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Article

Dietary Effect of a Plant-Based Mixture (Phyto AquaMeric) on Growth Performance, Biochemical Analysis, Intestinal Histology, Gene Expression and Environmental Parameters of Nile Tilapia (Oreochromis niloticus)

by
Abdel-Fattah M. El-Sayed
1,*,
Mahougnon Simeon Fagnon
2,*,
Amira M. Hamdan
1,
Thibaut Chabrillat
2,
Coralie Araujo
3,
Julie Bouriquet
2,
Sylvain Kerros
2 and
Salma M. S. Zeid
1
1
Oceanography Department, Faculty of Science, Alexandria University, Alexandria 21515, Egypt
2
Department of Innovation, Phytosynthese, 57 Avenue Jean Jaurès, 63200 Mozac, France
3
OxyLab, Phytosynthese, 57 Avenue Jean Jaurès, 63200 Mozac, France
*
Authors to whom correspondence should be addressed.
Fishes 2024, 9(9), 358; https://doi.org/10.3390/fishes9090358
Submission received: 9 August 2024 / Revised: 4 September 2024 / Accepted: 9 September 2024 / Published: 12 September 2024
(This article belongs to the Section Nutrition and Feeding)
Browse Figures
Figure 1
<p>Antioxidant potential of vitamin C and PAM in relation to the tested concentration in the reaction medium (0–100 mg L<sup>−1</sup>).</p> "> Figure 2
<p>Effect of dietary PAM on intestinal enzymes (mean ± SE; <span class="html-italic">n</span> = 3). ** indicates a significant difference between the two groups with <span class="html-italic">p</span> &lt; 0.01.</p> ">
Versions Notes

Abstract

:
This study aimed to assess the effect of dietary supplementation of a mixture of botanical compounds and essential oil (Phyto AquaMeric, PAM) on the growth, immune and antioxidant parameters and environmental benefits in Nile Tilapia. Two diets including a control and a PAM-supplemented diet at 0.5 g kg−1 were prepared for the trial. The diets were isonitrogenous (30% crude protein) and isoenergetic (17 MJ kg−1) extruded diets. Nile tilapia weighing initially 74.22 ± 1.96 g fish−1 were stocked in 2 m3 hapas at a density of 20 fish per m3 (40 fish per hapa). Each group was tested in triplicates over 80 days. The antioxidant potential of PAM was compared to vitamin C through KRL test, showing a higher value from 35 to 100 mg. The in vivo trial did not exhibit any significant growth performance improvement. However, the PAM group had a significantly improved feed conversion ratio (FCR) and protein efficiency ratio (PER). Moreover, the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), Malondialdehyde (MDA), immune parameters (ACH50, Lysozyme, phagocytic and phenoloxidase activities), digestive enzymes and hepatic enzymes were significantly improved in the PAM-fed group. An evaluation of eutrophication potential of PAM supplementation revealed a low input in the system. In conclusion, this mixture of botanicals and essential oil exhibited in vitro and in vivo antioxidant potential, improved health and digestive parameters and contributed to a reduced eutrophication risk in the tilapia production system.
Keywords:
botanical compounds; essential oils; phyto aquameric; antioxidants; health; feed efficiency; tilapia; eutrophication
Key Contribution: The mixture of plant and essential oil, hereby named PAM, improves feed efficiency and health parameters in Nile tilapia; PAM reduces the potential of eutrophication risk in Nile tilapia production.

1. Introduction

The Nile tilapia (Oreochromis niloticus) is one of the most cultivated fish species in the world; it is being farmed in more than 135 countries [1]. This species is currently ranked second in the list of top cultured fish species with a global production of 5.3 million tons and an estimated value of around 11.2 billion USD, representing 4.5% of the global value of cultured species [2]. Asia remains the top production continent of tilapia, which is mainly dominated by China with an average production of 1.62 million tons. Indonesia (Asia) and Egypt (Africa) are the second and third biggest tilapia producers over the world; contributing, respectively, 20.3 and 17.4% of the total tilapia production in 2022 [3].
The Nile tilapia is a fish species known for its capacity to resist to diverse environmental conditions and common pathogens, and it can achieve a rapid growth performance. However, there is increasing evidences that cultured Nile tilapia become more vulnerable to various abiotic and biotic stressful conditions, which may lead to important production loss [2]. For instance, in some countries, like Egypt, fish farmers observed unusual mortalities of Nile tilapia during the summer season, which might be due to the presence of pathogens or a weakened immune system [4]. Moreover, the efficiency of tilapia farming relies on different factors among which feed quality through a well-balanced feed represents 60–70% of the operational costs [5].
In recent years, apart from the feed cost, environmental issue and water quality have become a critical point to consider in the evaluation of fish farming profitability. Thus, addressing this issue requires consideration of different approaches, among which is the use of additives in fish farming. Additives are well known for their potential to optimize feed quality and utilization as well improve growth performance [6,7]. Nowadays, many additives are applied in fish farming with different purposes and modes of action. Among this diversity of solutions, botanicals or plant-based compounds (e.g., essential oils, powders of plants and plant extracts) are among the most documented in different production systems and for achieving different goals.
Botanicals refer to various preparations from herbs, spices, roots, stems and other plant materials [8]. These substances, which have been used since remotes times in phytotherapy, are well studied for their various bioactivities in different animals, including those of aquaculture [8]. Much evidence has demonstrated the potential effects of such compounds including by-products originating from plant sources, individually or in combination, on fish gut integrity, immune response, antioxidant parameters and environmental stress management [9,10]. Moreover, these botanical substances are usually considered as safe at the applied doses for animals, consumers and the environment, since they do not cause any detrimental problems [8,11]. Thus, Phyto AquaMeric (PAM), a blend of various botanical materials and by-products originating from bees, was formulated based on its richness in specific active molecules and bioactive properties. PAM is mainly formulated with turmeric, green propolis and essential oils. Turmeric, scientifically known as Curcuma longa, is a rhizomatous perennial herb of the botanical family of Zingiberaceae, which is rich in curcuminoids [9]. Curcuminoids, which represents 1.5–5% of the rhizome’s composition, are three main active compounds including curcumin (60–75%), desmethoxycurcumin (15–27%) and bisdemethoxycurcumin (5–15%) [12]. As polyphenolic compounds, curcuminoids are well documented for their potent antioxidant activity through in vitro [13] and in vivo approaches, especially in fish species [9]. The demonstration of these compounds in Nile tilapia improved growth performance and health status at different dosages [14,15]. Regarding propolis, it can be defined as a natural resinous substance produced by honey bees based on various plant materials such as leaves, buds and exudates [16]. Propolis, derived from a complex matrix of plants, has a variable chemical composition determined by different factors including bee species, plant species and geographical origins of production [17]. In addition, propolis is characterized by its diversity in term of colors, which may range from greenish-yellow to brown [16]. For instance, Brazilian green propolis is produced from an endemic Asteraceae plant species Baccharis dracuncufolia, commonly known as wild rosemary [16]. Brazilian green propolis is mainly rich in artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) [17]. This prenylated compound was well known for its immunomodulation and anti-inflammatory activities in vitro and in vivo biological models [18].
Thus, there is evidence of the beneficial effect of different types of propolis and turmeric on fish performance, individually. Furthermore, essential oils are a complex mixture of volatile compounds released as secondary metabolites of aromatic plants [19]. These substances are also well documented for their antimicrobial properties and their capacity to disturb pathogenic bacteria metabolism and promote the presence of beneficial bacteria [19]. For example, in Nile tilapia, lemon essential oils, rich in D-limonene exhibited bacteriostatic activity against Streptococcus agalactiae and Aeromonas hydrophila [20]. So far, there is no evidence in the literature combining different approaches mainly from antioxidant, anti-inflammatory and bacteriostatic compounds to evaluate their effect on fish performance and health condition, especially in the context of Nile tilapia under field production. In addition, the common dosages tested in the documented studies are relatively high according to some authority recommendations [21].
Despite the importance of health and growth performance in fish, especially tilapia, evidence had highlighted the importance of considering environmental parameters in the production system [22]. With the expected increase in tilapia production over the next years, it is critical to consider the sustainable and ecological impact of feed in formulation strategies. Intensive or semi-intensive farming can relatively lead to the accumulation of organic waste, excess nutrients and other residues in the fish environment. These discharges can contribute to the disturbance in water quality and sustainability performance of the overall production system. Thus, considering such an approach in the evaluation of additive performance in fish can contribute to mitigate environmental impacts.
Therefore, this research aimed at evaluating the beneficial effect of a specific mixture of compounds of botanical origin rich in artepillin C, curcuminoids and limonene (Phyto AquaMeric, PAM) on growth performance, health response and environmental parameters in Nile tilapia (O. niloticus) cultured under field conditions.

2. Materials and Methods

2.1. Experimental Design

This study was conducted in 2 m3 hapas (2 × 1 × 1.5 m) at Edku in Behaira Governate (Egypt). Each hapa was equipped with 50 cm of extension above the surface. The hapas were installed in a 2 ha pond with a depth of 1.5 m. Each hapa was installed into the pond bottom with 4 wooden legs at each corner. These hapas were covered with polyethylene netting to keep the fish from jumping out and to protect tilapia from bird predation.
Healthy monosex (all male) Nile tilapia (O. niloticus) with a mean weight of approximately 70 g were obtained from the same fish farmer who owns the farm. Fish were stocked into the hapas at stocking density of 20 fish m−3 (40 fish per hapa) and acclimatized over one week. After the acclimatization period, fish were netted, weighed and counted for recording of their initial average weight for the experimental trial. Thus, at the start of the trial, the average weight was 74.22 ± 1.96 g.

2.2. Botanical Mixture Description and Potential

2.2.1. Botanical Mixture Description

The tested supplementation is a mixture of standardized plant-based compounds and essential oils (Phyto AquaMeric, PAM), developed and formulated by Phytosynthese (Mozac, France). This mixture is rich in artepillin C from green propolis, curcuminoids from turmeric roots and D-limonene from natural essential oils.

2.2.2. In Vitro Antioxidant Potential of PAM through KRL Method

PAM was assessed for its potential to reduce oxidative stress with the KRL method (Kit Radicaux Libres). KRL test is a biological analytical method, which measures the potential of blood to resist free radical attack [23]. In this study, vitamin C was included in the analysis for comparison as it is a well-known antioxidant compound with a common application in fish for maintaining growth, health and achievement of physiological functions [24]. All analyses were performed in triplicates at OxyLab, Phytosynthese, Mozac (France). PAM and vitamin C were tested following the method described by Corino et al. [23]. Diluted blood (horse blood), in the presence of tested products or not, at different concentrations, was exposed to radical aggression using the method described by Corino et al. [23]. Hemolysis was performed using a 96-well microplate reader at 37 °C. The kinetic of resistance of the tested blood to hemolysis was monitored by measuring the absorbance change at 450 nm. The half time of hemolysis of blood in the presence of each product was determined (T1/2 hemolysis) as compared to the control. The obtained values were standardized into mg Trolox equivalent per g of the tested compounds and into mg of gallic acid per g of the tested compounds. Trolox® (Sigma-Aldrich, Saint Louis, MO, USA) concentrations ranged from 0 to 500 µmol L−1 (MW 250.29 G·mol−1) to standardize the overall antioxidant capacity of the tested products compared to Vitamin E [25].

2.3. Experimental Diet Preparation

Two experimental groups were considered according to the PAM supplementation in the diet. Thus, two isonitrogenous (30% CP), isocaloric (17 MJ kg−1) extruded (floating) diets were prepared with the inclusion of 0% PAM (control diet) and 0.5 g kg−1 PAM (tested group). The tested dosage (0.5 g/kg) was based on a previous trial performed on Atlantic salmon (unpublished data). The diets were produced by Makkah Aquafeed Mill, Kafr Elsheikh Governate, Egypt. The treatments were performed in triplicates (n = 3). Diet composition and proximate analysis are shown in Table 1.
During the trial, the feeds were distributed twice a day, between 8 and 9 am and 3 and 4 pm. Over the trial period (80 days), fish were fed at a rate of 3% of their body weight (BW) every day. The feeding rate was reduced to 2.5% when the temperature dropped to 25 °C and 2% when it reached 23 °C. Over the trial period, fish were weighed every 10 days, and the daily feed quantity was adapted accordingly.
The water quality parameters were measured during the whole trial and values were ranged as follows: temperature, 25 ± 3 °C; oxygen level, 5.1 ± 0.8 mg L−1; pH, 8.1 ± 0.45; and NH4-N, 0.05 ± 0.02 mg L−1.

2.4. Sampling

2.4.1. Growth and Feed Utilization Performance

At the end of the trial, fish were fasted for 24 h before harvest. Fish were netted, weighed and counted to record the final weight. Weight gain (WG), specific growth rate (%SGR), feed conversion ratio (FCR), protein efficiency ratio (PER) and survival were calculated from the following formulas:
-
WG (g) = final body weight (FBW) − initial body weight (IBW)
-
% WG (%) = 100 (FBW-IBW)/IBW
-
SGR (% Day−1) = 100 × [ln FBW − ln IBW]/feeding duration (days)
-
FCR = dry feed intake (g)/live WG (g)
-
PER = WG (g)/protein intake (g)
-
Survival (%) = 100 × [Total final fish number/total initial fish number]

2.4.2. Biochemical Analysis

To perform biochemical analysis, 5 fish from each hapa were sampled (n = 15 per group) and anesthetized using clove oil (0.5 mg L−1). Blood was collected from the caudal peduncle using sterile syringes. Samples from each hapa were separated into 2 groups. The first was stored in heparinized tubes for whole blood analysis (hemoglobin, total leukocyte and red blood cell counting), and the second group was kept in non-heparinized tubes for plasma analysis. Blood samples were preserved as described by Makled et al. [26]. Plasma was obtained from centrifugation at 6000× g for 10 min at 4 °C. Plasma was used for the analysis of lysozyme activity, immunoglobulin M level, superoxidase dismutase activity, glutathione peroxidase activity and ACH50.
All hematological parameters analyzed, including hemoglobin (Hb), red blood cells counting (RBC), total leukocyte counting (WBC), mean corpuscular volume (MCV) and mean corpuscular hemoglobulin (MCH), were determined using the methods described by Abdel-Tawwab et al. [27]. Phagocytic activity (PA) and lysozyme activity were determined using the method previously used by [28]. Phagocytic activity was calculated as follows:
PA = (Phagocytic leucocytes/total leucocytes)
Alternative complement activity (ACH50) was measured following the method outlined by Yano et al. [29] and superoxide dismutase activity (SOD) was analyzed according to the detailed method of Prieto et al. [30].
For digestive enzyme analysis, fish intestines were aseptically sampled and rinsed with cold distilled water. Intestine contents were collected, homogenized and centrifuged for assessing digestive enzymes. Protease, amylase and lipase activities were evaluated by following the standard methods as detailed by Sun et al. [31]. Results were expressed as specific activity (U mg−1 protein).
Livers were also sampled from the selected fish. Alanine aminotransferase (ALT), asparate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were analyzed according to the method described by Borges et al. [32].

2.4.3. Intestinal Histology Analysis

At the end of the trial, 3 fish were randomly sampled from each hapa (n = 9 fish per group). These fish were dissected, and intestines were removed, pooled together and preserved in ethyl alcohol (AR-95%) for intestinal histology evaluation. Analysis were performed following the protocol applied by Elsabagh et al. [33]. Briefly, the samples from the anterior gut, midgut and posterior gut were fixed in Bouin’s solution for 18–24 h. These samples were dehydrated in increasing concentration of ethanol and were prepared for histological analysis. Sections of 4–5 µm thickness were stained with hematoxylin and eosin for general morphometry. The length of intestinal villi were determined using NIH image analysis software (Version 1.49 NIH Image, Bethesda, MD, USA).
The liver was sampled from 3 fish per hapa, which equals 9 fish per dietary group. Each sample was labeled and immediately placed on dry ice whilst waiting for the end of the samples. After the sampling, livers were frozen at −80 °C until the final analysis as performed by Makled et al. [25]. Real-time PCR was used to assess the expression of the identified genes in fish livers. These genes include interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-12 (IL-12), interferon–gamma (IFN-γ), transforming growth factor–β (TGF-β), immunoglobulins (IGM) and tumor necrosis factor–α (TNF-α). The total RNA from liver tissues was isolated by using a Tri Pure Agent (Aidlab, Beijing, China) according to the manufacturer’s protocol, and their quantity and quality were tested using a NANODROP 2000 spectrophotometer (Thermo, Waltham, MA, USA). The cDNAs of the total RNA from liver tissues were analyzed using a Prime-ScriptTM, RT reagent Kit with a gDNA Eraser Eraser (Perfect Real Time, Takara, Japan). β-Actin was used as the reference gene. The PCR volume was 10 μL containing 1.0 μL of cDNA template, 5.0 μL of SYBR Premix Ex Taq, 1.0 μL of PCR primers (5 μM) and 3.0 μL of nuclease-free water. The qRT-PCRs were run with the following program: 95 °C for 30 s; 36 cycles of 95 °C for 5 s; and 60 °C for 20 s in the CFX Connect Real-Time System (Bio-Rad). To ensure specificity on intended genes, the primers for qRT-PCRs were designed to span an intron. qPCR efficiency was between 98 and 102%, and the correlation coefficient for each gene was over 0.97. The 2−ΔΔCt method was used to calculate the expression of target genes. The names and primer sequences of the genes are listed in Table 2.

2.5. Gut Microbiota Analysis

Gut microbiota analysis was performed following the procedures described by Yusefi et al. [34]. In summary, the intestines of 4 fish from each hapa (12 fish per dietary group) were aseptically excised and homogenized in 90 mL of 0.85% sterile physiological water (NaCl). The resulting suspension was filtered through a sterile nylon mesh (100 µm). Microbial cell screening and counts were expressed as log CFU. g-1 of intestine and analysis were performed in duplicate. Serial dilutions were prepared up to 10–4 using the same sterile physiological water (NaCl). Furthermore, total bacterial counts were assessed through incubation of the samples using plate count agar (TSA, Hi-Media). Beneficial bacteria, mainly Bacillus sp. and Lactobacillus sp., were counted by incubating diluted samples on de Man Rogosa and Sharpe (MRS) agar (Hi-Media). Pathogenic bacteria, especially Staphylococcus sp. and Streptococcus sp., were isolated and counted on blood agar. The incubation condition of plates was 37 °C for 48 h.

2.6. Environmental Impact on Farming System

The environmental impact of tilapia production in semi-intensive farming systems was analyzed based on the model determined by Yacout et al. [35]. In this study, the entire scope of life cycle analysis (LCA) of tilapia farming was not addressed, only the eutrophication potential (EP) of PAM was considered. The EP value for a semi-intensive farming condition was obtained from the study of Yacout et al. [35] as the baseline for comparison between the control and PAM groups. In the diet composition, the main difference was the substitution of corn amount by PAM.
The EP of the control diet including 0.5% of corn was obtained from the database of Agribalyse 3.1.1 (https://agribalyse.ademe.fr). Regarding PAM, its value was calculated from the same database and literature [36,37] as it contains various ingredients. The potential reduction of EP by incorporating PAM in the diet was compared with the control by considering their respective feed conversion ratio values (FCR). A negative difference in the EP value indicates a theoretical potential reduction of eutrophication in the presence of PAM in the diet when compared to the control under the same farming condition.

2.7. Data Statistical Analysis

Data were presented as the mean ± standard error (SE) of the 3 hapas (n = 3). For each variable, the normality was assessed using a Shapiro–Wilk test, and a Levene test was used to check the homogeneity of variances. When these assumptions were validated, Student’s t-test was applied to evaluate the difference between the control and PAM group. Otherwise, the non-parametric Wilcoxon signed-rank test was used to check the eventual difference with p value = 0.05. All statistical analyses were performed using R software (R Studio 2022.12.0, Boston, MA, USA).

3. Results

3.1. Antioxidant Status of PAM through a KRL Assay

Results from the KRL test revealed that PAM had a higher antioxidant capacity than vitamin C throughout the concentration range of 35–100 mg L−1 (Figure 1). However, before this concentration range, the KRL values of vitamin C was higher than PAM ones. The KRL values for vitamin C was 998 ± 34 mg of Trolox g−1 and 382 ± 13 mg of gallic acid g−1, while for PAM, it was 1025 ± 36 mg of Trolox g−1 and 405 ± 14 mg of gallic acid g−1 of the product at the concentration of 50 mg L−1. No significant difference was observed at this concentration (p > 0.05).

3.2. Growth Perfomance and Feed Effciency

The growth performance and feed efficiency parameters were illustrated in Table 3. The tested group did not show any significant difference regarding the final weight, weight gain, percentage of weight gain and specific growth rate (SGR). However, fish supplemented with PAM demonstrated a better feed conversion ratio (FCR) and protein efficiency ratio (PER) compared to the control (p < 0.05). No mortality was observed during the trial.

3.3. Hematological Parameters

The hematological parameters of sampled fish are summarized in Table 4. The analysis of blood parameters of each dietary group showed that the PAM group had a significantly higher value for red blood cells and hemoglobin compared to the control diet group (p < 0.05). No differences were observed between the groups regarding white blood cell count, the mean corpuscular hemoglobin and mean corpuscular volume (p > 0.05).

3.4. Immunological and Antioxidant Parameters

The effects of dietary PAM on the immune response and antioxidant parameters were summarized in Table 5. The data showed that all measured immune parameters, especially phagocytic activity (PA), phenoloxidase activity (PO), alternative complement activity (ACH50) and lysozyme activity (LSZ) were significantly enhanced (p < 0.05) in the group fed with PAM compared to the control group. Regarding the antioxidant enzymes measured, superoxide dismutase (SOD) was significantly increased, while malondialdehyde (MDA) was significantly reduced in the PAM group compared to the control (p < 0.05). However, glutathione peroxidase (GPx) was not significantly affected by the supplementation of PAM (p > 0.05).

3.5. Digestive and Liver Enzymes

Figure 2 shows the effect of PAM inclusion on digestive enzymatic activities. The supplementation of PAM in the diet significantly improved intestinal protease (82.20 ± 2.48 vs. 62.18 ± 2.74), lipase (66.80 ± 2.12 vs. 50.12 ± 2.44), and amylase (39.51 ± 1.55 vs. 21.58 ± 2.36) (p < 0.01). The results of liver enzymes are summarized in Table 6. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were significantly lower than the control (p < 0.05), while alanine aminotransferase (ALT) was not significantly affected by dietary treatment (p > 0.05).

3.6. Intestinal Histology

The histological parameters analyzed from fish intestines are presented in Table 7. The group supplemented with PAM showed an improvement of gut structure and integrity. The lengths of the intestinal folds and the number of goblet cells significantly increased in the anterior gut, midgut and posterior gut of Nile tilapia fed PAM in comparison to the control diet (p < 0.05). On the contrary, the width of intestinal folds and interfold space were significantly lower in the anterior, middle and posterior gut for the supplemented diet compared to the control (p < 0.05).

3.7. Gene Expression Analysis

The effect of PAM on the expression of gene in the liver was presented in Table 8. It was observed that FN-γ, TNF-α, TGF-β, IL1-β, IL-4, IL-12 and IgM were significantly up regulated in the PAM group.

3.8. Gut Microbiota

The results of the bacterial counting in this study were shown in Table 9. Total and beneficial microbiota (Bacillus sp.) count were significantly higher for the PAM group than the control group (p < 0.05). On the contrary, a significantly lower count of pathogenic bacteria (Staphylococcus sp.) was found in the group fed with PAM compared to the PAM-free group (p < 0.05) (Table 9).

3.9. Eutrophication Potential

The potential of eutrophication was evaluated in a simulated model of semi-intensive production. It was shown that PAM markedly reduced the eutrophication level in the production system (19.19 eq kg PO4 for control vs. 8.24 eq kg PO4 for PAM) compared to the control (Table 10).

4. Discussion

This study assessed the effect of a mixture of compounds from a botanical origin, bee by-products and essential oils on different parameters such as growth, feed efficacy, health and environmental parameters of Nile tilapia (O. niloticus) cultured under natural conditions. The key active compounds were curcuminoids from turmeric and artepillin C from green propolis are well documented for their antioxidant capacity in different models [9,17]. The KRL (Kit Radicaux Libres) test has emerged as a biological approach for testing the antioxidant status of plants and other biological models [37]. The KRL test assesses the resistance of a blood (supplemented with any material or not) to free radicals by measuring the time required for 50% hemolysis of red blood cells under radical attack [23,37]. In the current study, PAM showed a higher antioxidant capacity compared to vitamin C at the range of 35 to 100 mg L−1. Vitamin C is a well-known and powerful water soluble antioxidant, which can scavenge many free radicals [38]. Unlike to previous studies [23,37], this work revealed the antioxidant capacity of this specific compound blend through KRL. These findings highlighted the strong antioxidant ability of PAM, which can be attributed to the key molecules.
In the present study, Nile tilapia was not significantly affected by the supplementation of PAM in terms of growth performance. Similar results were observed by using such a bioactive technology, which includes artepillin C and curcuminoids in white leg shrimps (Liptopenaeus vannamei) [39]. From the literature, a numeric trend of growth improvement was also observed when Nile tilapia were supplemented independently with curcumin (from turmeric) or propolis at different levels [10,14]. A numerical increase in fish growth performance was found in Nile tilapia fingerlings fed with curcumin at 600 and 800 mg kg−1 diet, while a significant improvement was observed at 200 and 400 mg kg−1 by 21 and 27%, respectively, when compared to the control diet [14]. In addition, in another study on Nile tilapia juveniles, a significant increase was identified at 50 mg kg−1 of diet. Regarding propolis, in many trials, fish fed with diets supplemented with propolis revealed a significant growth enhancement compared to the control group [10]. However, in this study, the tested propolis was specifically the Brazilian green propolis. So far, there is no evidence of using green propolis alone on the assessment of fish growth performance. Another bioactive compound included in this PAM was limonene, a cyclic monoterpene derived mainly from the peel oil of oranges, grapefruits and lemons [40]. A significant increase in fish weight and SGR was pointed out in Nile tilapia in some studies [41]. However, it is noteworthy to mention that the inclusion rate of reviewed bioactive compounds from the literature was higher than their respective content in PAM.
In this study, PAM exhibited a significant reduction in FCR in comparison to the control group (1.46 ± 0.03 vs. 1.31 ± 0.06). A similar reduction of FCR was observed in various trials performed in Nile tilapia when fed separately on turmeric or curcumin [9,11], propolis [10] and limonene [41]. Such a reduction in FCR is usually determined by different factors, including digestive integrity.
As the intestine is the primary site for feed digestion and nutrient absorption, the efficiency of nutrient use by fish relies on the functional and integrity of this organ [41]. Ref. [14] showed that feeding Nile tilapia with curcumin at two levels (400–600 mg kg−1) increased villus height, crypt depth and goblet cell number in the mucosa and submucosa. It was observed the same trend in this study, with the PAM group exhibiting a significant increase in intestinal length folds in the anterior, middle and posterior guts. In addition, this increase was followed by the same significant trend of goblet cell numbers from the initial segment of the intestine to the end of intestine segment. Intestinal folds are identified as crucial sites for nutrient absorption and the extension of these tissues lead to the increase in surface area, therefore nutrient uptake [42]. The increase in these intestinal folds enhanced the contact with the fluids and digested nutrients in the lumen. Thus, the nutrients can diffuse from the intestinal folds into the bloodstream, which contributes to favoring of optimal efficient nutrition absorption and thereby feed utilization [41]. In another study in which propolis was tested in vitro on human intestinal epithelial cells, CaCo-2, ref. [43] demonstrated that cells treated with polyphenol-rich propolis extracts increased the expression of the tight junction proteins (occludin and zona occludens), which contribute to the strengthening of intestinal barrier function.
In this study, which is the first that involves green propolis extract in Nile tilapia diets, the combination of phenolic compounds from green propolis and curcuminoids from turmeric was thought to enhance the synergy of such compounds on gut integrity and health. The potential additive activity revealed by the current study on gut integrity was also partly demonstrated by Nishikawa et al. [44] on mice model. In addition, De Albuquerque et al. [45] demonstrated the interaction between different flavonoids and curcumin through various methods such as Caco-2 cell assays and molecular docking (in silico). From this study, it was shown that curcumin strongly interacts with flavonoids through non-covalent interactions (π-π stacking), which enhances their absorption and efficacy in the lipid bilayer of the cell membrane. Thus, the enhancement of the intestinal morphology may be explained by the capacity of these two compounds (curcuminoids and artepillin C) to maintain the integrity of gut through their synergetic activity. However, dietary limonene supplementation in Nile tilapia feed was also found to increase the length of intestinal folds, consequently increasing the surface area covered by these folds [46].
Endogenous enzymes are also crucial for nutrient digestion in fish, which thereby enhances their digestive ability [47]. In this current study, the activities of the digestive enzymes such as amylase, lipase and protease were significantly improved by this mixture of botanicals PAM. Similarly, curcumin and turmeric were demonstrated in many studies to improve enzymatic activities of protease, lipase and amylase [48,49]. A significant increase was observed in O. niloticus for the activities of α-amylase, protease and lipase fed with 5 and 10 g kg−1 curcumin supplementation in the feed [49]. In crucian carp, curcumin was demonstrated to enhance trypsin and lipase activities in the hepatopancreas and intestine as well as other enzymes such as natrium-kalium-adenosine triphosphatase (NKA), alkaline phosphatase (AKP), gamma-glutamyl transferase (γ-GT) and creatine kinase (CK) in fish intestine [48,50]. Thus, these studies are consistent with the key role of curcuminoids in the amelioration of digestive enzyme activities. However, limonene is also well-documented to exert such enzymatic activity improvement in various fish species such as O. niloticus and Catla catla [51,52]. A phytogenic mixture containing lemon (Citrus limon) fed to Nile tilapia showed an increase in the activity of digestive enzymes, such as chymotrypsin, trypsin, amylase, and lipase [52].
Moreover, the microbial population of the gut, in general, represents a highly important and diverse enzymatic potential. The enzymatic mass present in the digestive tract can significantly influence a large portion of the host animal’s metabolism, which could lead to better feed utilization [52]. Many studies demonstrated the capacity of essential oils to exert antimicrobial activity, and thereby regulate gut microbiota by decreasing the growth of pathogenic bacteria and colonizing beneficial bacteria [53]. In our study, this botanical and essential oil mixture significantly increased the beneficial gut microbiota count (ex. Bacillus sp.) and reduced the total number of pathogenic bacteria (ex. Staphylococcus sp.). Limonene, included in this botanical and essential oil mixture, was demonstrated to exhibit an antimicrobial effect on different pathogenic strains [54,55]. This bioactive compound also has the capacity to destroy the integrity and permeability of the cell wall and cell membrane of bacteria and can lead to the cell death of different bacterial strains (Staphylococcus sp., Listeria sp.) [54,55]. An in vivo trial on Labeo rohita fed citrus limon extract showed antibacterial activity and reduced mortality [56]. The same trend was observed in common carp (Cyprinus carpio) with a reduction in mortality and lowest intestinal total viable bacteria in the treated groups [57]. In the same line with limonene, curcumin supplementation was demonstrated to increase the relative abundance of lactic acid bacteria in the intestine of common carp (Cyprinus carpio) [58]. Similar results were also reported when curcumin was incorporated into the diets of various fish species, such as O. niloticus and O. mykiss [59,60].
In the present study, hematological parameters were also assessed in Nile tilapia supplemented with PAM. The PAM mixture significantly impacted hemoglobin count and red blood cell count over the feeding period. Thes findings were consistent with the results obtained on red tilapia [15] and other fish species fed with curcumin as supplement [59,61]. Red blood cells are the most common type of blood cells, primarily responsible for delivering oxygen to the body tissues. In addition, there is evidence that these cells are well involved in the support of immune system and health parameters. The observed increase in this study may indicate a positive effect on erythrogram indices, with no sign of anemia. Additionally, it may explain the potential of PAM to stimulate chemosynthesis and erythropoiesis in Nile tilapia [15]. Regarding the impact of PAM on the hematology of Nile tilapia, the capacity of PAM to boost the immune system can be highlighted as blood parameters are usually considered as health status indicators.
In the same vein as hematological parameters, immune parameters were analyzed in this study. Fish rely heavily on their innate immune system as the primary defense against pathogenic attacks, which is commonly more effective in fish compared to mammals [39]. From these results, it was observed that all parameters related to the immune system, including phagocytic activity, phenoloxidase activity, lysozyme activity and alternative complement activity were significantly enhanced. The same trend was observed in a mixture based on green propolis and turmeric on shrimps [40]. In addition, there is much evidence of the activity of such compounds (curcumin and artepillin C) on innate immune markers of fish species [9,10,11]. Furthermore, antioxidant capacity was higher in fish fed PAM than the control group. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are among key antioxidant enzymes that contribute to reduce oxidative stress in animal including fish. SOD is an enzyme known to convert harmful superoxide (O2−) into less toxic compound, hydrogen peroxide (H2O2) [61]. Furthermore, GPx, another enzyme that favors protection against free radicals, is well known to catalyze the reduction of hydrogen peroxide (H2O2) and organic hydroperoxide using reduced glutathione as a substrate [62]. Both endogenous enzymes, together, play a critical role in neutralizing excessive free radicals, maintaining oxidative balance in animals and thereby protecting animals and their tissues against oxidative damage [63]. In addition to these enzymes, MDA, a metabolite of lipoperoxidation, was significantly decreased in the PAM group compared to the control group. This significant reduction may indicate the extent of free generated radical reduction and less tissue damage due to oxidative stress. From these markers of antioxidant (SOD, GPx and MDA), it is evident that PAM has a potent antioxidant capacity as demonstrated in vitro through the KRL assay.
Gene expressions of immune-related genes, namely interferon, tumor necrosis factor, IL1-β, IL-12, IGM2 and TGF-β, were analyzed. The expression of these genes was significantly upregulated in the group fed with PAM. Propolis was known to exert an inflammatory and modulating effect on innate immunity. However, under some specific conditions, this compound, which is rich in artepillin C, could demonstrate an upregulation of some pro-inflammatory cytokines, suggesting its ability to act as immunomodulator and/or immune restoring compounds [63]. Similar findings were reported by Amer et al. [14], demonstrating that dietary curcumin supplementation in Nile tilapia feed stimulated the expression of proinflammatory cytokines such as TNF-α and apoptosis markers such as caspase-3. Midhun et al. [48] observed that supplementing Mozambique tilapia feed with 0.5% curcumin significantly increased the expression of IGF-1 and IGF-2 genes in fish muscle. This finding confirms that artepillin C and curcuminoids together have the capacity to modulate the immune responses in Nile tilapia.
The fish liver is a critical organ which plays an important role in the overall health of fish, and it is involved in various metabolic functions and detoxification processes. In this study, aspartate aminotransferase (AST) and lactic acid dehydrogenase (LDH), two key enzymes in hepatocytes, showed a significant reduction in the group fed with PAM as compared to the control group. This finding revealed a healthier liver in the group fed PAM compared to the control since any increase in such enzyme activities represent a biological marker of liver damage [64]. From these observations, it was suggested that fish in which PAM was included in the diet did not face liver stress and showed better hepatic status compared to the control. Similar results were reported by Amer et al. [14] in which a significant reduction of serum ALT and AST activities was observed in tilapia fed diets supplemented with curcumin. Thus, curcuminoids, a key compounds of PAM, showed their capacity to reduce certain liver markers such as ALT and AST, which have a huge potential as hepatoprotective against any toxic agent.
In aquaculture, the environmental evaluation of water quality has become a critical point that requires careful consideration. The assessment of water quality in fish production is important to guarantee good growth and health of fish. However, there are many factors that can interact with the production system among feed quality. As feed quality and quantity are two important factors that can impact water quality and be linked directly to eutrophication risk, this parameter was simulated in the present study. From this simulation, the potential for eutrophication with PAM supplementation (9.19 EP in eq kg PO4) was numerically lower than that of the feed without the PAM product (8.24 EP in eq kg PO4). This study provided a preliminary evaluation of the environmental benefit of PAM in a theoretical semi-intensive production system of tilapia. To achieve a comprehensive life cycle assessment of using this mixture in tilapia production, many other parameters need to be considered, such as acidification potential, water use, global warming potential and energy use [35]. These elements should be addressed in another study to perform a complete life cycle assessment of the product. Additionally, the low feed utilization required to achieve the same growth performance as the control group is crucial for economic purposes [65], highlighting the potential of such a mixture (PAM) to help achieve profitability (economic performance).

5. Conclusions

Phyto AquaMeric (PAM) resulted in better feed digestion and utilization, immune response and liver function than the control diet. In addition, the strong antioxidant capacity of PAM observed with the KRL biological method was confirmed through antioxidant parameters (SOD and MDA). The results highlighted through the potential of this PAM mixture is to reduce environmental impact (eutrophication) and contribute to economic benefits in the production system. To conclude, Phyto AquaMeric (PAM), a mixture of key compounds, mainly artepillin C, curcuminoids and Limonene, exhibited beneficial effects on Nile tilapia culture.

Author Contributions

The contribution of different authors are as follows: conceptualization, M.S.F., T.C. and A.-F.M.E.-S.; methodology, M.S.F., T.C. and A.-F.M.E.-S.; software, M.S.F., J.B. and A.-F.M.E.-S.; validation, M.S.F. and A.-F.M.E.-S.; formal analysis, A.M.H., S.M.S.Z. and C.A.; investigation, A.-F.M.E.-S.; resources, M.S.F., T.C. and S.K.; data curation, M.S.F.; writing—original draft preparation, M.S.F. and A.-F.M.E.-S.; writing—review and editing, M.S.F., T.C., A.M.H., S.M.S.Z., S.K., J.B., C.A. and A.-F.M.E.-S.; visualization, M.S.F.; supervision, A.-F.M.E.-S.; project administration, A.-F.M.E.-S.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Phytosynthese, France (Funding Number: N PGMET254).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board of University of Alexandrie (Egypt).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data availability statements are available under request via the above email.

Acknowledgments

We would like to thank the farmer who allowed us to perform the trial in his ponds. The authors are also thankful to Makkah AquaFeed Company, Kafrel-Shaikh Governorate, Egypt, for kindly manufacturing the test diets used in the study. This research is a scientific collaboration between the University of Alexandria (Egypt) and PHYTOSYNTHESE (Mozac, France).

Conflicts of Interest

M.S.F., T.C., C.A., J.B. and S.K. are employed by Phytosynthese (France). Other authors declare no conflict of interest.

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Fishes 09 00358 g001
Figure 1. Antioxidant potential of vitamin C and PAM in relation to the tested concentration in the reaction medium (0–100 mg L−1).
Figure 1. Antioxidant potential of vitamin C and PAM in relation to the tested concentration in the reaction medium (0–100 mg L−1).
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Fishes 09 00358 g002
Figure 2. Effect of dietary PAM on intestinal enzymes (mean ± SE; n = 3). ** indicates a significant difference between the two groups with p < 0.01.
Figure 2. Effect of dietary PAM on intestinal enzymes (mean ± SE; n = 3). ** indicates a significant difference between the two groups with p < 0.01.
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Table 1. Analyzed composition of the formulated diets (g/kg).
Table 1. Analyzed composition of the formulated diets (g/kg).
IngredientControlPAM
Fishmeal (54% crude protein)30.0030.00
PBM 1 (54% crude protein)75.0075.00
SBM 2 (54.6% crude protein)425.00425.00
Wheat bran287.00287.00
Rice bran115.00115.00
Corn51.1550.65
Soybean oil6.006.00
Monocalcium Phosphate4.004.00
Sodium bicarbonate1.001.00
Calcium carbonate1.001.00
Vitamin and mineral premix 32.002.00
Vitamin C0.500.50
Methionine0.500.50
Lysine0.500.50
Anti-toxin (Unic Plus)1.001.00
Emulsifier0.250.25
Phytase enzyme0.100.10
Phyto AquaMeric (PAM)-0.50
Total10001000
Proximate composition (% as fed)
Moisture7.807.58
Crude protein30.5030.10
Crude lipid5.606.10
Ash8.408.2
Crude fiber4.004.55
NFE 443.7043.47
Gross energy (MJ kg−1) 517.1017.17
1 PBM, poultry by-product meal. 2 SBM, Soy bean meal. 3 IU or mg kg−1: Vit A, 6,000,000 IU; Vit D3, 400,000 IU; Vit E, 60,000; Vit K3, 4000; Vit B1, 4000; Vit B2, 4000; Vit B3, 30,000; Vit B5, 10,000; Vit B6, 3000; Vit B12, 20; Biotin, 75; Folic Acid, 1500; Manganese, 7000; Iron, 20,000; Zinc, 30,000; Copper, 5000; Cobalt, 150; and Selenium, 1000. 4 Nitrogen-free extract (NFE) = 100 − (moisture + crude protein + crude lipid + ash). 5 Gross energy, calculated based on 23.64, 39.54 and 17.57 KJ g−1 for protein, lipids and carbohydrates, respectively.
Table 2. Primers used for the analysis of messenger RNA gene expression by quantitative real-time polymerase chain reaction (qRT-PCR).
Table 2. Primers used for the analysis of messenger RNA gene expression by quantitative real-time polymerase chain reaction (qRT-PCR).
GeneForward Sequence (5′->3′)Accession No.
IL-12F (5′->3′): GGGTGCGAGTCAGCTATGAG
R (5′->3′): GGTTGTGGATTGGTTGCGTC		XM_003437924.4
IL-1βF (5′->3′): GACACTGCTTCTGAACTACAAGT
R (5′->3′): TCAGCACTGGCTCTGAAGTG		XM_019365844.2
TNF-αF (5′->3′): GCAGCTGAATGAACCTCTCAC
R (5′->3′): GTTCTCAGTCTGTCCCCAGC		XM_019365844.2
TGF-βF (5′->3′): GTCCTGCAAGTGCAGCTAGA
R (5′->3′): CATGCCTGTGTGAAACGACTG		XM_005463992.4
IFN-γF (5′->3′): GGGTGGTGTTTTGGAGTCGT
R (5′->3′): CATCTGTGCCTGGTAGCGAG		XM_013266976.3
IL-4F (5′->3′): CAGCGAGAGAGAACTCGTGC
R (5′->3′): GGTTTCCTTCTCCGTCGTGT		NM_214123.1
IGM-2F: (5′->3′): CCACTTCAACTGCACCCACT XM_005463992.4
R (5′->3′): TGGTCCACGAGAAAGTCACC
β-actinF: TCAGGGTGTGATGGTGGGTATGEU887951.1
R: CTCAGCTCGTTGTAGAAGGTGT
IL-12, interleukin 12; IL-1 β, interleukin-1 beta; TNF-α, tumor necrosis factor alpha; TGF-β, transforming growth factor; IFN-γ, interferon-gamma; IL-4, interleukin-4; IGM, immunoglobulins; β-actin, beta-actin.
Table 3. Growth and feed efficiency parameters (mean ± SE; n = 3) of Nile tilapia fed the experimental and control diets. Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
Table 3. Growth and feed efficiency parameters (mean ± SE; n = 3) of Nile tilapia fed the experimental and control diets. Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
ParametersControlPAM
Initial weight (g fish−1)74.86 ± 2.19 a73.84 ± 1.27 a
Final weight (g fish−1)186.10 ± 3.96 a186.32 ± 3.12 a
Weight gain (g fish−1)111.24 ± 3.27 a112.48 ± 3.19 a
SGR (% day−1)1.15 ± 0.02 a1.16 ± 0.03 a
Feed intake (g)161.76 ± 3.51 a146.78 ± 3.94 b
FCR1.46 ± 0.03 a1.31 ± 0.06 b
PER2.25 ± 0.05 a2.48 ± 0.03 b
SGR, specific growth rate; FCR, feed conversion ratio; PER, protein efficiency ratio.
Table 4. Blood parameters of Nile tilapia fed the tested and control diets (mean ± SE; n = 3). Different letters on the same row indicate significant difference between the two groups (p < 0.05).
Table 4. Blood parameters of Nile tilapia fed the tested and control diets (mean ± SE; n = 3). Different letters on the same row indicate significant difference between the two groups (p < 0.05).
ParametersControlPAM
RBC count (106 mL−1)2.27 ± 0.08 a2.63 ± 0.16 b
WBC count (103 mL−1)177.52 ± 10.00 a195.37 ± 7.79 a
Hb (g dL−1)11.50 ± 0.62 a12.87 ± 0.49 b
MCH (pg·cell −1)50.63 ± 1.01 a48.90 ± 1.49 a
MCV (µm3)146.60 ± 5.73 a143.60 ± 1.82 a
RBC, red blood cells; WBC, white blood cell; Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV: mean Corpuscular Volume.
Table 5. Immune response and antioxidant capacity (mean ± SE; n = 3) of Nile tilapia fed the tested diets. Different letters on the same row indicate significant difference between the two groups (p < 0.05).
Table 5. Immune response and antioxidant capacity (mean ± SE; n = 3) of Nile tilapia fed the tested diets. Different letters on the same row indicate significant difference between the two groups (p < 0.05).
ParametersControlPAM
PA (µM mL−1)1.22 ± 0.04 a1.65 ± 0.05 b
PO (mU mL−1)98.63 ± 2.13 a108.0 ± 2.98 b
ACH50 (ng mL−1)44.16 ± 2.62 a53.90 ± 0.27 b
LSZ (ng mL−1)82.57 ± 0.55 a92.01 ± 3.04 b
SOD (mU mL−1)29.93 ± 2.06 a39.17 ± 3.54 b
MDA (mU mL−1)23.41 ± 2.51 a15.02 ± 2.33 b
GPx (mU mL−1)3.47 ± 0.12 a3.67 ± 0.14 a
PA, phagocytic activity; PO, phenoloxidase activity; LSZ, lysozyme; ACH50, alternative complement activity; MDA, malondialdehyde; SOD, superoxide dismutase; GPx, glutathione peroxidase.
Table 6. Dietary effect of PAM on liver enzymes (mean ± SE; n = 3) of Nile tilapia fed the test diets. Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
Table 6. Dietary effect of PAM on liver enzymes (mean ± SE; n = 3) of Nile tilapia fed the test diets. Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
ParametersControlPAM
LDH (U L−1)135.48 ± 4.52 a126.21 ± 2.18 b
AST (U L−1)16.67 ± 2.08 a12.67 ± 2.08 b
ALT (U L−1)10.67 ± 1.15 a8.67 ± 1.52 a
LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase.
Table 7. Effect of dietary AM on liver enzymes (mean ± SE; n = 3). Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
Table 7. Effect of dietary AM on liver enzymes (mean ± SE; n = 3). Different letters on the same row indicate a significant difference between the two groups (p < 0.05).
ParameterControlPAM
Anterior gut
Length of intestinal folds (µm)133.17 ± 9.75 a212.58 ± 3.74 b
Width of intestinal folds (µm)74.26 ± 5.18 a67.11 ± 7.67 b
Interfold space (µm)80.40 ± 1.36 a48.32 ± 2.35 b
Goblet cells number mm−251.99 ± 3.50 a80.04 ± 1.72 b
Midgut
Length of intestinal folds (µm)155.40 ± 13.12 a341.30 ± 32.36 b
Width of intestinal folds (µm)55.00 ± 6.50 a48.23 ± 8.82 b
Inter fold space (µm)61.00 ± 5.74 a45.72 ± 4.22 b
Goblet cells number mm−277.62 ± 6.20 a159.19 ± 10.33 b
Posterior gut
Length of intestinal folds (µm)80.60 ± 5.76 a165.59 ± 15.53 b
Width of intestinal folds (µm)112.33 ± 4.18 a54.07 ± 4.18 b
Inter fold space (µm)125.99 ± 24.61 a60.84 ± 5.54 b
Goblet cells number mm−228.27 ± 2.31 a82.40 ± 5.59 b
Table 8. Gene expression analysis of Nile tilapia fed the tested diets (mean ± SE; n = 3). Different letters on the same row indicate significant difference between the two groups (p < 0.05).
Table 8. Gene expression analysis of Nile tilapia fed the tested diets (mean ± SE; n = 3). Different letters on the same row indicate significant difference between the two groups (p < 0.05).
GenesControlPAM
IFN-γ (pg mL−1)128.23 ± 1.16 a137.60 ± 0.69 b
TNF-α (copies mL−1)338.47 ± 1.92 a350.57 ± 1.34 b
TGF-β (pg mL−1)65.60 ± 1.14 a 71.97 ± 1.60 b
IL-1β (copies mL−1)563.57 ± 1.32 a 585.1 ± 1.51 b
IL-4 (copies mL−1)81.33 ± 1.52 a 118.33 ± 4.04 b
IL-12 (copies mL−1)244.33 ± 1.15 a299.33 ± 2.51 b
IGM (µg mL−1)120.80 ± 0.55 a159.17 ± 1.37 b
Table 9. Bacterial counts (log CFU g−1) in the intestines of Nile tilapia fed the test diets (mean ± SE; n = 3) Different letters on the same row indicate significant difference between the two groups (p < 0.05).
Table 9. Bacterial counts (log CFU g−1) in the intestines of Nile tilapia fed the test diets (mean ± SE; n = 3) Different letters on the same row indicate significant difference between the two groups (p < 0.05).
CountControlPAM
Total bacterial counts142.66 ± 5.37 a201.33 ± 5.61 b
Beneficial bacteria44.33 ± 2.34 a151.00 ± 3.22 b
Pathogenic bacteria98.33 ± 4.42 a50.33 ± 3.18 b
Table 10. Eutrophication potential of the tilapia production system considering both groups (with PAM and without PAM).
Table 10. Eutrophication potential of the tilapia production system considering both groups (with PAM and without PAM).
Eutrophication Potential (EP in eq kg PO4)/Semi-Intensive System
ParametersControlPAM
Semi-intensive production6.36.3
EP of each product (wheat vs. PAM)1.8 × 10−44 × 10−3
FCR1.461.31
EP for each production system9.198.24
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El-Sayed, A.-F.M.; Fagnon, M.S.; Hamdan, A.M.; Chabrillat, T.; Araujo, C.; Bouriquet, J.; Kerros, S.; Zeid, S.M.S. Dietary Effect of a Plant-Based Mixture (Phyto AquaMeric) on Growth Performance, Biochemical Analysis, Intestinal Histology, Gene Expression and Environmental Parameters of Nile Tilapia (Oreochromis niloticus). Fishes 2024, 9, 358. https://doi.org/10.3390/fishes9090358

AMA Style

El-Sayed A-FM, Fagnon MS, Hamdan AM, Chabrillat T, Araujo C, Bouriquet J, Kerros S, Zeid SMS. Dietary Effect of a Plant-Based Mixture (Phyto AquaMeric) on Growth Performance, Biochemical Analysis, Intestinal Histology, Gene Expression and Environmental Parameters of Nile Tilapia (Oreochromis niloticus). Fishes. 2024; 9(9):358. https://doi.org/10.3390/fishes9090358

Chicago/Turabian Style

El-Sayed, Abdel-Fattah M., Mahougnon Simeon Fagnon, Amira M. Hamdan, Thibaut Chabrillat, Coralie Araujo, Julie Bouriquet, Sylvain Kerros, and Salma M. S. Zeid. 2024. "Dietary Effect of a Plant-Based Mixture (Phyto AquaMeric) on Growth Performance, Biochemical Analysis, Intestinal Histology, Gene Expression and Environmental Parameters of Nile Tilapia (Oreochromis niloticus)" Fishes 9, no. 9: 358. https://doi.org/10.3390/fishes9090358

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