Introduction

Genetically improved farmed tilapia (GIFT), an enhanced strain of Nile tilapia (Oreochromis niloticus), exhibits better performance rates than routinely available strains of tilapia [1]. Currently, there is increased interest in Egypt in the culture of the GIFT tilapia mainly because of its rapid growth rate, high fillet yield, and high disease resistance [2]. GIFT also can be farmed in different aquaculture systems. The intensive culture systems are commonly used for tilapia culture which causes stressful conditions that reduce fish growth and wellbeing. In addition, there are increasing concerns about the use of antibiotics in tilapia culture to overcome infectious diseases [3,4,5]. Key driving forces include restrictions or bans on the use of prophylactic antibiotics in various countries. The use of functional feed additives as bio-friendly agents is a sustainable way to improve cultured fish performance. The use of probiotics and/or functional ingredients with immune modulatory properties has become more prominent across the animal feed industry [6,7,8]. In a wide range of species, both terrestrial and aquatic, a substantial body of literature exists demonstrating the advantageous effect of feeding functional immune modulatory substances [9,10,11].

The impetus of using functional feed additives in aquafeeds is to promote intestinal health and improve fish performance [12, 13]. According to Ouwehand and Salminen [14], probiotics can be existed in both live and killed bacterial forms. The application of probiotics “live bacterial cells” might affect the aquaculture environment negatively by the interaction with the ecosystem. Dead beneficial cells remarkably help in increasing feed efficiency along with disease resistance by immune modulation and/or competitive exclusion for adhesion sites [15]. Many reports have concluded that the dietary inclusion of heat-killed Lactobacillus plantarum (HK strain L-137) can positively impact the performance and wellbeing of fish, shrimp, and sea cucumber [16,17,18,19,20,21].

It is well documented that the supplementation of β-glucan (BG) in aquafeed resulted in improved performance, physiological and oxidative status as well as the enhancement in the immune response by increasing the lysozyme and complement system levels which increase macrophages phagocytic activity [22,23,24]. Further, BG can be used as a decent preventive and restorative choice against infectious diseases in cultured fish [9, 24].

Using synergistically related functional supplements as bio-friendly agents is a sustainable way for improving the performance of aquatic species [25,26,27]. As far as we know, no published data about the detailed supplemental effects of HK L-137 or/and BG on tilapia’s growth-related gene expression, digestive enzymes activity, intestinal morphometry, hemato-immune response, and antioxidative status. Thus, the present study is done to examine the probability of feeding a novel HK L-137 or/and BG on several performances of GIFT tilapia.

Materials and Methods

Fish, Diet, and Experimental Protocol

GIFT tilapia fingerlings were obtained from a private farm located in Kafrelsheikh, Egypt, and transported to Sakha Aquaculture Research Unit, Kafrelsheikh, Egypt. After 2-week acclimation, 180 fish (15.94 ± 0.02 g) were put into 12 glass aquaria (70 L) (15 fish/tank) for 12 weeks. Feeding rate was fixed at 3% of body weight per day with two feeding times 08:00 and 15:30 and batch-weighed every 2 weeks to adjust daily feed input. The leftover feed was siphoned out after 3 h, and 50% of water was replaced daily. During the trial, the rearing conditions were 12:12 h light/dark photoperiod, water temperature 23.1 ± 0.8 °C, pH 6.8–7.5, DO 7.5–8 mg/l, and ammonium 0.04–0.08 mg/l.

Four experimental diets were tested in triplicate: the basal (control) diet and the basal diet supplemented with HK L-137 “House Wellness Foods Corp., Itami, Japan [28]” at 100 mg/kg, β-glucan “BG, Daigon do, Tokyo, Japan” at 100 mg/kg or 50 HK L-137 + 50 BG mg/kg (control, HK L-137, BG, and HK L-137/BG diets) (Table 1). The doses of HK L-137 and BG were selected by following Dawood et al. [2]. HK L-137, BG or HK L-137/BG additives were thoroughly mixed with lipid source before adding other ingredients. Fish oil was well mixed the additives, added to the dry ingredients and mixed for 15 min. All ingredients were combined well into a homogenous mixture to produce the test diets. Water was then added to achieve a uniform texture appropriate for pelleting (1 to 2 mm pellets) using an extruder machine, followed by air drying at room temperature. The nutritional profile for each diet was fixed by following AOAC [29] certified procedures (Table 1).

Table 1 Basal diet and proximate chemical composition (on dry matter basis)
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Sampling Schedule

Three fish per group (one fish per aquarium) were randomly caught and euthanized by “diluted tricaine methanesulfonate (MS-222; 1:2,500 ratio; Sigma-Aldrich, Egypt).” Fish were individually measured for final body-weight, length and the intestine sampled for morphometrical and digestive enzymes analysis. Liver and viscera were removed then weighed to get hepatosomatic index “HSI = weight of liver/weight of fish × 100” and viscerasomatic index “VSI = weight of viscera/weight of fish × 100,” respectively.

For digestive enzyme analysis, intestine from the control and treated fish were aseptically taken, washed with PBS (pH 7.5; 1 g per 10 ml), homogenized and centrifuged for 5 min at 8000 rpm. The supernatant then kept at 4 °C. Protease, lipase, and amylase enzyme activities performed according to Lowry et al. [30], Borlongan [31], Jin [32], and Worthington [33], respectively. Protease, lipase, and amylase activities were stated as “specific activity” (units per mg of protein) of intestine content [34, 35].

Samples from the different intestinal portions (anterior, middle, and posterior) were collected from the tested fish, then fixed in 10% formalin. After dehydration and clearance, the tissues were embedded in paraffin and sectioned in 5-μm thickness. The serial sections were subjected to H/E staining [36]. The villus height was performed using “Image J analysis software (National Institutes of Health, MD, USA).”

Tissue samples from different organs (liver and muscle) were collected from three fish per group (one fish per aquarium), immediately in 2-ml Eppendorf tubes and shocked in liquid nitrogen then stored at − 80 °C for RNA extraction.

Blood Assays

Blood was collected from the caudal vein of nine anesthetized fish per group (three fish per aquarium) to collect enough amount of blood. The collected blood was then put into either heparinized (with EDTA) or non-heparinized (for serum analysis) separate tubes. Blood hematology markers “hemoglobin, red blood cells (RBCs), white blood cells (WBCs), total and differential count of heterophil and lymphocyte” were analyzed according to Brown [37]. Hematocrit was determined using “the microhematocrit technique.” Blood smears were prepared for the determination of differential leukocyte counts [38]. The remaining samples (1 ml blood) were left for 30 min till blood clotting then serum separation by centrifugation at 3000 rpm for 10 min. Serum samples were stored at − 20 °C until further analysis. Blood biochemical investigations to all studied subjects “cholesterol, triglycerides, glutamyl oxaloacetic transaminase (GOT), and glutamic-pyruvate transaminase (GPT)” were carried out by RA-50 chemistry analyzer (Bayer) using readymade chemicals (kits) supplied by Spinreact Co. Spain, following manufacturer’s guidelines.

Total RNA Extraction and cDNA Synthesis

A total of 100 mg tissue was homogenized in liquid nitrogen, and total RNA from the tested samples (collected on liquid nitrogen) were extracted using easy RED total RNA extraction kits (iNtRON Biotechnology, Inc., Korea) according to the manufacturer’s instructions. The RNA concentration (OD 260 nm) and purity (OD 260 nm/OD 280 nm ratio, range 28.15–103.1) of each sample. The RNA integrity was verified by agarose gel electrophoresis while the concentration and purity of the samples were examined by NanoDrop spectrophotometer. The first-strand cDNA was synthesized using HiSenScript cDNA synthesis kit (iNtRON Biotechnology, Inc., Korea).

qRT-PCR Assay

The specific primers were used to amplify the selected genes of the Nile tilapia (O. niloticus) with β-actin as a housekeeping (internal standard) gene-primer sequence and references are shown in Table 2. Real-time quantitative (qRT-PCR) assay was carried out using Stratagene MX300P real-time PCR system (Agilent Technologies, USA), using TOP real™ preMIX SYBR Green qPCR master mix (Enzynomics, cat. RT 500) following the manufacturer’s recommendations. The thermocycling conditions for the reaction were as follows: 95 °C for 30 s, followed by 45 cycles of denaturation at 63 °C for 60 s and annealing at 72 °C for 60 s. MxPro QPCR Software was used for data collection. The relative gene expression levels were evaluated using the 2−ΔΔct method as described by Pfaffl [41]. All samples were analyzed in triplicate and along with non-template control and negative RT controls in each plate.

Table 2 Primers used for qRT-PCR analysis
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Growth Performance Calculations

During the final sampling, 15 fish per tank were weighed separately. Growth and feed efficiency were evaluated using weight gain (WG), specific growth rate (SGR), feed efficiency ratio (FER), and condition factor (CF). Calculations were made using the following formulae: WG (%) = (FBW − IBW) × 100/IBW; SGR (%BW/day) = 100((lnFBW − lnIBW)/T); FER = WG /FI; CF = BW/FL3, where FBW = body weight final (g), IBW = body weight initial (g), T = duration of the trial in days, WG = wet weight gain (g), FI = estimated feed intake (g), and FL = standard fork length (cm).

Statistical Analysis

Shapiro–Wilk and Levene tests confirmed normal distribution and variance homogeneity. All statistical differences (growth performance, digestive enzymes, intestinal morphometry, blood indices, immune, oxidative responses, and qRT-PCR data) were assessed by one-way ANOVA tests (SPSS version 22, SPSS Inc., IL, USA) with Duncan’s as post hoc test where differences in experimental groups occurred. The level of significance was accepted at P < 0.05. All data are presented as means ± standard error (SE).

Results

Growth Parameters Analysis

The different growth and feed utilization parameters are represented in Table 3. A significant (P < 0.05) enhancement of FBW and WG was observed in fish fed only HK L-137 and both (HK L-137/BG) over the control group. Further, specific growth rate (SGR) and feed efficiency ratio (FER) were significantly (P < 0.05) higher in fish fed both HK L-137 and BG (HK L-137/BG) than the control. No significant (P > 0.05) differences on the survival rate and somatic indices (CF, HSI, and VSI) were observed among all groups.

Table 3 Growth performance, nutrient utilization, survival and somatic indices of fish fed test diets for 12 weeks
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Digestive Enzyme Analysis and Intestinal Morphometry

Lipase and protease activities increased significantly (P < 0.05) in HK L-137/BG group over the control regime without no differences among the other group (Table 4). Amylase activity showed no significant differences among the groups (Table 4).

Table 4 Digestive enzymes activity in fish fed test diets for 12 weeks
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The results of the intestinal morphological analysis of GIFT fish fed test diets for 12 weeks are summarized in Table 5. The anterior intestine, mucosal, and villi lengths showed no significant differences (P > 0.05) among the groups. For the middle intestine, mucosal and villi lengths as well as the muscle thickness were increased significantly (P < 0.05) in HK L-137 or/and BG over the control. For the last section of the intestine (posterior), no changes were observed among all the experimental groups.

Table 5 Intestinal morphometry in fish fed test diets for 12 weeks
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Fish fed the basal diet showed normal thick and blunt ended anterior villi, while fish fed HK L-137 or/and BG diets showed mild increase of intestinal villi length (Fig. 1A). Middle intestine of fish fed with basal diet showed normal thick and blunt ended villi, while fish fed HK L-137 or/and BG diets showed clear increase of intestinal villi length and branches (Fig. 1B). Fish fed with basal diet showed normal thick and blunt ended villi, while fish fed HK L-137 or/and BG diets showed normal villi number and length (Fig. 1C).

Fig. 1
figure 1

Intestinal morphology ((A) anterior intestine, (B) middle intestine, and (C) posterior intestine) of fish fed control, HK L-137, BG, and HK L-137/BG diets (H&E, × 100) (scale bar = 50 μm). Fish fed the control diet showed normal thick and blunt ended anterior villi, while fish fed HK L-137 or/and BG diets showed mild increase of intestinal villi length (A). Middle intestine of fish fed with basal diet showed normal thick and blunt ended villi, while fish fed HK L-137 or/and BG diets showed clear increase of intestinal villi length and branches (B). Fish fed with basal diet showed normal thick and blunt ended villi, while fish fed HK L-137 or/and BG diets showed normal villi number and length (C)

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Gene Expression

Gene expression analysis revealed a significant upregulation (P < 0.05) in IGF-1 expression in fish fed HK L-137 or/and BG compared to the control with no differences among the supplemented groups. The expression of G6PD in the muscle and liver tissues were also upregulated in fish fed both HK L-137 and BG over the control with no differences between the other groups (Fig. 2). In contrast, fish fed HK L-137 or both HK L-137 and BG exhibited relatively down regulation in FAS gene expression compared to fish fed the control diet.

Fig. 2
figure 2

Relative expression of growth-related genes in the muscle and liver tissues of fish fed test diets for a period of 12 weeks. Values are expressed as mean ± SE from triplicate groups (n = 9). Bars with an asterisk are significantly different from those of control group (P < 0.05)

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Blood Markers

Hematocrit increased significantly (P < 0.05) in BG and HK L-137/BG groups over the control group (Table 6), while no differences between BG and HK L-137/BG or HK L-137. Further, the hemoglobin increased significantly (P < 0.05) in HK L-137/BG group over the control group without no differences with the other groups (Table 6). WBCs increased significantly (P < 0.05) in HK L-137 and HK L-137/BG groups over the control group without no differences with BG group (Table 6). Blood glucose level decreased significantly (P < 0.05) in HK L-137/BG in comparison with the control without no differences with the other groups (Table 6). Blood triglyceride decreased significantly (P < 0.05) in HK L-137/BG group, while no alterations were detected between the control, HK L-137, and BG groups (Table 6). Blood GPT also decreased significantly (P < 0.05) in fish fed HK L-137 or/and BG. No significant changes (P > 0.05) were observed in the remaining blood variables of tilapia in the current study (Table 6).

Table 6 Blood hematological and biochemical parameters of fish fed test diets for 12 weeks
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Discussion

Functional feed additives have got superior consideration in aquaculture recently due to its beneficial activity not only in growth promotion but also in disease resistance and immune modulation [18, 42, 43]. Among them, dead Lactobacillus sp. and BG which got greater attention because of their immunomodulation prosperities to overcome unfavorable conditions [16, 17, 24, 27]. In this study, we revealed detailed mechanistical effects of HK L-137 or/and BG on GIFT’s growth performance, digestive enzymes activity, intestinal morphometry, hemato-immune response, and antioxidative status.

Inclusion of HK L-137 or/and BG in GIFT feed were shown to increase FBW, WG, SGR, and FER significantly compared to control. It may be suggested that the beneficial bacterial cells modulated growth and caused total physiological changes [9]. The probiotic bacterium was analyzed for its growth-promoting effects by means of growth rate, feed utilization, modulation of digestive enzyme activity, antioxidant status, and upregulation of immune response in fish [16, 17, 24, 27]. It is well known that bacterial cells can colonize in the host intestine and then acting its effects by improving the digestion and absorption process as well as the supportive role to the intestinal digestive enzymes [14]. Similarly, tilapia fed probiotic showed improved growth rates and feed efficiency [20, 44]. Also, the results suggested that tilapia utilized test diets efficiently by HK L-137 resulting in improved FER, which would be one reason for the quicker growth in fish fed HK L-137. The improved feed utilization may affect feed protein and energy to be more offered for helping in the fish growth [45]. An improvement of growth has been detected in red sea bream [17], mirror carp [24], cyprinid rohu [46], and Nile tilapia [47] fed with BG. BG can be absorbed by fish to produce energy and proteins required for the growth and development [17, 23]. Nile tilapia fed BG exhibited high feed intake and accordingly quick growth rate [47]. Probiotics could enhance feed utilization and weight gain of aquatic animals through affecting the host’s appetite and feed digestion by breaking down indigestible components, increase production of vitamins, and detoxify compounds in the diet [3, 48]. Fermented prebiotics including BG can play an important role in improving the activity of intestinal microbiota [46, 49]. Further, BG has beneficial effects focused on growth performance and health, particularly prevention of the attachment and colonization of pathogenic bacteria in the gastrointestinal tract (GIT) as well as the modulation of intestinal microbiota and the promotion of intestinal integrity in fish [9].

The effects of probiotics have been linked to modulation of gut microbiota and total digestive enzyme activity in the brush-border membrane of GIT, which increases nutrient digestibility and feed utilization and ultimately improves growth performance [50, 51]. The major digestive enzymes produced by fish are protease, lipase, and amylase, which play roles in feed digestion and assimilation [52]. If the activity of these enzymes increases, overall body metabolism may increase [24, 27]. In this study, the obtained data showed enhanced activity of lipase and protease digestive enzymes in case of feeding HK L-137 or/and BG over the control. Similarly, use of probiotics caused the increase in the protease and lipase activity regardless of its concentration in sea bream [45]. Digestive enzymes exhibited elevated activity by HK L-137 or/and BG supplementation resulting in high feed efficiency. Synbiotics mode of action depends essentially on its content, dose, and period of supplementation. However, more studies are required to find the mechanistic role in improving the feed efficiency and growth (e.g., microbiome, proteome, and transcriptome studies).

Morphometric analysis of the intestine was conducted to enable thorough evaluation of the effects of HK L-137 or/and BG at the digestive organs level which provide information about the histological condition of the intestine and consequently the growth of the fish [24, 27, 53]. Supplementation of HK L-137 or/and BG resulted in improved length of villi in the current study especially in the middle portion. Increased villi length could cause an improvement in the absorptive surface area resulting in better nutrient utilization followed by improved growth performance [24, 27]. The inclusion of probiotics and prebiotics in aquafeeds has improved feed absorbance efficiency in fish due to increasing the absorptive area, microvilli density, and height. The authors concluded that the beneficial role of HK L-137 or/and BG possibly depends on the duration of feeding, the composition, and the dose. In this study, improved growth and feed efficiency is related to increased villi length and enhanced secretion and activity of digestive enzymes.

Although there are several assumptions regarding the growth benefits triggered by beneficial bacteria, the actual molecular mechanism behind the elevated growth is unclear [2]. In addition, the relationship between the “central nervous system (CNS)” and the bacteria inhabiting the gut remains a mystery. If bacteria can control the expression of local growth factors (IGF-1) and the receptor (GHR-I) responsible for binding growth factors/hormones (growth hormones and steroid hormones), mediation of such signaling may lead to better growth [1, 39, 40]. The expression of IGF-1 varies according to the type of tissue, feed intake, and environmental stress [40]. In this study, the results showed that IGF-1 expression was significantly upregulated over the control in fish fed HK L-137 or/and BG, which agrees with previous studies on European sea bass or Yellow perch [54, 55], respectively. In response to growth hormone, cellular level biological responses of IGF signaling such as growth, proliferation, survival, immune response, and cell migration have been reported to vary. The high level of glycolysis, as indicated by increased glucose-6-phosphate dehydrogenase (G6PD) expression, is responsible for maintaining energy requirements needed for fish growth [1, 39, 40]. The current study revealed that G6PD in liver and muscle tissues of fish fed HK L-137 and BG was significantly upregulated over the control regime. The increased expression of fatty acid synthase (FAS) is one of the reasons of elevated fat deposition such as triglyceride in the body [40]. In fish, expression of mRNA genes involved in triglyceride metabolism was downregulated by dietary probiotics [56]. In our study, a relatively reduction in FAS was observed in fish fed HK L-137 and BG. The downregulation of FAS might be the reason of low blood triglyceride levels in GIFT tilapia. The results also suggested that FAS have a positive feedback regulator on lipase activity by decreasing lipid droplet size and increasing fatty acid levels in the intestinal epithelium.

Blood hematological and biochemical markers could be utilized (as physiological biomarkers) to recognize probable enhancements in fish health condition up on supplementation of functional feed additives [57,58,59,60,61]. In this study, not only the growth rate was ameliorated by HK L-137 or/and BG feeding but also hematocrit, hemoglobin, and WBCs upregulated. It has been assumed that dietary probiotic can induce the blood biomarkers in fish as an indicator of improved health status. In addition, it is assumed that hemato-immunostimulating effects of HK L-137 or/and BG associated with its digested products that promote the innate immune reaction. Similarly, enhanced hematocrit by feeding HK L-137 or/and BG in red sea bream was observed [16, 17]. Supplementations of HK L-137 also improved hemoglobin as a general sign of healthy fish. Similarly, hemoglobin was increased in tilapia fed beneficial bacterial cells [62, 63]. In this study, a significant decreased glucose, triglycerides, and GPT by HK L-137 or/and BG, hypothetically signifying the influence of HK L-137 and BG in maintaining the wellbeing of GIFT tilapia. Low triglycerides in fish potentially indicate the role of HK L-137 in regulating the normal range of blood lipid derivatives of tilapia [56]. Similarly, blood lipids were influenced by probiotic supplementation in rainbow trout [64]. Blood GPT content is regularly used to assess the capacity of liver, while GPT is emitted into blood and the qualities expanded or diminished relying upon the injury of the hepatic tissue [45]. Low GPT in fish fed HK L-137 or/and BG showed that the secretion of GPT into blood was at normal level, representing enhanced health status of fish.

The reason of using functional feed additives in aquafeeds is to promote intestinal health and improve fish performance [65, 66]. The findings of this trial are in the line of previous studies that recommended the supplementation of probiotics, prebiotics, or both to improve the growth performance, feed utilization, and health status [49, 51, 67,68,69].

Conclusion

In conclusion, the findings of this trial have demonstrated that dietary inclusion of HK L-137 or/and BG can increase the performance of GIFT tilapia by promoting FBW, WG, SGR, FER, digestive enzyme activity, villi length, and increasing the intestinal surface area. For the first time, our results revealed an elevation in IGF-1, G6PD, and decreased FAS gene expression profiles. Dietary HK L-137 or/and BG also increased the hematocrit, hemoglobin, and WBCs and decreased triglyceride and glucose levels and could have significant effect on preserving a healthy fish in turn potentially enhance defense against infectious pathogens. In most of the studied parameters, fish fed both HK L-137 and BG exhibited the highest performances among the other groups which confirm the synergistic relation between both additives. This result merits further attention using mechanistic and disease challenge studies.