Effects of Bacillus subtilis as a single strain probiotic on growth, disease resistance and immune response of striped catfish (Pangasius hypophthalmus)

The present study investigated the potential role of Bacillus subtilis as probiotic in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 150.00±2.63g n = 180) were stocked in circular tanks. Four isonitrogenous (30%) and isolipidic (3.29%) diets were formulated having supplementation of B. subtilis at four different levels (P0; 0, P1: 1×106, P2: 1×108 and P3: 1×1010 CFU/g). Each treatment had three replicates, while each replicate had fifteen fish. The trial started on second week of July and continued for eight weeks. Growth, feed conversion ratio, crude protein content, the concentration of amylase and protease, the profile of both dispensable and non-dispensable amino acids in all four dietary groups increased with a gradual increase of B. subtilis in the diet. At the end of growth experiment, fish in all four groups were exposed to Staphylococcus aureus (5×105 CFU/ml). After S. aureus challenge, fish fed with B. subtilis responded better to damage caused by reactive oxygen species and lipid peroxidation and better survival rate. The catalase and superoxide dismutase level also increased in response to bacterial challenge in B. subtilis fed groups. On the other hand, the concentration of malondialdehyde gradually decreased in these groups (+ve P0 >P1>P2>P3). It is concluded that supplementation of B. subtilis as a probiotic improved the growth, protein content, antioxidant response and immunocompetency against S. aureus in striped catfish. The optimum dosage of B. subtilis, at a concentration of 1×1010 CFU/g, resulted in the most favorable outcomes in striped catfish. This single bacterial strain can be used as an effective probiotic in large scale production of aquafeed for striped catfish. Future studies can investigate this probiotic’s impact in the intensive culture of the same species.


Introduction
The Striped catfish (Pangasius hypophthalmus) is widely recognized as an exceptional aquaculture species which is ideally suited for warm climates.It occupies a prominent position as a primary aquaculture commodity in international markets, holding the status of the second most produced prominent species, surpassed only by tilapia [1].The global production of striped catfish was recorded as 2520.41thousand tons in 2022 [2].In South Asia, aquaculture is a rapidly expanding industry; therefore, species diversification is an indispensable measure for the advancement and sustainability of this sector.One of the finest opportunities to stimulate investment and foster the expansion of aquaculture in South Asia is the cultivation of striped catfish.This species flaunts a proven breeding, husbandry protocols, and already possesses an established market, making it an ideal choice [1].However, ensuring sufficient nutrition becomes paramount for augmenting striped catfish production since it constitutes 40-50% of the cumulative production expenditure [3].In intensive aquaculture, it is possible to provide fish with a diet of superior quality and adequately balanced [4].Moreover, it facilitates the cultivation of fish with high stocking biomass, necessitating minimal investment yet generating enhanced profitability [5].However, the rate of pathogen transmission could be heightened through increased stocking biomass [6].In fact, intensive farming environments are considered evolutionary hotspots, wherein the escalated transmission and frequency of infections could promote virulence in pathogen populations [7,8].
In recent decades, intensive systems of striped catfish have suffered substantial economic losses (US $60 million annually) due to disease outbreaks by pathogenic organisms [9,10].More than 92 genera of pathogens are responsible for this economic loss including, Aeromonas (60-70%), Staphylococcus (70%), Pseudomonas (50%), Edwardsiella ictaluri (50-70%), Shigella (32%) and Salmonella (3.22%) [11][12][13].Among the various species of freshwater and marine fish, S. aureus, is recognized as one of the most commonly encountered pathogens, with a prevalence rate of 40-60% in fish farming and an astonishing 87% in associated products [14,15].Fish handlers are the common vectors of this bacterium, transmitting the infection to fish at the stage of stocking, feeding, harvesting, and processing e.g., approximately 30% of exported striped catfish fillets to Poland were found to be contaminated with coagulase-positive Staphylococcus aureus [16].
To mitigate the risk posed by all pathogens, a range of antibiotics, pesticide residues, and chemical products have been employed [17].However, the excessive and unjustified use of antibacterials for pathogen prevention and growth stimulation has led to the emergence of antibiotic resistance.Nonetheless, instead of relying on antibiotics, several ecologically sustainable biological approaches have been developed, emphasizing the significance of probiotic administration as a central focus of aquaculture research [18,19].When administered in appropriate mixture and dose, probiotic bacteria are beneficial microorganisms that exert therapeutic effects in several species such as tilapia [20,21], grass carp [17], and goldfish [22].The effectiveness of probiotics in the aquaculture industry, including economic expansion, disease resistance (Bacillus subtilis-60-70%) and high yield (Bacillus licheniformis-50%) has been conclusively illustrated [23,24].
Probiotics empower fish to combat inherent stressors by reducing the quantities of reactive oxygen species (ROS) which are naturally produced during regular metabolic activities [25].ROS commonly act as redox messengers determining cellular fate, and acting as signaling molecules for oxidative stress [26].At low levels, antioxidant system can eliminate ROS.Nevertheless, when exposed to intense stimuli such as hypoxia, the excessive buildup of ROS disrupts the equilibrium within cells, resulting in oxidative stress and impairments in cellular functionality [27,28].Probiotic strains, such as Bifidobacterium animalis, Lactobacillus rhamnosus, and Bacillus spp.have exhibited substantial antioxidant potential and capabilities to alleviate oxidative damage [29,30].The potential mechanism underlying the antioxidant effects of probiotics encompass the autonomous secretion of antioxidant metabolites, adjustment of antioxidative activities, and suppression of enzyme activities implicated in the generation of ROS [31].
However, among all other probiotics, Bacillus subtilis is widely accepted in aquaculture due to its spore-forming ability [32], production of a broad spectrum of antibacterial substances [33], and the presence of high-antioxidant-activity substances such as superoxide dismutase (SOD) and glutathione (GSH) [34,35].Multiple studies substantiate the probiotic effects of B. subtilis, including the prevention of gastrointestinal disorders, leading to the improvement of pond water quality and the increased survival rate of animals in aquaculture [17,36].A study conducted by [37] examined the synergistic effects of Bacillus strains on the growth and immune response of striped catfish.However, to the best of our knowledge, the specific impact of a single strain, B. subtilis, has yet to be investigated in this species.The effectiveness of single strains was found to be comparable to that of multi-strains combinations.Single strain probiotic B. subtilis enhances survival rate (65-70%) [34], increases weight gain (50%) [38], improves digestive activity ( [33], mitigates enteric septicemia in catfish (70-80%) [39], control Aeromonas infection in Oreochromis mykiss [40] and increases innate immunity and intestinal microbial population [41].Therefore, the present investigation aims to study the potential effects of dietary supplementation of B. subtilis on growth efficiency, digestive enzyme activity, antioxidant mechanism, and immunological response in striped catfish.

Preparation of experimental diets
In this study, commercial probiotic (ECOSH, Estonian) was used as a source of B. subtilis, contained a concentration of 1×10 12 billion colony forming unit (CFU).One gram of probiotic containing 1×10 12 billion CFU was used to obtained, final concentrations of 1×10 10 , 1×10 8 , and 1×10 6 billion CFU of B. subtilis, respectively.The volume of desired probiotics was calculated, using following formulae and then mix with sterile distilled water.

Volume of probiotic ¼ Target CFU � Initial CFU � Initial volume
For confirmation of CFU in each concentration, plate count assay was performed using nutrient agar plate (HiMedia Ltd., Lahore, Pakistan).Plates were incubated for 24 hours at 37˚C and number of colonies was counted afterwards by using a digital colony counter (Model: AVI-35).Treatment diets were prepared by mixing the finely ground ingredients (grains were procured from local farmers in Pakistan while origin of soybean was USA) (Table 1) with four levels of probiotics (P0: 0, P1: 1×10 6 , P2: 1×10 8 , P3: 1×10 10 CFU/g of the B. subtilis and pellets (1 mm) were prepared using pellet machine (PCSIR, Pakistan).The pellets were air-dried at room temperature and stored at 4˚C.The treatment diets were formulated weekly to ensure the preservation of the actual bacterial count.Bacterial count in feed was performed after every three days using above mentioned plate count assay.

Growth experiment
Trial was started after ethical approval from Animal Ethics Committee (Zoo/LCWU/932).Fish were collected from a local hatchery and transported to the aquaculture facility at Lahore College for Women University.We acclimatized the fish in 600L tanks for a week.During acclimatization, the fish were fed with prepared feed without probiotics.After acclimatization, fish (initial weight = 150.00±2.63gn = 180) were stocked in 12 circular tanks (1.26 m 3 ).Each treatment had three replicates, while each replicate had fifteen fish.An additional thirty fish were fed with a diet without probiotic to be used as the negative control in the bacterial challenge trial.These fish were reared in 2 separate circular tanks, (15 fish each) under the same husbandry conditions as other fish.The fish in each treatment group were fed three times a day.A total of 10% water in tank was exchanged on daily basis.Daily ration was calculated based upon 2% of biomass in that treatment group.The water quality parameters including dissolved oxygen (DO) (7.51±0.21mg/L),pH (7.21±0.41)and temperature (29.00±1.00˚C)were monitored on a daily basis.

Sample collection
At the end of the growth experiment, fish were fasted for 24 hours and anesthetized using clove oil (Sigma Aldrich USA) (6ml/L).Five fish were randomly collected from each replicate of each treatment group.Total body weight and total body length, specific growth rate (SGR), feed conversion ratio (FCR), and weight of viscera, and liver were measured to calculate following parameters: Blood was collected from caudal vein and stored in pro-coagulation clot activator and EDTA coated tubes, respectively.Clot activator tubes were employed to obtain serum, while EDTA coated tubes were utilized for analysis of hematology and blood biochemistry.Blood samples were centrifuged at 5000 rpm for 20 min to extract plasma.It was stored at -20˚C until assayed.Muscle and intestine samples were collected and stored at -20˚C to determine chemical composition, profile of amino acids and digestive enzymes.

Chemical composition and amino acid analysis
The chemical composition of body muscles was analyzed using the protocol outlined by the Association of Official Analytical Chemists [42].Muscle samples were dried in an oven at 80˚C until a constant dry weight was achieved.These dried samples were then ground for further chemical analysis.The crude protein was determined using the Kjeldahl apparatus (PCSIR, Pakistan).Crude lipids were determined by following Folch method [43] in the Soxhlet apparatus (PCSIR, Pakistan).The ash content in the muscles were determined by using the furnace burning method.An amino acid analyzer (Biochrome 30+, Biochrome limited, Cambridge, UK) was used to quantify the amino acid contents of fish muscles and the analytical protocols followed by Ahmed et al [44].

Digestive enzymes assay
Crude enzymatic extracts from intestine samples were prepared Ding et al [45].Properly rinsed intestine samples were homogenized in the phosphate buffer saline (PBS) (pH 7.5) (1 g/ 10 ml and centrifuged at 5000 rpm for 20 minutes.The resultant supernatant was procured and preserved at 4˚C.All analyses were performed within a few hours following the extraction process.Protease activity of intestine samples was determined using Folin-phenol reagent, according to Jin [46].Quantification of amylase enzymes activity was carried out by utilizing iodine to detect the unhydrolyzed starch in samples, as followed by Jiang [47].Lipase enzymatic activity was assessed by measuring the fatty acids released through the enzymatic breakdown of triglycerides in a stabilized dispersion of olive oil droplets, as described by Borlongan [48].The enzymatic activities are expressed as intestine content units per liter (U/L).
The SOD activity [EC.1.15.1.1],was assessed utilizing the (SOD-1 ELISA Kit-PARS BIO-CHEM) (Cat No. PRS-02005 hu), providing a direct and kinetic method for quantifying SOD activity.The extent of inhibition is proportionate to the SOD concentration within a specified range (0.3ng/ml-10ng/ml).SOD activity was determined by measuring the auto-oxidation rates in the presence and absence of the sample, the results were expressed as μmol/L.The activity of catalase (EC:1.11.1.6)were determined spectrophotometrically (560nm) by using catalase colorimetric activity kit (Thermo Fisher Scientific, USA) (Cat No. EIACATC), as per manufacturer instruction.Malondialdehyde (MDA) (EC No. 202-974-4) concentration was determined using ELISA Kit (Cat No. PRS -00991hu).MDA level was measured within the range of 0.3nmol/ml-7nmol/ml at 450nm.

Histological analysis
At the end of bacterial challenge, the intestine, gills, liver, muscles, and kidney were collected from each group (n = 5 of each organ) and placed in sterilized tubes containing 3ml of Bouin's fluid solution (Solarbio, Beijing, China).Following this, the samples were undergoing standard dehydration procedures and were embedded in paraffin.Sections with a thickness of 5μm were then sliced from each sample and subjected to staining with hematoxylin and eosin [49].

Bacterial challenge
2.8.1 Isolation Staphylococcus aureus.S. aureus was obtained from diseased Labeo rohita fish originating from the University diagnostic laboratory, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore Pakistan.A 10-gram portion of the afflicted fish sample was blended with 90 ml of sterile peptone water, generating a 1:10 dilution, to facilitate the enrichment of the target bacterial species.Subsequently, this mixture was incubated at 37˚C for 6 hours following Akbar and Anal [50].From dilutions, 0.5 ml was inoculated on to Mannitol Salt Agar (MSA) and incubated at 37˚C for 24 hours.The emergence of colonies exhibiting a yellow hue was indicative of S. aureus and was subsequently validated through gram staining and coagulase production test.The purified subculture was duly preserved to facilitate subsequent analyses, in accordance Akbar and Anal [50].

Challenge with S. aureus
After the growth experiment, we challenged the fish with S. aureus for 15 days (September 15 until September 30).The S. aureus culture was prepared in 10 ml volume of nutrient broth (HiMedia Ltd., Lahore, Pakistan).Subsequently, the culture was vortexed, and incubated within a shaker incubator for a 24 hour at 37˚C.The culture was centrifuged (Micro Prime Centrifuge, Pocklington, UK) at 8000 rpm for 15min at 4C to get the hard pellet.The obtained pellet underwent several washings, employing sterile phosphate buffer saline (PBS).Following the thorough washing process, the pellet was re-suspended in PBS (pH 7.4).To ascertain the optical density of bacterial suspension, a UV spectrophotometer was utilized to obtained a corresponding concentration of 5×10 5 CFU/ml.The control group was split into two distinct subgroups: positive control (+ve P0) and negative control (-ve P0).Fish in -ve P0 was given bath with PBS only, whereas the other groups (+ve P0, P1, P2, and P3) (n = 15 for each group) were exposed to S. aureus (5×10 5 CFU/ml).Fish were bathed for 2 hours and the bath was repeated after seven days.Throughout the challenge period, all fish in different dietary groups were fed their appropriate diets, except for -ve P0 and +ve P0 fish, were specifically fed a diet with zero probiotics.Fig 1 illustrate step wise process of whole methodology.

Statistical analysis
The results were presented as mean ± standard error (S.E).Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) with a significance level set at P<0.05 to determine significant differences among groups.Based on the normality (Kolmogorov-Smirnov test) and homogeneity of variances (Levene test), any discrepancies between means were further examined using Duncan Multiple Range Test (DMRT).The parameters which showed significant variance after DMRT test have been mentioned with superscripts for all groups.All the Analyses were conducted using SPSS version 20.

Chemical composition and amino acid profile of muscles
Chemical composition (moisture content, crude protein, crude fat and crude ash) showed a substantial difference (P<0.05)among all dietary groups at the end of the growth experiment (Table 3).The level of crude protein between treatment groups directly correlated with a gradual increase in the concentration of probiotics.The highest concentration of crude protein (23.74±0.24%)was observed in the P3 group.The results showed a significant difference (P<0.05) between essential amino acids (EAA) and non-essential amino acids (NEAA) among all treatment groups (Table 4).P0 treatment group had substantially (P<0.05)lower concentrations of EAA and NEAA concentrations as compared to other treatments (P0<P1<P2<P3).

Digestive enzymes assay
Dietary supplementation of probiotics substantial (P<0.05)increased the levels of amylase lipase and protease in the intestine.The lowest levels of lipase were observed in fish fed with P0 diet.The highest level of digestive enzymes was observed in P3 dietary group at the end of the growth experiment (Table 5).

Hematology, blood biochemistry and antioxidant enzymes assay
All hematological and biochemical parameters showed substantial difference (P<0.05) between the four treatment groups both at the end of the growth experiment and after the bacterial challenge.Hematological parameters also showed a similar pattern between dietary groups, except that glucose gradually decreased with an increase in the probiotic (Table 6).These parameters were found to be lower in +ve P0 group as compared with those noted in -ve P0 group after bacterial challenge.The values of all blood biochemistry parameters increased with a gradual increase in the concentration of probiotic except triglycerides, ALT and AST at end of the growth experiment (Table 7).Similar results were observed at the end of the bacterial challenge.CAT, SOD, and MDA were substantially different (P<0.05)among all dietary groups (Table 8).The levels of CAT and SOD increased in response to bacterial challenge in B. subtilis fed groups.The highest level of CAT (2.55±0.01μmol/L)and SOD (0.54±0.03μmol/L) were observed in P3 group.On the other hand, the concentration of MDA gradually decreased with an increase in the probiotic (+veP0 >P1>P2>P3).

Histological study
The gut structure of different treatment groups showed several pathologies (Fig 2A -2E).Histopathological analysis of the -ve P0 showed a normal or less alterations of goblet cells, villi, and nuclei (Fig 2A).Meanwhile, the other treatment groups revealed structural anomalies such as excessive hypertrophy, the villi tended to fuse (FV), and the mucosal lining sloughed off, eventually leading to the large lumen (LL) (Fig 2B -2E).
Several histopathological alterations were observed in the structure of gills in all treatment groups (Fig 3A -3E).The histology of gills in the -ve P0 group exhibited the typical epithelial cell lining of lamellae (Fig 3A).In contrast, the groups exposed to S. aureus showed various structural changes, such as hemorrhage, intracellular oedema, disruption of gills with notable hypertrophy, loss of horizontal shaft with mucous membrane cellular proliferation (Fig 3B -3E).Liver in different treatment groups showed significant abnormalities (Fig 3F -3J).The group fed with zero probiotic (-ve P0) revealed normal hepatocytes, endothelium and serous membrane that contained blood vessels (Fig 3F).On the other hand, treatment groups showed pathologies such as necrosis, multinucleated nucleolus, oedema, hemosiderin, hematoma, intravenous tissue necrosis, edematous fluid intrusions (Fig 3G -3J).
Several anomalies were observed in the muscle's structures of different treatment groups after bacterial challenge (Fig 4A -4E).Muscle structures of the -ve P0 group showed less or no abnormalities (Fig 4A ) as compared to other treatment groups.Whereas, different treatment groups showed notable structural changes including, muscle fibers degeneration, vacuole

Discussion
The present study demonstrated a significant increase in various growth parameters, such as total body weight (%), SGR (%), K (%), and HSI (%) after feeding fish with different doses of Bacillus subtilis.The condition factor is closely linked to the weight-length ratio [51], reflecting fish's physiological and biological state.The fluctuations in the condition factor depends upon the feeding conditions, disease prevalence, and physiological factors [52].Condition factor in all probiotic fed groups indicate that the inclusion of B. subtilis ensured favorable health conditions and isometric growth throughout the growth period.Similar positive outcomes were observed in tilapia when administered with bacillus probiotic [21].
The findings of this study presented conclusive evidence that the substantial increase in weight gain resulting from probiotic supplementations can be attributed to an increased digestive enzyme functioning in striped catfish.The gastrointestinal enzymes were significantly increased in the treatment groups.These results are consistent with previous studies on freshwater species such as Nile tilapia [53], grass carp, and African catfish [54], B. subtilis possesses the capacity to improve the breakdown of nutrients in the gut, resulting in increased energy availability for fish growth.Previous studies have demonstrated that probiotics can generate a diverse array of exo-enzymes and enhance the functioning of the digestive enzymes within the gut [55].Furthermore, the inclusion of dietary probiotics can have an impact on the composition of the intestinal microbiota in fish.As a result, their administration can lead to the proliferation of advantageous microorganisms in the gut, ultimately enhancing the functioning of digestive [56].Table 6.Hematology from different dietary groups at the end of the growth experiment and after bacterial challenge.Different superscripts across the rows represent the significant variance between treatments were applied as a result of one-way ANOVA (Duncan multirange test) at P < 0.05.Other than elevation in digestive enzymes, the present study revealed that striped catfish had enhanced crude protein content (23.74±0.24%),which consequently led to an augmentation in muscle protein.The elevated protein content implies that incorporating probiotics in the feed resulted in a more effective conversion of nutrients into structural proteins, ultimately leading to better muscle production [57].The result coincides with findings in Nile tilapia and Table 7. Blood biochemistry of different dietary groups at the end of the growth experiment and after bacterial challenge.Different superscripts across the rows represent the significant variance between treatments were applied as a result of one way (Duncan multirange test) at P < 0.05.rainbow trout [58,59].An increase in body protein body protein levels demonstrated a significant rise in both dispensable and indispensable amino acids, particularly in P3 group.This study identified valine as an abundant amino acid, which had crucial role in cellular regeneration, muscle growth, and development.Furthermore, it serves as precursor in the production of antimicrobial agents.Dispensable amino acids showed significant increase in different treatment groups.These amino acids are essential for efficient utilization of essential amino acids and synthesis of various biological nitrogen containing molecules, including pyrimidines and purines, as well as antioxidant enzymes like glutathione [60].

Parameters
The present study demonstrated that the utilization of B. subtilis resulted in a significant enhancement of hematological parameters specifically in the counts of RBC and WBC.These cells play a crucial role in the circulation of oxygen within the respiratory system and blood flow regulation [61], as well in innate and adaptive immunity [62].Previous studies have demonstrated that probiotics containing a mixture of bacillus strains can improve the haematological profiles of O. niloticus [21,63] and rainbow trout [64].The +ve P0 group exhibited the highest glucose level compared to other treatments after bacterial challenge, indicating the increased tissue requirements to fuel the metabolic needs of osmoregulation and serves as the vital energy source for maintaining homeostasis [65] as well as assist fish in adapting to constant changes in metabolic demands [66].
Meanwhile, the current study demonstrated that the treatment groups supplemented with probiotics exhibited a significant improvement in the antioxidant response, as indicated by biomarkers (SOD, CAT and MDA).These results suggest that B. subtilis can stimulate the secretion of antioxidant enzymes in striped catfish, thereby enhancing the immune response, as observed in several other species [33,67].The SOD and CAT activities were observed to be lowest in the +ve P0 and -ve P0 groups, indicating a weakening of antioxidant defense, which could potentially lead to tissue damage caused by excessive free radicals.The persistence of free radicals can have detrimental effects on the normal functioning of cells.The excessive buildup of reactive oxygen species (ROS) can disrupt cellular metabolism and potentially lead to cell death [68].Reactive oxygen species, which include superoxide radical, hydroxide anion and peroxide (H 2 O 2 ), are generated during cellular phagocytosis and catabolism processes.To counteract the harmful effects of ROS, key biochemical factors i.e., superoxide dismutase, glutathione and catalase act as the body's first line of defense.These parameters modulate the presence of oxidative radicals and protect the body against oxidative pressure [69].Present results showed that S. aureus infection led to a significant augmentation in MDA levels in the +ve P0 group, which indicates damage in DNA, protein and cytoplasm.The redox imbalance resulting from lipid peroxidation by a microbe or an additive directly relates to MDA level [32].Whereas MDA level declined in groups fed diets containing B. subtilis and subsequently exposed to bacterial challenge.This decline could signify the presence of enzymatic regulators and non-enzymatic free radical quenchers that counteract the detrimental effects of ROS and reduce the rate of fatty acid peroxidation [18].The histological alterations during the bacterial challenge test correlated with haemato-biochemical and antioxidant enzyme data.This study elucidates notable variations in the various tissues, including muscles, gills, kidneys, liver, and gut.The greatest tissue damage was observed in the +ve P0 group.Histopathology, which is the study of tissue damage, is used to examine the effects of various chemicals or infections of biological origin [70,71].The gills, due to their perpetual exposure to the external environment, are particularly susceptible to waterborne pathogens [72,73].In +ve P0 group, the gills displayed a significant prevalence of histological abnormalities when compared to treatment groups.This result in erythrocytes congestion within the marginal channel [74].In contrast, the liver histology of probiotic treated groups showed characteristics reminiscent of those found in negative control group (-ve P0).The liver's impaired ability to efficiently remove foreign particles results in the degeneration of hepatocytes and congestion within sinusoid's [75].The presence of extracellular toxin generated by S. aureus might be the underlying factor responsible for the formation of lipid vacuoles and the occurrence of necrosis in the liver [76,77].Comparable hepatic irregularities, including the infiltration of lymphocytes, focal necrosis and the presence of cytoplasmic fat vacuoles, have been similarly observed in various species, such as carp [78].In fish exposed with S. aureus, the kidney tissues displayed severe necrosis and observable changes in the glomeruli.Notably, the glomerular epithelium in the kidney of catfish afflicted by S. aureus exhibited noticeable histological alterations [79].A pronounced elevation in the height of intestinal villi and reduction in adverse effects of S. aureus within the probiotic groups might be due to action of B. subtilis inhabiting the intestine, cause consequent reduction in pH and inhibit fermenting indigestible carbohydrates.Comparable investigation conducted [80] by supplementation of lactobacillus probiotic.Histopathological results support and confirm our examined hematological parameters and consistent with previous findings of pathological examination of S. aureus.

Conclusions
In conclusion, the present investigation exhibited that supplementation of B. subtilis could serves as optimal probiotic concerning growth performance, protein content, antioxidant response and immunocompetency against S. aureus in striped catfish.The optimum dosage of B. subtilis, at a concentration of 1×10 10 CFU/g, resulted in the most favorable outcomes in striped catfish.Moreover, the prospective utilization of B. subtilis presents a favorable opportunity to replace antibiotics in the context of aquaculture production.Further, the results of this study could suggest that this single bacterial strain probiotics have the potential for intensive farming to improve growth and immune responses in catfish farms, and effective probiotic in large scale production of aquafeed for striped catfish.

Table 2 . Summary of growth parameters in four dietary groups at the end of the growth experiment.
Different superscripts across the rows represent the variance between treatments were applied as a result of one-way ANOVA (Duncan multirange test) at P < 0.05.

Table 3 . Chemical composition of muscles in different dietary groups at the end of the growth experiment.
Different superscripts across the rows represent the significant variance between treatments were applied as a result of one-way ANOVA (Duncan multirange test) at P < 0.05.

Table 4 . Determination of essential amino acids (EAA) and non-essential amino acids (NEAA) from muscles of different dietary groups at the end of the growth experiment.
Different superscripts across the rows represent the significant variance between treatments were applied as a result of one-way ANOVA (Duncan multirange test) at P < 0.05.

Table 5 . Determination of digestive enzymes of intestine samples from different dietary groups at the end of the growth experiment.
Different superscripts across the rows represent the significant variance between treatments were applied as a result of one-way ANOVA (Duncan multirange test) at P < 0.05.

Table 8 . Determination of catalase (CAT), malondialdehyde (MDA) and superoxide dismutase (SOD) from serum of different dietary groups at the end of the growth experiment and after bacterial challenge.
Different superscripts across the rows represent the variance between treatments were applied as a result of one way (Duncan multirange test) at P < 0.05.