Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer)

Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer) Seyyad Mojtaba Emam, Babak Mohammadian, Takavar Mohammadian, Mohammad Reza Tabande This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3935430/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The study isolated two strains of intestinal autochthonous bacteria lactobacillus plantarum 1 (MH155966.1) (L1) and lactobacillus plantarum 2 (MH105076.1) (L2) from the Choobdeh Abadan region. To reveal the effects of these strains of bacteria on the growth performance, digestive enzyme activity, and histopathologic and histomorphometric characterization of the intestine, gut microflora, expression of immune and growth-related genes, and resistance against the disease of Lates calcarifer , examining 9 fish from each treatment, which after euthanasia, was placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium. The experimental design was completely randomized, with 3 treatments: pelleted feed without any probiotic (Diet 1); pelleted feed with Lactobacillus plantarum isolated 1 (L1), Lactobacillus plantarum isolated 2 (L2). For each treatment, 60 juveniles (75 ± 12 gr) were distributed in fiberglass tanks (1m 3 ) and fed for 45 days. Differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed feed with L1 had higher (P < 0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, Digestive enzyme activities were higher (P < 0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the Digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of EGF, TGFβ, GMCFC, and IL-10 genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 and L2 treatments than in the control. These results indicate that host-derived probiotics ( L. plantarum) have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of Lates calcarifer juveniles at 45 days of culture. Probiotic Lactobacillus plantarum Histopathologic Growth performance L. calcarifer V. alginolyticus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Prevention of diseases represents a primary objective in the field of aquaculture. The implementation of hygienic and preventive measures, such as fish health management, sanitation practices, and disease control procedures, plays a critical role in averting fish diseases (FAO. 2018). The expansion of aquaculture has led to intensified farming practices, resulting in the degradation of water quality, increased stress on fish, and heightened vulnerability to infectious diseases. Among bacterial pathogens, the genus Vibrio, particularly those belonging to the Vibrio clade, poses a potential global threat to mariculture (Reina et al., 2019). This poses a major concern for the sustainability of the aquaculture industry. As representatives of gram-negative bacteria, Vibrio belongs to the class Gammaproteobacteria and the family Vibrionaceae. Vibrio infections are primarily associated with stress conditions, notably fluctuations in water temperature and salinity, leading to significant losses in fish and shellfish cultured in marine and estuarine environments (Noga, 2010; Takemura et al.,2014). Several members of the Harveyi clade, including Vibrio harveyi , V. parahaemolyticus , V. alginolyticus , and V. campbellii , have been implicated in disease outbreaks among aquatic organisms, particularly in tropical regions (Darshanee Ruwandeepika et al.,2012; Mohamad et al.,2019a). Recently, V. alginolyticus and V. harveyi has been identified as a potential problem in the cultured gilt-head seabream ( Sparus aurata ) and European seabass ( Dicentrarchus labrax ) (Vendramin et al.,2016; Firmino et al.,2019; Zhang et al.,2020). The prevention of vibriosis in aquaculture primarily relies on the implementation of farm-level biosecurity measures and improved management practices (Ina-Salwany et al.,2019; Mohamad et al.,2019b). However, disease prevention strategies differ between open-ocean aquaculture and confined systems. Antibiotic therapy is commonly employed to treat vibriosis in affected fish, although the widespread use of antibiotics is no longer recommended due to concerns about antibiotic resistance, antibiotic residues in fish, environmental pollution, and ineffectiveness against bacterial biofilms (Defoirdt,2018; Assefa and Abunna,2018; Grenni et al.,2018) .Concerning Vibrio infections, there are currently no commercially available polyvalent vaccines that can protect against the diverse serotypes needed for broad cross-protection against different Vibrio species (Li et al.,2010; Powell et al.,2011; Galeotti et al.,2013; Peng et al.,2016). The limitations associated with the use of antibiotics in aquaculture have generated significant interest in probiotics as an alternative (Wang et al.,2008). Probiotics have gained popularity due to their environmentally friendly nature and their potential to replace antibiotics, improve animal health, and reduce diseases in aquatic animals (Gatesoupe,1999). Research has shown that the most effective probiotics for aquatic animals are bacteria isolated from the aquatic environment or the animals themselves. Therefore, various countries are working on isolating and producing native aquatic probiotics for commercial use. However, the introduction of non-native bacterial species into the aquatic environment by the aquaculture industry can have unintended harmful consequences (Balcazar et al.,2006). Autochthonous probiotics, such as Enterococcus faecium , Lactobacillus plantarum , Lactobacillus brevis , Bacillus subtilis , or Bacillus cereus , have been found to enhance weight gain, specific growth rate, feed conversion, and promote higher survival rates. They also exhibit beneficial immunological modulation, and hematological changes, and promote intestinal modulation in various fish species (Liu et al.,2013; Mohammadian etal.,2018). Lactobacillus plantarum is a commonly used lactic acid bacteria (LAB) species in aquaculture, renowned for its probiotic properties (Foysal et al.,2020). In recent years, numerous studies have demonstrated the potential use of L. plantarum as a probiotic in various aquatic species, including O. niloticus (Ruiz et al.,2020), Cyprinus carpio (Mohammadian et al.,2022), rainbow trout Oncorhynchus mykiss (Ahmadmoradi et al.,2023;Fregeneda et al.,2023), Pampus argenteus (Gao et al.,2016), shabot ( Tor grypus ) (Mohammadian et al.,2016), Lates calcarifer (Ghanei-Motlagh et al.,2021), as well as crustaceans such as Astacus leptodactylus (Didinen et al.,2016), Macrobrachium rosenbergii (Dash et al.,2015), Litopenaeus vannamei (Kongnum and Hongpattarakere,2012). One of the beneficial effects of probiotics in living organisms, as suggested by researchers, is the improvement of host nutrition through the production of digestive enzymes and growth supplements. This, in turn, increases survival, and food efficiency, prevents intestinal disorders, and enhances nutrient digestion. Additionally, probiotics help balance the microflora in the gastrointestinal tract, leading to better growth performance. Moreover, probiotics stimulate the proliferation of gastrointestinal epithelial cells (Ichikawa et al.,1999). Upon entering the intestine, probiotics start to multiply and utilize sugars to grow, producing short-chain unsaturated fatty acids, which may play a role in increasing the length of intestinal enterocytes (Pelicano et al.,2005). The evaluation of intestinal morphology and history is crucial for assessing the function of probiotics in fish. Ringo et al., (2007) emphasized the significance of intestinal morphology in selecting lactic acid bacteria as probiotics in fish. Several studies have also reported the beneficial effects of Lactobacillus species isolated from sources other than fish (Merrifield et al.,2010b). However, it is important to consider the potential impact of endogenous bacteria on intestinal morphology when using probiotics. Some studies have reported that certain bacteria isolated from non-fish sources had severe destructive effects on fish intestines (Salam et al.,2011). Thus, evaluating the effect of endogenous bacteria on intestinal morphology is a key factor in the use of probiotics. The selection of Asian sea bass ( L. calcarifer , Bloch) as the primary research focus is motivated by its high relevance to aquaculture practices in Iran. The species has been introduced in the southern provinces of Iran as a potentially suitable candidate for cage or earthen pond farming (Ghanei-Motlagh et al.,2021). By understanding and optimizing the supplementation of endogenous bacteria as probiotics, the efficiency and sustainability of sea bass farming can be significantly enhanced (Lim et al.,2019). However, Asian sea bass is highly susceptible to infections caused by Vibrio spp., particularly V. harveyi and V. alginolyticus (Dong et al.,2017;Mohamad et al.,2019c). Epidermal growth factor (EGF) is one of the growth factors with a protein structure of 53 amino acids. These proteins are involved in the growth and metabolism of many cells and are a type of cytokine. Transforming growth factor beta (TGF-β) is a multifunctional cytokine that belongs to the transforming growth factor superfamily and includes 4 isoforms and several other cell signaling proteins secreted by white blood cells. After activation, transforming growth factor-β combines with other factors and forms a serine/threonine kinase complex that binds to the transforming growth factor-beta receptor. One of the main functions of this protein is to regulate inflammatory processes, especially in the intestine. Interleukin 10 is one of the important interleukins of the body that is secreted from white blood cells and plays a role in inhibiting inflammatory and immune responses. Interleukin 10 is secreted from monocyte cells, as well as from helper T lymphocytes, macrophages, regulatory T lymphocytes. IL-10 is a cytokine with multiple functions in the regulation of immunity and inflammation. Interleukin 10 also increases the proliferation and survival of B lymphocytes and antibody production. IL-10 can block NF-κB activity. granulocyte-macrophage colony-forming cells (GMCFC) is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts that functions as a cytokine and specifically promotes neutrophil proliferation and maturation. Research suggests that probiotic bacteria can influence gene expression, including genes related to the immune system and growth factors. Some studies in aquatic animals, including fish, have explored the impact of probiotics on genes such as interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor, and Transforming Growth Factor Beta (TGF-β) (Mozanzadeh et al.,2023;Siddik et al.,2022;Huo et al.,2019;Jang et al.,2020).Probiotics may modulate the immune response and contribute to the regulation of growth factors, potentially promoting a more balanced and beneficial gene expression profile. However, the effectiveness can vary depending on factors like the specific probiotic strains used, the host species, and environmental conditions. The findings from this study will contribute significant knowledge on the potential benefits and risks associated with the incorporation of endogenous bacteria as probiotics in sea bass culture. Such insights are pivotal for the development of targeted strategies aimed at optimizing the growth, health, and overall performance of sea bass in aquaculture systems in Iran. The research aims to explore the effects of endogenous bacteria by evaluating various parameters throughout the culture period. One crucial indicator is intestinal pathology, which allows for the assessment of structural changes or damage in the sea bass intestines. Monitoring growth rates provides essential insights into the overall development and performance of the fish. Additionally, evaluating the activity of specific digestive enzymes offers valuable information on nutrient digestion and absorption efficiency in the digestive tract. 2. Materials and methods 2.1. Isolation and preliminary screening of bacteria in the intestine of Aquatic Animal Lithopeneus vanami specimens were obtained from Aquaculture Khuzestan, Choebdeh (Abadan, Iran) for sampling purposes. The intestinal contents were collected under aseptic conditions and subsequently diluted in a gradient manner using sterile PBS (phosphate-buffered saline). Serial dilutions of 10 − 3 , 10 − 4 , and 10 − 5 were prepared. The dilutions were evenly spread on de Man, Rogosa, and Sharpe (MRS) agar plates (BD, Sparks, MD, USA) (Ullah, 2020). The plates were then placed in a constant temperature incubator set at 37°C for 24 hours. After the colonies grew uniformly on the agar plates, individual colonies were carefully selected and purified until no other colonies were present on the plate. A purified single colony was inoculated into a 15-ml MRSB liquid medium and cultured at 37°C for 16 hours. The resulting bacterial solution was mixed with glycerin in a 1:1 volume ratio and stored at -80°C in a cryopreserved tube (Ullah, 2020). 2.2. Probiotic characteristic of isolates Two selected strains were subjected to the thermal growth experiment. To perform that, three dilutions from each colony (similar to McFarland No. 0.5, OD = 0.132 at 600nm) were inoculated in MRSB for 72h at different temperatures, including 10, 20, 30 and 20 \(℃\) . The bacterial growth rate was then recorded at 48 through turbidity measured by optical density (spectrophotometer, Jenway, 6400, UK) at 600 nm. Simultaneously, the number of colonies in each corresponding plate was also measured [ 47 ]. Two probionts were examined in vitro to check their antagonist effects against common pathogenic bacteria of trout, including A. hydrophila (AH04), L. garvieae , Y. ruckeri , and S. iniae (previously isolated). To do this, small amounts of fresh (18h-old) cultural media of each probiotic strain (colonies were between 20–30) were poured onto either an MRS or TSA plate, and incubated at 37 \(℃\) for 48h. Similarly, of the 18h-old cultural medium of each above-mentioned pathogen (on TSB), a peripheral bacterial culture was streaked crossly over the test bacteria inoculum in three triplicates. The plates were then incubated at 25 ℃ for 24h and finally, the inhibition zone of growth was measured (if any) and accounted as antagonist activity of each probation (Mohammadian et al.,2019). To investigate surface hydrophobicity, bacterial suspensions obtained, as previously described were adjusted to an OD600 of 1.0. Then, 5 mL of bacterial suspensions were mixed with 1 mL of toluene by vigorous vortexing for 1 min, allowing the separation of the aqueous and organic phases for 1 h at room temperature. Following phase separation, the final OD of the aqueous phase was determined. The cell surface hydrophobicity was calculated using the following formula: Surface hydrophobicity (%) = ([A0 − A1]/A0) × 100, where A1 and A0 are the ODs at the aqueous phase and the original suspension respectively. The assay was performed in triplicate (Ghanei-Motlagh et al.,2019) 2.3. Bacterial identification The identities of the isolated strains were confirmed through gram staining, followed by complementary biochemical identification tests and 16S rRNA gene sequencing. To determine the biochemical characteristics of the isolated strains, the biochemical trace reaction tubes of bacteria were utilized by the Manual for Systematic Identification of Common Bacteria. DNA was extracted from the bacteria using a commercial kit (SinaClon, Iran). PCR was performed on a PC 707 thermal cycler (Thermocycler, Mastercycler Gradient, Eppendorf, Germany) using universal bacterial primers, 27F (5ʹAGAGTTTGATCCTGGCTCAG-3ʹ) and 68R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ), which were synthesized by Comate Bioscience Co., Ltd (Jilin, China). The PCR process consisted of 2 minutes of initial denaturation at 92℃, followed by 35 cycles, consisting of 30 seconds of denaturation at 95℃, 45 seconds of annealing at 57℃, and 45 seconds of primer extension at 72℃. The process concluded with 5 minutes of final extension. Amplification products were analyzed via electrophoresis in 1.5% (w/v) Agarose gel containing Ethidium Bromide (1 mg ml-1). Upon obtaining aliquots of 15 µL of PCR product, sequencing was carried out using BioEdit, 7.2. The obtained sequence was then blasted using NCBI's online tool http://blast.ncbi.nlm.nih.gov/Blast.cgi . 2.4. Diet preparation A commercial feed containing 47% crude protein, 18% crude lipid, 2% crude fiber, 14% ash, 1.1% total phosphorus, 4200 kcal/kg feed digestible energy, and less than 12% moisture (Beyza Feed Mill, Shiraz, Iran) was used as the basal diet. Bacterial strains were grown aerobically on MRSB in a shaking incubator at 25 ℃, harvested by centrifugation (3000 rpm for 5 min), washed twice with normal saline, and re-suspended in the same solution. Bacterial suspensions were homogenized and adjusted to an OD600 of 2 using a Biophotometer (No 6131, Eppendorf, Germany). The Spread plate method was used to inoculate the adjusted serial dilutions of each suspension to commercial feed to achieve the desired concentration of probiotics. Experimental diets were prepared by gently spraying sterile normal saline pre-suspended with 1 x 10 9 CFU/g feed Isolated 1 (group 1) and 1 x 10 9 CFU/g feed Isolated 2 (group 2). The same amount of normal saline was added to the control diet, and control fish were fed with bacteria-free diet for the same duration. Food preparation was conducted under sterile conditions. The diets were air-dried at room temperature for 1 h, packaged, and stored in a refrigerator at 4°C until used. The viability of each probiont in the diet at the final concentrations mentioned earlier was confirmed using one gram of the food suspended in 9 mL sterile PBS, and the bacterial count was compared to the added probiotic bacteria. The supplemented diets were prepared twice per week (Mohammadian et al.,2019a) 2.5. Experimental design Healthy Asian Seabass L. calcarifer with normal appearance were purchased from a commercial fish farm located in Bushehr province, Iran. Before the commencement of the experiment, the specimens were kept in a recirculation system for a minimum of two weeks. A total of 25 fish, with an average weight of 70 ± 3.6 g and average length of 14.5 ± 0.25 cm, were randomly distributed into 300 L fiberglass tanks in triplicate for each treatment. The tanks were supplied with running seawater that had been sterilized using UV radiation. The fish were fed with assigned diets, based on 2.5% of their body weight, under a regular photoperiod cycle (16L:8D) three times a day for 45 days. One hour after feeding, any leftover feed was siphoned off, dried, and weighed to estimate the amount of feed consumed. Throughout the trial, the physicochemical parameters of the water were maintained within a normal range, with dissolved oxygen levels between 8–9 ppm, pH between 7.3–7.5, temperature between 27–30 ℃, un-ionized ammonia levels less than 0.05 ppm, and nitrate levels less than 0.1 ppm. All rearing conditions remained constant during the adaptation and experimental periods. After the acclimation period, 300 fish were divided into 3 groups, with three experimental units for each group (i.e., 9 tanks). To investigate the effects of different feeding treatments on the parameters studied below, probiotic-supplemented diets were conducted for 45 consecutive days. A group with no added probiotics was served as the control and administered the same diet ingredients. This study was approved by the institutional ethics committee of the Shahid Chamran University of Ahvaz, Iran, under approval number EE / 98.24.3.71483 / scu.ac.ir. All procedures on animals in this experiment were conducted according to the guide for the care and use of laboratory animals by the National Academy of Sciences (NIH publications No. 8023, revised 1978). 2.6. Sampling Before weighing or sampling, the fish were starved for 24 hours to minimize stress. Blood collection and gut sample removal were performed at the end of the trial (day 45; a total of 9 fish). The fish were anesthetized with 2-phenoxyethanol (0.3 ml/L), and blood was collected via the caudal vein using a 3 ml syringe (Mohammadian et al., 2019). An aliquot of the collected blood was immediately dispensed into a 0.5 ml Eppendorf tube previously coated with heparin sodium and was used for the analysis of hematological indices. The remaining portion was transferred to a 1.5 ml Eppendorf tube, left to clot at 4°C for 1 hour, and centrifuged at 3,000 g at 4°C for 10 minutes (Microliter Centrifuge, Mikro 220R, Hettich, Germany) to separate serum for the estimation of biochemical parameters (Mohammadian et al.,2019b). The sera were kept at -80°C for subsequent analysis. Afterward, the flank and ventral surfaces of the fish were disinfected using 70% ethanol. Once the fish were aseptically dissected, the specimens of the middle and posterior intestines were removed and placed in the respective volumes of sterile PBS for bacterial counts. Additionally, a portion of the pyloric ceca and foregut was sampled to assay the activities of digestive enzymes, as described in section 2.8 (Mohammadian et al.,2017). 2.7. Growth performance To determine the growth performance, all fish within each treatment were individually weighted at the beginning and 6th week of the trial. The weight of all fish in each tank was determined every two weeks and feed ratios were adjusted according to the fish weight. Growth parameters including absolute growth (Δw), relative growth rate (RGR), feed conversion ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), the feed efficiency ratio (FER), feed intake (FI) were calculated according to the following formula (Hopkins,1992): Δw = final body weight (FBW, g) - initial body weight (IBW, g) RGR = [Δw (g) / IBW (g)] × 100 FCR = net dry feed consumed per tank (g) / Δw (g) per tank SGR = [(ln FBW - ln IBW) / experimental period (d)] × 100 PER = Δw (g) per tank/protein intake (g) per tank FER = [Δw (g) per tank / net dry feed consumed per tank (g)] × 100 2.8. Digestive enzyme activities The activities of digestive enzymes including α-amylase, non-specific (total) protease, lipase, trypsin, chymotrypsin, and ALP were measured in triplicates per tank (using pooled samples from each replicate tank) after 45 days of feeding with strains. Intestinal samples in each tank were pooled, weighted and homogenized either in a hypotonic cold buffer (2 mM Tris–HCl buffer containing 50 mM mannitol, final pH = 7, 1:30 v/w) to measure intestinal ALP or an alkaline cold buffer (100 mM Tris-HCl, 0.1 mM EDTA and 0.1% Triton X-100, final pH = 7.8, 1:10 v/w) to quantify the other enzymes (Mohammadian et al., 2019c). The homogenates of intestinal ALP were treated with CaCl 2 (10 mM) and purified as described previously (Crane et al.,1979). The homogenates containing the other enzymes were centrifugated at 10,000 g for 12 min at 4°C and the obtained enzyme extracts were aliquoted and stored at -80°C for subsequent analyses. The concentration of total protein in the crude enzyme extracts was measured by the Bradford method using bovine serum albumin as standard (Bradford,1976). Total protease activity was measured using the casein hydrolysis method following the reaction between the liberated tyrosine and Folin-Ciocalteau reagent (Folin and Ciocalteau,1929;Anson,1938). the α-amylase activity was quantified using soluble starch as the substrate hydrolyzable to maltose reacting with a 3,5-Dinitrosalicylic acid solution (Bernfeld,1955). Standard solutions containing tyrosine and maltose were used to prepare the standard curves of total protease and α-amylase, respectively. Lipase activity was titrimetrically estimated using olive oil emulsion. The released fatty acids were titrated with sodium hydroxide solution using phenolphthalein as an indicator (Tietz and Fiereck,1966). The activities of chymotrypsin and trypsin were kinetically assayed using N-Benzoyl-L-tyrosine ethyl ester (BTEE) and N α -Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as substrates, respectively (Hummel,1959). Subsequently, the ratios of trypsin to chymotrypsin (T/C) and amylase to trypsin (A/T) were calculated. ALP activity was kinetically measured using 4-nitrophenyl phosphate (PNPP) as substrate by a commercial kit (Pars Azmoon Co., Tehran, Iran). 2.9. Oxidative Status Kurhaluk et al. method was modified for catalase (CAT) activity assay. The activity of serum SOD was determined and was estimated according to the method of Peixoto et al. (Peixoto et al.,2009). MDA in serum samples was assayed by the thiobarbituric acid reactive substances (TBARS) assay (Bradford,1976). The MDA concentration was estimated using the MDA molar extinction coefficient (156,000 M − 1 cm − 1 ) (Ghanei-Motlagh et al.,2021). 2.10. Intestinal histopathology and histomorphology To analyze the histopathology and histomorphological structure, the samples of the posterior intestine were collected. The samples were separately fixed in 10% buffered formalin (pH = 7.2) and processed using the standard protocol for histopathological examination. After mounting the samples with paraffin wax, three separate cross-sections with a thickness of ~ 5 µm were prepared using a microtome (Microtec CUT4050) and then stained with hematoxylin and eosin (H&E) for further histopathological investigations. The villi height, villi width, and the thickness of the epithelium, lamina propria, muscular layers, and the number of goblet cells were determined under a light microscope by using AxioVision microscope software, Carl Zeiss (Oberkochen, Germany) (Mohammadian et al.,2022). 2.10. Intestinal bacterial community The specimens of the posterior intestine were aseptically removed and homogenized with sterilized PBS (1:10 w/v). The homogenates were serially diluted and 100 µL of tenfold diluted suspensions were spread on different agar media including tryptone soy agar (TSA) supplemented with 2% NaCl, thiosulphate citrate bile sucrose (TCBS) and deman Regosa and sharp (MRS) in triplicates to determine the populations of total aerobic heterotrophic bacteria, Vibrio spp. and Bacillus spp. respectively. All plates were incubated for 48 h at 29°C and the counted colonies were expressed as denary logarithms of colony forming units (CFU) per ml homogenized suspension. 2.11. Gene expression 2.11.1. RNA isolation and cDNA synthesis Total RNA was extracted from 9 tissue samples of the intestine for each treatment on day 45, using the Tri Pure isolation reagent following the manufacturer's protocol (Roche, Canada). The concentration of the extracted RNA was determined at a wavelength of 260 nm using a nano-drop spectrophotometer (Eppendorf, Germany). The purity of RNA was assessed by determining the optical density (OD) absorption ratio at 260/280 nm, and only samples with a ratio above 1.8 were used for cDNA synthesis. Any potential DNA contamination was eliminated by treating RNA (1 µg) with DNase I (2 U µl − 1) for 1 hour at 37℃ (Vivantis, Malaysia). Reverse transcription was performed using the Rocket Script RT PreMix Kit, utilizing 1 µg of RNA and oligo dT as specified by the manufacturer's instructions (Bioneer Corporation, South Korea). 2.11.2. Real-time quantitative PCR To assess the intestinal expression levels of Epidermal growth factor (EGF), interleukin-10 (IL-10) and granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor and Transforming growth factor beta (TGF-β) mRNA, real-time PCR was conducted using the qPCRTM Green Master Kit for SYBR Green I® (Jena Biosciense, Germany) with the Light cycler® Detection System (Roche, USA). The relative expression levels of all transcripts were normalized against β-actin as the housekeeping gene. Specific primer sets (Bioneer, South Korea) were utilized based on O. mykiss (Table 1 ). Reactions were performed in triplicate in a 12.5 µl mixture, consisting of 6.25 µl qPCRTM Green Master Mix (2X), 0.25 µl of each primer (10 µM), 3 µl (100 ng) cDNA, and 2.75 µl nuclease-free water. The PCR protocol comprised of a denaturation stage at 94 ℃ for 5 min, followed by 45 cycles at 94 ℃ for 15 sec and 60 ℃ for 30 sec. Two separate reactions without cDNA or with RNA were employed as control groups parallel to the experimental groups. The relative quantification was conducted using Light cycler 96® software based on the comparative 2-ΔΔCt method. The validation of the assay was scrutinized to ensure that the chβ-actin and chCASQ2 primers had similar amplification efficiencies, as described before. All qPCR analyses were conducted following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline (Bustin et al.,2009). Table 1 Optical density of bacterial isolates with the best probiotic performance at different temperatures (Mean ± S.E) Isolation of bacteria OD at 10°C OD at 20°C OD at 30°C OD at 37°C L1 0.05 ± 0.001 aD 0.528 ± 0.01 aBC 0.631 ± 0.01 bB 0.827 ± 0.06 aA L2 0.051 ± 0.005 acD 0.427 ± 0.01 bC 0.739 ± 0.05 aB 0.861 ± 0.01 aA For each parameter, values (Mean ± SD, n = 9) bearing different lowercase letters or different uppercase letters represent significant differences within each column or each row, respectively ( p < 0.05). Table 2 Maximum inhibition zone (mm) for growth of each pathogenic bacteria on different probiotics cultural media Control-60d L1 L2 P value L. garvieae - 21.3 ± 2.29 b 32.6 ± 1.1 a < 0.001 A. hydrophila - 21.3 ± 1.1 a 23.3 ± 1.19 a < 0.001 Y. ruckeri - 31.3 ± 1.1 a 38.6 ± 4. 1 a < 0.001 V. harvie - 12.75 ± 2.21 a 13.3 ± 1.29 a < 0.001 Data are presented as mean ± SD (n = 5). The superscript alphabetic letters in each row indicate significant differences among different groups (ANOVA) 2.12. In vivo bacterial challenge and LD 50 measurement At the end of the 45-day experimental period, fish in each group were challenged with V. alginolyticus , which was isolated from a diseased barramundi fish reared in a cage and identified by 16s rDNA analysis before the challenge trial. Before utilizing V. alginolyticus for the challenge, we investigated whether the applied probiotics could protect the fish against pathogenic bacteria, thereby initially determining the lethal dose (LD) of V. alginolyticus . The 45 fish (weighing 43.2 ± 6.4 g) were injected with serial intraperitoneal doses of V. alginolyticus at 10 5 , 10 7 , and 10 9 CFU ml − 1, with 15 fish per dose. Mortality rates were recorded for four consecutive days at each dose level, and probit analysis (SPSS, 18, USA) was conducted to determine the exact LD50 of V. alginolyticus to Lates calcarifer . The V. alginolyticus was prepared for the challenging test as follows: the bacterium was grown in TSA for 48 h at 37 ℃, washed twice with PBS, and re-suspended in the same buffer. The bacterial concentration was adjusted to bacterial LD50 (1 ×10 8 CFU ml − 1) using a spectrophotometer, and the concentration of the bacterial suspension was determined using a bacterial counting chamber to verify the challenge dose. Anesthesia was induced with 2-phenoxyethanol (1:10,000) (Shanghai Reagent, China) in all 45 fish (15 from each treatment) before injection. All fish in each group were intraperitoneally injected with 0.2 ml of V. alginolyticus suspensions using a 1 ml sterile syringe. The control group was also injected with 0.2 ml of V. alginolyticus suspension. Mortalities were recorded daily for four days’ post-challenge, and all dead Lates calcarifer were examined bacteriologically to confirm the presence of the pathogen (Perez-Sanchez et al.,2011; Rahimnejad et al.,2018). 2.13. Statistical Analysis All data presented in the current study were expressed as mean ± standard deviation. Statistical analysis was performed using the SPSS program version 22. The normality of the data was checked by the Shapiro–Wilk test. One-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test was carried out to determine differences significant at the 5% probability level between the mean values of parameters. 3. Results and discussion 3.1. In vitro temperature–growth, antibacterial activity and hydrophobicity test of bacteria In Table 1 , the amount of optical density (OD) measured from the culture of bacteria with probiotic power at temperatures of 10, 20, 30, and 37°C for 48 hours is given. During the 48 hours, there was a significant difference between the growth of bacteria at different temperatures (p < 0.05). All bacterial isolates were able to grow from 10 to 37°C based on the measured optical density. At a temperature of 10°C, all the isolates could grow at a very low rate. The highest values of optical density at a temperature of 20°C correspond to L1, at a temperature of 30°C and 37°C respectively to L2 and L1. L1 and L2 had good growth ability at the investigated temperatures (p < 0.05). Based on the bacterial growth test at different temperatures, it was found that by increasing the temperature of the greenhouse for 48 hours, the growth rate of bacterial isolates increases based on optical density. During the 48 hours of the experiment, the amount of optical density resulting from the growth of bacteria at a temperature of 10°C was very low, but at temperatures of 20, 30, and 37°C, the optical density increased and the growth of bacteria increased significantly. It arrived. It can be said that the results of the present study show that the obtained bacterial isolates can grow at the tested temperatures and the best temperature range for the growth of bacteria is the temperature range for the culture of Asian sea bass. Riaz et al., (2010) investigated the growth rate of Lactobacillus acidophilus and Lactobacillus fermentum at temperatures of 15, 20, 25, and 35°C. The optimal growth temperature of Lactobacillus acidophilus was 35°C and 25°C was introduced for Lactobacillus fermentum . The optimal temperature for the growth of bacteria in the current study is 37°C, which indicates that these isolates are thermophilic. The antibacterial characteristic of each probiont, including L1 , and L2 was examined against pathogenic bacteria of trout (i.e., L. garvieae , A. hydrophila , Y. ruckeri , V. harvei ), by using an in vitro test in which the inhibition zone of the cross culture medium was measured. Table (2) shows the higher inhibition zone for L2 rather than L1 in most cases (P < 0.001). Antimicrobial tests are one of the common tests for choosing a suitable probiotic. The probiotic bacteria must have antimicrobial properties and the ability to produce antimicrobial compounds (Zokaeifar et al.,2012). This effect is applied in several ways, some of which are as follows: The secretion of extracellular metabolites with bactericidal power by probiotic cells is the competition in occupying the place to stick to the mucus. In the present study, noteworthy antimicrobial activity against pathogenic bacteria was observed in lactic acid bacteria. Within probiotic strains, lactic acid bacteria exhibit the production of various compounds, including organic acids, lactic acid, bacteriocin, and hydrogen peroxide. These compounds contribute to the activation of non-specific immunity in the host. The subjects under investigation in this research are likely to possess a heightened capacity for producing growth-inhibitory substances. Vieira et al., (2016) demonstrated that Lactobacillus plantarum strains, isolated from shrimp intestines, exert an inhibitory effect on a broad spectrum of both Gram-positive and Gram-negative pathogenic bacteria, including V. harveyi and A. hydrophila . Additionally, Zapata and Lara-Flores, (2013) reported that Leuconostoc mesenteroides , a lactic acid bacterium isolated from tilapia fish intestines, exhibited the ability to inhibit V. harveyi and Mycobacterium marinum , both recognized as pathogenic bacteria. The percentage of hydrophobicity of the strains ranged from 6.6 ± 0.45 to9.37 ± 0.95% (L1 and L2 respectively). 3.2. Growth performances In this study, the results of the tests indicate that the percentage of weight gain in fish fed with L1 probiotics was significantly higher than that of L2 probiotic treatments and the control group. Both probiotic treatments led to an improvement in the food conversion ratio and specific growth rate compared to the control group. Moreover, the protein efficiency ratio was also significantly improved in the probiotic treatments compared to the control group; however, this improvement was not seen to be significant between the two probiotic treatments. There was no significant effect of probiotic feeding on the condition factor. Additionally, no cases of mortality were observed during the test period in any of the groups. (Table 3 ). Bacterial pathogens are a significant contributor to disease damage in aquaculture, with vibriosis standing out as a major cause of extensive losses and damages in the marine aquaculture industry. This phenomenon is closely linked to the invasive capabilities of these bacteria, particularly under stressful conditions (Ina-Salwany et al.,2019). In this investigation, a notable observation was the 100% survival rate (SR) across all examined groups throughout the entire culture period, implying optimal maintenance conditions in each treatment. The study revealed that fish utilizing feed enriched with L 1 (to a greater extent) and L2 (to a lesser extent, compared to another probiotic group) over a 45-day culture period outperformed the control group, which had no added probiotics. Additionally, these treated groups exhibited superior growth indices, particularly in terms of final weight factors. Our results are those of Reda et al., (2018) ( Clarias gariepinus ), Yang et al., (2019) ( Carassius auratus) , Gisbert et al., (2013) ( Oncorhynchus mykiss) , Yang et al., (2015) ( Apostichopus japonicas) fed with host-associated probiotic compared to fish fed with commercial probiotics. Similar results have been reported in Asian seabass fed with Bacillus subtilis E20 and a mixture of Bacillus licheniformis and Bacillus subtilis (Lin et al., 2017; Adorian et al.,2019). In previous investigations, probiotic bacteria in aquaculture have been linked to enhanced growth by upregulating the insulin-like growth factor system. This is often associated with a concurrent decrease in myostatin gene expression and lower cortisol levels, contributing to improved overall health and growth in aquatic species (Carnevali et al.,2006; Hauville et al.,2016). It sounds like the researchers are proposing a hypothesis that suggests L1, in addition to its high probiotic potential, may enhance growth performance through the extracellular metabolites of protease, carbohydrase, and lipase. These metabolites could potentially break down complex dietary proteins, lipids, and carbohydrates into more usable forms, such as amino acids, peptides, fatty acids, and monosaccharides, ultimately improving digestibility and promoting growth. Table 3 Growth performance of Asian seabass fed either regular feed or feed supplemented with probiotics for 45 Day Parameters Treatments Control L1 L2 IBW (g) 75.55 ± 0.09 a 74.36 ± 1.2 a 74.91 ± 0.43 a FBW (g) 115.78 ± 2.05 b 123.67 ± 2 a 118.33 ± 1.5 ab FTL (cm) 19.06 ± 0.11 b 19.4 ± 0.2 a 18.9 ± 0.17 b RGR (% / 45 day) 53.19 ± 1.99 b 65.4 ± 2.5 a 57.95 ± 2.35 b FCR (g / g) 1.14 ± 0.12 a 1.002 ± 0.04 b 1.01 ± 0.04 b SGR (% / d) 1.41 ± 0.14 b 1.67 ± 0.05 a 1.52 ± 0.04 ab PER (g gain / g pro) 2.03 ± 0.16 b 2.32 ± 0.06 a 2.29 ± 0.09 a FER (%) 87.55 ± 6.2 b 99.77 ± 2.8 a 98.89 ± 4.07 a Survival (%) 100 a 100 a 100 a The data represent the Mean ± SD of three tanks per treatment (n = 3). Values with various lowercase letters in each row indicate significant differences ( p < 0.05). 3.3. Digestive enzyme activities Based on the results of the enzyme activity analysis presented in Fig. 1 . On the 45th day of sampling, a significant difference in the activity of α-amylase, alkaline phosphatase, trypsin, and chymotrypsin was observed between group 2 and the other experimental groups. The study conducted on sea bass over 45 days revealed that the administration of both types of endogenous probiotics led to a significant increase in various enzymes, including trypsin, chymotrypsin, amylase, lipase, alkaline phosphatase, and protease. The observed enhancement in enzyme levels could be attributed to two possible mechanisms. Firstly, probiotic bacteria may secrete a diverse range of enzymes (exoenzymes). Alternatively, the activity of the fish's digestive enzymes (endoenzymes) might increase. Furthermore, certain probiotic bacteria can produce foreign enzymes like cellulase, amylase, protease, lipase, and phytase. These foreign enzymes, in addition to the host's digestive enzymes, contribute to the digestion of organic matter and enhance the overall activity of the fish's digestive enzymes (Del’Duca et al.,2013). Our observations are in agreement with those of Ghanei-Motlagh et al., (2021), Adorian et al., (2019), and Reda et al., (2018), who reported increased activities of protease, lipase, and amylase in L. calcarifer and C. gariepinus nourished by diets containing Bacillus spp. for 60 days, respectively. The improvement of the activities of digestive protease, lipase, and α-amylase can be partly attributed to the potential of probiotic strains for the production of extracellular enzymes including protease, lipase, and amylase as confirmed in our previous study (Mohammadian et al.,2018). Marlida et al., (2014) found that probiotic bacteria from the humpback grouper's ( Chromileptes altivelis ) digestive tract positively influenced the growth factors and digestive enzymes in their study. This aligns with the idea that probiotics can enhance the digestive system, leading to better digestion of food particles and ultimately promoting growth in the fish. The findings from studies employing probiotics have demonstrated an augmentation in the breakdown of dietary proteins, fats, and starch (Wang and Xu,2006). Hence, it is plausible that the probiotic bacteria investigated in the current research have enhanced the utilization efficiency of proteins, fats, and carbohydrates within the sea bass diet. Comparable instances exist where gastrointestinal bacteria have the ability to produce extracellular digestive enzymes (Suzer et al.,2011; Son et al.,2009). These instances substantiate the outcomes of the present experiment, indicating an elevation in digestive enzymes specifically α-amylase, trypsin, and lipase in rainbow trout. Lara-Flores et al., (2003) explored the impact of administering a blend of Enterococcus faecium and Lactobacillus acidophilus alongside the yeast Saccharomyces cerevisiae on Nile tilapia, focusing on growth rate and changes in alkaline phosphatase enzyme. Their findings indicated superior growth factors in treatments involving either bacteria or yeast compared to the control treatment. Regarding the alkaline phosphatase enzyme, the yeast treatment exhibited the highest activity level. The authors suggested that the heightened enzyme activity likely signifies enhanced development of brush membranes in fish enterocytes. It is plausible that the probiotic bacteria in the current study exerted a substantial influence on the development of sea bass enterocyte brush-like appendages, contributing to increased fish growth through various mechanisms. In the current investigation, a significant disparity emerged between the groups administered probiotics and the control group to the study's conclusion. Given the optimal rearing conditions and minimal stress experienced by all fish groups during the cultivation period, noteworthy augmentation in the activity of digestive enzymes α-amylase, lipase, trypsin, and chymotrypsin was observed in the probiotic treatments during the final sampling. This effect is likely attributed to the enhanced retention of probiotic bacteria within the digestive system of the fed fish. L 2, and to a lesser extent L1 , seemingly heightened digestive enzyme activity and secretion within the sea bass digestive system, thereby enhancing the capacity for food digestion and absorption. Consequently, this led to an increased specific growth rate. Notably, despite its comparatively lower impact on digestive enzymes, the L1 probiotic exhibited superior growth indicators among the experimental treatments. The outcomes suggest that the L 2 treatment potentially established a dominant intestinal flora in sea bass, with a consequential elevation in lactic acid bacteria influencing growth indicators more profoundly than other bacteria. Another potential factor contributing to the superior performance of L1 may involve the influence of unidentified growth factors inherent in the growth process of sea bass within this specific probiotic treatment. 3.4. Innate immune parameters The data related to the serum innate immune response are summarized in Fig. 4 . The highest and lowest serum lysozyme, anti-trypsin, myeloperoxidase, Respiratory burst activity (NBT reduction), and bactericidal activity levels were in L2, L1, and Control, respectively ( P 0.05). Fish in the L1 group had higher serum total complement than the other treatments. The study demonstrated a significant enhancement in immune responses, including bactericidal, antiprotease, and respiratory burst activities in fish subjected to probiotic-supplemented diets compared to those on basal diets. Specifically, serum bactericidal activity, indicative of the serum's ability to resist bacterial infections, showed elevated levels in probiotic-fed fish over 45 days. Probiotics were found to enhance the host's immune competence by activating various protective mechanisms, as elucidated by Lazado et al., (2010). Furthermore, serum respiratory burst, a phagocyte-mediated killing mechanism, exhibited a significant increase in probiotic-fed fish. Antiprotease activity, impeding pathogen infiltration with protein-degrading enzymes, was significantly elevated in all probiotic-treated groups. Consistent with these findings, prior research by Newaj-Fyzul et al., (2007) indicated that incorporating Bacillus subtilis AB1 in rainbow trout's diet substantially heightened antiprotease activity. Similarly, Kim et al., (2012) reported increased serum antiprotease activity in olive flounder following dietary supplementation with E. fascium . Myeloperoxidase (MPO) plays a pivotal role in pathogen eradication, and in this investigation, there was a notable augmentation in MPO activity across L1 and L2 groups at this study. Mohammadian et al., (2018) demonstrated that endogenous probiotic supplementation resulted in a significant increase in lysozyme and MPO activities. Lysozyme, possessing antibacterial properties, exhibited heightened activity in fish receiving L1 and L2 in their diet. Lactic acid bacteria have been shown to stimulate immunoglobulin synthesis (Mohammadian et al.,2022; Gopalakannan and Arul,2011 Mohammadian et al.,2021). Similar enhancements in immune responses have been documented in various fish species, including Nile tilapia, pirarucu, catla, red seabream, striped catfish, snakehead, Mrigal, and Japanese eel (Makled et al.,2019; do Vale Pereira et al.,2019; Bhatnagar and Saluja,2019; Dawood et al.,2019; Thy et al.,2017; Talpur et al.,2014; Bhatnagar and Lamba,2017; Lee et al.,2013) 3.5. Intestinal histopathology and histomorphology In the present experiment, the height of the villus, average width of the villus, and epithelium thickness were evaluated across different treatment groups. Statistical analyses were performed to examine the potential differences in these parameters between the experimental groups at the end of the testing period. Concerning the height of the villus, width of the villus, number of goblet cells, thickness of the submucosal layer, and epithelium thickness by the end of the experiment, the L2 group exhibited a statistically higher height compared to other treatment groups. In the current investigation, pathological examinations revealed an absence of damage in the prepared sections. (Fig. 4 ) Groups receiving probiotics with food exhibited reduced damage in microvilli presence compared to the control group, along with decreased dead tissue in the lumen. Noteworthy is the absence of histological changes in intestinal epithelial tissue post probiotic exposure, with no indications of basement membrane connection rupture in intestinal cells. By the findings, Kristiansen et al., (2011) observed that the administration of food containing Carnobacterium divergens did not inflict damage on intestinal structure. Martinsen et al., (2011) demonstrate that C. maltaromaticum did not change and had adverse effects on the intestinal structure of cod. Probiotics can bind to the intestine and pass through it without damaging the structure of the intestinal wall, which indicates that their entry method is not destructive. Kristiansen et al., (2011) electron microscope observations indicate no visible difference between the groups in terms of the presence of cell debris in the lumen, increased amount of mucus, the number of bacteria-like particles in the lumen and between microvilli, disruption of microvilli order. and did not show a loss of integrity of strong joints. Normal and intact cell connections, along with a healthy zona adherence area, were observed in all groups, emphasizing the non-destructive impact of probiotics on intercellular junctions. This is crucial, as compromised junctions could serve as an entry point for potential pathogens. The study underscores the significance of intestinal morphology and histometry in assessing probiotic performance in fish, aligning with Ringo et al., (2007) emphasis on intestinal morphology in selecting lactic acid bacteria as fish probiotics. While Lactobacillus species from non-fish sources are generally considered beneficial for fish ( Bagheri et al.,2008) Salma et al., (2011) cautioned against the use of bacteria isolated from Iranian cheese due to severe destructive effects on Huso huso 's intestine. Evaluating endogenous bacteria's impact on intestinal morphology is deemed crucial in probiotic selection. Macroscopic and microscopic images indicate that endogenous probiotics enhance epithelium development in the intestine, confirmed by changes in epithelium surface and the growth of intestinal villi under the probiotic influence. Assessing intestinal tissue, particularly microvilli diameter and size, serves as a valuable indicator of the intestine's physiological state and digestive system health. Increased villi diameter and height signify enhanced absorption levels, indirectly improving food ration utilization efficiency. This information is pivotal for understanding fish's nutritional needs. The measured lengths of intestinal villi (Fig. 3 , 4 ) align with macroscopic observations of the intestine in this study. Histological examinations reveal variations in villi length, with the L2 group exhibiting the most significant increase in villi length after the sampling period. The use of L1 also positively influences villi growth, potentially contributing to the microscopic structural integrity of the intestine and fostering expansion of intestinal epithelial tissue. Over the probiotic supplementation period, villi length increased across all groups, with a more pronounced increase compared to the control group in the treatments receiving a ration containing probiotics. Similar results were noted for other factors such as villus width, thickness of the submucosal layer, epithelium thickness, muscle layer thickness, submucosa thickness, and the number of goblet cells. Consistent with findings from Nakandakare et al., (2013) in Nile tilapia fish, as well as Burrells et al., (2001) in salmon, treatments incorporating probiotics demonstrated a significant difference in villi height in the middle intestine compared to the control group. The augmentation in villi width and the amplification of intestinal goblet cell numbers are conspicuously evident in both probiotic treatments upon course completion. Additionally, parameters such as the thickness of the submucosal layer, epithelium thickness, and muscle layer thickness manifest an increment in both treatments by day 45. Numerous anatomical factors influence absorption levels within the digestive system. Notably, elongated, narrower, and more regular villi, coupled with an increased villus count per area, signify heightened activation of intestinal villi (Heidarieh et al.,2012). In this investigation, the observation of elongated villi in the group administered with a combination of L2 (to a greater extent) and L1 (to a lesser extent) appears promising. However, further scrutiny is warranted, particularly concerning intestinal villi width and epithelium layer thickness, necessitating additional studies to elucidate effects on fish subjected to probiotics. Comparing the effects of the shape of the intestine with the activity of digestive enzymes shows that the most changes in the administration of bacteria occur at the end of the period. The two treatment groups of L1 and L2 had better results compared to the control group, and it should be noted that each of the probiotic bacteria may have different morphological effects in each of the fish species (Ferguson et al.,2010). The increase in the height of villi in treatments containing probiotics is probably due to the improvement of growth conditions and proliferation of lactic acid bacteria under the positive effects of probiotics. In the present study, an increase in the thickness of different tissue layers was observed in the groups treated with probiotics, especially L2 treatment at the end of the period. The anterior and middle intestines, which are the main places of digestion and absorption of food in the intestine, probably indicate the adaptive response of fish to increase the ability to absorb nutrients inside the intestine, and this increase in the diameter of the covering muscle layer is caused by the production of chain fatty acids. Zahran et al., (2014) showed the effect of probiotic use on increasing the length of intestinal villi in tilapia fish. On the other hand, there was no significant effect on the thickness of the muscle layer. Contrary to the present results, Enferadi et al. (2018) investigated the effect of Lactobacillus plantarum on the intestinal morphology of salmon and did not observe a significant change in the height of the villus, the thickness of the epithelium layer, the width of the villus and the thickness of the muscle layer. In this study, after feeding with food containing a probiotic supplement, the number of goblet cells increased at the end of the period in the L2 group. Although there are studies on the increase of goblet cells in the skin and digestive tract after being challenged with infectious diseases (Buchmann and Bresciani,1998), there are few studies on increasing the density of mucous cells in fish. Ramos et al., (2015) investigated the effect of commercial probiotics including Bacillus, Pedicoccus, Enterococcus, and Lactobacillus in different amounts on the intestine of rainbow trout. They found an increase in the area of the anterior intestine and the number of goblet cells in the group containing 3 grams of probiotics per kilogram of food ration and also an increase in hair length in the group containing 1.5 grams of probiotics per kilogram of food ration. Goblet cells in the digestive tract act as the first sensitive layer of fish immune defense factors by forming a viscous watery coating on the surface of the mucus and providing protection against the damage caused (Ringø et al.,2003). 3.6. Intestinal microflora It has been evidenced that probiotics, particularly those derived from the host, can modulate the gut microbiota. However, functional probiotics require to be colonized in the host intestinal tract before exerting their beneficial effects. In this study, no significant alteration in populations of Bacillus spp., total cultivable bacterial counts, and Vibrio spp. was observed between the treatments ( p > 0.05). Moreover, LAB was significantly higher in the probiotic groups than in the other groups ( p < 0.05) (Table 4 ). This could be associated with the high survival of these strains within the gastrointestinal tract from which they isolated and their appropriate abilities to adhere to mucus as described in our previous study (Ghanei-Motlagh et al.,2021). Table 4 The intestinal populations of total cultivable bacteria, Bacillus spp. and Vibrio spp. in Asian seabass fed or un-fed with probiotics at different time intervals Parameters Treatments Initial (day 0) Day 45 Total aerobic heterotrophic bacteria (Log CFU/ml) Control 25.7 ± 4.05 a,A 27.13 ± 3.5 a,A L1 27.6 ± 6.75 a,A 26 ± 3.86 a,A L2 25.1 ± 6.006 a,A 26.7 ± 4.57 a,A Lactic Acid Bacteria (Log CFU/ml) Control ND ND L1 ND 40.3 ± 14.7 a,A L2 ND 45.3 ± 20.31 a,A Bacillus spp. (Log CFU/ml) Control 1.5 ± 0.57 a,A 1.16 ± 0.81 a,A L1 1.4 ± 0.54 a,A 1.66 ± 0.43 a,A L2 1.16 ± 0.4 a,A 1.28 ± 0.46 a,A Vibrio spp. (Log CFU/ml) Control 15.03 ± 2.53 a,A 14.8 ± 2.58 a,A L1 13.4 ± 2.45 a,A 12.5 ± 2.83 a,A L2 14.4 ± 2.4 a,A 12.8 ± 2.57 a,A ND: No detected. For each parameter, values (Mean ± SD, n = 9) bearing different lowercase letters or different uppercase letters represent significant differences within each row or each column, respectively ( p < 0.05). 3.7. Expression gens On the 45th day of sampling, fish fed with the L2 probiotics had higher GMCFC and IL-10 in the gut than those fed the L1 probiotic and control group (Fig. 5 ). Fish in L1 and L2 groups had higher gut EGF gene expression values than the control group. The highest TGFβ gene expression value in the gut was in L2, meanwhile, the lowest values were in the control group ( P < 0.05). Results of the present study showed that all probiotics, applied here, can up-regulate the immune-related gene expression of the L. calcarifer intestine. probiotic bacteria can influence gene expression, including genes related to the immune system and growth factors. Some studies in aquatic animals, including fish, have explored the impact of probiotics on genes such as interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor, and Transforming Growth Factor Beta (TGF-β). In the present study, feeding with L1 and L2 probiotics induced higher transcription levels of EGF, TGFβ, GMCFC, and IL-10 genes in the gut, which may correlate with better immune and hematological parameters in these groups. The increase in IL-10 mRNA expression may have led to the enhanced expression of pro-inflammatory cytokines such as GMCFC in the L. calcarifer fed L1 probiotic to balance the immune responses for better immunocompetence. The mechanism of action of probiotics in the expression of interleukin 10 genes is related to their effect on internal signaling pathways in cells. These processes usually involve molecular interactions between probiotics and the surface of host cells. One possible mechanism is that probiotics activate signaling communications by combining with various cells of the gastrointestinal tract. This interaction can lead to the activation of specific signaling pathways, which ultimately leads to increased expression of TGF and interleukin 10 genes. Additionally, probiotics may help improve the gut microbiome. A better microbiome balance can lead to different production of chemical compounds and molecular signals that regulate gene expression. Although the exact mechanism is not yet fully understood, these effects may improve cellular activity and balance the immune system. In this context, Siddik et al., (2022); Choi et al., (2022); Panigrahi et al., (2011) and Abarike et al., (2020) reported administration of a diet with probiotic significantly increased IL-10 gene expression in the head kidney of olive flounder and Nile tilapia. In the present study, the growth performance of sea bass fed with L1 probiotic was proportional to TGFβ gene expression. Probiotics may modulate the immune response and contribute to the regulation of growth factors, potentially promoting a more balanced and beneficial gene expression profile. However, the effectiveness can vary depending on factors like the specific probiotic strains used, the host species, and environmental conditions. 3.8. Relative survival rate after challenge with V. alginolytichus Seven days after the injection challenge of fish with V. alginolyticus bacteria, the percentage of survival in unchallenged control groups (100%), challenged control (64.95%), L1 treatment (76.2%), treatment L 2 (80.95%) was obtained (Table 5 ). Statistically, the relative survival percentage of fish fed with both Lactobacillus plantarum bacteria was significantly higher than the control group (p < 0.05). Probiotics increase survival and natural resistance against unfavorable factors. In the current investigation, a notable enhancement in the survival rates of sea bass fish exposed to V. alginolyticus bacterial challenge was evident in probiotic interventions, notably with L2. This finding aligns with prior research conducted on European migratory eel and Indian carp, where increased survival rates were observed against A. hydrophila infection after dietary supplementation with Bacillus probiotics (Das et al.,2011). The heightened resistance observed in probiotic treatments is likely attributable to an augmentation in non-specific immune responses influenced by the administration of endogenous probiotics. The augmentation in non-specific immune responses in probiotic-treated groups is evident from the results presented in Table 5 . Notably, the introduction of L2 and L1 into the diet of sea bass fish beyond day 45, followed by intraperitoneal injection of V. alginolyticus bacteria, resulted in enhanced resistance in both probiotic treatments compared to the control group. However, the reduction in losses observed in the group receiving L2 was particularly noteworthy (p < 0.05). Clinical signs and bacteriological examination demonstrated the infection of V. alginolytichus in the mortalities. Son et al. [ 83 ] investigated the effect of oral administration of Lactobacillus plantarum on common grouper fish for four weeks and then challenged with Streptococcus species. Probiotic treatment showed 10 8 CFU/gr compared to the control group. Balcazar et al.,( 2006) investigated the ability of three species of lactic acid bacteria ( Lactobacillus lactis , Lactobacillus plantarum , and Lactobacillus fermentum ) in inhibiting the adhesion of several fish pathogens ( Aeromonas hydrophila , Aeromonas salmonicida , Yersinia ruckeri , and Vibrio anguillarum ), and the results showed that Lactobacillus plantarum reduces the adhesion of A. hydrophila and A. salmonicida and Yersinia ruckeri , but it had no effect on V. anguillarum . Increasing the level of non-specific immune defense, reducing the penetration power of pathogenic agents, reducing access to essential nutritional factors, and producing antibacterial substances can be considered among the reasons for increasing resistance to the challenge of pathogenic agents. Table 5 Relative Survival percentage of fish of different treatments after challenge with Vibrio alginolyticus Parameters Treatments Control L1 L2 Survival (%) 64.95 b 76.2 a 80.95 a The data represent the Mean ± SD of three tanks per treatment. Values with various lowercase letters in each row indicate significant differences ( p < 0.05). 4. Conclusion The study indicates that Lactobacillus plantarum (L1 and L2) positively influences Asian sea bass by enhancing growth rates, establishing a stable natural flora in the digestive system, increasing digestive enzymes, inducing tissue structure alterations, particularly in the digestive tube, and promoting the expression of growth-related genes. Moreover, these bacteria stimulate the immune system, elevate the expression of immune genes, and enhance bacterial resistance, collectively suggesting potential benefits for fish health. In conclusion, incorporating L1 and L2 into the diet of Asian sea bass could improve growth and immune system functions, potentially enhancing overall fish production quality. The study recommends considering the combined effect of these two bacteria in the fish's diet. Declarations Competing Interests The authors declare no competing interests. Funding This research was financed by a grant from the Shahid Chamran University of Ahvaz Research Council (Grant No. vC98.299). The funding body had no role in the design of the study or interpretation of data. Author Contribution B.M. conceptualized the study. T.M. and B .M and and M.T. designed and supervised the study.S.M.E. wrote and T.M. and B.M. revised the manuscript draft. S.M.E. and B.M. performed in vitro experiments related to Acidifier. T.M. and B.M and M.T. conducted in vitro evaluations of test. T.M. and B.M and M.T. conducted data analysis. T. M. and B.M. and M.T. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgments This research was financed by a grant from the Shahid Chamran University of Ahvaz Research Council (Grant No. vC98.299). Availability of data and materials Data will be available on reasonable request. References Abarike, E. D., Jian, J., Tang, J., Cai, J., Sakyi, E. M., & Kuebutornye, F. K. (2020). A mixture of Chinese herbs and a commercial probiotic Bacillus species improves hemato-immunological, stress, and antioxidant parameters, and expression of HSP70 and HIF-1α mRNA to hypoxia, cold, and heat stress in Nile tilapia, Oreochromis niloticus . Aquaculture Reports , 18 , 100438.‏ Adorian, T.J., Jamali, H., Farsani, H.G., Darvishi, P., Hasanpour, S., Bagheri, T. and Roozbehfar, R., 2019. 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Histological effects on the kidney, spleen, and liver of Nile tilapia Oreochromis niloticus fed different concentrations of probiotic Lactobacillus plantarum . Tropical animal health and production , 52 , 167-176.‏ Salma, W. Zhou, Z., Wang, W., Askarian, F., Kousha, A., Ebrahimi, M. T., Myklebust, R., & Ringø, E. (2011). Histological and bacteriological changes in intestine of beluga ( Huso huso ) following ex vivo exposure to bacterial strains. Aquaculture, 314, 24–33. https://doi.org/10.1016/j.aquaculture.2011.01.047‏. Siddik MAB et al (2022) Probiotic yeast Saccharomyces cerevisiae coupled with Lactobacillus casei modulates physiological performance and promotes gut microbiota in juvenile barramundi, Lates calcarifer . Aquaculture 546: 737346. Son, V.M., Chang, C.C.,Wu, M.C., Guu, Y.K., Chiu, C.H. and Cheng,W. (2009) Dietary administration of the probiotic, Lactobacillus plantarum , enhanced the growth, innate immune responses, and disease resistance of the grouper Epinephelus coioides . Fish and Shellfish Immunology 26 , 691–698. Süzer, C., Kamacı, H. O., Çoban, D., Saka, Ş., Fırat, K., & Karacaoğlan, A. (2011). Early weaning of sea bass ( D. labrax ) larvae: effects on growth performance and digestive enzyme activities. Turkish Journal of Fisheries and Aquatic Sciences , 11 (3), 491-497.‏ Takemura, A.F., Chien, D.M. and Polz, M.F., 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front Microbiol, 5, 38. Talpur, A.D., Munir, M.B., Mary, A. and Hashim, R., 2014. Dietary probiotics and prebiotics improved food acceptability, growth performance, haematology and immunological parameters and disease resistance against Aeromonas hydrophila in snakehead ( Channa striata ) fingerlings. Aquaculture, 426, 14-20. Thy, H.T.T., Tri, N.N., Quy, O.M., Fotedar, R., Kannika, K., Unajak, S. and Areechon, N., 2017. Effects of the dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A, and Bacillus pumilus 47B on growth, innate immunity and stress responses of striped catfish ( Pangasianodon hypophthalmus ). Fish shellfish immun, 60, 391-399. Tietz, N.W. and Fiereck, E.A., 1966. A specific method for serum lipase determination. Clin Chim Acta, 13(3), 352-358. Vendramin, N., Zrncic, S., Padrós, F., Oraic, D., Le Breton, A., Zarza, C. and Olesen, N.J., 2016. Fish health in Mediterranean Aquaculture, past mistakes and future challenges. Bull. Eur. Assoc. Fish Pathol, 36(1), 38-45. Vieira, F. D. N., Jatobá, A., Mouriño, J. L. P., Buglione Neto, C. C., Silva, J. S. D., Seiffert, W. Q., ... & Vinatea, L. A. (2016). Use of probiotic-supplemented diet on a Pacific white shrimp farm. Revista Brasileira de Zootecnia , 45 , 203-207.‏ Wang, Y.B., and Xu, Z. (2006). Effect of probiotics for common carp ( Cyprinus carpio ) based on growth performance and digestive enzyme activities. Animal feed science and technology, 127: 283-292. Wang, Y.B., Tian, Z.Q., Yao, J.T. and Li, W.F., 2008. Effect of probiotics, Enteroccus faecium , on tilapia ( Oreochromis niloticus ) growth performance and immune response. Aquaculture, 277(3-4), 203-207. Yang, G., Cao, H., Jiang, W., Hu, B., Jian, S., Wen, C., Kajbaf, K., Kumar, V., Tao, Z. and Peng, M., 2019. Dietary supplementation of Bacillus cereus as probiotics in Pengze crucian carp ( Carassius auratus var. Pengze): Effects on growth performance, fillet quality, serum biochemical parameters and intestinal histology. Aquac. Res, 50(8), 2207-2217. Yang, G., Tian, X., Dong, S., Peng, M. and Wang, D., 2015. Effects of dietary Bacillus cereus G19, B. cereus BC-01, and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression of immune-related genes in coelomocytes and intestine of the sea cucumber ( Apostichopus japonicus Selenka). Fish shellfish immun, 45(2), 800-807. Zahran, E., Risha, E., AbdelHamid, F., Mahgoub, H. A., & Ibrahim, T. (2014). Effects of dietary Astragalus polysaccharides (APS) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of Nile tilapia ( Oreochromis niloticus ). Fish & shellfish immunology, 38(1), 149-157. Zapata, A. A. and Lara-Flores, M. (2013). Antimicrobial activities of lactic acid bacteria strains isolated from Nile tilapia intestine ( Oreochromis niloticus ). Journal of Biology and Life Science 4(1), 164-171. Zhang, X.H., He, X. and Austin, B., Vibrio harveyi : a serious pathogen of fish and invertebrates in mariculture., 2020. MLST, 1-15. Zokaeifar, H., Luis Balcázar, J., Kamarudin, M. S., Sijam, K., Arshad, A., & Saad, C. R. (2012). Selection and identification of non-pathogenic bacteria isolated from fermented pickles with antagonistic properties against two shrimp pathogens. The Journal of antibiotics , 65 (6), 289-294.‏ Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3935430","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273570102,"identity":"6eb0b4b8-96ac-4449-bf04-2ba9d19909bf","order_by":0,"name":"Seyyad Mojtaba Emam","email":"","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":false,"prefix":"","firstName":"Seyyad","middleName":"Mojtaba","lastName":"Emam","suffix":""},{"id":273570103,"identity":"80df51a5-561e-451e-9c40-415742ded8b7","order_by":1,"name":"Babak Mohammadian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFACHjDJuIG9ASrATLQWngMka5FIINJZuu29Bz/dzLGT3S75/OFnHgY7eQZ23gd4tZidOZcsnbst2Xjn7BxjaR6GZMMGZnYD/Fpu5BgAtTAnbridw8bMw8CcwMDMht9hQC3Gv3O31SduuHn8GVBLPVFazIC2HE7ccIPBDKjlMBFazpwxs87ddtx4Z0+OseQcg+OGbQS1HO8xvp27rVp2O/vxhx/eVFTL8/Mfw68FDQDDioAdo2AUjIJRMAqIAQAdhz4HU9GN5AAAAABJRU5ErkJggg==","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":true,"prefix":"","firstName":"Babak","middleName":"","lastName":"Mohammadian","suffix":""},{"id":273570104,"identity":"54e05d6f-30ae-40e3-b495-efcfb6a3f702","order_by":2,"name":"Takavar Mohammadian","email":"","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":false,"prefix":"","firstName":"Takavar","middleName":"","lastName":"Mohammadian","suffix":""},{"id":273570105,"identity":"18596895-1852-411a-9644-55a09e49bdb5","order_by":3,"name":"Mohammad Reza Tabande","email":"","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Tabande","suffix":""}],"badges":[],"createdAt":"2024-02-07 01:47:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3935430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3935430/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51399912,"identity":"5e2f5c2f-b97d-4305-bf9f-f42c802140b4","added_by":"auto","created_at":"2024-02-20 22:08:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322773,"visible":true,"origin":"","legend":"\u003cp\u003eThe activities of digestive enzymes in Asian seabass fed or un-fed with probiotics for 45 days. For each parameter, different lowercase letters between the treatments denote significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The data were expressed as Mean ± SD of 9 fish per treatment (n = 9).\u003c/p\u003e","description":"","filename":"FigureA1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/ba085681f678f6bea36f51f6.jpg"},{"id":51399871,"identity":"75dde85a-e41e-4c55-99ae-77e3021bf5b4","added_by":"auto","created_at":"2024-02-20 22:00:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294341,"visible":true,"origin":"","legend":"\u003cp\u003eThe innate immune parameters in Asian seabass fed or un-fed with probiotics for 45 days. For each parameter, different lowercase letters between the treatments denote significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The data were expressed as Mean ± SD of 9 fish per treatment (n = 9).\u003c/p\u003e","description":"","filename":"FigureA2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/00af527a431d570ce701dd29.jpg"},{"id":51399873,"identity":"a0c8ed6d-3169-47c0-8cd7-35182556a6eb","added_by":"auto","created_at":"2024-02-20 22:00:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":325697,"visible":true,"origin":"","legend":"\u003cp\u003eIntestinal histomorphometic in Asian seabass fed or un-fed with probiotics for 45 days. For each parameter, different lowercase letters between the treatments denote significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The data were expressed as Mean ± SD of 9 fish per treatment (n = 9).\u003c/p\u003e","description":"","filename":"FigureA3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/5df284d437e88359e360a055.jpg"},{"id":51399874,"identity":"1ebeff6b-a7df-4085-84b9-387ae2d13193","added_by":"auto","created_at":"2024-02-20 22:00:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":781779,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathologic view of Intestinal tissue sections of Asian seabass fed (A) or un-fed with probiotics (B) for 45 days. Paraffin-embedded tissue blocks were stained with H\u0026amp;E and examined with 10X magnification. \u0026nbsp;(A) and (B), Pay attention to the height of the villi (thin arrow) and the width of the villi (thick arrow). (C) Pay attention to the width of the villi and numerous of goblet cells (H\u0026amp;E.20X).\u003c/p\u003e","description":"","filename":"FigureA4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/f7459a740fe065ae1601718b.jpg"},{"id":51399875,"identity":"07dd2d00-17a7-4f7c-8345-01e407a51a4c","added_by":"auto","created_at":"2024-02-20 22:00:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":315835,"visible":true,"origin":"","legend":"\u003cp\u003eThe quantitative expressions of interleukin-10 (IL-10) and granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor and Transforming growth factor beta (TGF-β), and Epidermal growth factor (EGF) extracted from the intestine of Asian seabass fed or un-fed with probiotics for 45 days. For each parameter, different lowercase letters between the treatments denote significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The data were expressed as Mean ± SD of 9 fish per treatment (n = 9).\u003c/p\u003e","description":"","filename":"FigureA5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/29d80788efa9903bbd633ec2.jpg"},{"id":51400023,"identity":"9268190d-0cf3-4d92-8366-b032317239cf","added_by":"auto","created_at":"2024-02-20 22:16:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1076738,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3935430/v1/c7e6ca6b-dfcc-469a-9937-d9dbeff48134.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrevention of diseases represents a primary objective in the field of aquaculture. The implementation of hygienic and preventive measures, such as fish health management, sanitation practices, and disease control procedures, plays a critical role in averting fish diseases (FAO. 2018). The expansion of aquaculture has led to intensified farming practices, resulting in the degradation of water quality, increased stress on fish, and heightened vulnerability to infectious diseases. Among bacterial pathogens, the genus Vibrio, particularly those belonging to the Vibrio clade, poses a potential global threat to mariculture (Reina et al., 2019). This poses a major concern for the sustainability of the aquaculture industry. As representatives of gram-negative bacteria, Vibrio belongs to the class Gammaproteobacteria and the family Vibrionaceae. Vibrio infections are primarily associated with stress conditions, notably fluctuations in water temperature and salinity, leading to significant losses in fish and shellfish cultured in marine and estuarine environments (Noga, 2010; Takemura et al.,2014). Several members of the Harveyi clade, including \u003cem\u003eVibrio harveyi\u003c/em\u003e, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, \u003cem\u003eV. alginolyticus\u003c/em\u003e, and \u003cem\u003eV. campbellii\u003c/em\u003e, have been implicated in disease outbreaks among aquatic organisms, particularly in tropical regions (Darshanee Ruwandeepika et al.,2012; Mohamad et al.,2019a). Recently, \u003cem\u003eV. alginolyticus and V. harveyi\u003c/em\u003e has been identified as a potential problem in the cultured gilt-head seabream (\u003cem\u003eSparus aurata\u003c/em\u003e) and European seabass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e) (Vendramin et al.,2016; Firmino et al.,2019; Zhang et al.,2020). The prevention of vibriosis in aquaculture primarily relies on the implementation of farm-level biosecurity measures and improved management practices (Ina-Salwany et al.,2019; Mohamad et al.,2019b). However, disease prevention strategies differ between open-ocean aquaculture and confined systems. Antibiotic therapy is commonly employed to treat vibriosis in affected fish, although the widespread use of antibiotics is no longer recommended due to concerns about antibiotic resistance, antibiotic residues in fish, environmental pollution, and ineffectiveness against bacterial biofilms (Defoirdt,2018; Assefa and Abunna,2018; Grenni et al.,2018) .Concerning Vibrio infections, there are currently no commercially available polyvalent vaccines that can protect against the diverse serotypes needed for broad cross-protection against different Vibrio species (Li et al.,2010; Powell et al.,2011; Galeotti et al.,2013; Peng et al.,2016). The limitations associated with the use of antibiotics in aquaculture have generated significant interest in probiotics as an alternative (Wang et al.,2008). Probiotics have gained popularity due to their environmentally friendly nature and their potential to replace antibiotics, improve animal health, and reduce diseases in aquatic animals (Gatesoupe,1999). Research has shown that the most effective probiotics for aquatic animals are bacteria isolated from the aquatic environment or the animals themselves. Therefore, various countries are working on isolating and producing native aquatic probiotics for commercial use. However, the introduction of non-native bacterial species into the aquatic environment by the aquaculture industry can have unintended harmful consequences (Balcazar et al.,2006). Autochthonous probiotics, such as \u003cem\u003eEnterococcus faecium\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus brevis\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, or \u003cem\u003eBacillus cereus\u003c/em\u003e, have been found to enhance weight gain, specific growth rate, feed conversion, and promote higher survival rates. They also exhibit beneficial immunological modulation, and hematological changes, and promote intestinal modulation in various fish species (Liu et al.,2013; Mohammadian etal.,2018). \u003cem\u003eLactobacillus plantarum\u003c/em\u003e is a commonly used lactic acid bacteria (LAB) species in aquaculture, renowned for its probiotic properties (Foysal et al.,2020). In recent years, numerous studies have demonstrated the potential use of \u003cem\u003eL. plantarum\u003c/em\u003e as a probiotic in various aquatic species, including \u003cem\u003eO. niloticus\u003c/em\u003e (Ruiz et al.,2020), \u003cem\u003eCyprinus carpio\u003c/em\u003e (Mohammadian et al.,2022), rainbow trout \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (Ahmadmoradi et al.,2023;Fregeneda et al.,2023), \u003cem\u003ePampus argenteus\u003c/em\u003e (Gao et al.,2016), shabot (\u003cem\u003eTor grypus\u003c/em\u003e) (Mohammadian et al.,2016), \u003cem\u003eLates calcarifer\u003c/em\u003e (Ghanei-Motlagh et al.,2021), as well as crustaceans such as \u003cem\u003eAstacus leptodactylus\u003c/em\u003e (Didinen et al.,2016), \u003cem\u003eMacrobrachium rosenbergii\u003c/em\u003e (Dash et al.,2015), \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Kongnum and Hongpattarakere,2012). One of the beneficial effects of probiotics in living organisms, as suggested by researchers, is the improvement of host nutrition through the production of digestive enzymes and growth supplements. This, in turn, increases survival, and food efficiency, prevents intestinal disorders, and enhances nutrient digestion. Additionally, probiotics help balance the microflora in the gastrointestinal tract, leading to better growth performance. Moreover, probiotics stimulate the proliferation of gastrointestinal epithelial cells (Ichikawa et al.,1999). Upon entering the intestine, probiotics start to multiply and utilize sugars to grow, producing short-chain unsaturated fatty acids, which may play a role in increasing the length of intestinal enterocytes (Pelicano et al.,2005). The evaluation of intestinal morphology and history is crucial for assessing the function of probiotics in fish. Ringo et al., (2007) emphasized the significance of intestinal morphology in selecting lactic acid bacteria as probiotics in fish. Several studies have also reported the beneficial effects of Lactobacillus species isolated from sources other than fish (Merrifield et al.,2010b). However, it is important to consider the potential impact of endogenous bacteria on intestinal morphology when using probiotics. Some studies have reported that certain bacteria isolated from non-fish sources had severe destructive effects on fish intestines (Salam et al.,2011). Thus, evaluating the effect of endogenous bacteria on intestinal morphology is a key factor in the use of probiotics. The selection of Asian sea bass (\u003cem\u003eL. calcarifer\u003c/em\u003e, Bloch) as the primary research focus is motivated by its high relevance to aquaculture practices in Iran. The species has been introduced in the southern provinces of Iran as a potentially suitable candidate for cage or earthen pond farming (Ghanei-Motlagh et al.,2021). By understanding and optimizing the supplementation of endogenous bacteria as probiotics, the efficiency and sustainability of sea bass farming can be significantly enhanced (Lim et al.,2019). However, Asian sea bass is highly susceptible to infections caused by Vibrio spp., particularly V. \u003cem\u003eharveyi\u003c/em\u003e and V. \u003cem\u003ealginolyticus\u003c/em\u003e (Dong et al.,2017;Mohamad et al.,2019c). Epidermal growth factor (EGF) is one of the growth factors with a protein structure of 53 amino acids. These proteins are involved in the growth and metabolism of many cells and are a type of cytokine. Transforming growth factor beta (TGF-β) is a multifunctional cytokine that belongs to the transforming growth factor superfamily and includes 4 isoforms and several other cell signaling proteins secreted by white blood cells. After activation, transforming growth factor-β combines with other factors and forms a serine/threonine kinase complex that binds to the transforming growth factor-beta receptor. One of the main functions of this protein is to regulate inflammatory processes, especially in the intestine. Interleukin 10 is one of the important interleukins of the body that is secreted from white blood cells and plays a role in inhibiting inflammatory and immune responses. Interleukin 10 is secreted from monocyte cells, as well as from helper T lymphocytes, macrophages, regulatory T lymphocytes. IL-10 is a cytokine with multiple functions in the regulation of immunity and inflammation. Interleukin 10 also increases the proliferation and survival of B lymphocytes and antibody production. IL-10 can block NF-κB activity. granulocyte-macrophage colony-forming cells (GMCFC) is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts that functions as a cytokine and specifically promotes neutrophil proliferation and maturation. Research suggests that probiotic bacteria can influence gene expression, including genes related to the immune system and growth factors. Some studies in aquatic animals, including fish, have explored the impact of probiotics on genes such as interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor, and Transforming Growth Factor Beta (TGF-β) (Mozanzadeh et al.,2023;Siddik et al.,2022;Huo et al.,2019;Jang et al.,2020).Probiotics may modulate the immune response and contribute to the regulation of growth factors, potentially promoting a more balanced and beneficial gene expression profile. However, the effectiveness can vary depending on factors like the specific probiotic strains used, the host species, and environmental conditions. The findings from this study will contribute significant knowledge on the potential benefits and risks associated with the incorporation of endogenous bacteria as probiotics in sea bass culture. Such insights are pivotal for the development of targeted strategies aimed at optimizing the growth, health, and overall performance of sea bass in aquaculture systems in Iran. The research aims to explore the effects of endogenous bacteria by evaluating various parameters throughout the culture period. One crucial indicator is intestinal pathology, which allows for the assessment of structural changes or damage in the sea bass intestines. Monitoring growth rates provides essential insights into the overall development and performance of the fish. Additionally, evaluating the activity of specific digestive enzymes offers valuable information on nutrient digestion and absorption efficiency in the digestive tract.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Isolation and preliminary screening of bacteria in the intestine of Aquatic Animal\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLithopeneus vanami\u003c/em\u003e specimens were obtained from Aquaculture Khuzestan, Choebdeh (Abadan, Iran) for sampling purposes. The intestinal contents were collected under aseptic conditions and subsequently diluted in a gradient manner using sterile PBS (phosphate-buffered saline). Serial dilutions of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e were prepared. The dilutions were evenly spread on de Man, Rogosa, and Sharpe (MRS) agar plates (BD, Sparks, MD, USA) (Ullah, 2020). The plates were then placed in a constant temperature incubator set at 37\u0026deg;C for 24 hours. After the colonies grew uniformly on the agar plates, individual colonies were carefully selected and purified until no other colonies were present on the plate. A purified single colony was inoculated into a 15-ml MRSB liquid medium and cultured at 37\u0026deg;C for 16 hours. The resulting bacterial solution was mixed with glycerin in a 1:1 volume ratio and stored at -80\u0026deg;C in a cryopreserved tube (Ullah, 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003eProbiotic characteristic of isolates\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTwo selected strains were subjected to the thermal growth experiment. To perform that, three dilutions from each colony (similar to McFarland No. 0.5, OD\u0026thinsp;=\u0026thinsp;0.132 at 600nm) were inoculated in MRSB for 72h at different temperatures, including 10, 20, 30 and 20 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e. The bacterial growth rate was then recorded at 48 through turbidity measured by optical density (spectrophotometer, Jenway, 6400, UK) at 600 nm. Simultaneously, the number of colonies in each corresponding plate was also measured [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Two probionts were examined \u003cem\u003ein vitro\u003c/em\u003e to check their antagonist effects against common pathogenic bacteria of trout, including \u003cem\u003eA. hydrophila\u003c/em\u003e (AH04), \u003cem\u003eL. garvieae\u003c/em\u003e, \u003cem\u003eY. ruckeri\u003c/em\u003e, and \u003cem\u003eS. iniae\u003c/em\u003e (previously isolated). To do this, small amounts of fresh (18h-old) cultural media of each probiotic strain (colonies were between 20\u0026ndash;30) were poured onto either an MRS or TSA plate, and incubated at 37\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for 48h. Similarly, of the 18h-old cultural medium of each above-mentioned pathogen (on TSB), a peripheral bacterial culture was streaked crossly over the test bacteria inoculum in three triplicates. The plates were then incubated at 25 ℃ for 24h and finally, the inhibition zone of growth was measured (if any) and accounted as antagonist activity of each probation (Mohammadian et al.,2019). To investigate surface hydrophobicity, bacterial suspensions obtained, as previously described were adjusted to an OD600 of 1.0. Then, 5 mL of bacterial suspensions were mixed with 1 mL of toluene by vigorous vortexing for 1 min, allowing the separation of the aqueous and organic phases for 1 h at room temperature. Following phase separation, the final OD of the aqueous phase was determined. The cell surface hydrophobicity was calculated using the following formula: Surface hydrophobicity (%) = ([A0\u0026thinsp;\u0026minus;\u0026thinsp;A1]/A0) \u0026times; 100, where A1 and A0 are the ODs at the aqueous phase and the original suspension respectively. The assay was performed in triplicate (Ghanei-Motlagh et al.,2019)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Bacterial identification\u003c/h2\u003e \u003cp\u003eThe identities of the isolated strains were confirmed through gram staining, followed by complementary biochemical identification tests and 16S rRNA gene sequencing. To determine the biochemical characteristics of the isolated strains, the biochemical trace reaction tubes of bacteria were utilized by the Manual for Systematic Identification of Common Bacteria. DNA was extracted from the bacteria using a commercial kit (SinaClon, Iran). PCR was performed on a PC 707 thermal cycler (Thermocycler, Mastercycler Gradient, Eppendorf, Germany) using universal bacterial primers, 27F (5ʹAGAGTTTGATCCTGGCTCAG-3ʹ) and 68R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ), which were synthesized by Comate Bioscience Co., Ltd (Jilin, China). The PCR process consisted of 2 minutes of initial denaturation at 92℃, followed by 35 cycles, consisting of 30 seconds of denaturation at 95℃, 45 seconds of annealing at 57℃, and 45 seconds of primer extension at 72℃. The process concluded with 5 minutes of final extension. Amplification products were analyzed via electrophoresis in 1.5% (w/v) Agarose gel containing Ethidium Bromide (1 mg ml-1). Upon obtaining aliquots of 15 \u0026micro;L of PCR product, sequencing was carried out using BioEdit, 7.2. The obtained sequence was then blasted using NCBI's online tool \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Diet preparation\u003c/h2\u003e \u003cp\u003eA commercial feed containing 47% crude protein, 18% crude lipid, 2% crude fiber, 14% ash, 1.1% total phosphorus, 4200 kcal/kg feed digestible energy, and less than 12% moisture (Beyza Feed Mill, Shiraz, Iran) was used as the basal diet. Bacterial strains were grown aerobically on MRSB in a shaking incubator at 25 ℃, harvested by centrifugation (3000 rpm for 5 min), washed twice with normal saline, and re-suspended in the same solution. Bacterial suspensions were homogenized and adjusted to an OD600 of 2 using a Biophotometer (No 6131, Eppendorf, Germany). The Spread plate method was used to inoculate the adjusted serial dilutions of each suspension to commercial feed to achieve the desired concentration of probiotics. Experimental diets were prepared by gently spraying sterile normal saline pre-suspended with 1 x 10\u003csup\u003e9\u003c/sup\u003e CFU/g feed Isolated 1 (group 1) and 1 x 10\u003csup\u003e9\u003c/sup\u003e CFU/g feed Isolated 2 (group 2). The same amount of normal saline was added to the control diet, and control fish were fed with bacteria-free diet for the same duration. Food preparation was conducted under sterile conditions. The diets were air-dried at room temperature for 1 h, packaged, and stored in a refrigerator at 4\u0026deg;C until used. The viability of each probiont in the diet at the final concentrations mentioned earlier was confirmed using one gram of the food suspended in 9 mL sterile PBS, and the bacterial count was compared to the added probiotic bacteria. The supplemented diets were prepared twice per week (Mohammadian et al.,2019a)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experimental design\u003c/h2\u003e \u003cp\u003eHealthy Asian Seabass \u003cem\u003eL. calcarifer\u003c/em\u003e with normal appearance were purchased from a commercial fish farm located in Bushehr province, Iran. Before the commencement of the experiment, the specimens were kept in a recirculation system for a minimum of two weeks. A total of 25 fish, with an average weight of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 g and average length of 14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 cm, were randomly distributed into 300 L fiberglass tanks in triplicate for each treatment. The tanks were supplied with running seawater that had been sterilized using UV radiation. The fish were fed with assigned diets, based on 2.5% of their body weight, under a regular photoperiod cycle (16L:8D) three times a day for 45 days. One hour after feeding, any leftover feed was siphoned off, dried, and weighed to estimate the amount of feed consumed. Throughout the trial, the physicochemical parameters of the water were maintained within a normal range, with dissolved oxygen levels between 8\u0026ndash;9 ppm, pH between 7.3\u0026ndash;7.5, temperature between 27\u0026ndash;30 ℃, un-ionized ammonia levels less than 0.05 ppm, and nitrate levels less than 0.1 ppm. All rearing conditions remained constant during the adaptation and experimental periods. After the acclimation period, 300 fish were divided into 3 groups, with three experimental units for each group (i.e., 9 tanks). To investigate the effects of different feeding treatments on the parameters studied below, probiotic-supplemented diets were conducted for 45 consecutive days. A group with no added probiotics was served as the control and administered the same diet ingredients. This study was approved by the institutional ethics committee of the Shahid Chamran University of Ahvaz, Iran, under approval number EE / 98.24.3.71483 / scu.ac.ir. All procedures on animals in this experiment were conducted according to the guide for the care and use of laboratory animals by the National Academy of Sciences (NIH publications No. 8023, revised 1978).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.6. Sampling\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBefore weighing or sampling, the fish were starved for 24 hours to minimize stress. Blood collection and gut sample removal were performed at the end of the trial (day 45; a total of 9 fish). The fish were anesthetized with 2-phenoxyethanol (0.3 ml/L), and blood was collected via the caudal vein using a 3 ml syringe (Mohammadian et al., 2019). An aliquot of the collected blood was immediately dispensed into a 0.5 ml Eppendorf tube previously coated with heparin sodium and was used for the analysis of hematological indices. The remaining portion was transferred to a 1.5 ml Eppendorf tube, left to clot at 4\u0026deg;C for 1 hour, and centrifuged at 3,000 g at 4\u0026deg;C for 10 minutes (Microliter Centrifuge, Mikro 220R, Hettich, Germany) to separate serum for the estimation of biochemical parameters (Mohammadian et al.,2019b). The sera were kept at -80\u0026deg;C for subsequent analysis. Afterward, the flank and ventral surfaces of the fish were disinfected using 70% ethanol. Once the fish were aseptically dissected, the specimens of the middle and posterior intestines were removed and placed in the respective volumes of sterile PBS for bacterial counts. Additionally, a portion of the pyloric ceca and foregut was sampled to assay the activities of digestive enzymes, as described in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.8\u003c/span\u003e (Mohammadian et al.,2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Growth performance\u003c/h2\u003e \u003cp\u003eTo determine the growth performance, all fish within each treatment were individually weighted at the beginning and 6th week of the trial. The weight of all fish in each tank was determined every two weeks and feed ratios were adjusted according to the fish weight. Growth parameters including absolute growth (Δw), relative growth rate (RGR), feed conversion ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), the feed efficiency ratio (FER), feed intake (FI) were calculated according to the following formula (Hopkins,1992):\u003c/p\u003e \u003cp\u003eΔw\u0026thinsp;=\u0026thinsp;final body weight (FBW, g) - initial body weight (IBW, g)\u003c/p\u003e \u003cp\u003eRGR = [Δw (g) / IBW (g)] \u0026times; 100\u003c/p\u003e \u003cp\u003eFCR\u0026thinsp;=\u0026thinsp;net dry feed consumed per tank (g) / Δw (g) per tank\u003c/p\u003e \u003cp\u003eSGR = [(ln FBW - ln IBW) / experimental period (d)] \u0026times; 100\u003c/p\u003e \u003cp\u003ePER\u0026thinsp;=\u0026thinsp;Δw (g) per tank/protein intake (g) per tank\u003c/p\u003e \u003cp\u003eFER = [Δw (g) per tank / net dry feed consumed per tank (g)] \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Digestive enzyme activities\u003c/h2\u003e \u003cp\u003eThe activities of digestive enzymes including α-amylase, non-specific (total) protease, lipase, trypsin, chymotrypsin, and ALP were measured in triplicates per tank (using pooled samples from each replicate tank) after 45 days of feeding with strains. Intestinal samples in each tank were pooled, weighted and homogenized either in a hypotonic cold buffer (2 mM Tris\u0026ndash;HCl buffer containing 50 mM mannitol, final pH\u0026thinsp;=\u0026thinsp;7, 1:30 v/w) to measure intestinal ALP or an alkaline cold buffer (100 mM Tris-HCl, 0.1 mM EDTA and 0.1% Triton X-100, final pH\u0026thinsp;=\u0026thinsp;7.8, 1:10 v/w) to quantify the other enzymes (Mohammadian et al., 2019c). The homogenates of intestinal ALP were treated with CaCl\u003csub\u003e2\u003c/sub\u003e (10 mM) and purified as described previously (Crane et al.,1979). The homogenates containing the other enzymes were centrifugated at 10,000 \u003cem\u003eg\u003c/em\u003e for 12 min at 4\u0026deg;C and the obtained enzyme extracts were aliquoted and stored at -80\u0026deg;C for subsequent analyses. The concentration of total protein in the crude enzyme extracts was measured by the Bradford method using bovine serum albumin as standard (Bradford,1976). Total protease activity was measured using the casein hydrolysis method following the reaction between the liberated tyrosine and Folin-Ciocalteau reagent (Folin and Ciocalteau,1929;Anson,1938). the α-amylase activity was quantified using soluble starch as the substrate hydrolyzable to maltose reacting with a 3,5-Dinitrosalicylic acid solution (Bernfeld,1955). Standard solutions containing tyrosine and maltose were used to prepare the standard curves of total protease and α-amylase, respectively. Lipase activity was titrimetrically estimated using olive oil emulsion. The released fatty acids were titrated with sodium hydroxide solution using phenolphthalein as an indicator (Tietz and Fiereck,1966). The activities of chymotrypsin and trypsin were kinetically assayed using N-Benzoyl-L-tyrosine ethyl ester (BTEE) and N\u003csub\u003eα\u003c/sub\u003e-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as substrates, respectively (Hummel,1959). Subsequently, the ratios of trypsin to chymotrypsin (T/C) and amylase to trypsin (A/T) were calculated. ALP activity was kinetically measured using 4-nitrophenyl phosphate (PNPP) as substrate by a commercial kit (Pars Azmoon Co., Tehran, Iran).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Oxidative Status\u003c/h2\u003e \u003cp\u003eKurhaluk et al. method was modified for catalase (CAT) activity assay. The activity of serum SOD was determined and was estimated according to the method of Peixoto et al. (Peixoto et al.,2009). MDA in serum samples was assayed by the thiobarbituric acid reactive substances (TBARS) assay (Bradford,1976). The MDA concentration was estimated using the MDA molar extinction coefficient (156,000 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Ghanei-Motlagh et al.,2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Intestinal histopathology and histomorphology\u003c/h2\u003e \u003cp\u003eTo analyze the histopathology and histomorphological structure, the samples of the posterior intestine were collected. The samples were separately fixed in 10% buffered formalin (pH\u0026thinsp;=\u0026thinsp;7.2) and processed using the standard protocol for histopathological examination. After mounting the samples with paraffin wax, three separate cross-sections with a thickness of ~\u0026thinsp;5 \u0026micro;m were prepared using a microtome (Microtec CUT4050) and then stained with hematoxylin and eosin (H\u0026amp;E) for further histopathological investigations. The villi height, villi width, and the thickness of the epithelium, lamina propria, muscular layers, and the number of goblet cells were determined under a light microscope by using AxioVision microscope software, Carl Zeiss (Oberkochen, Germany) (Mohammadian et al.,2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Intestinal bacterial community\u003c/h2\u003e \u003cp\u003eThe specimens of the posterior intestine were aseptically removed and homogenized with sterilized PBS (1:10 w/v). The homogenates were serially diluted and 100 \u0026micro;L of tenfold diluted suspensions were spread on different agar media including tryptone soy agar (TSA) supplemented with 2% NaCl, thiosulphate citrate bile sucrose (TCBS) and deman Regosa and sharp (MRS) in triplicates to determine the populations of total aerobic heterotrophic bacteria, \u003cem\u003eVibrio\u003c/em\u003e spp. and \u003cem\u003eBacillus\u003c/em\u003e spp. respectively. All plates were incubated for 48 h at 29\u0026deg;C and the counted colonies were expressed as denary logarithms of colony forming units (CFU) per ml homogenized suspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Gene expression\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11.1. RNA isolation and cDNA synthesis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 9 tissue samples of the intestine for each treatment on day 45, using the Tri Pure isolation reagent following the manufacturer's protocol (Roche, Canada). The concentration of the extracted RNA was determined at a wavelength of 260 nm using a nano-drop spectrophotometer (Eppendorf, Germany). The purity of RNA was assessed by determining the optical density (OD) absorption ratio at 260/280 nm, and only samples with a ratio above 1.8 were used for cDNA synthesis. Any potential DNA contamination was eliminated by treating RNA (1 \u0026micro;g) with DNase I (2 U \u0026micro;l\u0026thinsp;\u0026minus;\u0026thinsp;1) for 1 hour at 37℃ (Vivantis, Malaysia). Reverse transcription was performed using the Rocket Script RT PreMix Kit, utilizing 1 \u0026micro;g of RNA and oligo dT as specified by the manufacturer's instructions (Bioneer Corporation, South Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.11.2. Real-time quantitative PCR\u003c/h2\u003e \u003cp\u003eTo assess the intestinal expression levels of Epidermal growth factor (EGF), interleukin-10 (IL-10) and granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor and Transforming growth factor beta (TGF-β) mRNA, real-time PCR was conducted using the qPCRTM Green Master Kit for SYBR Green I\u0026reg; (Jena Biosciense, Germany) with the Light cycler\u0026reg; Detection System (Roche, USA). The relative expression levels of all transcripts were normalized against β-actin as the housekeeping gene. Specific primer sets (Bioneer, South Korea) were utilized based on O. mykiss (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Reactions were performed in triplicate in a 12.5 \u0026micro;l mixture, consisting of 6.25 \u0026micro;l qPCRTM Green Master Mix (2X), 0.25 \u0026micro;l of each primer (10 \u0026micro;M), 3 \u0026micro;l (100 ng) cDNA, and 2.75 \u0026micro;l nuclease-free water. The PCR protocol comprised of a denaturation stage at 94 ℃ for 5 min, followed by 45 cycles at 94 ℃ for 15 sec and 60 ℃ for 30 sec. Two separate reactions without cDNA or with RNA were employed as control groups parallel to the experimental groups. The relative quantification was conducted using Light cycler 96\u0026reg; software based on the comparative 2-ΔΔCt method. The validation of the assay was scrutinized to ensure that the chβ-actin and chCASQ2 primers had similar amplification efficiencies, as described before. All qPCR analyses were conducted following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline (Bustin et al.,2009).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOptical density of bacterial isolates with the best probiotic performance at different temperatures (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolation of bacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOD at 10\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOD at 20\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOD at 30\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOD at 37\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003eL1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 \u003csup\u003eaD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.528\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eaBC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.631\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.827\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003eL2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.051\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 \u003csup\u003eacD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.427\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.739\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.861\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eFor each parameter, values (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;9) bearing different lowercase letters or different uppercase letters represent significant differences within each column or each row, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMaximum inhibition zone (mm) for growth of each pathogenic bacteria on different probiotics cultural media\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl-60d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL1\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eL2\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. garvieae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. hydrophila\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY. ruckeri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4. 1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eV. harvie\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5). The superscript alphabetic letters in each row indicate significant differences among different groups (ANOVA)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.12. In vivo bacterial challenge and LD\u003csub\u003e50\u003c/sub\u003e measurement\u003c/h2\u003e \u003cp\u003eAt the end of the 45-day experimental period, fish in each group were challenged with \u003cem\u003eV. alginolyticus\u003c/em\u003e, which was isolated from a diseased barramundi fish reared in a cage and identified by 16s rDNA analysis before the challenge trial. Before utilizing \u003cem\u003eV. alginolyticus\u003c/em\u003e for the challenge, we investigated whether the applied probiotics could protect the fish against pathogenic bacteria, thereby initially determining the lethal dose (LD) of \u003cem\u003eV. alginolyticus\u003c/em\u003e. The 45 fish (weighing 43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 g) were injected with serial intraperitoneal doses of \u003cem\u003eV. alginolyticus\u003c/em\u003e at 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e CFU ml\u0026thinsp;\u0026minus;\u0026thinsp;1, with 15 fish per dose. Mortality rates were recorded for four consecutive days at each dose level, and probit analysis (SPSS, 18, USA) was conducted to determine the exact LD50 of \u003cem\u003eV. alginolyticus\u003c/em\u003e to \u003cem\u003eLates calcarifer\u003c/em\u003e. The \u003cem\u003eV. alginolyticus\u003c/em\u003e was prepared for the challenging test as follows: the bacterium was grown in TSA for 48 h at 37 ℃, washed twice with PBS, and re-suspended in the same buffer. The bacterial concentration was adjusted to bacterial LD50 (1 \u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU ml\u0026thinsp;\u0026minus;\u0026thinsp;1) using a spectrophotometer, and the concentration of the bacterial suspension was determined using a bacterial counting chamber to verify the challenge dose. Anesthesia was induced with 2-phenoxyethanol (1:10,000) (Shanghai Reagent, China) in all 45 fish (15 from each treatment) before injection. All fish in each group were intraperitoneally injected with 0.2 ml of \u003cem\u003eV. alginolyticus\u003c/em\u003e suspensions using a 1 ml sterile syringe. The control group was also injected with 0.2 ml of \u003cem\u003eV. alginolyticus\u003c/em\u003e suspension. Mortalities were recorded daily for four days\u0026rsquo; post-challenge, and all dead \u003cem\u003eLates calcarifer\u003c/em\u003e were examined bacteriologically to confirm the presence of the pathogen (Perez-Sanchez et al.,2011; Rahimnejad et al.,2018).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data presented in the current study were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was performed using the SPSS program version 22. The normality of the data was checked by the Shapiro\u0026ndash;Wilk test. One-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple-comparison test was carried out to determine differences significant at the 5% probability level between the mean values of parameters.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1. In vitro temperature\u0026ndash;growth, antibacterial activity and hydrophobicity test of bacteria\u003c/h2\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the amount of optical density (OD) measured from the culture of bacteria with probiotic power at temperatures of 10, 20, 30, and 37\u0026deg;C for 48 hours is given. During the 48 hours, there was a significant difference between the growth of bacteria at different temperatures (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All bacterial isolates were able to grow from 10 to 37\u0026deg;C based on the measured optical density. At a temperature of 10\u0026deg;C, all the isolates could grow at a very low rate. The highest values of optical density at a temperature of 20\u0026deg;C correspond to L1, at a temperature of 30\u0026deg;C and 37\u0026deg;C respectively to L2 and L1. L1 and L2 had good growth ability at the investigated temperatures (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Based on the bacterial growth test at different temperatures, it was found that by increasing the temperature of the greenhouse for 48 hours, the growth rate of bacterial isolates increases based on optical density. During the 48 hours of the experiment, the amount of optical density resulting from the growth of bacteria at a temperature of 10\u0026deg;C was very low, but at temperatures of 20, 30, and 37\u0026deg;C, the optical density increased and the growth of bacteria increased significantly. It arrived. It can be said that the results of the present study show that the obtained bacterial isolates can grow at the tested temperatures and the best temperature range for the growth of bacteria is the temperature range for the culture of Asian sea bass. Riaz et al., (2010) investigated the growth rate of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e at temperatures of 15, 20, 25, and 35\u0026deg;C. The optimal growth temperature of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e was 35\u0026deg;C and 25\u0026deg;C was introduced for \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. The optimal temperature for the growth of bacteria in the current study is 37\u0026deg;C, which indicates that these isolates are thermophilic. The antibacterial characteristic of each probiont, including \u003cem\u003eL1\u003c/em\u003e, \u003cem\u003eand L2\u003c/em\u003e was examined against pathogenic bacteria of trout (i.e., \u003cem\u003eL. garvieae\u003c/em\u003e, \u003cem\u003eA. hydrophila\u003c/em\u003e, \u003cem\u003eY. ruckeri\u003c/em\u003e, \u003cem\u003eV. harvei\u003c/em\u003e), by using an \u003cem\u003ein vitro\u003c/em\u003e test in which the inhibition zone of the cross culture medium was measured. Table\u0026nbsp;(2) shows the higher inhibition zone for \u003cem\u003eL2\u003c/em\u003e rather than \u003cem\u003eL1\u003c/em\u003e in most cases (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Antimicrobial tests are one of the common tests for choosing a suitable probiotic. The probiotic bacteria must have antimicrobial properties and the ability to produce antimicrobial compounds (Zokaeifar et al.,2012). This effect is applied in several ways, some of which are as follows: The secretion of extracellular metabolites with bactericidal power by probiotic cells is the competition in occupying the place to stick to the mucus. In the present study, noteworthy antimicrobial activity against pathogenic bacteria was observed in lactic acid bacteria. Within probiotic strains, lactic acid bacteria exhibit the production of various compounds, including organic acids, lactic acid, bacteriocin, and hydrogen peroxide. These compounds contribute to the activation of non-specific immunity in the host. The subjects under investigation in this research are likely to possess a heightened capacity for producing growth-inhibitory substances. Vieira et al., (2016) demonstrated that \u003cem\u003eLactobacillus plantarum\u003c/em\u003e strains, isolated from shrimp intestines, exert an inhibitory effect on a broad spectrum of both Gram-positive and Gram-negative pathogenic bacteria, including \u003cem\u003eV. harveyi\u003c/em\u003e and \u003cem\u003eA. hydrophila\u003c/em\u003e. Additionally, Zapata and Lara-Flores, (2013) reported that \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e, a lactic acid bacterium isolated from tilapia fish intestines, exhibited the ability to inhibit \u003cem\u003eV. harveyi\u003c/em\u003e and \u003cem\u003eMycobacterium marinum\u003c/em\u003e, both recognized as pathogenic bacteria. The percentage of hydrophobicity of the strains ranged from 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 to9.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95% (L1 and L2 respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Growth performances\u003c/h2\u003e \u003cp\u003eIn this study, the results of the tests indicate that the percentage of weight gain in fish fed with \u003cem\u003eL1\u003c/em\u003e probiotics was significantly higher than that of L2 probiotic treatments and the control group. Both probiotic treatments led to an improvement in the food conversion ratio and specific growth rate compared to the control group. Moreover, the protein efficiency ratio was also significantly improved in the probiotic treatments compared to the control group; however, this improvement was not seen to be significant between the two probiotic treatments. There was no significant effect of probiotic feeding on the condition factor. Additionally, no cases of mortality were observed during the test period in any of the groups. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Bacterial pathogens are a significant contributor to disease damage in aquaculture, with vibriosis standing out as a major cause of extensive losses and damages in the marine aquaculture industry. This phenomenon is closely linked to the invasive capabilities of these bacteria, particularly under stressful conditions (Ina-Salwany et al.,2019). In this investigation, a notable observation was the 100% survival rate (SR) across all examined groups throughout the entire culture period, implying optimal maintenance conditions in each treatment. The study revealed that fish utilizing feed enriched with \u003cem\u003eL\u003c/em\u003e1 (to a greater extent) and L2 (to a lesser extent, compared to another probiotic group) over a 45-day culture period outperformed the control group, which had no added probiotics. Additionally, these treated groups exhibited superior growth indices, particularly in terms of final weight factors. Our results are those of Reda et al., (2018) (\u003cem\u003eClarias gariepinus\u003c/em\u003e), Yang et al., (2019) (\u003cem\u003eCarassius auratus)\u003c/em\u003e, Gisbert et al., (2013) (\u003cem\u003eOncorhynchus mykiss)\u003c/em\u003e, Yang et al., (2015) (\u003cem\u003eApostichopus japonicas)\u003c/em\u003e fed with host-associated \u003cem\u003eprobiotic\u003c/em\u003e compared to fish fed with commercial probiotics. Similar results have been reported in Asian seabass fed with \u003cem\u003eBacillus subtilis\u003c/em\u003e E20 and a mixture of \u003cem\u003eBacillus licheniformis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e (Lin et al., 2017; Adorian et al.,2019). In previous investigations, probiotic bacteria in aquaculture have been linked to enhanced growth by upregulating the insulin-like growth factor system. This is often associated with a concurrent decrease in myostatin gene expression and lower cortisol levels, contributing to improved overall health and growth in aquatic species (Carnevali et al.,2006; Hauville et al.,2016). It sounds like the researchers are proposing a hypothesis that suggests L1, in addition to its high probiotic potential, may enhance growth performance through the extracellular metabolites of protease, carbohydrase, and lipase. These metabolites could potentially break down complex dietary proteins, lipids, and carbohydrates into more usable forms, such as amino acids, peptides, fatty acids, and monosaccharides, ultimately improving digestibility and promoting growth.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance of Asian seabass fed either regular feed or feed supplemented with probiotics for 45 Day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIBW (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFBW (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e115.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e123.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFTL (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRGR (% / 45 day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCR (g / g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSGR (% / d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePER (g gain / g pro)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFER (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.55\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.89\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eThe data represent the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three tanks per treatment (n\u0026thinsp;=\u0026thinsp;3). Values with various lowercase letters in each row indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Digestive enzyme activities\u003c/h2\u003e \u003cp\u003eBased on the results of the enzyme activity analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. On the 45th day of sampling, a significant difference in the activity of α-amylase, alkaline phosphatase, trypsin, and chymotrypsin was observed between group 2 and the other experimental groups. The study conducted on sea bass over 45 days revealed that the administration of both types of endogenous probiotics led to a significant increase in various enzymes, including trypsin, chymotrypsin, amylase, lipase, alkaline phosphatase, and protease. The observed enhancement in enzyme levels could be attributed to two possible mechanisms. Firstly, probiotic bacteria may secrete a diverse range of enzymes (exoenzymes). Alternatively, the activity of the fish's digestive enzymes (endoenzymes) might increase. Furthermore, certain probiotic bacteria can produce foreign enzymes like cellulase, amylase, protease, lipase, and phytase. These foreign enzymes, in addition to the host's digestive enzymes, contribute to the digestion of organic matter and enhance the overall activity of the fish's digestive enzymes (Del\u0026rsquo;Duca et al.,2013). Our observations are in agreement with those of Ghanei-Motlagh et al., (2021), Adorian et al., (2019), and Reda et al., (2018), who reported increased activities of protease, lipase, and amylase in \u003cem\u003eL. calcarifer\u003c/em\u003e and \u003cem\u003eC. gariepinus\u003c/em\u003e nourished by diets containing \u003cem\u003eBacillus\u003c/em\u003e spp. for 60 days, respectively. The improvement of the activities of digestive protease, lipase, and α-amylase can be partly attributed to the potential of probiotic strains for the production of extracellular enzymes including protease, lipase, and amylase as confirmed in our previous study (Mohammadian et al.,2018). Marlida et al., (2014) found that probiotic bacteria from the humpback grouper's (\u003cem\u003eChromileptes altivelis\u003c/em\u003e) digestive tract positively influenced the growth factors and digestive enzymes in their study. This aligns with the idea that probiotics can enhance the digestive system, leading to better digestion of food particles and ultimately promoting growth in the fish. The findings from studies employing probiotics have demonstrated an augmentation in the breakdown of dietary proteins, fats, and starch (Wang and Xu,2006). Hence, it is plausible that the probiotic bacteria investigated in the current research have enhanced the utilization efficiency of proteins, fats, and carbohydrates within the sea bass diet. Comparable instances exist where gastrointestinal bacteria have the ability to produce extracellular digestive enzymes (Suzer et al.,2011; Son et al.,2009). These instances substantiate the outcomes of the present experiment, indicating an elevation in digestive enzymes specifically α-amylase, trypsin, and lipase in rainbow trout. Lara-Flores et al., (2003) explored the impact of administering a blend of \u003cem\u003eEnterococcus faecium\u003c/em\u003e and \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e alongside the yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e on Nile tilapia, focusing on growth rate and changes in alkaline phosphatase enzyme. Their findings indicated superior growth factors in treatments involving either bacteria or yeast compared to the control treatment. Regarding the alkaline phosphatase enzyme, the yeast treatment exhibited the highest activity level. The authors suggested that the heightened enzyme activity likely signifies enhanced development of brush membranes in fish enterocytes. It is plausible that the probiotic bacteria in the current study exerted a substantial influence on the development of sea bass enterocyte brush-like appendages, contributing to increased fish growth through various mechanisms. In the current investigation, a significant disparity emerged between the groups administered probiotics and the control group to the study's conclusion. Given the optimal rearing conditions and minimal stress experienced by all fish groups during the cultivation period, noteworthy augmentation in the activity of digestive enzymes α-amylase, lipase, trypsin, and chymotrypsin was observed in the probiotic treatments during the final sampling. This effect is likely attributed to the enhanced retention of probiotic bacteria within the digestive system of the fed fish. \u003cem\u003eL\u003c/em\u003e2, and to a lesser extent \u003cem\u003eL1\u003c/em\u003e, seemingly heightened digestive enzyme activity and secretion within the sea bass digestive system, thereby enhancing the capacity for food digestion and absorption. Consequently, this led to an increased specific growth rate. Notably, despite its comparatively lower impact on digestive enzymes, the \u003cem\u003eL1\u003c/em\u003e probiotic exhibited superior growth indicators among the experimental treatments. The outcomes suggest that the \u003cem\u003eL\u003c/em\u003e2 treatment potentially established a dominant intestinal flora in sea bass, with a consequential elevation in lactic acid bacteria influencing growth indicators more profoundly than other bacteria. Another potential factor contributing to the superior performance of L1 may involve the influence of unidentified growth factors inherent in the growth process of sea bass within this specific probiotic treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Innate immune parameters\u003c/h2\u003e \u003cp\u003eThe data related to the serum innate immune response are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The highest and lowest serum lysozyme, anti-trypsin, myeloperoxidase, Respiratory burst activity (NBT reduction), and bactericidal activity levels were in L2, L1, and Control, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, total immunoglobulin was significantly higher in the L2 compared to the L1 and control groups at day 45, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Fish in the L1 group had higher serum total complement than the other treatments. The study demonstrated a significant enhancement in immune responses, including bactericidal, antiprotease, and respiratory burst activities in fish subjected to probiotic-supplemented diets compared to those on basal diets. Specifically, serum bactericidal activity, indicative of the serum's ability to resist bacterial infections, showed elevated levels in probiotic-fed fish over 45 days. Probiotics were found to enhance the host's immune competence by activating various protective mechanisms, as elucidated by Lazado et al., (2010). Furthermore, serum respiratory burst, a phagocyte-mediated killing mechanism, exhibited a significant increase in probiotic-fed fish. Antiprotease activity, impeding pathogen infiltration with protein-degrading enzymes, was significantly elevated in all probiotic-treated groups. Consistent with these findings, prior research by Newaj-Fyzul et al., (2007) indicated that incorporating \u003cem\u003eBacillus subtilis\u003c/em\u003e AB1 in rainbow trout's diet substantially heightened antiprotease activity. Similarly, Kim et al., (2012) reported increased serum antiprotease activity in olive flounder following dietary supplementation with \u003cem\u003eE. fascium\u003c/em\u003e. Myeloperoxidase (MPO) plays a pivotal role in pathogen eradication, and in this investigation, there was a notable augmentation in MPO activity across L1 and L2 groups at this study. Mohammadian et al., (2018) demonstrated that endogenous probiotic supplementation resulted in a significant increase in lysozyme and MPO activities. Lysozyme, possessing antibacterial properties, exhibited heightened activity in fish receiving L1 and L2 in their diet. Lactic acid bacteria have been shown to stimulate immunoglobulin synthesis (Mohammadian et al.,2022; Gopalakannan and Arul,2011 Mohammadian et al.,2021). Similar enhancements in immune responses have been documented in various fish species, including Nile tilapia, pirarucu, catla, red seabream, striped catfish, snakehead, Mrigal, and Japanese eel (Makled et al.,2019; do Vale Pereira et al.,2019; Bhatnagar and Saluja,2019; Dawood et al.,2019; Thy et al.,2017; Talpur et al.,2014; Bhatnagar and Lamba,2017; Lee et al.,2013)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Intestinal histopathology and histomorphology\u003c/h2\u003e \u003cp\u003eIn the present experiment, the height of the villus, average width of the villus, and epithelium thickness were evaluated across different treatment groups. Statistical analyses were performed to examine the potential differences in these parameters between the experimental groups at the end of the testing period. Concerning the height of the villus, width of the villus, number of goblet cells, thickness of the submucosal layer, and epithelium thickness by the end of the experiment, the L2 group exhibited a statistically higher height compared to other treatment groups. In the current investigation, pathological examinations revealed an absence of damage in the prepared sections. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) Groups receiving probiotics with food exhibited reduced damage in microvilli presence compared to the control group, along with decreased dead tissue in the lumen. Noteworthy is the absence of histological changes in intestinal epithelial tissue post probiotic exposure, with no indications of basement membrane connection rupture in intestinal cells. By the findings, Kristiansen et al., (2011) observed that the administration of food containing \u003cem\u003eCarnobacterium divergens\u003c/em\u003e did not inflict damage on intestinal structure. Martinsen et al., (2011) demonstrate that \u003cem\u003eC. maltaromaticum\u003c/em\u003e did not change and had adverse effects on the intestinal structure of cod. Probiotics can bind to the intestine and pass through it without damaging the structure of the intestinal wall, which indicates that their entry method is not destructive. Kristiansen et al., (2011) electron microscope observations indicate no visible difference between the groups in terms of the presence of cell debris in the lumen, increased amount of mucus, the number of bacteria-like particles in the lumen and between microvilli, disruption of microvilli order. and did not show a loss of integrity of strong joints. Normal and intact cell connections, along with a healthy zona adherence area, were observed in all groups, emphasizing the non-destructive impact of probiotics on intercellular junctions. This is crucial, as compromised junctions could serve as an entry point for potential pathogens. The study underscores the significance of intestinal morphology and histometry in assessing probiotic performance in fish, aligning with Ringo et al., (2007) emphasis on intestinal morphology in selecting lactic acid bacteria as fish probiotics. While Lactobacillus species from non-fish sources are generally considered beneficial for fish ( Bagheri et al.,2008) Salma et al., (2011) cautioned against the use of bacteria isolated from Iranian cheese due to severe destructive effects on \u003cem\u003eHuso huso\u003c/em\u003e's intestine. Evaluating endogenous bacteria's impact on intestinal morphology is deemed crucial in probiotic selection. Macroscopic and microscopic images indicate that endogenous probiotics enhance epithelium development in the intestine, confirmed by changes in epithelium surface and the growth of intestinal villi under the probiotic influence. Assessing intestinal tissue, particularly microvilli diameter and size, serves as a valuable indicator of the intestine's physiological state and digestive system health. Increased villi diameter and height signify enhanced absorption levels, indirectly improving food ration utilization efficiency. This information is pivotal for understanding fish's nutritional needs. The measured lengths of intestinal villi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) align with macroscopic observations of the intestine in this study. Histological examinations reveal variations in villi length, with the L2 group exhibiting the most significant increase in villi length after the sampling period. The use of L1 also positively influences villi growth, potentially contributing to the microscopic structural integrity of the intestine and fostering expansion of intestinal epithelial tissue. Over the probiotic supplementation period, villi length increased across all groups, with a more pronounced increase compared to the control group in the treatments receiving a ration containing probiotics. Similar results were noted for other factors such as villus width, thickness of the submucosal layer, epithelium thickness, muscle layer thickness, submucosa thickness, and the number of goblet cells. Consistent with findings from Nakandakare et al., (2013) in Nile tilapia fish, as well as Burrells et al., (2001) in salmon, treatments incorporating probiotics demonstrated a significant difference in villi height in the middle intestine compared to the control group. The augmentation in villi width and the amplification of intestinal goblet cell numbers are conspicuously evident in both probiotic treatments upon course completion. Additionally, parameters such as the thickness of the submucosal layer, epithelium thickness, and muscle layer thickness manifest an increment in both treatments by day 45. Numerous anatomical factors influence absorption levels within the digestive system. Notably, elongated, narrower, and more regular villi, coupled with an increased villus count per area, signify heightened activation of intestinal villi (Heidarieh et al.,2012). In this investigation, the observation of elongated villi in the group administered with a combination of L2 (to a greater extent) and L1 (to a lesser extent) appears promising. However, further scrutiny is warranted, particularly concerning intestinal villi width and epithelium layer thickness, necessitating additional studies to elucidate effects on fish subjected to probiotics. Comparing the effects of the shape of the intestine with the activity of digestive enzymes shows that the most changes in the administration of bacteria occur at the end of the period. The two treatment groups of L1 and L2 had better results compared to the control group, and it should be noted that each of the probiotic bacteria may have different morphological effects in each of the fish species (Ferguson et al.,2010). The increase in the height of villi in treatments containing probiotics is probably due to the improvement of growth conditions and proliferation of lactic acid bacteria under the positive effects of probiotics. In the present study, an increase in the thickness of different tissue layers was observed in the groups treated with probiotics, especially L2 treatment at the end of the period. The anterior and middle intestines, which are the main places of digestion and absorption of food in the intestine, probably indicate the adaptive response of fish to increase the ability to absorb nutrients inside the intestine, and this increase in the diameter of the covering muscle layer is caused by the production of chain fatty acids. Zahran et al., (2014) showed the effect of probiotic use on increasing the length of intestinal villi in tilapia fish. On the other hand, there was no significant effect on the thickness of the muscle layer. Contrary to the present results, Enferadi et al. (2018) investigated the effect of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e on the intestinal morphology of salmon and did not observe a significant change in the height of the villus, the thickness of the epithelium layer, the width of the villus and the thickness of the muscle layer. In this study, after feeding with food containing a probiotic supplement, the number of goblet cells increased at the end of the period in the L2 group. Although there are studies on the increase of goblet cells in the skin and digestive tract after being challenged with infectious diseases (Buchmann and Bresciani,1998), there are few studies on increasing the density of mucous cells in fish. Ramos et al., (2015) investigated the effect of commercial probiotics including Bacillus, Pedicoccus, Enterococcus, and Lactobacillus in different amounts on the intestine of rainbow trout. They found an increase in the area of the anterior intestine and the number of goblet cells in the group containing 3 grams of probiotics per kilogram of food ration and also an increase in hair length in the group containing 1.5 grams of probiotics per kilogram of food ration. Goblet cells in the digestive tract act as the first sensitive layer of fish immune defense factors by forming a viscous watery coating on the surface of the mucus and providing protection against the damage caused (Ring\u0026oslash; et al.,2003).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Intestinal microflora\u003c/h2\u003e \u003cp\u003eIt has been evidenced that probiotics, particularly those derived from the host, can modulate the gut microbiota. However, functional probiotics require to be colonized in the host intestinal tract before exerting their beneficial effects. In this study, no significant alteration in populations of \u003cem\u003eBacillus\u003c/em\u003e spp., total cultivable bacterial counts, and \u003cem\u003eVibrio\u003c/em\u003e spp. was observed between the treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, LAB was significantly higher in the probiotic groups than in the other groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This could be associated with the high survival of these strains within the gastrointestinal tract from which they isolated and their appropriate abilities to adhere to mucus as described in our previous study (Ghanei-Motlagh et al.,2021).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe intestinal populations of total cultivable bacteria, \u003cem\u003eBacillus\u003c/em\u003e spp. and \u003cem\u003eVibrio\u003c/em\u003e spp. in Asian seabass fed or un-fed with probiotics at different time intervals\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInitial (day 0)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 45\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTotal aerobic heterotrophic bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Log CFU/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.006\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eLactic Acid Bacteria\u003c/b\u003e \u003cb\u003e(Log CFU/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;14.7\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;20.31\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003espp. (Log CFU/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eVibrio\u003c/b\u003e \u003cb\u003espp. (Log CFU/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57\u003csup\u003ea,A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eND: No detected. For each parameter, values (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;9) bearing different lowercase letters or different uppercase letters represent significant differences within each row or each column, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Expression gens\u003c/h2\u003e \u003cp\u003eOn the 45th day of sampling, fish fed with the L2 probiotics had higher GMCFC and IL-10 in the gut than those fed the L1 probiotic and control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Fish in L1 and L2 groups had higher gut EGF gene expression values than the control group. The highest TGFβ gene expression value in the gut was in L2, meanwhile, the lowest values were in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Results of the present study showed that all probiotics, applied here, can up-regulate the immune-related gene expression of the \u003cem\u003eL. calcarifer\u003c/em\u003e intestine. probiotic bacteria can influence gene expression, including genes related to the immune system and growth factors. Some studies in aquatic animals, including fish, have explored the impact of probiotics on genes such as interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor, and Transforming Growth Factor Beta (TGF-β). In the present study, feeding with L1 and L2 probiotics induced higher transcription levels of EGF, TGFβ, GMCFC, and IL-10 genes in the gut, which may correlate with better immune and hematological parameters in these groups. The increase in IL-10 mRNA expression may have led to the enhanced expression of pro-inflammatory cytokines such as GMCFC in the \u003cem\u003eL. calcarifer\u003c/em\u003e fed L1 probiotic to balance the immune responses for better immunocompetence. The mechanism of action of probiotics in the expression of interleukin 10 genes is related to their effect on internal signaling pathways in cells. These processes usually involve molecular interactions between probiotics and the surface of host cells. One possible mechanism is that probiotics activate signaling communications by combining with various cells of the gastrointestinal tract. This interaction can lead to the activation of specific signaling pathways, which ultimately leads to increased expression of TGF and interleukin 10 genes. Additionally, probiotics may help improve the gut microbiome. A better microbiome balance can lead to different production of chemical compounds and molecular signals that regulate gene expression. Although the exact mechanism is not yet fully understood, these effects may improve cellular activity and balance the immune system. In this context, Siddik et al., (2022); Choi et al., (2022); Panigrahi et al., (2011) and Abarike et al., (2020) reported administration of a diet with probiotic significantly increased IL-10 gene expression in the head kidney of olive flounder and Nile tilapia. In the present study, the growth performance of sea bass fed with L1 probiotic was proportional to TGFβ gene expression. Probiotics may modulate the immune response and contribute to the regulation of growth factors, potentially promoting a more balanced and beneficial gene expression profile. However, the effectiveness can vary depending on factors like the specific probiotic strains used, the host species, and environmental conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.8.\u003c/em\u003e Relative survival rate after challenge with \u003cem\u003eV. alginolytichus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSeven days after the injection challenge of fish with \u003cem\u003eV. alginolyticus\u003c/em\u003e bacteria, the percentage of survival in unchallenged control groups (100%), challenged control (64.95%), L1 treatment (76.2%), treatment \u003cem\u003eL\u003c/em\u003e2 (80.95%) was obtained (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Statistically, the relative survival percentage of fish fed with both \u003cem\u003eLactobacillus plantarum\u003c/em\u003e bacteria was significantly higher than the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Probiotics increase survival and natural resistance against unfavorable factors. In the current investigation, a notable enhancement in the survival rates of sea bass fish exposed to \u003cem\u003eV. alginolyticus\u003c/em\u003e bacterial challenge was evident in probiotic interventions, notably with L2. This finding aligns with prior research conducted on European migratory eel and Indian carp, where increased survival rates were observed against \u003cem\u003eA. hydrophila\u003c/em\u003e infection after dietary supplementation with Bacillus probiotics (Das et al.,2011). The heightened resistance observed in probiotic treatments is likely attributable to an augmentation in non-specific immune responses influenced by the administration of endogenous probiotics. The augmentation in non-specific immune responses in probiotic-treated groups is evident from the results presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Notably, the introduction of L2 and L1 into the diet of sea bass fish beyond day 45, followed by intraperitoneal injection of \u003cem\u003eV. alginolyticus\u003c/em\u003e bacteria, resulted in enhanced resistance in both probiotic treatments compared to the control group. However, the reduction in losses observed in the group receiving L2 was particularly noteworthy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Clinical signs and bacteriological examination demonstrated the infection of \u003cem\u003eV. alginolytichus\u003c/em\u003e in the mortalities. Son et al. [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e83\u003c/span\u003e] investigated the effect of oral administration of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e on common grouper fish for four weeks and then challenged with Streptococcus species. Probiotic treatment showed 10\u003csup\u003e8\u003c/sup\u003e CFU/gr compared to the control group. Balcazar et al.,( 2006) investigated the ability of three species of lactic acid bacteria (\u003cem\u003eLactobacillus lactis\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e) in inhibiting the adhesion of several fish pathogens (\u003cem\u003eAeromonas hydrophila\u003c/em\u003e, \u003cem\u003eAeromonas salmonicida\u003c/em\u003e, \u003cem\u003eYersinia ruckeri\u003c/em\u003e, and \u003cem\u003eVibrio anguillarum\u003c/em\u003e), and the results showed that \u003cem\u003eLactobacillus plantarum\u003c/em\u003e reduces the adhesion of \u003cem\u003eA. hydrophila\u003c/em\u003e and \u003cem\u003eA. salmonicida\u003c/em\u003e and \u003cem\u003eYersinia ruckeri\u003c/em\u003e, but it had no effect on \u003cem\u003eV. anguillarum\u003c/em\u003e. Increasing the level of non-specific immune defense, reducing the penetration power of pathogenic agents, reducing access to essential nutritional factors, and producing antibacterial substances can be considered among the reasons for increasing resistance to the challenge of pathogenic agents.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative Survival percentage of fish of different treatments after challenge with \u003cem\u003eVibrio alginolyticus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eThe data represent the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three tanks per treatment. Values with various lowercase letters in each row indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe study indicates that \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (L1 and L2) positively influences Asian sea bass by enhancing growth rates, establishing a stable natural flora in the digestive system, increasing digestive enzymes, inducing tissue structure alterations, particularly in the digestive tube, and promoting the expression of growth-related genes. Moreover, these bacteria stimulate the immune system, elevate the expression of immune genes, and enhance bacterial resistance, collectively suggesting potential benefits for fish health. In conclusion, incorporating L1 and L2 into the diet of Asian sea bass could improve growth and immune system functions, potentially enhancing overall fish production quality. The study recommends considering the combined effect of these two bacteria in the fish's diet.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was financed by a grant from the Shahid Chamran University of Ahvaz Research Council (Grant No. vC98.299). The funding body had no role in the design of the study or interpretation of data.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.M. conceptualized the study. T.M. and B .M and and M.T. designed and supervised the study.S.M.E. wrote and T.M. and B.M. revised the manuscript draft. S.M.E. and B.M. performed in vitro experiments related to Acidifier. T.M. and B.M and M.T. conducted in vitro evaluations of test. T.M. and B.M and M.T. conducted data analysis. T. M. and B.M. and M.T. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis research was financed by a grant from the Shahid Chamran University of Ahvaz Research Council (Grant No. vC98.299).\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eData will be available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbarike, E. D., Jian, J., Tang, J., Cai, J., Sakyi, E. M., \u0026amp; Kuebutornye, F. K. (2020). 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MLST, 1-15.\u003c/li\u003e\n\u003cli\u003eZokaeifar, H., Luis Balc\u0026aacute;zar, J., Kamarudin, M. S., Sijam, K., Arshad, A., \u0026amp; Saad, C. R. (2012). Selection and identification of non-pathogenic bacteria isolated from fermented pickles with antagonistic properties against two shrimp pathogens. \u003cem\u003eThe Journal of antibiotics\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e(6), 289-294.\u0026rlm;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotic, Lactobacillus plantarum, Histopathologic, Growth performance, L. calcarifer, V. alginolyticus","lastPublishedDoi":"10.21203/rs.3.rs-3935430/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3935430/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study isolated two strains of intestinal autochthonous bacteria \u003cem\u003elactobacillus plantarum\u003c/em\u003e1 (MH155966.1) (L1) and \u003cem\u003elactobacillus plantarum\u003c/em\u003e2 (MH105076.1) (L2) from the Choobdeh Abadan region. To reveal the effects of these strains of bacteria on the growth performance, digestive enzyme activity, and histopathologic and histomorphometric characterization of the intestine, gut microflora, expression of immune and growth-related genes, and resistance against the disease of \u003cem\u003eLates calcarifer\u003c/em\u003e, examining 9 fish from each treatment, which after euthanasia, was placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium. The experimental design was completely randomized, with 3 treatments: pelleted feed without any probiotic (Diet 1); pelleted feed with \u003cem\u003eLactobacillus plantarum\u003c/em\u003e isolated 1 (L1), \u003cem\u003eLactobacillus plantarum\u003c/em\u003e isolated 2 (L2). For each treatment, 60 juveniles (75\u0026thinsp;\u0026plusmn;\u0026thinsp;12 gr) were distributed in fiberglass tanks (1m\u003csup\u003e3\u003c/sup\u003e) and fed for 45 days. Differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed feed with L1 had higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, Digestive enzyme activities were higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the Digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of EGF, TGFβ, GMCFC, and IL-10 genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 and L2 treatments than in the control. These results indicate that host-derived probiotics (\u003cem\u003eL. plantarum)\u003c/em\u003e have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of \u003cem\u003eLates calcarifer\u003c/em\u003e juveniles at 45 days of culture.\u003c/p\u003e","manuscriptTitle":"Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 22:00:11","doi":"10.21203/rs.3.rs-3935430/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"265e02a6-55bb-4093-9ac2-3b94dc548b3b","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-31T02:14:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-20 22:00:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3935430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3935430","identity":"rs-3935430","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}