Dietary mycotoxin contamination is an area of great concern for different organisms (Abdelhamid, 2019a & b). It negatively affects the immune system, gut development (Mass, 2018), digestion and performance (Abdelhamid et al., 1992), and may lead to death (Abdelhamid et al., 2006); so, leads to economic loss (Abdelhamid, 2000; 2004 and 2009). Particularly aflatoxins, since their producing fungi are detected in various food-and feedstuffs that found also in diseased fish and fish diets (Abdelhamid et al., 2006 & 2008; Abdelhamid, 2010 and Abdallah et al., 2017). Therefore, many attempts were made to prevent growth of toxigenic fungi (Abdelhamid et al., 2008 and 2009) and mycotoxin production (Abdelhamid et al., 1985 and Kumar et al., 2018) and to control mycotoxicoses (Abdelhamid and Mahmoud, 1996; Abdelhamid et al., 2002a, b &c; 2004a, b &c and 2007). All these attempts concluded that exact overcome of mycotoxicoses is impossible, so recommended the prevention (prophylaxes) that is better than curing. Hence, the aim of the present research was to study the effects of a new commercial feed additive aiming in ameliorating fish aflatoxicosis.
MATERIALS AND METHODS
Unsexed Nile tilapia fingerlings collected from Lake Manzala in September 2018 and transferred to Elmataria Station for Aquatic Research, Dakahlia Governorate, National Institute of Oceanography and Fisheries, Egypt in order to investigate the effect of aflatoxin B (AFB) in the diets of Nile tilapia (Oreochromis niloticus) and to examine the detoxification activity of a commercial anti-mycotoxin ARCAVIT Bioacid Forte (Italy Product) distributed in Egypt by ALLgaeuvet Egypt. The experimental period lasted 70 days (10 weeks). The experiment started on 15th of September 2018 and finished on the 2nd of December 2018. After 2 weeks adaptation period in a 3m3 fiberglass tank; during that period, fish were fed a basal diet from Bio Feed Factory (Asafra Industrial zone, Dakahlia governorate, Egypt), fish (40 g initial body weight) were redistributed randomly into three treatments in six glass aquaria (each had dimensions of 30 x 60 x 40cm, filled with 60 L of dechlorinated tap water, supplied by air stone and electric glass heater to keep water temperature constant at 25-28ºC and stocked with 10 fingerlings), each treatment was applied in two aquaria (replicates). One third of the water in each aquarium was replaced daily and totally once weekly after removing the wastes by siphoning. Photoperiod was natural by the sunlight.
Formulation and composition (according to the manufacturer Bio feed factory) of the basal diet used are illustrated in Table1. The experimental diets (treatments) were T1: control basal diet, T2: basal diet artificially contaminated with aflatoxin B (AFB) at a 5 ppb level, and T3: basal diet artificially contaminated with AFB at a 5 ppb level and included 0.5g/Kg diet of ARCAVIT Bioacid Forte. Aflatoxin was produced by growing Aspergillus parasiticus (standard toxigenic strain, NRRL 2999 culture, Lyophilized strain, was kindly obtained from Vet. Med. Microbiology Dept., Iowa State University, USA) on rice fermentation. The moldy rice was steamed to kill the fungus, dried, milled and analyzed for aflatoxin determination using the AflaTest Fluorometer Procedure (Isaka et al., 2011). The AFB-extract of chloroform was sprayed on T2 and T3 diets to reach AFB level of 5 ppb (5 ng/g or 5 μg/ Kg diet). Diets were let in dry and dark place to chloroform volatilization then the fed stored in cool and dark place till be offered to fish. The ARCAVIT is one of the commercial anti-mycotoxin in the local market; it is the first test for it in aquaculture. ARCAVIT composition according to the company's advertising brochure is cell wall of Saccharomyces extract 100%, β-glucans mannan oligosaccharide, lactic acid, propionic acid, calcium propionate, sodium citrate, potassium citrate, chestnut extract tannins, seaweed meal (Ascophyllum nodosum), silicon dioxide and sepiolite. One gram of ARCAVIT was dissolved in one liter of distilled water then sprayed on two kilograms of feed which contains 5ppb AFB to be treatment 3 (T3). All the feed of the experiment stored in a dark place under low temperature.
All the experimental groups were fed the experimental diets at a daily rate of 3% of the live body weight of the fish, at two meals. The feed quantity was revalued monthly on the basis of the actual average biomass of the fish in each treatment. The feed residues were collected daily from each aquarium, and then calculated as the average weight of the dry feed to calculate the actual feed consumed by the fish.
Water quality parameters:
Air stones connected by an electric air blower were used for aerating the aquarium; it was enough to reach the dissolved oxygen (DO) to the optimum range (6-8 mg/l), it was measured by HANNA HI 9146-04 – Romania. The water temperature was adjusted between 25 and 28º C by an electric glass heater. The pH values were in the optimum range in all treatments (7-8), it was measured by Consort C860 – Belgium. because of changing and cleaning the water on each treatment daily there are no troubles came from water quality parameters.
Growth performance and feed utilization:
At the beginning of the experiment, randomly samples of fishes were measured for its weight and total and standard length. At the end of the experiment, samples of each cages' fish were weighted to calculate the growth performance and feed utilization parameters according to NRC (1993), Toguyeni et al. (1997) and Abdelhamid (1996 and 2009) in form of final weight (FW), total weight gain (TWG), average daily gain (ADG, %), relative growth rate (RGR), specific growth rate (SGR, %/d), feed intake (FI), feed conversion ratio (FCR), feed efficiency (FE, %), protein efficiency ratio (PER), protein productive value (PPV, %), energy utilization (EU, %), and lipid retention efficiency (LR, %).
Internal organs indices and dressing and boneless meat:
At the end of the experiment, three fish per treatment were randomly taken, individually weighed and total and standard lengths were measured; to calculate the condition factors (Kt and Ks); then the liver, gonads and viscera with associated fat tissue were individually removed and weighed, to calculate the hepatosomatic index (HSI), the gonadosomatic index (GSI) and viscerosomatic index (VSI), respectively according to Clement and Lovell (1994) and Abdelhamid (2009). Dressing and boneless meat (fillet) percentages were calculated too (Clement and Lovell, 1994).
Proximate analysis, water holding capacity and lean meat of fish:
At the start of the experiment, 5 fish were taken and kept frozen until carrying out the chemical analysis. At the end of the experiment, a random sample of five fish collected from each treatment was weighed and minced, then dried at 65°C for 12 hours, ground, then assayed to determine the moisture, crude protein, ether extract and ash contents by using standers methods (AOAC, 1990). Gross energy was calculated according (NRC, 1993). Water holding capacity (WHC, %) was calculated as cited from Abdelhamid (1983). Lean meat (LM, %) was also estimated.
Blood sampling and parameters' estimations:
Blood samples were collected from all tested fishes' groups (Fig. 1) at the end of the experiment from the caudal peduncle by special syringe in small plastic vials containing EDTA as an anticoagulant and used to obtain the blood serum by centrifuge at 3500 rpm for 15 min. Blood serum samples were used for determination of cortisol (Zaki and Fawzy, 2012), uric acid and glucose (Elboshy et al., 2008 and Shalaby, 2009), creatinine (Tietz, 1986), triglycerides (McGowan et al., 1983), total protein (Henry, 1964 and Tietz, 1990), high density lipoprotein (HDL), low density lipoprotein (LDL) and albumin (Wotton and Freeman, 1982) concentrations as well as the activity of enzymes aspartate amino transferase (AST) and alanine amino transferase (ALT) using commercial test kits (Robonik Prietest touch, Biochemistry Analyzer, Made in India). Globulin level was calculated by subtracting albumin from total protein. The other samples of blood were used to determine the blood hematology as hemoglobin (Hb), total erythrocytes (RBCs), total leukocytes (WBCS) (Natt and Herrick, 1952) and hematocrit (Hct) using (Horbia ABX micros 60 Auto blood hematology analyzer, made in USA) (Decie and Lewis, 2006). The other hematological parameters were mathematically calculated.
At the end of the experiment, all fish were sacrificed to take the liver samples. The samples were fixed at 10% neutralized formalin solution followed by washing with tab water, then dehydrated by different grades of alcohol. Samples were cleared by xylene and embedded in paraffin wax. The wax blocks were sectioned to six microns. The sections were stained by hematoxyline and eosin and then subjected to a histological examination.
Data were statistically analyzed by one way ANOVA using the Statistical Package for the Social Sciences (SPSS, 2017). Duncan's multiple range test (Duncan, 1955) was used to separate differences between treatment means at the probability level of 5%.
Morphology and gross pathology:
Normal morphology was observed for T1 and T3 fed fish concerning the healthy external appearance (shape, fins, color, eyes, gills,… etc.), revealing that ARCAVIT in T3 succeeded in overcoming the AFB toxicity symptoms (Figs. 2-4). Yet, fish fed the AFB-contaminated diet T2 reflected bad appearance (discoloration, scattered-destructed fins, scales fall, dive eyes). Concerning the gross pathology, it showed normal post-mortem examination for fish fed either T1 or T3; whereas, AFB only fed fish (T2) reflected pale internal organs, particularly liver (Figs. 5-7).
The initial body weight of the experimental fish was identical in all experimental groups (Table 2). The AFB-included diets (T2 and T3) were responsible for significantly (P≤0.05) lower values of all calculated measurements, where final weight (FW), total weight gain (TWG), average daily gain (ADG), relative growth rate (RGR), or specific growth rate (SGR), but reduced the survival rate (SR) also significantly (P≤0.05). Yet, dietary inclusion of ARCAVIT in T3 significantly (P≤0.05) improved all these parameters than in D2.
Internal organs indices:
The internal organs indices including as percentage hepato-somatic (HSI), gonado-somatic (GSI), and viscera-somatic indices were calculated and presented in Table 3. It cleared the presence of significant (P≤0.05) increases of both HIS and VSI percentages with AFB-included diets (T2 and T3) comparing with control (T1). Yet, ARCAVIT (T3) alleviated significantly (P≤0.05) the negative effect of AFB on these indices. On the other hand, AFB alone (T2) significantly lowered the GSI %, but ARCAVIT did not improve it.
Aflatoxin alone included diet (T2) significantly (P≤0.05) lowered the feed intake (FI), feed conversion, and feed efficiency (FE) than the control (T1), but T3 improved this picture (Table 4). The same negative effect of AFB-diet (T2) was calculated for the nutrients utilization criteria; since it reduced (P≤0.05) each of protein efficiency ratio (PER), protein productive value (PPV), energy utilization (EU), and lipid retention (LR) than in T1 and T3. Yet, ARCAVIT (T3) alleviated significantly (P≤0.05) the negative effect of AFB on these parameters. Diets containing AFB reduced the appetite of fish, i.e. lowered the consumed feed and consequently also reduced all growth performance parameters (Table 2) and negatively affected all indices of the internal organs too (Table 3).
Proximate chemical analysis of the fish:
Chemical analysis on dry matter basis of the fish body is given in Table 5, which cleared that dry matter (DM) content at the end of the experiment was higher than at the start, therefore and consequently also crude protein (CP) and energy contents were higher at the end of the experiment than at the start. However, DM, ether extract (EE) and ash percentages in T2 and T3 were higher (P≤0.05) than in T1; yet, CP % was significantly lower, particularly in T2 than T3.
At the end of the experiment, some physical properties were measured and calculated for the experimental fish (Table 6). This Table cleared the negative effects of the toxicated diet (T2) on fish, although the very low level of AFB (5 ppb) in this diet. Since it was responsible for significant (P≤0.05) decrease of Kt, BLM, and LM, besides high WHC. These refer to lower weight of each of the fish body, flesh, lean meat and dry matter of this group's fish. That refer to the unsuitability of these group's fish for preservation or filleting nor processing, i.e. it has poor or bad properties and thus has low price and economic value. Dietary addition of ARCAVIT (T3) improved, to some extent, these quality parameters.
The aflatoxic diet (T2) led to significant (P≤0.05) increase (Table 7) in values of hemoglobin (Hg), red blood cells (RBC) and white blood cells (WBC); but did not affect (P>0.05) all other parameters [i.e. hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT) and all WBC-differentiations]. Dietary addition of ARCAVIT (T3) improved, to some extent, these affected criteria (Hg, RBC, and WBC). That means that the dietary treatments were of limited effect, perhaps for the low level of AFB tested.
Serum biochemical parameters (hepatic function):
Table 8 presents mean values of some biochemical parameters measured for experimental fish at the end of the experiment. AFB alone (T2), and even T3 (AFB + ARCAVIT), increased (P≤0.05) the values of different parameters of the liver function (ALT, AST, and AST/ALT, referring to liver damage) but decreased the albumin (AL) level (P≤0.05) and globulin (P>0.05) too, which is important for good immunity.
Serum biochemical parameters for liver and kidney functions as well as immunity:
Some other serum biochemical parameters for liver function (triglyceride, cholesterol, HDL, LDL, and glucose), kidney function (creatinine and uric acid), and immunity (cortisol) were measured (Table 9) to clear to what extent these function were affected by the dietary treatments. There were significant (P≤0.05) effects of the AFB diets (T2 and T3), although the very low AFB and ARCAVIT concentrations in these experimental diets. Since T2 increased triglyceride, cholesterol, low density lipoprotein (LDL, bad cholesterol), glucose, and cortisol besides uric acid, but decreased high density lipoprotein (HDL, sound or good cholesterol). That means that this very low of AFB is harmful for liver, kidney and immunity, since cortisol is an indicator for suffering from different stress factors. The dietary inclusion of ARCAVIT alleviated one of the toxic effects of AFB (lowered significantly the triglyceride level than in T1 and T2 and LDL than T2), but even strengthen the other toxic symptoms (increased uric acid, cholesterol and glucose and lowered HDL levels significantly than in T1 and T2).
Histologically, O. niloticus fed free AFs-basal diet (BD, as a control group, T1) showed intact hepatic lobular architecture with normal hepatocytes (h) arrangement around the central vein (CV), and with normal nucleus (N) (Fig. 8 A and B). However, O. niloticus fed 5 ng AFs / g BD (T2) showed enlargement and severe congestion of the portal blood vessel (PBV), severe degeneration of the hepatocytes, and severe congestion of the blood vessels (BVs), besides severe congestion and dilatation, thickening of BV (Fig. 8 C and D). From other side, O. niloticus fed 5 ng AFs / g BD supplemented with 1g ARCAVIT® / Kg diet (T3) showed intact hepatic lobular architecture with slight degenerative changes, moderate necrotic of the hepatocytes, moderate congestion of BV (Fig. 8 E and F).
From the forgoing results, it was clear (although the very low level of AFB used herein) that 5 ppb AFB in tilapia diet (fed for 70 days) led to external disorders (bad appearance), post-mortem (gross pathological) alterations, lower growth performance, increased internal organs' indices, lower feed and nutrients utilization, lower fish carcass protein content, bad physical properties of the fish (Kt, BLM, LM, and WHC), blood profile changes (liver, kidney and immune-disfunctions).
Such morphological and gross pathological alterations were reported too in aflatoxicated fish by Abdelhamid et al. (2002b & c, 2004b, and 2007). However, AFB is well known as of hepatorenal (Newberne et al., 1964), chromosomal and histological (2002c) and carcinogenic (Ahamad et al., 2015) effects. It is a risk to human and animal health, and it is responsible for significant economic losses (Abdallah et al., 2015 and Anater et al., 2016).
In a long-term trial (20 weeks), Deng et al. (2010) studied the toxic effects of 19-1641 ppb aflatoxin B1 on tilapia, that was in a dose- and duration-dependent manner. It was of harmful effects with 245 ppb or higher doses reducing the growth and lipid content, induced hepatic disorder, hepatosomatic index, cytochrome P450 A1 activity, elevated plasma ALT activity and abnormal hepatic morphology. They added that such dietary AFB1 doses did not affect the survival rate. The AFB1 residue was only detected in liver, in a dose-dependent manner, but not in edible flesh. No toxic effects of AFB1 were found during the first 10 weeks, but by 20 weeks under good culture conditions, tilapia is a rather tolerant species for dietary AFB1 exposure up to 1641 μg/kg diet during 20 weeks. Yet, Abdelhamid et al. (2004c) found aflatoxin residues in fish muscles.
The obtained negative effects of AFB on fish skin, gut, liver, and kidney are reflecting the low immunity, since these organs belonging to fish immune system (Press and Evesen, 1999) and blood proteins are non-specific immune factors (Shehata and Goda, 2000 and Hussein and Kobeisy, 2001). Moreover, Abdelhamid et al. (2004a, b, & c) evaluated the toxicity symptoms in tilapia, whether on fish performance, feed and nutrients utilization, clinical, blood, and histologically. Abdelhamid et al. (2006) reported that stress factor (e.g. toxicants) negatively affect blood picture, i.e. led to decrease in hemoglobin content, hematocrite %, red blood cells and platelets count, hemolytic activity, total protein, but increase blood glucose.
Recently, Mehrim et al. (2016) reported that 150 ppb negatively affected fish growth, feed efficiency, body composition, condition factor, hepato-somatic index, blood profile, liver histology. Aflatoxin can carryover from food to fish tissues. Additionally, Hussain et al. (2017) described the toxicity symptoms of aflatoxicated (2-4 ppm) fish (reduced weight gain, feed efficiency ratio, hepatosomatic index, muscle ratio, whole-body crude lipid and protein retention efficiency). Saei et al. (2017) registered some changes in the blood picture of aflatoxicated rainbow trout fingerlings than the control. Therefore, considerable investigations are being performed to diminish aflatoxin harmful effects and to prevent its formation.
ARCAVIT addition not often succeeded in overcoming or improving (to some extent alleviation) the AFB toxicity symptoms, but even strengthen some other toxic symptoms. In this field of study, i.e. attempts of prevention, control, and/ detoxification), Abdelhamid et al. (2002b) emphasized to hygienic control of fish diets to avoid toxigenic fungal invasion, since prophylaxis is better than medication. Also, Abdelhmid et al. (2004a) used egg shell, betafin, clay and silica to detoxify aflatoxin effect on Nile tilapia. They found egg shel and clay were the best for that purpose. Mehrim et al. (2006) came to the conclusion that ginger was the best detoxifying agent of aflatoxin (100 ppb) by fish, followed by aspirin and chamomile flowers, respectively among the tested supplements. Perhaps the positive effects of ARCAVIT are due to its containing on active yeast and probiotics, that has positive effects on fish performance, nutrient utilization, body composition, and blood constituents (Abdelhmid et al., 2000; El-Ebiary and Zaki, 2003; Abdelhamid et al., 2002b; Khattab et al., 2004 and Aly et al., 2008, respectively).
Zychowski et al. (2012) used NovaSil to overcome the aflatoxicosis by fish. Additionally, Mehrim et al. (2016) tested Glutathione-Enhancer™ against foodborne aflatoxicosis (150 ppb) by Oreochromis niloticus. It ameliorated the aflatoxicosis severity. Magouz et al. (2016) reported that black pepper, Filofeed plus and cap T2 could be usedfor aflatoxin detoxification. Also, Hussain et al. (2017) used clay-based binders (calcium bentonite clay) to adsorb 2-4 ppm aflatoxin B1 in fish food. They reported improved some of aflatoxic-fish responses. Saei et al. (2017) tried to bind dietary 1 ppm aflatoxin (to prevent its absorption by fish) via 0.2% Biotoxin. They realized increased survival rate. Also, Ayyat et al. (2018) alleviated aflatoxin residues by Nile tilapia fed aflatoxic diet (2 ppm) using some dietary additives (clay, coumarin, curcumin, vitamin C, probiotics and prebiotics). Kovac et al. (2018) reached to nanoparticles capable to possess a great potential of modifying secondary metabolites biosynthesis of aflatoxin B1 in Aspergillus flavus. Moreover, Neeratanaphan and Tengjaroenkul (2018) studied the protective effect of dietary bentonite on aflatoxicated fish.
Generally, the negative correlation between protein and fat percentages is a fact (Soltan et al., 2006; Saad, 2010; Ali, 2008; Salem et al., 2008 and Farrag et al., 2013). Yet, other researchers found a positive relation between CP and EE contents of fish body (Eweedah et al., 2006 and Soltan et al., 2008). Others did not find an effect of dietary treatments on fish body composition (Soltan et al., 2002 and El-Dakar, 2004).
Histopathological alterations have been usually used as biologically markers in the assessment of the health status of fish exposed to pollutants or contaminants (Thophon et al., 2003). Especially, liver is considered to be the primary target organ of aflatoxins (AFs).
From the obtained histological characteristics, it could be noted the adverse effects of AFs on liver tissue of treated O. niloticus. These findings are similarly obtained by Mehrim et al. (2006 & 2016); El-Barbary and Mehrim (2009); Mehrim and Salem (2013); Zychowski et al. (2013). Additionally, Mahfouz and Sherif (2015) reported the same histopathological effects of AFB1 on the liver of O. niloticus treated fish. Also, they stated that the observed alterations in fish status, especially in the liver coincide well with the expected oxidative stress related with the toxicity of AFB1. In a recent study, Shahafve et al. (2017) stated that Cyprinus carpio fed AFs-contaminated diet, even in low concentrations (≤ 1.4 mg / Kg diet) caused histopathological damages, especially for liver tissue and disturbed their physiological balance. In this regard, severely hepatic lesions in the AFB1-injected O. niloticus were recently reported by El-Barbary (2018). Moreover, Anikuttan et al. (2018) also clearly indicated the strict hepatotoxicity of AFB1 on tropical estuarine teleost fish, Etroplus suratensis.
A number of studies are attentive on the prevention or detoxification of mycotoxins, especially AFs from food and feed. Consequently, it is of utmost importance to develop a safe and suitable detoxification technique without conceding the nutritional value of food (Aiko and Mehta, 2015). In the present findings, addition of 0.5g ARCAVIT® / Kg of AFs- contaminated diet (T3) as an anti-AFs agent led to protective effects against the aflatoxicosis of O. niloticus compared with those fed AFs- contaminated BD diet (5 ng / g diet, T2). These currently promising effects of ARCAVIT® against the toxicity of AFs on treated fish are comparable with those previously well documented by Mehrim et al. (2006 & 2016); El-Barbary and Mohamed (2014). Recently, Abdel Rahman et al. (2017) found that the aflatoxicated O. niloticus treated with Fennel essential oil (FEO) or Saccharomyces cerevisiae or their mixture revealed significant improvement of mostly measured parameter, as well as they reported that FEO can successfully relieve AFB1 noxious effects compared with S. cerevisiae. In this respect, a degree of protection of aflatoxicated O. niloticus fed garlic and curcumin supplemented diets was recently detected by El-Barbary (2018). Inversely with the obtained findings herein, Abdelhamid et al. (2002a) suggested that no significant effects of dietary Biogen of detoxification of AFB1 of treated O. niloticus. These differences may be related with the concentration of AFs, the exposure time, type of the detoxification method and experimental management.