Introduction

The aquaculture industry has a key role in human nutrition and in meeting the future demand for aquatic food products and currently accounts for 50% of global fish consumption. The rapid increase in the human population has compelled the aquaculture industry to intensify its efforts to sustainably meet growing fish demand (Boyd et al. 2020). However, culturing a large number of fish in limited space leads to frequent collisions, competition for food, and degradation of water quality, which lead to oxidative stress in fish. Stress causes fish to exhibit depressed growth performance, a weakened immune system, and vulnerability to pathogens, which leads to frequent disease occurrence and causes huge economic losses in aquaculture (Martos-Sitcha et al. 2020). In the past, antibiotics were utilized to prevent bacterial diseases; however, due to serious health concerns such as antibiotic resistance and bioaccumulation, antibiotics have been banned in aquaculture (Manage 2018). Therefore, researchers have been actively seeking cost-effective, promisingly safe, and environmentally friendly alternatives to replace antibiotics in aquafeed. To address these concerns, dietary intervention with additives such as probiotics is considered the best approach (Mishra et al. 2015).

Probiotics, whether in viable or nonviable states, refer to nonpathogenic bacteria administered at a specific duration and optimal concentration that confer health benefits to their host. In fish farming, the incorporation of probiotics into feed offers a valuable nutritional approach and has become vital for various purposes (Nayak 2010). Probiotics help to maintain a healthy microbial balance by establishing beneficial microbiota in the gut. Probiotics help in disease prevention, reduce stress, support immunomodulation, and contribute to bioremediation efforts (El-Saadony et al. 2021; Tachibana et al. 2020)

Aquaculture researchers have successfully evaluated the use of single-strain probiotics (SSPs) as valuable supplements (Kwoji et al. 2021). However, the effect of probiotics on the host immune response depends on various factors, such as their source, type, strain, and species, which means that supplementing SSPs to a specific host may not necessarily yield a beneficial impact on the host immune system. Therefore, combining diverse species and genera of probiotics as multi-strain probiotics (MSPs) can be used in harmony and synergistically strengthen the host’s immune system (Ouwehand et al. 2018). Currently, in the fish farming industry, there has been a growing trend in the utilization of a large range of bacterial species, such as gram-positive spore-forming bacteria belonging to the genus Bacillus and lactic acid bacteria (LAB) (Abdel-Tawwab et al. 2020; Ringø et al. 2018). Among all of the species, Bacillus species have gained much importance, especially Bacillus subtilis, which has shown efficiency in various fish species. It has been used to enhance the growth, nutritional profile, immunity, and overall health status of fish. It exhibits the ability to prevent pathogen colonization and promote a balanced gut microbial environment (Kuebutornye et al. 2019; Telli et al. 2014; Zaineldin et al. 2018). Likewise, Bacillus licheniformis has emerged as a highly promising probiotic known for its endospore production, which enhances the immunity of fish and promotes the production of enzymes and the synthesis of antimicrobial compounds (Darafsh et al. 2019; Dawood et al. 2018; Gobi et al. 2018). Furthermore, LAB such as Enterococcus faecium can be effectively used in aquaculture (Ringo et al. 2020; Ringø et al. 2018). E. faecium, commonly found in the gastrointestinal tract, is a potential probiotic because it acts as a growth promotor, performs immunomodulatory activities, and produces bacteriocins to counteract the risk of infection (Costa Sousa et al. 2019; Tarkhani et al. 2020).

Clarias batrachus is an economically important freshwater air-breathing fish that belongs to the family Clariidae. Although it is considered an invasive species, its adaptability has led to its introduction in many countries beyond its native range for aquaculture purposes (Paul et al. 2015). In Asia, it is famous for its good nutritional content, taste, and consistent availability throughout the season and has gained immense popularity with high market demand among consumers (Gupta and Verma 2020; Sinha et al. 2014). Furthermore, it has a high growth rate, an efficient feed conversion ratio, and the capacity to survive in oxygen-depleted water conditions, which make it a desirable culture species. Therefore, this experiment was planned to assess the effect of MSP on growth performance, blood biochemistry, and digestive and antioxidant enzyme activities in C. batrachus.

Materials and methods

Preparation of probiotic-based diets

A commercial MSP powder (Compro NaproTM Aqua Probiotics (PR-24), China) was used, and each 1.0 kg of this product contained Bacillus subtilis (5.0 × 109 CFU/gram), Enterococcus faecalis (5.0 × 108 CFU/gram), and B. licheniformis (5.0 × 109 CFU/gram). Five experimental diets with 40% CP were formulated by incorporating different levels of MSP powder: 0.0, 0.5 g/kg, 1.0 g/kg, 1.5 g/kg, and 2.0 g/kg, labelled T0 (control diet), T1, T2, T3, and T4, respectively. Ingredients were taken from the local market in Lahore and analyzed for proximate composition following AOAC (2016). All the ingredients (Table 1) were weighed and ground to a particulate size of 0.05 mm and mixed in an electrical mixer (km 280, Kenwood) with the gradual addition of oil. Then, MSP powder was added according to the formulation described in Table 1. The ingredients were combined and mixed thoroughly by adding 15% distilled water to make a stiff dough. The dough was then passed through a meat mincer (Ag 3060, Anex) for pellet formation. Pellets were air dried with 10% moisture, and the experimental diets were packed in well-sealed plastic-labelled zipper bags in a refrigerator at 4 °C and then used throughout the research period. The samples of formulated feed were also analyzed for proximate analysis according to the method followed by AOAC (2016). The moisture content was estimated by using a hot-air oven (Wise Ven). Crude protein (CP) was analyzed using the micro-Kjeldahl method with an N percent of 6.25 by using the Kjeltec autoanalyzer (KjeltecTM 8100), crude fat (CF) was assessed through the use of a Soxhlet apparatus (Behro Test 901745) with diethyl ether extraction (40–60 °C), and the ash contents were determined by using a muffle furnace (Vulcan D-550) at 660 °C for 5 h.

Table 1 Feed formulation and proximate composition of the experimental diets used in the present study
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Fish acclimatization

Two hundred twenty-five C. batrachus fingerlings (10.13 ± 0.01 g; mean + SD) were procured from the local fish farm of the Sindhwan fish hatchery, Head Balloki, Department of Fisheries, Government of Punjab, and transferred to the Fish Seed Rearing Unit, University of Veterinary and Animal Sciences, Ravi Campus, Pattoki , Pakistan. Fingerlings were placed in a KMnO4 bath (5 g/L) for 1–2 min and stocked into a cemented flow through circular tanks with 0.3 L/mint water exchange for acclimatization. Fish were fed a control diet (T0) during a 15-day acclimatization period.

Experimental design, fish rearing, and husbandry

At the beginning of the feeding trial, fingerlings were weighed and randomly distributed into glass aquaria (315 liter capacity) with dimensions 89 × 58 × 61 (cm) (length × width × depth) and a stocking density of 15 fish per aquarium. The fish were hand fed with test diets in triplicate at 5% of their body weight, and feed was given to fingerlings five times (every 4 h) a day for 90 days. On a daily basis, any uneaten feed was siphoned daily to calculate feed intake (g) and then feed conversion ratio. Physicochemical parameters such as dissolved oxygen, temperature, and pH (7.1 ± 0.2 mg/L, 28.5 ± 0.3 °C, and 7.1 ± 0.1, respectively) were monitored on a daily basis.

Growth indices

After the termination of the 90-day feeding trial, the fish weight was measured, and the number of fish in each replicate was also recorded. Growth parameters, such as weight gain percent (WG%), feed intake (FI), feed conversion ratio (FCR), specific growth rate (SGR), and survival rate (SR), were determined using the following formulae:

$$WG\%=\frac{final\ body\ weight- initial\ body\ weight}{initial\ body\ weight}\times 100$$
$$FI\left(g/ fish\right)= feed\ given\ (g)- unconsumed\ feed(g)$$
$$\textrm{FCR}=\frac{\textrm{FI}\ \left(\textrm{g}\right)\ }{\textrm{WG}\ \left(\textrm{g}\right)}$$
$$SGR\ \left(\%/ day\right)=\frac{\mathit{\ln}\left( average\ final\ weight\right)-\ln \left( average\ final\ weight\right)}{Number\ of\ days}\times 100$$
$$SR\ \left(\%\right)=\frac{Number\ of\ fish\ at\ the\ end\ to\ trial}{Number\ of\ fish\ at\ the\ initiation\ of\ trial}\times 100$$

Whole-body composition

The four fish were arbitrarily selected for whole-body proximate analysis in terms of moisture content, crude protein, crude fat, and ash content following the protocol AOAC (2016) described above in the feed proximate analysis.

Digestive enzyme activities

Intestinal digestive enzyme analysis, such as amylase activity, was evaluated using starch solution as a substrate at 2% (w/v) (Bernfeld 1955). Lipase activity was measured using the spectrophotometric technique with the substrate p-nitro phenyl palmitate (pNPP) (Mahadik et al. 2002). The activity of protease was determined using the García-Carreño (1992) casein digesting technique. At 37 °C, the enzymatic unit hydrolyzes casein and produces a color identical to 1 mol/mint tyrosine (pH = 7.5).

Antioxidant enzyme activities

Each 2-g liver sample was mixed with 6 ml phosphate buffer (pH 7.4), homogenized, filtered using Whatman filter paper no. 1, and centrifuged at 10,000 × g for 15 min. The supernatant was separated, and all enzyme isolation steps were carried out at 4 °C. The activity of CAT was estimated by using the method of Maehly and Chance (1954). Briefly, the reduction in H2O2 concentration at a wavelength of 240 nm was measured using an Analytik Jena Specord 200 Plus UV/VIS spectrophotometer. SOD activity was measured as described by Giannopolitis and Ries (1977). The activity of SOD was determined by measuring its ability to inhibit the photoreduction of nitroblue tetrazole (NBT). The activity of GPx was determined by calculating its capacity to decrease the H2O2 concentration at a wavelength of 470 nm following the method of Civello et al. (1995).

Thiobarbituric acid reactive substance contents in the liver and muscles were determined by following Gatta et al. (2000). Briefly, samples were homogenized in a solution of KCl and Tris-maleate, followed by the addition of ascorbic acid and incubation at room temperature. Thiobarbituric acid (TBA) and HCl were added to the samples, which were boiled for 25 min and then cooled and centrifuged after the addition of trichloroacetic acid. Finally, TBA values, expressed as l g malondialdehyde equivalents/mg tissue, were determined photometrically at 530 nm.

Blood analysis

Five live fish were randomly selected from each aquarium, and their blood was collected from the caudal vein by using 3-ml syringes and stored in EDTA vacutainers. Hematological parameters such as red blood cells (RBCs), white blood cells (WBCs), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HB), mean corpuscular hemoglobin (MCH), and platelets (PLT) were determined by an autohematological analyzer (Celltac α, MEK-6550 Ltd., Japan) in the general laboratory, Department of Fisheries and Aquaculture, UVAS Ravi Campus, Pattoki.

Aside from hematological testing, blood samples were also collected in Eppendorf tubes without anticoagulant and centrifuged at 3000 rpm for 15 min within 30 min of collection. The resulting samples were then stored at − 80 °C for analysis. Serum was used for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alanine phosphatase (ALP) by using the kit method (BioScien) followed by Hossain et al. (2016). Serum was also used for stress parameters such as cortisol and glucose assays for the determination of stress by an automatic biochemical analyzer (Hitachi 7600-110 Ltd., Japan).

Statistical analysis

Data presented in the description and tables are the means and standard deviation of the three replicates. Data obtained in this study were subjected to one-way ANOVA in SAS (version 9.1). Significant parameters (p < 0.05) were compared using Duncan’s multiple range (DMR) test (Duncan 1955). Linear regression analysis (best fitter model based on R2) was performed on WG% data.

Results

Growth and feed utilization

Dietary supplementation with MSP significantly (p < 0.05) enhanced the growth performance and feed utilization of C. batrachus compared to the control group (Table 2). Significantly higher (p < 0.05) weight gain % was observed in fish fed with 0.5 (340.04 ± 5.68), 1.0 (393.92 ± 6.42), 1.5 (430.56 ± 5.69), and 2.0 (493.98 ± 6.39) g/kg MSP-supplemented diets compared with the control group (299.73 ± 5.17). The highest weight gain was observed in fish fed 2.0 g/kg MSP based on linear regression analysis (295.842x + 95.808; R2 = 0.99) (Fig. 1). Similarly, significantly (p < 0.05) higher SGR (%/day) was observed in fish fed with 0.5 (1.64 ± 0.01), 1.0 (1.77 ± 0.01), 1.5 (1.85 ± 0.01), and 2.0 (1.97 ± 0.01) g/kg MSP-supplemented diets compared with the control group (1.53 ± 0.01). Dietary supplementation significantly reduced (p < 0.0.5) the FCR linearly in fish fed with 0.5–2.0 g/kg MSP-supplemented diets in comparison to the control group. The lowest FCR was observed in the 2.0 g/kg MSP treatment (1.72 ± 0.01), and the highest FCR was observed in the control group (2.28 ± 0.05). Dietary supplementation of MSP showed significantly higher (p < 0.05) feed intake (g/fish) of 0.5 (73.88 ± 0.58), 1.0 (81.78 ± 0.58), 1.5 (85.51 ± 0.51), and 2.0 (86.30 ± 0.56) g/kg compared with the control group (69.35 ± 0.57) after 90 days of the feeding trial. However, the highest feed intake was observed in the 1.5 and 2.0 g/kg MSP-supplemented groups, which showed nonsignificant (p > 0.05) differences.

Table 2 Growth performance and feed utilization of C. batrachus fed diets supplemented with MSP
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Fig. 1
figure 1

Linear regression analysis on the WG% data in response to multi-strain probiotics

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Whole-body proximate analysis

Dietary supplementation of MSP in the diet did not influence (p > 0.05) the whole-body composition, as presented in Table 3. Values of proximate composition were observed as moisture (74.41–74.91%), crude protein (16.75–17.64%), crude fat (4.98–5.31%), and ash contents (3.69–3.93%).

Table 3 Whole-body proximate analysis of C. batrachus fed diets supplemented with MSP
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Intestinal digestive enzyme activity

Dietary incorporation of MSP in the diets significantly improved (p < 0.05) the digestive enzyme activities (U/mg protein)) in the intestine of C. batrachus (Table 4). Amylase activities were significantly enhanced (p < 0.05) with increasing levels of MSP such as 0.5 g/kg (1.35 ± 0.03), 1.0 g/kg (1.55 ± 0.03), 1.5 g/kg (1.78 ± 0.03), and 2.0 g/kg (1.96 ± 0.02) compared with the control group (1.20 ± 0.01). A similar trend (p < 0.05) was observed in protease and lipase activities. The highest protease activities (p < 0.05) were observed in the 2.0 g/kg treatment group (10.51 ± 0.06), and the lowest activity was observed in the control group (8.61 ± 0.01). Similarly, fish fed the 2.0 g/kg MSP-supplemented diet showed the highest (p < 0.05) lipase activities (2.26 ± 0.02), and the lowest activities were observed in the control group (1.55 ± 0.02).

Table 4 Intestinal digestive enzyme activity of C. batrachus fed diets supplemented with MSP
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Antioxidant enzyme activity

Dietary supplementation of MSP in diets of C. batrachus demonstrated a significant enhancement (p < 0.05) in liver SOD, CAT, and GPH-X activities (Table 5). Significantly higher (p < 0.05) activities of CAT (U/mg prot) were observed in fish fed with 0.5 g/kg (73.86 ± 0.56), 1.0 g/kg (79.0 ± 0.59), 1.5 g/kg (83.09 ± 0.57), and 2.0 g/kg (85.54 ± 0.58) compared with the control group (70.24 ± 0.53). Similarly, significantly higher (p < 0.05) activities of SOD (U/mg prot) were also observed in fish fed with 0.5 g/kg (4.98 ± 0.09), 1.0 g/kg (5.21 ± 0.08), 1.5 g/kg (5.64 ± 0.09), and 2.0 g/kg (5.86 ± 0.04) MSP-supplemented diets compared with the control group (4.54 ± 0.08). Dietary supplementation of MSP at 2.0 g/kg showed the highest (p < 0.05) activities (μU/mg prot) of GPH-x (85.54 ± 0.58), and the lowest activities were observed in the control group (89.61 ± 0.32). Supplementation with MSP significantly reduced (p < 0.05) the MDA concentrations (mg/g prot) with increasing levels of MSP such as 0.5 g/kg (2.56 ± 0.03), 1.0 g/kg (2.43 ± 0.02), 1.5 g/kg (2.28 ± 0.03), and 2.0 g/kg (2.13 ± 0.06) in comparison to the control group (2.77 ± 0.02).

Table 5 Antioxidant enzyme activity and MDA concentrations of C. batrachus fed diets supplemented with MSP
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Hematological parameters

The hematological parameters of C. batrachus in response to MSP in the diets are shown in Table 6. Values of WBC, RBC, HB, and HCT showed a significant linear increase (p < 0.05) with increasing levels of MSP in diets compared to the control group, and the highest values were observed in 2.0 g/kg MSP-supplemented diets. Moreover, MCH showed a significant decrease (p < 0.05) in response to increasing MSP supplementation in diets compared to the control group. Dietary supplementation with MSP did not influence (p > 0.05) the MCV, MCHC, or PLT values.

Table 6 Hematological parameters of C. batrachus fed diets supplemented with MSP
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Serum biochemistry

Dietary MSP supplementation significantly (p < 0.05) increased the activities (U/L) of ALP and reduced the activities of AST and ALT in C. batrachus (Table 7). The activities of ALP were significantly (p < 0.05) increased in fish fed with 0.5 g/kg (82.82 ± 0.31), 1.0 g/kg (83.91 ± 0.16), 1.5 g/kg (86.90 ± 0.15), and 2.0 g/kg (90.30 ± 0.45) compared with the control group (80.79 ± 0.42). Significant (p < 0.05) increases in AST activities were observed in fish fed 0.5 g/kg (13.11 ± 0.23), 1.0 g/kg (12.10 ± 0.24), 1.5 g/kg (11.17 ± 0.24), and 2 g/kg (10.68 ± 0.29) MSP-supplemented diets compared with the control group (13.95 ± 0.23). Similarly, a significantly (p < 0.05) decreasing trend was also observed in activities, and the lowest activities were observed in the 2.0 g/kg (24.05±0.38) MSP group, while the highest activities were observed in the control group (36.23 ± 0.38).

Table 7 Serum biochemistry of C. batrachus fed diets supplemented with MSP
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Stress parameters

The effect of dietary MSP supplementation on the stress biomarkers is given in Table 8. Dietary supplementation with MSP decreased (p > 0.05) the glucose level with increasing levels of MSP, and the lowest glucose level was observed in the 2.0 g/kg (47.85 ± 0.84 g/dL) MSP group, while the highest glucose level was observed in the control group (47.26 ± 0.94 g/dL). Cortisol levels were observed to decrease (p > 0.05) with increasing supplementation of MSP, and the lowest level was observed in the 2.0 g/kg (8.33 ± 0.18 ng/mL-1) treatment group, while the highest level was observed in the control group (8.58 ± 0.10 ng/mL-1). Both glucose and cortisol showed nonsignificant responses (p > 0.05) with MSP supplementation.

Table 8 Effect of dietary MSP on the stress biomarkers of C. batrachus fingerlings
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Discussion

Single-strain probiotics are usually used in aquaculture; however, with advancements in aquaculture, multi-strain probiotics (MSPs) may provide additional benefits due to the synergistic effects of different probiotic strains (Mohammadi et al. 2021; Puvanasundram et al. 2021). In the current study, the supplementation of MSP in the diet significantly (p < 0.05) improved the growth performance in terms of FW, WG%, SGR, and FCR with increasing levels up to 2.0 g/kg. Consistent with our results, MSP has been reported to enhance growth performance, such as MSP consisting of B. subtilis, L. plantarum, and E. faecium in Pangasianodon hypophthalmus (Abdel-Latif et al. 2023); B. subtilis and B. licheniformis in O. niloticus (Tachibana et al. 2020); B. subtilis, B. licheniformis, and E. faecium in Oncorhynchus mykiss (Merrifield et al. 2010); Lactobacillus plantarum and Pediococcus pentosaceus in O. niloticus (Muhammad et al. 2022); and Lactobacillus and Bifidobacterium in Clarias gariepinus (Ayoola et al. 2013). Probiotics have been reported to enhance digestive enzyme activities in the fish gut, which leads to improved nutrient absorption and growth (El-Haroun et al. 2006). Therefore, the enhancement of growth might be due to improvements in growth factors, absorption, and assimilation in fish (Balami et al. 2022).

In the present study, a significant (p < 0.05) improvement in digestive enzyme activities (amylase, lipase, and protease) was observed in C. batrachus fed MSP-supplemented diets. The findings of this study are consistent with previous investigations in L. rohita (Mukherjee et al. 2019; Saravanan et al. 2021; Ullah et al. 2020), P. hypophthalmus (Abdel-Latif et al. 2023; Akter et al. 2019), O. mossambicus (Yaqub et al. 2021), O. niloticus (Liu et al. 2021), and O. mykiss (Adel et al. 2017). Probiotics help to boost the activation and secretion of digestive enzymes (Vazirzadeh et al. 2020). Moreover, bacteria have the ability to release protease enzymes to breakdown peptide bonds within proteins, facilitating the breakdown of proteins into component monomers and free amino acids, which can help the animal’s nutritional condition (Macfarlane and Cummings 1991). The majority of probiotics may secrete lipase enzymes, which stimulate the digestion of lipids, resulting in increased growth and immunity in fish (Sharma et al. 2010).

Antioxidant enzymes (SOD, GPx, CAT) are involved in the defense mechanism of fish and play an important role in protecting fish from oxidative stress. Moreover, MDA serves as an indicator of lipid peroxidation in fish species (Hoseinifar et al. 2020). The increased SOD, CAT, and GPx activities and decreased MDA content in our study are consistent with previous research studies reported in different fish species fed probiotic-supplemented diets (Giannenas et al. 2015; Gobi et al. 2018; Kuebutornye et al. 2020; Wang et al. 2017). Probiotics are capable of producing metabolites such as glutathione, butyrate, folate, and exopolysaccharides, which are known for their antioxidant properties (Hoseinifar et al. 2020). Furthermore, probiotics also have the ability to produce or stimulate the release of GSH from the intestines, which has a favorable effect on the production of antioxidants in the liver of fish species (Mishra et al. 2015).

MSP supplementation showed nonsignificant (p > 0.05) results on the whole-body proximate composition of C. batrachus. These findings are aligned with previous studies on probiotic supplementation in O. mykiss (Ramos et al. 2015), P. hypophthalmus (Boonanuntanasarn et al. 2019), O. niloticus (El-Haroun et al. 2006), and Paralichthys olivaceus juveniles (Niu et al. 2019). In contrast, Mukherjee et al. (2019) reported a significant increase in the crude protein and crude lipid content of L. rohita fed a diet containing a Bacillus strain. The variation in the results could be attributed to differences in probiotic composition, fish species, experimental design, feed formulations, and rearing conditions.

The hematological parameters of fish are known to fluctuate in response to changes in physiological, nutritional, and environmental conditions and can serve as valuable indicators of the overall health of fish (Fazio 2019). The current results revealed a significant (p < 0.05) improvement in the hematological profile of C. batrachus. Similar to our study, previous investigations on MSP supplementation in fish diets have also reported enhanced levels of Hb, RBCs, MCH, and WBCs (Dahiya et al. 2012; Tabassum et al. 2021; Yaqub et al. 2021). Furthermore, previous research has also demonstrated that the use of MSP consisting of L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, and S. cerevisiae in C. mrigal (Sharma et al. 2010), as well as L. acidophilus and B. subtilis in O. niloticus (Aly et al. 2008), can lead to an increase in WBCs, which is also consistent with the findings of the present study. Furthermore, improvements in MCV, MCH, and MCHC suggest that fish fed the MSP diet were healthier, similar to the study reported by Gabriel et al. (2004). It can be inferred that MSP may play a role in the increase in HB and RBC content through the promotion of iron absorption in the gut. This could be attributed to the release of organic acid, which enhances the availability of iron, thus facilitating the production of more RBCs and HB (Dahiya et al. 2012). Furthermore, the observed increase in WBC levels may indicate improved innate immunity (Rajikkannu et al. 2015).

In serum biochemistry, AST, ALP, and ALT are key enzymes that serve as important biomarkers for evaluating liver and kidney functions. The reduced ALT and AST levels in our study are in line with previous studies, which demonstrated a significant reduction in AST and ALT levels in different fish species fed a combination of probiotics, i.e., B. subtilis, B. licheniformis, and E. faecalis (Liu et al. 2021; Wang et al. 2017). Dietary supplementation enhanced ALP activities in the current study, which is consistent with previous studies on probiotics (Gobi et al. 2018; Panigrahi et al. 2004; Sangma and Kamilya 2015; Sheikhzadeh et al. 2012). ALP activity is commonly associated with the increased production of enzymes by macrophages.

The blood glucose level is thought to be a sensitive indicator of stress (Abdel Rahman et al. 2020). Cortisol is classified as a chronic stress hormone, and its level has been used to measure stress in aquatic animals (Sadoul and Geffroy 2019). In the current study, no significant differences (p > 0.05) in the levels of glucose and cortisol were observed in fish fed MSP, which indicates the absence of any stress condition in fish during the rearing period. Our results are in line with the study of Tachibana et al. (2020).

Conclusion

In conclusion, MSP supplementation up to 2.0 g/kg significantly enhanced the growth performance and feed utilization of C. batrachus. Furthermore, MSPs also enhanced hematology, serum biochemistry, and digestive enzyme activities in fish fed MSP-supplemented diets, which indicates better health conditions. Moreover, antioxidant enzyme activities were also enhanced with the MSP-supplemented diet, indicating better stress coping capabilities. Therefore, it is recommended to supplement the diet of C. batrachus with 2.0 g/kg MSP to enhance growth performance and health.