Effects of Addition of a Mycotoxin Detoxifier in Poultry Feed Containing Different Levels of Aflatoxins on the Performance of Broilers

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INTRODUCTION
Aflatoxins are the most important mycotoxins constituting a considerable health hazard for commercial poultry.Cases of acute aflatoxin poisoning have been reported worldwide (Heathcote and Hibbert, 1978).However, these cases represent a small percentage of losses caused by aflatoxins since majority of cases are sub-clinical.These sub-clinical levels of aflatoxins not only reduce production performance of birds (Howorth and Wyatt, 1976;Verma et al., 2003) but also cause immunosuppression resulting in susceptibility to infections (Sims et al., 1970;Sohane and Chaturvedi, 2001).Aflatoxins have been reported from various animal feedstuffs in Pakistan (Afzal et al., 1979;Chaudhry et al., 1981;Bhatti et al., 2001) and their involvement in disease and mortality among commercial poultry has also been documented (Siddique et al., 1987;Sabri et al., 1989;Anjum, 1990;Azim et al., 1990).
The best strategy to avoid the build-up of aflatoxins in feedstuffs is to avoid contamination with aflatoxin producing fungi both in the field and during harvesting and storage (Heathcote and Hibbert, 1978).However, it is a difficult proposition particularly in developing countries where harvesting and storage conditions are far below the required standards.There are many methods that can be used to neutralize ill-effects of aflatoxins in the poultry feeds.Addition of organic acids on inert support (e.g.vermiculite, zeolite, etc.) in feed results in fungistatic actions and thus reducing the chances of aflatoxin production (Heathcote and Hibbert, 1978;Piva et al., 2000).Aflatoxins in feedstuffs can be degraded and destroyed by the use of oxidizing agents, chlorinating agents, organic and inorganic acids and alkalis (Heathcote and Hibbert, 1978;Sohane and Chaturvedi, 2001).However, most of these chemicals are unsafe to use and impractical.Detoxification of aflatoxins in the feed has been achieved commercially by ammonia treatment (Neal et al., 2001).However, this proposition is not suitable for the developing countries due to technology adoption problems and higher initial cost.Absorption of aflatoxins from feed can be effectively achieved by substances like bentonite, zeolite, hydrated calcium sodium aluminosilicate, kaolin, etc. or synthetic ion exchange resins (Piva et al., 2000;Rosa et al., 2001;Tozaki, 2001) but these substances show poor efficacy when contamination is large and also absorb vitamins and other nutrients from the feed resulting in poor feed conversion ratio.Enzymes and microorganisms can also degrade aflatoxins (Liu et al., 2001;Suman et al., 2001;Raju and Devegowda, 2002;Byun and Yoon, 2003) but cost is prohibitive and treatment methods have still not been developed.
Mycotox  is a medicated premix for the treatment of mycotoxicosis in poultry.It contains oxyquinol, dichlorothymol and micronized yeast.All these ingredients are known to have antifungal and antimycotoxin properties.The present study was designed to determine the detoxifying effect of the mycotox on aflatoxins in poultry feed and its effects on the productive performance of the commercial broilers.

Experimental birds
Three hundred hatched broiler chicks were purchased from a commercial hatchery (Islamabad Poultry Breeders, Rawalpindi).These birds were randomly divided into 6 groups of 50 each and kept in separate pens.The birds were reared under electric brooders.The birds were kept on floor and given starter feed for first 4 weeks and finisher ration for remaining 3 weeks.Feed and water were offered ad lib throughout 7 weeks of study period.

Poultry feeds
Three poultry feeds were used in the experiment.All feeds were isonitrogenous (starter CP 21%, finisher CP 18.5%) and isocaloric (starter ME 2,900 kcal/kg, finisher ME 3,000 kcal/kg) and only varied in the aflatoxin contents.Aflatoxin contents in the feed were varied by incorporating different batches of corn having different levels of aflatoxins.Feed composition is shown in the Table 1.Feeds contained different levels of aflatoxins and were classified as normal feed (containing 28 ppb aflatoxin), contaminated feed (78 ppb of aflatoxins) and highly contaminated feed (containing 170 ppb aflatoxins).

Experimental protocol
Chickens of various groups were given the following feeding treatments: Group 1: Chicks were given normal feed throughout study period.Group 2: Chicks were given alfatoxin contaminated feed throughout 7 weeks study period.Group 3: Chicks were given normal feed for first 2 weeks, highly contaminated feed for next 2 weeks and then normal feed for the rest of study period.Group 4: Chicks were given mycotox  (1 kg/ton) mixed contaminated feed throughout 7 weeks of study period.Group 5: Chicks were given normal feed for first 2 weeks, highly contaminated feed for next 2 weeks and then mycotox mixed (3 kg/ton) normal feed for remaining 3 weeks.Group 6: Chicks were given normal feed for first 2 weeks, highly contaminated feed for next 2 weeks followed by mycotox mixed (5 kg/ton) normal feed for remaining 3 weeks.Vaccination schedule was uniform for all groups and included Newcastle disease (ND) vaccine on day 5, infectious bursal disease vaccine on day 12, hydropericardium syndrome vaccine on day 18 and ND on day 21 of age.
Randomly selected 10 birds from each group were  Age (weeks) Figure 1.Weight of chicken given various levels of aflatoxins and mycotoxin detoxifier.G-1 represents chicken given normal feed throughout 7 weeks; G-2 represents chicken given contaminated feed throughout 7 weeks; G-3 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7; G-4 represents chicken given contaminated feed added with Mycotox (1 kg/ton) throughout 7 weeks; G-5 represents chicken given normal feed for first 2 weeks, highly contaminated feed for week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (3 kg/ton), and G-6 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (5 kg/ton).
weighed weekly.At the end of the experiment all birds were weighed.Blood samples were collected from 7 birds from each group at the age of day 5, 15 and 28 of age for determining the antibody titres against ND.Serological titres against ND were determined using a commercial ELISA Kit (IDDEX) and titers were expressed as geomeans.
The data were analyzed statistically using computer package SPSS.

RESULTS
Body weight of the birds was measured weekly and is shown in Figure 1.Chickens given contaminated feed throughout the experiment (group 2) had the lowest weight gain followed by chicken belonging to group 3 which were given highly contaminated feed during 3 rd and 4 th week.Treatment of contaminated feed with mycotoxin detoxifier resulted in the improvement of weight gain.Body weight of chicken given detoxifier mixed contaminated feed (group 4) for 7 weeks was similar to chicks given normal feed (group 1).Higher levels of mycotoxin detoxifier (5 kg/ton) proved better than lower level (3 kg/ton) in alleviating the effects of highly contaminated feed.
FCR was similar in groups 1, 4 and 6 showing that mycotoxin detoxifier did not increase the FCR (Table 2).FCR was higher in groups given contaminated feeds but no detoxifier when compared with group given normal feed.Dressing percentage (without skin and giblets) did not differ among various groups and ranged from 56.73 to 58.77%.
The broiler chicks had good maternal antibody titres as on day 5 geomean ELISA titre against ND was 6,491.At 15 days of age, aflatoxins at lower level did not seem to depress immune response to ND and birds of all six groups had good immune response and geomean ELISA titres varied from 6,235 in group 6 to 7,484 in group 5.However, at 28 days of age antibody levels against ND were significantly lower in chicken given aflatoxin contaminated feed than those given detoxifier treated contaminated feed or normal feed (Figure 2).Detoxifier given in higher dose (5 kg/ton) after feeding highly contaminated feed for 2 weeks (group 6) resulted in better humoral antibody titre than those given feed containing medium level (3 kg/ton) of detoxifier (group 5).
Mortality was highest in group 2 which was given contaminated feed throughout 7 weeks and it reached 52% by the end of the experiment (Figure 3).In fact in this group an outbreak of infectious bursal disease was seen in 4 th week of age which resulted in heavy mortality in 5 th week.This outbreak spread to all other groups also.Significant mortality was also recorded in group 3 which was given highly contaminated feed for 2 weeks.Mortality was lowest in group given normal feed.Mortality did not differ Figure 2. Antibody titres of chicken given various levels of aflatoxins and mycotoxin detoxifier against Newcastle Disease virus.The figure shows antibody titres on day 15 and day 28 of the age of chicken.Each bar shows geomean titre of 7 chicken.G-1 represents chicken given normal feed throughout 7 weeks; G-2 represents chicken given contaminated feed throughout 7 weeks; G-3 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7; G-4 represents chicken given contaminated feed added with Mycotox (1 kg/ton) throughout 7 weeks; G-5 represents chicken given normal feed for first 2 weeks, highly contaminated feed for week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (3 kg/ton), and G-6 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (5 kg/ton).
significantly between groups given normal feed and that given detoxifier mixed contaminated feed throughout 7 weeks.
The study shows that mycotoxin detoxifier containing oxyquinol, dichloro-thymol and micronized yeast can effectively neutralize ill effects of aflatoxins (the most important mycotoxin) in poultry feed.This detoxifier can also be used to off-set the ill effects of poultry feed containing higher levels of aflatoxins.

DISCUSSION
Mycotoxins in general, and aflatoxins in particular are of great economic concern in commercial poultry production in all developing countries.This is particularly important in Pakistan where mycotoxicosis has been ranked as the third most important disease factor in broiler poultry (Bhatti, 1989;Sabri et al., 1989;Anjum, 1990).The problem has also been reported in layers resulting in up to 37.8 per cent reduction in egg production (Siddique et al., 1987).The aflatoxins have been reported from almost all commonly used poultry feedstuffs and commercial poultry rations with wide range of concentration (Afzal et al., 1979;Bhatti et al., 2001).The aflatoxins have also been detected in different organs of poultry (Azim et al., 1990;Begum et al., 2001).
Although a large number of methods have been researched to detoxify aflatoxins in poultry feedstuffs (Heathcote and Hibbert, 1978;Piva et al., 2000;Sohane and Chaturvedi, 2001), a few have found commercial application and those too only in industrialized countries.This study evaluates the effects of a mycotoxin detoxifier on the performance of broiler chicken.The study results showed that this detoxifier can alleviate the negative effects of aflatoxins in broilers as indicated by improved weight gain, better humoral immune titres and lower mortality.This detoxifier has antifungal activity that can check further production of aflatoxins but exact mechanism of action for detoxification of already present aflatoxins has still not been elucidated.
The study indicates that the mycotoxin detoxifier containing oxyquinol, dichloro-thymol and micronized yeast can be used by the poultry farmers or the feed manufacturer for improving the quality of aflatoxin containing feed without any ill-effects.The graph shows cummulative mortality for 7 weeks.G-1 represents chicken given normal feed throughout 7 weeks; G-2 represents chicken given contaminated feed throughout 7 weeks; G-3 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7; G-4 represents chicken given contaminated feed added with Mycotox (1 kg/ton) throughout 7 weeks; G-5 represents chicken given normal feed for first 2 weeks, highly contaminated feed for week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (3 kg/ton), and G-6 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (5 kg/ton).

Figure 3 .
Figure3.Mortality in chicken given various levels of aflatoxins and mycotoxin detoxifier.The graph shows cummulative mortality for 7 weeks.G-1 represents chicken given normal feed throughout 7 weeks; G-2 represents chicken given contaminated feed throughout 7 weeks; G-3 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7; G-4 represents chicken given contaminated feed added with Mycotox (1 kg/ton) throughout 7 weeks; G-5 represents chicken given normal feed for first 2 weeks, highly contaminated feed for week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (3 kg/ton), and G-6 represents chicken given normal feed for first 2 weeks, highly contaminated feed during week 3 and 4 followed by normal feed for week 5, 6 and 7 added with Mycotox (5 kg/ton).

Table 1 .
Composition of broiler starter and finisher feed used in Note: Different aflatoxin levels in diets were achieved by including batches of contaminated corn having various levels of aflatoxins

Table 2 .
Body weight, feed conversion ratio (FCR) and dressing percentage of broilers given various treatments of aflatoxins and mycotox Different letters in the column show that figures are statistically different (p<0.01).* Dressing weight was taken after removing the skin and without giblets i.e. gizzard, liver, spleen and heart.