Restoring Activity of Milk Thistle (Silybum marianum L.) on Serum Biochemical Parameters, Oxidative Status, Immunity, and Performance in Poultry and Other Animal Species, Poisoned by Mycotoxins: A Review

Simple Summary Mycotoxins are secondary metabolites produced naturally by toxigenic fungi, which elicit a toxic response in humans and animals. The livestock sector is showing a greater focus on phytoextracts for their limiting and controlling the toxic effects of mycotoxins with safeguarding animals’ health and performance. Milk thistle and its bioactive compound silymarin are considered useful for this scope. This review underlines the efficacy of milk thistle to counteract mycotoxins toxicity on organs, biochemical and immunological functions, and performance in poisoned poultry and livestock. The use of milk thistle as a whole plant, seed, and its standardized silymarin extract in mycotoxicosis cases produce positive effects on the maintenance of the animals’ performance, restoring liver functionality due to its known hepatoprotective and antioxidant effects, reducing organ lesions caused by intoxication. The use of milk thistle in animal farming can be useful since the bioactive compounds, also if present in variable amounts, can help the animals to counteract the effects of mycotoxins. The use of silymarin, due to its cost, can be useful if it reported the specific bioactive compounds it contained. Abstract Grains are major farm animals’ diet ingredients, and one of the main concerns is when are mycotoxin (MyT) contaminated, compromising animals’ health, performance, and product safety. Among the natural phytocompounds that are being used to prevent MyT damage, silymarin (SIL), an extract from the seed of the milk thistle (MT) is a suitable candidate. This review aims to examine the scientific evidence concerning the anti-MyT toxicity effects of MT/SIL in poultry and livestock. In vitro and in vivo studies (n = 27) showed that MT whole plant, seed, or SIL-standardized extract had positive effects on animal health, performance, and restoring the hepatic activity, with a reduction of organ lesions caused by MyT. Furthermore, showed utility for combating MyT-immunodepression, improving intestinal health, and limiting the excretion of toxins residues in food of animal origin, although in some cases, MT/SIL supplementation does not produce appreciable effects. The use of MT in animal nutrition can be useful since the bioactive compounds, also if present in variable amounts, can help the animals to counteract the effects of MyT. The use of the phytoextract of SIL, due to its cost, can be useful if it reported the specific bioactive compounds, recognize for their pharmacological activities.


Introduction
Mycotoxins (MyT) are secondary toxic metabolites naturally produced by fungal species, that contaminate agricultural commodities before or under post-harvest conditions, and during their storage [1]. There are about 200 species of MyT-producing fungi [2], but Aspergillus, Fusarium, Penicillium, Alternaria, and Claviceps genres, are the main ones responsible for the production of MyT [3,4]. The most important MyT for the safety of The pharmacological potential of MT is well documented by existing data that confirm the safety and tolerability of its herbal preparations in various settings related to hepatic disorders, and in farm animals to support their performance and health [29]. Detailed information on the multifunctional and multitarget activity and the potential mechanism of action of MT and derivative products were reported in the European Medicines Agency (EMA) report [25] and by Tedesco and Guerrini [29]. In the liver, SIL suppresses the increase in the hepatic enzymes, such as ALT, AST, and ALP, during liver damage, associated with the ribosomal RNA synthesis induced by SIL, improving liver regeneration and preventing the transformation of stellate hepatocytes into myofibroblasts [30][31][32], conducing to the restoration of normal liver functions and enzymes production at a normal value [33,34]. These main other properties, regarding the antioxidant and anti-inflammatory activity, are due to SIL's capacity to act as both free radical scavenging (reactive oxygen species-ROS), and lipid peroxidation (i.e., malondialdehyde-MDA) inhibitors [35]. It acts, in fact, through the suppression of pro-inflammatory signals, derived from nuclear factor-κB (NF-κB) activation, involved in the induction of the synthesis of cytokines such as tumor necrosis factor-α (TNF-α), interleukins (IL-1, IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) [36,37]. To date, for this effect, SIL can be useful in the treatment of poisoned episodes in animals caused by drugs, heavy metals, pesticides, and toxins, including MyT, that negatively involve liver functionality. SIL interferes with the activity of CYP450 in the cells, reducing its activation system [38,39]. The same mechanism of detoxification can occur for AFs [40]. In the case of AFs poisoning, SIL limits the AFs damage interfering with CYP450 activity, so reducing epoxide production, as also demonstrated for other bioactive compounds, such as curcumin [41]. Additionally, the major AFs detoxification route is via conjugation of its AFs-epoxide to hepatic glutathione (GSH), catalyzed by the detoxification glutathione S-transferases (GSTs) enzyme. SIL would interfere positively with the regulation of this route, with the minor consumption of hepatic glutathione [42].
Various experimental studies were performed to demonstrate the hepatoprotective action of MT/SIL as a feed ingredient or feed additive, and their activity to restore health parameters and performance in farm animals, as previously described in "Use of Milk Thistle in Farm and Companion Animals: A Review" [29]. The present review aims to discuss the role and the effects of MT in MyT-poisoned animals in restoring the serum biochemical, and oxidative parameters, immunity, and performance, analyzing in vitro and in vivo studies.

Research Methodology
This review analyzes scientific papers reporting evidence of the use of MT and its phytocompound, the SIL, against MyT toxic effects. To explore the topic systematically in detail, a literature search was performed by entering keywords in specific scientific databases, including Minerva, PubMed (including ToxNet platform), Prime PubMed App, Medline Plus, Google Scholar, Scopus-Elsevier, Scifinder-n, Web of Science, ResearchGate and Scientific Information Database (SID). The Minerva database is the access point to the bibliographic resources available from the University of Milan.
The same papers consulted provided by the database search were also used for a cross-comparison, using the reference list cited. In this way, it was verified whether or not the databases also provided all the studies cited in the consulted papers. No specific timeframe of publication years or language was considered for the research in the databases. The search, using the same keywords, was performed two consecutive times at a distance of 1 month from each other, due to the continuous updating of the database. The literature search was performed from July to November 2022.
The selection of the studies was conducted following specific criteria. To be included, publications had to provide at least one abstract written in English. The publications considered were in vivo studies including field trials on farms, experimental controlled trials (i.e., intoxicated-challenge animals), and in vitro/ex vivo studies on cells. Publications regarding case reports were not excluded. Instead, studies involving the use of toxin binders (TB), including the use of MT individually tested in combination with TB in the experimental trial, or with concomitant tests with other nutrients (i.e., vitamins, lycopene, etc.) were included. Additionally, considering the publications that investigated the effects of a mixture of different MyT. Contrarily, studies regarding the use of MT in a plant or extract mix were excluded.

Results from Databases
From the overall search on each database, with the use of the above-indicated keywords, the scientific publications found were 44. Excluding the same publications provided as duplicates from one or more databases, and applying the exclusion and inclusion criteria, the usefulness number of the final publications was 27. No publications relating to laying hens, turkeys, swine, goat, sheep, fish, and their all categories emerged from the research. Tables 1-3 summarize the data obtained and presented from the studies considered in chickens, other poultry species, and livestock, respectively. The following sections thus summarize the outcomes of identified experimental studies investigating the AFs toxicity and the effects of MT to contrast and restore the negative impact on production, biochemical and immunological effects in poultry and other livestock animal species. MyT + MT group: <FCR. Immunological parameters MyT group: <ND, IB, IBD antibody titers. MyT + MT group: >ND, IB, IBD antibody titers compared also to MyT + TB group. Histopatology/organ lesions MyT group: <thymus and bursa weight but no differences in other groups. MyT + MT group: weight of bursa restored and high weight of spleen compared to other groups. [44]  Digestibility of nutrients AFB 1 (500 ppb) group: <ileal digestibility of dry matter, Ca, crude protein, and apparent digestible energy. MT (5-10 g) group: >digestibility of crude protein and Ca, AFB 1 (250-500 ppb) + MT (5-10 g) group: no differences in Ca and crude protein digestibility.
Biochemical parameters (before and after the challenge) AFB 1 group: <Ca and HDL; >ALT, AST. AFB 1 + MT group: >Ca; <ALT, AST. Redox parameters (before and after the challenge) AFB 1 group: >MDA in muscle. AFB 1 + MT: <MDA in muscle. Immune system responses (before and after the challenge) No differences after the challenge.

Protective Effects of SIL Supplementation in Poultry against MyT Anatomopathological and Cyto-Histological Modification
As widely known and reported by several studies and scientific reviews, MyT intoxication in chickens produces adverse effects resulting in typical evident anatomopathological (macroscopic) and histological observations. However, the lesions and their severity vary based on chronic or acute exposure, but especially by MyT dose-dependent. An in vitro study reported that MT seed was able to diminish AFB 1 , in particular against its density and absorbency, probably due to its rich source of fiber able to bind the aflatoxin [43]. This could be directly useful to reduce the amount of MyT absorbed and metabolized and to maintain animal health. From the anatomopathological point of view, in an early study, broiler chicks fed with a contaminated diet with 0.8 mg/kg of AFB 1 and SIL-phytosome, at a dose of 600 mg/kg-BW (SIL-Indena standardized extract) by gavage, it was found that liver weights were not different from the liver from the control group and the diet did not influence the organ's weight nor restore it [40]. On the contrary, broilers fed with a diet supplemented with MT seed at a dose of 10 g/kg/diet after being exposed to AFB 1 contaminated feed (80-520 µg/kg/diet), restored the negative effects of AFB 1 on atrophy and the smaller weight of lymphoid organs such as the bursa of Fabricius, spleen, and thymus, counteracting the AFs immunosuppressive effect [44,45]. In quail, fed with diets containing 2.2 mg/kg of AFB 1 , at 35 days of age the testis proportional weight decreased, and the weight of the liver and spleen increased, damages restored with the supplementation of 1000 mg/kg/diet of SIL [63]. Conversely, Jahanian and colleagues [46], in AFB 1 -poisoned broiler denoted an increase in only heart weight, but not in the liver and other organs (including the pancreas and gallbladder), which were not restored by the supplementation with a dose of 500-1000 ppm/kg/diet of SIL (composition not reported) [46]. In broiler exposed to 3 mg/kg/diet of OTA, slight congestion and enlargement of the liver and kidneys with gallbladder distension was observed, which was associated with small hemorrhages on the epicardium and duodenal mucosa, and with catarrhal enteritis and hyperemia of the mucosal surface, without pathomorphological changes of internal organs [47]. Additionally, in this case, the lesions caused by OTA were not mitigated by the supplementation of a 1%/kg/diet of SIL for 32 days, at least macroscopically. The same observation was evidenced by Grizzle and co-workers [65], with the administration of 10-100 mg/kg-BW of SIL (SIL 80%, analytical composition not reported) in pigeons poisoned with 3 mg/kg-BW of AFB 1 , administered by gavage. The lesions caused by AFs such as macroscopic liver degeneration, necrosis, inflammation, and hemorrhage, were not alleviated by SIL.
However, in most cases, even when the effects on organs were not macroscopically visible, histological evaluations are considered mandatory due to the anti-MyT effects of SIL on target organs, such as the intestine, lymphoid organs, liver, and kidneys. On the intestine, the morphometric analysis performed in a study conducted by Armanini and co-workers [48], it showed positive effects only in chickens fed with SIL alone, in a greater villus height and crypt depth, compared to the control and in MyT mix (AFB 0.05 ppm/kg/diet + FUM 20 ppm/kg/diet) plus SIL group (100 mg/kg/diet, commercial product 84.16% SIL-based). A lower intestine length was measured in broilers receiving AFB 1 (500 ppb), and the MT administration improved the length of the ileum and duodenum plus jejunum when administered alone, at a dose of 1.0%/kg/diet, but not when administered combined with MyT [49]. The exposure to 2 ppm of AFB 1 , resulted in decreases in villus height (VH), villi height and villus height-to-crypt depth (CD) ratio (VH/CD), villi surface area, apparent villus absorptive area, muscular diameter in jejunal sections, increases in CD, goblet cell count (GCN) and lymphoid follicular count, at days 28 and 42 of age [46]. In this study, it was observed that the jejunal CD, GCN, lymphoid follicular count, and diameter were aggravated, as AFs levels increased. This effect was mitigated with the addition of SIL to the diet. Furthermore, at a maximum dose tested (1000 ppm/kg/diet) plus AFB 1 , a depression of the VH/CD ratio was observed [63]. In broiler poisoned by AFB 1 , a linear increase in CD, and a linear decrease in villus length (VL) and villus width (VW) was observed [50]. Despite this, also if the VL/CD ratio in the ileum was decreased significantly, the interaction between VW and GCN was unaffected by the consumption of AFB 1 plus MT [46].
Histologically, a very in-depth investigation was conducted by Stoev and colleagues [47]. They evidenced that the pathomorphological alterations of the organs were seriously degenerative in chicks treated with OTA, at a dose of 3.0 mg/kg/diet, followed by those treated with OTA plus SIL (1%/kg/diet, 60% purity). In SIL-treated animals, the intestinal mucosa showed slight degenerative changes and depletion of cells in the lymph follicles, with a slight mononuclear cell infiltration. In the lymphoid organs, such as the bursa of Fabricius and the thymus, the main degenerative damages were found in chicks exposed to OTA, followed by those treated with OTA plus SIL (60% of purity). In the liver, the main target organs of the toxic effects of MyT, but also hepatoprotection of SIL, its addition revealed a reduction in cloudy swelling, granular or vacuolar degeneration of hepatocytes and a slight activation of the capillary endothelium and Kupffer's cells in OTA-exposed chicks [47]. These data were also supported by in vitro study [51], where the cytotoxicity of OTA in primary chicken hepatocytes was investigated. Testing a dose of 1 µg/mL of OTA, the cell number and density decreased markedly and ruptured and necrotic hepatocytes confirmed that OTA induced a significant increase in apoptosis and related expression genes, with the activation of a mitochondrion-dependent pathway. When the cell cultures were treated with a dose of 0.1, 1, or 10 µg/mL of SIL (analytical composition not reported) for only 24 h, the cell viabilities increased, and there was a reduction in the apoptosisassociated genes expression, hepatic enzymes activity, and MDA level [51]. In a study conducted by Tedesco and co-workers [40], in the liver tissue of AFB 1 -treated chickens, were observed necrosis and multifocal portal infiltration of mononucleates, granulocytes, and eosinophils diffused in the parenchyma, especially at the portal areas. However, less severe lesions were evidenced when SIL-phytosome was administered. The same lesions in the parenchyma were observed when DON and ZEA were administered at a dose of 4.9 mg/kg/diet and 0.66 mg/kg/diet, respectively, in a high-sensitivity species, the ducks, for 47 days (chronic exposure) [60]. The lesions were represented by a highgrade vacuolar degeneration of hepatocytes cytoplasm, solitary hepatocyte necrosis, and also individual cell deaths of the mononuclear phagocyte system. Focal lymphocytic and histiocytic interstitial infiltrates and mild interstitial fibrosis was noted, nevertheless, these observations and lesions were milder in the treated group with MT seed (analytical composition not reported) at a dose of 0.5%/kg/diet [60]. At a lesser dose of AFB 1 (3 mg/kg-BW) in pigeons' liver tissue, necrosis with multifocal portal infiltration of mononucleates, granulocytes, and eosinophils, fatty infiltration, and vacuolization, bile-duct hyperplasia, cell necrosis, and inflammation were noted and were not alleviated with SIL (10-100 mg/kg-BW, SIL 80%, standardized extract) administration [65].

SIL Effects on Serum-Biochemical Parameters on MyT-Poisoned Poultry
The hepatoprotective activity of SIL induces different modifications in the production and activity of the hepatic enzymes and their correlated biochemical parameters. In a study conducted by Amiridumari and colleagues [52], MT seed supplementation (0.5-1%) to AFB 1 -contaminated feed (500 ppb/kg/diet) significantly increased serum glucose and decreased creatinine and AST levels, compared to AFB 1 (500 ppb/kg/diet). At the same dose of AFB 1 , Fanni Makki and co-workers [53] denoted a significantly decreased AST enzyme but also uric acid, glucose, and gamma-glutamyl transferase (γ-GT) enzymes compared to the control group with the addition of 0.5-1% of MT seed. Similar results were obtained by Raei and co-workers [54] in chicks fed for 21 days with 2 mg/kg of AFB 1 , which showed a significantly declined Ca and HDL, as well as an increase in ALT and AST activity. Adding MT seed (10 g/kg/diet) enhanced the levels of Ca and HDL, reducing the activity of ALT and AST enzymes, without considerable variations in total serum protein, lowdensity lipoproteins (LDL), uric acid, and γ-GT levels. Even the addition for 41 days of a dose of 100 mg/kg/diet of SIL (SIL 84.16%) plus MyT mix including FUM + AFB, prevents the increase of ALT in broiler [48]. Evidence for this was also observed by Feshanghchi and colleagues [55], where MT seed powder (10 g/kg/diet: silichristin 2851 mg/kg, silybin B 8864 mg/kg, silymarin 100 mg/kg) significantly decreased AST and ALT activity. A significant decrease in AST activity was detected in growing quail fed with 500 mg/kg/diet of SIL, compared to the control, with no effects on ALT activity [64]. The effects of MT were investigated using the whole plant, compared to the effect of the seed administration, specifically on the influences of ALT enzyme activity. The addition of 0.5% MT seed powder, 1% MT plant powder, and 1000 mg/kg MT plant extract to the contaminated broiler diets with 500 ppb/kg/diet of AFB 1 , decreased ALT at 35 days of treatment, compared to the contaminated control [56]. At a dose of SIL even 20 times higher, the same effect was noted in quails, with the administration of 1000-2000 mg/kg/diet of SIL (analytical composition not indicated) plus AFs mix (2.2 mg/kg/diet, including AFB 1 68.19%, AFB 2 4.57%, AFG 1 24.96%, AFG 2 2.28%). SIL reduced ALT, AST, and ALP activity with a low value of glucose, LDL, and triglycerides (TG), at 35 days of age [63]. With an alternating dose of AFs in broilers chicks, poisoned with AFB 1 (80 µg/kg/diet) during the first week of age and 520 µg/kg/diet in the remaining experimental period (other 4 weeks), resulting in a significant decrease in serum total protein, and an increase in ALP and ALT enzymes. ALT enzyme was significantly higher compared to the control and chicks fed with SIL or TB (3 g/kg/feed) supplementation, while AST was significantly lower in the group treated with AFB 1 plus MT dried seed [45].
Some studies used lycopene, beta-glucans (extracted from Saccharomyces cerevisiae yeast), and sodium bentonite to compare the effects with MT. According to the study conducted by El-Sheshtawy and co-workers [61], on Pekin ducklings, ALT, AST, γ-GT, and ALP enzyme activities were significantly elevated in the Afs-intoxicated group (30 ppb/kg/diet, naturally contaminated). In comparison, they decreased significantly in groups treated with lycopene (100 mg/kg/diet) plus SIL (SIL 80%, analytical composition not reported) at a dose of 600 mg/kg-BW, as well as creatinine, with an associated increase of serum total protein and albumin. These latter parameters were altered in quail treated with AFB 1 at a dose of 1500 µg/kg/diet [62], but not when fed with 250-500 mg/kg/diet of SIL [64]. No changes were observed in biochemical parameters in quail treated for 60 days with beta-glucans (1 kg/ton/diet) or SIL (500 g/ton/diet) plus AFB 1 [62]. The administered dose of AFs (containing: AFB 1 : 84.64%; AFB 2 : 4.28%; AFG 1 : 11.07%) with any changes for AST, GGT, and CK values, showed that at this percentage, the additives were not able to mitigate the negative effects of the toxin on quail metabolism [62]. Otherwise, the combination of MT seed (0.5%/kg/diet) plus 0.5% sodium bentonite in the AFs-contaminated diet significantly increased the albumins level. At the same time, MT and sodium bentonite, separately or in combination, significantly decreased the AST level in AFs contaminated diet. Only the administration of MT seed plus AFB 1 decreases the level of ALT and LDH [57]. In the trial conducted by Tedesco and co-workers [40] in AFB 1 -treated chickens, a lower level of ALT enzyme was observed. In SIL-treated chicks, the ALT enzyme did not decrease following AFB 1 treatment. The serum levels of glucose, uric acid, AST, and ALT increased in response to OTA treatment (noted that glucose decrease usually in AFB 1 intoxication), consistent with the common OTA intoxication [69]. The SIL treatment was found to protect against these increases [47]. Pigeons [65] treated with AFB 1 by gavage for 2 consecutive days showed an increase in bile acid levels, AST, ALT, LDH, and creatine phosphokinase (CPK), with any variation of the levels of γ-GT. However, in the treatment with a high dose of SIL, these parameters were mostly unchanged. Furthermore, only pigeons treated with SIL at a dose of 10 mg/kg-BW, showed significant reductions in LDH, ALT, and CPK [65].

Influence of SIL on Serum and Tissues Antioxidant Parameters in MyT-Poisoned Poultry Species
Of the studies analyzed, some of these investigate the antioxidant effects of SIL in MyT-poisoned poultry to evaluate its antioxidant potential, and the activity to restore the antioxidant defense of the tissues, in particular in the hepatic and muscle tissue. The study conducted by Egresi and co-workers [60], in ducks, evidenced that acute exposure to MyT (DON + ZEA) caused oxidative stress, which induced an effective antioxidant defensive response, indicated by the decrease in the hepatic MDA and diene conjugate content, not observed in the untreated group, and MT seed (0.50%/kg/diet) + MyT group. They observed also an increase in Al, Ca, Cu, and Fe, and a decrease in Mg, Mn, P, S, and Zn in both treated groups (MyT and MyT + MT). Contrary, always in ducks, was reported that on hepatic tissue there was a significant reduction in MDA level in the SIL (600 mg/kg-BW) plus MyT-treated group [61], showed also an improvement in the antioxidant levels of catalase (CAT), and glutathione S-transferase (GST) significantly decreased in the AFsintoxicated group with a dose of 30 ppb/kg/diet (feed naturally contaminated).
As reported in an in vitro study, hepatocytes treated with OTA (1 µg/mL) for 24 h, reduced the superoxide dismutase (SOD), and GSH activity with an increase in MDA compared with the untreated cells [51]. The SIL treatment exhibited the largest antioxidation activity compared with the other three hepatoprotective agents tested in their study, glycyrrhizin, L-arginine, and glucorolactone [51]. At the same dose of OTA, in vivo study on intoxicated quails treated with SIL (500 mg/kg/diet), showed the highest total antioxidant capacity (TAOC), and GSH-Px and recorded the significantly lowest MDA values [64].
The antioxidant parameters were also measured in chicken muscle. The AFB 1 treatment with a dose of 2 mg/kg/diet in E. coli-challenge broiler for 21 days, gave rise to the enhancement in thigh muscle's MDA concentration, restored by the addition of MT dried seed (10 g/kg/diet) [54]. Following the effects on MDA concentrations, no differences in the thigh muscle's ferric-reducing antioxidant power levels were noted in all experimental groups. Conversely, the study of Armanini and colleagues [48], reported no differences regarding the thiobarbituric acid reactive substances (TBARS) levels among the SIL-treated group with 100 mg/kg/diet (SIL 84.16% purity), plus AFB (0.05 ppb/kg/diet) + FUM (20 ppb/kg/diet), but showed a lower level of ROS in meat derived from the SIL-treated animals. The antioxidant defense systems of the animals are upregulated to counteract the excessive production of free radicals, including ROS observed in poisoned animals. Despite this, the addition of SIL restored ROS levels and GST activity evidencing the potent antioxidant capacity of its compound.

Influence of SIL on Immunological Parameters in MyT-Poisoned Poultry Species
Immunosuppression in chickens can be caused by several factors such as natural, nutritional, managemental, diseases, stress, and vaccination, including also the interferences derived from MyT feed contaminations. MyT has a significant effect on poultry health, due to their interference with the immune response and response to the vaccination [70]. For example, OTA immunosuppression may be manifested as depressed T and B lymphocyte activity, suppressed immunoglobulin, antibody production [71], and complement activity in broiler chickens [72], including in the first life phase in ovo [73]. The same effects were noted in ducks when exposed to AFB 1 [74]. On broiler, Fanni Makki and co-workers [49] investigated the trend of the Newcastle disease virus (NDV) or Avian Influenza (AI) antibody titers after 34 days of treatment with 250-500 ppb/kg/diet of AFB 1 plus MT seed (0.5-1%/kg/diet). The administration of MT (at both doses) reduces the 500 ppb/kg/diet AFs effects with less reduction in the antibody titers, against NDV and AI. Chand and colleagues [44] found similar results studying the titers trends of NDV, infectious Bronchitis virus (IBV), and infectious Bursitis virus (IBv), evidencing that the contaminated diet with AFB 1 , significantly decreased serum mean antibody titers for these diseases. Again, the addition of MT seed at a dose of 10 g/kg/diet plus AFB 1 protected from the reduction in humoral immune response in AFs poisoned broiler. Instead, in another study, the addition of MT seed powder at a dose of 10 g/kg/diet plus AFB 1 increased the antibody titers (IgT) followed by the stimulation with the injection of sheep red blood cells (SRBC) in the broiler [55]. The administration of SIL at a dose of 1%/kg/diet showed also an increase in lysozyme concentration in animals poisoned by OTA, at concentrations very similar to the control group [58]. However, in Leghorn cockerels treated with SIL (10 g/kg/diet) in OTA contaminated diet (1 mg/kg/diet), no differences were noted for Ig titers at day 7 post-primary SRBC injection (p.i.) and 14 days p.i. The same effect was noted 24, 48, and 72 h p.i. after tubercolin administration [59]. The results obtained by Kathoon and co-workers [59] suggest that SIL possessed the ability to prevent titer decline caused by OTA-induced immunotoxic effects in chicks (1 mg/kg). Similarly, Raei and colleagues [54], showed the ability of the addition of 10 g/kg/diet of MT seed to counteract the decline of the total antibody titers in E. coli-challenged broiler.

Influence of SIL on Performance and Products Quality Parameters in MyT-Poisoned Poultry Species
Mycotoxicosis inhibits poultry health, and consequently their performance, such as growth reducing feed consumption, digestion, absorption, enzymatic activity, and protein metabolism. In some studies, the MT potential to improve or prevent further worsening of the performance and quality parameters of products, such as poultry meat [29,75], was investigated when the performance can be worsened by MyT intoxication.
MT seed powder, whole plant powder, or an MT extract addition in feed, in MyTintoxicated broiler, maintained a normal increase in BW compared to poisoned animals [56]. In this regard, the addition of MT seed powder (0.5%/kg/diet, analytical composition not reported) in an AFB 1 (500 ppb/kg/diet)-contaminated diet, positively reduced the FCR compared to broiler fed with AFB 1 alone, from 1 to 7 days of age. The addition of MT extract (600-1000 mg/kg/diet) had no significant effect on FCR. Overall, the addition of 1% of MT whole plant powder plus AFB 1 in the diet proves to deliver the best results in terms of BW and FCR [56]. Chand and co-workers [44] reported an optimal BWG observed in the untreated broiler group, but also in the group poisoned with AFB 1 (80-520 µg/kg/diet) plus MT seed powder (10 g/kg/diet, analytical composition not reported) showing the ability of MT to contrast the negative effects of AFs. These results on performance were similar to the findings by Tedesco and colleagues [40], with the administration of a dose of 600 mg/kg-BW of SIL-phytosome to an AFB 1 -poisoned broiler. They observed an increase in BW, compared to AFs-intoxicated broiler, and showed that SIL action was constant and positive to birds receiving only AFB 1 , considering BW and FI. These results suggest that treatment with SIL can be effective in counteracting the negative effects of AFB 1 intoxication on FI and BW in growing broilers. In other studies, the BW was maintained with the administration of MT seed at a dose of 0.5-1%/kg/diet in a contaminated diet with AFB 1 [53], and with MT seed powder at a dose of 10 g/kg/diet (silichristin 2851 mg/kg, silybin B 8864 mg/kg, SIL 100 mg/kg) [55]. Muhammad and co-workers [45] showed similar results, as an improved FI, BW, and best FCR using 10 g/kg/diet of dried MT seed added to AFB 1 contaminated broiler diet. However, Raei and co-workers [54], evidenced no significant discrepancies between FI, BWG, and FCR. Furthermore, FCR was lower in the period between 1 to 7 days of age, for the MT-treated group (10 g/kg/diet), compared to the AFB 1 -poisoned group (2 mg/kg/diet). Even broilers exposed to a MyT mix (AFB + FUM) showed that SIL prevented the impairment of WG and FI compared to the MyT-treated group up to 21 days of the age of 42 days of trial, which evidenced that the addition of SIL minimizes the toxic effect of MyT on the broiler performance [48]. A dose ranging from 0.5 to 2 ppm/kg/diet of AFB 1 , resulted in a decrease in daily FI and DWG, with poor FCR, but the addition of 500 or 1000 ppm/kg/diet, especially at 1000 ppm (analytical composition not reported) of SIL contrast these effects, improving the WG, FI, and FCR [46]. In quails, a dose of 1000-2000 mg/kg/diet of SIL (analytical composition unknown) plus AFs (2.2 mg/kg/diet, as AFB 1 : 68.19%, AFB 2 : 4.57%, AFG 1 : 24.96%, AFG 2 : 2.28%), showing an increase in BWG correlated with a good FCR, with a minor rate of mortality compared to the poisoned group, and control group [63]. Similarly, Youssef and colleagues [64] evidenced an improvement, increasing supplementation levels of SIL to 500 mg/kg/diet plus AFB 1 (19 ppb/kg/diet), in BW and BWG but not in FCR. Even in quails, the addition of 500 g/ton/diet of SIL in a diet contaminated with AFs (1500 µg/kg/diet, as AFB 1 : 84.64%; AFB 2 : 4.28%; AFG 1 : 11.07%), for 60 days of treatment, demonstrated that SIL was not able to mitigate the negative effects of AFs. In addition, the SIL plus AFB 1 -treated quail group produced 0.58% more eggs than the control group [62]. MT seed, at a dose of 5-10 g/kg added to the broiler diet containing AFB 1 (250-500 ppb/kg), improved the ileal digestibility of dry matter, crude proteins, and Ca, evidenced that MT seed reduced the toxic effects of AFB 1 , facilitated the absorption of nutrients, and reduced the metabolic demands of the intestinal tract in broiler chickens [50]. Moreover, Armanini and co-workers [48] underline that the consumption of SIL affected the meat's pH, increasing it. At the same time, the addition of SIL in a contaminated diet prevents cooking loss, showing a high water retention capacity and shear strength. Further, the PUFA was higher in the groups that consumed SIL and in the MyT+ SIL group. According to the study conducted by Jahanian and colleagues [46], the authors evidenced microbiota modifications; the ileal E. coli, Salmonella spp., Klebsiella spp. enumerations and total negative intestinal bacteria were markedly increased in poisoned chickens, both at 28 and 42 days of age, but not in the SIL-treated animals with a dose of 500 ppm/kg/diet. A strong suppressive effect of pathogenic microflora was evidenced in SIL plus AFB 1 (0.5 ppm) group, with a dose of 1000 ppm of SIL, which may have contributed to indirectly improving the performance.

Influence of SIL on MyT-Poisoned Livestock
From the literature search, few studies on other farms or companion animals were detected, reporting similar positive effects observed in poultry species. In farm animals, a study was conducted on calves, and one on dairy cows. In calves [66] fed with 1.0 mg/kg AFB 1 for 10 days, and subsequently treated with 600 mg/kg-BW of SIL (analytical composition not reported), and choline chloride (500 mg/kg-BW) for only 7 days, showed that the aflatoxicosis caused a worsening of the FI, and average daily weight gain (ADWG), associated with an increase in hepatic enzymes activity, such as AST, ALT, and renal function parameters, BUN and creatinine. The supplementation with SIL was comparatively more efficient to ameliorate the effects induced by AFB 1 than choline chloride. With the use of an SIL-phytosome complex, SIL was effective in reducing the excretion of AFM 1 , the main metabolite of AFB 1 toxin, in dairy cows. Tedesco and co-workers [67] investigated at the same time the use of SIL-phytosome and SIL standardized extract, at a dose of 30 g/day for 17 days, and 10 g/day (76% pure extract) for 9 days, respectively, in an organic dairy farm with high excretion of AFM 1 in milk. In this farm, the results showed that the administered feed was naturally contaminated by AFB 1 . Both treatments do not completely inhibit AFM 1 excretion in milk but contributed to reducing drastically its level. In other species such as the rabbit, raised as a pet, commercial choline (Epocler) plus SIL, turned out to be a valid association in the clinical treatment of AFB 1 intoxication [68]. In a young female rabbit with ascites and hepatic damage caused by intoxication with 300 mg/kg/ feed of AFB 1 , the treatment for 1 month with SIL at a dose of 50 mg/kg/orally plus Epocler commercial product (1 mL/q) restore its hematological and biochemical parameters.

Conclusions
Cereals are the major ingredients for the nutrition of farm animals and provide primarily energy and proteins to the diet. One of the main concerns when using grain ingredients in feed formulation is MyT contamination. Overall, from the analyzed literature, several aspects emerged regarding the use of MT and the bioactive compound, SIL, against MyT toxicity, mainly evaluated in poultry species. The results, sometimes contradictory, display, in general, a broad range of MT/SIL effects finalized on the maintenance (due to its known important pharmacological properties) of animal health, restoring of the liver enzymes activity altered by the MyT-intoxication, reducing at the same time, the organs lesions caused by MyT. These latter effects, in many studies, were also supported and confirmed by histological evaluations. On immunity parameters, the treatment with MT can be effective in counteracting the negative effects of AFs intoxication with a decrease in antibody titers. On intestinal health, the use of MT in intoxicated animals showed an improvement in gut functionality. However, in some cases, the SIL supplementation does not produce appreciable effects to contrast ones caused by MyT. This can be due to the dose of MyT administered (range of administration), the time of exposition, and the dose of MT/SIL tested in the reported trials. Furthermore, it is necessary to underline the importance of the effective bioactive compounds content tested during MyT-poisoning. It is not clear if MT/SIL acts directly against MyT-intoxication, or if the positive effects observed can be related to the specific pharmacological properties of MT itself, for example acting as a hepatoprotector or as an antioxidant, and further trial can be useful. Nevertheless, the results showed that the use of MT/SIL, as a feed ingredient or feed additive, is a helpful natural dietary supplement to maintain the animals' health, since the bioactive compounds, also if present in variable amounts, can help the animals to counteract the effects of MyT. The use of the phytoextract of SIL, due to its cost, can be useful if it is reported the specific bioactive compounds recognized for their pharmacological activities.