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Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae
Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx
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Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae Afaf N. Abdel Rahman a,⇑, Suhair A. Abdellatief b, Heba Hassan H. Mahboub a a b
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, Egypt Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, Egypt
a r t i c l e
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Article history: Received 17 April 2017 Revised 30 August 2017 Accepted 25 September 2017 Available online xxxx Keywords: Aflatoxin B1 Nile tilapia Fennel essential oil Saccharomyces cerevisiae Phagocytic assay Malondialdehyde
a b s t r a c t Significant losses in ration industry especially aquaculture resulting from aflatoxins. The effect of supplementing Nile tilapia diet with Fennel essential oil (FEO) with or without Saccharomyces cerevisiae on negative impacts of aflatoxin B1, AFB1 (200 ppb) for one month was studied. Two hundred and forty Nile tilapia of 26.6 ± 0.12 g (mean ± SE) were divided into eight groups (30 fish/group) with three replicates. G1 was fed on a basal diet only (D1), while G2 was fed on AFB1 (200 ppb) contaminated diet (D2). G3 and G4 were fed on FEO supplemented diets (1 ml/kg diet) with or without AFB1 (200 ppb) (D3 and D4), respectively. G5 and G6 were fed on S. cerevisiae supplemented diets (1 g/kg diet) with or without AFB1 (200 ppb) (D5 and D6), respectively. G7 and G8 were fed on supplemented diets with mixture from FEO (1 ml/kg diet) and S. cerevisiae (1 g/kg diet) with or without AFB1 (200 ppb) (D7 and D8), respectively. The phagocytic assay, protein profile, serum alanine aminotranferase (ALT) and creatinine were measured at the end of feeding period (30 days). The oxidative stress biomarker as catalase activity and malondialdehyde (MDA) and AFB1 residues in liver and musculature were also measured. At the end of feeding period, the phagocytic activity, total blood proteins and catalase activity showed lower significant values in AFB1 exposed fish. Also, AFB1 induced significant rising in levels of ALT, creatinine and MDA with higher residual levels recorded. Meanwhile, the aflatoxicated groups treated with FEO or S. cerevisiae or their mixture revealed significant improvement of mostly all the measured parameters. FEO can successfully relieve AFB1 noxious effects compared with S. cerevisiae in Nile tilapia. Ó 2017 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction In aquaculture, the extensive use of cereal ingredients in rations formulation to minimize the cost, increases of aflatoxicosis occurrence (Zychowski et al., 2013) as high percent of world’s crops are contaminated with mycotoxins before or post-harvest due to incorrect storage (Rodrigues and Nährer, 2012). Aflatoxins are toxic metabolites of genus Aspergillus which frequently noted in several areas in the world (Militßa˘ et al., 2010). Aflatoxins are about 20 types, but there are four main types (B1, B2, G1 and G2). The most dangerous and toxic one is aflatoxin B1 (AFB1) (Amiri et al., 2013). Each aquatic species vary in their tolerance to aflatoxins.
Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding author. E-mail addresses: [email protected] (A.N. Abdel Rahman), S.abdellatief777@ gmail.com (S.A. Abdellatief), [email protected] (H.H.H. Mahboub).
Nile tilapia (O. niloticus) is an important cultured fish species in Egypt and one of highly sensitive fish species to AFB1 (Kenawy et al., 2009). The exposure of fishes to AFB1 causes many risks such as the decrease in growth performance, immunosuppression, increase susceptibility to disease and high mortality (Santacroce et al., 2008). As fish exposed for long time, AFB1 accumulates in fish flesh and organs led to great dangers such as human poisoning, liver tumors and even death resulted from their consumption (Murjani, 2003; Shephard, 2008). Using of natural plants or its essential oils (EOs) to control of mycotoixns has been applied recently as they have antifungal, anti-aflatoxin and antioxidant activities. EOs gain high acceptance as feed additives as they are safe, low toxic and biodegradable (Gupta et al., 2011). In vitro, essential oils of some plants suppress growth of Aspergillus flavus and Aspergillus parasiticus and production of aflatoxins (Rasoli et al., 2008) such as fennel essential oil (Gemeda et al., 2014). Fennel (Foeniculum vulgare) belongs to
https://doi.org/10.1016/j.ejar.2017.09.006 1687-4285/Ó 2017 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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Apiaceae family is an herbal, aromatic and long lasting plant and cultivated in most parts of the world (Gulfraz et al., 2008). The fennel essential oil (FEO) has more than 87 volatile compounds, but tanethole Limonene, fenchone and vitamin C are the major components (Zoubiri et al., 2010). FEOs are valuable as they have hepatoprotective, antioxidant, anti-inflammatory and antimicrobial activities (El Ouariachi et al., 2014). Adding some microorganisms as a binding agent to AFB1 contaminated diets can be helpful in controlling of AFB1 (Oguz, 2012). Saccharomyces cerevisiae has many beneficial effects in fish such as improving growth performance, feed efficiency, immunity and controlling aflatoxins in foodstuffs through adsorption (AbdelTawwab et al., 2008; Selim et al., 2014). There are many researches on aflatoxins in fish and their control, but the information about using of FEO with S. cerevisiae is rare. Therefore, the present work was planned to evaluate the capability of dietary additions of FEO and/or S. cerevisiae additions to lessen the hazards of AFB1 in Nile tilapia. Some immunological and biochemical parameters as well as AFB1 residues in fish liver and musculature were assessed. Material and methods
plemented with fennel essential oil (FEO, Royal herbs company, Bayad el Arab, Beni-Suef, Egypt) at 1 ml/kg diet with or without AFB1 (200 ppb), respectively. D5 and D6 were basal diets supplemented with S. cerevisiae (Levitan, Dox-al Italia S.P.A., Italy, each gram contains 10 109 cells) at 1 g/kg diet with or without AFB1 (200 ppb), respectively. Meanwhile, D7 and D8 were basal diets in which mixture from FEO (1 ml/kg diet) and S. cerevisiae (1 g/kg diet) with or without AFB1 (200 ppb), respectively were incorporated. The prepared diets were air-dried at room temperature for 8 h and then kept in dark plastic bags at 4 °C. Experimental design The fish were randomly divided into eight equal experimental groups (G1–G8) in triplicates aquaria (30 fish/group). G1 was fed on D1 and kept as the negative control while, G2 was fed on D2 and kept as the positive control. G3, G4, G5, G6, G7 and G8 were fed on D3, D4, D5, D6, D7 and D8, respectively. The diets were offered for one month at the rate of 3% of fish biomass twice daily and adjusted every two weeks according to fish weight.
Blood collection
Fish Two hundred and forty apparently healthy Nile tilapia, Oreochromis niloticus (26.6 ± 0.12 g) obtained from Fish Research Unit at Faculty of Veterinary Medicine, Zagazig University, Egypt. Fish were kept in glass aquaria (80 40 30 cm) supplied with dechlorinated tap water and continuous aeration. The fish were acclimated to the new environment for 15 days before starting the experiment. The water of each glass aquaria was exchanged daily at a rate of 25% for waste removal. The water dissolved oxygen, salinity, temperature and pH were monitored daily but, ammonia-N, nitrate-N and carbonate hardness were monitored weekly according to APHA (1998). The water parameters were kept within the recommended ranges during the experimental period according to Lim and Webster (2006). Diets for fish
After one month of feeding, fish were anesthetized with 250 mg/l tricane methanesulfonate (MS222). The blood samples were collected from the caudal blood vessels using sterile syringes and then transferred into sterile heparinized tubes to determine the phagocytic assay. Other blood samples were collected without anticoagulants and centrifuged at 3000 rpm for 15 min for serum separation. The serum samples were stored at 20 °C for biochemical analysis.
Determination of the phagocytic assay The phagocytic activity% and index were performed using heatinactivated Candida albicans according to methods of Kawahara et al. (1991).
Determination of some biochemical parameters
The fish basal diet (D1) formed at the Fish Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt was shown in Table 1. Seven experimental diets were also prepared; D2 was a basal diet incorporated with aflatoxin B1 (AFB1 produced from Aspergillus flavus, was kindly supplied by Department of Biochemistry, Toxicology and Feed deficiency, Animal Health Research institute, El- Dokki, Egypt) at the dose of 200 ppb after dissolved in chloroform (Selim et al., 2014). D3 and D4 were basal diets supTable 1 Basal diet composition and its chemical analysis.
The serum samples were used to measure the total serum protein and albumin spectrophotometrically according to Doumas et al. (1981) and Reinhold (1953), respectively. Serum globulin was determined by subtracts serum albumin from total serum protein (Coles, 1986). Also, serum alanine aminotransferase (ALT) and creatinine were determined colorimetrically according to the methods described by Varley (1976) and Henry et al. (1974), respectively.
Determination of the oxidative stress biomarkers
Ingredients
Percentage of diet
Fish meal Soy bean meal Ground yellow corn Meat meal Fish oil Vitamin and mineral mixture (premix)
25 22 26.5 20 5 1.5
Calculated chemical analysis Crude protein Crude lipid Crude fiber Moisture Ash
39.9 10.89 3.68 10.58 9.11
Catalase activity concentration was determined according to methods of Aebi (1984). The lipid peroxidation was measured as malondialdehyde (MDA) level according to the methods of Ohkawa et al. (1979).
Detection of AFB1 residues in tissue samples Liver and musculature samples from the fish at the end of the experiment were pooled and thoroughly homogenized in a mortar. Aflatoxin B1 extraction and analysis by HPLC with a reverse phase column were done according to AOAC (2000).
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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A.N. Abdel Rahman et al. / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx Table 2 Effects of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on phagocytic activity% (P%), phagocytic index (PI) and protein profile of Nile tilapia. Groupsa
Diets
P%
PI
Total protein (g/dl)
Albumin (g/dl)
Globulin
(g/dl) G1 G2 G3 G4 G5 G6 G7 G8
D1 D2 D3 D4 D5 D6 D7 D8
55.5 ± 0.5b 34 ± 1d 57.5 ± 2.5b 65 ± 1a 53.5 ± 1.5b 63 ± 2.5a 41 ± 1c 60 ± 0.5a
5.5 ± 0.05b 3.4 ± 0.1d 5.6 ± 0.15b 6.1 ± 0.1a 5.4 ± 0b 6.2 ± 0.2a 4.1 ± 0.1c 6 ± 0.1a
3.01 ± 0.04c 1.45 ± 0.04d 3.56 ± 0.03b 4.25 ± 0.06a 3.12 ± 0.04c 3.74 ± 0.06b 2.98 ± 0.07c 4.11 ± 0.06a
1.4 ± 0.3a 0.75 ± 0.01c 1.54 ± 0.04a 1.45 ± 0.05a 1.43 ± 0.01a 1.44 ± 0.04a 1.08 ± 0.03b 1.51 ± 0.01a
1.61 ± 0.02c 0.7 ± 0.01d 2.02 ± 0.04b 2.8 ± 0.03a 1.69 ± 0.01c 2.3 ± 0.02b 1.9 ± 0.01c 2.6 ± 0.06a
Total number of fish/group = 30 fish in triplicates. Means within the same column carrying different superscripts (a–d) are significant at p value p 0.05. D1 (control diet), D2 (basal diet + aflatoxin B1 200 ppb), D3 and D4 (basal diet + fennel essential oil 1 ml/kg with or without AFB1, respectively), D5 and D6 (basal diet + Saccharomyces cerevisiae 1 g/ kg with or without AFB1, respectively), D7 and D8 (basal diet + FEO 1 ml/kg +S. cerevisiae 1 g/kg with or without AFB1, respectively).
Statistical analysis
Serum ALT and creatinine
All data were statistically analyzed by one-way Analysis of Variance (ANOVA) using SPSS version 14 (SPSS, Chicago, IL, USA). Duncan’s multiple range tests was used to determine differences among means at significance level of 0.05 (Duncan, 1955).
The levels of ALT and creatinine were significantly higher in G2 than other groups as shown in Fig. 1(A and B). There were significant reductions (P < 0.05) in levels of ALT and creatinine in aflatoxicated groups exposed to FEO or S. cerevisiae compared with G2, but there were no any changes in other groups.
Results The oxidative stress biomarkers Phagocytic assay The phagocytic activity% and index results of Nile tilapia after 30 days of feeding AFB1 contaminated diets with or without addition of FEO or S. cerevisiae or their mixture were shown in Table 2, where G2 showed the lowest values compared with all groups. The phagocytic activity% and index exhibited significant increases in G3 and G5 followed by G7 compared with G2. Also, there were significant increases in FEO or S. cerevisiae only supplemented groups (G4, G6 & G8) compared with control fish.
Catalase activity showed the lowest significant value in G2, but G3 had the highest significant value compared with G5 and G7 (Fig. 2A). The fish of G4, G6 and G8 showed higher values of catalase activity compared with G1. Aflatoxicated fish (G2) showed the highest value of MDA among other groups (Fig. 2B). There was significant reduction (P < 0.05) in MDA value according the pattern: G3 > G5, G7 > G2. Meanwhile, MDA did not significantly differ between other groups. AFB1 residues
Protein profile Aflatoxicated group (G2) exhibited the lowest values of total blood protein, albumin and globulin (Table 2). The total blood protein and globulin values showed high significant differences (P < 0.05) in aflatoxicated groups according the pattern: G3 > G5, G7 > G2 and in non aflatoxicated groups according the pattern: G4, G8 > G6 > G1. G3 and G5 had higher values of albumin than G7 and G2. The level of serum albumin was not affected by supplementation of either FEO or S. cerevisiae alone.
At the end 30 days of feeding, the residues of AFB1 were measured in liver and musculature of control fish and aflatoxicated fish with or without treatment (G1, G2, G3, G5 and G7) as shown in Fig. 3 (A and B). The control fish (G1) was free from AFB1 residues but, the higher residual levels were revealed in G2 in liver (5 ± 0.5 ppb) and musculature (3.7 ± 0.1 ppb). The residues in liver (Fig. 3A) were significantly decreased in G3 (0.5 ± 0.5 ppb) and G7 (0.8 ± 0 ppb) followed by G5 (2 ± 0.5 ppb) compared with G2. On the other hand, the residues in musculature (Fig. 3B) were significantly reduced in
Fig. 1. Effect of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on (A) serum ALT (IU/L) and (B) creatinine (mg/dl) of Nile tilapia. The bars with different superscripts (a–c) are significantly different (P < .05, one-way ANOVA).
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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Fig. 2. Effect of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on (A) catalase activity (CAT, U/L) and (B) malondialdehyde (MDA, nmol/ml) of Nile tilapia. The bars with different superscripts (a–d) are significantly different (P < 0.05, one-way ANOVA).
Fig. 3. Effect of Fennel essential oil and Saccharomyces cerevisiae on residues of aflatoxin B1 in (A) Liver and (B) musculature of Nile tilapia. The bars with different superscripts (a–c) are significantly different (P < 0.05, one-way ANOVA).
G5 (0.5 ± 0.2 ppb) compared with G2 while, no residues were recorded in the other groups.
Discussion In Egypt, fish commercial diets are contaminated with high level of AFB1, which have bad effects on fish health and production (Shehata et al., 2003). Therefore, effective control methods must be done in aquaculture to get rid of aflatoxins hazards. In this study, AFB1 led to the lowest phagocytic activity values. Similarly, Sherif et al. (2013) reported the phagocytic activities of Nile tilapia decreased by AFB1 at different doses and exposure time. The phagocytic cells are important for phagocytosis process which activates the inflammatory response firstly before antibody production (Nayak, 2010). Addition of FEO or S. cerevisiae enhanced phagocytic activity in Nile tilapia exposed to AFB1. This can be attributed to the immunostimulant effects of FEO as reported by Hassaan and Soltan (2016) and Mahdavi and Yeganeh (2014) and beta-glucan and nucleotides components of S. cerevisiae as reported by Kumari and Sahoo (2006). The phagocytic activities of aflatoxicated O. niloticus and treated with beta-glucans or garlic, onion and black seed were enhanced (El-Boshy et al., 2008; Saad et al., 2013). Similar results were recorded in Labeo rohita by Tewary and Patra (2011), addition S. cerevisiae of alone induced significant rising in phagocytic activities. Serum proteins including albumin and globulin help in maintenance of osmotic balance and transporting many exogenous chem-
icals, endogenous metabolites and some immune parameters against infections (Jha et al., 2007). In our study, AFB1 caused significant reductions in total serum protein, albumin and globulin. These may be attributed to aflatoxins metabolites that bind with cellular macromolecules and inhibit protein synthesis in liver as reported by Cagauan et al. (2004). Significant reductions in total proteins by AFB1 were recorded in sea bass (Dicentrarchus labrax), O. niloticus and Labeo rohita (El-Sayed and Khalil, 2009; Hessein et al., 2014; Mohapatra et al., 2011). Supplementation of the diet with FEO against AFB1 succeeded to increase total protein and globulin more than S. cerevisiae and these reflect strong immunity and improved fish health. Similar improvements were recorded in rainbow trout exposed to mixed oil of thyme and fennel and in Nile tilapia exposed to FEO with B. licheniformis supplemented diets (Gulec et al., 2013; Hassaan and Soltan, 2016). Selim et al. (2014) reported that in Nile tilapia, the total serum proteins improved in aflatoxicated fish exposed to S. cerevisiae. The liver is the principle target organ for AFB1 causing damage and dysfunction (Santacroce et al., 2008). In this study, AFB1 caused significant elevations of serum ALT and creatinine. The results confirm the hepatotoxicity and renal tissue dysfunction resulted from AFB1 exposure (Sherif et al., 2013). In contrast, Hessein et al. (2014) reported that serum creatinine of Nile tilapia was not affected by AFB1. Using dietary FEO and S. cerevisiae lowered ALT and creatinine compared with aflatoxicated group. This proves that FEO or S. cerevisiae protect fish target organs from destructive effect of AFB1. Addition of black pepper or 0.25% S. cerevisiae to diets was able to protect Nile tilapia liver and kidney from AFB1 dangers
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
A.N. Abdel Rahman et al. / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx
(Salem et al., 2010; Selim et al., 2014). Ozbek et al. (2003) reported protection of rat liver from carbon-tetrachloride (CCl4) toxicity due to-limonene and b-myrcene of FEO. The oxidative stress causes damages and degenerative changes to body tissues resulted from high level of reactive oxygen species (ROS) (Ben Ameur et al., 2012). AFB1 can generate ROS during processing in liver leads to lipid peroxidation cytotoxicity and DNA damage (Marin and Taranu, 2012). There are defense systems in fish like other vertebrates to protect from these damages named antioxidant enzymes. Catalase enzyme is one of them, which destroys hydrogen peroxide free radicals (Gülçin et al., 2009). In this study, catalase activity exhibited the lowest significant value while MDA was highly increased in AFB1 exposed fish. This low level of catalase resulted from liver damage which enhanced fish exposure to oxidative damages. AFB1 decreases the antioxidant capacity in rats and broiler (Sivanesan and Hazeena Begum, 2014; Wang et al., 2013). The high level of MDA might indicate increasing in lipid peroxidation or suppression of antioxidant defense. Confirmations to these results are reported in Nile tilapia by El-Barbary and Mohamed (2014). Meanwhile, FEO improved these parameters more than S. cerevisiae. These are because of the strong antioxidant and free radical scavenging capabilities of FEO natural antioxidants (Shahat et al., 2011). In addition, S. cerevisiae has antimycotoxic effect through binding with AFB1 and prevention their absorption in alimentary tract or increasing antioxidant enzymes (Selim et al., 2014). Reduction of MDA with increasing of glutathione and superoxide dismutase activities in rat received S. cerevisiae before exposure to aflatoxins (Darwish et al., 2011). Presence of mycotoxins residues in fish flesh is very dangerous problem for food safety (Wild and Gong, 2010). The higher AFB1 residual levels were measured in aflatoxicated fish liver and musculature in this study. Similar to these findings were recorded in Nile tilapia and sea bass (Abd EL-Ghany et al., 2014; El-Sayed and Khalil, 2009). In contrast, Deng et al. (2010) reported that after feeding tilapia with different levels of AFB1, no residue revealed in fish flesh. Aflatoxicated fish that received FEO, exhibited more significant reduction or no AFB1 residues than S. cerevisiae. These reductions prove the antimycotoxic effects of FEO or S. cerevisiae. Salem et al. (2010) and Selim et al. (2014) reported that no or decreases in AFB1 residues in different tissues of O. niloticus treated with Piper nigrum or S. cerevisiae respectively. The combination between FEO and S. cerevisiae were effective also in reduction of danger effects of AFB1 in fish, but not significantly differ from use them separately. This indicates slow synergism between them, so from economic point, it is recommended to use FEO or S. cerevisiae separately against AFB1 rather than combined use. In conclusion, AFB1 (200 ppb) has unfavorable effects on Nile tilapia phagocytic activity, total blood proteins, ALT, creatinine, catalase activity and MDA with high residual levels in liver and musculature. Dietary incorporation of FEO (1 ml/kg diet) was more effective than S. cerevisiae (1 g/kg diet) in amelioration of these drastic effects and enhancement fish immunity.
Acknowledgements The authors tend to thanks Prof. Dr. Mahmoud Arafa Mohamed, Chief Researcher of Biochemistry, Toxicology and Feed deficiency Department, Animal Health Research institute, El- Dokki, Egypt for providing them with aflatoxin B1 and analysis of AFB1 residues. Great thanks to Prof. Dr. Rasha Mohammed Reda, Assistant professor of Fish Diseases and Management Department, Faculty of Veterinary Medicine, Zagazig University, Egypt for revision of this research.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial or not for profit sectors. Conflict of interest The authors declared that there is no conflict of interest.
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Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae Afaf N. Abdel Rahman a,⇑, Suhair A. Abdellatief b, Heba Hassan H. Mahboub a a b
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, Egypt Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, Egypt
a r t i c l e
i n f o
Article history: Received 17 April 2017 Revised 30 August 2017 Accepted 25 September 2017 Available online xxxx Keywords: Aflatoxin B1 Nile tilapia Fennel essential oil Saccharomyces cerevisiae Phagocytic assay Malondialdehyde
a b s t r a c t Significant losses in ration industry especially aquaculture resulting from aflatoxins. The effect of supplementing Nile tilapia diet with Fennel essential oil (FEO) with or without Saccharomyces cerevisiae on negative impacts of aflatoxin B1, AFB1 (200 ppb) for one month was studied. Two hundred and forty Nile tilapia of 26.6 ± 0.12 g (mean ± SE) were divided into eight groups (30 fish/group) with three replicates. G1 was fed on a basal diet only (D1), while G2 was fed on AFB1 (200 ppb) contaminated diet (D2). G3 and G4 were fed on FEO supplemented diets (1 ml/kg diet) with or without AFB1 (200 ppb) (D3 and D4), respectively. G5 and G6 were fed on S. cerevisiae supplemented diets (1 g/kg diet) with or without AFB1 (200 ppb) (D5 and D6), respectively. G7 and G8 were fed on supplemented diets with mixture from FEO (1 ml/kg diet) and S. cerevisiae (1 g/kg diet) with or without AFB1 (200 ppb) (D7 and D8), respectively. The phagocytic assay, protein profile, serum alanine aminotranferase (ALT) and creatinine were measured at the end of feeding period (30 days). The oxidative stress biomarker as catalase activity and malondialdehyde (MDA) and AFB1 residues in liver and musculature were also measured. At the end of feeding period, the phagocytic activity, total blood proteins and catalase activity showed lower significant values in AFB1 exposed fish. Also, AFB1 induced significant rising in levels of ALT, creatinine and MDA with higher residual levels recorded. Meanwhile, the aflatoxicated groups treated with FEO or S. cerevisiae or their mixture revealed significant improvement of mostly all the measured parameters. FEO can successfully relieve AFB1 noxious effects compared with S. cerevisiae in Nile tilapia. Ó 2017 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction In aquaculture, the extensive use of cereal ingredients in rations formulation to minimize the cost, increases of aflatoxicosis occurrence (Zychowski et al., 2013) as high percent of world’s crops are contaminated with mycotoxins before or post-harvest due to incorrect storage (Rodrigues and Nährer, 2012). Aflatoxins are toxic metabolites of genus Aspergillus which frequently noted in several areas in the world (Militßa˘ et al., 2010). Aflatoxins are about 20 types, but there are four main types (B1, B2, G1 and G2). The most dangerous and toxic one is aflatoxin B1 (AFB1) (Amiri et al., 2013). Each aquatic species vary in their tolerance to aflatoxins.
Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding author. E-mail addresses: [email protected] (A.N. Abdel Rahman), S.abdellatief777@ gmail.com (S.A. Abdellatief), [email protected] (H.H.H. Mahboub).
Nile tilapia (O. niloticus) is an important cultured fish species in Egypt and one of highly sensitive fish species to AFB1 (Kenawy et al., 2009). The exposure of fishes to AFB1 causes many risks such as the decrease in growth performance, immunosuppression, increase susceptibility to disease and high mortality (Santacroce et al., 2008). As fish exposed for long time, AFB1 accumulates in fish flesh and organs led to great dangers such as human poisoning, liver tumors and even death resulted from their consumption (Murjani, 2003; Shephard, 2008). Using of natural plants or its essential oils (EOs) to control of mycotoixns has been applied recently as they have antifungal, anti-aflatoxin and antioxidant activities. EOs gain high acceptance as feed additives as they are safe, low toxic and biodegradable (Gupta et al., 2011). In vitro, essential oils of some plants suppress growth of Aspergillus flavus and Aspergillus parasiticus and production of aflatoxins (Rasoli et al., 2008) such as fennel essential oil (Gemeda et al., 2014). Fennel (Foeniculum vulgare) belongs to
https://doi.org/10.1016/j.ejar.2017.09.006 1687-4285/Ó 2017 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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Apiaceae family is an herbal, aromatic and long lasting plant and cultivated in most parts of the world (Gulfraz et al., 2008). The fennel essential oil (FEO) has more than 87 volatile compounds, but tanethole Limonene, fenchone and vitamin C are the major components (Zoubiri et al., 2010). FEOs are valuable as they have hepatoprotective, antioxidant, anti-inflammatory and antimicrobial activities (El Ouariachi et al., 2014). Adding some microorganisms as a binding agent to AFB1 contaminated diets can be helpful in controlling of AFB1 (Oguz, 2012). Saccharomyces cerevisiae has many beneficial effects in fish such as improving growth performance, feed efficiency, immunity and controlling aflatoxins in foodstuffs through adsorption (AbdelTawwab et al., 2008; Selim et al., 2014). There are many researches on aflatoxins in fish and their control, but the information about using of FEO with S. cerevisiae is rare. Therefore, the present work was planned to evaluate the capability of dietary additions of FEO and/or S. cerevisiae additions to lessen the hazards of AFB1 in Nile tilapia. Some immunological and biochemical parameters as well as AFB1 residues in fish liver and musculature were assessed. Material and methods
plemented with fennel essential oil (FEO, Royal herbs company, Bayad el Arab, Beni-Suef, Egypt) at 1 ml/kg diet with or without AFB1 (200 ppb), respectively. D5 and D6 were basal diets supplemented with S. cerevisiae (Levitan, Dox-al Italia S.P.A., Italy, each gram contains 10 109 cells) at 1 g/kg diet with or without AFB1 (200 ppb), respectively. Meanwhile, D7 and D8 were basal diets in which mixture from FEO (1 ml/kg diet) and S. cerevisiae (1 g/kg diet) with or without AFB1 (200 ppb), respectively were incorporated. The prepared diets were air-dried at room temperature for 8 h and then kept in dark plastic bags at 4 °C. Experimental design The fish were randomly divided into eight equal experimental groups (G1–G8) in triplicates aquaria (30 fish/group). G1 was fed on D1 and kept as the negative control while, G2 was fed on D2 and kept as the positive control. G3, G4, G5, G6, G7 and G8 were fed on D3, D4, D5, D6, D7 and D8, respectively. The diets were offered for one month at the rate of 3% of fish biomass twice daily and adjusted every two weeks according to fish weight.
Blood collection
Fish Two hundred and forty apparently healthy Nile tilapia, Oreochromis niloticus (26.6 ± 0.12 g) obtained from Fish Research Unit at Faculty of Veterinary Medicine, Zagazig University, Egypt. Fish were kept in glass aquaria (80 40 30 cm) supplied with dechlorinated tap water and continuous aeration. The fish were acclimated to the new environment for 15 days before starting the experiment. The water of each glass aquaria was exchanged daily at a rate of 25% for waste removal. The water dissolved oxygen, salinity, temperature and pH were monitored daily but, ammonia-N, nitrate-N and carbonate hardness were monitored weekly according to APHA (1998). The water parameters were kept within the recommended ranges during the experimental period according to Lim and Webster (2006). Diets for fish
After one month of feeding, fish were anesthetized with 250 mg/l tricane methanesulfonate (MS222). The blood samples were collected from the caudal blood vessels using sterile syringes and then transferred into sterile heparinized tubes to determine the phagocytic assay. Other blood samples were collected without anticoagulants and centrifuged at 3000 rpm for 15 min for serum separation. The serum samples were stored at 20 °C for biochemical analysis.
Determination of the phagocytic assay The phagocytic activity% and index were performed using heatinactivated Candida albicans according to methods of Kawahara et al. (1991).
Determination of some biochemical parameters
The fish basal diet (D1) formed at the Fish Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt was shown in Table 1. Seven experimental diets were also prepared; D2 was a basal diet incorporated with aflatoxin B1 (AFB1 produced from Aspergillus flavus, was kindly supplied by Department of Biochemistry, Toxicology and Feed deficiency, Animal Health Research institute, El- Dokki, Egypt) at the dose of 200 ppb after dissolved in chloroform (Selim et al., 2014). D3 and D4 were basal diets supTable 1 Basal diet composition and its chemical analysis.
The serum samples were used to measure the total serum protein and albumin spectrophotometrically according to Doumas et al. (1981) and Reinhold (1953), respectively. Serum globulin was determined by subtracts serum albumin from total serum protein (Coles, 1986). Also, serum alanine aminotransferase (ALT) and creatinine were determined colorimetrically according to the methods described by Varley (1976) and Henry et al. (1974), respectively.
Determination of the oxidative stress biomarkers
Ingredients
Percentage of diet
Fish meal Soy bean meal Ground yellow corn Meat meal Fish oil Vitamin and mineral mixture (premix)
25 22 26.5 20 5 1.5
Calculated chemical analysis Crude protein Crude lipid Crude fiber Moisture Ash
39.9 10.89 3.68 10.58 9.11
Catalase activity concentration was determined according to methods of Aebi (1984). The lipid peroxidation was measured as malondialdehyde (MDA) level according to the methods of Ohkawa et al. (1979).
Detection of AFB1 residues in tissue samples Liver and musculature samples from the fish at the end of the experiment were pooled and thoroughly homogenized in a mortar. Aflatoxin B1 extraction and analysis by HPLC with a reverse phase column were done according to AOAC (2000).
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
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A.N. Abdel Rahman et al. / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx Table 2 Effects of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on phagocytic activity% (P%), phagocytic index (PI) and protein profile of Nile tilapia. Groupsa
Diets
P%
PI
Total protein (g/dl)
Albumin (g/dl)
Globulin
(g/dl) G1 G2 G3 G4 G5 G6 G7 G8
D1 D2 D3 D4 D5 D6 D7 D8
55.5 ± 0.5b 34 ± 1d 57.5 ± 2.5b 65 ± 1a 53.5 ± 1.5b 63 ± 2.5a 41 ± 1c 60 ± 0.5a
5.5 ± 0.05b 3.4 ± 0.1d 5.6 ± 0.15b 6.1 ± 0.1a 5.4 ± 0b 6.2 ± 0.2a 4.1 ± 0.1c 6 ± 0.1a
3.01 ± 0.04c 1.45 ± 0.04d 3.56 ± 0.03b 4.25 ± 0.06a 3.12 ± 0.04c 3.74 ± 0.06b 2.98 ± 0.07c 4.11 ± 0.06a
1.4 ± 0.3a 0.75 ± 0.01c 1.54 ± 0.04a 1.45 ± 0.05a 1.43 ± 0.01a 1.44 ± 0.04a 1.08 ± 0.03b 1.51 ± 0.01a
1.61 ± 0.02c 0.7 ± 0.01d 2.02 ± 0.04b 2.8 ± 0.03a 1.69 ± 0.01c 2.3 ± 0.02b 1.9 ± 0.01c 2.6 ± 0.06a
Total number of fish/group = 30 fish in triplicates. Means within the same column carrying different superscripts (a–d) are significant at p value p 0.05. D1 (control diet), D2 (basal diet + aflatoxin B1 200 ppb), D3 and D4 (basal diet + fennel essential oil 1 ml/kg with or without AFB1, respectively), D5 and D6 (basal diet + Saccharomyces cerevisiae 1 g/ kg with or without AFB1, respectively), D7 and D8 (basal diet + FEO 1 ml/kg +S. cerevisiae 1 g/kg with or without AFB1, respectively).
Statistical analysis
Serum ALT and creatinine
All data were statistically analyzed by one-way Analysis of Variance (ANOVA) using SPSS version 14 (SPSS, Chicago, IL, USA). Duncan’s multiple range tests was used to determine differences among means at significance level of 0.05 (Duncan, 1955).
The levels of ALT and creatinine were significantly higher in G2 than other groups as shown in Fig. 1(A and B). There were significant reductions (P < 0.05) in levels of ALT and creatinine in aflatoxicated groups exposed to FEO or S. cerevisiae compared with G2, but there were no any changes in other groups.
Results The oxidative stress biomarkers Phagocytic assay The phagocytic activity% and index results of Nile tilapia after 30 days of feeding AFB1 contaminated diets with or without addition of FEO or S. cerevisiae or their mixture were shown in Table 2, where G2 showed the lowest values compared with all groups. The phagocytic activity% and index exhibited significant increases in G3 and G5 followed by G7 compared with G2. Also, there were significant increases in FEO or S. cerevisiae only supplemented groups (G4, G6 & G8) compared with control fish.
Catalase activity showed the lowest significant value in G2, but G3 had the highest significant value compared with G5 and G7 (Fig. 2A). The fish of G4, G6 and G8 showed higher values of catalase activity compared with G1. Aflatoxicated fish (G2) showed the highest value of MDA among other groups (Fig. 2B). There was significant reduction (P < 0.05) in MDA value according the pattern: G3 > G5, G7 > G2. Meanwhile, MDA did not significantly differ between other groups. AFB1 residues
Protein profile Aflatoxicated group (G2) exhibited the lowest values of total blood protein, albumin and globulin (Table 2). The total blood protein and globulin values showed high significant differences (P < 0.05) in aflatoxicated groups according the pattern: G3 > G5, G7 > G2 and in non aflatoxicated groups according the pattern: G4, G8 > G6 > G1. G3 and G5 had higher values of albumin than G7 and G2. The level of serum albumin was not affected by supplementation of either FEO or S. cerevisiae alone.
At the end 30 days of feeding, the residues of AFB1 were measured in liver and musculature of control fish and aflatoxicated fish with or without treatment (G1, G2, G3, G5 and G7) as shown in Fig. 3 (A and B). The control fish (G1) was free from AFB1 residues but, the higher residual levels were revealed in G2 in liver (5 ± 0.5 ppb) and musculature (3.7 ± 0.1 ppb). The residues in liver (Fig. 3A) were significantly decreased in G3 (0.5 ± 0.5 ppb) and G7 (0.8 ± 0 ppb) followed by G5 (2 ± 0.5 ppb) compared with G2. On the other hand, the residues in musculature (Fig. 3B) were significantly reduced in
Fig. 1. Effect of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on (A) serum ALT (IU/L) and (B) creatinine (mg/dl) of Nile tilapia. The bars with different superscripts (a–c) are significantly different (P < .05, one-way ANOVA).
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Fig. 2. Effect of aflatoxin B1, Fennel essential oil and Saccharomyces cerevisiae on (A) catalase activity (CAT, U/L) and (B) malondialdehyde (MDA, nmol/ml) of Nile tilapia. The bars with different superscripts (a–d) are significantly different (P < 0.05, one-way ANOVA).
Fig. 3. Effect of Fennel essential oil and Saccharomyces cerevisiae on residues of aflatoxin B1 in (A) Liver and (B) musculature of Nile tilapia. The bars with different superscripts (a–c) are significantly different (P < 0.05, one-way ANOVA).
G5 (0.5 ± 0.2 ppb) compared with G2 while, no residues were recorded in the other groups.
Discussion In Egypt, fish commercial diets are contaminated with high level of AFB1, which have bad effects on fish health and production (Shehata et al., 2003). Therefore, effective control methods must be done in aquaculture to get rid of aflatoxins hazards. In this study, AFB1 led to the lowest phagocytic activity values. Similarly, Sherif et al. (2013) reported the phagocytic activities of Nile tilapia decreased by AFB1 at different doses and exposure time. The phagocytic cells are important for phagocytosis process which activates the inflammatory response firstly before antibody production (Nayak, 2010). Addition of FEO or S. cerevisiae enhanced phagocytic activity in Nile tilapia exposed to AFB1. This can be attributed to the immunostimulant effects of FEO as reported by Hassaan and Soltan (2016) and Mahdavi and Yeganeh (2014) and beta-glucan and nucleotides components of S. cerevisiae as reported by Kumari and Sahoo (2006). The phagocytic activities of aflatoxicated O. niloticus and treated with beta-glucans or garlic, onion and black seed were enhanced (El-Boshy et al., 2008; Saad et al., 2013). Similar results were recorded in Labeo rohita by Tewary and Patra (2011), addition S. cerevisiae of alone induced significant rising in phagocytic activities. Serum proteins including albumin and globulin help in maintenance of osmotic balance and transporting many exogenous chem-
icals, endogenous metabolites and some immune parameters against infections (Jha et al., 2007). In our study, AFB1 caused significant reductions in total serum protein, albumin and globulin. These may be attributed to aflatoxins metabolites that bind with cellular macromolecules and inhibit protein synthesis in liver as reported by Cagauan et al. (2004). Significant reductions in total proteins by AFB1 were recorded in sea bass (Dicentrarchus labrax), O. niloticus and Labeo rohita (El-Sayed and Khalil, 2009; Hessein et al., 2014; Mohapatra et al., 2011). Supplementation of the diet with FEO against AFB1 succeeded to increase total protein and globulin more than S. cerevisiae and these reflect strong immunity and improved fish health. Similar improvements were recorded in rainbow trout exposed to mixed oil of thyme and fennel and in Nile tilapia exposed to FEO with B. licheniformis supplemented diets (Gulec et al., 2013; Hassaan and Soltan, 2016). Selim et al. (2014) reported that in Nile tilapia, the total serum proteins improved in aflatoxicated fish exposed to S. cerevisiae. The liver is the principle target organ for AFB1 causing damage and dysfunction (Santacroce et al., 2008). In this study, AFB1 caused significant elevations of serum ALT and creatinine. The results confirm the hepatotoxicity and renal tissue dysfunction resulted from AFB1 exposure (Sherif et al., 2013). In contrast, Hessein et al. (2014) reported that serum creatinine of Nile tilapia was not affected by AFB1. Using dietary FEO and S. cerevisiae lowered ALT and creatinine compared with aflatoxicated group. This proves that FEO or S. cerevisiae protect fish target organs from destructive effect of AFB1. Addition of black pepper or 0.25% S. cerevisiae to diets was able to protect Nile tilapia liver and kidney from AFB1 dangers
Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006
A.N. Abdel Rahman et al. / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx
(Salem et al., 2010; Selim et al., 2014). Ozbek et al. (2003) reported protection of rat liver from carbon-tetrachloride (CCl4) toxicity due to-limonene and b-myrcene of FEO. The oxidative stress causes damages and degenerative changes to body tissues resulted from high level of reactive oxygen species (ROS) (Ben Ameur et al., 2012). AFB1 can generate ROS during processing in liver leads to lipid peroxidation cytotoxicity and DNA damage (Marin and Taranu, 2012). There are defense systems in fish like other vertebrates to protect from these damages named antioxidant enzymes. Catalase enzyme is one of them, which destroys hydrogen peroxide free radicals (Gülçin et al., 2009). In this study, catalase activity exhibited the lowest significant value while MDA was highly increased in AFB1 exposed fish. This low level of catalase resulted from liver damage which enhanced fish exposure to oxidative damages. AFB1 decreases the antioxidant capacity in rats and broiler (Sivanesan and Hazeena Begum, 2014; Wang et al., 2013). The high level of MDA might indicate increasing in lipid peroxidation or suppression of antioxidant defense. Confirmations to these results are reported in Nile tilapia by El-Barbary and Mohamed (2014). Meanwhile, FEO improved these parameters more than S. cerevisiae. These are because of the strong antioxidant and free radical scavenging capabilities of FEO natural antioxidants (Shahat et al., 2011). In addition, S. cerevisiae has antimycotoxic effect through binding with AFB1 and prevention their absorption in alimentary tract or increasing antioxidant enzymes (Selim et al., 2014). Reduction of MDA with increasing of glutathione and superoxide dismutase activities in rat received S. cerevisiae before exposure to aflatoxins (Darwish et al., 2011). Presence of mycotoxins residues in fish flesh is very dangerous problem for food safety (Wild and Gong, 2010). The higher AFB1 residual levels were measured in aflatoxicated fish liver and musculature in this study. Similar to these findings were recorded in Nile tilapia and sea bass (Abd EL-Ghany et al., 2014; El-Sayed and Khalil, 2009). In contrast, Deng et al. (2010) reported that after feeding tilapia with different levels of AFB1, no residue revealed in fish flesh. Aflatoxicated fish that received FEO, exhibited more significant reduction or no AFB1 residues than S. cerevisiae. These reductions prove the antimycotoxic effects of FEO or S. cerevisiae. Salem et al. (2010) and Selim et al. (2014) reported that no or decreases in AFB1 residues in different tissues of O. niloticus treated with Piper nigrum or S. cerevisiae respectively. The combination between FEO and S. cerevisiae were effective also in reduction of danger effects of AFB1 in fish, but not significantly differ from use them separately. This indicates slow synergism between them, so from economic point, it is recommended to use FEO or S. cerevisiae separately against AFB1 rather than combined use. In conclusion, AFB1 (200 ppb) has unfavorable effects on Nile tilapia phagocytic activity, total blood proteins, ALT, creatinine, catalase activity and MDA with high residual levels in liver and musculature. Dietary incorporation of FEO (1 ml/kg diet) was more effective than S. cerevisiae (1 g/kg diet) in amelioration of these drastic effects and enhancement fish immunity.
Acknowledgements The authors tend to thanks Prof. Dr. Mahmoud Arafa Mohamed, Chief Researcher of Biochemistry, Toxicology and Feed deficiency Department, Animal Health Research institute, El- Dokki, Egypt for providing them with aflatoxin B1 and analysis of AFB1 residues. Great thanks to Prof. Dr. Rasha Mohammed Reda, Assistant professor of Fish Diseases and Management Department, Faculty of Veterinary Medicine, Zagazig University, Egypt for revision of this research.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial or not for profit sectors. Conflict of interest The authors declared that there is no conflict of interest.
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Please cite this article in press as: Abdel Rahman, A.N., et al. Protection of Nile tilapia, Oreochromis niloticus from aflatoxin B1 toxicity by dietary supplementation with Fennel essential oil and Saccharomyces cerevisiae. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.006