Egypt
Author to whom correspondence should be addressed.
Fishes 2023, 8(2), 105;
Submission received: 31 December 2022 / Revised: 26 January 2023 / Accepted: 8 February 2023 / Published: 10 February 2023
(This article belongs to the Special Issue Disease Control in Fish and Shrimp Aquaculture)


The potential effects of dietary supplementation with β-1,3 glucan and fructooligosaccharides (β-1,3 GF) on antioxidant activities, immunological response, and growth performance of Pacific white shrimp (Litopenaeus vannamei) was investigated. Four diets (iso-energetic and iso-nitrogenous) with different levels of β-1,3 GF (0, 0.5, 1.0, and 1.5 g kg−1) were fed to healthy shrimp juveniles weighing 3 ± 0.5 g for 75 days. Shrimps were randomly distributed into 12 net enclosures at a density of 30 shrimp/net, and the experiment was performed in triplicate. The results revealed that long-term supplementation with 1.5 g kg−1 β-1,3 GF significantly improved shrimp weight gain, feed conversion ratio, and digestive enzyme profiles compared to the control diet group. However, there were no substantial variations in the contents of moisture, crude protein, total lipids, and ash in the muscles of shrimp fed on different diets. Surprisingly, all antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and immune biomarkers (lysozyme, total hemocyte count, phenol oxidase, and respiratory burst) activities were significantly elevated with increasing levels of β-1,3 GF in the shrimp diet, and the highest values were recorded in the 1.5 g kg−1 diet groups. Challenge test results revealed that F. solani could cause a high mortality rate (86.7%) in a group fed a normal basal diet within 14 days at a dose of 5 × 104 conidia mL−1. Surprisingly, all dietary treated groups with different doses of β-1,3 GF showed high resistance against F. solani, represented by lower cumulative mortality rates (20–43.3%) compared to the control group. Moreover, most of the infected shrimp showed a typical black to brown gill lesion similar to that observed in the natural infection, where an identical fungus was successfully re-isolated from infected gills and muscles. Overall, this study recommends an appropriate incorporation level of β-1,3 GF that could enhance growth performance and improve the antioxidant activities, non-specific immunity, and disease resistance of L. vannamei, with an optimal level of 1.5 g kg−1.

1. Introduction

Aquaculture is one of the most promising food sectors to provide the world’s population with animal protein sources and tackle the problem of food scarcity that is caused by overpopulation [1,2]. Shrimp farming is one of the fastest-growing economic sectors in the Pacific Coast region, accounting for more than 58% of total shrimp production worldwide in 2016 [3]. One of the most economically significant penaeid shrimp currently being produced in several nations, including Egypt, is the Pacific white shrimp (Litopenaeus vannamei) [4]. Recently, numerous diseases have emerged in cultured L. vannamei due to further intensification and environmental water deterioration [5,6], resulting in a significant impact on the global shrimp farming business. Among these diseases, black spot disease (BSD) caused by Fusarium spp., is considered a major issue in shrimp-producing sectors [7]. Fusarium spp. belong to the family Nectriaceae and are widespread fungi commonly found on plants, in soil, freshwater, and brackish water [8,9]. The fungi have caused several diseases in plants and animals, resulting in decreased crop yields, lethal mycotoxins, and disease emergence [10,11]. L. vannamei is highly susceptible to BSD, with most naturally infected shrimp exhibiting black gill symptoms and serious tissue damage with high mortality [7]. F. solani was identified as the main causative organism [12], causing molting failure and rapid onset of BSD outbreaks among individuals [7]. The pathogen affects a wide range of cultured shrimp, including Penaeus semisulcatus in Israel [13], Penaeus. japonicus in Japan [14], and Pacific white shrimp in China [7].
Antibiotics, insecticides, synthetic herbal-based phenols, and other chemicals have been used extensively in shrimp culture systems to control pathogens and treat diseases [15,16,17]. However, overuse and indiscriminate use of these medicines could result in the emergence of drug resistant pathogens of public health significance [18,19,20]. Furthermore, these substances can negatively influence healthy bacteria or the host’s normal flora and have direct impacts on consumer food safety [21,22]. Consequently, shrimp farming requires sustainable alternative non-classical strategies, including phage therapy, dietary plant-based medications, probiotics, prebiotics, and synbiotics administration [23,24]. Prebiotics are gaining popularity as an alternative to antibiotics and pesticides in aquaculture [25]. The term “prebiotic” was first defined as “non-digestible foodstuffs found in the digestive tract that stimulate growth or development of beneficial bacteria and thereby improving the intestinal balance of the organism” [26]. There are various prebiotics that have been evaluated in-depth in the aquaculture sector, such as fructooligosaccharides (FOS), short-chain FOS, galactooligosaccharides, inulin, mannan oligosaccharides, arabinoxylooligosaccharides, β-glucan xylooligosaccharides, and isomaltooligosaccharides [27,28]. Prebiotics are effective in preventing infectious infections from spreading during shrimp farming, as well as having the ability to increase growth efficiency, innate immunity, and disease resistance [19,29]. Prebiotics have also been demonstrated to improve the host’s ability to absorb nutrients and to boost the performance of probiotics in the form of synbiotics during growth in the digestive system [30]. Among these, FOS and β-1,3 glucan are promising prebiotics that have been demonstrated to be immunomodulators in aquaculture [31]. Previous studies have assured these compounds are worthy and auspicious immunostimulant that support the growth of the gut microbiota, improve growth performance, boost immune responses, and monitor diseases in aquaculture [32,33]. However, there is no concrete evidence from the past about the use of these combination substances in shrimp. Therefore, the current study aimed to evaluate the potential use of dried β-1,3 glucan and fructooligosaccharide (β-1,3 GF) in shrimp diet, highlighting their main effects on Pacific white shrimp L. vannamei growth performance, immune response, antioxidants, and resistance to F. solani infection.

2. Materials and Methods

2.1. Preparation of Experimental Diets

Although dried β-1,3 GF is widely utilized in fish diets as synthetic commercial prebiotics, there were no intelligible databases to determine the required dosages in shrimp-producing sectors. The β-1,3 GF (Aquastem™ V) was purchased from Kemin Industries, Inc., USA, and was given at different concentrations of 0.0 (control), 0.5, 1.0, and 1.5 g kg−1 diet, corresponding to four dietary groups (C1–C4, respectively). AquastemTM V is a unique combination of an algal source of linear 1,3-beta glucan, enriched with vitamin C and fructooligosaccharides. Brief information about the main compositions, features, and instructions for use are available on the company website (, accessed on 26 January 2023). The compound was diluted in 100 mL water and thoroughly mixed with the basic components of the control diet for 30 min. The feed is formulated from commercial ingredients (shrimp meal, fishmeal, rice bran, wheat flour, rice bran, soybean meal, fish oil, vitamin and mineral mixture). The composition and chemical analysis of the experimental diets are shown in Table 1. The dry ingredients were passed through a sieve (aperture of 1.5–2.0 mm in diameter) before being mixed into the diet. Emulsified oil was added with equal quantity of water with 0.7% phosphatidylcholine (lecithin) according to [34], to the experimental rations. Mixtures were homogenized in a model SNFGA forage mixer (St. Joseph Kitchen Assistant, M149085 USA). The boiling water was then mixed with the mixtures at a rate of 50% for pelleting. Diets were pelleted (1.5–2.0 mm in diameter) using Kitchen Assistant meat mincers and stored in a refrigerator (4 °C) until used.

2.2. Shrimp Rearing and Feeding Trial

This study was carried out on a private shrimp farm in Damietta, Egypt, where Litopenaeus vannamei juveniles were reared at 22.36‰ salinity in a rectangular pond fitted with 12 identical hapas; each was built with green fine mesh and had a 175-L holding capacity. Shrimp were fed with basal diet (~39% CP kg−1 diet) thrice daily until visual satiety and left to acclimatize for three weeks. Subsequently, 360 apparently healthy juveniles weighing 3 ± 0.5 g were randomly relocated into hapas in triplicate at a density of 30 shrimp/hapa, corresponding to the above four dietary treatments (C1–C4 groups). Shrimp were fed twice daily (9 a.m. and 3 p.m.) at a rate of 6% of their body weight, and the trial lasted for 75 days. The shrimps were weighed in bulk every two weeks, and the feeding amount was adopted accordingly. All water quality parameters were maintained close to the permissible levels for shrimp culture; meanwhile, the trial following [37] recommended levels as follows: water temperature (25.6 ± 1.3 °C), pH (7.6 ± 1.2), and dissolved oxygen (6.4 ± 1.4) mg L−1. Water temperature, pH, and DO were measured using a handheld digital meter (YSI Pro1020), while salinity was measured by an ATC salinity refractometer. Ammonia and nitrite were evaluated twice per week using API test commercial kits, and never exceeded 0.05 and 0.25 mg L−1, respectively. Periodic water exchange of 20% was performed every two days during the adaptation period to keep ammonia levels constant.

2.3. Growth Performance

All shrimp groups fasted for 24 h and were counted and weighed in bulk to determine the survival and growth rates at the onset and the end of the feeding trial. In order to determine the feed efficiency index, the consumed feed was also weighed and reco
The following equations were used:
Weight gain (WG) = final weight (g) − initial weight (g).
Survival rate (SR) % = (Final No./Initial No.) × 100.
Feed conversation ratio (FCR) = feed intake (g)/body weight gain (g).
Specific growth rate (SGR) %/day = (ln final weight (g/shrimp) − ln initial weight (g)) 100/n
Since [n: number of days).
Weight gain rate (WGR %) = [final weight (g) − initial weight (g)] × 100/initial weight (g).

2.4. Sample Collection

At the end of the feeding trial, all groups were starved for 24 h prior to sampling. Six shrimp/net were randomly harvested and kept at −4 °C for carcass analysis. Muscles were collected after dissecting shells and shrimp heads. Samples of hemolymph were collected from the first abdominal segment in 1.5 Eppendorf tubes containing EDTA using a 1-mL sterile syringe. The collected samples were centrifuged at 3000× g for 10 min, and the supernatants were separated and kept at −80 °C for further analyses.

2.5. Carcass Chemical Analysis

At the start and the end of the experiment, the approximate chemical composition of six shrimp muscles and diets were determined following [38]. Moisture content was determined after drying to constant weight in an oven at 105 °C. The crude protein was assessed using a micro-Kjeldahl analyzer. Soxhlet apparatus was used with petroleum ether for 16 h to measure the total lipid content. Ash content was estimated using weight loss following 6 h burn at 550 °C in a muffle furnace. All the chemical analyses were performed in triplicate, and the data were expressed in a dry matter basis.

2.6. Digestive Enzymes Analysis

The intestinal samples (each was pooled from six biological replicates and weighing 1 g) were eviscerated and blended in a 10 mL Eppendorf tube with 4 mL phosphate buffer saline (PBS) solution following the method of [39]. Subsequently, the samples were emulsified using an electric homogenizer in an ice water bath for 15 s and centrifuged at 12,000 rpm at 4 °C for 20 min. The supernatant was then collected for digestive enzyme analysis. The activities of trypsin and chymotrypsin were evaluated according to the protocol of [40] and calculated from the increase in absorbance at 410 nm for 10 min at 25 °C using p-nitro aniline as a standard. Amylase, protease and lipase activities were measured following the method of [41]. The weight (mg) of maltose released during the course of 10 min at 30 °C was used to compute one unit of amylase activity. Tyrosine released in 15 min under the experiment conditions was used to measure unit protease activity. The amount of 0.025 NaOH needed to neutralize the fatty acids released during 18-h incubation period at pH 6.9 and 30 °C was used to measure unit lipase activity. Digestive enzymes were calculated as enzyme unit per gram tissue.

2.7. Antioxidants and Immune Biomarkers

All antioxidant enzymes were measured from collected hemolymph samples. Superoxide dismutase (SOD) activity was assessed following the protocol of [42]. Catalase (CAT) activity was determined using H2O2 reaction according to [43]. Glutathione peroxidase (GPx) activity was measured using methyl catechol reaction according to [44]. Malondialdehyde (MDA), as a biomarker of lipid peroxidation, was detected using a thiobarbituric acid reaction following the protocol of [45]. Hemolymph lysozyme activity was assessed using the turbidimetric method described elsewhere by [46]. The respiratory burst activity was determined using nitro blue tetrazolium (NBT) following the method of [47]. Phenol oxidase activity (PO) was measured based on the method of [48]. Total hemocyte count (THC) was performed by adding diluted hemolymph samples to a Neubauer hemocytometer and calculating as follows:
THC (cells mL−1) = count No × 104 × dilution factor.

2.8. Fusarium solani Challenge Trial

The tested strain of F. solani was kindly provided by the Microbiology Unit, Fish Diseases Department, Animal Health Institute, Dokki, Giza. The fungal strain was routinely cultured on Potato Dextrose Agar (PDA) at 25 °C for 7 days. To obtain the conidial mass, 20 mL of sterile distilled water was added to each culture plate, and the suspension was collected in 30 mL sterile tubes. The suspensions were purified using two layers of sterile medical dressing to ensure that the filtrate contained only fungal conidia. The fungal conidia concentration was determined by an erythrocyte-counting chamber and was finally adjusted to 5 × 104 conidia mL−1 in sterile distilled water.
At the end of the feeding trial, 30 shrimp per each treated group were randomly collected and reassigned in triplicate into 120-L aquariums (10 shrimp/aquarium). Shrimp were injected intramuscularly (IM) in the third abdominal segment with 0.1 mL of 5 × 104 conidia suspension according to [14]. Another group of shrimp was injected IM with 0.1 mL of PBS and served as a negative control group. Mortality rates were recorded daily for 14 days. Moribund and freshly dead shrimp were regularly harvested for clinical and mycological examinations to ascertain the cause of death. Mortality was considered only when the injected strain was retrieved from experimentally infected shrimp (Koch’s postulates).

2.9. Statistical Analysis

All trials were performed in triplicate. Data were analyzed for both normality and homogeneity of variance using Kolmogorov–Smirnov and Levene’s tests, respectively. When needed, they were transformed before being treated. Duncan’s multiple range tests (SPSS, version 22.0) and one-way analysis of variance (ANOVA) were used to analyze the data. The resulting values were expressed as means ± standard error, and the value of p < 0.05 was considered significant.

3. Results

3.1. Growth Performance and Proximate Carcass Composition

All values of growth performance parameters are presented in Table 2. As indicated, The C4 group showed no significant difference from the C3 group in FW and WG. However, these groups showed higher values compared to C1 and C2. The feed conversion ratio (FCR) displayed lower values in all dietary-treated groups compared to the control (p < 0.05) and was more exceptional in the C4 group. The survival rate exhibited no notable differences (p > 0.05) among the tested groups during the feeding trial, and it ranged from 92.5 to 96.3 ± 0.1%. In addition, there were no significant variations (p > 0.05) in the contents of moisture, total lipids, crude protein, and ash in the muscles of tested shrimp following supplementation with different concentrations of β-1,3 GF (Table 3).

3.2. Digestive Enzymes Analysis

The values of all digestive enzyme activities showed significant differences (p < 0.05) between the dietary treated groups and the control (Figure 1). Lipase activity was higher in shrimp fed the diet with a high dose of β-1,3 GF in C3 and C4 groups for 75 days than those fed only the lower dose in the C2 group and basal diet C1 group for the same period. Similarly, chymotrypsin, trypsin, amylase, and protease activities were significantly increased (p < 0.05) following dietary supplementation with a high dose of β-1,3 GF compared to other treatments.

3.3. Antioxidants and Immunity Biomarkers

The kinetics of antioxidants and immunity biomarkers following supplementation with different concentrations of β-1,3 GF are presented in Table 4. SOD, CAT, and GPx activities increased with increasing β-1,3 GF levels, with the highest values recorded in the C4 group. Likewise, there was a remarkable improvement in lysozyme and respiratory burst activities in all treated groups in comparison to the control. In contrast, the level of MDA was significantly (p < 0.05) decreased in shrimp that received a higher dose of β-1,3 GF when compared to the control group. The total hemocyte count of the control group was consistently lower (p < 0.05) than the treated groups. However, the highest values were observed in the C4 group. Moreover, the PO activity of shrimp in the C4 group was notably higher compared to the other treatments.

3.4. Fungal Challenge Trial

Colonies of Fusarium solani grew well on PDA media, with dense aerial white to creamy mycelium, which were abundant and woolly, and the reversal color was brownish. After 3 days, microconidia in fresh isolates were abundant, ellipsoidal, fusiform, or kidney-shaped. Within 4–7 days, the macroconidia were abundantly developed and thick-walled, having three to four septa, straight and parallel-sided for most of the length. The apical cell blunt was end rounded.
At the end of the trial, no mortality was observed in the negative control group that was intramuscularly injected with PBS (C-ve), whereas different levels of mortality were observed in challenged shrimp groups (C1–C4) depending on the dosages of the experimental diet. Herein, the dietary control group exposed to F. solani (C1 group) started to die one day post-challenge (Figure 2). Shrimp-fed different levels of β-1,3 GF (C2–C4 groups) showed lower cumulative mortality (20–43.3%) than those fed a normal basal diet (86.7 %) (C1 group). SR% was clearly higher for shrimp fed the diet with 1.5 g kg−1 β-1,3 GF (C4 group). Most of the infected shrimp showed marked clinical signs similar to those of the natural infection. Within a few days of the challenge, the infected shrimp showed black to brown collapsed gills with severe necrosis and atrophy (Figure 3), whereas the negative control group displayed normal dusky white gills. Nonetheless, the fungal isolate was successfully recovered on PDA from the gills and muscles of infected shrimp and verified as described above. The fungal was also seen under a microscope in wet mount preparations from the same specimens.

4. Discussion

Prebiotics are widely regarded as one of the most important and practical dietary supplements for sustainable shrimp farming [27]. Incorporating synthetic prebiotics into shrimp meals is now one of the most widely used alternative disease control and preventive strategies in aquaculture. However, their efficacy may vary depending on the species, dosage, and synergism of the combined compounds [49,50]. In the present study, β-1,3 GF enhanced the growth performance and feed utilization of white shrimp at an inclusion level of 1.5 g kg−1. Additionally, it exhibited remarkably lower FCR and higher WG than the control. These findings were in accordance with earlier findings that revealed that the feeding of β-glucans augmented the growth and feed utilization of Pacific white shrimp [51,52], red seabream [53], and tilapia [54]. Similarly, O. niloticus fed a synbiotic containing high levels of β glucans and fructooligosaccharides showed a noticeable increase in weight gain and SGRs than non-treated fish [55]. Nevertheless, some studies did not elucidate the growth-promoting effect of β-glucans when administered to the white shrimp diet. It is still unclear how β-glucans work to improve this growth [56], but it is believed that the growth-enhancing impact of this compound could depend on its concentration, solubility, and structures, as well as the examined species.
In aquaculture, β glucans and fructooligosaccharides (FOS) are one of the main prebiotics mostly selected to be integrated with probiotics [57]. Several studies in the literature have elucidated the positive influence of both compounds on the gut microbiota and have definitively verified their roles as growth promoters in aquaculture sectors [58,59]. The principle of prebiotics is that these compounds are degraded to their respective sugars in the shrimp gut and used by beneficial bacteria as carbon sources [49]. Shrimp rely on the actions of digestive enzymes for nutrient breakdown and absorption. The intestines and the hepatopancreas are crucial organs for the secretion of digesting enzymes [60]. Shrimp growth is dependent on nutrient uptake, which is correlated with the activity of digestive enzymes in the hepatopancreas and intestine, including chymotrypsin, amylase, lipase, and trypsin [28]. According to the current study, feeding fish with 1.0 and 1.5 g kg−1 of β-1,3 GF considerably increased the digestive enzyme activity of shrimp. The outcomes were comparable to those of earlier research on Pacific white shrimp (L. vannamei) fed on different prebiotics such as β-glucan, dietary glycerol monolaurate, and microbial lysozyme [61], crude polysaccharides, arabinoxylan-oligosaccharide, inulin, and fructooligosaccharide [62]. Herein, the significant increase in the activities of digestive enzymes in L. vannamei fed on β-1,3 GF suggests that the prebiotics may encourage the growth of endogenous probiotic bacteria, followed by an increase in the secretion of exogenous hydrolytic enzymes in the shrimp, which improved the digestion of ingested foods [62].
The most likely explanation may be the changes in the composition of the intestinal microflora increase the amount of bifidobacteria and lactobacilli while decreasing the growth of harmful bacteria such as Escherichia coli and Clostridium difficile. The intestinal microbiota is essential for many physiological and nutritional processes, because it lowers the loss of digesting enzymes, enhances their activity, and fosters growth [33]. Additionally, the digestive gland of shrimp has glucanases that may break down the glucose polymer-glucan to release energy and use the UDP-glucose route to turn glucose into glycogen [63]. Shrimp can obtain energy by digesting glucan through this mechanism, which enables quicker growth [32]. Contrarily, gut bacteria of the genera Bifidobacterium and Lactobacilli selectively digest the oligosaccharide FOS, which promotes the growth and activation of these bacteria’s metabolic processes. This enhances the prebiotic property of FOS and benefits the host’s health [58].
Regarding antioxidants and immunity biomarkers, it was known that SOD, CAT, and GPx are crucial enzymes that work through enzymatic pathways to reduce oxidative stress [64]. These enzymes can enhance the ROS imbalance and preserve the normal redox balance in biological systems [65]. In this study, the activities of SOD, CAT, and GPx were significantly increased; meanwhile, MDA levels were substantially lower in groups fed β-1,3 GF compared to the control group. These results reflect the main role of β-1,3 GF as a potential antioxidant due to its high phenolic compound content, which is likely responsible for its potent antioxidant activity [66]. Our results were in agreement with those reported by [67], who mentioned that dietary inclusion of β-glucans and other oligosaccharides could mitigate and improve shrimp antioxidant enzymes and innate immune parameters.
Lysozyme activity is one of the most essential indicators of the immune system and its action by the activation of the complement system and phagocytosis [68]. Further, it possesses bactericidal activity against a wide range of Gram-negative and other threatening pathogens [69]. The current study surmised that the dietary addition of β-1,3 GF to white shrimp significantly enhanced lysozyme activity compared to the control group. These results can be attributed to the complex structure of β-1,3 GF and its ability to activate the immunity of white shrimp [70].
Indeed, shrimp circulating hemocytes are the main sources of lysozyme [71]. Hence, the surge in hemocytes in this study was concomitant with elevated lysozyme activity. The results of the present investigation are consistent with the previous findings of [72,73]. The NBT assay primarily measures the production of free oxidative radicals produced by leukocytes in the defense in the face of fish pathogens [74,75]. Our results revealed that there was a remarkable improvement in lysozyme and respiratory burst activities of shrimp hemocytes following supplementation with β-1,3 GF in comparison with the control diet group. Similar findings were reported by [76], who observed an increase in respiratory burst and phenol oxidase activities in white shrimp hemocytes following in vitro culture with a hot water extract from the leaves and branches of the Cinnamomum kanehirae. In addition, [77] found that the hemocytes of prawns (Macrobrachium rosenbergii) displayed up-regulation of PO and RB activities after incubation of leaf extract of Morinda citrifolia.
Assessment of hematological profiles is an essential tool for shrimp health evaluation [78]. Hemocytes are critical components of cellular defense. In crustaceans, a lower number of circulating hemocytes corresponds well with decreased susceptibility to infections [79]. With regard to hematological profiles in this study, Total hemocyte values were proportionally increased as the level of β-1,3 GF increased; the highest values were recorded in the group fed a high dose (1.5 g Kg−1). Similar enhancing patterns have been identified in Fenneropenaeus indicus shrimp fed on a diet supplemented with different levels of β-1,3 glucan [80]. Likewise, [81] observed an increase in total hemocyte count in F. indicus fed on a yeast-incorporated diet.
In this study, shrimp experimentally infected with F. solani showed remarkable clinical signs similare to those of natural infection, which appeared as black to brown-colored gills and collapsed and necrotized gill lamellae, consistent with the previous findings of [7]. The characteristic lesions may be related to granuloma formation or possibly due to mycotic enzymes and toxins causing severe degeneration of shrimp gills [82]. Following the challenge with F. solani, shrimp fed different levels of β-1,3 GF showed lower cumulative mortality than those fed a normal basal diet. The survival rate was clearly higher for shrimp fed the diet with 1.5 g kg−1 β-1,3 GF. Our results are consistent with previous studies, where herb administration enhanced the immunity of L. vannamei after one week of dietary inclusion [83,84]. Based on our findings, β-1,3 GF therapy may be a suitable alternative strategy for controlling fungal infection in the shrimp culture system.

5. Conclusions

Supplementation with β-1,3 GF has a pronounced effect on the immune response of Pacific white shrimp, Litopenaeus vannamei, through the enhancement of total hemocytes and immune-related enzymes. The favorable effect of these compounds depends mainly on their content, dosage, and days of supplementation. Lastly, dietary inclusion of β-1,3 GF at an optimum level (1.5 g/kg diet) in commercial shrimp farms is recommended as it enhances the growth parameters and immune responses, and increases shrimp resistance to invading fungi, especially F. solani. In this study, our results showed an improvement in the growth, immunity, and Fusarium resistance of shrimp following supplementation with β-1,3 glucan at concentrations of 1–1.5 g/kg diet. However, the impact of supplementing with β-1,3 glucan over 1.5 g/kg diet is still not fully elucidated, and requires further investigations.

Author Contributions

E.-S.H.E., conceptualization, methodology, data curation, and investigation; R.A.A., conceptualization, methodology, and investigation; N.A.A.E., conceptualization, methodology, and investigation; A.E., conceptualization, methodology, and investigation; S.S., conceptualization, methodology, and investigation; N.H.A., conceptualization, methodology, and investigation; S.E.-S.S., conceptualization, methodology, and investigation; G.B.D. data curation, review, and editing; C.P.S.T., data curation, review, and editing; A.A.C.A., data curation, review, and editing; M.M., data curation, writing—original draft, review, and editing; H.F.A., conceptualization, methodology, data curation, investigation, supervision, writing—original draft, review, and editing. All authors have read and agreed to the published version of the manuscript.


This research received no external funding.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Review Committee of Suez Canal University (AERC-SCU), Egypt, with approved No. 2022037 on 9 November 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. Digestive enzymes analysis of the intestine ((A), chymotrypsin; (B), trypsin; (C), amylase; (D), lipase; (E), protease (U/g tissue)) of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and Fructooligosaccharides for 75 days. Data are presented as means ± SD (n = 3). Superscripted letters denote significant differences between experimental groups (ANOVA; p < 0.05).
Figure 1. Digestive enzymes analysis of the intestine ((A), chymotrypsin; (B), trypsin; (C), amylase; (D), lipase; (E), protease (U/g tissue)) of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and Fructooligosaccharides for 75 days. Data are presented as means ± SD (n = 3). Superscripted letters denote significant differences between experimental groups (ANOVA; p < 0.05).
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Figure 2. The cumulative mortality (%) over time (Days 0–14) for Pacific white shrimp, Litopenaeus vannamei, after challenge with Fusarium solani; shrimp were fed with β-1,3 glucan and fructooligosaccharides (β-1,3 GF) supplemented diets (0, 0.5, 1, 1.5 g/kg; C1–C4, respectively). C-ve, refer to shrimp groups that received a normal basal diet and were intramuscularly injected with PBS. Means (SD±) with different letters are significantly different ANOVA test, at p  ≤  0.05.
Figure 2. The cumulative mortality (%) over time (Days 0–14) for Pacific white shrimp, Litopenaeus vannamei, after challenge with Fusarium solani; shrimp were fed with β-1,3 glucan and fructooligosaccharides (β-1,3 GF) supplemented diets (0, 0.5, 1, 1.5 g/kg; C1–C4, respectively). C-ve, refer to shrimp groups that received a normal basal diet and were intramuscularly injected with PBS. Means (SD±) with different letters are significantly different ANOVA test, at p  ≤  0.05.
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Figure 3. Gills of experimentally infected Litopenaeus vannamei by Fusarium solani showing noticeable black to brown melanated gills (black arrows).
Figure 3. Gills of experimentally infected Litopenaeus vannamei by Fusarium solani showing noticeable black to brown melanated gills (black arrows).
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Table 1. Ingredients and proximate chemical analysis of the control diet (g/kg on dry matter basis).
Table 1. Ingredients and proximate chemical analysis of the control diet (g/kg on dry matter basis).
Ingredients (g/kg)β-1,3 Glucan and Fructooligosaccharides Levels (g/kg Diet)
Wheat flour120120120120
Shrimp meal250250250250
Rice bran7069.569.068.5
Soybean meal150150150150
Fish meal300300300300
Fish oil60606060
β-1,3 GF
* CMC10101010
** Vit & Min Mix40404040
Proximate analysis (% dry weight)
Dry mater90.6290.9391.2190.65
Crude protein (N × 6.25)38.8238.7738.7638.77
Crude fat10.9610.8410.8310.77
Crude fiber1.741.511.241.62
Carbohydrate (NFE)232.97533.08733.29133.017
*** Gross energy (GE) kcal/100g3459.467458.56459.22457.61
Analysis was performed according to Official Methods of Analysis, Association of Official Analytical Chemists, Arlington [35]. * CMC, carboxymethylcellulose as a thickening agent. ** Vit & Min Mix, Vitamin and Mineral Mix; each 100 g contained minerals (Zn, 2.50 mg; Mn, 16.00 mg; Fe, 31.50 mg; Cu, 5.50; I, 0.55 mg; Ca, 1.15 gm and P, 450 mg). Vitamins (Vit A, 7,500,000 Iu; Bi, 100 mg; B3, 500 mg; B6, 150 mg; B12, 2.5 mg; E, 100 mg; K, 100 mg; pantothenic acid, 275 mg; folic acid, 100 mg; vit. D3, 7500 Iu). *** GE (kcal/100 g DM) = CP × 5.64 + EE × 9.44 + NFE × 4.11, calculated according to [36]. NFE = 100 − [% Ash + % lipid + % protein].
Table 2. Growth performance and survival rate of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and fructooligosaccharides for 75 days.
Table 2. Growth performance and survival rate of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and fructooligosaccharides for 75 days.
Parametersβ-1,3 G F Levels (g/kg Diet)
Initial weight (g)3.0 ± 0.092.90 ± 0.263.03 ± 0.033.10 ± 0.06
Final weight (g)13.1 ± 0.26 c15.83 ± 0.32 b17.93 ± 0.41 a18.30 ± 0.26 a
Weight gain (g)10.1 ± 0.21 c12.93 ± 0.30 b14.90 ± 0.42 a15.20 ± 0.29 a
Weight gain rate (%)336.67 ± 1.70 c445.86 ± 11.20 b491.74 ± 15.90 a490.32 ± 16.20 a
SGR 1.97 ± 0.007 c2.26 ± 0.026 b2.369 ± 0.035 a2.367 ± 0.036 a
FCR2.02 ± 0.018 a1.96 ± 0.015 b1.57 ± 0.025 c1.55 ± 0.02 c
Survival rate (SR%)96.3 ± 0.1092.5 ± 0.1092.5 ± 0.1093.8 ± 0.10
Data are presented as means ± SD (n = 3). Superscripted letters denote significant differences between experimental groups (ANOVA; p < 0.05).
Table 3. Proximate chemical composition (% on a dry matter basis) of muscles of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and Fructooligosaccharides for 75 days.
Table 3. Proximate chemical composition (% on a dry matter basis) of muscles of Pacific white shrimp, Litopenaeus vannamei, fed various concentrations of β-1,3 glucan and Fructooligosaccharides for 75 days.
Parameter (%)β-1,3 G F Levels (g/kg Diet)
Moisture 79.18 ± 0.2378.85 ± 0.1079.32 ± 0.3379.30 ± 0.34
Protein15.85 ± 0.1916.59 ± 0.1915.46 ± 0.0516.32 ± 0.17
Lipid1.98 ± 0.031.62 ± 0.371.99 ± 0.051.95 ± 0.04
Ash3.06 ± 0.083.22 ± 0.182.96 ± 0.133.19 ± 0.04
Data are presented as means ± SD (n = 3). No significant difference was recorded.
Table 4. Antioxidants and non-specific immune response of white shrimp, Litopenaeus vannamei, fed different levels of β-1,3 glucan and fructooligosaccharides for 75 days.
Table 4. Antioxidants and non-specific immune response of white shrimp, Litopenaeus vannamei, fed different levels of β-1,3 glucan and fructooligosaccharides for 75 days.
Parameterβ-1,3 Glucan and Fructooligosaccharides Levels (g/kg Diet)
SOD (U/L)21.01 ± 0.57 d25.69 ± 0.25 c28.31 ± 0.25 b30.45 ± 0.57 a
GPX (U/L)22.63 ± 0.38 c26.44 ± 0.56 b27.24 ±0.27 ab28.50 ± 0.39 a
CAT (U/L)3.11 ± 0.05 c3.98 ± 0.26 b4.82 ± 0.14 a5.34 ± 0.25 a
MDA (nmol/mL)18.13 ± 0.12 c24.12 ± 0.03 b25.21 ± 0.11 ab26.03 ± 0.11 a
Lysozyme activity (μg/mL)0.95 ± 0.02 d1.24 ± 0.04 c1.62 ± 0.03 b1.85 ± 0.01 a
Respiratory burst activity (mg/mL)0.23 ± 0.12 d0.43 ± 0.12 c0.61 ± 0.12 b0.67 ± 0.12 a
THC (×104 cells/mL)21.01 ± 0.57 d25.69 ± 0.25 c28.31 ± 0.25 b30.45 ± 0.57 a
Phenol oxidase (U/min/mg)18.13 ± 0.12 c24.12 ± 0.03 b25.21 ± 0.11 ab26.03 ± 0.11 a
Data are presented as means ± SD (n = 3). Superscripted letters denote significant differences between experimental groups (ANOVA; p < 0.05).
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MDPI and ACS Style

Eissa, E.-S.H.; Ahmed, R.A.; Abd Elghany, N.A.; Elfeky, A.; Saadony, S.; Ahmed, N.H.; Sakr, S.E.-S.; Dayrit, G.B.; Tolenada, C.P.S.; Atienza, A.A.C.; et al. Potential Symbiotic Effects of β-1,3 Glucan, and Fructooligosaccharides on the Growth Performance, Immune Response, Redox Status, and Resistance of Pacific White Shrimp, Litopenaeus vannamei to Fusarium solani Infection. Fishes 2023, 8, 105.

AMA Style

Eissa E-SH, Ahmed RA, Abd Elghany NA, Elfeky A, Saadony S, Ahmed NH, Sakr SE-S, Dayrit GB, Tolenada CPS, Atienza AAC, et al. Potential Symbiotic Effects of β-1,3 Glucan, and Fructooligosaccharides on the Growth Performance, Immune Response, Redox Status, and Resistance of Pacific White Shrimp, Litopenaeus vannamei to Fusarium solani Infection. Fishes. 2023; 8(2):105.

Chicago/Turabian Style

Eissa, El-Sayed Hemdan, Ragaa A. Ahmed, Nadia A. Abd Elghany, Amal Elfeky, Saadea Saadony, Norhan H. Ahmed, Salah El-Sayed Sakr, Geraldine B. Dayrit, Charlene Princess S. Tolenada, Adlene Anne C. Atienza, and et al. 2023. "Potential Symbiotic Effects of β-1,3 Glucan, and Fructooligosaccharides on the Growth Performance, Immune Response, Redox Status, and Resistance of Pacific White Shrimp, Litopenaeus vannamei to Fusarium solani Infection" Fishes 8, no. 2: 105.

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