Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Abstract

The quality of avian semen is an important economic trait in poultry production. The present study examines the in vitro effects of non-thermal dielectric barrier discharge plasma on chicken sperm to determine the plasma conditions that can produce the optimum sperm quality. Exposure to 11.7 kV of plasma for 20 s is found to produce maximum sperm motility by controlling the homeostasis of reactive oxygen species and boosting the release of adenosine triphosphate and respiratory enzyme activity in the mitochondria. However, prolonged exposure or further increase in plasma potential impairs the sperm quality in a time- and dose-dependent manner. Optimal plasma treatment of sperm results in upregulated mRNA and protein expression of antioxidant defense-related and energetic metabolism-related genes by increasing their demethylation levels. However, 27.6 kV of plasma exerts significant adverse effects. Thus, our findings indicate that appropriate plasma exposure conditions improve chicken sperm motility by regulating demethylation levels of genes involved in antioxidant defense and energetic metabolism.

Figure 1

Effect of plasma on chicken sperm morphology. Semen of 60-week-old cocks was exposed to increasing potentials of plasma for 20 s. (a) 0 kV, (b) 11.7 kV, (c) 16.4 kV, (d) 22.0 kV, and (e) 26.7 kV. Semen smears were stained with Wright-Giemsa. Representative sperm morphology was photographed using a Leica DM 2500 microscope. Black arrows show normal spermatozoa and red arrows show abnormal spermatozoa. Scale bar: 5.0 μm. (f) Schematic of non-thermal DBD plasma treatment system.

Figure 2

Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of (a) NRF2, (b) KEAP1, (c) PRDX4, (d) ATP5A1, (e) AMPKα2, and (f) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of (g) NRF2, (h) KEAP1, (i) PRDX4, (j) ATP5A1, (k) AMPKα2, and (l) mTOR.

Figure 3

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: (a) NOX4, NRF2, and KEAP1; (b) SOD, CAT, and GPx; (c) PRDX1, PRDX3, PRDX4, and PRDX6; (d) ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5G1, ATP5G3, ATP5H, ATP5I, ATP5J, ATP5J2, ATP5L, and ATP5S; and (e) AMPKα2, AMPKβ2, AMPKγ3, and mTOR. (f) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1. The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of (g) NRF2, KEAP1, PRDX4, (h) ATP5A, (i) p-AMPKα/AMPKα, and (j) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. ^*p < 0.05 versus control; ^**p < 0.01 versus control, according to one-way ANOVA and LSD test.