Synergistic effects of Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1 as a probiotic functional feed (PFF) in Nile tilapia: Insights into growth, hemato-biochemical, and immune responses in the face of Vibrio challenges

doi:10.21203/rs.3.rs-5202048/v1

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Synergistic effects of Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1 as a probiotic functional feed (PFF) in Nile tilapia: Insights into growth, hemato-biochemical, and immune responses in the face of Vibrio challenges

https://doi.org/10.21203/rs.3.rs-5202048/v1

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Probiotics offer significant health advantages as they enter the digestive system via diet or water intake, playing a crucial role in enhancing immunity, growth, gastrointestinal microbiota, and feed attribute. The main objective of study to focusing in the impact of Probiotic Functional Feed (PFF) on Nile tilapia (Oreochromis niloticus) exposed to challenges from Vibrio harveyi and Vibrio parahaemolyticus. The investigation aims to analyze the genes linked to immunity, hemato-biochemical indices, and the immunological response in tilapia. PFF is a vital component of fish feed production, providing suitable nutrition for various ages and stages to promote healthy growth. The study comprises five treatments: Negative Control (NC), Positive Control (PC), Rossellomorea marisflavi sp. DAS-SCF02, Agrococcus sp. RKDAS1, and a dual probiotic mix. A total of 150 Nile tilapia juveniles, weighing 2.56 ± 1.26 g, were administered PFF in triplicates. Significant improvements were observed in hematological indices, encompassing white blood cells (WBC), hemoglobin (Hb), red blood cells (RBC), hematocrit (Htc), and blood performance (BP) in probiotic-treated groups compared to control. Biochemical analysis revealed lower levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in fish fed with probiotics, accompanied by increased total protein, albumin, and globulin levels. Furthermore, probiotic-fed fish exhibited heightened blood glucose, total cholesterol, and triglyceride levels. Immunological assessments demonstrated increased lysozyme activity, intracellular superoxide anion production, reactive nitrogen species synthesis, and myeloperoxidase activity in probiotic-fed groups. Immune gene expression analysis revealed up-regulation of stress response, cytokine signaling, and immune defense-related genes (HSP70, IL-1β, I C3, IFN-α, IFN-γ, GF1, GH, IL-1, and Lyz). In a Vibrio challenge study, probiotic-fed fish exhibited improved survival rates, underscoring the protective effects of probiotics against bacterial infections. Overall, this research underscores the multifaceted benefits of probiotic supplementation in enhancing the health and immunity of tilapia.

Biological sciences/Biotechnology

Biological sciences/Microbiology

Biological sciences/Molecular biology

Probiotic Functional Feed

Nile tilapia

Hemato-biochemical indices

Immunological response

Vibrio challenge

The fish farming sector is experiencing steady growth in response to the world's increasing population. The adoption of intensive aquaculture strategies aims to meet the rising demand, with the Nile tilapia (Oreochromis niloticus), currently ranking as the third-highest-farmed fish globally in terms of volume (4.5 Mmt) due to continuous advancements in farming practices [1]. Despite the success of tilapia farming, challenges have emerged, particularly the excessive feed requirements [2–4]. Consequently, there is a growing focus on researching feed formulations and feeding methods to enhance fish gastrointestinal and health while continuing a balanced inhabitation of stomach bacteria. These approaches aim to boosting productivity in the sector of fish farming.

As aquaculture intensifies, the facility's capacity to address various illnesses and health issues becomes crucial. Ongoing investigations into fish immune responses and physical development seek to develop sustainable and environmentally friendly methods to ensure the well-being of the fish [5–8]. Notably, research on immune-stimulating probiotics for fish healthcare shows promising potential in boosting immunity and treating diseases. These immunopotentiators can enhance fish growth, immunological response, intestinal health, and fillet quality [5, 8, 9]. Probiotics, active microbes, can thrive and proliferate in the animal's stomach, contributing to its overall well-being [10–13]. They promote fish development by improving gastrointestinal architecture and microbial diversity, influencing the gastrointestinal ecology through the secretion of extracellular digestive enzymes like protease and amylase and the production of compounds such as short-chain fatty acids [14].

Researchers have proven the efficacy of functional dietary additives, highlighting the detrimental effects of excessive excreta levels on fish and shrimp. Evidence suggests that nutritive yeast hydrolysate and baker's yeast supplements can improve the hostility of Litopenaeus vannamei to nitrogen toxicity [15]. In laboratory-cultured common carp (Cyprinus carpio) juveniles, a probiotic functional feed containing Pediococcus acidilactici and formic acid influenced growth, blood biochemical parameters, immune gene upregulation, and survival [16]. Rossellomorea marisflavi, an aerobic Gram-positive organism, exhibits non-mobile filaments, non-swollen spores, and terminally oval endospores [17]. When cultured on TSA (tryptic soy agar, M1968-Himedia, Mumbai, India) plates complemented with 2.5% NaCl (w/v) at pH 7.0 and 37°C for 24 hours, this strain demonstrated robust growth conditions, thriving between pH 6.0 and 9.0, at temperatures ranging from 15 to 45°C, and tolerating NaCl concentrations from 0 to 25% (w/v) [17].

The genus Rossellomorea, proposed by [18], is closely related to the Lactobacillaceae genus of lactic acid-producing bacteria and belongs to the Bacillaceae family within the phylum Firmicutes. Notably, Rossellomorea was part of a bacterial consortium that enhanced the potential of the halophyte Arthrocaulon macrostachyum for phytoremediation applications by improving seed development, growth, and heavy metal preservation in the root system, particularly in heavy metal-polluted soils [17, 19, 20, 21]. Agrococcus lahaulensis, an aerobic, non-acid-fast, Gram-positive bacterium, forms distinctly margined, opaque, round, lemon-colored colonies on TSA media. The colonies range in size from 0.7 to 3.0 mm, exhibit resilience to up to 7.0% NaCl, and thrive optimally at temperatures ranging from 25 to 37°C. Growth occurs within the pH range of 6.0 to 10.0, with the ideal pH being 8.0 [22]. Agrococcus sp. SD01-s17, a bioactive variant of Agrococcus, possesses diverse pharmacological and biocidal activities. It functions as an antioxidant, demonstrates anticancer, antibiotic, and antifungal properties, and has the potential to inhibit the growth of harmful bacteria, as reported by [23]. The antibacterial properties of Agrococcus lahaulensis against S. aureus and B. cereus [24]. Notably, the combination of Rossellomorea marisflavi sp. and Agrococcus lahaulensis sp. has not been previously reported for improving fish health. This groundbreaking discovery indicates that the production of sufficient quantities of antagonistic bioactive properties against pathogenic bacteria and their infections can enhance the aquatic environment, promoting fish health by boosting immunity.

Preparation of Nutrient-Enriched Probiotic Feed

The probiotic functional diet, designed to provide essential nutrients for juvenile tilapia, utilized feed materials sourced from CP Aquaculture (India) Pvt Ltd at 104, GNT Road, Nallur, Red Hills, Chennai, Tamil Nadu, India. The materials underwent crushing and sieving before meticulous blending. Dried ingredients were thoroughly mixed before the addition of liquid components. Employing a feed processing machine, the feed was produced, cut into appropriate sizes, and subjected to a two-hour oven-drying at 60°C. After cooling, the feed was sealed in plastic bags for storage until required (Table 1).

Table 1

PFF nutritional preparation and organic composition (g/kg).
Ingredients	CPF1	PFF2	PFF3	PFF4
Soybean meal	448	448	448	448
Yellow corn	170	170	170	170
Corn gluten	60	60	60	60
Rice bran	100	100	100	100
Wheat bran	150	150	150	150
Fish meal	40	40	40	40
Soya oil	1	1	1	1
Fish oil	1	1	1	1
Vitamin C	5	5	5	5
Vitamin & Mineral mix	25	25	25	25
Chemical analysis (g/kg)
Dry matter	895.3	895.3	895.3	895.3
Crude Protein	293.2	293.2	293.2	293.2
Crude Lipid	60.6	60.6	60.6	60.6
Fiber	257	257	257	257
Ash	50.7	50.7	50.7	50.7
200g of Rossellomorea marisflavi sp. (DAS-SCF02)	0	1×10⁴	0	0
200g of Agrococcus sp. (RKDAS1)	0	0	1×10⁶	0
200 + 200g of Dual strain mix (Rossellomorea marisflavi sp. (DAS-SCF02) + Agrococcus sp. (RKDAS1)	0	0	0	1×10⁷

Probiotics culture preparation

The probiotics were derived from two novel strains of bacteria: Agrococcus spp. RKDAS1 and Rossellomorea marisflavi spp. DAS-SCF02, which were isolated from Indian Snakehead fish (Channa straiata) in freshwater lakes and the Tamiraparani River at Tirunelveli, and sludge from the Muttukadu boat house in Kanchipuram, Tamil Nadu. Isolates of Agrococcus spp. RKDAS1 and Rossellomorea marisflavi spp. DAS-SCF02 were cultivated for a full day in NB (nutrient broth) to produce the probiotic cultures. The probiotic cells were centrifuged, cleaned, and suspended in 0.85% saline solution. Using a spectrophotometer, their concentrations were then adjusted to an absorbance OD of 600. Then, the washed probiotic strain suspensions were added to the basal feeds in a ratio of 20: 100 (w/w) between probiotic strain suspensions and basal supplementations for sustaining probiotics viability [25, 26].

Experimental Grouping and Preparation of Probiotic-Enriched Feeds

The study involved the production of four distinct feed types: Probiotic feeds PFF2, PFF3, and PFF4 incorporated probiotic isolates, namely Agrococcus spp. RKDAS1 and Rossellomorea marisflavi spp. DAS-SCF02, along with dual probiotic strains mix containing Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1, respectively. The control feed (CF1) served as the baseline without probiotic additives. Each experimental group consisted of three replicates, with each tank accommodating ten tilapia fish. The base feed underwent autoclaving, and subsequent blending with the prepared probiotic culture was followed by serial dilution with a 0.85% sterile saline solution to quantify the probiotic concentration in the feed. To enumerate viable probiotics, 100 µL samples from specified dilutions were cultured on nutrient agar plates and incubated for 24 hours. After preparation, all feeds were stored under refrigeration to maintain the probiotic dosage integrity [27].

Experimental design

Fish collection

Juvenile Oreochromis niloticus was obtained from the Freshwater Aquaculture Sector of the University of Fisheries, weighing 2.56 ± 1.26 g. Before the experiment, the general health of each animal was evaluated based on its swimming ability in the tank, regular feeding patterns, the absence of blemishes, the presence of undamaged and vibrant scales, the absence of protruding eyes, and the integrity of fins without tears or ragged edges

Feeding Practices and Maintenance Protocols in a Juvenile Fish Study

As part of Probiotic functional feed (PFF) trial, the fish were hand-fed a basal diet twice a day after being placed into ten twenty/100-L plastic tanks. They underwent a seven-day acclimatization phase to adapt to the experimental environment. The experiment divided into four groups, CPF-1 (control group, diet included solely of basal fish feed), the 20% of PFF2 (Rossellomorea marisflavi sp. (DAS-SCF02–1×10⁴), PFF3 (Agrococcus sp. (RKDAS1-1×10⁶), and PFF4- (DAS-SCF02–1×10⁴ + RKDAS1 (1×10⁷) respectively, were equally distributed among each treatment group. Each tank housed twenty juvenile fish in static water, with a routine refresh of approximately half the water volume through a flow-through aquarium system and clearing of accumulated excrement was conducted. The probiotics functional feeds (PFF2, PFF3, and PFF4) were administered for eight weeks, providing the animals with 3% of their daily feed allocation in three meals at 9:00, 15:00, and 21:00 [28].

Comprehensive Assessment of Hematological, Biochemical, and Environmental Parameters in Tilapia during Long-Term PFF Supplementation

At 20-days intervals, various hematological and biochemical indices were assayed, including total albumin, globulin, hemoglobin (Hb), albumin–globulin ratio, total serum protein, total leukocyte count (TLC), total erythrocyte count (TEC), acetylcholine esterase (AChE), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and total adenosine triphosphatase (ATPase). The water quality was maintained within acceptable limits, and environmental indices such as dissolved oxygen (O₂), pH, ammonia, and nitrite were assessed using readily available kits (Water Quality Test Kit - HI3817) during the investigation. The water temperature was maintained between 28 and 29°C.

Hemato-biochemical indices assessment

In order to collect plasma and perform enzyme assays, the fish were anesthetized with 100 mg/L of 3-aminobenzoic acid ethyl ester (MS-222, Sigma-Aldrich, St. Louis, MO, USA) on days 20, 40, and 60 of the experiment. The first portion of the blood sample was obtained using an anticoagulant 10% ethylene diamine tetra acetate (EDTA) to estimate the hematocrit (Htc) and white blood cells (WBCs). To harvest plasma samples, five fish from each treatment duplicate were euthanized. Tissue homogenization was carried out in a cold sucrose buffer (0.25 M) using a Teflon-coated mechanical tissue homogenizer (Remi, India), the samples were centrifugation for 10 minutes at 5000 g at 4°C followed by lysis. The enzyme activity was assessed using the supernatant obtained as the enzyme resource. All steps of the enzyme synthesis process were conducted in a refrigerated environment, with sample dilution performed where necessary.

For blood extraction, a sterilized 2-milliliter BD syringe cleaned with EDTA buffer (2.7%) was utilized. Blood was drawn into small glass vials containing 20 µl of EDTA buffer (2.7%) as an anticoagulant.

The TEC and TLC were determined according to the method outlined by [29]. A hemocytometer (Feinoptik, Germany) was employed for cell counting, with results reported as follows:

\(\:RBC\:Count=\:\frac{NrX\text{10,000}}{{mm}^{3}}\)\(\:WBC\:Count=\:\frac{500}{{mm}^{3}}\)

Here, Nr represents the total number of RBC measured in each square of the hemocytometer, and Nw indicates the total amount of WBC determined in each square. The factor acquired after accounting for the initial dilution factors is 10,000.

The blood Hb content was determined using Darbkins fluid and the Cyanmethemoglobin technique with a commercial kit (Qualigens, Mumbai, India).

Hematic indices

The blood collection procedure involved using a 2 mL BD sterilized syringe with 0.2 mL of anticoagulant, drawing blood from the caudal part of the fingerling fish, and transferring it to a fresh 1.5 mL Eppendorf centrifuge tube. The blood was permitted to clot for 45 minutes at room temperature with the tube tilted, followed by a 30-minute incubation at 4°C. Subsequently, the tube was centrifuged for 10 minutes at 3000g at 4°C. Blood plasma was then collected in sterile Eppendorf centrifuge tubes and analyzed using Qualigens diagnostic kits in a semi-automated analyzer (AR 601, Qualigens, Mumbai, India) for various serum indices. The examined serum indices included LDL, HDL, triglyceride, albumin (using the bromocresol green binding method) (ALB), cholesterol (CHO), and total serum protein (using the biuret method employing biuret reagent and buffered dye reagent). Globulin - albumin ratios were calculated by dividing albumin concentration by globulin content, and globulin content by albumin concentration.

Immunological evaluates

Lysozyme activity

Lysozyme performance was evaluated with slight modifications following the procedure outlined by Parry et al., 1965 [30]. In brief, a 96-well microplate was filled in triplicate with 25 µL of plasma. Subsequently, each well received 0.2 mg mL^− 1 of Micrococcus lysodeikticus solution in a buffered sodium phosphate solution (pH 6.2), totaling 175 µL. The reaction was monitored using a spectrophotometer set to measure reactions at 540 nm, with readings taken and recorded every minute for ten minutes. The blood activity of lysozyme was quantified as a 0.001 min^− 1 reduction at 540 nm and reported in units of mL^− 1.

Intracellular superoxide anion (SOA)

The assessment of intracellular superoxide anion (SOA) and respiratory burst activity involved conducting NBT (nitroblue tetrazolium) reduction reactions, adapted from the Secombes method (Secombes, 1990). In brief, microplates (96-well) were utilized to contain three batches of WBCs (6 × 10^6 cells). To each well, 25µL of NBT was added, and the plates were protected at room temperature for two hours. After the incubation, 150µL of absolute methanol was introduced to each well, and the residual fluid was discarded. Subsequently, the wells underwent repeated cleaning with a 70% methanol solution. Finally, 100µL of DMSO and 150µL of 2M KOH were added to each well. Following thorough mixing, the absorbance value, indicative of the reaction, was measured at 540 nm using a UV-Vis spectrophotometer (201/220, Thermo Scientific).

Production of reactive nitrogen species (RNS) analysis

The Griess reagent technique, which is related to the change of nitrite from nitric oxide, was used to assess the nitric oxide (NO) in the plasma of tilapia [31]. Using a conventional curve representing the level of nitrate in the serum of tilapia, the amount of nitrite present was determined.

MPO (myeloperoxidase)

The MPO activity in plasma was assessed using [32]. An absorption change was considered to be one unit, and the activity was represented as U mg plasma^− 1.

Immune-related gene expression

Isolation of RNA and cDNA Construction

For total RNA isolations, liver samples were dissected from three animals per treatment group. A concentration of 20 ng µL^− 1 was targeted for liver RNA. Total RNA was isolated using a commercial Kit (RNA mini kit, Cat No. 74,104, Qiagen, Germany) according to the manufacturer's guidelines. The purity of the RNA was assessed through gel electrophoresis (1.2% agarose gel) and NanoDrop spectrophotometry (NanoDrop 2000, Thermo Scientific). Subsequently, cDNA synthesis was performed using a cDNA RT Kit (Applied Biosystems, Cat# no. 4368813, USA), adhering to the manufacturer's instructions.

Quantitative (qPCR) RT-PCR examination

Quantitative RT-PCR analysis was conducted using the Applied Biosystems 96 Real-time qPCR System, USA, to assess the expression of genes, including β-actin household genes, hsp70, IL-1β, IC3, TNF-α, IFN-γ, GF1, GH, IL-1, and Lyz. The DNA primer sequences used for amplification are presented in Table 2. The SYBR green technique with the SensiFast SYBR Lo-Rox kit (Bioline) was employed for RT-PCR. Amplification conditions consisted of 45 cycles: 10 seconds at 95°C, 30 seconds at 63°C, and 30 seconds at 72°C. Subsequently, the 2^−∆∆CT method was applied to determine the relative expression levels of the target genes.

Table 2

List of primers employed to quantify immune relative gene expression.
Gene	Sequence (5′-3′)	GB. Accession No.	Annealing Temp. (°C)	R²	Efficiency (%)	A. size (bp)
β-actin	F:CAGCAAGCAGGAGTACGATGAG R: TGTGTGGTGTGTGGTTGTTTTG	XM_003455949.2	62	0.994	19.2	136
Interleukin-1β (IL-1β)	F:CAAGGATGACGACAAGCCAACC R: AGCGGACAGACATGAGAGTGC	XM_019365844.2	60	0.991	96.66	149
Interleukin-IL1	F:CTGTGAAGGCATGGGTGTGGAG R:TCGCAGTGGGAGTTGGGAAG	NM_001279704.1	60	0.979	96.49	111
complement component (IC3)	F: GGTGTGGATGCACCTGAGAA R: GGGAAATCGGTACTTGGCCT	XM_013274267.2	60	0.998	97.22	196
Heat shock protein 70 (Hsp70)	F: CATCGCCTACGGTCTGGACAA R: TGCCGTCTTCAATGGTCAGGAT	FJ207463.1	62	0.995	99.15	238
Tumor necrosis factor- α (TNF-α)	F:AAGCCAAGGCAGCCATCCAT R:TTGACCATTCCTCCACTCCAGA	NM_001279533.1	58	0.993	98.67	184
Interferon (IFN-γ)	F: TGGGTGGTGTTTTGGAGTCG R: TAGCGAGCCTGAGTTGTTGG	NM_001287402.1	60	0.998	99.54	138
Growth factor (GF1)	F-AAGGGAAGCAGCAGCAGTTGTG R-CGTCCATGCCGTTAGCCTTGAG	NM_001279708.4	58	0.989	98.96	151
Growth hormone (GH)	F:ACATCATCAGCCCGATCGAC R:TCAGCAGCAAGATTCCCGTT	XM_003442542.5	62	0.994	98.38	183
Lysozyme (Lyz)	F-AGGGAAGCAGCAGCAGTTGTG R-CGTCCATGCCGTTAGCCTTGAG	XM_003460550.2	60	0.997	94.98	107
F: forward; R: Reverse; GB: Gene bank; Temp: Temperature

Vibrio challenge

The Vibrio strains of V. harveyi and V. parahaemolyticus were isolated from the infected Tilapia fish at Ramayanpatti, in the Tamil Nadu district of Tirunelveli, India. The conventional morphological, biochemical, and pathogenicity assays identified the Vibrio bacterial isolates. The isolates were pre-enriched with an alkaline peptone solution (APS) before being diluted in conventional saline (0.85% NaCl w/v). Each isolate was surface dispersed on three agar media: TCBS (thiosulphate citrate bile salt sucrose agar), SWC (seawater complex agar), and Vibrio specific agar medium (VSAM). A dark room at 30°C was the perfect temperature for finding bio-luminous colonies on SWC agar. The Vibrio isolates were compared to strains of V. harveyi (MTCC 3438) and V. parahaemolyticus (MTCC 443) as positive and negative controls, respectively, and then further confirmed by PCR. The PCR confirmed Vibrio isolates were used in this study. To prepare separately fresh V. harveyi and V. parahaemolyticus, a single colony of Vibrio was inoculated into Nutrient Broth with 2% of NaCl and cultured for 24 h at 30°C. Cell harvesting was performed by centrifuge at 5,000 rpm and 4°C for 10 minutes, after that three washing and re-suspending of the cells in a 0.85% saline buffer. The suspension of V. harveyi and V. parahaemolyticus was modified to 106 CFU/ml with 0.85% saline buffer before injection. After the feeding trial, ten fish from each group were randomly selected and intraperitoneally injected with 0.1 ml of V. harveyi and V. parahaemolyticus (10⁶ CFU/ml) based on the procedure outlined by Fatima et al., 2022 [33].

Statistical analyses

To assess variances among treatments, we employed an ANOVA (one-way analysis of variance) along with the Duncan multiple range test. A significance level of 0.05 was set for the analyses. The standard deviation was calculated using a similar approach.

Hematological indices

The Fig. 1 depicts each of the four treatment groups and their respective hematological indices findings. The evaluated hematological indices exhibited significantly increased levels (P < 0.05) in the comparison of the fish treated with all three types of probiotics to those treated with an untreated diet. Fish raised in the Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1 (PFF3) treatment demonstrated much greater levels (P < 0.05) of Hb, WBC, RBC, Htc, and BP relation to the control group (CF). Likewise, after the trial, adding PFF4 to the feed led to a substantial increase (P < 0.05) in the numbers of neutrophils, lymphocytes, and monocytes compared to the control group. The present study results are agreed well with the reports of [34, 35, 36].

Hematological Biochemical Indices

In tilapia supplemented with probiotics, specifically Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. DASRK1 (PFF3) and Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. DASRK1 (PFF4), serum levels of ALT, and AST were lower (Fig. 2a). However, the CF3 diet resulted in the least significant (P < 0.05) values of AST and ALT. Fish-fed PFF2, PFF3, and PFF4 exhibited higher serum concentrations of total protein, albumin, and globulin compared to other diets (P < 0.05). The CF4 diet, containing Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. DASRK1 showed the highest levels of total globulin, albumin, and protein.

Furthermore, tilapia fish treated with three types of probiotics functional feed (PFF) additive mixed probiotics had expressively (P < 0.05) greater glucose, total cholesterol, and triglyceride levels than the control group. In comparison to fish fed the CP diet, all groups administered dietary PFF showed significantly lower plasma TG levels (P < 0.05), and all PFF-fed fish exhibited little variation in CHO or TG (P > 0.05). Additionally, with dietary supplementation of PFF, plasma LDL content was lowered, although no significant difference was observed (P > 0.05). The dual probiotics mix added to the feed dramatically raised albumin, globulin, and total protein levels in the serum, with PFF2 and PFF3 showing superiority over the CF1 control group (Fig. 2b). The present study results are well agreed with [37, 38] studies. Furthermore, one plausible explanation for this lowering effect on plasma lipid profiles could be the fermentation of indigestible carbohydrates derived from intestinal food to produce short-chain fatty acids, inhibiting the synthesis of cholesterol in the liver and/or returning cholesterol to the liver [39].

Immunological indices

Lysozyme Activity

Lysozyme, a bactericidal peptide crucial for the fish's innate immune response, plays a significant role in inhibiting biofilm formation by promoting phagocytes and the complement system [40]. It also prevents microbial adhesion and colonization [41]. In this study, during the 20th, 40th, and 60th days of dietary probiotic feeding, tilapia serum exhibited a substantial enhance (p < 0.05) in lysozyme related to the untreated experiment (Fig. 3a). The PFF4 group, in particular, demonstrated a significant (p < 0.05) boost in serum lysozyme activity after the 60th day of dietary probiotic feeding. Notably, plasma exhibited the highest lysozyme activity after the 60th day when compared to the 40th day.

Intracellular superoxide anion (SOA)

Following the 20th, 40th, and 60th day of dietary probiotic feeding, the SOA production in serum improved significantly (p < 0.05) in the dietary probiotic experiments in assessment with the untreated fish (Fig. 3b). In comparison with serum, SOA generation had been boosted in all dietary probiotic experimental feeds. In both the dietary probiotic experiments, the highest SOA generation occurred following the 60th day of feeding instead of the 20th day. The PFF4 group had the greatest enhancement, whereas the CP1 showed minimal enhancement. [42, 43].

Production of Reactive Nitrogen Species (RNS) Analysis

On the 20th, 40th, and 60th days of the dietary probiotic feeding experiment, there was a significant improvement in RNS synthesis in the serum of tilapia (p < 0.05), as compared to the untreated fish (Fig. 3c). Aligned with the formation of Reactive Oxygen Species (ROS), all dietary probiotic experiments exhibited stronger plasma RNS production on the 60th day than on the 40th day. The PFF4 feed demonstrated the highest activity of RNS generation, while the untreated fish showed a limited quantity. Similar results were observed by [44].

MPO (Myeloperoxidase)

After the 20th, 40th, and 60th days of dietary probiotic experiment feeding, dramatic improvement in MPO activity in plasma was observed (p < 0.05), contrasting with the untreated group (Fig. 3d). The MPO activity in serum increased for dietary PFF2, PFF3, and PFF4. Across all PFF2, PFF3, and PFF4 groups, the peak MPO activity was observed after the 60th day of feeding compared to the 40th day. These findings align well with the study conducted by [34].

Immune gene expression

Probiotics have a well-established ability to non-specifically alter the immune system [45, 46]. In the present study, supplementation with Rossellomorea marisflavi spp. DAS-SCF02, Agrococcus spp. DASRK1, Rossellomorea marisflavi spp. DAS-SCF02, and Agrococcus spp. DASRK1 increased the expression of HSP70, IL-1β, IC3, IFN-α, IFN-γ, GF1, GH, IL-1, and Lyz genes, with the highest expression observed in fish fed a PFF4 diet. Figure 4 (a-i) presents the transcript of immune-related gene expression experiments conducted on tilapia liver. In the liver of tilapia fed CF4 and those fed Rossellomorea marisflavi spp. DAS-SCF02, Agrococcus spp. DASRK1 additive diets (CF2, CF3, and CF4), HSP70, IL-1β, IC3, IFN-α, IFN-γ, GF1, GH, IL-1, and Lyz genes were significantly up-regulated (P < 0.05) (Fig. 4a-i). Compared to CP1 tilapia fingerlings, those fed with PFF3 and PFF4 showed up-regulated expression of the immune gene SOD (P < 0.05). The levels of TNF-α gene expression in fish-fed probiotic feed and all Rossellomorea marisflavi spp. DAS-SCF02, Agrococcus spp. DASRK1 treatment groups were considerably higher (P < 0.05) when compared to the other treatment groups and control group (CF2 and G3). Additionally, fish-fed CF4 exhibited increased TFN-γ gene expression compared to the control group and all treatment groups (P < 0.05).

Vibrio challenge study

Twenty-five days after PFF2, PFF3, and PFF4 were exposed by intraperitoneal administration of fingerling tilapia fish with respectively V. harveyi and V. parahaemolyticus, the Vibrio challenge test was carried out, as illustrated in Fig. 5a & b. Seven days’ post-challenge, the relative percentage survival (RPS) and cumulative mortality were recorded. Fish fed with dietary probiotics PFF2, PFF3, and PFF4 for 24 days exhibited a significantly reduced cumulative mortality rate compared to those fed with CF1. Cumulative mortality rates for fish fed CF1, PFF2, PFF3, and PFF4 were 92.44%, 13.65%, 11.18%, and 10.77%, respectively, at the end of the challenge test. A comparison of Tilapia fish that received functional probiotics versus those that did not show no significant difference in survival rates (P > 0.05). Fingerling fish with the infection displayed abnormal diving, darker pigmentation, and a lack of appetite. Moreover, it was observed that the hemorrhages on their bodies, both in flesh and livers, were more pronounced than those observed in typical fish. The results of the Vibrio challenge test indicate that the dietary probiotics PFF2, PFF3, and PFF4 can significantly reduce the cumulative mortality rate in fingerling tilapia fish infected with V. harveyi and V. parahaemolyticus. This was consistent with previous studies that have shown the beneficial effects of probiotics in enhancing shellfish health and disease resistance [47]. According to a recent study by [48], intraperitoneal exposure to kill V. harveyi enhanced the resistance and antibody response of marine red hybrid tilapia to Vibriosis. However, it is worth noting that there were no significant differences in survival rates between fish that received probiotics and those that did not, suggesting that other factors may also influence fish survival in the presence of these pathogens. Further research is needed to explore the potential mechanisms underlying the observed effects of probiotics on fish health and to optimize their application in aquaculture practices.

Survival rate after Vibrio challenge (SR)

On days five and six following the V. harveyi and V. parahaemolyticus challenge, the tilapia fish began to perish. The affected fish exhibited increased mucus discharges, scale detachment, and hemorrhages on numerous areas of their external body surface. Autopsy investigation revealed a pale, swollen liver with colorless nodules dispersed across its surface and a bloated gallbladder. The internal organs of the diseased fish were used to re-isolate Vibrio sp. In a dose-dependent manner, tilapia fish raised with any of the probiotic feed treatments-PFF2, PFF3, and PFF4-showed greater survival levels than those raised in the un-treatment fish group. Significant differences in survival rates were observed between the PFF administrated groups and the control after two weeks of the Vibrio sp. challenge [49, 50]. Notably, a dual probiotic mixed feed (PFF4) led to an increased survival rate among tilapia fish exposed to Vibrio sp. compared to the control fish. Fish fed with PFF4 exhibited the highest survival rate (89.23%), followed by those fed with PFF3 (88.82%), PFF2 (86.35%), and CF1 (7.56%), as shown in Fig. 5b. Figure 6 showed the schematic representations of Probiotic Functional Feed (PFF) on Nile tilapia (Oreochromis niloticus) exposed to challenges from Vibrio harveyi and Vibrio parahaemolyticus.

In conclusion, the supplementation of Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1 probiotics significantly improved hematological, biochemical, and immunological parameters in tilapia. The observed increases in hematological indices, including Hb, WBC, RBC, Htc, and BP, suggest enhanced overall fish health. Biochemical analysis indicated improved liver function, as reflected by lower ALT and AST levels and increased total protein, globulin, and albumin concentrations. Furthermore, the immunological responses, as evidenced by increased lysozyme activity, superoxide anion production, reactive nitrogen species synthesis, and myeloperoxidase activity, point towards enhanced immune defenses in probiotic-fed fish. The gene expression analysis revealed the up-regulation of genes associated with stress response, cytokine signaling, and immune defense, indicating the activation of key pathways (hsp70, IL-1β, I C3, IFN-α, IFN-γ, GF1, GH, IL-1, and Lyz) in response to probiotic supplementation. Importantly, the probiotic-fed fish exhibited improved survival rates in a Vibrio challenge study, demonstrating the practical relevance of these findings in disease resistance. Overall, this study provides valuable insights into the comprehensive benefits of probiotic supplementation in tilapia aquaculture, emphasizing its potential to enhance fish health, immunity, and resilience against bacterial challenges. These findings contribute to the growing body of knowledge supporting the sustainable and effective use of probiotics in aquaculture practices

Ethics approval and consent to participate

The experiment was conducted following the protocol involving animal use approved by the experiment was ethically reviewed and approved by the Department of Animal Science and Animal Ethical Committee, through the Animal and Welfare Ethical Review Body by the Manonmaniam Sundaranar University Animal Care and Use Committee MSU-ACUC (BP, PhD, Reg. No. 17214012272124). All fish handling procedures and regulations followed the ARRIVE guidelines for Animal Care and Use. Furthermore, all relevant organizational and government rules and regulations governing the ethical use of the experimental animals were followed. Written informed consent was obtained from the owners of all animals involved in the study.

Competing Interests

No competing interest

Author Contribution

B.P.: Formal and experimental analysis, Documentation & Formal analysis, Writing– original draft. R. T.: Investigation, Writing, review & editing – original draft, Conceptualization, Investigation, Visualization, Project administration. R. P.: Data curation & Review, K. S.: Supervision, Project administration, editing & review. K. A. A.G. & C. K.: Formal analysis, writing, review, & editing. All authors have read and agreed to the published version of the manuscript.

Acknowledgement

The authors acknowledge the Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India for their constant support and the grant support from UGC DR. D. S. Kothari Postdoctoral Fellowship (Normal Fellowship): No. F. 4-2/2006 (BSR)/BL/19-20/0298 New Delhi. The authors express their sincere appreciation to the Researchers supporting Project Number (RSP2025R48), King Saud University, Riyadh, Saudi Arabia.

Data Availability

The results presented are adequate to support the conclusion of this study. However, the lead author (B. P.) can provide extra data upon request.

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No competing interests reported.

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Synergistic effects of Rossellomorea marisflavi spp. DAS-SCF02 and Agrococcus spp. RKDAS1 as a probiotic functional feed (PFF) in Nile tilapia: Insights into growth, hemato-biochemical, and immune responses in the face of Vibrio challenges

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Preparation of Nutrient-Enriched Probiotic Feed

Probiotics culture preparation

Experimental Grouping and Preparation of Probiotic-Enriched Feeds

Experimental design

Fish collection

Feeding Practices and Maintenance Protocols in a Juvenile Fish Study

Comprehensive Assessment of Hematological, Biochemical, and Environmental Parameters in Tilapia during Long-Term PFF Supplementation

Hemato-biochemical indices assessment

\(\:RBC\:Count=\:\frac{NrX\text{10,000}}{{mm}^{3}}\)\(\:WBC\:Count=\:\frac{500}{{mm}^{3}}\)

Hematic indices

Immunological evaluates

Lysozyme activity

Intracellular superoxide anion (SOA)

Production of reactive nitrogen species (RNS) analysis

MPO (myeloperoxidase)

Immune-related gene expression

Isolation of RNA and cDNA Construction

Quantitative (qPCR) RT-PCR examination

Statistical analyses

Results and Discussion

Hematological indices

Hematological Biochemical Indices

Immunological indices

Lysozyme Activity

Intracellular superoxide anion (SOA)

Production of Reactive Nitrogen Species (RNS) Analysis

MPO (Myeloperoxidase)

Immune gene expression

Conclusion

Declarations

Ethics approval and consent to participate

Competing Interests

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

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