Effects of in-ovo injection of silver nanoparticles on immune system function in broiler chickens under oxidative stress induced by lipopolysaccharide

Stress, as a response to adverse stimuli, is difficult to define and understand mostly due to its nebulous perception. As Selye (1976) pointed out, “stress is the nonspecific response of the body to meet any demand”, whereas stressor is defined as “an agent that produces stress at any time”. Hence, stress represents the reaction of the animal organism (i.e., a biological response) to stimuli that disturb its normal physiological equilibrium or homeostasis. Thanks to their antioxidants, nutrients and anti-inflammatory properties silver nanoparticles are used in many developed countries (Bhanja et al 2015). The list potential applications of nanotechnology are very wide and diverse, but it is undoubtedly one of its most valuable applications in the development of therapeutic and pharmaceutical cases. Also, the use of silver nanoparticles in the treatment of deadly diseases like avian influenza supports further research on biological systems (Xiang et al 2011). Among inorganic materials, metal oxides such as TiO2, ZnO, MgO CaO, and Ag are of particular interest because they are stable in hard conditions and generally considered to be safe substances for humans and animals.

A total number of 592 eggs with an average weight of 50 g were purchased from a commercial Hubbard F15 broiler breeder flock aged 50 wk. The eggs, allotted to 4 treatments of 4 replicates with 37 eggs each, were set on the same floor to provide similar incubation conditions. Treatments including 2 doses of silver nanoparticles (20 and 40 mg) was injected via the egg holes using 1 mL insulin syringes equipped with disposable needles (21 gauge). The control group did not receive any treatment, and the sham control was injected with 1 ml phosphate-buffered saline (Triplett et al 2018). The injection holes were immediately covered using paraffin. At the end of d 18 of incubation, the eggs were transferred to the hatching cabinet. On the 21d of incubation, the hatched chicks were taken out of the incubator and after counting (checking for hatching) and weighting, half of the chicks were killed on the same day to check Hemoral immunity (G and M),white blood cell count and study of the desired genes (TNF-α, IL-6 and TGF-β). Among other chickens, those that were more uniform in weight with the desired treatment were transferred to the breeding period for 42 day and again in 4 treatments (treatments applied during incubation) and 4 replications with 20 broilers per replication. During 42 day, broilers were provided with unlimited water and food. To induce oxidative stress during the breeding period, 500 μg/kg live weight Salmonella lipopolysaccharide injected at 12, 24 and 48 hours before killed (Xi et al 2000).

While ovo injection, silver nanoparticles was significantly decreased hatchability in comparison to the control group (P<0/05). In line with our results (Goel et al 2017) found that the hatchability of the eggs injected with nanoparticles was significantly lower than the control group. The reason can be attributed to the smaller size of younger embryos at the 7th ED, injection method, location injection, injection depth, injection time, genetics, hen age, egg size and hatching conditions (Sun et al 2018; Pilarski et al 2005). In case of ovo injection of 20 and 40 mg silver nanoparticles, significantly increased carcass percentage compared to the control group at hatching and post-hatch period, respectively (P<0/05). (Bhanja et al 2015; Pineda et al 2012) proved that silver nanoparticles had positive effects on embryo weight. Due to the significant amount of antioxidant in the silver nanoparticles inside the egg and its antioxidant effect on energy efficiency during embryonic life, silver nanoparticles receiving groups had a higher body weight at hatching time. Higher birth weight makes it possible to increase feed intake and weight gain in these treatments.Upon ovo administration of 20 mg silver nanoparticles, spleen was increased significantly compared to the control group at hatching period (P<0/05). This is in line with a previous study of Goal et al (2017), who observed higher liver and spleen weights in 40 mg/kg silver nanoparticles group. The development of B and T lymphocytes initiates during embryogenesis in the bursa of Fabricius and thymus, respectively, and matures in the spleen until post-hatch (Erf 1997). These organs play an important role in imparting immunity. The cells produced in these organs differentiate into cellular immunity and humoral immunity, thus imparting immunity against different pathogens. Therefore, increased liver and spleen weight indicates a better immunological health status of in ovo silver nanoparticles supplemented birds. Ovo treatments did not affect the concentrations of immunoglobulin (IgG), (IgM), WBC counts and H/L ratio (P>0/05). Our results are consistent with previous studies of Pineda et al (2012) who showed the concentration of IgG and IgM were not affected by ovo injection of 10 and 20mg/kg silver nanoparticles. (Salari et al 2016; Saki and Salari 2015) showed silver nanoparticles increased in serum IgM and IgE, and increased in blood neutrophilic granulocytes. Rezaei Zarchi et al (2012) reported, feeding the rats for 28 days at doses of 25, 50, 100 and 200 mg / kg of silver nanoparticles had no significant effect on WBC and H/L. Differences in results may be due to differences in injection method, location injection, injection depth, injection time, genetics, hen age, egg size and hatching conditions. In comparison with the control and 40 mg silver nanoparticles group at hatching, there was significant up-regulation of TNF-α, IL-6 and TGF-β gene expression in 20 mg silver nanoparticles injected embryos,. Tumor necrosis factor-α (TNF-α) is a key cytokine involved in inflammation and immunity and the pro-inflammatory cytokine IL-6 induces the final maturation of B cells into antibody-secreting plasma cells, thereby increasing the secretion of immunoglobulins (Balkwill 2009; Mosmann 1989). Silver nanoparticles can interact with the immune system by binding and reacting with cells or proteins, thereby modulating the immune response. In the present study, higher expression of TNF-α gene in the livers was observed in ovo injected silver nanoparticles embryos. These findings also support earlier studies by Khan et al (2013), who showed the gene expression of IL-6 and TNF-𝛼 were affected by 50nm GNPs in the kidneys of rats. Also Vadalasetty et al (2018) reported that expression of TNF-α and NF-kB at mRNA were significantly up-regulated in the 50ppm silver nanoparticles group.

The results of this study suggest that silver nanoparticles improve the immune response of broilers by improving growth and increasing the relative expression of genes involved in immune function.