Eggs have been an important commodity in international trade; however,
it provides unique well balanced nutrients for persons of all ages. Their
high nutrient content, low caloric value and ease of digestibility make
egg valuable in many therapeutic diets for adults (Oliveira et al.,
2003; Heranz et al., 2007; Ebubekir et al., 2008).
Presence of fungi and their toxic metabolites (mycotoxin) in poultry
ration is virtually inevitable particularly in tropic areas. Mycotoxins
are unavoidable because they are naturally occurring compounds. They contaminate
crops before harvest or invade feedstuffs of laying hen during processing,
transport or storage (Liau et al., 2007; Yaling et al.,
Aspergillus species are common soil fungi; they are recognized
as major contaminant of many grains used for poultry diets. The aflatoxins
are produced by two molds, Aspergillus flavus and A. parasiticus.
Its specific forms are designated as B1, B2, G1,
G2, M1 and M2. Aflatoxin B1
is the most potent naturally occurring carcinogen known (Moss, 1991; Coulombe,
1993 and Binder et al., 2007). Aflatoxin affects all poultry species.
Although it generally takes relatively high levels to cause mortality,
low levels can be detrimental if continually fed. As a general rule, growing
poultry should not receive more than 20 ppb aflatoxin in the diet. However,
feeding levels lower than 20 ppb may still reduce their resistance to
disease, decrease their ability to withstand stress and bruising and generally
make them unthrifty. Laying hens generally can tolerate higher levels
than young birds, but levels should still be less than 50 ppb (Jones et
al., 1994 and Yaling et al., 2008). Aflatoxin contamination
can reduce the birds' ability to withstand stress by inhibiting the immune
system. This malfunction can reduce egg size and possibly lower egg production.
In addition, one must pay special attention to the use of contaminated
corn in layer rations because eggs are promptly used as human food and
aflatoxin metabolites have been found in egg yolks (Barly and Vadehara,
1999 and Bray and Ryan, 2006).
The presence of aflatoxins in egg is a potential threat to the health
of the consumer. Growing children are more sensitive than adults, as egg
is one of their main sources of nutrients. Aflatoxin is known to be human
carcinogens based on sufficient evidence of carcinogenicity in humans
(IARC, 1987, 1993 and Yaling et al., 2008).
Aflatoxins, especially B1, have been tested extensively for
gene-toxicity. It induces DNA damage, gene mutation, chromosomal anomalies
and cell transformation in mammalian cells in vitro. For this type
of carcinogen, it is generally felt that there is no threshold dose below
which no tumor formation would occur. In other words, only a zero level
of exposure will result in no risk (FAO/WHO, 2004). It is considered as
the cause of acute hepatitis, immuno-suppression, plays a role in Kwashiorkor,
increases neonatal susceptibility to jaundice and may have relevance to
hepatitis B and AIDS (Hsieh and Atkinson, 1999; Bintvihok et al.,
2002; Kuper-Goodman, 2003 and Kovacs, 2004).
Owing to the continuous consumer demand for fresh eggs, periodical assessment
is required to offer good and safe eggs for consumption. Therefore, this
investigation was planned to evaluate the effect of naturally contaminated
feed with aflatoxin on performance of laying hens and evidence of AFB1
residues in table eggs of laying chicken as well as the stability of AFB1
in contaminated eggs to boiling process.
Materials and Methods
Experimental design: Forty, 30-wk-old, vaccinated White Leghorn
laying hens, with a mean body weight of 2.2kg were randomly assigned into
4 experimental groups of 10 birds each. The birds were housed at average
22oC under 16 hr lighting in two batteries of eight wire cages
each having linear feed troughs and V-shaped troughs for running water.
Initially the hens were maintained for 2 weeks for adaptation and during
this period they were fed a conventional maize and soybean meal basal
diet previously screened for mycotoxins and formulated to meet or exceed
all the nutritional requirement of laying hens.
Naturally contaminated feed: Layer's commercial feed contaminated
with 100μg aflatoxin/kg feed (100ppb) was subjected for dilution with
mycotoxin-free diet to adjust the required limits of treatment for this
study. The dietary treatment was: zero (control), 25, 50 and 100 μg
total aflatoxins/kg feed (ppb). The laying hens were exposed to the naturally
contaminated feed at 32-wk-old of age and throughout the study (60 days)
this contaminated feed and clean water were provided ad libitum.
Production performance: Egg production and feed intake/bird/gram
was recorded daily. Eggs were identified for each group, dated, weighed
and stored at 3oC until analyzed for AFB1.
Statistical analysis was done according to Ingelfinger et al.,
(1994). The results (cage mean) were subjected to one day ANOVA and treatment
means were compared by the Tukey test. Statistical significant was accepted
Determination of aflatoxins in feed: Total aflatoxin was detected
in naturally contaminated layer's feed, using immunoaffinity method which
is applicable for mycotoxins that have fluorescence (Trucksess et al.,
1991). Series-4 Fluorometer (VICAM) was used in this procedure which
is summarized as follows:
Sample extraction: 50 g sample + 5 g. NaCl + 100 ml. methanol
(80%), Blended at high speed (1 min.), Filtered with fluted filter paper.
Ten ml. extract was diluted with 40 ml. distilled water and filtered with
glass microfibre filter paper.
Column chromatography: Ten ml. (= 1g. sample equivalent) of filtered
extract was passed through AflaTest-p affinity column with a rate of 1-2
drops/second. Column washed twice with 10 ml. distilled water. The toxin
was eluted with 1 ml.HPLC methanol, to which 1 ml. of freshly prepared
aflatoxin developer was added spontaneously. Reading of total aflatoxin
was obtained after 60 second as part per billion (ppb).
Analysis of AFB1 in eggs: The AFB1 concentration
was determined in the eggs collected from the hens in each experimental
group (20 eggs/treatment) on day (1-7), 10, 20, 30, 40, 50 and 60 of the
intoxication period according to the method recommended by the Association
of Official Analytical Chemists (AOAC, 1995) including modifications described
by Wolzak et al. (1985).
Each lot of eggs was pooled (egg white and yolk) to make approximately
100-g samples in a blender jar and blended for one minute at moderate
speed. Analytical homogenate samples (50 ml) were transferred to another
jar and mixed with 42 ml sodium chloride saturated solution. Samples were
placed in water bath at 60 o for 25 min. Each sample was blended with
10g citric acid and 300 ml acetone and filtered. A lead acetate solution
was added to the filtrate to remove interfering substances and then filtered
again with the aid of sodium sulphate and celite powder. The filtrate
was cleaned up by liquid-liquid extraction with hexane (discarded) and
chloroform (collected). Further purification of chloroform extracts was
performed using solid phase column from which aflatoxin B1
was eluted with chloroform-acetone (9:1) according to AOAC (1995). The
final extract was evaporated near dryness and diluted with 500 μl acetonitrite-benzene
(98:2 v/v) for final quantification of aflatoxin B1 by using
indirect Enzyme Linked Immunosorbent Assay method (ELISA) according to
the method applied by Riedel de Haen (1997). The detection limit was 0.01
Stability of aflatoxin B1 in naturally contaminated eggs Freshly
laid eggs were collected and used within 48 hours after collection. Whole
egg were manually separated from the shell and mixed to obtain a suspension.
The suspension was heated up to 100°C for 5, 10, 15 and 20 minutes
using controlled water bath Dual chamber 5/10 L Model 28 L, catalogue
No. 040661 according to Fabien and Ulrich (2007). The suspension was previously
tested for aflatoxin B1 with a mean value of 0.06 μg/kg.
The suspension was then extracted and quantified using ELISA.
Results and Discussion
Effect of aflatoxin-contaminated diet on performance of laying hens:
Table 1 and Fig.1 showed that
egg production and egg weight were not significantly affected (P >
0.05) by dietary treatment of 25, 50 and 100 aflatoxin ug/kg feed respectively.
||Effect of aflatoxin-contaminated diet fed to laying
hens for 60 days on some production parameters (mean values + SD)
|abMeans within the same row with different
letters are significantly different (P < 0.05).
||Effect of aflatoxin contaminated feed on feed intake
of laying hens
||AFB1 residue levels in laying eggs
These results agree with Oliveira et al., 2000 and Oliveira et
al., 2003, but with considering that the feed was contaminated with
AFB1. On another way, the results were disagree with Wolzak
et al., 1985; Kim et al., 2003; Rizzi et al., 2003;
Zaghini et al., 2005; Pandey and Chauhan, 2007; who found a significant
decrease in egg production and egg weights of laying hens fed on AFB1-treated
ration. Regarding feed intake/bird/day, a significant decrease in feed
intake (p<0.05) was appeared in the 2 groups fed on 50 and 100 aflatoxin
ug/kg feed and this similar to results determined by Pandey and Chauhan,
Aflatoxin residue in eggs: As shown in Table 2
and Fig. 2, aflatoxin B1 was detected in the eggs of
all groups receiving aflatoxin contaminated rations after 10 days. The
control egg pools showed no aflatoxin B1 during 60 days of the experiment.
The concentrations of aflatoxin B1 in individual egg samples ranged from
0.03 to 0.09 ug/kg, during 60 days of treatment, with mean values of 0.04,
0.05 and 0.07 ug/kg for groups fed 25, 50 and 100 ug/kg aflatoxin respectively.
Nearly similar findings were reported by Oliveira et al., 2000
and Bintvihok et al., 2002. However, previous studies reported
relative lower level of aflatoxin B1 in the examined egg samples (Fernandez
et al., 1994; Micco et al., 1999; Wolzak et al.,
The average feed to egg transmission for aflatoxin B1 after
60 day exposure to contaminated diets at levels of 25, 50 and 100 μg
aflatoxin/kg feed were 625:1; 500:1 and 1428:1 respectively. These values
are considerably below the ratio mentioned by Trucksess et al.,
1983, Wolzak et al., 1985, Park and Pohland, 1986; Oliveira et
al., 2000. The variation of aflatoxin residue in the examined eggs
confirm that only small quantities of the aflatoxins are likely to be
deposited, while the majority of aflatoxins detoxified and /or stored
in liver and other poultry tissues such as ovary, kidney, crops, breast
muscles, thigh and excreted in excreta (Trucksess et al., 1983;
Micco et al., 1988; Madden and Stahr 1995; Bintvihok et al.,
2002; Rizzi et al., 2003; Rauber et al., 2007).
So our data indicate that the carry over of aflatoxin B1 residues
is relatively most probable to occur in laying hen when the birds are
continuously exposed for long period of low level of aflatoxin in the
diet. This fact may be related to the lower capacity of laying hen in
detoxifying aflatoxin B1 (Mathes, 1984; Wolzak et al.,
1985; Hassan, 1995; Del Bianchi et al., 2005). Aflatoxins incorporated
into the feed of laying hens may cause relevant lesions in liver and in
kidneys, heart and ovaries. The ovaries showed follicular atresia which
has a detrimental effect on egg production (Hafez et al., 1982;
Del Bianchi et al., 2005; Pandey and Chauhan, 2007). Results also,
indicated that prolonged administration of aflatoxins, may cause economic
losses to egg producers, besides aflatoxins in egg even in small amounts
may cause public health problems due to its cumulative effects for egg
consumers as concluded by Chowdhury and Smith, 2004; Ogido, et al.,
Stability of AFB1 in eggs: Table 3 showed
high stability of AFB1 in contaminated eggs after boiling for
5,10,15 and 20 minutes, with a negligible mean reduction %, ranged from
0.2 -1.0%. Aflatoxin B1 was almost stable in egg for up to
20 minutes of boiling. Nearly similar findings were reported with Samarajewa,
et al. (1990) Rustom (1997) and Soliman (2002). Heat processing
is a common procedure in egg cooking, as regards the safety and nutritive
value of egg. It appears to be an effective method for controlling, or
even eliminating, contamination with Salmonella and Escherichia
coli (Tony et al., 2008). Thermal processing was not effective
for detoxification of aflatoxin B1 in egg. Therefore, avoiding
aflatoxin B1 transmission to egg appears to be the only practical
way to ensure the safety of egg for human consumption (Wood, 1989 and
Bong et al., 2007).
|| The mean aflatoxin B1 in eggs during 60 days exposure
of laying hen to different level of total aflatoxins in ration (μg/kg)
|ND: not detected (determination limit of the analytical
method: 0.01 ug/kg for aflatoxin B1)
||Stability of aflatoxin B1 in contaminated egg samples
(0.06 μg/kg) after heat treatment.
So laying hens, should not receive more than 20 ppb aflatoxin in the
diet (Ayesh et al., 1997; Dhand et al., 1998 and Bray and
The aflatoxin B1 levels used in the experimental rations in
the present investigation were in the range of natural occurrence of the
toxin in the contaminated grains and cereals in Egypt. Thus the carryover
of the aflatoxin metabolites into hens' eggs is possible. Therefore, the
control of aflatoxin B1 contamination in rations of laying
hens is recommended in order to avoid the occurrence of aflatoxin B1
in hen eggs intended for human consumption.
In conclusion, food safety has become and increasingly important issue
for all sectors of the poultry industry. For eggs, the food safety focus
has been on mycotoxins, especially aflatoxins, which cause human illness.
To promote safety, a growing number of egg producing companies are adopting
egg quality assurance programs, which stimulate actions for all aspects
of egg production to reduce the risk of egg becoming contaminated with
aflatoxins. These actions include making efforts to ensure that poultry
feeds are mycotoxin free.