Introduction
Mycotoxin-contaminated feed causes reduced growth rates, feed consumption, poor feed conversion ratio (FCR), suppression of the immune system and mortality in poultry. Mycotoxins are secondary fungal metabolites of structurally diverse groups occurring worldwide as contaminants of grains (Hussein and Brasel, Reference HUSSEIN and BRASEL2001). Exposure of birds to mycotoxins occurs mainly by ingestion of contaminated grains and other raw materials added in the preparation of animal feed (Chu, Reference CHU1991). Different types of mycotoxins such as aflatoxin, zearalenone, ochratoxin A, vomitoxin, T-2 toxin and fumoninsin, naturally contaminate feed materials (Arafa et al., Reference ARAFA, BLOOMER, WILSON, SIMPSON and HARMS1981; Hussein and Brasel, Reference HUSSEIN and BRASEL2001). The deleterious effects of mycotoxins include hepatotoxic, mutagenic and hepatocarcinogenic properties which affect many animal species including poultry (Chand et al., Reference CHAND, DIN, DURRANI and SAHIBZADA2011). Aflatoxins are a group of heterocyclic toxic metabolites of toxigenic fungi such as Aspergillus flavus and A. parasiticus (Arafa et al., Reference ARAFA, BLOOMER, WILSON, SIMPSON and HARMS1981). A total of 18 different types of aflatoxins are known, but only aflatoxin B1, B2, G1 and G2 have been reported as natural contaminants of feedstuffs (Chand et al., Reference CHAND, DIN, DURRANI and SAHIBZADA2011). Aflatoxin B1 is the most common form of aflatoxin found in maize mostly used in poultry feed (Ahsan et al., Reference AHSAN, BHATTI, ASI, BHATTI and SHEIKH2010). Its maximum permissible level is 20 ppb in diets for growing poultry (Sabri et al., Reference SABRI, SIDDIQUE, KHAN and SAMAD1989). Aflatoxicosis in poultry causes chronic illness which is characterised by high mortality, reduced growth rate, anorexia, poor FCR, fatty liver and increased chances of susceptibility to different diseases (Yunus et al., Reference YUNUS, RAZZAZI-FAZELI and BOHM2011). Biochemically, aflatoxicosis disturbs energy, protein, lipid and nucleic acid metabolism (Ellis et al., Reference ELLIS, SMITH, SIMPSON and OLDHAM1991). Aflatoxins cause immunosuppression (Kalorey et al., Reference KALOREY, KURKURE, RAMGAONKAR, SAKHARE, WARKE and NIGOT2005), reduce total serum protein, albumin (Oguz et al., Reference OGUZ, KECECI, BIRDANE, ONDER and KURTOGLU2000), uric acid (Kececi et al., Reference KECECI, OGUZ, KURTOGLU and DEMET1998) and increased aspartate aminotransferase and alanine aminotransferase (Santurio et al., Reference SANTURIO, MALLMANN, ROSA, APPEL, HEER and DAGEFÖRDE1999; Madheswaran et al., Reference MADHESWARAN, BALACHANDRAN and MURALI MANOHAR2004). Various methods are used for inactivation or detoxification of mycotoxins in contaminated foodstuffs. Most of these techniques are ineffective or impractical (Piva et al., Reference PIVA, GALVANO, PIETRI and PIVA1995; Indresh et al., Reference INDRESH, DEVEGOWDA, RUBAN and SHIVAKUMAR2013). One method for the detoxification of mycotoxins is the use of non-nutritive binding materials in the diets, which prevent the absorption of mycotoxin from the gastrointestinal tract.
Milk thistle as a medicinal herb is extensively used for treating of liver diseases. Milk thistle extract (silymarin) contains various flavonolignans such as silybin. The active compounds of milk thistle are mostly present in the seed, which contain approximately 70-80% silymarin., which is a mixture of a number of active compounds such as silybin, silychristin, dehydrosilybin, silydianin and isosilybin (Pradhan and Girish, Reference PRADHAN and GIRISH2006). Silybin is the major component and shows better biological activities (Ludovico et al., Reference LUDOVICO, CAPASSO, MILIC and CAPASSO2010), including in birds affected by aflatoxin B1 (Tedesco et al., Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004). Chand et al. (Reference CHAND, DIN, DURRANI and SAHIBZADA2011) reported that silymarin has anti-inflammatory, cytoprotective, hepatoprotective, and anti-carcinogenic properties.
Several aspects of milk thistle such as antioxidant, cell regenerating and increased protein synthesis have been suggested to be important for proposed mechanisms of action (Surai, Reference SURAI2015). Silymarin provides protection due to four important properties. It maintains membrane permeability and stability in the presence of xenobiotic damage (Yunus et al., Reference YUNUS, RAZZAZI-FAZELI and BOHM2011), prevents the transformation of stellate hepatocytes into myofibroblasts which lead to the deposition of collagen fibre and cirrhosis (Fraschini et al., Reference FRASCHINI, DEMARTINI and ESPOSTI2002) and disrupt free radicals, including hydroxyl radicals, superoxide and lipid peroxide cause liver disease (Miguez et al., Reference MIGUEZ, ANUNDI, SAINZ-PARDO and LINDROS1994).
Free radicals cause peroxidation of polyunsaturated fatty acid in the cell membrane bilayer, which cause a chain reaction of lipid peroxidation (Chand et al., Reference CHAND, MUHAMMAD, KHAN, ALHIDARY and RAHMAN2016). Silymarin may be able to prevent the reduction of the detoxification by decreasing the free radical and increasing antioxidant enzymes (Wisemann, Reference WISEMANN1996). The cytoprotective effects of silymarin are considered to be mainly due to its antioxidant potential. Silymarin acts directly on cell membrane and prevents abnormal changes in the contents of lipid, which maintain normal fluidity (Muriel and Mourelle, Reference MURIEL and MOURELLE1990). It enters the nucleus and acts on enzyme RNA polymerase 1 to increase the transcription of rRNA, and ribosomal formation, leading to the larger synthesis of proteins (Sonnenbichler and Zetl, Reference SONNENBICHLER and ZETL1986). This action has an important therapeutic implication in repairing damaged hepatocytes and restoration of normal function of the liver.
Silybinin reduces the leukotriene (B4) synthesis and inhibits 5-lipoxygenase pathway and leukotrien synthesis, which is an important pharmacological property (Saller et al., Reference SALLER, MEIER and BRIGNOLI2001). It suppresses the activity of NF-κB and DNA binding activity (Saliou et al., Reference SALIOU, RIHN, CILLARD, OKAMOTO and PACKER1998). The reduction in performance of birds is linked with a reduction in ileal digestibility of dry matter and ileal morphology and poor absorption of important nutrients (Alzawqari et al., Reference ALZAWQARI, AL-BADDANY, AL-BAADANI, ALHIDARY, KHAN, AQIL and ABDURAB2016). In addition, aflatoxicosis reduces lipase, amylase and chymotrypsin activities (Matur et al., Reference MATUR, ERGUL, AKYAZI, ERASLAN and CIRAKLI2010).
Hepatoprotective effects of milk thistle
Aflatoxin in poultry depresses liver function (Hussain et al., Reference HUSSAIN, KHAN and HASSAN2008). Affected birds have abnormal colouration and damage to the liver (Denli and Okan, Reference DENLI and OKAN2006). Liver inflammation and immune dysfunction have been reported in the literature (Asim et al., Reference ASIM, KHAN, CHEEMA, MIR and AFZAL1990). The severity of harmful effects of aflatoxin in broilers depends on the concentration and exposure of aflatoxin contaminated feed. Diseased livers show microscopic and macroscopic lesions (Hussain et al., Reference HUSSAIN, KHAN and HASSAN2008). These strong and stable toxins attach to the fatty acid and protein molecules in the liver resulting in necrosis. To detoxify the effects of aflatoxin B1 in liver by silymarin is a complicated process, largely controlled by cytochrome P450 enzyme (Yunus et al., Reference YUNUS, NASIR, FAROOQ and BÖHM2008; Omid et al., Reference OMID, OMID, AFZALI, SARI, FROUZANMEHR and SHIBAK2014). Baer-Dubowska et al. (Reference BAER-DUBOWSKA, SZAEFER and KRAJKA-KUZNIAK1998) found that it reduces the cytochrome P450 system, and, as a result, limits aflatoxin B1 activation.
Free radicals including hydroxyl radicals, superoxide and lipid peroxide cause liver disease (Miguez et al., Reference MIGUEZ, ANUNDI, SAINZ-PARDO and LINDROS1994). The free radicals bind to proteins and unsaturated fatty acid of cell membranes. The end result is hepatocyte death triggered by the failure of calcium in the cytosol and dysfunction of mitochondria (Wisemann, Reference WISEMANN1996; Kosina et al., Reference KOSINA, KREN, GEBHARDT, GRAMBAL, ULRICHOVA and WALTEROVA2002; Abudabos et al., Reference ABUDABOS, ALYEMNI, DAFALLA and KHAN2017). Silymarin fortifies cellular as well as mitochondrial membranes against xenobiotic injury (Munter et al., Reference MUNTER, MAYER and FAULSTICH1986). Silymarin binds toxins and prevents their absorption into the hepatocyte by occupying the binding sites (Faulstich et al., Reference FAULSTICH, JAHN and WIELAND1980). Aflatoxins cause immune suppression (Kalorey et al., Reference KALOREY, KURKURE, RAMGAONKAR, SAKHARE, WARKE and NIGOT2005), and silymarin protects the birds against the harmful effects of aflatoxin B1 (Chand et al., Reference CHAND, DIN, DURRANI and SAHIBZADA2011). Aflatoxin B1 contaminated diets significantly increased serum hepatic enzymes such as alanine amino-transaminase (ALT), aspartate amino-transferase (AST) and alkaline phosphatase, which were reversed by the supplementation of silymarin. Similar results have been reported by Abou-Shehema et al. (Reference ABOU-SHEHEMA, RAWIA, KHALIFAH and ABDALLA2016).
Growth and feed efficiency in broiler
Silymarin has been reported to have positive effects on feed consumption and utilisation in broilers (Tedesco et al., Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004; Gazak et al., Reference GAZAK, WALTEROVA and KREN2007). Feed contaminated with aflatoxin causes reduced growth rate, feed efficiency and increased mortality in broiler (Malekinejad et al., Reference MALEKINEJAD, AFZALI, MOHAMMADI and SARIR2015). Omid et al. (Reference OMID, AFZALI and OMIDI2013) reported that feed consumption and body weight were improved by supplementation of silymarin alone compared with the group given feed contaminated with aflatoxin B1, with no effect on feed conversion ratio in broiler. Muhammad et al. (Reference MUHAMMAD, CHAND, KHAN, SULTAN, MUSHTAQ and RAFIULLAH2012) reported that silymarin supplementation in diets significantly increased feed consumption and growth in the presence of aflatoxin B1 in feed of broilers. Chand et al. (Reference CHAND, DIN, DURRANI and SAHIBZADA2011) showed that feed intake was improved and weight gain was increased in response to milk thistle supplementation fed to broilers with aflatoxin B1 contaminated feed. Kalorey et al. (Reference KALOREY, KURKURE, RAMGAONKAR, SAKHARE, WARKE and NIGOT2005) found that silymarin increased body weight and feed intake in the presence of aflatoxin B1 in feed, and had no effect on the FCR (Tedesco et al., Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004). Tedesco et al. (Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004) reported that feed intake was improved (by 22%) and weight gain was increased (by 15%) in broilers by the presence of silymarin phytosome in the feed at 600 mg/kg in an aflatoxin B1 contaminated feed.
Similar results were reported in other studies (Chakarverty and Parsad, Reference CHAKARVERTY and PARSAD1991; Gowda and Sastry, Reference GOWDA and SASTRY2000). Abou-Shehema et al. (Reference ABOU-SHEHEMA, RAWIA, KHALIFAH and ABDALLA2016) showed higher dressing weight in broiler supplied with different levels of Silybum marianum compared with an unsupplemented control group. Similarly, Chand et al. (Reference CHAND, DIN, DURRANI and SAHIBZADA2011) found that broiler fed silymarin increased dressing percentage (6%), while the highest breast muscles and drumstick were found in birds consuming 1% silymarin, when compared with the groups exposed to aflatoxin B1. Similarly, Zahid and Durrani (Reference ZAHID and DURRANI2007) reported 4% gain in dressing percentage in broilers supplemented with milk thistle. Reductions in feed intake, weight gain in broiler fed with aflatoxin was restored by silymarin (Morovat et al., Reference MOROVAT, CHAMANI, ZAREI and SADEGHI2016; Jahanian et al., Reference JAHANIAN, MAHDAVI, ASGARY and JAHANIAN2017).
Immunoprotective effects
Chand et al. (Reference CHAND, DIN, DURRANI and SAHIBZADA2011) found that feeding an aflatoxin B1 contaminated diet significantly decreased serum mean antibody titre in broilers against Newcastle (ND), infectious bronchitis (IB) and infectious bursal disease (IBD). Silymarin supplementation significantly increased antibody titre against infectious diseases in the presence of aflatoxin B1 in feed. Other researchers reported that silymarin acts as an immunostimulant (Thyagarajan et al., Reference THYAGARAJAN, JAYARAM, GOPALAKRISHNAN, HARI, JEYAKUMAR and SRIPATHI2002; Wilasrusmee et al., Reference WILASRUSMEE, KITTUR, SHAH, SIDDIQUI, BRUCH, WILASRUSMEE and KITTUR2002), as it enhances lymphocyte proliferation, which is related to increased interferon gama, interlukin (IL)-4 and IL-10 cytokines (Wilasrusmee et al., Reference WILASRUSMEE, KITTUR, SHAH, SIDDIQUI, BRUCH, WILASRUSMEE and KITTUR2002). Basaga et al. (Reference BASAGA, POLI, TEKKAYA and ARAS1997) found that silymarin improved the immune system through quenching of free radicals. It has the ability to preserve the delivery of glutathione and decreases its oxidation (Muhammad et al., Reference MUHAMMAD, CHAND, KHAN, SULTAN, MUSHTAQ and RAFIULLAH2012) and has a direct effect on immune cells. Silymarin stabilises hepatocytes and other cell membrane and stimulates macromolecular histamine discharge, lipoxygenase and prostaglandin synthetase and leukotriene synthesis.
Chand et al. (Reference CHAND, DIN, DURRANI and SAHIBZADA2011) found that aflatoxicosis caused reduced bursa, spleen and thymus weight and increased mortality in broiler. The post-mortem lesions included enlarged friable liver, swollen kidneys, hydro pericardium and haemorrhagic enteritis, however, the post-mortem lesions were reversed in milk thistle supplementation. Similarly, supplementation restored the normal weight of bursa, spleen and thymus. Tedesco et al. (Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004) found decreased bursa, spleen and thymus weight in broilers in response to aflatoxin contaminated feed, while supplementation of milk thistle restored normal morphology and increased the weight of bursa, spleen and thymus. Kalorey et al. (Reference KALOREY, KURKURE, RAMGAONKAR, SAKHARE, WARKE and NIGOT2005) reported that a feed including milk thistle restored the morphological changes and weight of the lymphoid organs (bursa, spleen and thymus) in birds with aflatoxicosis.
Blood biochemistry
Consumption of aflatoxin B1 in the diet (500pbb) significantly reduced high density lipoprotein (HDL), calcium and glucose while serum creatinine was increased, however, supplementation with milk thistle seed reduced these effects (Amiridumari et al., Reference AMIRIDUMARI, SARIR, AFZALI and FANIMAKKI2013). Muhammad et al. (Reference MUHAMMAD, CHAND, KHAN, SULTAN, MUSHTAQ and RAFIULLAH2012) reported that feeding an aflatoxin B1 contaminated diet significantly decreased serum protein, while silymarin supplementation (10 g/kg) increased serum protein. In contrast, Dumari et al. (Reference DUMARI, SARIR, MAKKI and AFZALI2014) found that milk thistle seed supplementation had no significant effects on iron, phosphorus, cholesterol, triglycerides, uric acid and low-density lipoprotein (LDL) compared to free aflatoxin contaminated diet. Similarly, Tedesco et al. (Reference TEDESCO, DOMENEGHINI, SCIANNIMANICO, TAMENI, STEIDLER and GALLETTI2004) found that aflatoxin B1 and silymarin treatments did not influence serum glucose, urea nitrogen content, protein, bilirubin, calcium, aspartate amino transferase and phosphorus.
Conclusions
Aflatoxicosis adversely affects feed intake, weight gain, feed efficiency, serum biochemistry and immune status in broiler and supplementation of diets with silymarin helps to reduce such detrimental effects, thereby improving the productivity and health performance. Future studies should be focused on the adverse effects of milk thistle under different doses in varying experimental conditions. In addition, the dose, duration and the mechanism of action should be further elucidated under different experimental conditions. Furthermore, research studies should be focused on larger and longer randomised trials, compliance, dropout and surveillance.
Acknowledgement
This study was supported by King Saud University, Deanship of Scientific Research, College of Food and Agriculture Sciences, Research centre
