Effect of Biogen® and Myco-Ad® on the Growth Performance of Common Carp (Cyprinus carpio) Fed a Mycotoxin Contaminated Aquafeed
A commercial probiotic (Biogen®) was tested against a commercial mycotoxin binder (Myco-Ad®) for their effects on survival, growth, body composition and hematological picture of common carp (cyprinus carpio) fed a naturally contaminated aquafeed with aflatoxin and ochratoxin. A total number of 150 apparently healthy fingerlings common carp cyprinus carpio were divided into 5 triplicate groups. G-1 (control) received naturally ration found contaminated with aflatoxin (22 ppb) and ochratoxin (15 ppb). The other four groups supplemented with Biogen® at a rate of 0.2 and 0.4% (G-2 and G-3) and Myco-Ad® at a rate of 0.15 and 0.25% (G-4 and G-5). Results showed a significant reduction (p≤0.05) in aflatoxin recorded in aquafeeds of G-2 and G-3, while ochratoxin level showed a significant reduction in G-3. The four groups received Biogen® and Myco-Ad® showed a significant improve in the final weight, feed intake, FCR, survival rate, protein efficiency, body composition represented an increase in crude protein and reduction of ether extract and also improve hematological picture represented in erythrocyte counts, hematocrite and hemoglobin. It can be concluded that aflatoxin and ochratoxin contamination of fish diets can cause many drastic effects on performance parameters, feed utilization, hematological picture and body composition of common carp. Hence, there is always a demand for risk assessment regarding mycotoxins especially with the moving to plant protein sources in the aquafeeds. Commercial probiotic Biogen® at a level of 0.4 and 0.2% has a good improvement effect among common carp fed mycotoxin contaminated aquafeed, followed by the commercial mycotoxin binder Myco-Ad® at a level of 0.25 and 0.15%.
Received: November 07, 2010;
Accepted: January 11, 2011;
Published: February 07, 2011
The first documented incidences of aflatoxicosis affecting fish health occurred
in the 1960s in trout hatcheries where, rainbow trout were fed a pelleted feed
prepared with cottonseed meal contaminated with aflatoxins, developed liver
tumors (Motalebi et al., 2008). Interest in the
toxic effects on cultured warm-water fishes, such as tilapia and channel catfish,
has increased as diets for these species are now being formulated to contain
more plant and less animal ingredients. This increases the potential for development
of aflatoxicosis in these species because, as noted earlier, plant ingredients
have a higher potential than animal ingredients for contamination with aflatoxins
(Royes and Yanong, 2008).
Mycotoxins cause a wide variety of adverse clinical signs among fishes depending
on the nature and concentration of mycotoxins present, duration of exposure,
the fish species, its age, nutritional and health status at the time of exposure
to contaminated feed (Tuan et al., 2002). Jantrarotai
and Lovell (1990) and Lovell (1992) showed that
rainbow trout are extremely sensitive to AFB1, while channel catfish are much
less responsive. Even the production systems of aquaculture found affect the
tolerance levels for tilapia. In green water and flow-through systems, the presence
of aflatoxins at 25 to 30 parts per billion (ppb) in the water decreased growth
without any noticeable signs of mortality. However, in cage culture, concentrations
of aflatoxins above 5 ppb in the water caused an increase in mortality rates
(Royes and Yanong, 2008).
Mycotoxins have been shown to negatively affect production, growth and immune
system function among different aquaculture species (Lee
et al., 1978; Mahmoud et al., 1994;
Bautista et al., 1994; Marzouk
et al., 1994; Chavez-Sanchez et al., 1994;
Abdelhamid et al., 1998; Bailey
et al., 1998; Horvath, 1998; Aruke
et al., 1999; Ottinger and Kaattari, 2000;
Lim and Webster, 2001; Manning et
al., 2003). Also, Carlson et al. (2001)
mentioned that mycotoxins act synergistically so that the negative effects of
two mycotoxins are worse than the effects of each individually.
Probiotics are pure cultures of one or more living microorganisms given in
feed that proliferates in the host gastrointestinal (GI) tract. They ensure
that the host maintains a beneficial microbial population in the GI tract (Linge,
2005). The research of probiotics for aquatic animals is increasing with
the demand for environment-friendly aquaculture (Wang et
al., 2008). Most probiotics have been undertaken by isolating and selecting
strains from aquatic environment (Gatesoupe, 1999).
Also, probiotics have found use in aquaculture as a means of disease control,
supplementing or even in some cases replacing the use of antimicrobial compounds
(Irianto and Austin, 2002; Sahu et
Many physical, chemical and biological techniques to neutralize mycotoxins
have been developed and were reported in the literature throughout the years
(Doyle et al., 1982; Samarajeewa
et al., 1990). Binders have been used to neutralise the effects of
mycotoxins by preventing their absorption from the animals digestive tract
through bind toxins by adhesion or by electrostatic charge or cation exchange
capacity. The effectiveness of smectite clays (HSCAS) as adsorbents of aflatoxin
has been investigated and found successful for many farm animals over the past
20 years (Grim, 1962; Bluthgen and
Schwertfeger, 2000; CAST, 2003; Dixon
et al., 2008).
Therefore, the aim of this paper is to study the effect of feeding a naturally fish diets found contaminated with aflatoxin and ochratoxin on the performance and health status of common carp (cyrpinus carpio) fingerlings and a trial to control such effect through supplementation of Biogen® (probiotic-immunostimulant) and Myco-Ad® (smectite clay, HSCAS for mycotoxin binding) in this contaminated diet.
MATERIALS AND METHODS
This study was carried out in the Central Laboratory of Agriculture Research, Abbassa, Sharkia governorate, Agricultural Research center, Ministry of Agriculture, Egypt during summer season of 2009.
Experimental fish and management: A total number of 150 apparently healthy fingerlings of common carp Cyprinus carpio obtained from the hatchery of Agriculture Research at Abbassa, Sharkia, Egypt. The fish maintained in glass aquaria (each of 70x50x40 cm) filled with dechlorinated tap water which continuously aerated. They divided into 5 triplicate groups (10 fingerlings/aquarium) and named G-1 for control group and from G-2 to G-5 for the other four experimental groups. They were acclimatized to the laboratory conditions for 2 weeks before starting the experiment. The water temperature was kept at 25±2°C throughout the experiment. About half of the water was changed daily in all the experimental aquaria. The average weight of fingerlings was 15 gram/fish at the start of the experiment. The fingerlings weighted every 2 weeks and fed the experimental diets at 3% of the total biomass along the period of the experiment (90 days). The feeding rate was adjusted according to the last fish weight.
Aquafeeds and feeding regimes: A naturally ration found contaminated with aflatoxin (22 ppb) and ochratoxin (15 ppb) divided into five experimental diets. The first part used as a control (G-1). The other four parts supplemented with Biogen® (China-Way Corp; Taiwan) at a rate of 0.2% (G-2) and 0.4% (G-3) and Myco-Ad® (Special Nutrients, Inc. USA) at a rate of 0.15% (G-4) and 0.25% (G-5). The five experimental diets formulated to contain about 30% cp and 4700 GE Kcal kg-1 (Table 1). Each diet mechanically mixed, pressure pelleted by using meat mincer, air dried at room temperature, broken into small pieces, sieved to obtain appropriate size and stored at -5°C. The experimental fishes received the tested diets twice daily at 8 a.m. and 2 p.m.
Biogen®: A synthetic probiotic immunostimulant product from China Way Corp; Taiwan. It contains allicin (garlic extract) not less than 0.247 micromil g-1, Bacillus subtilis nato 6x107 cells g-1, high-unit hydrolytic enzymes 3690 units g-1 (proteolytic, lipolytic, amylolytic and cell separating enzymes), germanium (Ginseng extract 41.98 ppm) and organic selenium.
Myco-Ad®: Commercial smectite clay composed of an activated, broad spectrum, hydrated, sodium/calcium aluminosilicate (HSCAS) from Special Nutrients, Inc., USA specially formulated to adsorb major mycotoxins.
Determination of the levels of aflatoxins and ochratoxins in aquafeed samples:
A stored aquafeed, suspected to be contaminated with mycotoxins, was subjected
to determine the presence of aflatoxin and ochratoxin, after feed supplementation
with Biogen and Myco-Ad, using immunoaffinity method which is applicable for
mycotoxins that have fluorescence (Trucksess et al.,
1991). Series-4 Fluorometer (VICAM) was used in this procedure. Reading
of total aflatoxin or ochratoxin was obtained as part per billion (ppb = μg
Water quality: Water parameters (temperature, pH, salinity and dissolved
oxygen) were measured twice daily (10 a.m. and 10 p.m.). Dissolved oxygen measured
by oxygen meter as mg L-1. (yellow sparing Instrument Co. model 57).
Water samples were collected weekly from aquaria to detect total ammonia (ionized
and non-ionized) and total hardness. Ammonia measured by Direct-Nesslerization
method, while Total hardness measured by the EDTA-tetrimetric method (APHA,
et al., 1989).
||Body weight gain (W.G.): Total weight determined to
the nearest gram according to (Annet, 1985)
Hematological investigation: At the end of the experiment blood samples
were collected from the fish caudal peduncle of the different groups. Adequate
amounts of whole blood in small plastic vials containing heparin were used for
the determination of:
||Total erythrocytes (RBCs) and total leucocytes (WBCs) counted
on an Ao Bright-Line Haemocytometer model (Neubauer improved,
Precicolor HBG, Germany) according to Dacie and Lewis
||The packed cell volume carried out in small hematocrite graduated
tubes using Hematocrite centrifuge at 3000 rpm for 15 min
||Hemoglobin (Hb) estimated by Bochringer munnheim kit according
to Wintrobe (1965)
Statistical analysis: Data listed, computed and analyzed using Analysis
of Variance (ANOVA) and differences between means (Duncan,
1955). Multiple range test was done to determine differences between treatment
(mean at significance level of (p<0.05). Standard errors were also estimated.
All analyses were run on the computer using the SAS program.
The examined aquafeed samples as shown in Table 2, found
contaminated with a mean value of 22 ppb for aflatoxin and 15 ppb for ochratoxin.
A significant reduction (p<0.05) in aflatoxin found in G-2 and G-3 groups,
while ochratoxin reduced in G-3. No significant reduction in both aflatoxin
and ochratoxin recorded in G-4 and G-5 groups.
|| Mean values of mycotoxins detected in the examined aquafeed
|a-c: Means in the same column with different superscripts
are significantly (p≤0.05) different
|| Water quality parameters
|a-c: Means in the same column with different superscripts
are significantly (p≤0.05) different, Values are presented as Mean±SE
|| Growth performance and feed utilization of common carp fingerlings
|a-d: Means in the same row with different superscripts are
significantly (p≤0.05) different, Values are presented as Mean±SE
|| Body composition at the end of the experiment for common
|a-c: Means in the same column with different superscripts
are significantly (p≤0.05) different, Values are presented as Mean±SE
|| Hematological parameters from common carp fingerlings
|a-c: Means in the same column with different superscripts
are significantly (p≤0.05) different, Values are presented as Mean±SE
Table 3 illustrated that dissolved oxygen, pH and temperature
estimated in all aquaria showed nearly the same readings along the 90 days of
the experiment. Salinity increased in all treated groups (from G-2 to G-5).
Ammonia was highest in G-2 and 3 (1 mg L-1), followed by 0.08 mg
L-1 in G-4 and 5. Also, total hardness showed significant increase
in G-2 and 3 (about 440 mg L-1), followed by G-4 and 5 (270 and 290
Results in Table 4, showed a significant improvement in the final weight of cultured fish in G-2 and G-3 (58.37 and 55.08 g fish-1) and followed by G-5 and G-4 (53.55 and 51.7 g fish-1). This improvement in final weight reflected on the total weight gain and specific growth rate which improved by the same supplements. A significant reduction in feed intake and consequently in FCR recorded in G-3 and G-4, followed by G-2 and G-5. Protein efficiency significantly improved in G-3 and G-5, Followed by G-4 and G-2. Survival rate improved in the 4 groups of supplemented feed in comparison with G-1. In Table 5, all treated groups (from G-2 to G-5) showed improvement in final body composition as the increase in crude protein from about 51% in control to the maximum value (56.78) in G-3. Ether extract reduced in fish bodies of treated groups.
Results in Table 6, showed improvement in erythrocyte counts, hematocrite and hemoglobin in all treated groups (from G-2 to G-5), white blood cells which rose as an immune response in control group, showed a significant reduction in all treated groups (from G-2 to G-5).
The natural contamination of aquafeeds with aflatoxin (22 ppb) and ochratoxin
(15 ppb) indicate the demand for risk assessment regarding mycotoxins especially
with the moving to plant protein sources in aquafeeds (Ali
et al., 1998; Lim et al., 2001; Tacon,
In Egypt, feeds may expose to bad hygienic condition, during transportation and storage from wetting condition, insects infestation besides the long period of storage leading to the feed becoming mouldy and contaminated with mycotoxins.
Although, the little research studies on mycotoxins and mycotoxicoses in cultured
fish and still absence of definite regulation for mycotoxin levels of aquafeeds
(single or multi-mycotoxin contamination), there is studies on the effect of
mycotoxins on aquaculture which ascertained the deleterious effect on fish health,
immunity and consequently on their performance (Bailey et
al., 1998; Aruke et al., 1999; Lim
and Webster, 2001; Sahoo and Mukherjee, 2001; Tuan
et al., 2002 ,2003;
Abdelhamid et al., 2004a, b; 2002b,
c; Salem, 2002; Manning
et al., 2003).
The significant reduction of aflatoxin in G-2 and G-3 and also the reduction
of ochratoxin in G-3, may be attributed to the presence of garlic extract (allicin)
and the presence of Bacillus subtilis in component of Biogen®,
both are known that they have the characteristics of mould inhibition and mycotoxin
detoxification (Azzouz and Bullerman, 1982; Yin
and Cheng, 1998; Galvano et al., 2001; Petchkongkaew,
2007). This finding disagrees with Abdelhamid et
al. (2002b) who found that dietary Biogen® supplementation
was not useful in AFB1 detoxification.
The values of water parameters recorded in this study are within the acceptable
ranges recommended for fish culture (APHA et al., 1989;
Boyd and Tucker, 1993; Chapman, 2000;
Abdelhamid, 2009). The total concentration of all ions
in the water is its salinity. The increase in salinity in Biogen®
and Myco-Ad® groups may be attributed to the content of feed
additives from minerals and salts. The increase in ammonia in Biogen®
and Myco-Ad® groups may be attributed to the increase in feed
intake and fish growth rates, which consequently reflected on the increase of
the nitrogenous wastes.
An acceptable range for free calcium in culture waters is 25-100 mg L-1
or 63-250 mg L-1 CaCO3 hardness (Wurts,
1989). Calcium has an important role in the biological processes of fish.
Fish can absorb calcium directly from the water or food. The increase in total
hardness in Biogen® and Myco-Ad® groups may be
attributed to the content of feed additives from minerals and salts.
Biogen® at the recommended level of 0.4% and even the half of
that level (0.2%) improved the performance parameters and feed utilization of
the common carp fingerlings which are being stressed from feeding on mycotoxin
contaminated aquafeed. The explanation of this finding may be due to that the
commercial probiotic Biogen® consists of Bacillus licheniformis
and Bacillus subtilis. These spore-forming bacteria which survive the
pelletization process, can enhance the metabolism and energy of fish body cells,
raise the efficiency of feed utilization and balance the secretion of various
secretory glands. Moreover, it increases the vitality of cells by supplying
oxygen to whole body, improves the immune responses, helps to excrete heavy
metals, inhibits aflatoxin and maintains the normal endocrine system. Biogen®
has bactericidal effects and increases the palatability of feed, promotes the
secretion of digestive fluids and stimulates the appetite (EL-Dakar
et al., 2007; Diab et al., 2008; Eid
and Mohamed, 2008; Mehrim, 2010).
Also, Myco-Ad® comes after Biogen® in the improvement
of performance parameters and feed utilization of the cultured common carp fed
mycotoxin contaminated ration. This result may be attributed to the composition
of Myco-Ad® which is hydrated sodium calcium aluminosilicates
(HSCAS) which capable to the alleviation of mycotoxicosis through adsorbtion
capacity of mycotoxins (Barrer, 1989; Mumpton,
1999). Phillips et al. (1990) interpreted
the binding mechanism as the formation of a complex by the β-carbonyl system
of the aflatoxin with uncoordinated edge sitealuminium ions. Thus,
HSCAS can be used as an inorganic sponge sequestering aflatoxins
in the gastro-intestinal tract of farm animals. Regarding the applicability
of aluminosilicates for the binding of mycotoxins, it can be concluded that
they are very effective in preventing aflatoxicosis, but their efficacy against
zearalenone, ochratoxin and trichothecenes is limited (Huwig
et al., 2001). This finding disagrees with Abdelhamid
et al. (2002a) who mentioned that mycotoxin adsorbents did not significantly
reduce the aflatoxicity.
Survival rate in control group was lower (88%) compared with the other groups
supplemented with Biogen® and Myco-Ad® (about
96 and 93%, respectively). The sensitivity to mycotoxins varies and the exact
susceptibility of different fish species could not be determined up till now
as described by Lovell (1992) who found that warm water
fish such as channel catfish are reported to be less sensitive to aflatoxin
than rainbow trout.
In practical terms, the improvement in performance and feed utilization parameters means that the use of probiotic, Biogen® and the mycotoxin binder, Myco-Ad® in case of feeding carp fish a mycotoxin contaminated ration can decrease the amount of feed necessary for animal growth which could result in reductions of production cost.
The improvement in body composition of carp fish in groups supplemented with
Biogen® as found by Khattab et al.
(2004b) and also groups supplied with Myco-Ad® in the ration
contaminated with mycotoxin, is a significant evidence of the improvement in
general health condition of the cultured fish. These positive effects in body
composition of experimental fish may be due to the dietary supplementation with
Biogen® which caused the good growth performance, enhance the
metabolism and energy of fish body cells and raise the efficiency of feeds (Mehrim,
2001). Also, these results are in close agreement with Srour
(2004), EL-Haroun et al. (2006) and Mohamed
et al. (2007) for tilapia and EL-Haroun (2007)
for catfish. Moreover, Eid and Mohamed (2008) found that
no statistical differences were observed in whole body moisture, crude protein,
ether extract and ash of mono-sex O. niloticus fingerlings fed diets
containing different levels of commercial feed additives (Biogen®
Regarding the results from G-4 and G-5, the improvement in body composition
is a good indicator for the efficiency of Myco-Ad® containing
HSCAS against aflatoxin and ochratoxin to alleviate body from their effect.
This finding agree with Zaki et al. (2008) who
mentioned that dietary HSCAS clay act as an aflatoxin enterosorbant that tightly
and selectively binds the toxin in the gastrointestinal tract of fish, so that
diminishing the clinical signs of aflatoxicosis.
Improvement in erythrocyte count, hematocrite and hemoglobin in groups supplemented
with Biogen® agreed with the findings of Khattab
et al. (2004a), EL-Gohary et al. (2005)
and Mehrim (2010). White blood cells which raised as
an immune response in control group showed a significant reduction in treated
groups with Biogen® and Myco-Ad® and this finding
disagree with Marzouk et al. (2008) who found
increases in RBCs count, Hb value, PCV%, WBCs count and differential of leukocytic
count in the two fish groups fed the diets supplemented with probiotics (dead
Saccharomyces cerevisae yeast and both of live Bacillus subtilis
and Saccharomyces cerevisae). On the other hand, Abdelhamid
et al. (2002b) found that Biogen® reduced blood hemoglobin
of aflatoxicated O. niloticus fish.
From the foregoing results it could be concluded that aflatoxin and ochratoxin contamination of fish diets can cause many drastic effects on performance parameters, feed utilization, hematological picture and body composition of common carp. Hence, there is always a demand for risk assessment regarding mycotoxins especially with the moving to plant protein sources in the aquafeeds. Commercial probiotic Biogen® at a level of 0.4 and 0.2% has a good improvement effect among common carp fed mycotoxin contaminated aquafeed, followed by the commercial mycotoxin binder Myco-Ad® at a level of 0.25 and 0.15% which also can alleviate the effect of mycotoxin. The detoxification of aflatoxin and ochratoxin recorded after the addition of Biogen® need more research to investigate this result deeply.
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