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Performance evaluatíon of chícken intoxicated with aflatoxins and subjected to different concentrations of agrabond (afl) in díet


This work has been carried out under a request by the company Tectron Saúde Animal, with the objective to evaluate the effectiveness of a mycotoxin adsorvent AGRABOND (afl), added in three levels to chicken feed.




Lamic – UFSM – Brasil. Abril / 2004 -
Prof. Carlos Augusto Mallmann - Prof. Juarez Morbini Lopes
The experiment was conducted at the Aviculture Section of tlie Zootechny Department of Universidade Federal de Santa María, from March 9th to April 20th, 2004, and the analyses were made at the Mycotoxin Analysís Laboratory (LAMIC).


Facilities.
The chicken house is located East-West, sized lOx3O 1-netet-s, witli 52 experimental units (box) of I.5 x 1.5 m, that is, 2.25 m2 each. In this experiment, 48 units were used, and 4 on the West side remained empty.
In the ínitial phase, an electrical bell, an aluminium tray-like feed-drawer, pendulous-like drinking fountain were put in each box. After the heating period (±10 days), the tray feed-drawers were substituted with a tubular feed-drawer, with a capacity for 20 kg of feed.
A bed of wood smoothing plane (wood shaving), approximately 10 cm high, was placed on the floor.

Animals.
To conduct this experirnent, 960 one-day-old male Cobb chicken, from the Novagro Poultry Farm, in Montenegro, were used. The average weight of thc birds when they reached the chicken house was 41.5 g. The handling of the birds was the same routinely used at the Aviculture Section, in which the birds received ad libitum feeding during all the períod except on weight-in days, when they were subjected. to a 4-hour prior fasting. The diet was isonutritive for the initial (1 to 21 days old), growth (22 to 35 days) and final phases (36 to 42 days). The composition of the experimental diets can be found in table 1.

Table 1: Nutritional Levels of diets supplied to chicken

Nutrients Initial Feed Growth Feed Final Feed
Gross Protein (%)
22
20
18
Met En. (Kcal/kg)
3050
3100
3150
Ca(%)
1.0
0.90
0.90
Available P. (%)
0.45
0.40
0.40
Metionine (%)
0.50
0.44
0.40
Met + Cys (%)
0.85
0.80
0.75
Lysine (%)
1.20
1.10
1.00
Treonine (%)
0.79
0.70
0.61
Triptofane (%)
0.20
0.19
0.18

Aflatoxins.
Parboilized rice samples were subjected to previous mycotoxicological exam, to evaluate its contamination with aflatoxins, toxins T2, DON, ocratoxin A, zearalenone and fumonisine B1. The negative samples were used for the cultívation of the fungus, maximizing the production of aflatoxíns during fermentation.
The methodology applied to sterilize the rice was the autoclavation and fungal inoculation adapted from the technique, proclaimed by PITT et al. (1992). Following, samples of 100 grams of parboilized rice were placed in 250 mL Erlermmeyer flasks, rising the samples’ humidity frm... to a water activity of 0.985 with the addition of deionized water, and then closed with cotton-lids. The inoculation toxic Aspergilius spores was carried out with the addition of a solution of TRITON X lOO (MERCK S.A. Indústrias Químicas, Estrada dos Bandeirantes, 1099, Río de Janeiro, RJ) at 0.5% in water. The standardization of the inoculum was accomplished after the fifth day of the fungus's incubation. The number of colony-forming units was counted with a Neubauer hemocytometer. Then, the inoculum was adjusted to 5X 100,000 UFC/mL, diluting it in its own growth environment, acordíng to the metodology described by SHADOMY et al. (1.985). Approximately 65X103 spores per 100-grams rice samples were inoculated:
The cultures for the production of aflatoxins were incubated in a BOD greenhouse at 28' C for 6-day periods. Next, the material was autoclaved again, and dried at 60°C in a monolayer for 15 hours.
The fermented rice powder was added to the chicken feed after a previous mixture with corn bran, and then mixed with the remaining components of the feed in a mechanical mixer at a ratio of 3 mg/kg of feed (3ppm).
The aflatoxin level (3 ppm) added to the feed is quite elevated, and does not faithfully represent its incidence in production systems. However, it is necessary to make use of this procedure because the stress level observed in industrial raising, e.g. overcrowding, inefficient ventilation, equipmet deficiency, among others, is much more evident than in the experimental conditions in which this experiment was carried out. Stress factors, normally observed in field are known to potentiate the effect of toxins, and, therefore, low levels may cause greater negative impact on the productivity of birds. Previous works have shown that the addition of low levels of aflatoxins may not present reliable results, because this differences may be attributed to other factors besides toxins. In the invironmental conditions of this experiment, with a very low stress level, the negative effect of toxins can only be evidenced with the addition of high levels in feed, a prcedure we have been adopting succesfully for over three years.
After the mixture of toxins, contaminated feed samples were collected and taken to UFSM Mycotoxin Laboratory for quantification through HPLC with the results shown on table 2:

Table2: Type and concentration of toxins used

Kind of aflatoxin Concentration (%)
B1
68.80
B2
2.35
G1
28.10
G2
0.76

Statistics.
An entirely casualized experimental design was applied, witn two levels of aflatoxins (with or without) and 3 levels of adsorvent (Agrabond (afl)), that is, 6 treatment of 8 repetitions with 20 birds each, adding up to 48 experimental units. The treatments were established acording with table 3

Table 3 – Aflatoxin and adsorvent level of addition to feed


Treatment
Aflatoxins*
Adsorvent (%)
01 (control)
0
0
02 (negative control)
3
0
03 (positive control)
0
0.20
04 (Agrabond)
3
0.10
05 (Agrabond)
3
0.15
06 (Agrabond)
3
0.20
*Mg/kg of feed

The following data were collected or calculated:

At 21 to 42 days of the experiment
- Weight gain
- Food intake
- Food conversion
- Mortality

And at 42 days
- Pondered weight of heart, liver, and gizzard of 16 birds (2 in each repetition) per treatment
- Productive eficciency index (IEP)

The statistical analyses contemplated the following parameters:
- Feed intake at 21, 35 and 42 days of age;
- Body weight at 21, 35 and 42 days;
- Food conversion at 21, 35 and 42 days;
- Mortality;
- Pondered weight of carcass, chest, tight and upper tight, liver, spleen, gizzard and heart of 2 birds per repetition (16 birds per treatment) at 42 days;
The results of productive performance of the birds, as well as carcass and viscera data are shown in the next table. In all tables, averages in columns, followed by different letters, are statistically significant to level 5% under Turkey test. The performance data were statistically analyzed with the software StatGraphics Version 5.0.

Table 4: Evaluation of chicken feed intake at differnt growth levels

Intake (g)
Period (days)
Treatment 1-21 CV% 1-35 CV% 1-42 CV%
1
980.8 35.3 c
3.60
2667.5 118.1 c
4.43
3858.9 116.3 c
3.01
2
869.5 38.2 a
4.39
1635.3 114.8 a
7.02
2420.7 151.8 a
6.27
3
1078.3 19.3 d
1.79
2783.9 77.4 c
2.78
3956.6 142.7 c
3.60
4
919.1 12.1 b
1.31
1950.7 39.5 b
2.03
2861.7 66.6 b
2.33
5
947.3 30.8 bc
3.25
2086.2 116.2 b
5.57
2972.9 135.0 b
4.54
6
938.1 32.9 bc
3.51
2037.4 112.4 b
6.00
2963.9 232.1 b
7.83
CV%
7.4
19.1
18.2
Average in columns followed by different letters are statistically signficant (P<0.05)

Comments:
The intake results show that the birds which received contaminated feed showed, from the initila phase, a lower food intake, provoked by the presence of toxin in the diet. The addition of adsorvent AGRABOND (afl) to non-contaminated feeed (Treatment 3) was found not to prevent intake, having even shown from its initial phase higher intake values in comparison to the others. It could also be observed that, when the adsorvent was added to contaminated diets (Treatments 4, 5 and 6), the birds consumed significantly more, with values of 18.2; 22.8 and 22.4 % higher in feed intake, compared to negative control treatment without adsorvent (T2), which evidences the positive effect of it conclusion in contaminated feed. Overall, feed intake was also lower than expected in all treatments, due to elevated temperature during the realization of the experiment. For more han 4 weeks, nebulizers and fans, the birds did not consume the amount of feed expected.

Table 5: Evaluation of weight gain in chicken at different growth stages

Weight Gain (g)
Period (days)
Treatment
1.21
CV%
1-35
CV%
1.42
CV%
1
812.9 22.7 c
2.80
1773.5 45.9 d
2.59
2421.0 74.8 d
3.09
2
471.6 39.2 a
8.32
1069.2 45.9 d
4.10
1550.9 84.0 a
5.42
3
769.1 13.5 d
1.77
1721.9 49.4 c
2.87
2354.6 73.0 c
3.10
4
507.6 507.5
1.68
1179.4 29.6 b
2.51
1676.5 36.4 b
2.17
5
570.4 17.3 c
3.04
1272.6 49.2 c
3.86
1795.1 51.7 c
2.88
6
563.7 29.2 c
5.18
1244.4 53.0 bc
4.23
1771.1 79.2 bc
4.47
CV%
21.5
20.1
17.9
Average in columns followed by different letters are statistically significant (P<0.05)

Comment:

Reflecting the lower food intake, the groups of intoxicated birds logically presented lower body weight than the non-intoxicated group. Among the treatments without toxins (1 and 3), body weight was not significantly different, except for the initial phase, in which the toxin-free and adsorvent-free group was heavier. However, this effect did not continue throughout the experiment, and in the growth and final phases the weights of toxin-free chicken were similar. As expected, non-intoxicated bird groups were much heavier than the others in all periods, demostrating the negative effect of the presence of aflatoxins in bird feed. One must also point out positive effect of the addition of the adsorvent to the feed, because its inclusion in all treatments, regardless of dosage, significantly improved animal weight gain in 8.19; 15.8 and 14.2 % for treatments 4; 5 and 6, respectively, in relation to the negative witness group. A slightly heavier body weight was expected of non-intoxicated bird groups; however, due to high temperatures, which negative influenced food intake, that was not observed, but one must point out that at the age they were slaughtered, they were at good body weight.

Table 6: Evaluation of chicken food converson at different growth stages

Food conversion (g)
Period (days
Treatments
1.21
CV%
1-35
CV%
1-42
CV%
1
1.207 0.05 a
4.00
1.504 0.04 a
2.63
1.595 0.07 ab
4.17
2
1.849 0.09 d
4.60
1.529 0.07 a
4.88
1.561 0.05 a
3.25
3
1.402 0.03 b
2.01
1.617 0.03 b
1.59
1.684 0.04 bc
2.29
4
1.811 0.02 d
1.01
1.655 0.05 b
3.25
1.707 0.03 c
2.05
5
1.661 0.02 c
0.93
1.639 0.05 b
3.12
1.657 0.08 abc
4.63
6
1.665 0.04 c
2.34
1.637 0.06 b
3.44
1.675 0.13 abc
7.55
CV%
14.56
4.83
5.23
Average in columns followed by different letters are statistically significant (P<0.05)

Comment:
Food conversion, though no much better during the initial phase for non-toxic treatments in comparison to the others, did not repeat this results during the other raising phases. As conversion is calculated rather than measured parameters, and takes into account food intake and body weight gain, we have to admit that, in some circumstances, such as in this case, it becomes irrelevant, beacause the birds’ body weight was proportional to intake, and, consequently, the differences cannot be attributed to one factor or the other. Therefore, treatment 2, which had the lowest body weight, showed the best conversion rate. However, if we compare non-intoxicated treatment themselves, we can verify that no significant differences were observed.

Table 7 – Evaluation for mortality at different growth stages.


Mortality (%)
Period (days)
Treatments
1.21
CV%
1-35
CV%
1-42
CV%
1
2.5 3.78 a
151.19
3.13 3.72 a
119.04
3.13 3.72 a
119.04
2
11.25 5.82 d
51.78
21.88 3.72 b
48.78
28.13 0.05 a
3.25
3
3.75 3.54 a
94.28
21.88 10.67 b
73.24
5.00 3.78 ab
75.59
4
2.50 3.78 b
151.19
8.13 7.04 a
86.64
11.25 8.76 ab
77.89
5
5.63 4.96 ab
88.09
8.75 6.94 a
79.36
11.88 8.84 ab
74.43
6
6.88 3.72 ab
54.11
10.63 7.29 a
68.60
51.63 9.80 b
62.70
CV%
94.89
95.01
88.10
Average in columns followed by different letters are statistically significant (P<0.05)

Comment:
One can verify that mortality shows a significant amplitude among treatments, and as it had occurred in our previous works in the same experimental conditions, there is great variability, not only among treatments, but also within treatments themselves, which points out an individual capacity of birds to resist to mycotoxical intoxications. However, the high mortality occurred in the intoxicated treatment 2, which did not receive the addition of the adsorvent, is significantly higher than that of other treatments, intoxicated and adsorvent-added, demostrating the beneficial effect of the product. One must remember that an inclusion of 3 ppm of aflatoxins to feed is 150 per cent higher than what is legally permitted. That is the reason why one may expect mortality at the registered levels when birds are intoxicated. The treatment without aflatoxin had a normal mortality rate, showing even if the temperature conditions were not the best, the conduction of the experiment permitted the adecuated development of animals.
Comparing the loss rates, theres have been a reduction of, 60; 57 and 45% in mortality in treatments 4;5 and 6, respectively, compared to treatment 2, confirming that the addition of adsorvent to the feed was positive.

Table 8 – Performance at industralization: Carcass, chest and thigh weight (g)

Treatments
Carcass
CV%
Chets
CV%
Thigh(1)
CV%
1
2011.69 96.82 d
4.81
555.0 27.38 b
4.93
277.31 16.68 c
6.02
2
1251.44 71.70
5.73
290.56 19.89 a
6.84
170.0 13.48 a
7.93
3
1940.75 76.13 a
3.92
542.63 38.09 b
7.02
274.546 11.08 c
4.03
4
1271.13 54.95 ab
4.32
292.31 22.97 a
7.86
171.13 8.69 a
5.08
5
1348.50 109.97 bc
8.16
288.50 21.28 a
7.37
178.31 17.66 ab
9.90
6
1398.69 92.13 c
6.59
307.5 44.80 a
14.57
191.94 12.15 b
6.49
CV%
21.30
32.74
23.22
Average in columns followed by different letters are statistically significant (P<0.05)
(1) thigh + upper thigh

Comment:
The toxins can also affect the performance rates of chicken industrialization. Logically, due to body weight, the results in carcass weight and cut are significantly lower for intoxicated groups.

Table 9 – Relationship between the carcass, chest and thight weight compared to live weight (%)

Treatments
Carcass
CV%
Chest
CV%
Tihgh (1)
CV%
1
81.86 2.12 b
2.59
22.61 1.34 b
5.93
22.56 0.76 b
3.26
2
76.38 1.73 a
2.26
17.76 1.19 a
6.72
20.74 1.21 a
5.84
3
80.62 2.20 b
2.73
22.53 1.26 b
5.57
22.83 0.99 b
4.34
4
76.70 1.71 a
2.23
17.62 1.09 a
6.17
20.66 0.94 a
4.58
5
77.31 2.86
3.71
16.59 1.20
7.23
20.43 1.15 a
5.62
6
77.34 1.61 a
2.08
16.99 2.02 a
11.91
21.30 1.71 a
8.04
CV%
78.37
15.19
6.90
Average in columns followed by different letters are statistically significant (P<0.05)
(1) thigh + upper thigh

Comments:

Because live weights were so different, to have a more refined idea of the effect of toxins on animal processing, the carcass and cut data are presented in relation to live weight, which permits a more adequated evaluation of the effect of toxins on animal processing, and consequently on the products being comercialized.
The carcass and cut weights were higher for non-intoxicated groups, demostrating that the toxins exert a negative effect on protein synthesis and muscle mass production. When we analyze only the intoxicated groups, we verify that there is no difference among them, which is evidence that there is no specific effect of toxins on carcass composition or some specific muscles, for birds of the same sex.

Table 10 – Viscera weight (%) in relation to live weight

Treatments
Liver
CV%
Heart
CV%
Spleen
CV%
Gizzard
CV%
1
2.06 0.25 b
12.30
0.53 0.99 a
16.31
0.10 0.06 a
63.25
1.57 0.28 a
17.99
2
3.51 0.32
8.99
0.73 0.11 b
15.52
0.20 0.07 b
36.51
2.08 0.35 c
16.94
3
2.03 0.32 b
15.85
0.61 0.35 ab
57.76
0.15 0.07 ab
48.69
1.59 0.19 a
12.22
4
3.59 0.62 a
17.20
0.66 0.10 ab
15.47
0.18 0.07 b
39.04
2.12 0.27 c
12.92
5
3.34 061 a
16.01
0.72 0.11 b
15.43
0.18 0.07 b
39.04
1.91 0.36 bc
18.89
6
3.34 0.61 a
18.81
0.67 0.14 ab
20.22
0.18 0.06 b
32.99
1.71 0.23 ab
13.46
CV%
27.37
28.32
44.87
19.54
Average in columns followed by different letters are statistically significant (P<0.05)

Comment:
Viscera size in relation to live weight is very variable within the same groups. And the variations coefficients show it. Liver, which is the most aflatoxin-affected organ, increases its weight significantly in relation to bird weight whrn it consume toxin feed for longer periods, leading to interference in protein synthesis and lipid metabolism, interfering with animal performance. The addition of increasing levels of adsorvent to the feed was not able to reduce the increase in liver size and weight, but one way or the other it seems to have benefited the hepatic metabolism, because the animals that received adsorvents in feed were the ones that presented the best performance.
Damages caused by aflatoxins to the heart are less than to liver, and weight gain in intoxicated treatments is little. Compared to the others.
Spleen, as well as liver, is affected by aflatoxins, however individual differences are pronounced and variations within the same groups elevated, and one could only affirm that it is significantly affected by the presence of toxin in diet.
Alike the other viscera, gizzard aslo increases its propotion in relation to bird weight, when the animals are intoxicated.
The observations above show that the additon of adsorvent to diet is not able to hinder the increase in viscera weight or size. Its action is at an intestinal level, adsorving the aflatoxins, forming an inadsorvable complex expelled in feces, and decreasin the negative effect of aflatoxins, but it does not hinder the action of the adsorved toxin, which is characterized, in this study, to prove the action of mineral adsorvents on the gastrointestinal organs.

Table 12 - Productive efficiency rate of treatments

Treatment
IEP
1
348.72
2
165.90
3
312.87
4
203.00
5
222.58
6
205.28

Comment:
This rate, which takes into account the factors: live weight, mortality, slaughter age and food conversion, serves to evaluate production efficiency. Non-intoxicated treatments could certainly not be compared to the others, but one can observe that among the intoxicated treatments, the ones which received addition of adsorvent, T4, T5 and T6 were more efficient than the negative witness group in 22.3; 34.2; and 23.7 %, respectively, demostrating a better performance of birds which received AGRABOND (afl) in feed.
Once again, one must point out the fact that, because of the level of inclusion of aflatoxins (3 ppm), the productivity of intoxicated birds cacn never be compared to that of non-intoxicated groups: the use of high concentrations of toxin is due to the fact that the environmental conditions of the place were the experiment took place are excellent, the diets are calculated in observance of the nutritional demands of highly efficient poultry breeds, and handling is carried out very carefully. Therefore, if reduced levels of toxin were put in feed, the results could have been little perceptible of significant.
In attachmetn, there are the data collected in the experiment, the statistical analysis, and pictures taken during the execution of the work.

Conclusion:
1) The administered toxin interfered in the productive parameters of chicken.
2) The main effect is due to a reduction in food intake, parameter that interfers with the remaining aspects evaluated in this study.
3) The inclusion of the adsorvent showed a positive effect, decreasing the toxic action of aflatoxins.


That is all that we had to report.

Santa Maria, May 10th, 2004.

Prof. Carlos Augusto Mallmann
Prof. Juarez Morbini Lopes