The effect of concomitant use of ethanolic mixture extractions of Thyme and Oregano on performance and morphology of gastrointestinal tract in broilers fed contaminated feed with Aflatoxin B1

Aflatoxins are a class of secondary metabolites of fungi, which are present in the form of B (B1 and B2), G (G1 and G2), and M (M1 and M2) and are distinguished from each other through chromatography and fluorescent light. Aflatoxin B1 is more abundant in the feedstuff and more toxic than all other types of toxins (Manafi et al. 2011). Aflatoxins are the result of the metabolic activity of Aspergillus fungi, and in particular Aspergillus flavus, which can cause food contamination of living creatures. Aflatoxicosis has caused severe economic losses in the poultry industry and has had a negative impact on duck, broilers, laying hens, turkeys and quails (Deabes et al. 2011). The sensitivity of chickens to aflatoxin negative effects depends on a wide range of factors such as breed, aflatoxin concentration, age, nutritional status, as well as the capacity of liver microsomal enzymes to decontaminate aflatoxin. Mycotoxins normally affect intestinal secretions. The use of aflatoxin is proven to reduce the production of pancreatic secretions and changes in the intestinal morphology (the depth of the intestinal crypts), and the special activity of disaccharide and maltase in broiler’s intestine (Applegate et al. 2009). Increasing free radical formation or reducing the level of antioxidants in the body through aflatoxicosis leads to oxidative stress, which can lead to tissue destruction through the physical, chemical and physiological conditions (Eralsan et al. 2005). Reducing the toxicity of aflatoxin in broiler chickens as an important source of animal-based proteins for human beings by utilizing a combination of plant derivatives is considered vital. It has been suggested that the antioxidant properties of essential oils and plant extracts due to the presence of phenolic groups as a hydrogen supplier to peroxide radicals produced during oxidative stress, which will delay or deny peroxide formation (Roquia 2012). The current experiment is designed in order to study effects of ethanolic extractions of thyme and oregano on performance, blood biochemical parameters and small intestinal properties of broilers fed diets contaminated with aflatoxin B1. The total number of 300 Ross 308 chicks (mixed sex) were used in completely randomized design with 4 treatments and 5 replicates with 15 birds in each replicate. Treatments consisted of: 1- basal diet without aflatoxin B1 (control); 2- Basal diet with Aflatoxin group (basal diet + 600 ppb aflatoxin B1); 3- Basal diet with mixture of ethanolic extraction of thyme and oregano (either 500 ppm) and 4- Basal diet with Aflatoxin + mixture of ethanolic extraction of thyme and oregano (either 500 ppm). In the current study, performance including mortality, feed intake, and weight of birds were recorded weekly till the end of the rearing period. At the end of production period, from each experimental unit, 2 birds were selected and after killing by humanized method, sampling for intestinal morphology from the ileum part (with a distance of 2 cm from Meckl's appendix) with the length of 5 cm was done. Also, in order to check the blood parameters from each experimental group, two birds were randomly selected and blood samples were collected. Then, blood biochemical parameters included liver enzymes (ALT, AST and ALP), triglycerides, cholesterol, LDL and HDL were measured using the Pars Azmon Company kits and the Autoanalyzer apparatus (US, Technicon RA-1000). Aflatoxin B1 was purchased from Sigma Aldrich Company and was prepared according to the manufacturer's instructions for the preparation of the poison. Six mg of poison was mixed with 100 cc of 96% ethanol and then, sprayed on 1 kg of feed to obtain a homogeneous mixture of aflatoxin B1 contaminated feed. Ethanolic extracts of thyme and oregano were prepared by the Macceration method in the laboratory of the Department of Animal Science, Malayer University. The data obtained from this experiment were analyzed by SAS software. Mean comparison was done using Duncan's multiple -domain test at 5% error level. The results of the experiment showed that the weight of birds receiving aflatoxin B1 contaminated rations were reduced compared to the control group (P<0.05). Weight gain of broiler chicks in the experimental group receiving aflatoxin with a mixture of thyme and oregano extract increased compared to the experimental group receiving aflatoxin alone in the diet (P<0.05). This is in accordance with the reports of Manafi et al. (2009) who showed that consumption of one mg per kilogram of aflatoxin reduced the yield and growth of broiler chickens up to 5 percent. Decreased feed intake and impaired metabolism of energy and fat due to the use of aflatoxin in the diet due to changes in taste and disturbance in digestion and absorption mechanisms have been reported in various studies (Raju and Devegowda 2002). The presence of mannan oligosaccharide compounds and antioxidant compounds in thyme and oregano such as thymol and carvacrol may have antioxidant properties against negative activity of aflatoxin and inhibitory effects on metabolic changes caused by aflatoxin such as liver toxicity and contribute to reducing weight gain in broilers (Burt, 2004). A mixture of thyme and oregano extracts, and the consumption of feed in the experimental group receiving aflatoxin in comparison to the control group showed a significant decrease. Addition of plant extracts to aflatoxin contaminated diets increased significantly feed intake compared with control group (P<0.05). The increased in feed intake is due to the addition of herbal extracts. Thyme have appetitive factors like thymol and carracorol and other growth factors, which stimulates digestive enzymes and increases daily feed intake of broiler chickens (Cabuk et al. 2006). The group receiving herbal extracts in the 6th week had a lower FCR than the control group and the aflatoxin received group (P<0.05). Cinnamon-containing compounds and Thymol and Carvacrol have probably a role in improving the chicken feed conversion rate (Cabuk et al. 2006). Also addition of thyme and oregano extracts in aflatoxin challenged broilers caused a significant decrease in ALT, AST, ALP, LDL, and HDL (P<0.05). This might be due to the antioxidant properties of two plants, which reduce the toxic effects of oxidative toxicity on the liver and reduce cholesterol, triglyceride and liver enzymes. The reason for this decrease is the inhibitory effects of these extracts on the action of key enzymes such as HMG-COA reductase, which is present in the production of lipids and cholesterol (Sarica et al. 2005). Chickens receiving thyme and oregano extract had the highest villus height and showed a significant differences in comparison with other experimental groups (P<0.05). The villus height to crypt depth ratio was the highest in the group receiving the herbal extracts and the lowest amount was observed in the group receiving the aflatoxin and the addition of herbal extract to the contaminated diet improved this index significantly (P<0.05). Increasing the crypt depth in the ileum region and reducing digestion and absorption in this region might be due to the damage in the intestinal mucosal tissues of chickens fed contaminated diets. The results obtained from this research states that dietary supplementation with plant extracts of thyme and oregano reduces the negative effects of aflatoxin on performance, gastrointestinal index and improves the biochemical parameters in broiler chickens challenged with aflatoxicosis.