Effects of Phytobiotic Curcuma longa, Allium sativum and Zingiber officinale-Supplemented Diets on Growth, Utilisation of Feed and Nile Tilapia (Oreochromis niloticus) Resistance Against Streptococcus agalactiae
Abstract
The study investigated how phytobiotic-supplemented diets impact the growth performance, feed utilisation and resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus agalactiae. Fish with a total mean initial mass (37.6 g) of 180 and a random stocking of 15 fingerlings per 150 L tank in triplicate were divided into four groups and fed isonitrogenous (299 g kg−1 crude protein) and isoenergetic (15.7 kJ g−1 gross energy) control diets supplemented with 1% turmeric (Curcuma longa; TUD), garlic (Allium sativum; GAD) and ginger (Zingiber officinale; GID) powder for 56 days. After the trial period, growth performance, feed utilisation and blood health were measured. Ten fish from each replicate were infected with S. agalactiae and mortality was observed for 14 days. The results showed a significantly higher weight gain (g), specific growth rate (SGR; %/day) and average daily growth (ADG; g) in GAD (80.3 ± 1.0, 2.04 ± 0.05 and 1.434 ± 0.02, respectively) and GID (77.03 ± 0.8, 2.0 ± 0.04 and 1.376 ± 0.01, respectively) compared to the control group (60.4 ± 2.5, 1.71 ± 0.02 and 1.079 ± 0.04, respectively). However, these metrics were not significantly higher in TUD (63.8 ± 2.2, 1.8 ± 0.05 and 1.139 ± 0.04, respectively) when compared to the control group. TUD, GAD and GID feed conversion ratio (FCR) and efficiency, as well as protein efficiency ratio (PER), were improved as compared to the control. The haemoglobin (HGB), haematocrit (HCT), platelets (PLTs), white blood cells (WBCs), serum biochemistry and respiratory burst activity (RBA) of TUD, GAD and GID were significantly better than the control. Lysozyme and bactericidal activities were also significantly improved in TUD, GAD and GID as compared to the control. Following the S. agalactiae infection, the fish survival rates of GAD (67.7%), GID (53.3%) and TUD (46.7%) were better than those of the control (26.3%). Dietary supplementation with the above-mentioned phytobiotics in Nile tilapia culture can help to increase production in view of the current challenges posed by the increased incidence of disease in fish farms. They are recommended to enhance growth, immunity and resistance to disease.
1. Introduction
Aquaculture, a rapidly growing industry, currently provides almost 50% of the global fish supply [1]. The growth and expansion are due to the new direction of diversification and intensification [2]. However, the focus on aquaculture intensification has resulted in several problems, including reduced growth rates and increased susceptibility to disease [3]. The treatment of fish diseases with chemical additives is associated with environmental problems such as pollution of water bodies, the emergence of pathogen stains resistant to treatment and the chemical residues accumulating in the tissue of fish that adversely affect human consumers [4]. Therefore, there is increasing interest in the search for sustainable alternatives to improve fish health and performance [3].
Phytobiotics, or plant-based products, have proven to be possible natural alternatives to synthetic additives in aquaculture. Their use in fish farming or culture is relatively inexpensive and environmentally friendly compared to chemotherapeutic approaches to disease control [5]. Plants including turmeric (Curcuma longa), garlic (Allium sativum) and ginger (Zingiber officinale) are widely used phytobiotics known for their numerous bioactive compounds, including phenols, flavonoids, tannins and saponins, which have antimicrobial, immunostimulant, anti-inflammatory and growth-promoting properties [6, 7]. The inclusion of these phytobiotics in fish feed has shown promising results in enhancing growth performance, disease resistance and overall health [8]. Turmeric, for instance, has been shown to have growth-promoting and immune-boosting effects, as well as rainbow trout’s (Oncorhynchus mykiss) ability to resist infection with Aeromonas salmonicida [9]. Garlic and ginger were also included in the diet to promote growth, feed efficiency and resistance to Vibrio harveyi in black rockfish (Sebastes schlegelii) [10], thereby, enhancing their defence mechanisms against Streptococcus agalactiae, a confirmed pathogen associated with an unusual mass mortality of cage-cultured tilapias in the Volta Lake, Akosombo, Eastern Ghana.
The Nile tilapia (Oreochromis niloticus) is the second most economically valuable global freshwater species [11]. However, like any other cultured fish species, the Nile tilapia is susceptible to a number of diseases, including bacterial infections [12]. Streptococcus agalactiae is an important disease agent in tilapia production and causes significant economic losses in aquaculture production worldwide [13]. The disease leads to considerable financial losses due to reduced growth rates, mass mortality and treatment costs [14]. To meet the rising demand for Nile tilapia, it is important to promote its growth performance, immune response and disease resistance to ensure sustainable aquaculture production [15]. Therefore, the search for effective and ecologically friendly strategies to enhance the resistance of tilapia to S. agalactiae is of utmost importance. Self-immunity activation in fish to defend against disease infection has become a practical and reliable approach in aquaculture production. In recent years, the incorporation of medicinal herbs, spices or phytobiotics into fish feed to combat disease outbreaks has become an integral part of aquaculture [16, 17].
Turmeric, garlic and ginger powders have been included as feed additives in various contexts in aquaculture [18–20]. However, information available in the scientific literature on how they affect the immune response and serum biochemical parameters, particularly the liver and kidney functions of O. niloticus, during the period before and after S. agalactiae infection is scanty. These plant additives were incorporated into the diet at an optimal dose of 1%, aiming to improve the growth, feed utilisation and immunity to better resist disease in common carp (Cyprinus carpio) as reported by Giri, Sukumaran and Park [18], amur carp (C. carpio haematopterus) as indicated by Chesti, Chauhan and Khati [21] and Sobaity sea bream (Sparidentex hasta) as documented by Jahanjoo et al. [22]. Previous research has also indicated that feeding fish a supplemented diet for 56 days can improve growth, immunity and health status [23, 24]. It was hypothesised that the inclusion of the same amount of the above phytobiotics or spice powders in the diet of O. niloticus and feeding for 56 days would enhance growth and immune response for better resistance to infection with S. agalactiae. Therefore, this study aimed at investigating the potential benefits of diets incorporated with 1% garlic, turmeric and ginger powder on the growth, haematology, serum biochemistry, immunological parameters and Nile tilapia resistance to S. agalactiae. The powder of the above phytobiotics was used instead of the extraction method as it is easy to prepare and requires fewer resources [25]. It is also easier for local fish farmers, especially in developing countries, to adapt them. Understanding the effects of these medicinal herbs or phytobiotics on the health and productivity of tilapia will contribute to the development of sustainable aquaculture practices and provide valuable insights to local fish farmers and researchers. By elucidating the potential effects of these natural additives on tilapia, this research will provide a scientific basis for their use in aquafeed formulations, leading to improved fish health, increased productivity and reduced reliance on chemical additives.
2. Research Materials and Methods
2.1. Preparation of the Feed Supplements
Fresh turmeric, garlic and ginger were bought at a local market. Before use, the outer husks of the garlic were removed. They were thoroughly washed, sliced and oven-dried at 50°C to a moisture content of 10% before using a household blender to grind each into powder. The powder was passed through a fine sieve to get rid of the remaining fibres. They were kept in sealed and labelled plastic bags before being stored at a temperature of 4°C until use.
2.2. Phytochemical Analysis of the Supplements
| Bioactive compound | Curcuma longa | Allium sativum | Zingiber officinale |
|---|---|---|---|
| Alkaloids (%) | 14.7 | 12.2 | 11.7 |
| Flavonoids (%) | 4.6 | 6.1 | 6.9 |
| Tannins (mg/100 g) | 4.5 | 5.8 | 5.03 |
| Saponin (%) | 6.02 | 7.9 | 7.5 |
2.3. Experimental Diet Preparation
A control diet and three test diets were formulated to obtain four isonitrogenous diets. Turmeric, garlic and ginger powders were added to the test diets at a concentration of 10 g/kg of a basal diet. This dosage, adapted from the studies by Giri, Sukumaran and Park [18],Mahmoud et al. [19] and Chesti, Chauhan and Khati [21], has been previously applied in varying contexts to enhance fish growth, bolster immune response and improve disease resistance. The control was a basic feed without any supplements. The mixture was formed into pellets by using a meat grinder with a 3 mm perforated plate attached after being thoroughly mixed with 300 mL of water to moisten it. It was then dried at room temperature. Before use, the feeds were stored at 4°C in clearly labelled zip-lock plastic bags. The experimental diet ingredients and the proximate composition are listed in Table 2.
| Ingredients | Control | TUD | GAD | GID |
|---|---|---|---|---|
| Fishmeal | 100 | 100 | 100 | 100 |
| Soybean meal | 320 | 320 | 320 | 320 |
| Wheat bran | 410 | 400 | 400 | 400 |
| Soybean oil | 50 | 50 | 50 | 50 |
| Cassava flour | 80 | 80 | 80 | 80 |
| Vitamins premix | 20 | 20 | 20 | 20 |
| Minerals premix | 20 | 20 | 20 | 20 |
| Allium sativum | 0 | 0 | 10 | 0 |
| Zingiber officinale | 0 | 0 | 0 | 10 |
| Curcuma longa | 0 | 10 | 0 | 0 |
| Proximate composition | ||||
| Dry matter (%) | 91.5 | 91.3 | 91.3 | 91.1 |
| Crude protein (%) | 29.9 | 30.0 | 29.3 | 29.7 |
| Lipid (%) | 9.8 | 9.5 | 9.5 | 9.8 |
| Ash (%) | 11.9 | 11.6 | 11.7 | 12.0 |
| Gross energy (kJ g−1) | 15.7 | 16.7 | 17.7 | 15.7 |
- Note: Vitamin premix composition (per kg of premix): vitamin A/Retinyl acetate 3a672a: 3400000 IU; Vitamin D 3a671: 1000000 IU; choline chloride 3a890: 80,000 mg, vitamin E 3a700 (all-rac-alfa-tocopheryl acetate): 4000 mg; niacinamide 3a315: 6000 mg; calcuim D-pentothenate 3a841: 2420 mg; vitamin B2 3a825: 800 mg; vitamin B6/pyridoxine hydrochloride 3a831: 400 mg; vitamin B1/thiamine mononitrate 3a821: 400 mg; folic acid 3a316: 80 mg; vitamin B12/cyanocobalamin: 4 mg. Mineral premix composition (per kg of premix): manganese (II) oxide—manganese 3b502: 30,000 mg; zinc oxide—zinc 3b405: 20,000 mg; iron (III) sulphate monohydrate—iron 3b103: 20,000 mg; copper (II) sulphate (pentahydrate)—copper 3b405: 4000 mg; calcium iodate (anhydrous)—iodine 3b202: 600 mg; sodium selenite 3b801—selenium: 80 mg. Gross energy (KJ g−1), estimated as: crude protein% × 23.66 + crude lipid% × 39.5 + nitrogen-free extract × 17.2; nitrogen-free extract, calculated as: 100 − (crude protein% + crude lipid% + ash% + moisture%). GAD, Allium sativum supplemented diet; GID, Zingiber officinale supplemented diet; TUD, Curcuma longa supplemented diet.
2.4. The Experimental Diets’ Proximate Chemical Analysis
The proximate chemical composition of the experimental feeds was analysed according to the methodology of [27]. In brief, each feed sample underwent a 24-h oven-drying process at 105°C (conducted in a Gallenkamp hot air oven CHF097 XX2.5, Gemini B.V., Apeldoorn, The Netherlands) to measure the dry matter. Subsequently, to evaluate the ash content, the samples were burned in a muffle furnace (K1252, Heraeus Instruments GmbH, Hanau, Germany) for 6 h at 550°C. The crude protein concentration was measured using the Kjeldahl method (Foss Kjeltec 2200, Hillerod, Denmark), while the [28] method was also used to estimate the crude lipid concentration.
2.5. Research Design and Fish Culture Conditions
All guidelines and procedures pertaining to the care and use of live animals established by the Ethics Committee of Kwame Nkrumah University of Science and Technology (KNUST) were adhered to during the experiment. Juvenile Nile tilapia, exclusively male and weighing between 24 and 28 g, were sourced from a well-established hatchery located in Akosombo, within the Eastern Region of Ghana. The fish, enclosed in plastic bags enriched with oxygen, were transported to the fish farm of the Department of Fisheries and Watershed Management at KNUST, where the study was conducted. They were routinely examined clinically, parasitologically and bacteriologically at random to determine the presence of infection, infestation and pathological lesions, respectively. However, to ensure that they were free from Streptococci and other infections, 20 fish were selected randomly from the original population, and their livers, brains and skin were dissected and streaked onto tryptic soy agar (TSA) plates for bacterial isolation and inoculation. For a 2-week period, fish were acclimated in 12-cylindroconical tanks within a recirculating aquaculture system (RAS) holding 150 litres and fed with the control diet. The total of 180 fish (average weight: 37.6 ± 0.3 g) were weighed in bulk, counted and randomly allotted into four groups (45 fish per group) with three replicates each (15 fish per replicate) in the RAS-12 tanks after the acclimatisation period. During the experimental period, fish were hand-fed until they appeared satiated and administered two times a day (9:00 am and 4:00 pm). The feeding of each group of replicates was recorded. The subjects were fish fed the control diet containing 1% C. longa (TUD), 1% A. sativum (GAD), or 1% Z. officinale (GID; Table 1). The study was conducted in an indoor RAS and filtered both biologically and mechanically. The fluorescent tubes were controlled with a digital timer so that each day consisted of a 12-h cycle of light and darkness. The fish in each tank were weighed every fortnight using an electronic scale (Constant 5000 g/11LB, China). On the days when the fish were weighed, feeding was suspended and faeces were removed from the tanks daily using a syphon. Each treatment had its own equipment, such as syphons and sampling nets. Water quality variables such as pH, temperature and dissolved oxygen (DO) were measured twice a week in each treatment tank using a HACK multi-parameter probe (HQ40D, Loveland, Colorado, USA). Concurrently, water samples for ammonia analysis were assessed photometrically within the laboratory. Temperature, pH, DO and ammonia nitrogen did not vary in all treatment tanks and ranged between 27.8–28.2°C, 6.50–6.70, 5.4–5.8 mg L−1, and 0.03–0.04 mg L−1, respectively, throughout the trial.
2.6. Sampling of Blood and Analyses
At the conclusion of a 56-day period, three fish per replication (totaling nine per treatment group) were randomly selected. These chosen fish underwent sedation in aerated water using a propofol buffer (10 mg/L) from NEOROF Laboratory Limited, India, following a 24-h period of feed deprivation. Blood samples from fish were collected by using a sterile plastic syringe and needle through a non-lethal puncture of the caudal vein. The fish were placed in several tanks of aerated freshwater to recover after blood sampling. A set of blood samples was collected and immediately placed in test tubes coated with a solution of ethylene-diamine-tetra-acetic acid. This set of blood samples was used to determine the full blood count and respiratory burst activity (RBA) adapted by Mehrabi et al. [23] and Shin et al. [29].
The blood samples were subjected to analysis using an automated blood analyser (Sysmex XP 300 model, Japan). The measured parameters included haemoglobin (HGB), haematocrit (HCT), platelets (PLTs), red blood cells (RBCs), white blood cells (WBCs), mean corpuscular volume (MCV), mean corpuscular HGB (MCH), MCH concentration (MCHC) and differential WBC counts, such as lymphocyte counts, neutrophil counts and mixed difference (basophil, monocyte and eosinophil) counts. The evaluation of RBA was conducted by assessing the nitroblue tetrazolium (NBT) index, following the method outlined by Sahoo, Kumari and Mishra [30] with a partial modification. Briefly, 100 μL of freshly collected heparinised blood was dispensed into a 96-well microplate with a flat bottom, and 100 μL of 0.2% NBT solution (Sigma, St. Louis, MO, USA) was added. Thirty minutes of incubation were carried out at 25°C. Then 50 μL of the resulting mixture was mixed with 1 mL of N,N-dimethyl-formamide in a test tube, and 5 min of centrifugation were carried out at 3000 rpm. The supernatant liquid was measured with a spectrophotometer (Micro Plate Read, Sr. No.: RT0400814GDM, Germany) at 620 nm.
A second batch of blood samples, three fish per replicate (nine fish per treatment group), was taken from another batch of fish samples. The samples were placed in gel-containing serum tubes, allowed to coagulate and subjected to centrifugation at 4000 rpm (Eppendorf 5804) for 10 min to separate the serum from the cellular components. Following this, the serum were stored at −20°C until needed. Using a spectrophotometer (Jenway 6305, Cole Palmer, Staffordshire, UK), photometric analysis was performed on the serum samples to assess levels of total proteins (PROB-0250, BIURET, 12 × 20 mL, 684), albumin (ALBU-0259, BROMOCRESOL GREEN, 12 × 20 mL, 576), glucose (GHSL-0250, GO-PAP, 12 × 20 mL, 684), cholesterol (CHSL-0250, CHOD-PAP, 12 × 20 mL, 684), urea (URSL-0250, UREASE-GLDH, 8 × 20 mL, 528), creatinine (CRSL-0250, EXZYMATIPAP, 12 × 21 mL, 664), alanine aminotransferase (ALT; ALSL-0250, IFCC, 12 × 20 mL, 528), alkaline phosphatase (ALP; PASL-0230 DGKC/SCE-DEA, 4 × 20 mL, 320) and aspartate aminotransferase (AST; ASSL-0250, IFCC, 12 × 20 mL, 528). Concentrations of the serum samples were assessed according to the guidelines provided by the manufacturer, making use of commercially available diagnostic reagent kits from ELITech Diagnostics, ELITech Group, Puteaux, France. Some of the serum samples were also used to measure lysozyme and bactericidal activities.
The activity of lysozyme was evaluated using the turbidimetric procedure outlined by Kumari et al. [31], with slight modifications. A volume of 25 μL of blood serum was dispensed into a flat-bottomed 96-well microplate, followed by 175 μL of bacterial suspension (Micrococcus lysodeikticus, Sigma M3770) in addition. The OD was then recorded at 450 nm after incubation at 25°C for 0 and 30 min. A standard curve was established using hen egg white lysozyme from Sigma.
2.7. Streptococcus agalactiae Isolation for Bactericidal Activity and Disease Challenge
We aseptically collected tissue samples from the brain, kidney, liver and spleen of O. niloticus, which was exhibiting clinical signs of streptococcosis, from a cage farm on Volta Lake in Akosombo, Ghana. Bacterial isolation and quantification were performed using the pour plate method, with growth observed on TSA (Oxoid Ltd., Basingstoke, UK) supplemented with 2% NaCl. Sterile petri dishes with the prepared media were incubated at 30°C for 24 h, using a rotating shaker incubator set at 110 rpm. Initially, samples were inoculated in tryptic soy broth, then streaked onto TSA plates for a 24-h incubation at 30°C. After the incubation, isolates showing typical Streptococcus morphology—gram-positive cocci in pairs or chains with non-motility—underwent gram staining and biochemical tests for identification and characterisation. For this purpose, isolates were cultured on brain heart infusion agar (BHIA; Oxoid Ltd., UK) with 2% NaCl and incubated at 35°C for 24 h. Identification of the bacterial species (S. agalactiae) was performed using the API 20 Strep Kit (bioMerieux Inc., Durham, NC), with results cross-referenced against the manufacturer’s analytical profile index.
The bactericidal activity (BA) was assessed based on the serum’s capacity to kill S. agalactiae. The bacteria (S. agalactiae) culture was centrifuged at 15,000 rpm at 4°C for 15 min. In order to measure the BA, the pellet of S. agalactiae bacterial cultures was purified and diluted in phosphate-buffered saline (PBS) with the OD adjusted to 0.5 at 546 nm. The samples’ serial dilution was done five times with PBS (1:10). In a micro-vial, a combination of 20 µL of each fish treatment group serum and 2 µL of the bacterial sample, which had been previously diluted, underwent a 1-h incubation at 37°C. In the group of bacterial controls, phosphate PBS was used instead of serum. Following the incubation period, the colonies were inoculated on TSA plates at 37°C for a duration of 24 h to enumerate the viable bacterial count.
An inoculum of S. agalactiae prepared was used to conduct the disease challenge. The bacterial suspension was diluted in a sterile saline solution (0.75% NaCl) to achieve a target concentration of 1.0 × 107 CFU/mL through a series of tenfold serial dilutions. The cell density was determined using standard plate count techniques, with colony counts performed on TSA plates. The pathogenicity of the prepared bacterial inoculum was verified by administering it to a group of 10 naïve O. niloticus, each weighing approximately 50 g on average. To establish a control group, 10 additional fish received an intraperitoneal injection of 0.1 mL of 0.9% saline solution as a sham treatment.
2.8. Statistical Analysis
GraphPad Prism Version 8 software was employed to generate the graph and conduct data analysis. Variations among the four treatment groups across all parameters were assessed through one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons. A significance level of p < 0.05 was used to measure differences between treatment groups. Before analysis, percentages underwent arcsine transformation and data normality was verified by the use of the Kolmogorov–Smirnov test. The results were displayed as the mean ± standard deviation of the mean.
3. Results
3.1. Fish Growth Performance
GAD and GID had significantly improved (p < 0.05) growth performance, characterised by greater weight gain (g), percentage weight gain (%), specific growth rate (SGR; % day−1) and average daily growth (ADG; g) than the control group (Table 3). Although the growth performance metrics in TUD were high, no significant variances were observed (p > 0.05) compared to the control. Additionally, TUD recorded a significant (p < 0.05) low feed intake value (77.37 ± 1.0 g/fish) compared to that of the control (86.24 ± 3.8 g/fish), GID (94.81 ± 1.5 g/fish) and GAD (97.49 ± 1.0 g/fish). For the feed conversion ratio (FCR), the values for GAD (1.22 ± 0.01), GID (1.23 ± 0.01) and TUD (1.23 ± 0.06) were significantly better than the control (1.44 ± 0.1; p < 0.05). Protein efficiency ratio (PER) and feed conversion efficiency (FCE) in GAD, GID and TUD exhibited notable improvements compared to the control. Additionally, there were no discernible differences in the survival percentages across all treatment groups.
| Parameter | Control | TUD | GAD | GID | p value |
|---|---|---|---|---|---|
| Initial weight (g) | 37.6 ± 1.1 | 38.2 ± 0.5 | 37.5 ± 0.5 | 37.1 ± 1.0 | 0.53 |
| Final weight (g) | 98.03 ± 3.5b | 102.0 ± 2.6b | 117.6 ± 1.3a | 114.1 ± 1.3a | <0.0001 |
| Weight gain (g) | 60.4 ± 2.5b | 63.8 ± 2.2b | 80.3 ± 1.0a | 77.03 ± 0.8a | <0.0001 |
| Weight gain (%) | 160.4 ± 3.2b | 167.0 ± 4.7b | 214.2 ± 4.3a | 207.7 ± 5.7a | <0.0001 |
| SGR (% day−1) | 1.71 ± 0.02b | 1.8 ± 0.05b | 2.04 ± 0.05a | 2.0 ± 0.04a | <0.0001 |
| ADG (g) | 1.079 ± 0.04b | 1.139 ± 0.04b | 1.434 ± 0.02a | 1.376 ± 0.01a | <0.0001 |
| Feed intake (g/fish) | 86.24 ± 3.8b | 77.37 ± 1.0c | 97.49 ± 1.0a | 94.81 ± 1.5a | <0.0001 |
| FCE (%) | 70.2 ± 5.7b | 82.5 ± 3.9a | 82.4 ± 0.9a | 81.3 ± 0.7a | 0.007 |
| FCR | 1.44 ± 0.1b | 1.23 ± 0.06a | 1.22 ± 0.01a | 1.23 ± 0.01a | 0.02 |
| PER | 2.4 ± 0.2b | 2.8 ± 0.2a | 2.8 ± 0.04a | 2.8 ± 0.05a | 0.007 |
| Survival rate (%) | 93.3 ± 6.7 | 95.5 ± 3.9 | 97.8 ± 3.9 | 95.5 ± 3.9 | 0.73 |
- Note: Different superscript letters in the same row indicate significant differences (p < 0.05) based on analysis of variance followed by Tukey’s multiple comparison test. GAD, Allium sativum supplemented group; GID, Zingiber officinale supplemented group; TUD, Curcuma longa supplemented group.
- Abbreviations: ADG, average daily growth; FCE, feed conversion efficiency; FCR, feed conversion ratio; PER, protein efficiency ratio; SGR, specific growth rate.
3.2. Haematology of Oreochromis niloticus in Response to Allium sativum, Zingiber officinale and Curcuma longa-Supplemented Diets
Higher RBCs were observed in GAD (1.7 ± 0.1), GID (1.7 ± 0.2) and TUD (1.5 ± 0.2) compared to the control (1.3 ± 0.2; Table 4). Nonetheless, it was the GAD and GID that observed significant differences as compared to the control (p < 0.05). Also, O. niloticus that fed the supplemented diets had considerably higher HGB, HCT, WBC and PLT values in comparison to the control fish group (p < 0.05). There were no significant differences in MCH, MCV or MCHC values among the treatments (p > 0.05). In all treatment groups, the fish showed a high percentage of lymphocytes, with notably low levels of neutrophils and mixed differences (basophils, monocytes and eosinophils). Specifically, the lymphocyte percentages in Nile tilapia-fed diets enriched with turmeric (94.5 ± 1.6), garlic (94.7 ± 1.5) and ginger (94.7 ± 1.0) were significantly greater (p < 0.05) than those in the control group (90.4 ± 2.8). In contrast, the control group exhibited significantly greater proportions of neutrophils and mixed cells (p < 0.05) compared to the supplemented diet groups.
| Parameter | Control | TUD | GAD | GID | p value | ||
|---|---|---|---|---|---|---|---|
| RBCs (×106/µL) | 1.3 ± 0.2c | 1.5 ± 0.2bc | 1.7 ± 0.1ab | 1.7 ± 0.2ab | 0.0002 | ||
| HGB (g/dL) | 6.8 ± 0.7b | 9.2 ± 0.6a | 9.5 ± 1.0a | 9.9 ± 0.8a | <0.0001 | ||
| HCT (%) | 22.0 ± 3.5b | 28.2 ± 4.6a | 32.1 ± 4.9a | 32.9 ± 5.0a | <0.0001 | ||
| MCV (fL) | 177.2 ± 34.5 | 193.3 ± 31.5 | 192.6 ± 32.6 | 201.9 ± 40.9 | 0.51 | ||
| MCH (pg) | 54.9 ± 10.0 | 64.6 ± 9.1 | 56.7 ± 7.2 | 60.7 ± 8.2 | 0.11 | ||
| MCHC (g/dL) | 31.5 ± 5.7 | 33.2 ± 4.5 | 30.2 ± 4.1 | 30.6 ± 4.4 | 0.6 | ||
| PLT (×103/µL) | 35.5 ± 7.8b | 48.7 ± 3.2a | 48.2 ± 5.8a | 46.6 ± 5.0a | <0.0001 | ||
| WBCs (×103/µL) | 20.0 ± 3.3c | 26.3 ± 3.4b | 30.8 ± 2.4a | 28.7 ± 4.2ab | <0.0001 | ||
| Lymphocytes (%) | 90.4 ± 2.8b | 94.5 ± 1.6a | 94.7 ± 1.5a | 94.7 ± 1.0a | <0.0001 | ||
| Neutrophils (%) | 4.3 ± 0.9a | 2.7 ± 0.7b | 2.7 ± 0.8b | 2.8 ± 0.5b | <0.0001 | ||
| Basophils, monocytes and eosinophils (%) | 5.3 ± 2.0a | 2.8 ± 1.0b | 2.6 ± 0.9b | 2.5 ± 0.6b | <0.0001 | ||
- Note: Different superscript letters in the same row indicate significant differences (p < 0.05) based on analysis of variance followed by Tukey’s multiple comparison test. GAD, Allium sativum supplemented group; GID, Zingiber officinale supplemented group; TUD, Curcuma longa supplemented group.
- Abbreviations: HCT, haematocrit; HGB, haemoglobin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; PLT, platelets count; RBC, red blood cells count; WBCs, white blood cells count.
3.3. Oreochromis niloticus Biochemical and Immunological Parameters in Response to Allium sativum, Zingiber officinale and Curcuma longa-Supplemented Diets
Fish-fed diets supplemented with phytobiotics exhibited significantly elevated total protein and albumin levels, as well as decreased glucose levels (p < 0.05; Table 5). That notwithstanding, the inclusion of supplements in the diet had no discernible impact on fish cholesterol levels (p > 0.05). Additionally, GAD, GID and TUD recorded significant reductions in both kidney function (creatinine and urea) and liver function (ALP, ALT and AST) parameters in comparison with the control (p < 0.05). Furthermore, Nile tilapia-fed diets enriched with spices or phytobiotics displayed significantly higher RBA when compared to the control (p < 0.05). Moreover, GAD and GID exhibited significantly higher lysozyme and bactericidal activities as compared to the control (p < 0.05). Although TUD also observed enhanced lysozyme and BA values, there were no significant differences as compared to the control (p > 0.05).
| Parameter | Control | TUD | GAD | GID | p value |
|---|---|---|---|---|---|
| Total protein (g/L) | 36.5 ± 4.4b | 43.9 ± 5.4a | 47.1 ± 3.5a | 45.3 ± 4.1a | <0.0001 |
| Albumin (g/L) | 13.3 ± 1.0b | 23.2 ± 2.1a | 22.7 ± 1.2a | 21.9 ± 1.9a | <0.0001 |
| Glucose (mmol/L) | 4.0 ± 0.4b | 2.9 ± 0.5a | 2.4 ± 0.4a | 2.5 ± 0.6a | <0.0001 |
| Cholesterol (mmol/L) | 5.7 ± 1.4 | 5.2 ± 1.2 | 5.0 ± 1.0 | 4.9 ± 1.3 | 0.53 |
| Urea (mmol/L) | 23.8 ± 1.4c | 18.3 ± 1.4b | 14.4 ± 1.1a | 15.8 ± 1.3a | <0.0001 |
| Creatinine (µmol/L) | 23.4 ± 1.8c | 12.4 ± 1.3a | 13.3 ± 0.9a | 15.2 ± 1.1b | <0.0001 |
| ALT (U/L) | 22.8 ± 3.4b | 14.8 ± 2.7a | 15.7 ± 3.0a | 13.1 ± 1.1a | <0.0001 |
| AST (U/L) | 42.2 ± 8.0b | 34.8 ± 4.1a | 31.0 ± 4.8a | 33.2 ± 5.5a | 0.002 |
| ALP (U/L) | 51.5 ± 8.1b | 43.0 ± 3.3a | 38.8 ± 4.0a | 42.0 ± 5.6a | 0.0002 |
| RBA (OD at 450 nm) | 0.776 ± 0.12c | 1.102 ± 0.08b | 1.392 ± 0.09a | 1.306 ± 0.07a | <0.0001 |
| LA (U/mL) | 118.4 ± 3.1b | 121.1 ± 4.5b | 132.9 ± 3.9a | 130.1 ± 8.6a | <0.0001 |
| BA (CFU) | 11.3 ± 1.2c | 10.4 ± 0.7c | 6.02 ± 1.5b | 4.4 ± 1.1a | <0.0001 |
- Note: Different superscript letters in the same row indicate significant differences (p < 0.05) based on analysis of variance followed by Tukey’s multiple comparison test. GAD, Allium sativum supplemented group; GID, Zingiber officinale supplemented group; TUD, Curcuma longa supplemented group.
- Abbreviations: ALP, Alkaline phosphatase; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; BA, Bactericidal activity; LA, Lysozyme activity; OD, optical density; RBA, Respiratory burst activity.
3.4. Disease Challenge
The highest survival of fish following the S. agalactiae challenge was observed in GAD (67.7%), followed by GID (53.3%) and TUD (46.7%) as against the control (26.7%; Figure 1). The control fish group displayed severe clinical signs, such as erratic and circular swimming, visceral cavity distension, isolation, exophthalmia, loss of appetite and skin haemorrhages. While some fish lost their balance and collapsed unexpectedly, others were observed swimming close to the water’s surface. The fish displayed a range of clinical signs after contracting the disease. Severe signs were noted in the control group, moderate signs in the TUD group and mild signs in both the GAD and GID groups. The naïve Nile tilapia that were tested to confirm the pathogenicity recorded a survival percentage of 20% and severe disease signs similar to those already described above. Nevertheless, a control group injected with the saline solution (sham treatment) observed no mortality or disease signs.
3.5. Oreochromis niloticus Serum Biochemistry and Lysozyme Activity in Response to the Streptococcus agalactiae Challenge
The serum biochemical parameters and LA of O. niloticus infected with S. agalactiae and fed diets enriched with C. longa, A. sativum and Z. officinale for 56 days are shown in Table 6. GAD, GID and TUD observed significantly higher serum protein and albumen levels as compared to the control (p < 0.05). Also, fish that fed the supplemented diets had a significant decrease in glucose, cholesterol, urea, creatinine, ALT and AST (p < 0.05). Again, diets supplemented with the above-mentioned phytobiotics significantly improved the activity of lysozyme in fish compared to the control following the disease challenge.
| Parameter | Control | TUD | GAD | GID | p-Value |
|---|---|---|---|---|---|
| Total protein (g/L) | 25.8 ± 4.2b | 39.1 ± 6.1a | 44.02 ± 2.6a | 39.2 ± 4.1a | <0.0001 |
| Albumin (g/L) | 10.2 ± 1.1b | 21.8 ± 1.8a | 20.1 ± 1.4a | 19.5 ± 2.1a | <0.0001 |
| Glucose (mmol/L) | 6.1 ± 0.5b | 4.4 ± 1.0a | 3.8 ± 0.4a | 4.01 ± 0.6a | <0.0001 |
| Cholesterol (mmol/L) | 8.1 ± 1.1b | 6.5 ± 1.0a | 6.3 ± 0.5a | 6.7 ± 0.7a | 0.007 |
| Urea (mmol/L) | 32.7 ± 1.4c | 23.2 ± 1.4a | 19.6 ± 1.4b | 20.7 ± 1.4b | <0.0001 |
| Creatinine (µmol/L) | 30.4 ± 1.4b | 17.9 ± 1.6a | 17.9 ± 1.0a | 20.9 ± 1.3a | <0.0001 |
| ALT (U/L) | 36.3 ± 3.8c | 27.7 ± 2.8a | 25.8 ± 3.8ab | 23.2 ± 1.1b | <0.0001 |
| AST (U/L) | 51.2 ± 3.5b | 43.1 ± 3.7a | 37.2 ± 5.3a | 38.9 ± 4.8a | <0.0001 |
| LA (U/mL) | 159.7 ± 2.8c | 181.6 ± 4.0b | 196.8 ± 2.9a | 200.6 ± 7.8a | <0.0001 |
- Note: Different superscript letters in the same row indicate significant differences (p < 0.05) based on analysis of variance followed by Tukey’s multiple comparison test. GAD, Allium sativum supplemented group; GID, Zingiber officinale supplemented group; TUD, Curcuma longa supplemented group.
- Abbreviations: ALT, Alanine aminotransferase; AST, aspartate aminotransferase; LA, Lysozyme activity.
4. Discussion
This study indicated that feeding O. niloticus with diets enriched with garlic and ginger powders significantly enhanced growth performance compared to the control group. However, the addition of 1% turmeric powder to the fish diet did not lead to a significant growth improvement. The high alkaloid levels (14.7%) in turmeric powder might have contributed to the lack of improvement in tilapia growth. In contrast to our previous study [33], which reported remarkable improvements in both feed intake and growth in O. niloticus-fed diets supplemented with 1% phytobiotic mantle button (Tridax procumbens), wild peanut (Calopogonium mucunoides) and clove basil (Ocimum gratissimum) leaf meals containing alkaloid concentrations of 9.3%, 9.4% and 12.3%, respectively, the present study observed enhanced growth only in the GAD and GID. Improved feed intake was also noted in the GAD, GID and control fish groups, but not in the TUD. This suggests that the alkaloid levels of 12.2% in garlic powder and 11.7% in ginger powder did not negatively impact Nile tilapia’s feed intake and growth performance. Conversely, the 14.7% alkaloid level in turmeric powder likely affected palatability, leading to reduced feed intake and growth. Nevertheless, feed conversion and utilisation were remarkably enhanced in all fish that fed the supplemented diets. The improved growth performance leads to higher production yields. Efficient feed utilisation not only reduces production costs, but also promotes sustainable practices as fewer resources are wasted. The improvement in growth performance in GAD and GID and the better feed utilisation in fish fed phytobiotic-enriched diets may be attributed to the effect of phytochemicals including alkaloids, flavonoids, saponins and tannins contained in garlic, ginger and turmeric, which improve feed digestion and nutrient absorption. According to Ahmadifar et al. [34], these phytochemical components improve fish growth by secreting enzymes that promote digestion and the absorption of feed. The improved growth performance observed in GAD and GID could also be due to the beneficial effect of garlic and ginger on the intestinal microbiota, leading to improved feed digestion and utilisation [22]. The authors also explained that ginger speeds up the digestion of food and helps maintain a healthy balance of intestinal bacteria by stimulating the release of liver bile and pancreatic enzymes. Studies have shown the positive impact of garlic and ginger-enriched feed on fish growth performance. Mohammadi et al. [35] noticed that common carp fed 0.1%, 0.2% and 0.4% ginger-containing feed exhibited significant enhancement in growth rate and feed utilisation as compared to the control. Furthermore, the 2% garlic added to the O. niloticus feed over a period of 84 days significantly enhanced fish growth and feed efficiency [36]. Moreover, the mixture of 1% garlic, 1% ginger and 1% thyme (Thymus vulgaris) reportedly improved growth in S. hasta [22]. In contrast to this study, Yusuf et al. [37] observed significantly enhanced feed intake and growth of Nile tilapia fed a 0.2% turmeric powder-incorporated diet for 8 weeks. Giri, Sukumaran and Park [18] also reported significantly improved growth performance when fed a C. carpio 1.0% or 1.5% turmeric powder-enriched diet for 56 days. The results of this study showed improved feed utilisation but no significant growth performance for O. niloticus fed a diet containing 1% turmeric powder. The contradictory results could be due to the differences between the species and the phytochemicals, such as the alkaloid concentration in the turmeric included in the fish diet.
Recently, haematology, serum biochemistry, respiratory burst, lysozyme and bactericidal activities have become effective parameters to assess fish innate immune response and health [38, 39]. The present study results indicated that a diet incorporated with 1% garlic, ginger and turmeric significantly increased the HGB, HCT, PLTs and WBC levels in the blood of O. niloticus. This was attributed to the above-mentioned bioactive compounds in the spices contained in the fish diet. Fish with higher levels of HCT, RBC and HGB in the blood have a better oxygen supply to the tissues, which has a positive effect on growth and health [40]. Previous research have demonstrated that herbal supplements promote growth in fish with elevated blood levels of RBC, HCT and HGB [41, 42]. The improvement in RBCs, HGB and HCT observed in this study could also be due to the increased antioxidant capacity resulting from supplementation with garlic, ginger and turmeric powder. Antioxidants reduce haemolysis and erythrocyte destruction by protecting erythrocyte membrane lipids from oxidative stress [40]. According to Jomeh, Chitsaz and Akrami [39], the ability of fish to function properly under stress and resist infection improves as the WBC count increases. Diets supplemented with garlic, ginger and turmeric have been reported to increase WBC counts, possibly enhancing innate immune response and thus reducing the production of inflammatory cells [35, 43, 44]. Since PLTs are cells with multiple functions involved in the blood clotting process and support defence mechanisms, the increase in PLT numbers shown in this study also contributes to fish immunity [44].
Interestingly, GAD, GID and TUD were shown to have improved protein profiles, glucose, cholesterol, kidney and liver function compared to control groups during the pre- and post-disease periods. This reflects the enhanced fish humoral defence system and innate immunological response of the fish due to the beneficial effects of the above-mentioned bioactive compounds on fish metabolism, thus, reducing stress levels [33, 44]. The reduced serum glucose and cholesterol levels also indicate improved regulation of glucose and lipid metabolism due to the increased short-chain fatty acid production, which minimised stress in the fish [45, 46]. In addition, a significant decrease in urea, creatinine, ALT, AST and ALP in GAD, GID and TUD suggests that herbal compounds such as alkaloid, flavonoid, saponin and tannin contained in garlic, ginger and turmeric have a protective effect on the liver and kidneys. Similar research has shown that the incorporation of garlic, ginger and turmeric or their mixture in the diet significantly improves the immune parameters in the blood serum of S. hasta [22], Tambaqui (Colossoma macropomum) [44] and O. mykiss [9]. In this study, the observed improvement in LA, both pre- and post-disease infection, as well as RBA before disease infection in fish fed phytobiotics might be a result of the beneficial effects of the bioactive compounds in the powders of garlic, ginger and turmeric. Previous research has demonstrated that dietary supplementation with garlic, ginger and turmeric—whether individually or in combination—can enhance fish immunity. These benefits include increased activity in respiratory burst and lysozyme levels, indicating strengthened immune responses [22, 47].
The BA of O. niloticus-fed diets supplemented with A. sativum and Z. officinale was higher than that of those fed the control diet. This could be attributed to the beneficial effects of alkaloids, flavonoids, saponins and tannins present in the phytobiotics, which stimulated the fish’s immune responses and thereby, enhanced their antibacterial activity [33]. The study observed a significant relationship between the BA of peripheral blood in fish and their survival rates following infection with S. agalactiae. Specifically, Nile tilapia with higher BA in their peripheral blood exhibited higher survival rates after infection with the disease. GAD and GID exhibited significantly greater BA and higher survival rates compared to the control group, suggesting a more regulated and balanced immune response. This regulated fish immunity, due to the antioxidant, antimicrobial and anti-inflammatory benefits of bioactive compounds in garlic, ginger and turmeric, protected the fish against S. agalactiae infection, resulting in higher survival rates [47]. In addition, the synergistic effect of the phytochemicals in the supplements could enhance the immune systems of GAD and GID, potentially improving BA and survival rates [33].
The improved disease resistance and minimised signs of streptococcosis observed in the O. niloticus groups fed phytobiotic-enriched diets were attributed to the improved humoral defence system and innate immune response due to the phytochemical-rich additives in the fish feed. The additives enhanced the fish’s resistance to disease due to their bactericidal effect. The bioactive compounds, including alkaloid, flavonoid, saponin and tannin, of the supplements, which possibly improved resistance to S. agalactiae in GAD, GID and TUD, have been reported to possess antibacterial potential [48]. They inhibit the growth of microorganisms by deactivating the functions of microbial enzymes, cell envelope transport proteins and adhesion, as well as the formation of polysaccharide compounds [48, 49]. The addition of A. sativum, Z. officinale and C. longa to the diet of Nile tilapia enhances their immune response, enabling the fish to better resist S. agalactiae infections. This approach improves the survival rate, thus increasing fish yield, enhancing food security and promoting sustainable and profitable aquaculture. The findings of this study support other studies that have reported that diets supplemented with ginger, garlic and turmeric promote fish resistance to bacterial infection. Naliato et al. [25] found that incorporating 1% ginger powder into the diet of O. niloticus enhanced the fish’s resistance to infections caused by A. hydrophila. Abdel-Tawwab et al. [50] also noted that supplementing the European sea bass (Dicentrarchus labrax) diet with 3% garlic stimulated its immune response to better resist Vibrio alginolyticus infection. In another study, diets enriched with 0.5%–2% turmeric improved the ability of Labeo rohita to better resist infection with Aeromonas veronii [51]. The incorporation of 1% garlic, 1% ginger and 1% thyme mixture in the diet significantly improved S. hasta resistance to Photobacterium damselae [22]. The enhanced resistance of O. niloticus against S. agalactiae infection noted in this research could be attributed to the phytochemicals like alkaloids, flavonoids, saponins and tannins present in the phytobiotics. These compounds exhibit antimicrobial, anti-inflammatory, antioxidant and immunostimulatory properties, positively influencing fish health status [52].
5. Conclusions
The study has shown that feed-enriched diets supplemented with 1% ginger, garlic and turmeric powder for O. niloticus beneficially affect fish growth, feed utilisation, immunity and resistance to S. agalactiae. The phytobiotics improved innate immune responses, contributing to fish overall health and vitality. In addition, the antimicrobial properties of phytobiotics or spices increase the fish’s resistance to disease and reduce the mortality rate. The aquaculture industry can harness the potential of phytobiotics to promote sustainable and healthy fish production and thus address concerns about antibiotic-resistant bacteria and chemical residues in fishery products. According to the study, natural aquafeed supplements such as ginger, garlic and turmeric powders can help enhance fish growth and overall health and are therefore recommended in aquaculture for sustainability reasons.
Ethics Statement
The authors followed ethical committee guidelines for the care and use of animals at the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding
No funding was received for this manuscript.
Acknowledgments
The authors appreciate the contribution of a microbiology technician, Mr. Acheampong Eric at the Department of Theoretical and Applied Biology, during bacteria inoculation.
Open Research
Data Availability Statement
The data that supports the findings of this study are available from the corresponding author upon reasonable request.
