Preparation of the experimental diets
Moringa oleifera leaves and seeds were obtained from the Scientific Society of Moringa, Dokki, Giza, Egypt. The processing of M. oleifera leaves was done following the method of Mishra et al. [28], while the seeds were processed according to the method of Ijarotimi et al. [29], with minor modifications. The seeds were sorted, dehulled, and oven dried at 50 °C for 10 h then ground using an electric grinder (Moulinex, Grenoble, France) and sieved through 60 mm sieve to obtain raw moringa seed powder. The powder was subsequently packed in plastic container sealed with aluminum foil until being used for diet formulation. Both M. oleifera leaf and seed powder were sterilized at 121ºC for 20 min and cooled to room temperature. Thereafter, the powder fermentation was made by multi-strain microbial fermentation using probiotics (Lactobacillus acidophilus (ATCC 4356), Bacillus subtilis (BEST195) and Saccharomyces cerevisae), obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University. The strains were preserved in glycerol at -80ºc and then were revived by streaking samples of each culture on its corresponding media. Lactobacillus acidophilus was cultured in De Man–Rogosa–Sharpe (MRS) agar (Thermo Scientific™, USA) at 37ºC for 16 h using anaerobic jars containing Anaerocult® A gas packs. Bacillus subtilis was cultured in tryptic soy agar (TSA) with polymyxin (Oxoid Australia Pty Limited ©) at 35ºC for 10 h. Saccharomyces cerevisiae was inoculated in yeast extract-peptone-dextrose (YPD) broth containing conical tube followed by overnight incubation in an orbital shaker at 30 °C with agitation at 200 rpm. The grown yeast cells were then streaked on a petri dish containing 2% YPD agar (Sigma-Aldrich, USA) and incubated at 30 °C until colonies showed growth. Culture suspensions of the three microbial strains were prepared and adjusted to a concentration of 108 CFU/mL with sterilized physiological saline solution. The three microbial strains were used at a ratio of 1:1:1 for the inoculation of moringa’ leaf and seed powder. The optimized co-culture parameters were as follows: total inoculation size, 24%; temperature, 32 °C; fermentation time, 6 days; and initial water content, 60%.
Fermentation of seeds and leaves was carried out separately by the method described by Honghui et al. [25]. Following fermentation, fermented moringa leaf and seed samples were oven dried at 60 °C for 12 h. After that, oven dried samples were further sieved then mixed at a ratio of 1:1 (w/w) in form of fermented mixture (FMO). Raw powdered leaves and seeds were also mixed at a ratio of 1:1(w/w) and referred to as non-fermented mixture (MO). Both MO and FMO were used for diet formulation each in two different concentrations (5 and 10%).
Fish management
The trial was experimentally designed using a completely randomized group of Nile tilapia fingerlings in agreement with the standard procedures and policies approved by the local Institutional Animal Care and Use Committee of Zagazig University in Egypt (permission number: ZU-IACUC/2/F/388/2023). The National Institutes of Health (NIH)-approved ethical guidelines were followed for the handling and use of laboratory animals during all experimental procedures. A total of apparently healthy 180 O. niloticus fingerlings (30 ± 0.5 g average of initial weight) were acquired from a local hatchery (Abbassa, Sharqiyah, Egypt), and then they were transported to the Fish Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt, where the experimental study was conducted. Initially, fish were transferred and acclimatized for two weeks in fiberglass aquaria before starting the actual experiment, in which they were immersed in a potassium permanganate (2 mg/L) and NaCl 2.5% bath for three days. Each aquarium (80 × 40 × 30 cm) was filled with 60 L of chlorine-free tap water, provided with aerators, and thermostatically controlled. Water parameters were measured and kept within the specified ranges throughout the experiment, according to the American Public Health Association (APHA) [30]. During the acclimatization period, fish were fed a basal diet (no additives) which was prepared to meet the basic nutrient requirements of Nile tilapia, according to the National Research Council (NRC) [31].
Experimental design and feeding trial
After the acclimatization period, the fish were randomly divided into five experimental fish groups (36 each, in triplicate) and were distributed in 15 aquaria (3 aquaria per group, each of 12 fish) in a 30-day feeding trial. The five groups were designated as four moringa-fed groups where the fish were fed a basal diet containing a nonfermented M. oleifera leaf and seed mixture at a concentration of 5% (MO5%) and 10% (MO10%) and fermented at 5% (FMO5%) and 10% (FMO10%) in addition to the control group which was fed a basic diet free from M. oleifera (Table 1). The five isonitrogenous and isoenergetic experimental diets were formulated to meet the dietary requirements of Nile tilapia, O. niloticus, based on the recommendations of the NRC [31]. Their approximate chemical composition was presented in Table 1, which was determined following the protocols of the Association of Official Agricultural Chemists [32].
Chemical analysis of both MO and FMO mixtures was performed. Total phenolic concentration in the MO and FMO samples was determined using the Folin-Ciocalteu colorimetric method [33], and expressed as milligrams of gallic acid equivalent (GAE) per gram (g) dry weight. Total flavonoid content was analyzed using the aluminum chloride colorimetric method [34, 35], then estimated using the quercetin standard calibration curve, and the obtained results of flavonoids were expressed as micrograms of quercetin equivalent (Qu) per 1 g of dry weight. Tannin concentration was determined by using tannic acid as a reference compound, following the method described by Broadhurst et al. [36], while total saponin content (TSC) was estimated by using the modified vanillin-sulphuric acid TSC assay, a spectrophotometric method proposed by V. Le, Anh, et al. [37], and it was expressed as mg aescin equivalents per gram dry weight of powder (mg AE/g).
Fish were fed twice daily at 8:00 a.m. and 4:00 p.m., six days per week, for thirty days at a rate of 3% of their biomass. The daily feed intake was modified based on changes in fish weight every 15 days. During the 30-day feeding trial, fish were monitored at regular intervals, with records kept for any clinical symptoms, mortality, and postmortem findings.
Sample collection post-M. oleifera feeding
Blood and serum samples
At the end of the feeding trial, the fish were starved for 24 h. At day 32nd and directly before collecting blood samples, fish were first anesthetized with 100 mg/L benzocaine solution (Al-Nasr Pharmaceutical Chemicals Co., Egypt). Then blood samples were drawn at random from the fish’s caudal vein (4 samples per group) using sterile, disposable syringes rinsed with heparin. The blood samples were divided into two portions. One portion was transferred to tubes without anticoagulant, followed by centrifugation at 3000 rpm for 10 min at 4 °C, then the serum was carefully separated, and a portion was stored at − 80 °C for analysis of the lysozyme activity, while the other portion was freshly used for the preparation of pooled homologous fish sera (used in phagocytosis assay). The other whole blood portion was transferred to tubes containing anticoagulant agent (EDTA-coated tubes) (Al-Nasr Pharmaceutical Chemicals Co., Egypt), and it was used to assess the peripheral blood mononuclear cells’ (PBMCs) phagocytic activity.
Liver samples
Fish liver samples (6 fish per group), collected post-the 30-day feeding trial, were used for estimation of M. oleifera antioxidant activity via detection of hepatic antioxidant enzymes [(super oxide dismutase, SOD) and reduced glutathione, GSH)], as well as malondialdehyde (MDA).
In-vivo challenge with A. hydrophila
At the end of the feeding trial, the fish were rested for 24 h. Consequently, all experimental groups, in addition to part of the control (positive control), were challenged with A. hydrophila, while the other part of the control remained unchallenged (the negative control). The A. hydrophila strain (ATCC 7966) was obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University. It was identified by its 16S rRNA gene sequence and screened for the presence of some virulence genes, including hemolysin (hlyA), aerolysin (aer), lipase (lip), and cytotonic heat-stable enterotoxin (ast) [38]. Its median lethal dose (LD50 = 1.5 × 107) was determined (data not shown). Ten fish per group were intraperitoneally injected with 0.2 mL of virulent A. hydrophila suspension 1.5 × 107 CFU/mL (LD 50) [39]. During the challenge period (14 days), the fish continued to feed on the same respective feeding regimes, and clinical signs, mortalities, and PM lesions were reported. The survival rate (SR) was calculated using the following formula: (Number of surviving fish post-challenge/Number of A. hydrophila-injected fish) × 100 at the 14th day of the bacterial challenge.
Sample collection post-A. hydrophila challenge
Head kidney samples (HK)
Fish head kidney (HK) samples (n = 6) were randomly harvested from 6 fish per group on the 5th day of the bacterial challenge. Tissue samples were kept in TRIzol (Invitrogen) and were used for analysis of the mRNA expression levels of pro-inflammatory cytokines (TNF-α and IL-1β), anti-inflammatory cytokine (IL-10), and IgM genes via RT-qPCR.
Liver and spleen samples
The freshly dead fish’ liver and spleen were aseptically collected and kept frozen at -80˚C. For the still-live fish, several fish were randomly caught from the aquaria, representing each group. The fish were anesthetized, dissected and A. hydrophila was re-isolated from the challenged fish’ liver and spleen samples (n = 3 each/group), and the viable bacterial count (CFU/g) was calculated via the streak plate method.
Immunological parameters evaluation
Phagocytic activity (percentage and index)
The phagocytic activity of PBMCs in the different fish groups was evaluated post-M. oleifera feeding following the method described by Ainsworth and Chen [40]. The peripheral blood immune cells were isolated according to the method of Waterstrat et al. [41], and their viability was determined by trypan blue exclusion assay [42]. Candida albicans yeast obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, was used as a phagocytosis microbial model. It was prepared and heat-inactivated according to the method described by Wood et al. [43]. The phagocytic activity percentage (PA%) was estimated as the percentage of phagocytic cells that engulfed one or more yeast cells. The phagocytic index (PI) was determined at the same time, and it equaled the total number of engulfed yeast cells divided by the number of phagocytes with engulfed yeast [43].
Evaluation of the lysozyme activity
Assessment of the serum lysozyme activity was performed according to the turbido-metric assay following the methods [44] and [45], with some modifications. This assay detects lysozyme activity using 0.75 ml of lyophilized Micrococcus lysodeikticus cells (0.2 mg/ml PBS, pH 6.2) (Catalog Number LY0100, Sigma Aldrich, USA) with 0.25 ml of serum. Lysozyme splits the peptidoglycan of the M. lysodeikticus cell wall, resulting in subsequent lysis of the bacterial cells and change in the solutions’ optical density during incubation of the serum and the bacterial cells. The reaction was carried out at room temperature, and the absorbance at 450 nm was measured after 0 and 10 min using spectrophotometer (BM Co. 5010, Germany). The serum lysozyme concentrations were calculated using a calibration curve of standard lysozyme with known units’ activity, and then a linear-regression equation, obtained from the calibration curve, was used to calculate the lysozyme activity of the tested sera samples. One unit of lysozyme activity in the standard was defined as the amount of lysozyme causing a decrease in absorbance of 0.001 optical density.
Immune-related cytokines and IgM mRNA relative expressions by Real-Time PCR (RT-qPCR)
The head kidney samples were cleaned in cold PBS buffer (pH 7.2). The primer sets for the selected immune-related cytokines (IL-1β, TNF-α, and IL-10) and IgM gene expression together with the β-actin reference gene (for normalization) were designed according to the NCBI reference sequence accession number presented in Table 2. Total RNA was extracted from head kidney tissues using the easy-RED kit (17,063, iNtRON Biotechnology), according to the instructions. The QuantiTect Reverse Transcription Kit (205311, Qiagen, Germany) was used in accordance with the manufacturer’s instructions to produce cDNA from a total of 1.0 μg of RNA, and cycling was done using the Rotor-Gene Q 2 plex. Real-Time PCR System using TopReal SYBR green master mix (RT500S, Enzynomics, Korea) following the manufacturer’s instructions. The PCR cycling conditions included an initial denaturation at 95 °C for 12 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. All gene tests were done in duplicate. PCR amplification was performed under standard conditions. A melting curve analysis was performed following PCR amplification to determine the specificity of the amplified product. After RT-qPCR was conducted and the threshold cycle (Ct) values of each sample were obtained, the relative mRNA expression levels of the tested cytokines and IgM were normalized using the mRNA expression of a known housekeeping gene, β actin. Results were expressed as fold-changes [46].
Determination of hepatic antioxidants activity
The fish’ liver samples, collected from Nile tilapia in all tested groups, were homogenized in ice-cold 50 mM sodium phosphate buffer (pH 7) containing 0.1 mM ethylene diamine tetra acetic acid (EDTA) to obtain 10% (W/V) homogenate. The homogenates were centrifuged at 4 000 g for 30 min at 4 °C, and the supernatant was aliquoted and kept at − 20 °C for estimation of oxidative stress’ marker (MDA) and antioxidant enzymes (SOD and GSH).
Determination of super oxide dismutase (SOD)
Hepatic SOD was measured according to the method described by Nishikimi et al. [47], a colorimetric method that detects SOD in the liver sample depending on the ability of the enzyme (SOD) to inhibit phenazine methosulphate (Catalog No.: SD 25 21, Bio-diagnostics Co., Egypt) PMS-mediated reduction of Nitro blue Tetrazolium (NBT) dye, which then reduces NBT. The change in absorbance at 560 nm, related to the amount of NBT present, was then spectrophotometerically measured for 5 min, which is directly proportional to the amount of SOD present in the sample.
Determination of reduced glutathione (GSH)
The reduced glutathione (GSH) was detected in the liver sample according to the colorimetric method reported in Beutler et al. [48], which depends on the ability of GSH to reduce a chromogen (DNTB) (Catalog No: GR 25 11, Bio-diagnostics Co., Egypt) with subsequent production of a yellow compound (reduced chromogen) when present in an acidic medium. The resultant, yellow-colored reduced chromogen was directly proportional to GSH concentration, and its absorbance was measured spectrophotometerically at 405 nm. SOD levels in each fish group were expressed as U per gram of hepatic tissue, while GSH activity was expressed as mg per gram of hepatic tissue.
Determination of Malondialdehyde (MDA)
The liver homogenates, collected and prepared as described in SOD and GSH, were used to assess MDA in the different fish groups. Hepatic MDA was measured according to the colorimetric method reported in previous studies [49, 50]. The assay depends on the principle that Thio barbituric acid (TBA) (Catalog No: MD 25 29, Bio-diagnostics Co., Egypt), a chromogen, reacts with MDA, present in liver sample, in acidic medium at temperature of 95 °C for 30 min to form TBA reactive product of a pink color whose concentration was directly proportional to the MDA concentration in the sample. The absorbance of the resultant pink product was measured at 534 nm via a thermostated spectrophotometer (VAR-CARY-400, Canada). The MDA levels in each group were expressed as nmol/g tissue.
Growth performance
At the end of the 30-day feeding trial, fish in all groups were fasted for 24 h and anesthetized with 95 mg/L clove oil water bath. Five fish were randomly collected from the aquaria of each experimental group, and each fish was individually weighed on a digital meter to evaluate their final weight. Then, the mean was calculated. In order to evaluate the growth-promoting activity of M. oleifera, the fish’s weight gain rate (WGR), specific growth rate (SGR), average daily gain (ADG), and feed efficiency (FE) were determined using the formulas reported in Zhang et al. [26].
Statistical analysis
The data were edited in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). A Shapiro–Wilk test was conducted in order to check for normality, as described by Razali and Wah Data [51]. The significant effects of the treatments were examined according to the one-way ANOVA [52], with the level of significance set at α = 0.05. Results were expressed as means ± SE. Turkey’s’ test was used to perform pairwise comparisons between means in cases where a significant effect was detected. The significant differences between survival rates were examined according to the chi-square test. The statistical significance between means was set at a p-value less than 0.05. Figures were fitted by the GraphPad Prism software 9.0 (GraphPad, USA).
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