The protective effect of zinc oxide nanoparticles on boar sperm during preservation at 17 °C

Abstract

More than 90% of spermatozoa of boars in pork producing countries is stored in liquid at 17 °C; however, the quality of these spermatozoa is affected by bacterial breeding and oxidative damage. This study analyzed sperm quality and sperm capacitation after storage to study the effects of the effects of ZnO nanoparticles (ZnO NPs) supplementation on seminal plasma (SP)-free sperm preservation. We investigated the effects of adding 20, 50, 100 and 200 μg/mL of ZnO NPs to a seminal free boar sperm diluent over a 7-day period at 17 °C to assess the changes in non-capacitated/capacitated sperm quality parameters, antioxidant capacity, ATP content and extent of protein tyrosine phosphorylation. The addition of different doses of ZnO NPs to stored sperm did not induce significant effects on the sperm motility and ATP content when compared to the sperm without ZnO NPs treatment. However, the addition of 50, 100, 200 μg/mL ZnO NPs to stored sperm improved total antioxidant capacity (T-AOC) and CuZn-superoxide dismutase (CuZn-SOD) (p < 0.05). ZnO NPs also reduced the malondialdehyde (MDA) content of the preserved sperm (p < 0.05). Moreover, our results indicate that the supplementation of 50 μg/mL ZnO NPs to preserved sperm improved the sperm membrane integrity (p < 0.05). ZnO NPs exerted protective effects on protein tyrosine phosphorylation, especially with regards to membrane proteins. Following incubation and capacitation, sperm exhibited good levels of protein tyrosine phosphorylation and ATP levels with high T-AOC and CuZn-SOD activity and low MDA content. ZnO NPs exerted protective capacity to a preservation extender used for SP-free boar sperm during storage at 17 °C. The optimal concentration of ZnO NPs for preservation extender was 50 μg/mL.

Keywords: capacitation; oxidative stress; protein; sperm.

Figure 1. The effects of ZnO NPs on the total motility (A) and plasma membrane integrity (B) of boar semen preserved at 17 °C. The data are expressed as the mean ± SD. Different lowercase letters demonstrate significant differences (p < 0.05), whereas the same lowercase letters denote insignificant differences. N-Cap and Cap refer to sperm under respectively non-capacitating conditions and capacitating conditions.

Figure 2. The effects of ZnO NPs on the ATP content (A a), T-AOC (B b), CuZn-SOD activity (C c) and MDA content (D d) of boar semen preserved at 17 °C for 7 days. The data are expressed as the mean ± SD. Different lowercase letters demonstrate significant differences (p < 0.05), whereas the same lowercase letters denote insignificant differences. N-Cap and Cap refer to sperm under respectively non-capacitating conditions and capacitating conditions.

Figure 3. Western blot analysis of PTP on total protein when exposed to different concentrations of ZnO NPs. Western blot analysis was performed using an anti-phosphotyrosine antibody (A, a, C, c) and (B, b) is protein gray value. β-actin was used as an internal control. The experiment was performed in triplicate (n = 3, p < 0.05). Immunolocalization of tyrosine-phosphorylated protein in boar sperm stored at 17 °C with different concentrations of ZnO NPs (C, c). N-Cap and Cap refer to sperm under respectively non-capacitating conditions and capacitating conditions.

Figure 4. Western blot analysis of PTP on triton-insoluble protein when exposed to different concentrations of ZnO NPs. Western blot analysis was performed using an anti-phosphotyrosine antibody (A, a) and (B, b) is protein gray value. β-actin was used as an internal control. The experiment was performed in triplicate (n = 3, p < 0.05). N-Cap and Cap refer to sperm under respectively non-capacitating conditions and capacitating conditions.

Figure 5. Western blot analysis of PTP on triton-soluble protein when exposed to different concentrations of ZnO NPs. Western blot analysis was performed using an anti-phosphotyrosine antibody (A) and (B) is protein gray value. β-actin was used as an internal control. The experiment was performed in triplicate (n = 3, p < 0.05). N-Cap refer to sperm under non-capacitating conditions.