IntroductionTop
Brazilian fish farming has increased significantly over the last few decades as the result of adequate climate, high availability of water, and excellent nutritional value of fish, in general, causing this market to emerge as one of the main activities of Brazilian agribusiness (Oliveira, 2015). One of the most cultivated species in the country is the Nile tilapia (Oreochromis niloticus), because it presents good adaptation to intensive culture, rapid growth, rusticity and excellent meat quality (Figueiredo-Júnior & Valente, 2008).
The presence of high storage densities, high concentrations of ammonia, inadequate feeding, mismanagement, and the presence of bacteria and ectoparasites are common factors that lead to increased stress and incidence of diseases (Karunasagar et al., 1991) when intensive culturing practices are used, consequently affecting the growth and physiological performance of the fishes. The use of antibiotics to face possible diseases has been adopted, however, risks related to their use, such as cumulative effects on the environment and humans, and the development of resistant pathogens must be seriously considered (Holmstrom et al., 2003).
One promising method for improving fish health performance and reducing the occurrence of diseases is the prophylactic administration of diets supplemented with immunostimulants (Robertsen, 1999) from natural sources (Santos et al., 2013) to enhance fish natural defense mechanisms. Some substances derived from natural sources have the added advantage of not harming either the environment or consumers (Sforcin, 2007).
Propolis is among the various phytotherapeutic sources (Talas & Gulhan, 2009) with potential to be used as a natural diet supplement. It is produced by Apis mellifera L. bees from resinous and balsamic material collected from leaf shoots and cracks in the bark of several plants (Burdock, 1998; Toreti et al., 2013). Propolis has a complex chemical composition including compounds such as flavonoids (galangin, techtochrysin, pinocembrin, kaempferol, and quercetin), aromatic and phenolic acids (caffeic, ferulic, cinnamic, and coumaric), terpenoids, aldehydes, alcohols, aliphatic acids, and esters, amino acids, steroids, and sugars (Bankova et al., 1995; Marcucci, 1996).
Propolis has been widely studied due to its antimicrobial activity (Sforcin et al., 2000). In fish, the propolis extract has a growth-promoting and immunostimulatory effects, benefiting liver physiology and resistance to bacteria (Abd-El-Rhman, 2009; Meurer et al., 2009b). In addition, anti-fungal (Uzel et al., 2005), anti-inflammatory (Mirzoeva & Calder, 1996; Song et al., 2002), immunostimulant (Burdock, 1998), immunomodulator (Fisher et al., 2008), antiviral (Marcucci, 1996; Burdock, 1998), antioxidant (Cabral et al., 2009), and cytostatic (Banskota et al., 2001) properties have also been reported. Moreover, the scientific literature highlights several other activities such as hepatoprotective effects promoted by chemical compounds (Basnet et al., 1996), and hypoglycemic (Matsui et al., 2004) and hypotensive (Burdock, 1998) effects.
These biological and pharmacological properties make propolis an attractive supplement for nutritious diets, being hypothesized the benefit of diets containing propolis providing growth and fish homeostasis. Thus, this study evaluated the responses of tilapia fingerlings (O. niloticus) to the inclusion of propolis alcoholic extract in their diet through the analysis of growth performance, carcass proximate composition, blood parameters, and liver histopathological alterations.
Material and methodsTop
This study was carried out at the State University of Western Paraná in Toledo Campus along with the Aquaculture Management Study Group (GEMAq). It was evaluated and approved by the Ethics Committee on Animal Use under Protocol number 03/15 - CEUA.
Experimental diets
Six isoenergetic (3083 kcal digestible energy/kg of ration) and isoproteic (29.73% crude protein) diets were formulated based on the nutrient and energy requirements for Nile tilapia (O. niloticus) according to Furuya et al. (2010). The experimental diets included a control diet without the addition of propolis extract (0%), and five treatments with different supplementation levels of alcoholic propolis extract (APE), 2%, 4%, 6%, 8% and 10% (Table 1).
The Laboratory of Research & Development of Drug Delivery Systems, Dept. of Pharmacy, State University of Maringa, Brazil, donated the ethanolic extract of green propolis. The extractive solution was prepared by turbo-extraction and using propolis/ethanol ratio of 30/70 (w/w) (Bruschi et al., 2003a,b). Afterwards, the following characteristics of the extract were analyzed: pH (5.4 ± 0.00), density (0.87 ± 0.00 g mL-1), dryness residue (16.93 ± 0.63%, w/w), and total polyphenol content (1.83 ± 0.09%, w/v). This propolis research was registered in Brazil with SISGEN No AC7A2F5. The polyphenol concentrations were 0.01%, 0.02%, 0.03%, 0.04% and 0.06% the levels of inclusion of propolis extract diets in this study. Quality assays were carried out according to Bruschi et al. (2003a).
Dietary ingredients were individually ground in a hammer mill to prepare 0.5 mm diameter particles. The particles were weighed, mixed in the "V" equipment, and extruded (Ex-Micro® Extruder). The diets were dried in a forced circulation oven at 55ºC for 24 h and subsequently stored and protected from light in an airy location. The granulometry of the diets was adequate according to the fish growth phase. Fish were fed to apparent satiation four times a day (8 am, 11 am, 2 pm and 5 pm).
Table 1. Composition of the experimental diets with different levels of propolis supplementation
[1] Composition values based on Boscolo et al. (2001) and Furuya et al. (2001). [a] Guaranteed levels
per kg of product - Premix (DSM-Roche®): Vit. A, 24,000 IU; Vit. D3, 6,000 IU; Vit. E, 300 mg;
Vit. K3, 30 mg; Vit. B1, 40 mg; Vit. B2, 40 mg; Vit. B6, 35 mg; Vit. B12, 80 mg; Folic acid, 12 mg;
Ca Pantothenate, 100 mg; Vit. C, 600 mg; Biotin, 2 mg; Choline, 1,000 mg; Niacin; Iron, 200 mg;
Copper, 35 mg; Manganese, 100 mg; Zinc, 240 mg; Iodine, 1.6 mg; Cobalt, 0.8 mg. [b] BHT, butyl
hydroxy toluene.
Animals and experimental design
Three hundred Nile tilapia fingerlings (O. niloticus), with an initial weight of 0.61± 0.02 g, were distributed in 30 plastic mesh hapas (experimental unit) with 0.15 m3 of useful volume (40 cm × 40 cm × 70 cm in length, width, and depth respectively), arranged in a concrete tank with a capacity of 25 m3 of constantly aerated water. Each experimental unit consisted of ten fish. The fish were evaluated during 90 days in a random experimental design based on feeding of six experimental diets previously described.
The water quality was monitored for temperature (24.07 ± 1.66°C), dissolved oxygen (5.84 ± 0.97 mg/L), pH (7.67 ± 0.69), and conductivity (91.03 ± 4.97 μS/cm) using multiparameter mater, YSI Professional model, YSI- EUA. The temperature was measured daily and other parameters were monitored weekly. The variables were in the optimal levels for O. niloticus growth (Ridha & Cruz, 2001).
Animals and experimental design
Three hundred Nile tilapia fingerlings (O. niloticus), with an initial weight of 0.61± 0.02 g, were distributed in 30 plastic mesh hapas (experimental unit) with 0.15 m3 of useful volume (40 cm × 40 cm × 70 cm in length, width, and depth respectively), arranged in a concrete tank with a capacity of 25 m3 of constantly aerated water. Each experimental unit consisted of ten fish. The fish were evaluated during 90 days in a random experimental design based on feeding of six experimental diets previously described.
The water quality was monitored for temperature (24.07 ± 1.66°C), dissolved oxygen (5.84 ± 0.97 mg/L), pH (7.67 ± 0.69), and conductivity (91.03 ± 4.97 μS/cm) using multiparameter mater, YSI Professional model, YSI- EUA. The temperature was measured daily and other parameters were monitored weekly. The variables were in the optimal levels for O. niloticus growth (Ridha & Cruz, 2001).
Sample collection and growth performance evaluation
At the end of the experiment, all the fish were fasted for 12h to empty the gastrointestinal tract. Fish were subsequently anesthetized with benzocaine (100 mg/L) according to Gomes et al. (2001) for measurements of weight (g) and total length (cm). Growth performance was evaluated by estimation of the following parameters and indexes:
− Weight gain (WG) = Final body weight (g) – Initial body weight (g).
− Feed conversion rate (FCR) = Consumed diet (g) / Weight gain (g).
− Specific growth rate (SGR) = [(ln Final weight (g) – ln Initial weight (g)) / time] × 100.
− Protein efficiency ratio (PER) = (Weight gain (g) / Protein consumed (g)).
− Survival (SU) = (Final number of fish / Initial number of fish) × 100.
− Batch uniformity (BU) = (Number of fish with body weight within the mean / Total number of fish) × 100.
Three fish from each experimental unit were euthanized with benzocaine (250 mg/L) for the collection of visceral fat and liver. For hematological and proximate samples, three fish from each experimental unit were used for tissue collection. The following indexes were estimated:
− Hepatosomatic index (HSI) = [Liver weight (g) / Final body weight (g)] × 100.
− Viscerosomatic fat index (VFI) = [(Visceral fat weight (g) / Final body weight (g)] × 100.
Proximate composition
Three fish from each experimental unit were sent to the Laboratory of Food Quality (LQA) of the Aquaculture Management Study Group (GEMAq) for the analysis of proximate composition; these fish were frozen (-20°C) until analysis. Samples were subsequently ground, homogenized, and processed according to AOAC (2000) for the following analyses: moisture (pre-drying at 55ºC for 72 h followed by drying at 105ºC for 8 h), proteins (Kjeldahl method, Modle MA-036, Piracicaba-SP, Brazil), ethereal extract (Soxhlet extractor with petroleum ether as the solvent; Model TE-0,44, Piracicaba-SP, Brazil), and ash (calcination of samples at 550ºC for 6 h; Modle 2000B, Belo Horizonte-MG, Brazil).
Hematologic evaluations
Blood samples were collected by caudal puncture using a 1.0 mL syringe with anticoagulant (10% EDTA) from three fish in each experimental unit. Total erythrocytes were counted using the Neubauer chamber and Hayen solution. The hemoglobin rate was calculated by Collier´s (1944) method, the hematocrit percentage was calculated by Goldenfarb et al.´s (1971) method. Hematimetric indexes, such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Winthrobe (1933):
Blood smears were prepared on frosted glass slides, air dried. These slides were read in an optical microscope at 100x magnification using immersion oil; the total count of leukocytes and thrombocytes was performed by the indirect method of Ranzani-Paiva et al. (2013). The staining of blood extensions was performed according to Rosenfeld (1947).
The leukocyte differential counting consisted in determining the ratio (as a percentage) between the different leukocyte forms: lymphocytes, neutrophils and monocytes. One hundred leukocytes were counted on the smear area by traversing the entire material and moving the slide in a "zig-zag" motion.
Histology of the liver
Two livers from each replicate (10 fish per treatment) were washed with distilled water and samples from the medial portion of the left lobe were fixed in aqueous Bouin solution and transferred to vials containing 70% alcohol (Tokumaru et al., 1968). This material was dehydrated through an increasing alcohol series, diaphanized in xylol, and embedded in paraffin to obtain 6-μm-thick semi seriate cross sections using a rotary microtome (Microm HM 340 E Thermo Scientific, Germany).
These cuts were mounted on slides, stained with hematoxylin and eosin for general morphological analysis and determination of the number of hepatocytes per area (counting area: 2048.1532 μm2 ). The histological analyses of the liver were performed using images captured with an optical microscope (P1 Olympus BX 50, Manila, Philippines) coupled to a camera (Olympus PMC 35 B, Berlin, Germany) and used a 40x objective; 10 images were taken per slide (30 images/animal), totaling 300 images/treatment. These measurements were performed using the Image-Pro Plus® image analysis software (vers 4.5, Media Cybernetics, USA). These analyses were performed at the Histology Laboratory of State University of Western Paraná, Unioeste, Campus Toledo, PR, Brazil.
Statistical analyses
The data were submitted to normality tests (Shapiro-Wilk) and homoscedasticity, and subsequent analysis of variance. The Tukey’s test (5%) was applied when the ANOVA was significant for the variables’ means (p<0.05). The Bernet index was performed through the non-parametric Kruskal-Walis analysis. These analyses were all conducted on Statistica 7.1® program.
Results Top
Growth performances and proximate composition
Fish fed with the 10% propolis alcohol extract diet showed significantly the worst feed conversion and survival (p<0.05) compared to values observed in fish fed with the other experimental and control diets (Table 2). On the other hand, the increasing levels of propolis in the diets studied for Nile tilapia fingerlings did not influence the final weight, weight gain, final length, specific growth rate, visceral fat, hepatosomatic index, protein efficiency rate and batch uniformity (Table 2). Proximate composition did not show significant differences between treatments (Table 3).
Table 2. Productive performance of Nile tilapia (Oreochromis niloticus) fingerlings fed with increasing levels of propolis extract in the diet
[[1] FW: final weight, WG: weight gain FL: final length, FCR: feed conversion rate, SGR: specific growth rate, VFI: viscerosomatic fat
index, PER: protein efficiency ratio, SU: survival, BU: batch uniformity, HSI: hepatosomatic index. Values are presented as mean ±
standard deviation. Different letters indicate significant differences among treatments, Tukey’s test (p<0.05).
Table 3. Proximate composition of Nile tilapia (Oreochromis niloticus) fingerlings fed with increasing levels of propolis extract. Values are means ± standard deviation
Hematological parameters
The different levels of propolis alcoholic extract in the diet did not show influence (p>0.05) on the hematological parameters of the red blood cells represented by mean erythrocyte values of 1.7 106 μL, hematocrit with a percentage of 29 to 31%, and hemoglobin between 6.6 and 7.1 g/dL. The hematimetric indexes showed values of MCV above 171 fL, MCH averages between 39 and 48 μg, and MCHC between 22.1 and 24.08 g/dL (Table 4).
Total leukocytes and total thrombocytes levels were increased with propolis supplementation in diet. According to leukocytes (p<0.05) fish fed with 2% and 8% of propolis extract showed significant higher values (56.226 and 55.169 μL, respectively). Total thrombocytes showed the highest significant values (p<0.05) in fish fed with 6% and 8% of propolis extract (56.954 and 56.138 μL, respectively). The mean values of lymphocytes were 94.18±0.66%, the percentage of neutrophils were 2.89±0.52% and monocytes were 0.99±0.22%; in these parameters no significant difference (p>0.05) was observed between treatments.
Table 3. Hematological parameters, mean values of white blood cells and thrombocytes of tilapia (Oreochromis niloticus) fingerlings fed with diets containing increasing levels of alcoholic propolis extract
[1] RBC: red blood cells, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin
concentration. Values are presented as mean ± standard deviation (p<0.05). Different letters indicate significant differences among treatments,
Tukey’s test (p<0.05).
Liver histology
The macroscopic evaluation of the liver showed well-developed organs, homogeneous, with a slightly brownish reddish-brown color and absence of striations or whitish lesions. The microscopic qualitative evaluation of hepatocytes showed hepatic parenchyma composed of hepatocytes arranged in continuous cords permeated by sinusoids. Typical hepatocytes were characterized with round nuclei of central position, with evident nucleoli and slightly acidophilic and vacuolated cytoplasm. In all treatments containing propolis as well as in the control diet.
However, higher number of hepatocytes was observed in fish fed 2% and 6% propolis extract diets (p<0.05) compared to those fed control diet (Fig. 1).
Figure 1. Hepatocytes in the hepatic tissue of Nile tilapia
(Oreochromis niloticus) fingerlings fed with diets containing
increasing levels of alcoholic propolis extract. Counting area:
2048.1532 µm2
. Different letters above the histogram bars indicate significant differences between treatments by the Tukey’s
test (p<0.05%)
Discussion Top
Growth performance
An increasing number of studies have evaluated the effects of non-conventional products on animal nutrition. The use of propolis as an additive has been of great interest because its complex composition may yield improved results in productive performance and animal health (AbdEl-Rhman, 2009; Velotto et al., 2010; Sforcin & Bankova, 2011; Abbas et al., 2012; De la Cruz-Cervantes et al., 2018).
Deng et al. (2011) reported different results using an alcoholic propolis extract supplemented in the diet of rainbow trout (Oncorhynchus mykiss); these authors observed a significant improvement in growth rates, food efficiency, and protein efficiency that promoted improvement in performance. Meurer et al. (2009b) evaluated increasing levels of brown propolis extract used as growth promoters in Nile tilapia (O. niloticus) and reported improved results in final weight, weight gain, and feed conversion using between 1.83 and 2.74 g of brown propolis extract/kg of diet.
In this study, final weight, weight gain, final length, specific growth rate, and protein efficiency in tilapia fingerlings showed no significant differences between propolis alcoholic extract diet supplementation and the control. Santos et al. (2013) found similar results using residues of red propolis extract in the concentrations of 0.5, 1.0, and 1.5% for Nile tilapia fingerlings. Uczay et al. (2014) did not observe a significant difference in final length, and specific growth rate in silver catfish juveniles (Rhamdia quelen) fed with 0.5, 1.0, 1.5, and 2.0% propolis. Similarly, the use of 0.1, 0.2, 0.3 and 0.4% of alcoholic propolis extract in the diet of the common carp (Cyprinus carpio) did not result in differences in final weight and specific growth rate compared to the control (Uczay et al., 2011).
According to Santos et al. (2013), the fish digestive system is developing during the initial phase of life and has small amounts of microorganisms compared to adult fish. Therefore, the capacity of propolis to modulate the quality of the intestinal microbiota and promote the growth of beneficial microorganisms, influencing the expression of its effect on the absorption of nutrients available in diets (Guo et al., 2004), is still inconsistent.
The contradictory results described for growth performance using alcoholic propolis may be related to the complexity of its chemical composition. Although propolis is classified as an opotherapeutic substance,in the case of substances obtained from glands, other organs, tissues, and animal secretions, the composition is determined by the individual plant source and the effects of seasonality which influence the synthesis and concentrations of bioactive compounds and, therefore, potentially affect activity (Marcucci, 1996).
Another important factor related to the response of productive performance in this study is the absence of challenge. Specifically, the favorable environmental conditions used in this study ensured the fish’s well-being. Some studies on non-ruminant animals demonstrated that growth promoters such as prebiotics show improved results when challenged (Meurer et al., 2009a). The effects of crude propolis and propolis extract on the diet of tilapia juveniles infected by Aeromonas hydrophila were evaluated by Abd-El-Rhman (2009). Their results showed improved productive performance, immunity, and resistance to bacteria with the use of alcoholic propolis extract followed by crude propolis when compared to the control group.
According to Bae et al. (2012), the inclusion of propolis levels above 10% in the diet of juvenile Anguilla japonica may negatively affect fish physiology. In this study levels assayed were up to 10% (10 g/kg). In fact, the lowest survival rate (62.5%) was observed in fish fed with 10% of propolis extract suggesting that this concentration did not favor productive performance and fish health. This would explain the increased food conversion values in this experimental group (Table 2) probably due to the lower fish survival rate associated the feeding at libitum in this experiment.
As reported, propolis contains several chemical substances that are responsible for its therapeutic activities. Nevertheless, some compounds can provide negative side effects. According to Ramos & Miranda (2007), propolis-induced cinnamic acid derivatives can lead to hypersensitivity and intoxication in sensitive organisms. Therefore, the use of propolis alcoholic extract at concentrations of 10% in the 90-day period influenced the physiological response of tilapia fingerlings in this study, suggesting that this concentration must be used with caution in order to maintain the optimal supplementation level at this stage of development.
Proximate composition
In this study, the proximate composition was not influenced by the different concentrations of alcoholic propolis extract in the diet (Table 3). Uczay et al. (2014) found decreased values of fat in carcass in silver catfish (R. quelen) juveniles fed on diets supplemented with propolis (0.5, 1.0, 1.5, and 2.0%). The presence or absence of specific components (Haard, 1992) could explain the lack of differences under different propolis supplementation when compared with other studies (Meurer et al., 2009b; Uczay et al., 2011, 2014; Santos et al., 2013). Although we did not find any difference in proximate composition, Bae et al. (2012) stated that propolis has the ability to improve the proximate composition in fish but the active ingredient and the mechanism involved in this beneficial effect are still unknown.
Hematological parameters
The effect of propolis as an additive in animal nutrition showed varied responses to its biological and pharmacological activities. According to Arauco et al. (2007) it was observed an immunostimulating effect when using low concentrations of propolis in diets; however, Kashkooli et al. (2011) found no effect of propolis when used in the long-term diet and Talas & Gulhan (2009) found that in high concentrations propolis has a negative effect on hematological variables.
The evaluation of hematological parameters is important for understanding the homeostatic and pathological conditions in fish (Barton & Iwama, 1991; Ranzani-Paiva et al., 2004). The hematological parameters evaluated in this study (Table 4) verified the influence of the different levels of propolis (p<0.05). However, our results are within the recommended reference for the species. The percentages of hematocrit, hemoglobin and concentrations of erythrocytes observed in this study are in good agreement with those described in literature (Tavares-Dias & De-Moraes, 2007; Tavares-Dias et al., 2009).
Hematimetric indexes are used in the evaluation and classification of anemia, in general. It is a condition in which the blood's ability to transport oxygen to tissues is reduced (Ranzani-Paiva et al., 2013). The values obtained for MCV, MCH, and MCHC (Table 4) were not altered (p>0.05) by the concentration of propolis in the diet. These results are in agreement with the variation considered normal for this species as proposed by Hrubec et al. (2000), and the use of propolis alcohol extracts up to 10% may be indicated, as observed in this study.
In general, the levels of alcoholic propolis extract provided an increase in total leukocytes and thrombocytes (Table 4), despite results within the range of variation for tilapias (Hrubec et al., 2000). Leukocytes together with thrombocytes form a cellular population of organic defense (Tavares-Dias et al., 2002) and present important phagocytic function in the regulation of the immune system (Harikrishnan et al., 2011). Increased levels of leukocytes and thrombocytes in the vascular blood of fish could be an indicative of a rapid renewal and an increased protection against different pathogenic microorganisms (Ranzani-Paiva et al., 2004).
The analysis of leukocyte differentiation showed no significant differences between treatments (Table 4), which consequently justify the absence of pathological and stressful agents during the experiment. These results are in line with those previously reported by Ledic-Neto et al. (2014), who did not find significant differences in monocytes and neutrophils with the addition of 2% propolis in the diet for tilapia fed during 15 and 21 days.
The efficacy of the heterogeneous composition of propolis that has flavonoids as the main group of compounds responsible for therapeutic activities (Marcucci, 1996) can interfere in several physiological processes and act as immunostimulants in fish. Such compounds, in turn, may increase resistance to infectious diseases by enhancing nonspecific defense mechanisms (Zhang et al., 2009), which are normally identified by their ability to activate leukocytes in in vitro tests (Bricknell & Dalmo, 2005; Mouriño et al., 2012).
Histological alterations
The macro and microscopic evaluations of the hepatic histology observed in the present study are in line with those described by Vicentini et al. (2005), allowing the use of the structural pattern for the species for characterization of the liver histology of the Nile tilapias in this study.
Fish liver, as well as that of other vertebrates, is responsible for the animal's physiology as well as the execution of many vital activities. Fish liver is formed by hepatocytes, which are cells with high mitotic potential (Costa et al., 2012), that function in metabolism of proteins, lipids, and carbohydrates, storage of some nutrients, hematopoiesis, antibody production (Takashima & Hibiya, 1995) and the capacity of biotransformation and excretion of xenobionts (Bernet et al., 1999).
The liver analysis in this study identified a higher number of hepatocytes in fish fed with 2%, 6% and 8 % propolis extract compared to those in the control, 4% and 10 % in the diet (Fig. 1). This result suggests that these levels of propolis promote a possible hepatoprotective activity as demonstrated by Bhadauria et al. (2008) in studies with rats.
The histopathological changes observed are part of a spontaneous tissue regenerative process (Baldisserotto, 2013). The qualitative variation of fish hepatocytes showed subtle changes in the liver structure, with mildly acidophilic cytoplasm and central nuclei, indicating normal metabolic activity (Bernet et al., 1999), with no significant liver changes in the concentrations of propolis extract used. These results are similar to those found by Honorato et al. (2014) in tilapia fed with diets containing biological fish silage and in traíras (Hoplias malabaricus) exposed to methyl mercury (Mela et al., 2007).
As report
As report
Conclusion Top
The use of propolis alcoholic extract in the range of 2% to 8% provided an increase in the number of leukocytes, total thrombocytes, and hepatocytes, suggesting that inclusion within this range leads to healthier fish with increased immunity in the evaluated culture conditions. Therefore, the inclusion of up to 8% of propolis in the diet for Nile tilapia fingerlings is recommended.
ReferencesTop
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