EFFECTSOFAFLATOXINB1ONLY OR CO-ADMINISTERED WITH MYCOTOX NG ON LIVER FUNCTION IN TURKEY BROILERS

Ivan Valchev 1 , Nelly Grozeva 2 , Vanija Marutsova 1 And Yordan Nikolov 1 . 1. Department of Internal Non-Infectious Diseases, Faculty of Veterinary Medicine, 6000 Stara Zagora, Bulgaria. 2. Department of General and Clinical Pathology; Faculty of Veterinary Medicine, 6000 Stara Zagora, Bulgaria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The toxic effects of aflatoxins in domestic poultry are manifested with anorexia, apathy, reduced growth performance, reduced feed intake, increased feed conversion ratio, reduced egg production, altered visceral organs relative weights, increased death rates (Bailey et al., 1998;Kubena et al., 1998 The sensitivity of the different fowl species to the toxic effects of aflatoxins is different. Most sensitive are growing ducklings, goslings, pheasants and broiler chickens (Leeson et al., 1995). From public health point of view, aflatoxins are dangerous for the health of poultry meat consumers as they accumulate and could provoke neoplastic growths (Ishfaq et al., 2014).
Different methods for elimination, inactivation and reduction of bioavailability of aflatoxins in feeds have been implemented: physical (separation, heat, radiation), chemical (ammonia, ozone, sulfites, hypochlorites), biological (bacteria, yeasts) and nutritional (vitamins, minerals). Most of them have at least two disadvantages: high cost of decontamination of feeds and failure to ensure a complete removal of aflatoxins without losing a great part of the nutritional value of feeds (Méndez-Albores et al., 2007;Eshaket al., 2010). One of the most practical methods nowadays is the use of non-nutritional mycosorbents as aluminosilicates, bentonites, clinoptiolites (Oguz et al., 2003;Valchev et al., 2013;Valchev et al., 2014), activated charcoal (Khadem et al., 2012)  Blood samples were collected from v. metatarsalis medialis on post treatment days 21 and 42 in sterile heparinised containers (FL medical, Italy) for analysis of enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma-glutamyl transferase (γ GT), lactate dehydrogenase (LDH) and blood total protein, albumin, , cholesterol, triglycerides,total bilirubin, blood glucose. Samples were centrifuged within 30 min after collection at 1500×g at 4°С for 10 min. Plasma was harvested and stored at -20°С until analysis. The blood chemical parameters were assayed on an automated biochemical analysed BS-120, Mindray, China.
Liverspecimenswerecollectedformorphologicalexamination after euthanasia by cervical dislocation as per Ordinance 20 on the minimum requirements for the protection and welfare of experimental animals and requirements to objects for use, cultivation and / or supply (State Gazette 87/9/11/2012). They were fixed in 10% formalin, embedded in paraffin after an ethanol series. Cross sections were stained with haematoxylin/eosin. The experiments were approved by the Bulgarian Food Safety Agencypermit No 19218/06.11.2014. Data were statistically processed using single-factor Anova, and the level of statistical significance was determined with the Tukey-Kramer test (p0.05).

Blood chemistry analysis:-
The data from Table 1 showed the effects of AFB 1 or/and Mycotox NG on blood plasma total protein, albumin and glucose on treatment days 21 and 42. By the 21 st day, turkeys receiving only AFB 1 (at 0.2 and 0.4 mg/kg feed) had statistically significant lower blood total protein (32.53±0.83 g/l and 28.53±1.19g/l) and albumin (16.8±0.91 and 15.4±0.60g/l) respectively(p<0.001) than control birds (46.31±1.46 g/l and 21.6±0.72 g/l). By the 42 nd day, the changes were more pronounced with total protein concentrations of 28.01±0.83 g/l and 25.09±1.47 g/l respectively(p<0.001) vs controls (45.59±1.36 g/l) and mean albumin levels in blood of 13.8±0.94 g/l and 12.6±0.52 g/l (p<0.001) respectively as compared to untreated turkeys (21.3±0.61 g/l). In Groups V and VI which were supplemented with 0.2 and 0.4 mg/kg AFB 1 plus 0.5 mg/kg mycosorbent, the values were also reduced by day 21 and day 42 (p<0.05, p<0.001 respectively). Blood total protein levels were 35.89±1.08 g/l and 30.09±0.94 g/l respectively by the 21 st day, whereas corresponding albumin concentrations -19.2±0.77 g/l and 17.7±0.54 g/l. By the 42 nd day, the tendency towards lower total protein and albumin levels in blood was preserved as seen from respective total protein (33.74±0.69 g/l; 28.49±1.39 g/l) and albumin concentrations (17.2±0.72 g/l; 16.2±0.80 g/l).
The changes in plasma aminotransferase activities (AST, ALT and γ GT) as well as activities of LDH and AP are presented in Tables 3 and 4 Histopathological studies:-Birds fed the standard feed only (control group) and receiving only Mycotox NG (Group II) did not exhibit gross changes in normal liver architectonics.
Turkey poults treated with 0.2 mg/kg AFB 1 , demonstrated congestion in their liversstrongly dilation of capillaries with activation of the endothelium, pericapillary oedema, granular degradation of the cytoplasm of hepatocytes (Fig.  1). In some areas, round cell proliferations were visible (Fig. 2).

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The birds treated at 0.4 mg/kg AFB 1 showed strongly impaired liver structure. Apart the congestive events in blood vessels and round cell proliferations, necrobiotic areas and vacuolar degeneration in many hepatocytes were present (Fig. 3). A part of the biliary duct epithelium was hyperplastic (Fig. 4).
Turkeys treated at 0.4 mg/kg фураж AFB 1 and 0.5 g/kg фураж Mycotox NG exhibited widened sinusoidal spaces and reduced size of hepatocytes (Fig. 7). At the same time, fibrous connective tissue proliferation was observed in perilobular spaces (Fig. 8).

Discussion:-
Fungi alter the colour of cereal crops, their nutritional and chemical characteristics, reduce seed germination and contaminate the feed with mycotoxins, which are highly toxic for men and animals. Aspergillus flavus and Aspergillus parasiticus are among the most important fungal producers of aflatoxins in storehouses, commonly present in cereals and cereal animal feeds (Paster et al.,1993).
Aflatoxins are of particular importance for the poultry industry due to their high toxicity and frequent contamination of feeds. Feeds contaminated with aflatoxins reduce the activity of numerous enzymes involved in the metabolism of carbohydrates, proteins, fats and nucleic acids (Azizpour and Moghadam, 2015). Chronic and subchronic aflatoxicosis could be detected through analysis of changes in blood serum biochemical and haematological parameters before any clinical signs become visible. TheturkeysareespeciallysusceptibletotoxiceffectsofAFB 1. The liver glutathione S-transferases alpha class (GSTA) in this species is not able to detoxify AFBO, which is probably the main factor for their high sensitivity (Rawaletal., 2010). Aflatoxins impair the protein synthesis by forming adducts with DNA, RNA and proteins (Busby and Woganq 1984), RNA synthesis inhibition, reduction of DNA-dependent RNA polymerase activity, degranulation of granulated endoplasmatic reticulum (Verma and Nair, 2001), all of them mechanisms through which the structure of various tissues (liver, kidneys, skeletal muscles, heart, pancreas) is altered (Sharma et al., 2011). On the other hand, reduced blood total protein concentrations are probably a result of liver dystrophy and hence, reduced rate of protein synthesis. The lower protein biosynthesis and dystrophic changes of liver cause reduction in plasma protein concentrations observed in this study. The enhanced elimination of proteins through the kidneys is another cause for low plasma values (Shahzad et al., 2014). Hypoproteinaemia can probably is a result of reduced food intake (El-Shewy and Ebrahem, 2004). Lower blood levels of total protein and albumin observed in the present experiments agree with the results of others (Shi et al., 2009;Valchev et al., 2014). Disturbed protein synthesis is a cause for lower concentrations of proteins in skeletal muscle (Verma & Chaudhari, 1999), the heart, liver, and kidneys (Verma and Kolhe, 1997) in animals fed aflatoxin-contaminated feeds.

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Bilirubin is a secondary product of haemoglobin conversion, filtered through kidneys and then, conjugated to glucuronic acid in the liver and excreted with the bile. Increased total bilirubin results from impaired liver function in aflatoxicosis (Rustemeyer et al., 2010). Hyperbilirubinaemia is a very sensitive test for detection of the functional integrity of the liver, as well as for assessment of dystrophy severity consequently to aflatoxicosis (Rizvi et al., 2000;Banu et al.,2010).
The present study established lower plasma cholesterol and triglycerides in turkey broilers treated with AFB 1 . They result from aflatoxin-impaired lipid metabolism in the liver (Shi et al., 2009).
Lower blood glucose in the present experiments was due to lower intake of food and/or reduced activity of enzymes involved in carbohydrate metabolism and glycogenolysis (glucose-6-phosphatase and fructose-1,6-bisphophatase) as a result of liver dystrophy or overproduction of insulin (Soliman et al., 2008;Zao et al., 2010;Valchev et al., 2014). Aflatoxins inhibit the protein synthesis and thus, the excess of amino acids could be used to meet the energy needs of the body and maintenance of blood glucose levels, bit impaired gluconeogenesis could cause lower blood glucose levels (Soliman et al., 2008). Hypoglycaemia could probably result from enhanced glycolysis or reduced absorption of glucose through the intestines (Semenov et al., 2016).  2006). The liver is a target organ for the toxic effect of AFB 1 , as in the liver aflatoxins undergo bioactivation to reactive 8,9-epoxide, which binds to DNA and proteins. As a result, the normal liver structure is damaged (Pasha et al., 2007). Aflatoxin В 1 is cytotoxic for hepatocytes and inhibits their proliferation (Abdel-Wahhab et al., 2007). The observed histopathological changes in the liver parenchyma are due to the accumulation of fats in hepatocytes following impaired lipid metabolism, which in turn is caused by inhibited synthesis of phospholipids and cholesterol causing impairment of liver lipid transport (Espada et al., 1992).
Bile duct epithelium hyperplasia results from the direct toxic effects of AFB 1 on biliary epithelium or from overproduction of prostaglandins following AFB 1  Compared to control group of birds, the addition of 0.5 g/kg Mycotox NG to the feed of Groups V and VIreducedpartlythedeleteriouseffectsofaflatoxinВ 1 on blood concentrations of studied biochemical blood parameters and the extent of histological liver changes. It is presumed that most of aflatoxin molecules are absorbed by feed ingredients in the gastrointestinal tract causing less adverse effects on the histostructure of target organs and blood biochemical parameters (Azizpour and Moghadam, 2015).

Conclusion:-
The results of the present study showed that the supplementation of turkey broiler feed with AFB 1 had a negative effect on liver function as seen from the increased blood enzyme activities of AST, ALT, γ GT, LDH and AP and of total bilirubin. At the same time, the tested doses of aflatoxin B 1 provoked reduction of plasma total protein, albumin, blood glucose, triglycerides and cholesterol, Increased doses of AFB 1 (0.2 or 0.4 mg/kg feed) induced specific histological changes in the liver. In this study, the addition of 0.5 g/kg Mycotox NG to poultry diets contaminated with 0.2 or 0.4 mg/kg AFВ 1, was capable to alleviate the severity of changes in analysed parameters and to reduce the severity of histological lesions induced by aflatoxicosis.