Cardioprotective and Hepatoprotective Activity of Silymarin in BroilerChickens Fed on Mash and Pellet Diets

Introduction

The genetic selection has led to a fast growth rate in modern strains of broiler chickens (Ahmadipour et al., 2015). In fast-growing broilers, the metabolic rate and the requirement of oxygen are increased, and the heart and lungs are not capable of providing enough oxygen, which causes metabolic disorders (Wideman et al., 2013). Right ventricular failure (RVF) and ascites syndrome are the most prominent metabolic disorders, commonly seen in broiler chickens with a high-growth rate (Ahmadipour et al., 2019).

Diet form (pellet, mash, and crumble) is one of the most important effective factors on the incidence of RVF and ascites in broilers (Sahraei, 2014). Broilers fed on the pellet diet present higher weight gain and better feed conversion ratio compared with those fed the mash diet (Amerah et al., 2008). Several studies have stated that susceptibility to RVF and ascites was higher in birds fed pelleted diets than in birds received mash diets (Jafarnejad et al., 2010; Sahraei, 2014). The heart and liver are two major damaged organs in RVF and ascites syndrome (Jafarnejad et al., 2010). Improving the health of the heart and liver using herbal extracts is a benefit and can be a clinical strategy to control RVF lesions and decrease RVF-induced mortality. Milk thistle is one of the more common medicinal herbs with protective effects on the liver and heart.

Milk thistle (Silybum marianum L., Asteraceae) is one of the most common herbs for the treatment of liver diseases (Schrieber et al., 2008). The seed and fruit of milk thistle contain silymarin that shows a variety of pharmacological activities, as well as antioxidant, anti-inflammatory, antibacterial, and antiviral properties in many experimental and clinical studies (Koçarslan et al., 2016).

Some studies have evaluated the positive effects of silymarin extract on growth performance and hepatic disorders in broilers (Schiavone et al., 2007; Kralik et al., 2015; Hosseinian et al., 2020). Further, various researches have established that silymarin had cardioprotective activity due to its antioxidant content in laboratory animals (Rao & Viswanath, 2007; Al-Rasheed et al., 2014; Koçarslan et al., 2016). However, there is a lack of knowledge about the positive effects of silymarin on broiler’s heart health. Evaluating heart electrical activity by electrocardiogram (ECG) is a simple method to assess heart health in birds (Yogeshpriya et al., 2018).

In veterinary medicine, there are several diagnostic tools for the evaluation of cardiac abnormalities, but ECG is one of the most useful and non-invasive techniques, which gives significant information about heart rate (HR), cardiac arrhythmias, and electrical conductance abnormalities (Cushing et al., 2013; Yogeshpriya et al., 2018).

In avian medicine, ECG is a useful tool utilized to measure HR and detect cardiac arrhythmias and cardiac chamber enlargement (Sharifi et al., 2015; Hosseinian et al., 2019). Several studies have indicated that RVF and ascites syndrome induce a change in the morphology of ECG waves in broilers, and they used ECG to detect and diagnosis the ascites syndrome (Hassanpour et al., 2009; Yousefi et al., 2013).

In this study, broilers were fed with two different forms of diet (mash and pellet), and heart and liver health was evaluated by assessing some serum biochemical and ECG induces. Also, the effects of two different doses of dietary silymarin (500 and 2500 ppm) were assessed on some serum parameters and ECG. In this research, this hypothesis was assessed that pellet diet can be led to heart and liver dysfunction in broilers, and silymarin could attenuate the cardiac and hepatic damages.

The results of the present study may clarify the protective effects of silymarin on the liver and heart in fast-growing broilers, and silymarin can be used as a cardioprotective and hepatoprotective compound during the rearing period of susceptible broilers to ascites syndrome.

Materials and Methods

Birds and Treatment Groups

This experiment was performed at the Veterinary College of Shiraz University. A total of 120 1-day-old Arbor Acres broiler chicks were used for the experiment and reared for 42 days. During study, broilers were raised in environmentally controlled rooms under standard environmental conditions suggested by commercial recommendations of the chick producer company (Aviagen, 2018).

On day 14 of the experiment, birds were weighed and randomly assigned into 6 equal groups (n=20) with five replicates. The treatment groups consisted of supplementation with silymarin extract in the mash and pelleted diets as follows: the control mash group (basal mash diet [CM], fed on a mash diet without silymarin), control pellet (basal pellet diet [CP], fed on a pellet diet without silymarin), two doses of silymarin in mash feed (silymarin at 500 ppm of mash [M500] and silymarin at 2500 ppm of mash [M2500]), and two doses of silymarin in pellet feed (silymarin at 500 ppm of pellet [P500] and silymarin at 2500 ppm of pellet [P2500]).

In the present study, the basal diet was corn-soybean meal-based and formulated to meet or exceed the minimum National Research Council (NRC; 1994) Standards. The starter (0-10 days), grower (10-25 days), and finisher (25-42 days) feeds were used during the experiment. All birds were fed on a pellet diet during the first and second weeks of the experiment, and then two forms of diet (mash and pellet) with a similar composition of ingredients (see Table 1) were used from the third to sixth weeks of the experiment.

Table 1. Ingredients and composition of mixture feed (gkg^-1)

* Vitamin and mineral content per kilogram of premix: vitamin A: 3,600,000 IU; vitamin D3: 800,000 IU; vitamin E: 7,200 IU; vitamin K3: 0.8 g; vitamin B1: 0.71 g; vitamin B2: 2.64 g; vitamin B3: 3.92 g, vitamin B5: 11.88 g; vitamin B6: 1.176 g; vitamin B12: 6 mg; folic acid: 0.4 g; biotin: 40 mg; choline chloride: 100 g; selenium: 80 mg; cobalt: 100 mg; iodine: 396 mg; copper:4 g; zinc: 33.88 g; iron: 20 g; manganese: 39.68 g.

The used diets in this study was prepared in the livestock affairs of the Faculty of Veterinary Medicine, Shiraz University. Firstly, the feed was supplied based on NRC Standards for broilers. Then, a part of the feed was finely grounded, mixed, and used as a mash form in mash treatments during the entire the experiment, and the other part of the feed was pelleted in a steam pellet mill. After pelleting, the pelleted feed was dried and cooled to an average temperature of 37°C and used as a pellet form in pellet treatments during the entire the experiment. Feed and freshwater were supplied ad libitum during the entire experiment.

Silymarin powder was purchased from BarijEsans Company (Iran), containing 43.87% of silibinin and 96.76% dry matter and was added to mash and pellet diets from days 14 to 42 of the experiment. All diets contained neither anticoccidials nor any other medications.

The temperature was maintained at 32°C during the first week and reduced gradually until a constant temperature of 24°C was achieved. A 24-hour lighting schedule was used during the first week and then reduced gradually until 20 h light: 4 h darkness (20L:4D).

Blood Sampling

Ten apparently healthy birds of each group were selected, and blood sampling (5 mL/birds) was done from the brachial vein on days 21, 28, 35, and 42. Blood samples were kept at room temperature for 30 minutes, and then the clotted blood samples were centrifuged at 3000g for 10 minutes to separate sera. The clear sera were collected and stored at -20℃ until the biochemical analysis.

Evaluating Hepatic Parameters

The hepatic parameters, including total protein, albumin, globulin, uric acid, triglyceride, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), were measured by an automated analyzer (Alpha Classic, Sanjesh Company, Iran) using commercial clinical investigation kits (Pars Azmoon, Tehran, Iran). Moreover, the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated using a commercial kit (Pars Azmoon, Tehran, Iran).

Evaluating Cardiac Parameters

Biochemical Assays

Calcium, magnesium, phosphorous, chloride, and creatine kinase MB (CK-MB) were measured using commercial kits (Pars Azmoon, Iran) and biochemical auto analyzer (Alpha Classic, Sanjesh Company, Iran). Sodium and potassium were determined using a flame photometer (Clinical flame photometer, Fater Company, Iran).

Electrocardiographic Studies

From each group, 7 apparently healthy chicks were randomly selected on 21, 28, 35, and 42 days, and ECGs were recorded by a single channel ECG machine (Kenz-line EKG 110, Suzuken Company, Japan). The ECG measurements were obtained from unanesthetized and restrained birds in a standing position. Before ECG recording, the chickens rested for about 5 minutes to calm down. ECG gel was applied to the skin, and then alligator clip electrodes were positioned at the base of the right and left wings and gastrocnemius muscle of the right and left limbs (Yogeshpriya et al., 2018).

All ECGs were recorded using a calibration of 10 mm/mV and a paper speed of 50 mm/sec (Reddy et al., 2016). ECGs were recorded by different leads, including I, II, III, and aVR, aVL, and aVF for every chicken. In birds, lead II is commonly used to evaluate waves' morphology in ECG (Reddy et al., 2016). For this reason, the amplitude and duration of P, T, R, and S waves, as well as the duration of QT, R-R, PR, and ST intervals, in each bird of the present experiment were measured from lead II as a standard lead (Reddy et al., 2016). Finally, HR was measured in each bird.

Statistical Analysis

The statistical analysis was performed using SPSS 22 (SPSS Inc., Chicago, Ill., USA). In the present study, the data from various serum parameters and ECG indices were analyzed by 1-way analysis of variance (ANOVA). The data were presented as mean ± SE. The significance level was set as P-value<0.05.

Results

The results of serum hepatic parameters are presented in Tables 2 and 3.

As seen in Table 2, total protein and albumin significantly decreased in CP compared with CM at some times of the experiment (P<0.05). P500 and P2500 significantly had a higher total protein compared with CP on days 28, 35, and 42. Circulating uric acid increased significantly in CP and P500 compared with CM. In this experiment, P2500 had a lower level of uric acid compared to CP and P500. The serum activity of ALT and AST enzymes in the CP group was significantly higher than in CM, and the activity levels of ALT and AST decreased significantly in P2500 compared to CP on day 42 (P<0.05) (Table 2).

Table 2. Circulating hepatic parameters (mean±SE) of broiler chickens (n=20) following dietary supplementation of pellet and mash diets by silymarin extract.

CM: control mash diet (0 ppm silymarin); CP: control pellet diet (0 ppm silymarin); M500: mash diet + 500 ppm silymarin; M2500: mash diet + 2500 ppm silymarin; P500: pellet diet + 500 ppm silymarin; P2500: pellet diet + 2500 ppm silymarin; ^a,b,c Different letters in the superscripts of the same row indicate significant differences (P <0.05).

As seen in Table 3, the serum levels of total triglyceride, cholesterol, and LDL in supplemented groups (i.e., M500, M2500, P500, and P2500) decreased significantly compared to CM and CP groups (P<0.05). The CP group had lower concentrations of VLDL and LDL compared to the CM group.

Table 3Circulating hepatic indices (mean±SE) of broiler chickens (n=20) following dietary supplementation of pellet and mash diets by silymarin extract.

CM: control mash diet (0 ppm silymarin); CP: control pellet diet (0 ppm silymarin); M500: mash diet + 500 ppm silymarin; M2500: mash diet + 2500 ppm silymarin; P500: pellet diet + 500 ppm silymarin; P2500: pellet diet + 2500 ppm silymarin; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; VLDL: very low-density lipoprotein cholesterol; ^a,b,c Different letters in the superscripts of the same row indicate significant differences (P <0.05).

Serum Cardiac Parameter Analysis

The results of serum cardiac parameters are presented in Table 4.

The circulating levels of calcium and phosphorus were significantly lower in the CP group than in other groups on 28, 35, and 42 days (P<0.05). P500 and P2500 had significantly higher concentrations of calcium and phosphorus compared with CP (P<0.05). CP had a higher activity of CK enzyme compared with CM on day 42, and P500 and P2500 had a lower serum activity of CK compared with CP (P<0.05) (Table 4).

Table 4Circulating electrolytes and cardiac parameters (mean±SE) of broiler chickens (n=20) following dietary supplementation of pellet and mash diets by silymarin extract.

CM: control mash diet (0 ppm silymarin); CP: control pellet diet (0 ppm silymarin); M500: mash diet + 500 ppm silymarin; M2500: mash diet + 2500 ppm silymarin; P500: pellet diet + 500 ppm silymarin; P2500: pellet diet + 2500 ppm silymarin; CK-MB: creatine kinase MB; ^a,b,c Different letters in the superscripts of the same row indicate significant differences (P <0.05).

Electrocardiographic Analysis

All ECG leads (6 leads) of the broilers in various groups seen in Figure 1. The durations and amplitudes of all waves in lead II are presented in Table 5.