Reduction of Toxic Effects of Aflatoxin B1 by Using Baker Yeast (Saccharomyces cerevisiae) in Growing Broiler Chicks Diets

The occurrence of mycotoxins in foods and feeds is a problem of major concern in all over the world. Profitability of poultry production can be greatly affected due to the frequency of feed contamination and the detrimental effects of these toxins on the performance (Hamilton, 1984). Aflatoxins, a group of closely related and biologically active mycotoxins, are produced by strains of Aspergillus flavus and Aspergillus parasiticus. They commonly occur as natural contaminant of poultry feeds (Edds & Bortell, 1983). Domestic animal species such as chickens, ducks, cattle, swine and turkeys consuming sublethal doses of aflatoxins for several days developed a toxic syndrome in which liver damage was the most significant change. According to Who (1979), the biological effects of aflatoxins could be categorized into two groups, long-term and short-term effects. Long-term effects included chronic toxicity, cancer, birth defects and genetic alterations (Hayes, 1978). Aflatoxins affected all poultry species, although they generally take relatively high levels to cause mortality, low levels can be detrimental if continually fed. Young poultry, especially ducks and turkeys, are very susceptible. As a general rule, growing poultry should not receive more than 20 µg of aflatoxin in the diet. However, feeding levels lower than 20 µg may still reduce their resistance to disease, decrease their ability to withstand stress and bruising, and generally make them unthrifty (Coelho, 1990). Numerous strategies for the detoxification-inactivation of mycotoxins contaminated feed have been proposed (e.g., physical separation, thermal inactivation, irradiation, microbial degradation and treatment with a variety of chemicals). Practical methods to detoxify aflatoxins contaminated feed on a large scale and in a cost-effective manner are not current (Norag, 1995). A new approach to the detoxifying of aflatoxin is the use of organic and inorganic adsorptive and high protein compounds in the diet of farm animals and one of these organic materials is baker yeast (Saccharomyces cerevisiae). Yeasts have been fed to animals for more than a hundred years, and commercial yeast products specifically produced for animal feeding (Reed & Nagodawýthana, 1991). Few species of yeast are commercially used. Saccharomyces cerevisiae, also known as ‘’bakers yeast’’, is one of the most widely commercialized species and one of the effective adsorbent, rich in protein (40-45%), whose biological value is high and is also rich in vitamin B complex. Several vitamins were first extracted and characterized from yeast, including biotin, niacin, pantothenic acid and thiamin (Reed & Nagodawýthhana, 1991). With the advent of using yeast cultures as growth promoters in poultry diets, several beneficial effects have been recorded.Mannan oligosaccharide (MOS) is a product designed to influence microbial ecology. It is derived from yeast cell walls and it consists primarily of phosphorylated glucomannans. Two modes of action are now recognized: (1) It binds to lectins on the cell walls of certain undesirable bacteria. These bacterial lectins normally bind to the intestinal epithelial cells and aid the bacteria in colonization of the gut. However, if the lectins are bound to MOS, they cannot bind to the epithelial cells and undesirable bacteria are eliminated from the gut lumen. (2) It enhances certain actions of the immune system. These modes of action enable MOS to help to protect animals from pathogens. On the other hand, chlorotetracycline is a therapeutic antibiotic in poultry, effective in controlling signs of synovitis (Olson, 1976), in reducing numbers of Clostridium perfringens isolated from the ceca of birds infected with Eimeria tenella (Arakawa, 1975) and in reducing shed of Salmonella typhimurium in turkey poults (Nývas, 1976). Due to AFB₁ does increase the susceptibility of chicks to infections, antibiotics would likely be given to birds experiencing an infection along with a mycotoxicosis. This study was conducted to determine the effect of dietary AFB₁ (200 ng/g) when given baker yeast and CTC in growing broiler chicks.

One hundred Ross PM 3 (5 treatments of 20 animals each) day-old male broiler chicks were obtained from a commercial hatchery, individually weighed, wing-banded and housed in experiment room and continuous fluorescent lighting. The birds were randomly assigned to the following treatment groups. Control diet without additives (treatment 1), aflatoxin B₁ AFB₁ (treatment 2), baker yeast (BY 3,44 x 10⁸ CFU/g for 37oC of 3 days) +AFB₁ (treatment 3), chlortetracycline (CTC)+AFB₁ (treatment 4) and BY+CTC+AFB₁ (treatment 5). Feed and water were provided for ad libitum consumption. Chicks were reared in individual wire cages for 37 days and fed a typical broiler diet with 22.0% crude protein and 12.8 metabolizable energy (MJ/kg) for starter phase (1-28 days) and 18.1% crude protein and 13.4 MJ/kg for growing phase (28- 37 days). Diets were designed to satisfy the recommendations of the NRC (1984). BY and CTC were calculated and 2 kg per t and 2.5 ng/g feed, respectively, were added. The toxin was measured by spectrophotometric methods and it was estimated to be 200 ng/g. Feeds were analyzed for aflatoxins by thin layer chromatography, according to Howel (1983). Feed consumption efficiency and body weight were weekly determined. Dead animals were daily recorded. All chicks were sacrificed at the end of 37 days and 3-4 mL blood sample was taken and stored frozen at (-20oC) until assayed for blood enzymes, such as Glutamic Pyruvic Transaminase (GPT), Glutamic-Oxalacetic Transaminase (GOT), Alkaline Phosphatase (ALP) and Alphapheto protein (AFP). These parameters were evaluated by a clinical laboratory by SNA-12 method (Anonymous, 1974). Liver portions were checked by routinely tissue analyses method. Pure crystalline AFB₁ was obtained from Sigma-Makor Chemical Corp., Jerusalem-Israel. AFB₁ was weighed and dissolved in warmed chloroform under a hood. Feed and water were fed ad libitum until the end of the study. Statistical variables, except for mortality and feed efficiency, that were evaluated by SAS (1986). Broiler chicks were daily examined in the morning and at evening for any sign of toxicosis and mortality. Liver portions (10 g) were collected from all sacrificed broiler chicks and frozen for analysis of mycotoxicosis and pathological examinations.

Data presented in Table 1 showed the effect of AFB₁ baker yeast, chlortetracycline and the combination of feed intake, feed efficiency and body weight, serum GOT, GPT, ALP and AFP of broilers. Feed intake and body weights of broilers receiving AFB₁ for the entire period (5 weeks) were significantly decreased in treatment 2 and increased in treatment 3 (P<.05). The birds that received AFB₁ at 200 ng/g level in treatment 2 had a significantly lower average body weight than others (P<.05). The birds fed dietary toxins with BY had a significantly higher feed intake than others (P<.05). Devedowda et al. (1997) clearly indicated the beneficial effects of viable yeast culture when supplemented to aflatoxin-contaminated diets in poultry. Besides the positive effects on body weight, feed efficiency and mortality, the most significant contribution was on the ability to modify immune response, which was reflected in the improvement in size of the bursa of Fabricius and increased levels of serum protein and albumin, thereby enhancing the levels of circulating immunoglobulins. In this research, AFB₁ resulted in a significant decrease both feed intake and body weight in treatment 2 (P<.05). Birds receiving AFB₁ 200 ng/g dietary in treatments 3, 4 and 5 had a significantly higher body weight than in treatment 1 and 2 (P<.05). Feed efficiency was better in treatment 4 than others. There were no mortalities attributed to AFB₁ in the 37 days of experiment in any of the groups. Control chickens were free of gross lesions and histologically sound. At the end of study post-mortem examinations were performed on a total of 100 birds 8 from each group were removed at 37 days of age. Changes were clearly apparent in the livers of treatments 2 and 4. Most of livers were yellow and had pethecial hemorrhages and had enlarged. Liver section from birds group receiving 200 ng/g AFB₁ treatments 2 and 1 chicks showed individual hepatocytes to be swollen and fatty degenerations. The livers were swollen and congested in treatments 2 and 4. In the AFB₁ group of broilers acute hepatitis was observed. This histologic pattern was characterized by hepatocyte injury and necrosis accompanied to varying degrees by lobular and portal inflammation. The mildest alterations consisted of scattered acidophilic bodies and foci of hepatocyte necrosis with minor inflammatory infiltration, yielding a picture of nonspecific reactive hepatitis. More substantial involvement yielded a distinctive viral hepatitis like appearance, diffuse lobular disarray with liver cell damage in the form of ballooning and acidophilic generation, spotty hepatocyte necrosis and variable inflammatory infiltration, predominantly by mononuclear cells. Canalicula cholestosis was absent in treatments 2 and 4, but not others. ALP appears to be significantly increased in the treatments 2 and 4 (P<.05).

Serum glutamic oxalacetic transaminase (GOT) had increased (P<.05) in treatments 2, 3 and 4. Serum glutamic pyruvic transaminase (GPT) significantly increased (P<.05) in treatments 2, 3 and 4. No statistical differences were found between groups for serum AFP levels in all groups (P<.05). Considerably research had demonstrated that severe chronic deficiencies of most nutrients impaired the immune response and increased susceptibility to mycotoxicosis and some infectious diseases. Severe nutrient deficiencies were particularly deleterious to the immune system when they occurred early life. This is when the primary lymphoid organs and the maturation of the immune system were developed (Cook, 1991). This may occur when the dietary levels were varied over ranges that were marginally below to well above to those required to meet typical dietary recommendations and in chronic poisoning of mycotoxicosis. There was evidence that the high carcinogenic potential of AFB₁ was related to a varied response in susceptible species (Chattopadhyay, 1985). AFB₁ in feed rations has been reported to affect livers, spleen, kidneys, bodyweight, feed intake, feed efficiency and some biochemical parameters in all species (Smýth, 1984; Hamýlton, 1984). Both GOT and GPT enzymes are indicators of hepatocellular damage (Kubena, 1990), Serum gamma glutamyl transferase activity, which is sensitive indicator of liver dysfunction, indicating liver inflammation, space space occupying lesions or obstruction of the biliary tract, was significantly increased by feeding diets containing AFB₁ to broilers Kubena (1990) and Bilgiç et al. (1998), studying broiler chicks and had pethecial hemorrhages in liver and kidneys. This was in agreement with results obtained by Çelik et al. (1999). In the present investigation, the data from the results demonstrated that dietary aflatoxin significantly lowers the feed intake and body weight of the birds. Similar results has been reported by Cova et al. (1994). Sell et al. (1998), studying ducks, had similarly reported decreased feed intake and lower body weight. Our results indicated a significant increasing of GOT, GPT and ALP. Increase in these enzyme concentration may result from many kind of degenerations of livers. However, varying amounts of fatty degeneration was detected in liver cells, confirming the observations of others (Lanza, 1980). For parameters monitored and tabulated in Table 1, abnormal values were observed for all enzymes except ALP except control group. Hayes (1978), studying swine, had similarly reported elevated GOT and GPT (Garlých, 1973). Same results has been reported by Brown (1965). Data of this research showed that AFB₁ levels as low as 200 ng/g could affect liver enzymes of broiler chicks. Giambrone (1985) showed that the development of acquired immunity in turkeys and broilers was significantly when given 200 ng/g of pure AFB₁ capsules. According to Wogan (1974), dietary aflatoxin levels 100 ng/g induced liver carcinoma at an incidence greater than 50%, when feeding was continued up to 80 weeks. The interrelationship between the immune system and carcinogenesis has been reported by Sun (1984). These data, as well as previously reported data by Huff & Doerr (1981). No carcinoma was found in AFB₁ treatment (no additives) even at 200 ng/g levels during 5 weeks in this research. Our results indicated the hemorrhagic anemia syndrome caused by AFB₁ was characterized by pethecial and larger hemorrhages into the musculature and internal organs in treatment 2 and 4 of animals. This is in agreement with results obtained Muller (1970). Lipids in liver increased in broiler chicks fed diet with AFB₁ (Smýth, 1970). Similar results were found in this research in treatments 1 and 2. Aflatoxin in feed rations has been reported to affect body weight by Doerr (1983). Our data show similar results in this research. This is in agreement with results obtained by Sahoo (1993) and Adav (1997) & Sell (1998). Miller (1984) has been reported that chickens suffering from aflatoxicosis have been shown to be hypoproteinemic. The use of a protein-sparing antibiotic that also enhances intestinal absorption of essential nutrients, in this research was found to improve feed conversion in diets containing antibiotic and baker yeast.

The findings of this research suggested that baker yeast (2, 0%) could partly counteract some of the toxic effects of AFB₁ in growing chicks. In conclusion, further investigations may be necessary in long run and other organic-inorganic compounds against the effects of aflatoxins. The use of viable yeast cultures in poultry diets has given promising results and research has taken one step further by identifying the yeast cell wall as the active component which aids in counteracting mycotoxicosis. Mannanoligosaccharides provide new insights into counteracting several pathogens and toxins besides their major impact on modifying the immune response.

We wish to thank Prof. Dr. F. DORAN, for pathological assistance.

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