Nano silver in the diet is beneficial to the nutrition of broilers

This study was performed to investigate the growth regulatory effects of dietary supplementation with silver nanoparticles (AgNPs) and silver nitrate (AgNO3) on broilers. Thirty 15-day-old chicks were evenly divided into the control group (fed the baseline diet), the nano-silver group (fed the basal diet supplemented with 50 ppm / kg of AgNPs for 12 days) and the AgNO3 group (given the basic dietary supplement 100 ppm / kg Ag nitrate for 12 days).

Chickens fed with nano silver were shown to increase body weight and muscle weight, improve feed efficiency and increase ash digestibility, while Ag digestibility tended to increase but not significantly. Plasma triiodothyronine, muscle Ag and nitrogen content as well as a significant increase in the following mRNA levels in muscle tissue: insulin-like growth factor-1 (IGF1), glucose transporter (Glut1, Glut3) , citrate synthase (CS), and glutathione peroxidase (GPx), while atrogin-1 mRNA levels, synthetic fatty acids (FAS), acetyl CoA carboxylase (ACC), lactate dehydrogenase (LDH) and carnitine palmitoyl transferase 1A (CPT1A) constant. However, these chicks exhibited decreased plasma cholesterol, triglyceride and glucose levels. Supplementing the diet with AgNPs improved broiler growth performance.

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INTRODUCE

Silver (Ag) has been highly valued throughout history for its many properties beneficial to humans and animals. Compounds that exist, such as silver nitrate (AgNO3) and silver oxide (Ag2O), differ in solubility from easily soluble in water to difficult to dissolve in water.

Silver nanoparticles (AgNP) are small particles of metallic silver that are at least less than 100 nm in size. One of the most beneficial uses of AgNPs is as a potent antimicrobial agent because they are toxic to fungi, bacteria, viruses and algae (Cho et al. 2005; Percival et al. 2007).

Therefore, AgNPs are widely used in food preservation, household products, disinfectants, textiles and medical devices (Furno et al. 2004; Poynton et al. 2012).

Several theories have described established uses of nano-silver in the poultry industry:

• (1) The antibacterial properties of AgNPs affect the microbial population without inducing resistance and increasing the anabolic activity of the animals, helping to stimulate their growth and development [1]
• (2) nano-silver contributes to oxygen demand and increases metabolic rate, which further improves growth (Loghman et al. 2012);
• (3) nano-silver affects the gene expression of fibroblast growth factor (FGF), stimulates the proliferation and differentiation of vascular, muscle, and fibroblast cells; promotes angiogenesis through an increase in vascular endothelial growth factor (VEGF), required for the proliferation and organization of endothelial cells; increased levels of the pair box transcription factor (Pax7); promote growth of muscle satellite cells; and increase proliferation of cell nucleus antigens (PCNA) and DNA synthesis and repair; and these factors are essential in embryogenesis and are important for postnatal development (Sawosz et al. 2009); and
• (4) Silver nanoparticles increase cell immunity by stimulating heat shock protein synthesis (HSP) without activating the proinflammatory pathway (Atiyeh et al. 2007). Because the bioavailability of molecules increases as they are decomposed into nanoscale AgNP particles may be a promising new approach for better livestock efficiency. Large-scale studies have investigated the effect of nano-silver on chicken performance; However, the results obtained are inconsistent (Ahmadi et al. 2013; Ognik et al. 2016; Pineda et al. 2012; Sawosz et al. 2007). Therefore, this study was performed to investigate the effects of AgNPs and AgNO3 in the diet on growth performance, blood parameters and growth-related gene expression in broilers.

Materials and methods

Design and test samples

This experiment was conducted under the guidance of the Universities of Kagoshima and Kafrelsheikh. One hundred day old chicks were housed in an electrically heated battery incubator and supplied with water and a commercial diet (23% crude protein at 12.9 MJ / kg). On day 12, 30 male birds of similar body weight were selected and placed individually in an armored aluminum cage (49 × 39 × 59 cm). The birds were pre-adjusted for 3 days prior to treatment. Chicks were divided into three equal groups: control group (baseline diet), nano-silver group (baseline diet supplemented with 50 ppm / kg Ag NP) and silver nitrate group (basal diet supplemented). 100 ppm / kg Ag nitrate supplement). AgNPs supplied from Sigma are described as follows (Silver, Sigma-Aldrich, 576,832-5G, Lot # MKBN3581V, particle size <100 nm nanoparticles containing PVP as dispersant, 99.5% base metal, molecular weight 107.87 g / mol, surface area 5.0 m2 / g). Experimental diets were formulated using yellow corn and soybean meal (Table 1) and provided between 16 and 27 days old. The experiment was conducted in a temperature controlled room with a cycle of 14 am, 10 pm. Room temperature is kept at 25 ° C with a relative humidity between 50 and 70%. Body weight is recorded every 3 days and food intake is recorded daily. At the end of the experiment period, the birds were slaughtered and then slaughtered to measure the weight of the sternum and thigh muscles as well as belly fat. Blood samples were collected in liverized tubes, centrifuged at 6000 × g for 10 minutes at 4 ° C for plasma separation and stored at 20 ° C until analysis.

Digestibility

Feed and feces were dried in an oven at 105 ° C. After drying, samples were ground and passed through a 0.5 mm screen to facilitate analysis of dry matter content. Chromic oxide content was analyzed according to the procedure described by Guzman-Cedillo et al. (2017). Nitrogen content was determined by the Kjeldahl method. The ash digestibility was analyzed according to the procedure described by AOAC (2007). The following equation is used to calculate the digestibility of nutrients: Digestion rate (%) = 100 – [100 × (Cr2O3 diet / Cr2O3 manure) × (feces / nutrients in the diet eat)].

Mineral-vitamin premix provided per kg of feed: 154 mg Mn, 121 mg Zn, 176 mg Fe, 33 mg Cu, 1.1 mg I, 0.7 mg Se, 3784 mcg vitamin A, 0.066 mcg vitamin D, 110.11 mcg vitamin E, 12 mg vitamin B12, 1.37 mg retinol, 0.13 mg cholecalciferol, 6.50 mg riboflavin, 2.60 mg thiamine hydrochloride, 1.30 mg pyridoxamine hydrochloride, 0.03 mg cyanocobalamin, 10.40 mg D-pantothenic acid, 26.00 mg nicotinic acid, 1.05 mg vitamin K3, 0.52 mg pteroylglutamic acid, 0.78 mg choline chloride, 0.07 mg biotin and 2.54 g sucrose

Biochemical analysis

Total cholesterol (TC), triglycerides (TG), HDL, glucose, total protein and AST in plasma were measured using the Fuji DRY-CHEM 3500 automatic machine (Fuji Medical Systems, Tokyo, Japan) using the kits. Commercial available. Triiodothyronine plasma concentrations (T3) were measured using a commercial enzyme immunoassay kit (Elisa-T, International Reagents Corp., Kobe, Japan). Thiobarbituric acid reagent (TBARS) content in muscle and total muscle fat were measured using commercially available kits (BioDiagnostic, Egypt). The concentration of α-tocopherol in muscle tissue was determined using Shimadzu HPLC model LC6A (Tokyo, Japan) with Shim-Pack CLC-ODS column (6.0 × 150 mm) according to Faustman et al. (1989).

Real-time RNA extraction and PCR

Total RNA was extracted from the superficial muscle of the chest using the RNeasy® Fibrous Tissue Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s procedure. The concentration and purity of RNA was determined by spectrophotometric method (BioPhotometer, Eppendorf, Hamburg, Germany) to obtain the values ​​A260 and A280. The A260 / A280 ratio for all models is between 1.8 and 2.0. cDNA was synthesized with 800 ng RNA per 20 mL of reaction solution using the PrimeScript® RT Reagent Kit (Perfect Real Time, Takara, Shiga, Japan) and PC320 Program Temperature Control System (Astec, Fukuoka, Japan) under the following conditions: reverse transcription 37 ° C for 15 minutes, reverse transcriptase inactivation at 85 ° C for 5 seconds and refrigeration at 4 ° C for 5 minutes. Real-time PCR primers have been prepared as described previously. Gene expression was measured via real-time PCR using a 7300 Real Time PCR (Applied Biosystems, Foster, USA) system with SYBR® Premix Ex Taq ™ (Perfect Real Time, Takara, Shiga, Japan) and segments primers are specific to candidate genes (Table 2). The thermal recirculation conditions are as follows: 1 cycle at 95 ° C for 10 seconds and then 30 cycles at 95 ° C for 5 seconds and 60 ° C for 30 seconds. The expression of mRNA glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard, and it did not differ significantly between experimental groups. Gene expression results are displayed as% of the control value.

Statistical analysis

The magnitude of the difference was assessed by testing multiple ranges of ANOVA and Tukey. All statistical analyzes were performed using SAS V9 (SAS Inst. Inc., Cary, NC, USA). Data are reported as mean ± standard error of mean value (SEM). P value <0.05 is considered statistically significant.

Results

The effects of nano silver and Ag nitrate on body weight gain, food intake, food metabolism, muscle weight and belly fat, and digestibility are summarized in Table 3.

Table 3. Effects of feeding AgNPs and Ag nitrate on growth performance and digestibility in broilers

Values ​​bearing different letters on the same row have different meanings at P <0.05; value expressed as the mean ± standard error of the mean; n = 7 BWG increased body weight, FCR feed conversion ratio, FI feed amount, IBW initial body weight.

Feed intake (FI) and body weight gain (BWG) increased significantly (P <0.05), while the feed conversion ratio (FCR) decreased significantly (P <0.05) in chickens. The animals were fed Ag NP or Ag nitrate compared with the control group. Weight of sternum and thigh muscle increased significantly (P <0.01) in chicks fed Ag NP or Ag nitrate, although belly fat weight was not significantly different. The digestibility of ash was increased in chicks fed Ag nitrate compared with chicks fed Ag NP and in the control group. Crude protein and Ag digestibility increased in chicks fed AgNP or Ag nitrate, although these differences were insignificant. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C) cholesterol, glucose, aspartate amino transferase (AST), total protein (TP) and triiodothyronine (T3) are submitted shown in Table 4.

Table 4 Effects of nano silver and Ag nitrate feeding on plasma parameters in broiler chickens

Values ​​bearing different letters on the same row have different meanings at P <0.05; value expressed as the mean ± standard error of the mean; n = 7 AST aspartate amino transferase, HDL-C high density lipoprotein cholesterol, T3 triiodothyronine, total cholesterol TC, triglycerides TG, total TP protein

The plasma TG and TC were significantly lower in chicks fed nano silver and Ag nitrate than in the control group. Plasma glucose decreased significantly, and plasma T3 concentrations increased in chicks fed nano silver compared to chickens in the control group and the Ag nitrate group. HDLC, AST and total plasma protein were not affected by the Ag NP or Ag nitrate diet. Breast muscle fat, muscle α-tocopherol and TBARS were not affected by the Ag NP or Ag nitrate diet; however, the concentration of Ag in muscle tissue increased slightly (Table 5).

Table 5 Effects of feeding nano silver and Ag nitrate on muscle chemistry in broiler chickens

Values ​​bearing different letters on the same row have different meanings at P <0.05; value expressed as the mean ± standard error of the mean; n = 7

However, the nitrogen content in muscle tissue increased significantly (P <0.05) in chicks fed Ag NP and Ag nitrate compared with the control group. Insulin-like growth factor-1 (IGF1) and glucose transporter The level of mRNA Glut1 and Glut3 increased significantly in skeletal muscle (P <0.05) of the Ag NPs group compared to those in the other groups (Figure 1). . Insulin receptor (IR) and mRNA Glut8 levels increased, although this difference was insignificant. In contrast, the relative expression of the atrogin-1 gene was not significantly changed in the NP Ag or the Ag nitrate group (Figure 1).

Figure 1 Effect of nano silver and Ag nitrate provision on relative expression of IGF1 (a), atrogin-1 (b), insulin receptor (c), GLUT1

The genes (d), GLUT3 (e) and GLUT8 (f) in broiler muscles. The GAPDH gene is used as an internal reference for normalization. Results are expressed as a percentage relative to the expression of genes in the control group, with expression level equal to 100% and a mean reflection of ± SD for six birds. The mean values ​​in the columns with the above letters differ significantly from the control (P ≤ 0.05).

The mRNA levels of glutathione peroxidase (GPx) and citrate synthase (CS) increased significantly (P <0.05) in chicks fed Ag NP compared with the control group and the Ag nitrate group, while in the synthetic group. fatty acids (FAS), acetyl CoA carboxylase mRNA (ACC), lactate dehydrogenase (LDH) and carnitine palmitoyl transferase 1A (CPT1A) levels were unchanged in the Ag NP and Ag nitrate groups (Figure 2).

Figure 2 Effects of applying nano silver and Ag nitrate on relative expression of FAS (a), ACC (b), GPx (c), CS (d), LDH (e), and

CPT1A (f) genes in broiler muscles. Results are expressed as a percentage relative to the expression of genes in the control group, with expression level equal to 100% and a mean reflection of ± SD for six birds. The mean values in the columns with the above letters differ significantly from the control (P ≤ 0.05).

Discussion

The purpose of this study is to investigate the growth regulatory effects of nano silver and Ag nitrate in broiler diets and provide insights into the underlying mechanism. Our results showed a significant improvement in growth performance (weight gain, feed intake, feed conversion ratio, thymus and thigh weight) when introducing nano silver in the diet.

Andi et al. reported a similar improvement in growth performance in broilers fed with nano silver (Andi et al. 2011). However, many researchers did not find any significant changes after feeding AgNPs to birds (Ahmadi and Kurdestani 2010; Ognik et al. 2016; Pineda et al. 2012; Sawosz et al. 2007).

These conflicting results may be related to changes in nano silver size, dose, exposure time and method of preparation (electrical, electrochemical or chemical). The improvement in growth performance could be attributed to the bactericidal effect of AgNPs on the harmful gut bacteria; This effect promotes healthy gut tissues and improves the absorption of nutrients.

Silver nanoparticles also have anti-inflammatory effects because they modulate the expression of metallo-proteinase substrates, which are important proteolytic enzymes in various inflammatory and repair processes (Nadworny et al. 2010).

Another possible mechanism for the nano silver growth stimulating effect is the stimulation of the activity of digestive enzymes. To support this hypothesis, our results show improved digestibility of ash and Ag and increased muscle Ag concentrations of broilers fed AgNPs.

A similar effect was reported by Ahmadi and Rahimi (2011), who found an increase in Ag concentrations in the chest muscles of broilers fed Ag NP. Although the mechanism by which Ag stimulates digestive enzymes is unclear, chemically, Ag is similar to other metals, such as zinc and copper, and may have a similar effect.

Zinc and copper stimulate the growth of intestinal villi, thereby improving pancreatic absorption and enhancing metabolic activity (Li et al. 2001). Compared with nano silver, Ag nitrate showed a smaller improvement in growth performance and this result could be due to the small size of AgNPs (less than 100 nm), which allows bacteria to penetrate and accumulate effectively ( increased antimicrobial activity) and in the intestinal mucosa epithelium (enhances absorption) (Atiyeh et al. 2007). Despite these beneficial effects, Ag NP can induce ROS inducing oxidative damage to cells (Zhang et al. 2014).

This oxidative stress can trigger inflammatory cytokines and can cause DNA damage and mutation. Ahmadi and Mehrdad (2009) observed mild necrotic changes in the liver of chickens treated with high dose silver nanoparticles. However, Ognik et al. (2016) did not find a deviation from normal liver structure when using larger silver nanoparticles with lower dosage.

Indeed, this treatment strategy did not induce lipid peroxidation as evidenced by no significant changes in breast fat content, muscle α-tocopherol content, and TBARS in broilers fed brochures. Silver nano mode. However, Ahmadi and Kurdestani (2010) found a significant increase in the total number of oxidative stress enzymes and erythrocyte MDA in broilers fed with smaller NPs (5, 10 and 15 ppm). ).

In addition, LDH gene expression was unaffected by Ag NPs, indicating normal cell membrane integrity. In other studies using smaller Ag NPs over a longer exposure period, LDH activity was significantly reduced in chicken plasma (Ognik et al. 2016). Oxidative stress caused by Ag NP often induces a decrease in mitochondrial function, manifested by a decrease in LDH activity (Carr et al. 2000). Furthermore, in this study, the use of nano-silver caused the regulation of the GPx gene in muscle. This gene encodes the antioxidant enzyme GPx, which is needed to reduce ROS production and prevent cell damage caused by oxidative stress.

To further confirm the normal integrity of cellular organelles, in particular the mitochondria, which are the primary source of ROS and oxidative stress, the change in the expression of the mitochondrial intact marker is citrate synthase. (CS) was discovered after using nano-silver.

Interestingly, CS expression in the nano-silver group was significantly higher than in the control group, indicating the presence of intact mitochondria. Another evidence for the normal integrity of cells is the normal plasma concentration of the liver functional enzyme AST and the concentration of total protein (TP). In accordance with our data, Andi et al. (2011) showed that nano silver had no effect on AST, ALT and ALP. In contrast, Ahmadi (2012) reported that broilers fed Ag NP had significant changes in liver function enzymes, TP, albumin and gamma globulin, suggesting that oxidative stress induces chemical peroxidation. fat and damages the cell membranes, where enzymes are released in the liver. and albumin into the blood.

Reduced total plasma cholesterol and triglycerides while plasma HDL was not affected in this study indicated that the use of nano-silver can alter plasma lipid profile and this is an important result because of the positive correlation. between increased TC or TG and incidence of diseases such as coronary artery disease, hypertension and diabetes, as well as certain inflammatory diseases such as arthritis and dermatitis in humans.

The decrease in plasma glucose, while the unaffected plasma AST in this study meant that the use of nano-silver or Ag nitrate did not cause any stress to poultry and liver function was not affected. Treatment at this dosage is safe for poultry. Total protein in plasma may be indicated for immunity but was not affected by AgNPs or Ag nitrate in this study. Triiodothyronine in plasma is increased by Ag NPs; This could be a good explanation for improving body weight, as there is a correlation between this hormone and growth.

The use of silver nanoparticles helps to modulate the expression of fibroblast growth factors (FGF2) and VEGF, which are essential for proliferation, differentiation, angiogenesis and angiogenesis in defense cell tissue. (Hotowy et al. 2012). Because the increase in body weight increased primarily due to increased muscle weight (revealed by increased pectoral and thigh muscle weight) and not fat (no significant change in belly fat), there are the possibility that Ag NP could induce expression of growth genes. To test this ability, expression levels of genes (IGF1, IR, atrogen-1, Glut1, Glut3 and Glut8) involved in muscle growth were studied.

IGF1, Glut1 and Glut3 were increased, while atrogen-1 was not affected. In line with our findings, Bhanja et al. (2015) found a similar increase in relative gene expression of IGF1 when chicken embryos were injected with nano silver. An increase in IGF1 and glucose transporters was associated with a significant decrease in glucose levels and an increase in T3 concentrations in the plasma of nano-silver supplied broilers.

In contrast, Sawosz et al. (2009) reported that Ag NPs did not affect blood glucose concentrations in chickens. Elevated IGF1, Glut1, Glut3 and T3 may account for an increase in muscle weight and hence body weight. We also examined changes in the expression of fat metabolism-related genes (FAS, ACC, CPT1A) after using nano-silver and found no significant changes in these genes or in belly fat weight. However, there is a significant decrease in TC and TG in plasma. In contrast, previous studies showed a significant increase in belly fat and blood lipids (Ahmadi 2012) or no significant effect after chicks were fed a diet containing Ag NP (Sawosz et al. 2009). It was concluded that the addition of Ag NP in the diet improved growth performance of broilers.

Reference source: Beneficial effects of dietary silver nanoparticles and silver nitrate on broiler nutrition

Ahmed A. Saleh1 & Mohammed A. El-Magd2