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Effect of Adding Milk Thistle Silybum marianum Cereal to Naturally Contaminated Ration by Mycotoxins on Productive and Physiological Performance of Laying Hens.
Mahdi Salih Jsasim1 and Taghreed Hadi M. Al-Jurany 1,*
1  University of Diyala / college of agriculture
* Correspondence: [email protected].
Available from.

The current study aimed to evaluate the effect of adding different milk thistle cereal powder levels to the ration naturally contaminated by mycotoxins on the productive and physiological performance of laying hens fed. A total of 108 laying hens at 49 weeks old were used in this study. Birds were randomly distributed to four treatments, each with three replicates and nine birds in each replicate. The experimental treatments were as follows: Treatment 1 (Negative control) fed standard ration, treatment 2 (Positive control) fed normal ration its content of yellow corn was naturally contaminated by Mycotoxins (aflatoxins 87.58 ppb, T-2 toxin 1.13 ppb and Ochra Toxin 2.0 ppb ), treatment 3 fed as treatment 2 + Milk thistle 7.5 gm/kg feed, treatment 4 provided as treatment 2 + Milk thistle 15 gm/kg feed. The experimental period was 112 days. The results obtained showed that the supplementation of milk thistle of contamination ration alleviated the adverse effects of mycotoxins significantly (P≤0.05) on egg production, egg weight,  egg mass and feed conversion ratio, as well as on blood biochemical  (Immunity, Protein, ALT, AST, Cholesterol)  and histological indices of intestinal (Villi high, Crypts depth), while the experimental treatments had no significant effect on the concentration of uric acid in the blood. Results from this study suggested that adding of milk thistle to the contamination ration by mycotoxins alleviated the adverse effects of mycotoxins on the productive and physiological performance of laying hens.
Keywords: Milk Thistle; Mycotoxicosis; Laying hen; Productive; Physiological.


Contamination of poultry diet by mycotoxin is one of the significant challenges related to poultry industry1. Mycotoxins are toxic compounds, biologically active with low molecular weight, naturally produced by the metabolism of some fungi, such as Aspergillus, Fusarium, and Penicillium species 2, 3. Many types of mycotoxins, such as aflatoxin, Ochratoxin A, T-2 toxin, zearalenone, vomitoxin, and fumonisins, naturally contaminate feed materials 4. Some factors are necessary for fungi to generate mycotoxins, including high temperature and humidity, physical condition of grain, storage time, and presence of fungi in grain 5. Mycotoxins are found in all types of cereals, which are used in the food of animals and humans, mainly in corn, and cause vast economic losses by reducing birds' performance, health and immune response 6, 5. Mycotoxins cause various adverse clinical signs, depending on the concentration and nature of toxins in the diets, the animal species, their age, and health status at the time of exposure to contaminated diet7. High concentrations of mycotoxins in the diet result in the appearance of acute mycotoxicoses and a high death rate, while lower concentrations cause chronic mycotoxicoses without showing clinical signs but followed by a considerable decrease in production performance, health status and immunosuppressive effects8. Due to the high cost of poultry feed, which represents more than 70% of the price of the production chain, it is necessary to reduce or eliminate mycotoxin in animal feed; many natural, physical, biological, and chemical methods are used for this purpose. Unfortunately, all these methods significantly reduced the amount of mycotoxin, but they did not eliminate it.
Recently, medical plants have received much attention as feed additives for the elimination of the adverse effects of mycotoxins; milk thistle (Silybum marianum) is one of the primary medicinal plants that are commonly used for the treatment of liver disease, and different flavonolignans, such as silybin are present in milk thistle extract, the main bioactive compound in milk thistle is present in seeds, which contains about70%-80% silymarin, in addition to its hepato-protective effect, silymarin acted as antioxidant, anti-inflammatory, antifibrotic, anti-lipid peroxidative, cell membrane stabilization and liver restoring effects10. Metabolically, silymarin stimulates the hepatic cells and induces ribosomal RNA synthesis to promote protein production11. The current research aimed to evaluate the influence of supplementation of different milk thistle Silybum marianum cereal powder levels on the productive and physiological performance of laying hens fed a ration naturally contaminated by mycotoxins.

The study used one hundred and eight, age 49 weeks, laying hens (Lohmann white). Layer hens were randomly divided into four treatments of 27 birds. Each treatment has three replicates. The hens were reared in floor cages 1.5×1m(9 birds/ pen). The birds were fed on the following treatments: T1 (Negative control) was fed on a free mycotoxin basal diet (Table 1); T2 (Positive control ) was fed on a basal diet its content of yellow corn was naturally contaminated by mycotoxin.T3 fed as T2 + Milk thistle cereal powder 0.75 gm/ kg feed, T4  fed as T2 + Milk thistle cereal powder 1.5 gm/ kg feed. Naturally, contaminated yellow corn by mycotoxins was obtained from a private feed factory and examined at the master lab. (Netherlands),
Moreover, recorded these results: aflatoxins 87.58 ppb, T-2 toxin 1.13 ppb and Ochra Toxin 2.0 ppb. Milk thistle cereal was obtained from the local market and ground before being added to the diet. The lighting program was 16 hours per day. Drinking Water was ad libitum, whereas diet was constrained (110 gm/day/bird) during the experiment. The experiment lasted 122 days at the age of 49-64 weeks.
Egg production (H.D%) was documented daily, and egg weight, egg mass, and feed conversion ratio (FCR) were recorded weekly. On the last day of the experiment (age 64 weeks), 12 layer hens were slaughtered (3 birds from each treatment); blood samples were collected by a jugular vein of the neck and placed in non-additive blood collection tubes to produce serum blood samples were collected randomly in a nonheparinized tube. The measure of biochemical blood serum was separated by centrifugation at 1800g for 15 minutes. It analyzed Newcastle titers using the ELISA technique,  uric acid, total protein, ALT, AST, and cholesterol by a biochemical analyzer using commercially available kits (Spanish company SPINRECT). After extracting the bird's intestine, a histological examination was conducted; as a sample of the jejunum, two cm was taken for each of the treatment birds, and they were washed with physiological saline. Then, the pieces were placed in formalin 10%, and the tissue slides were prepared according to 12. All the prepared slides were examined using a light microscope at a magnification of 40x. Measurements were recorded using the ocular micrometer after calibrated with the stage micrometer. The villi length and crypt depth were both estimated, where the villi length extends from the tip of the villi to its attachment to the crypt, and the crypt depth the distance from the base of the villi to the end of the crypt 13, was determined.
Statistical analysis of data using complete random design (CRD), Duncan's multiple range test14 was used to test the significance of the differences between the treatment averages at the probability P≤0.05 level. Statistical analyses were performed using SPSS software.

Table 1. Composition and calculated chemical analysis of the basal experiment diets used in the study.

Productive performance
Results of Egg production (H.D%), Egg weight (g), Egg mass (g/hen/day) and  Feed conversion ratio  (g feed/ g egg mass) are presented in Table 2. According to this table, the productive performance parameters decreased in the group fed mycotoxins contaminated diets (T2) compared with control (T1), while supplementation of contaminated diets with different levels of milk thistle seeds (7.5, 15 g /kg feed) (T3 and T4 respectively ) alleviated the adverse effects of mycotoxins on productive performance. Egg production (H.D%), Egg weight (g),  Egg mass (g/hen/day) and feed conversion ratio (FCR) were significantly lower (p≤0.05) in groups fed mycotoxins contaminated diets T2 compared with control (T1 ).

Table 2. Effect of adding Milk Thistle Silybum marianum cereal to naturally contaminated ration by mycotoxins on productive performance of laying hens (mean ± standard error).
Serum biochemistry and some liver enzymes
The effects of mycotoxins and different levels of Milk thistle powder seeds on serum biochemical parameters are shown in Table ​3. The results showed that Newcastle disease Virus titer (NDV) showed a significant reduction (p≤ 0.05) in birds fed mycotoxins contaminated diets (T2) compared with the control group (T1), the birds fed mycotoxins contaminated diets containing about 7.5 and 15 gm/kg feed milk thistle (T3 and T4) did not show a significant change in titer of NDV compared to the control group (T1). There were no significant differences in the concentration of Uric acid in all treatments of the experiment. Feeding mycotoxins contaminated diets caused a significant decrease (p≤0.05) in concentration of total protein compared with the control group (T1). In the group fed mycotoxins diet (T2), the concentration of ALT, AST and Cholesterol was significantly higher (p≤0.05) compared with the control group (T1). The concentration of total protein, ALT, AST. and cholesterol did not differ substantially (p≤0.05)  in treatments containing different levels of milk thistle seeds T3 and T4  (7.5 and 15 g/kg feed) compared to control group T1.        

Table 3. Effect of adding Milk Thistle Silybum marianum cereal to naturally contaminated ration by mycotoxins on serum biochemistry of laying hens(mean ± standard error).
Histological indices of intestinal
As shown in Table 4. the Results of statistical analysis of Villi height and Crypts depth showed a significant reduction (p≤ 0.05) in birds fed mycotoxins-contaminated diets (T2) compared with the control group (T1). The birds fed mycotoxins-contaminated diets containing about 7.5 and 15 gm/kg feed milk thistle (T3 and T4) did not show a significant change in villi height and crypts compared to the control group (T1).

Table 4. Effect of adding Milk Thistle Silybum marianum cereal to naturally contaminated ration by mycotoxins on histological indices of intestinal (mean ± standard error).

A contaminated diet alone caused lower egg production, egg mass, and weight. Moreover, that reflected negatively on the fed conversion ratio (FCR) due to the negative effect of mycotoxin. The reduction in productive performance due to mycotoxins effect in the current study may be attributed to many factors:- firstly, the hepatotoxic effects of mycotoxins suppressing protein synthesis through reduced functionality of enzymes and substrates; secondly, mycotoxins interfere with DNA, RNA, and protein synthesis and affects carbohydrate metabolism, particularly glucogenolysis16. Moreover, the effect of aflatoxins in reducing villus height and porous surface area, causing a reduction in nutrient absorption, affects productive performance 17, as confirmed by this study(table 4).
Adding milk thistle into mycotoxin-contaminated diets alleviated the adverse effects of mycotoxin on the productive performance of laying hens due to the multiple actions of milk thistle and its active compound (Silymarin), which act as a hepatoprotective agent and has antioxidant activity that stimulates protein synthesis by the bird's enzymatic system, and prevent it is damaging in liver cells18 as confirmed by this study(table 3).
The lower Newcastle disease titers were recorded in groups fed aflatoxin b1 contaminated diets compared to the control group. It was clear that mycotoxins caused severe immuno-suppression due to reduced phagocytic activity of blood monocytes, depressed complement activity, and hence depressed opsonization and phagocytic activity8. However, treatment with milk thistle protected the reduction of humoral immune response in layer hens due to mycotoxins in feed 19. Milk thistle supports the immune system through its powerful antioxidant, free-radical scavenging action, its ability to preserve the supply of another important antioxidant, glutathione, and its direct effects on immune cells 20.   
The decrease in serum total protein in birds fed mycotoxins contaminated diets may be due to the inhibition of protein synthesis that happened by competitive inhibition of phenylalanine-t-RNA-synthesis16. Supplementation of different levels of milk thistle to mycotoxins contaminated diet improves the total protein concentration. This improvement was related to adding milk thistle containing a compound named silymarin, which has antioxidant properties and prevents free radical-induced hepatocyte damage to liver cells. Silymarin also promotes liver cell protein synthesis and decreases glutathione oxidation 19.  
In the current study, the AST and ALT levels in mycotoxin-treated birds increased significantly compared with control birds. It was evident from the findings that mycotoxins caused liver damage and leakage of enzymes, resulting in elevated ALT and AST levels. However, supplementation of bird feed with milk thistle seeds prevented the rise in values of ALT and AST by keeping the liver healthy due to its anti-oxidative properties, 21.   
The lower cholesterol concentration was observed in birds fed mycotoxins contaminated diet. Mycotoxins are stressful, and the stress in birds causes cortisone to be secreted by the adrenal cortex, which has negative feedback on thyroid activity, causing an increase in cholesterol and triglycerides16. Birds fed a mycotoxin diet with 7.5 and 15 gm/kg milk thistle showed an enhancement in cholesterol concentration. This enhancement was related to active milk thistle compounds, which have antioxidant properties and a protective effect against mycotoxin20.
The decreased villi height and crypt depth recorded in the contaminated diets group may be related to impaired epithelial protein synthesis and reduced cell proliferation16. Supplementation of different levels of milk thistle to mycotoxins diet enhanced the villus height and crypt depth in the jejunum of laying hens; the observed effects of milk thistle on intestinal morphology may result from the removal of invasive pathogenic microorganisms that shift the bacterial community towards the predominance of the fermentative taxa 19, 22, 23.
The groups fed a mycotoxins diet containing different levels of 7.5 and 15 g/kg feed of milk thistle were non-significantly different compared with the control group. It indicates amelioration from the negative effect of mycotoxins with milk thistle. The current study's findings are supported by many studies, such as studies of  24-27, which found that milk thistle and its extract significantly help keep the levels of serum biochemical parameters in the normal range. It can be used efficiently to decrease the toxic and suppressive effects of mycotoxicosis.     

Adding 7.5 or 15 g/kg feed milk thistle seeds into naturally contaminated diets enhanced productive performance and blood biochemical Parameters and indicated a protective effect of milk thistle against mycotoxin. Milk thistle may be used at level 7.5 mg/kg feed to ameliorate mycotoxin's adverse impact on Productive Performance and serum blood biochemical parameters of laying hens.

The author would like to express his gratitude to the Department of Animal Production, College of Agriculture, University of Diyala, Diyala, Iraq
Conflicts of Interest:
The author declares that no conflict of interest exists

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Received: 25 June 2023/ Accepted: 26 August 2023 / Published:15 September 2023
Citation:  Jsasim M. , Al-Jurany T. L. F. Effect of Adding Milk Thistle Silybum marianum Cereal to Naturally Contaminated Ration by Mycotoxins on Productive and Physiolog-ical Performance of Laying Hens. Revis Bionatura 2023;8 (3) 130
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