Performance, intestinal microbial population, immune and physiological responses of broiler chickens to diet with different levels of silver nanoparticles coated on zeolite

Abstract The aim of this study was to evaluate the effects of different levels of silver nanoparticles (Ag-NPs) coated on zeolite on performance, gastrointestinal microflora, heterophile to lymphocyte ratio and humoral immune system responses of broiler chickens. Three hundred seventy-five chicks (Cobb 500) in a completely randomised design were distributed to five diets: 1) basal diet, 2) basal diet containing 1% zeolite and 3, 4 and 5) basal diet containing 1% zeolite coated with 25, 50 and 75 ppm Ag-NPs. Performance traits were recorded and blood samples were taken to determine antibody response against Newcastle on day 14, infectious bursal disease on day 21 and serum immunoglobulins (Igs) were determined on day 42. Heterophile to lymphocyte ratio was evaluated on days 28, 35 and 42. The result showed that feed conversion ratio (FCR) was significantly decreased in Ag-NP treatments compared with control group (p < .05). Total anaerobic bacteria and the population of Escherichia coli in ileum and caecum were decreased (p < .05). The highest number of lactobacillus was observed in NS50 (p < .05). Antibody response against Gumboro was decreased as well as the amount of IgY antibody in Ag-NPs treatments. Significant differences were observed in heterophile to lymphocyte ratio on day 28 and 35 and the highest amount was observed in NS50 group (p < .05). In conclusion, although this study showed that using Ag-NP dietary coated on zeolite could improve gut microflora, but Ag-NPs could also have side effects on the immune mechanism of broiler chickens. Highlights Silver nanoparticles (Ag-NPs) coated on zeolite improved feed efficiency at different levels. Ag-NPs (50 ppm) coated on zeolite reduced Escherichia coli number and decreased lactobacillus number. Ag-NPs (50 ppm) coated on zeolite decreased antibody response against Gumboro and amount of IgY antibodies.


Introduction
Intensive poultry farming and industrial poultry production have been achieved from the second half of the 20th by shortening the productive cycle and increasing the production capacity. In the past 50-60 years, the most important goal of broiler breeding is to increase profitability of broiler meat production and most achieved in poultry have been on performance traits. High-intensity poultry production requires fast-growing strains, usually at high stocking densities. With this type of husbandry, flocks are highly susceptible to disease. Studies with wild birds showed that increased productive effort reduces specific immune responses, parasite resistance and parent survival (Van der Klein et al. 2015). It was determined that genotypes with higher body weight have weaker antibody response than lower body weight line of broilers (Boa-Amponsem et al. 2000). On the other hand, gastrointestinal tract and the gut microbial ecosystem with 10 7 -10 11 bacteria per each gram play vital roles in nutrition absorption, development of immunity and disease resistance (Pickard et al. 2017). Alterations in the microbial community may have adverse effects on feed efficiency, productivity and health of chickens (Wu and Wu 2012). IgA production is highly sensitive to the presence of intestinal microbes because regarding the mechanisms of IgA production, it is clear that it is highly dependent on bacteria, because mucosal IgA is almost absent in germ-free animals and is rapidly induced upon bacteria colonisation and IgA has able to neutralise viruses and exotoxins (Macpherson et al. 2015). Gut microbial population was also found to be the major driving force to produce mucosal IgA (Crabb e et al. 1968). During the last few decades, antibiotics have been widely used as growth promoters to improve feed efficiency and animal growth through the modulation of gut microbial population and the host's immune system. The ban on antibiotics for growth promotion within the European Union in 2006, caused more interest in finding alternatives to antibiotics in poultry production (Torok et al. 2011). Therefore, any factor and approach that can improve this microbial ecosystem could be considered as immunomodulators and also, one of several approaches to increase immune responsiveness in high-intensity production in poultry is to supplement rations with feed additives (Rauw 2012;Hashemi and Davoodi 2012). The common feed additives used in poultry industry are herbal plants, antioxidants, emulsifiers, binders, enzymes, pH control agents, probiotics and prebiotics .
Recently, using silver as an alternative antimicrobial agent has been noticed by researchers. Metallic silver compounds and silver ions have been known to have unique antibacterial properties. It has been used since ancient times to treat burns, wounds and bacterial infections (Rai et al. 2009). Nanoparticles (NPs) are promising antimicrobial agents, but the studies on livestock species used as antimicrobial growth promoters are scarce. More studies are required to recommend the nanomaterials as new generation antimicrobial feed additives in farm animals as a substitute for in-feed antibiotics and other antimicrobial agents (Patra 2019). Nanotechnology is one of the newest branches of science that make it possible to manufacture silver in the nano size (1-100 nm). NPs are more reactive that larger particles because of the larger surface area and exposing more atoms on the surface of them. Furthermore, silver NPs (Ag-NPs) have unique biological properties and broad-spectrum antibacterial activity against microorganisms (Lara et al. 2010). Because of these strong abilities of Ag-NPs to eliminate bacteria, and disability of pathogens to resist these particles, it is supposed that Ag-NPs may affect microbial populations of gastrointestinal tract and hereby may improve immune system and productive purposes (Varner et al. 2010). Nanobiotechnology is a growing field in animal and veterinary sciences for various practical applications including diagnostic, therapeutic and nutritional applications. Recently, nanoforms or NPs of essential minerals have been explored for growth performance, feed utilisation and health status of animals (Patra and Lalhriatpuii 2020).
Also, it has been reported that the use of the Ag-NPs in poultry has great potential (Sawosz et al. 2007) but there are only a few investigations regarding the use of Ag-NPs and zeolite in poultry nutrition. Also, zeolites are crystalline aluminosilicates with physicochemical effects such as permitting ion exchange, absorption, diffusion, molecular sieving, dehydration, reversible dehydration and catalysis that caused using of these products in animal nutrition (Eleroglu and Yalçin 2005). Mallek et al (2012) reported that supplementing zeolite to broiler diets has positive effects on performance, organoleptic parameters and mainly increased level of Omega 3 fatty acid. Besides, Ag-NPs coated on zeolite were demonstrated to use as feed additives in several studies because of their positive effects on meat quality, feed conversion ratio (FCR) and hepatic enzymes of broiler chickens .
Thus the aim of this experiment was to study the effects of Ag-NPs coated on zeolite on microbial community composition of ileum and caecum, heterophile to lymphocyte ratio as an index of stress, humoral immunity and morphometric analysis of the gut in broiler chickens.

Diet and feeding
A total of 375 one-day-old (Cobb 500) broiler chicks (half male and half female) were obtained from a local commercial hatchery. Broilers were vaccinated for infectious bronchitis (on d 1), Newcastle disease (on day 7) and Gumboro live vaccine (on day 14). Chickens were randomly allocated in 5 experimental treatments for 6 weeks. Each treatment is arranged in 5 replicated of 15 broilers each. All procedures were approved by the ethics committee for animal experiments at the Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. The temperature was regulated at 32 ± 1 C during the first week and it decreased 1 C every 3 d. The temperature was fixed at 23 ± 1 C for the rest of the experiment. The experimental treatment diets were: 1. Basal diet without any addition (C). 2. Basal diet containing 1% zeolite (Z). 3. Basal diet containing 1% zeolite coated with 25 ppm Ag-NPs (NS25). 4. Basal diet containing 1% zeolite coated with 50 ppm Ag-NPs (NS50). 5. Basal diet containing 1% zeolite coated with 75 ppm Ag-NPs (NS75).
The zeolite that used in this experiment obtained from well-defined zeolitic stratigraphic units from the region of Semnan province, Iran. Zeolitic rock was pulverised and sieved to make particles in the size of 1-2 mm. distilled water was used to wash and remove contaminations from the particles and subsequently dried at 105 C all over the night in the oven. The chemical formula of pure zeolite was (K 2 , Na 2 , Ca and Mg) 3 Al 6 Si 30 O 72 . 24H 2 O.
Nanosized silver particles (Ag-NPs) with a maximum diameter of 50 nm were coated on zeolite and received as a gift from Dr. D. Davoodi at Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran. Briefly, the preparation of coating method on zeolite with desired properties was that the zeolite was first stirred in distilled water with a stirrer, and then, the prepared nanosilver (20-50 nm) with the desired percentage added to the mixture after adjusting the pH and Stirring was continued for a further 30 min at 15 C. The stabilisers were gradually added to the mixture and stirred until the resulting mixture colour became brown. After settling of the precipitate dried at ambient temperature.
Zeolites were analysed by X-ray Fluorescence (XRF) technique to determine their chemical composition and the XRF data collected on a PHILIPSPW1480 XRF spectrometer with Rh tube (Nikpey et al. 2013) ( Table  1). The morphology studied by field emission scanning electron microscopy (FESEM, Mira, 3-XMU) and the materials composition investigated by energy-dispersive X-ray spectroscopy (EDX) and elemental mapping analyses at Razi Metallurgical Research Centre, Iran ( Figure 1). To measure the silver (Ag) content of zeolite we used the method described by Kulthong et al (2010). Briefly, 0.2-0.3 g of sample digested in 5 mL of 14.4 M HNO 3 by a microwave digestion system to dissolve all the silver content. The microwave irradiation cycles were 250 W (5 min), 400 W (5 min) and 600 W (5 min). Then the digested sample was cooled and diluted up to 25 mL with deionised water to enable quantification of silver by a graphite furnace atomic absorption spectroscopy or GFAAS (Perkin Elmer Analyst 300, Waltham, MA).
The basal diet was in mash form and formulated for starter (1-21 d), and grower (22-42 d) periods. The <5 ppm a XRF PHILIPS PW1480 XRF spectrometer with Rh tube. b LOI: loss on ignition. c The silver content was measured by a graphite furnace atomic absorption spectros-copy (GFAAS). composition and ingredients of basal diets are shown in Table 2. Birds had free access to feed and water.

Sample collection and measurement
Feed consumption was recorded a weekly basis, by monitoring the feed offered and amounts remained at feeders at the end of the week. Individual bird weight was also recorded weekly. FCR was calculated as the following: FCR ¼ average feed consumed/average live weight. On day 21 and 42, two birds of each replication were killed. The gastrointestinal tract was removed and immediately the contents of the caecum and ileum were diluted 10-fold using sterile phosphate buffer solution (BPS) (pH 7.0) and homogenised on a vortex for 3 min. The dilution continued serially for each sample from 10 À1 to 10 À9 and then subsequently plated to the enumeration of bacterial groups.
Total anaerobes, Lactobacillus and Escherichia coli were enumerated respectively using duplicated plate count agar (PCA), MRS agar and Mac Conkey agar according to Tuohy et al (2002). Anaerobic incubation was achieved using sealed anaerobic jars. The plates were incubated at 37 C for 24-72 h and then the numbers of colonies were counted. The results of the experiment were expressed as log 10 colony-forming units per each gram of digested content of the intestine.
A total of 50 birds (10 birds per treatment) was used for determining the proportion of heterophile to lymphocyte (H/L) as an index of stress on day 28, 35 and 42. Blood samples (1-2 mL) were collected from the wing vein and immediately transferred to heparinised containers. Two drops of blood samples placed on the slide and a blood smear were made. After drying stained with Wright-Giemsa stain. Samples were examined under a compound microscope (Nikon, Eclipse E200-LED, Tokyo, Japan) (Carroll et al. 1996). Standard avian guidelines for manual haematology were used to identify cells. Heterophils and lymphocytes were counted to a total of 100 cells.
The titre of antibody activity against Newcastle disease was measured by conventional hemagglutination inhibition (HI) assay on day 14 (7 d after postimmunisition). HI assay was done according to the procedure of OIE and the titres of antibodies against Gumboro were measured on day 21 (7 d after postimmunisition) using a commercial Enzyme-linked immunosorbent assay (ELISA) kit (Razi Company; Arak, Iran). To measure the amount of plasma immunoglobulin (Ig), blood samples were collected from the wing vine on day 42 and centrifuged at 2000 rpm for 10 min. Blood plasma stored at À20 C for further analysis. The concentrations of immunoglobulin IgY, IgM and IgA (Y, M and A) were measured by commercial ELISA kits (Kamiya Biomedical Company, Tukwila, WA).

Statistical analysis
All data were initially checked for normality and homogeneity of variance using Bartlett and Kolmogorov-Smirnov tests. All bacteria enumeration data were transformed to log 10 CFU/mL. Statistical analyses were subjected to ANOVA using the general linear model procedure (GLM) (SAS Institute Inc. 2003). When significant differences occurred (p < .05), means were separated by Tukey's Studentized range test (SAS Institute Inc.1989).

Results
The effect of dietary treatments on performance traits including body weight and feed intake indicates that diet containing Ag-NPs and zeolite have not significant difference with the control group (p >.05). Moreover, FCR was significantly higher in Ag-NP treatments compare with the control group (Table 3) (p < .05). The population of total anaerobes bacteria of ileum on day 21 did not show any significant difference between treatments, while it significantly decreased on day 42 in The ratio of H/L on day 28 increased in both NS50 and NS75. It was also increased on day 35 in NS50 significantly.
Experimental treatments did not have any significant effect on the titre of antibody against Newcastle but Ag-NPs significantly decreased antibody against Gumboro compared to the control diet (p < .05). Moreover, the concentration of IgY and IgA of plasma significantly decreased by increasing the level of Ag-NPs (p < .05) while the amount of IgM did not show any significant difference (

Discussion
Out of all the metals with antimicrobial properties, it was found that Ag has the most effective antibacterial action and is least toxic to animal cells (Ahmed et al. 2016). Many studies have demonstrated the antibacterial activity of Ag-NPs even in low concentrations (Rai et al. 2009). The mechanisms of action of Ag-NPs on bacteria are not totally investigated yet but it is known that silver binds with sulphur-containing proteins of the bacterial cell wall and cell membrane and inhibits the respiration process (Slavin et al. 2017). Moreover, silver ions interact with the phosphoruscontaining compounds like DNA and inhibit replication (Silver et al. 2006). It has also been attributed that Ag-NPs can inactive phosphomannose isomerase enzyme. This enzyme catalyses the conversion of mannos-6-phosphate to fructose-6-phosphate which is an important intermediate of glycolysis as the most  common pathway in sugar catabolism of bacteria. Moreover, in the E. coli bacteria, some holes in the cell membranes were observed after contact with Ag-NPs (Choi and Hu 2008).
In addition, Pineda et al. (2012) reported that in ovo injection of Ag-NPs reduced feed intake and body weight gain in chickens however, no concurrent effect on FCR was observed. Although there are no negative effects on feed intake has been reported as a result of Ag-NP supplementation in the feed or via the drinking water (Ahmadi 2012;Pineda et al. 2012).
Exact mechanism of NP synthesis in biological systems has not been completely described. The mechanism of intracellular and extracellular production differs from one organism to another. In ovo administration of Ag-NP in chickens reduced the counts of lactic acid bacteria and lactose-negative enterobacteria, but did not affect numbers of anaerobic, coliform bacteria, enterococci and Clostridium perfringens in the caeca (Pineda et al. 2012).
It has been suggested that Ag-NP would exhibit their greatest antimicrobial activity when birds are exposed to stressful conditions, e.g. when levels of pathogenic bacteria are high (Patra 2019).
Therefore, as it was observed in this study, the population of anaerobic bacteria was decreased which are sensitive to oxygen concentration while the colonies of lactic acid bacteria (non-obligatory anaerobic), increased in competition with anaerobes (Singh et al. 2008). Moreover, it was shown that Gram-negative bacteria are more sensitive to treatment with NPs (Coccini et al. 2013) that can explain the reason for significant reduction in E. coli. Moreover, in this study, the amount of H/L ratio increased significantly in NS50 (Days 28 and 35 of age) and NS75 (Day 35 of age).
Moreover, Ag-NPs can cause damage to different organs and tissues like liver cells.
It was demonstrated that stressors, such as food or water deprivation, temperature extremes, constant light and exposure to novel social situations increase a number of heterophils while decrease lymphocytes.
The mechanisms of these changes in the number of immune cells of birds are not clearly known, but it may due to the effects of stress on the adrenal corticotropic hormone and corticosterone and cytokines.
The results of this study indicated that Ag-NPs is a stressor factor in young chickens (day 28 and 35) but it did not affect the H/L ratio at the age of 42 d. Toxicity effects of Ag-NPs were studied in the zebrafish embryonic model. The results showed that all sizes of colloidal Ag-NPs (3, 10, 50 and 100 nm) could induce toxicity and the rate of mortality increased by size reduction of particles (80%, 64%, 36% and 3%, respectively). Overall, there are other different parameters that can be responsible for toxicity of Ag-NPs, such as, concentration, stability and chemistry.
Similar to the results of this study in humoral immunity parameter, Pineda et al. (2012) reported that the concentration of plasma IgY was reduced by using 10 and 20 ppm of Ag-NPs. Vadalasetty et al. (2018) also showed that the concentration of IgY and IgM were lower in the chickens received Ag-NPs compared to the control group. There are several possible explanations for this result. The reason for reduction in the levels of IgY and IgA in this study may be explained by the fact that the changes in microbial colonisation occurred by Ag-NPs received from the diet. Another possible explanation for this is that Ag-  .0088 1 Control (Basal diet); 2 Diet containing 1% zeolite; 3 Diet containing 1% zeolite coated with 25 ppm nanosilver; 4 Diet containing 1% zeolite coated with 50 ppm nanosilver; 5 Diet containing 1% zeolite coated with 75 ppm nanosilver; 6 Standard error of means. Values with different superscripts in the same column for each section are significantly different (p < .05).
NPs may impair intestinal transportation of nutrients such as sugar, amino acids, trace elements and vitamins and the deficiency of nutrients can decrease antibody formation. It should be noted that formulation of broiler diets consists of an array of ingredients that match a desired nutrient profile at the minimum cost. The nutrient profile used is based on research or field observations evaluating the economically important production function of interest. This production function is typically BW, feed conversion, or breast meat accretion, not immunity or disease resistance (kidd 2004). There are, however, other possible explanations.

Conclusions
In conclusion, the results of this study investigated that Ag-NPs improved the gastrointestinal microflora properly. Despite its potential effect on digestive microbial biodiversity and function, other effects of nanosilver related to host physiological statuses, such as the immunological status and stress factors may negatively be influenced by silver NPs.