44 Exploring the use of silver and diamond nanoparticles on sperm cell in vitro and chicken embryo in ovo

Abstract

In poultry industry, chick viability is a crucial factor determining profitability from fertilized egg to placement at the farm. However, decreases in fertility and hatchability have been observed. Recently, there has been renewed interest in the use of silver nanoparticles (Ag-NPs) due to their antimicrobial properties and growth-promoting ability, and diamond nanoparticles (D-NPs) due to their biocompatibility properties. The aim of the study was to evaluate the effect of silver and diamond nanoparticles on chicken embryo oxidative status, biochemical indices, and expression of immune-related genes and on sperm cell viability. The experiment was conducted in Ross 308 chicken embryos and Ross 308 cockerels. One hundred and fifty fertilized eggs were divided randomly into 5 groups (5 × 30). Fertilized eggs were injected with 50 mg/L Ag-NPs at volumes of 100 μL (group 1), 200 μL (group 2) or 50 mg/L D-NPs at volumes of 100 μL (group 3) or 200 μL (group 4), or received no nanoparticles (control; group 5) and incubated at 37°C and 55% humidity for 20 days. Then, chicken blood was collected and centrifuged to evaluate alkaline phosphatase (ALP), alanine transaminase (ALT), lactate dehydrogenase (LDH), glucose, urea, and free haemoglobin. Chicken embryo liver was used to evaluate antioxidant capacity (TAC) and chicken embryo spleen was used to evaluate expression of the immune-related genes interleukin-1β (IL-1β), toll-like receptor (TLR)4, TLR2, and TLR15. Semen was randomly divided into 1 control and 8 treatment groups and treated with 50 mg/L Ag-NPs: group A (0.1 ppm), group B (1 ppm), group C (5 ppm), group D (10 ppm) or 50 mg/L D-NPs: group E (1 ppm), group F (5 ppm), group G (10 ppm), and group H (20 ppm). Sperm viability was analysed using prestoblue metabolic assay. Data were analysed using PROC in GLM procedure of SAS 2014 (SAS Institute Inc.). Decrease in sperm cell viability was recorded in a dose-dependent manner. Sperm cell viability decreased (P < 0.005) as the concentration of Ag-NP or D-NP increased. Addition of 100 μL of Ag-NPs increased the growth rate of chicken embryo but not 200 μL of Ag-NPs or addition of D-NPs. Increases in ALP, ALT, LDH, glucose and urea enzyme were observed in a dose-dependent manner in both Ag-NPs and D-NPs. Addition of 50 mg/L Ag-NPs or 50 mg/L D-NPs increased (P < 0.001) TAC of chicken embryo as the volume increased. Additions of 200 μL of Ag-NPs, 100 μL of D-NPs, and 200 μL of D-NPs were haemolytic (P < 0.001) but addition of 100 μL of Ag-NPs was not. Additions of 100 or 200 μL of Ag-NPs or 100 μL of D-NPs downregulated IL-1β and 200 μL of D-NPs upregulated IL-1β compared with the untreated control group. Additions of 100 or 200 μL of Ag-NPs or 200 μL of D-NPs induced expression of TLR4 and TLR15. Furthermore, addition of Ag-NPs did not result in expression of TLR2. We concluded that administration of 50 mg/L Ag-NPs and 50 mg/L D-NPs in ovo improve immune status and administration of 100 μL of Ag-NPs improved the growth rate of chicken embryo. However, toxicity associated with 50 mg/L Ag-NPs and 50 mg/L D-NPs remains a concern and need to be addressed before use.