Nutritious attempts to detoxify aflatoxic diets of tilapia fish (Fish performance, feed and nutrients utilization, organs indices, residues and blood parameters)
The Food and Agricultural Organisation (FAO) estimated that 25% of the world’s crops are affected by mycotoxins, of which the most notorious are aflatoxins. Aflatoxins are considered the most carcinogenic, mutagenic and teratogenic poisonous by-products of the growth of the molds Aspergillus flavus and Aspergillus parasiticus, and are important contaminants of certain foods and animal feeds because of their ability to produce aflatoxins (Farr et al., 1989). As well as, Gradelet et al., (1997) suggested thatthese metabolites cause liver damage to humans and to most experimental animal species tested. Also, aflatoxins were caused the economic and health problems because of their ability to contaminate human food and animal feeds, in particular cereals, nuts and oilseeds (Arim 1995 and Njapau et al., 1998). Aflatoxin losses to livestock and poultry producers from aflatoxin-contaminated feeds include death and more subtle effects of immune system suppression, reduced growth rates, and losses in feed efficiency (Vincelli et al., 1995 and FAO 1997 & 2002).
Therefore, some scientific efforts were conducted to use the herbs or natural plants which, detoxification the drastic effects of mycotoxins or aflatoxins on some animals such as, glucomannan (Karaman et al., 2005) or yeast cell wall mannanoligosaccharide (MOS) (Devegowda et al., 1998), or Saccharomyces cerevisiae which were found to have beneficial effects in poultry during mycotoxicosis (Raju and Devegowda 2000), chamomile (Abdelhamid et al., 1985; Soliman and Badeaa 2002 and Ibrahim, 2004), ginger (Vimala et al., 1999 and Abdelhamid et al., 2002e).
Nile tilapia Oreochromis niloticus may represent such a model (as a sensitive model for mycotoxicosis), since this fish is highly susceptible to nutritional deficits and is extremely vulnerable to toxic insult from various chemicals and poisons including aflatoxin B1 (AFB1). Therefore, the present work aim to study the drastic effects of AFB1 on growth performance and survival, feed and nutrients utilization, some organs indices, carcass composition, residues of AFB1 and some parameters of blood hematology and biochemistry of the experimented fish O. niloticus. Also, this study conducted to evaluate the ability of some nutritious agents namely, Bio-Buds-2x, chamomile flowers, aspirin and ginger (at a level of 0.5%) to detoxify the drastic effects of this dangerous toxic AFB1 on the Nile tilapia fish for 14 weeks.
MATERIALS AND METHODS
This study was conducted to evaluate the ability of some nutritious agents namely Bio-Buds-2x (T3), chamomile flowers (T4), aspirin (T5), and ginger (T6), (at a level of 0.5%), to detoxify the drastic effects of this dangerous toxic AFB1 on the Nile tilapia fish for 14 weeks. A group of 180 of mono-sex Nile tilapia O. niloticus fingerlings (obtained from the private fish farm at Tolombat 7, Kafr El-Sheikh), with an average initial body weights of 10g were used in this study. Fish were maintained in the aquaria for one month before the beginning of the experiment for acclimatization purpose. The fish in all experiments were distributed into the aquaria at stocking rate of 15 fish per aquarium. The experimental treatments were tested in two aquaria for each.
A basal diet (30.38% crude protein, 8.79% ether extract, 4.40% crude fiber, 6.24% ash, 478.4 Kcal/100g DM gross energy and 63.5mg cp/Kcal GE, P/E ratio) was formulated from the commercial ingredients (fish meal 10%, soybean meal 38%, yellow corn 35.5%, sun flower oil 4%, wheat bran 12% and vit. & min.0.5%). The basal diet was considered as a control (T1). These ingredients were pressed by manufacturing machine (pellets size 1mm), they were milled and toxin AFB1 was added at a concentration of 100ppb to all diets (T2,T3,T4,T5,T6), except control (T1). Anti-toxin was added at a concentration of 0.5%.The ingredients and supplements were bought from the local market, aflatoxin B1 was produced through pellets fermentation using Aspergillus parasiticus NRRL 2999 according to the method described by Abdelhamid and Mahmoud (1996).Concentration of the produced aflatoxin B1 was calculated and incorporated into the experimental diets at a rate of 100 ppb.
The experiment continued for 14 weeks. During the experimental period the fish were fed the experimental diets at a rate of 3% of the live body weight daily, Six days a week. The diet was introduced twice daily, at 8 a.m. and 2 p.m.. The amount of food was adjusted bi-weekly based on the actual body weight changes. Light was controlled by a timer to provide a 14h light: 10h dark as a daily photoperiod.
At the end of the experiment, one fish from each aquarium was taken immediately for determination the residues of AFB1 in the whole fish body. Also, the remained fish were sampled from each aquarium and kept frozen for chemical analysis. The chemical analyses of the basal diet and whole fish body were carried out according to the AOAC (2000). Aflatoxin B1 determinations in the media extract and the basal diet were determined as described by Abdelhamid (1996). Water quality parameters were measured weekly (Abdelhamid, 1996) including temperature (via a thermometer), pH (using Jenway Ltd., Model 350-pH-meter) and dissolved oxygen (using Jenway Ltd., Model 970- dissolved oxygen meter).
Body weight of individual fish was measured biweekly to point feed quantity and to calculate growth performance and feed utilization (Abdelhamid, 2000b) in form of: Average weight gain (g/fish) AWG = Average final weight (g) – Average initial weight (g), Average daily gain, (g/fish/day) ADG = AWG (g)/Experimental period (days), Specific growth rate (SGR, %/day) = [In final weight – In initial weight] x 100/Experimental period (d), Feed conversion ratio (FCR) = Feed intake (g)/Live weight gain (g), Protein efficiency ratio (PER) = Live weight gain (g)/Protein intake (g), Protein productive value (PPV %) = Retained protein (g)/Protein intake (g) x 100, and Survival rate (SR%) = End number of the alive fish/The beginning number of the fish x 100. Total tissue residues of aflatoxin B1 were estimated by TLC (Thin Layer Chromatography) method described by Abdelhamid (1981). At the end of the experiment, the liver, spleen, kidneys and gonads were removed and weighted individually. The liver, spleen, kidneys and gonads indices were calculated, where: Hepato-somatic index (HSI) = Liver weight (g) x 100/Gutted fish weight (g) (Jangaard et al., 1967), Spleeno-somatic index (SSI) = Spleen weight (g) x 100/fish weight (g), Kidney – somatic index (KSI)=Kidneys weight (g) x 100/fish weight (g) (Alabaster and Lioyd, 1982) and Gonado-somatic index (GSI) = Gonads weight (g) x 100/fish weight (g) (Tseng and Chan, 1982).
Blood samples from the fish of the different groups were collected from the caudal peduncle. Adequate amounts of whole blood in small plastic vials containing heparin were used for the determination of hemoglobin (Hb) by using commercial kits (Diamond Diagnostic, Egypt). Also, total erythrocytes count (RBCs) and total leucocytes count (WBCs) were measured on an Ao Bright –Line Haemocytometer model (Neubauer imroved, Precicolor HBG, Germany). Other blood samples were collected and transferred for centrifugation at 3500 rpm for 15 min to obtain blood plasma for determination of total protein according to Gornall et al., (1949), albumin according to Weichsebum(1946), globulin by difference according to Doumas and Biggs (1977), cholesterol according to Richmond (1973), uric acid according to Barhamd (1972), alkaline phosphatase (AlP) according to Belfield and Goldberg (1971), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) according to Varley (1976) using a spectrophotometer (model 5010, Germany) and commercial kits.
The data collected statistically analyzed using general linear models procedure adapted by SAS (1997) for users guide, with a one-way ANOVA. Means were statistically compared for the significance (p ≤ 0.05) using Duncan (1955) multiple range test.
RESULTS AND DISCUSSION
1- Quality parameters of rearing water:
All tested water quality criteria were suitable for rearing mono-sex Nile tilapia O. niloticus fingerlings as cited by Abdelhamid (2000b) and Abd El-Hakim et al. (2002). Since water temperature ranged between 22 and 25oC, pH values 6.9 – 7.8 and dissolved oxygen 6.71 – 8.77 mg/l. Also, Abdelhamid et al. (2002c) suggested that these values are suitable for rearing Nile tilapia O. niloticus. In the same trend, Abdelhamid et al. (2004 b&c) found that all the tested water quality (temperature °C, pH value, conductivity mg/l and dissolved oxygen mg/l) criteria were suitable for rearing Nile tilapia fish O. niloticus.
2- Growth performance and survival rate:
Data presented in Table (1) showed that there were no significant (P≥0.05) differences among the initial body weights and survival rate of the different dietary groups of fish. Yet, there were significant (P≤0.05) differences among various group of fish concerning final body weight, AWG, ADG and SGR of the experimented fish. Being the best values in favor of T6 (aflatoxin-contaminated diet plus 0.5% ginger), which seem even more better than the control (uncontaminated diet,T1) and significantly (P≤0.05) better than the aflatoxin-contaminated diet (T2) and aflatoxin-contaminated diets plus 0.5% Bio-Buds-2x (T3) or 0.5% chamomile flowers (T4). The aflatoxin-contaminated diet plus 0.5% aspirin gave performance values not significantly (P≥0.05) differ than those given by T1 and T6 groups. But, T3 was the worst one, even than T2.
In this context, similar negative effects of AFB1 on different growth performance parameters and survival rate of tilapia fish were recorded by other authors (Hussein et al., 2000; Abdelhamid et al., 2002 b&c and 2004 b&c ; Nguyen et al., 2002 and Shehata et al.,2003). Recently, also Abdelhamid et al., (2004 b&c) found that the effects of aflatoxins B1 (AFB1) were significant decreases on growth performance and survival rate of O. niloticus fish. Yet, the effects of mycotoxins on fish depend on potency of mycotoxin, dose, species and strain of the fish, state of health, stage of life, temperature of the water and presence or absence of substances that can modify the toxicity (El-Said, 1997 and Abdelhamid, 2000a).
The positive effects to alleviate the toxic effects of AFB1 on growth performance and survival rate of tilapia fish by dietary addition of Nigella sativa seeds was recorded by Hussein et al. (2000). As well as, Shehata et al.(2003) found that adding the adsorbent agents significantly (P ≤ 0.05) reduced the toxic effect of aflatoxin on growth performance and mortality rate of Nile tilapia fish. Recently, Abdelhamid et al. (2004 b&c) found that the best feed additives led to significant overcoming the aflatoxic symptoms on growth performance and mortality were egg shell and clay, respectively. Also, they added that the effects of either adsorbents namely, egg shells and shrimp wastes at levels of 1 and 2%, respectively, were useful to reduce the toxic effects of AFB1 on O. niloticus fish via adsorbing the toxin from the fish diets. On the other side, Abdelhamid et al. (2002e) suggested that no one of the tested medicinal herbs (thyme, safflower, ginger, black cumin and/ or garlic) completely overcome the effects of foodborne aflatoxicosis.
Meanwhile, in the present study useful effects of some nutritious agents which, used to detoxify aflatoxic diets of tilapia fish, namely ginger and aspirin due to their chemical and physical properties and/or their positive effects on immune system. Ginger stimulates digestion as it influences positively on the terminal enzymes of digestive process (Ahmed and Sharma, 1997 and Platel and Srinivasan, 2000). However, aspirin (acetylsalicylic acid) is known to inhibit the cyclooxygenases and enhancement of cellular immune response, or induction of apoptosis (Shiff and Rigas, 1999 and Subongkot et al., 2003).
Table (1): Means* ± standard errors of the growth performance of the experimented tilapia fish as affected by the dietary treatment for 14 weeks
| Treat. No. |
Body weight (g/fish) | Body gain | SGR(%/day) | SR % | ||
| Initial | Final | AWG (g/fish) | ADG (g/fish/d) | |||
| 1 | 10.00 ± 0.10 |
25.34 ab ±1.13 |
15.34 ab ±1.03 |
0.15 ab ±0.01 |
0.94 ab ±0.03 |
100.00 ±0.00 |
| 2 | 10.00 ±0.00 |
23.45 b ±0.05 |
13.45 b ±0.05 |
0.13 b ±0.00 |
0.86 b ±0.00 |
93.33 ±0.00 |
| 3 | 9. 95 ±0.05 |
22.35 b ±1.65 |
12.40 b ±1.60 |
0.12 b ±0.01 |
0.82 b ±0.07 |
100.00 ±0.00 |
| 4 | 10.10 ±0.10 |
23.82 b ±0.28 |
13.72 b ±0.37 |
0.13 b ±0.00 |
0.87 b ±0.02 |
86.66 ±13.33 |
| 5 | 10.00 ±0.00 |
25.38 ab ±0.62 |
15.38 ab ±0.62 |
0.15 ab ±0.00 |
0.95 ab ±0.02 |
100.00 ±0.00 |
| 6 | 10.00 ±0.00 |
29.25 a ±1.97 |
19.25 a ±1.97 |
0.19 a ±0.02 |
1.09 a ±0.06 |
89.99 ±3.34 |
Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
T1= Control diet.T2= Diet1 + AFB1 (100ppb).
T3= Diet1 + AFB1 (100ppb) + 0.5% Bio-Buds-2x.
T4= Diet1 + AFB1 (100ppb) + 0.5%Chamomile flowers.
T5= Diet1 + AFB1 (100 ppb) + 0.5% Aspirin.
T6= Diet1 + AFB1 (100 ppb) + 0.5% Ginger
All criteria studied and presented in Table (2) showed that once again, T6 was the best (P≤0.05) treatment (even than the control, T1) concerning FI, FCR, PER and PPV in tilapia fish; then followed by T5. There were no significant (P≥0.05) differences among T1, T5 and T6 for FCR and PPV. While, there were no significances between T6 and T5 in data of FCR, PER and PPV. Again, T3 was the worst one, even than the aflatoxin-contaminated diet without additives (T2).
Similar negative effects of AFB1 on feed and protein utilization parameters of tilapia fish were recorded by Hussein et al. (2000), Abdelhamid et al. (2002 b&c and 2004 b&c), Nguyen et al. (2002) and Salem (2002). This negative effect of AFB1 may be attributed to its causative pathological alterations in the gastro-intestinal tract (Murjani, 2003). Also, the present results agree with the finding of Nguyen et al. (2002) who suggested that fish fed diets containing 10 and 100 mg AFB1/kg were observed to expel feed after ingestion. As well as, the authors added that because fish fed the highest levels of aflatoxin-B1did not consume all of the feed, the AFB1 dose was not directly related to the concentration in the feed. Therefore, fish fed the 100 mg AFB1/kg diet consumed a total of 59 mg AFB1/kg of body weight, which was only three times than the amount for fish fed the 10 mg AFB1/kg.Yet, Salem (2002) indicated that feed and protein utilization parameters significantly (P<0.05) reduced by O. niloticus fed the dietary AFB1. Similar results were obtained by Abdelhamid et al. (2002 b&d).
Moreover, Kasper et al. (2002) found that Betafin® did not significantly change feed efficiency and protein utilization. Also, Abdelhamid et al., (2002b) found that dietary Biogen® supplementation was not useful in AFB1 detoxification. As well as, Abdelhamid et al., (2002a) mentioned that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce the aflatoxicity. On the other side, recently Abdelhamid et al. (2004 b& c) found that the best feed additives led to significant overcoming the aflatoxic symptoms on growth, mortality, feed and protein utilization were egg shell and clay, respectively. However, in the present results, better results of ginger and aspirin used may be due to their chemical and physical properties and/or its positive effects on immune system. Ginger stimulates digestion as it influences positively the terminal enzymes of digestive process (Ahmed and Sharma, 1997 and Platel and Srinivasan, 2000). However, aspirin (acetylsalicylic acid)is known to inhibit the cyclooxygenases and enhancement of cellular immune response, or induction of apoptosis (Shiff and Rigas, 1999 and Subongkot et al., 2003).
Table (2): Feed intake and conversion as well as protein utilization in the experimented tilapia fish (X* ± SE) as affected by the dietary treatments during the 14 weeks experiment
|
Treat. No. |
FI (g/fish) | FCR | Protein utilization | |
|
|
PER | PPV % | ||
| 1 | 37.26 ab ±0.46 |
2. 43 ab ±0.13 |
1.35 b ±0.07 |
24.18 ab ±1.69 |
| 2 |
33.39 c |
2.48 ab |
1.32 b |
21.97 b |
| 3 |
33.53 c |
2.73 a |
1.21 b ±0.10 |
21.28 b ±1.01 |
| 4 | 33.74 c ±1.01 |
2.46 ab ±0.01 |
1.34 b ±0.00 |
23.15 b ±1.13 |
| 5 | 34.93 bc ±0.78 |
2.27 ab ±0.03 |
1.45ab ±0.02 |
24.48 ab ±1.00 |
| 6 | 38.44 a ±0.81 |
2.01 b ±0.16 |
1.65 a ±0.12 |
28.48 a ±1.92 |
*Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
| FI= Feed intake | FCR= Feed conversion ratio |
| PER= Protein efficiency ratio | PPV= Protein productive value |
4- Internal organs indices:
The only significant (P≤0.05) differences were found among the dietary treatments for gonado-somatic index (GSI), but not (P≥0.05) for hepato (HSI), kidney (KSI), and spleen-somatic index (SSI) as presented in Table (3).Treatment No.5 (aflatoxin-contaminated diet plus 0.5% aspirin) reflected significantly (P≤0.05) higher value for GSI than those of the contaminated diet (T2) and contaminated diet plus 0.5% Bio-Buds-2x (T3).
Generally, from these results in the present study, the aflatoxic diets caused negative effects on the internal organs indices significantly (P≤0.05) in GSI or not significantly (P≥0.05) in other indices (HSI, KSI and SSI) comparing with the control diet (T1). This means that AFB1 not only reduced growth performance of the tested fish, but also negatively altered internal organs function as a consequence of increasing their relative weights, which may be due to increasing their cells number or volume or elevating their water and/or blood contents (Glaister, 1986). The same negative effects of AFB1 on internal organs indices of O. niloticus were recorded too by Hussein et al. (2000) and Abdelhamid et al. (2002c and 2004 a, b & d).
In this context, recently, Abdelhamid et al. (2004a) suggested that the aflatoxic diets increased obviously relative weights of all tested organs (liver, kidneys, spleen, testes, heart and lungs) of rats comparing with the aflatoxin free diets. As well as, Abdelhamid et al. (2004 b &d) reported that the aflatoxic diet (100 ppb AFB1) led to significant increases (P<0.05) all organs indices comparing with the control diet ( zero ppb AFB1).
Anyhow, AFB1 effects are variable depending on level and exposure time of/ to the toxin as well as animal species, age, sex and physiological and nutritional states (Abdelhamid, 2000a). Moreover, although AFB1 is a strong hepatic mycotoxin (Hussein et al.,2000 and Nguyen et al., 2002), it has also nephritic (Abdelhamid and Saleh, 1996) as well as sexual negative effects (Constantini et al., 1999), therefore, it affected either of the tested indices.
Hussein et al. (2000) reported that Nigella sativa seeds reduced the negative effect of 1.0µg AFB1/kg BW on internal organs indices of O. niloticus fish. Also, Abdelhamid et al. (2004 b &d) reported that the best feed additives led to significant overcoming the aflatoxic symptoms on organs indices were egg shell and clay, respectively. Also, they added that the effects of either adsorbents namely, egg shells and shrimp wastes at levels of 1 and 2%, respectively, were useful to reduce the toxic effects of AFB1 on O. niloticus fish via adsorbing the toxin from the fish diets. On the other side, Abdelhamid et al. (2002a) found that adsorbents, e.g. Antitox plus, Fix-a-tox and tafla did not significantly reduce aflatoxicosis symptoms. As well as, Abdelhamid et al. (2002b) reported that dietary Biogen® supplementation was not useful in AFB1 detoxification. Also, Abdelhamid et al. (2004a) added that the additives (tafla, ammonia and hydrogen peroxide) did not alter the organs weight; yet, they slightly diminished- to some extent- the negative effect of dietary aflatoxin inclusion on the relative weights of all tested organs. However, in the present study, the effects of ginger and aspirin may be due to their adsorbative characteristics as mentioned before, so prevented or reduced the absorption of AFB1 and hence hide its negative effects on internal organs indices of fish.
Table (3): Means * ± standard errors of the internal organs indices of the tilapia fish at the end of the 14-weeks period as affected by the experimental diets.
| Treat. | HSI% | KSI% | SSI% | GSI% |
| 1 | 3.85 ±1.25 |
0.51 ±0.02 |
0.26 ±0.08 |
0.85 ab ±0.20 |
| 2 | 0.59 b ±0.12 |
0.47 ±0.15 |
0.41 ±0.06 |
4.34 ±0.11 |
| 3 | 0.79 b ±0.04 |
0.24 ±0.05 |
0.38 ±0.03 |
3.32 ±0.71 |
| 4 | 1.37 ab ±0.47 |
1.44 ±1.04 |
0.35 ±0.03 |
3.68 ±0.85 |
| 5 | 1.69 a ±0.27 |
0.25 ±0.03 |
0.39 ±0.07 |
3.72 ±0.16 |
| 6 | 0.91 ab ±0.07 |
0.25 ±0.01 |
0.50 ±0.11 |
3.02 ±0.09 |
*Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
5- Biochemical analysis and AFB1 residues of the whole body:
Data of the proximate chemical analysis of the whole body of the tested tilapia fish are given in Table (4). Dry matter (DM) content increased by age advance, but not significantly (P≥0.05) affected by the dietary treatments. Also, crude protein (CP), ether extract (EE), and ash contents increased by aging, but reflected significant (P≤0.05) differences among the dietary treatments. The highest CP and the lowest EE and energy contents were determined in the fish group of T6, which was even more better than the uncontaminated control group (T1). The best treatment (T6) did not differ significantly (P≥0.05) in ash content of the fish than in the other dietary treatments. The fish group of T3 reflected the lowest CP and the highest energy contents among the dietary treatments tested.
Similar results were recorded by Hussein et al. (2000) and Abdelhamid et al. (2002 b&c) concerning fish carcass analyses. The same adverse effects of AFB1 on carcass composition of O. niloticus were recorded too by Abdelhamid et al. ( 2004 b & c) and Salem (2002).
The present results agree with the findings of Salem (2002) who found that the control group of fish had the highest (P < 0.05) DM and CP values and the lowest (P < 0.05) EE percentage. The latest author added that percentages of DM and CP decreased as the levels of the dietary aflatoxin B1 increased, while the values of EE and ash increased with increasing the levels of AFB1. Also, Hussein et al. (2000) found that AFB1 administration led to reduce fish muscular protein, but it was improved by feeding fish on 1-2% Nigella sativa seeds. Yet, Abdelhamid et al. (2004 b&c) found that aflatoxin B1 significantly reduced DM and CP contents of the O. niloticus fish carcass but, it significantly increased EE and ash contents of the fish. Meanwhile, they added that dietary addition of clay or egg shell and shrimp wastes to the AFB1 including diets improved this picture.
In accordance with the present findings, Abdelhamid et al. (2002b) reported that the aflatoxic diets significantly (P < 0.01) reduced the fish flesh crude protein content but increased its fat and ash contents proportional to the dietary levels of the aflatoxin. The opposite trends were recorded with the Biogen® inclusion, since it increased crude protein and decreased fat contents (P < 0.01) of the whole fish body, without significant (P > 0.05) effect on the ash percentage. However, the dietary Biogen® addition improved these parameters. Anyhow, Abdelhamid et al. (2002 c) confirmed that adsorbents still neither obstacle nor sufficient mean for removing AFB1 and its toxic effects. Yet, the positive effects of ginger and aspirin used in the present study may be due to their adsorbative characteristics as mentioned before, so prevent or reduce absorption of AFB1 and hence hide its negative effects on carcass composition of O. niloticus fish.
Table (4): Proximate chemical analysis and energetic value of the whole tilapia body as affected by the experimental diets (X* ± SE).
|
Treat. No. |
DM % | % On Dry matter basis | |||
| CP | EE | Ash |
EC** | ||
| At the start of the experiment: | |||||
| 24.80 | 54.30 | 8.70 | 22.10 | 388.38 | |
| At the end of the experiment: | |||||
| T1 | 26.55 + 0.18 |
60.61 ab + 0.17 |
15.26 e + 0.07 |
24.09 a + 0.17 |
486.05 c + 0.86 |
| T2 | 25.68 + 0.12 |
59.37 d + 0.07 |
16.82 a + 0.06 |
23.85 ab + 0.03 |
493.71 a + 0.35 |
| T3 | 26.19 + 0.21 |
60.05 c + 0.18 |
16.49 b + 0.13 |
23.61 b + 0.07 |
494.35 a + 0.78 |
| T4 | 25.95 + 0.43 |
60.37 bc + 0.25 |
15.99 c + 0.12 |
23.71 ab + 0.11 |
491.43 b + 0.37 |
| T5 | 26.11 + 0.30 |
60.26 bc + 0.10 |
15.66 d + 0.05 |
24.09 a + 0.10 |
487.72 c + 0.67 |
| T6 | 26.18 + 0.35 |
60.99 a + 0.15 |
15.24 e + 0.09 |
23.83 ab + 0.23 |
487.21 c + 1.05 |
*Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
** Calculated after Macdonald et al. (1973).
However, the control fish were free from the aflatoxin residues; whereas, T2 reflected the highest level being 28.95 ppb aflatoxin B1, followed by T3, T4, T5 and T6, being 27.50, 26.72, 21.21 and 15.64 ppb respectively. So, T6 was the best treatment in reducing the level of the residues, followed by T5.
In this respect, similar results were recorded by (Soliman et al., 1998 and 2000).Yet, Abdelhamid et al. (1998) recorded high level of aflatoxin-B1 (246-303 ppb) in whole body of Nile tilapia fish. As well as, Abdelhamid et al. (2004d) found that residues of AFB1 in the whole body of the aflatoxicated O. niloticus fish directly at the end of the experiment were high and tended to decrease after freezing periods. On the other hand, Abdelhamid et al. (2002 b&c and 2004 b) reported that there were no AF- residues in O. niloticus body. They added that the absence of AFB1 residue may be attributed to the lost appetite of fish to feed. Thus, AF may be mobilized or excreted from the fish. These variable results may be due to AF level and exposure time as well as to sensitivity variation among fish species to AF.
Moreover, Soliman et al. (1998) found that the presence of Fix-A-tox in the contaminated diet led to a significant decreases in aflatoxin residue (p < 0.05) in O. niloticus fish. Also, recently Abdelhamid et al. (2004d) found that AFB1 levels were reduced by going on the freezing time of the fish samples in all treatments of aflatoxicated fish. As well as, they reported that addition of 1% egg shell and 2% shrimp wastes to aflatoxicated diets led to adsorptive effects of the dietary aflatoxin and reduced its residue in fish carcass. However, in the present results the effects of ginger and aspirin may be due to their adsorbative characteristics as mentioned before, so prevent or reduce absorption of AFB1 and hence there were no AFB1 residues in the fish body and muscles.
6- Blood analysis:
Data of some hematological parameters are illustrated in Table (5) which presented significant (P≤0.05) differences among the tested dietary treatments. The ginger diet (T6) led to higher (P≤0.05) hemoglobin content (Hb), red blood cells (RBCs) count and white blood cells (WBCs) count at all, followed by the aspirin diet (T5).The first three treatments (T1,T2 and T3) did not differ significantly (P≥0.05) in these hematological parameters tested.
Table (6) presents some biochemical parameters in fish plasma known as kidney function indicators. There were significant (P≤0.05) differences among the dietary treatments for concentrations of uric acid, total protein, albumine and globuline, but not (P≥0.05) for albumine/ globuline ratio. The lowest values were recorded for T6 followed by T5.
Liver function indicators were studied in the treated fish plasma. Data of these plasma biochemical criteria are given in Table (7). All toxic diets with different additives (T3, T4, T5 and T6) used elevated the values of the tested parameters [aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and cholesterol] than in the toxic diet without additives (T2).Yet, there were no significant (P≥0.05) differences among T6 and T5 on one side and T1, on the other side concerning activity of AST and ALT and concentration of cholesterol.
Similar negative effects of AFB1 on blood parameters of tilapia fish were recorded also (Soliman et al., 2000; Abdelhamid et al. 2002 c, d &e and Nguyen et al.,2002). Moreover, the present findings confirm those reported by Abdelhamid et al. (2002b) who mentioned that the aflatoxin contaminated diets reduced the blood values of PCV, Hb and total protein (P <0.01). Also, the authors added that the dietary addition of Biogen® did not improve this blood picture of O. niloticus fish.
Moreover, Hussein et al. (2000) suggested that AF negatively affected blood parameters of tilapia fish. Also, Shehata et al. (2003) found that the activity of AST and ALT enzymes increased significantly (P≤ 0.05) in the fish fed aflatoxin-B1 contaminated diet. Also, in the present results; ALP, AST and ALT activity increased by the AFB1 treatments indicating a damage of the liver and probably also the kidney. Evidence for acute aflatoxin B1 nephrotoxicity was provided by distended gall bladder indicating disrupte osmoregulation (i.e. water retention) as reported by Carpenter et al. (1995).
In the same trend, recently Abdelhamid et al., (2004 b&d) found that AFB1 caused significant decrease in hemoglobin concentration, red blood cells count and uric acid and significantly increase in white blood cells count and alkaline phosphatase and transaminases activity of aflatoxicated O. niloticus fish.
Yet, Hussein et al. (2000) added that Nigella sativa seeds (1 and 2%) alleviated the negative effects of aflatoxicosis by fish. Recently, Abdelhamid et al. (2004 b&d) found that the best feed additives led to significant overcoming the aflatoxic symptoms on blood hematological and biochemical parameters were egg shell and clay, respectively. As well as, the positive effects of some nutritious additives used in the present study, namely Bio-Buds-2x, chamomile flowers, aspirin and ginger may be due to their adsorbative characteristics as mentioned before, so prevent or reduce absorption of AFB1 and hence hide its negative effects on blood parameters of O. niloticus fish.
This positive effects of the best nutritious additives [namely 0.5% ginger (T6) and 0.5% aspirin (T5)], may be due to their chemicals and physicals properties and/or its positive effects on immune system. Ginger stimulates digestion as it influences positively the terminal enzymes of digestive process. (Ahmed and Sharma, 1997 and Platel and Srinivasan, 2000). However, aspirin (acetylsalicylic acid) is known to inhibit the cyclooxygenases and enhancement of cellular immune response, or induction of apoptosis (Shiff and Rigas, 1999 and Subongkot et al., 2003)
Table (5): Means* and standard errors of some hematological parameters of the tilapia fish at the end of the 14- weeks experimental feeding.
| Treat. No. |
Hb (g/dl) | RBCs (x106/mm) | WBCs (x103/mm) |
| 1 | 7.70 b ±0.10 |
28.50 bc ±0.50 |
200.00 b ±0.00 |
| 2 | 7.60 b ±0.10 |
27.50 c ±0.50 |
200.00 b ±0.00 |
| 3 | 7.89 b ±0.02 |
30.50 abc ±0.50 |
200.00 b ±0.00 |
| 4 | 6.60 c ±0.30 |
27.50 c ±1.50 |
300.00 ab ±99.99 |
| 5 | 8.45 b ±0.45 |
31.00 ab ±1.00 |
400.00 ab ±0.00 |
| 6 | 9.70 a ±0.20 |
33.50 a ±0.50 |
500.00 a ±99.99 |
* Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
Table (6): Means* and standard errors of some plasma biochemical (kidney function) parameters at the end of the 14-weeks experimental feeding of the tilapia fish.
| Treat. No. |
Uric acid mg/dl |
Total protein g/dl |
Albumine g/dl |
Globuline g/dl |
Al/Gl ratio |
| 1 | 2.85 a ±0.05 |
4.50 a ±0.00 |
2.26 a ±0.05 |
2.25 a ±0.05 |
1.11 ±0.35 |
| 2 | 2.75 ab ±0.15 |
4.20 a ±0.20 |
2.20 a ±0.00 |
2.00 ab ±0.20 |
1.11 ±0.11 |
| 3 | 2.43 bc ±0.13 |
3.60 b ±0.20 |
1.70 b ±0.20 |
1.90 b ±0.00 |
0.90 ±0.11 |
| 4 | 2.15 c ±0.15 |
3.33 bc ±0.08 |
1.44 bc ±0.04 |
1.89 b ±0.04 |
0.77 ±0.01 |
| 5 | 1.65 d ±0.50 |
3.18 bc ±0.03 |
1.40 bc ±0.05 |
1.78 b ±0.03 |
0.80 ±0.04 |
| 6 | 1.40 d ±0.10 |
3.14 c ±0.04 |
1.33 c ±0.03 |
1.81 b ±0.01 |
0.74 ±0.02 |
* Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
Table (7): Means* and standard errors of some plasma biochemical (liver function) parameters at the end of the 14-weeks experimental feeding of the tilapia fish.
| Treat. No. |
AST UL |
ALT U/L |
ALP U/L |
Cholest. mg/dl |
| 1 | 26.00 b ±1.00 |
20.50 b ±0.50 |
17.00 c ±2.00 |
104.50 ab ±25.50 |
| 2 | 29.50 ab ±1.50 |
27.00 ab ±3.00 |
23.00 bc ±3.00 |
87.50 b ±7.50 |
| 3 | 34.00 ab ±4.00 |
35.00 a ±1.00 |
27.50 ab ±1.00 |
99.50 ab ±9.50 |
| 4 | 37.00 a ±3.00 |
34.00 a ±5.00 |
34.00 a ±2.00 |
115.50 ab ±3.50 |
| 5 | 34.00 ab ±2.00 |
29.00 ab ±1.00 |
29.50 ab ±2.50 |
122.00 ab ±2.00 |
| 6 | 34.00 ab ±2.00 |
31.00 a ±1.00 |
32.00 a ±1.00 |
132.50 a ±2.50 |
* Means (in the same column) superscripted with different letters significantly (P≤0.05) differ.
CONCLUSIONS
From the foregoing results it could be concluded that aflatoxin contamination of fish diets caused many drastic effects on all the tested parameters. Also, AFB1 is very dangerous from the view point of fish production and public health. It could be recommended for the beneficial using of 0.5% ginger and/or 0.5% aspirin to alleviate the toxic effects of AFB1 contaminated fish diets. Also, it is a must to conduct a lot of scientific efforts in this trend to use the medical herbs and other natural agents to detoxify the aflatoxic diets of fish. But, the wisdom still right, that prophylaxis, from toxic effects of mycotoxins especially AFB1, is more useful than treatments.
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Authors: A.M. Abdelhamid*, M.F.I. Salem**, A.I. Mehrim*, M.A.M. El-Sharawy*
* Department of Animal Production, Faculty of Agriculture, Mansoura University, Egypt.
**Central Laboratory for Aquaculture Research, Kafr El-Shiekh Aquaculture Research Unit, Egypt.
