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

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.

Sperm quality of male chickens directly affects fertility and hatchability 1 , which are the most important economical requisites of poultry breeding because they determine the profitability of production. Chicken spermatozoa acquire the motility as soon as they leave the testis. Sperm requires high levels of adenosine triphosphate (ATP) as the energy source to ensure motility, which shows the ability of sperm to move properly and efficiently through the female's reproductive tract to reach the egg 2 . The middle region of sperm contains numerous mitochondria to supply energy for flagellum movement. These biological features indicate a central role of energetic metabolism in sperm motility to ensure the successful fertilization and reproduction of male chickens.
Non-thermal dielectric barrier discharge (DBD) plasma is generated at atmospheric pressure in gas when a high voltage of time-varying waveform or short duration pulses is applied between two electrodes, which produces electrically safe plasma without substantial gas heating [3][4][5] . Advancements in non-thermal DBD plasma systems have promoted the potential for various medical and biological applications including sterilization, blood coagulation, wound healing, tissue regeneration, dental treatment, promotion of cell proliferation and transfection, and cancer treatment [5][6][7][8][9] . Our previous studies apply the non-thermal DBD plasma in the promotion of soybean sprout growth 10 and the embryonic development of chicken during the early stage of incubation 11 . However, studies on the effects of non-thermal DBD plasma on sperm quality remain to be conducted. Thus, the present study addresses whether plasma treatment can affect chicken sperm parameters.
When cells or tissue surfaces are exposed to non-thermal DBD plasma a variety of biologically active reactive species are generated, particularly, reactive oxygen species (ROS) 4,12,13 . DBD plasma selectively initiates and amplifies ROS signaling to enhance cell proliferation and differentiation 14 ; however, excessive intracellular ROS formation after DBD plasma treatment leads to cell deficiency 15 . ROS homeostasis plays an important role in sperm physiological processes, whereas elevated concentrations of ROS result in sperm morphological pathology, ATP depletion, lipid peroxidation, and loss of motility and viability 16 . Thus, the balance between ROS generation and scavenging activity is required to ensure optimum sperm quality. Here, we hypothesize that non-thermal DBD plasma may affect chicken sperm quality by mediating the ROS balance and energetic metabolism.
Sperm DNA methylation is a key regulator of transcription and contributes to altered gene expression that deteriorates semen properties, sperm counts, and motility 17,18 . Genes that promote development are generally hypomethylated in the sperm 19 , whereas hypermethylation can lead to impaired spermatogenesis and poor sperm quality 20 . Changes in sperm methylation patterns in response to toxic exposure can have severe consequences on chromatin integrity and gene expression profiles 21,22 . The pronounced hypermethylation observed in the radiation-exposed spermatozoa implies defective chromatin condensation, which in turn produces morphologically abnormal spermatozoa 23 . Hence, these findings prompt that changes in sperm quality are potentially influenced by changes in DNA methylation levels following plasma exposure.
Therefore, the effects of non-thermal DBD plasma on chicken sperm quality and the mechanisms that regulate the antioxidant defense and energetic metabolism system of sperm through cytosine methylation remain to be elucidated.

Results
Sperm quality. Our results show that sperm motility is improved within 40 s of non-thermal plasma exposure to 11.7 kV, peaking at 20 s (Table 1). There are no significant differences in sperm viability, acrosome integrity, DNA integrity, and total fertility between the control group and the plasma-treated groups within 40 s. However, plasma exposure for 60 s or longer has significant impairments on the sperm quality. These indicate a time-dependent effect of plasma treatment on sperm quality. Moreover, plasma is found to influence sperm motility in a dose-dependent manner. At 20 s of exposure, maximum sperm motility is obtained at 11.7 kV, with an increase of 0.23-fold (p < 0.001) compared with that of the control group (Table 2). However, sperm motility starts to decrease at plasma potentials higher than 16.4 kV. In addition, spermatozoa exposed to plasma at higher than 16.4 kV for 20 s exert inhibitory effects on viability, integrity of acrosome and DNA, and total fertility. However, exposure to 11.7 kV of plasma shows no significant changes in the abovementioned sperm properties ( Table 2).
The optical microstructure of spermatozoa exposed to different plasma potentials for 20 s is determined. Spermatozoa from the plasma-treated group at 11.7 kV and the control group show no significant differences in the ratio of abnormal spermatozoa, 84.44% of spermatozoa in the control group and 85.71% in the 11.7 kV group are normal and complete (Fig. 1a,b). However, exposure to potential higher than 16.4 kV exerts harmful effects on sperm morphology and produces a significant number of crooked spermatozoa (Fig. 1c-e). Plasma exposure at 27.6 kV shows the highest ratio of abnormal spermatozoa (94.44%; Fig. 1e), which increases by 5.07-fold compared with that of the control group (15.56%).  Table S1). Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities increase within 40 s of plasma treatment. However, prolonged plasma exposure shows contrary effects on those parameters. Plasma treatment shows a dose-dependent effect on ROS and MDA levels and antioxidant enzyme activity in chicken spermatozoa (Table 3). At 20 s of exposure, minimal levels of ROS and MDA and maximum activities of antioxidant enzyme are observed at 11.7 kV, with decreases of 0.10-(p = 0.019) and 0.33-fold (p = 0.004) in ROS and MDA, respectively, and increases of 0.71-(p < 0.001), 0.60-(p < 0.001), and 0.42-fold (p < 0.001) in SOD, CAT, and GPx compared with those of the control group. On the other hand, further increases in plasma potential (16.4 to 27.6 kV) result in continuously elevated ROS and MDA levels but decreased SOD, CAT, and GPx levels ( Table 3).   ATP and mitochondria respiratory enzyme. ATP and nicotinamide adenine dinucleotide hydrogen (NADH) levels, and enzymatic activities of cytochrome c oxidase and ATP synthase increase within 40 s of plasma exposure at 11.7 kV, but longer plasma exposure duration decreases ATP production and respiratory enzyme activity (see Supplementary Table S2). A dose-dependent effect on ATP, NADH, and mitochondria respiratory enzyme in the spermatozoa is observed in the plasma-treated group. Maximum ATP, NADH, and respiratory enzyme levels are observed at 20 s of exposure at 11.7 kV, with increases of 0.61-(p < 0.001), 0.38-(p < 0.001), 0.63-(p < 0.001), and 0.45-fold (p < 0.001). Further increases in plasma potential decrease the ATP, NADH, and respiratory enzyme levels ( Table 4).  (Table 5). However, methylation levels of NRF2, PRDX4, ATP5A1, and mTOR in the group treated with 27.6 kV of plasma show corresponding increases of 0.16-, 0.14-, 0.12-, 0.10-fold, whereas KEAP1 and AMPKα2 methylation levels decrease 0.13-and 0.11-fold, respectively (Table 5). In addition, the variations in average methylation levels of ATP5A1, mTOR, and AMPKα2 for CG type are found to be more pronounced than those of CHG and CHH in the spermatozoa following plasma exposure ( Fig. 2g-l).

Discussion
Our results show that plasma exposure at 11.7 kV within 40 s results in an increase of sperm motility, without corresponding significant changes in sperm morphology, viability, integrity of acrosome and DNA, and total fertility. However, exposure time longer than 1 min or treatment with higher potentials reduces sperm parameters. In the previous study, we find that non-thermal DBD plasma affects chicken embryonic development in a dose-dependent manner during the early stage of incubation 11 . In vitro experiments using fibroblasts 24 , endothelial cells 3 , epithelial cells 5 , myoblast cells 25 , and tumor cells 26 demonstrate the dose-dependent effects of plasma. Furthermore, longer durations of plasma exposure increase apoptotic cell number 27 , decrease cell viability 28 , and cause endothelial cell toxicity 3 . Based on these findings, we conclude that plasma affects chicken sperm motility in a time-and dose-dependent manner. We also show that optimal plasma conditions for increasing sperm motility are plasma potential of 11.7 kV and exposure time of 20 s. Sperm quality is affected by ROS directly in a dose-and time-dependent manner 29 . Diffusion of plasma-generated ROS or stimulation of intracellular ROS-generating mechanisms as a result of non-thermal DBD plasma treatment 30 has been suggested to regulate sperm quality and physiology 31 . ROS homeostasis between generation and scavenging activity is favorable to the sperm quality 16 . Low physiological concentrations of ROS in vivo are known to stimulate sperm capacitation and acrosome reaction 1,31 , whereas excessive ROS accumulation results in lipid peroxidation of sperm membrane, which in turn leads to impaired viability, motility and acrosome integrity, DNA damage, and infertility 16,32,33 . Our results show that plasma exposure at 11.7 kV for Exposure potential (kV) ATP (nmole/10 9 spz) NADH (U/10 9 spz) Cytochrome c oxidase (mU/10 9 spz) ATP synthase (mU/10 9 spz) 20 s maintains relatively low ROS level and suppresses MDA activity, which are mediated by the upregulation of antioxidant enzyme activity of SOD, CAT, GPx, and PRDX, protecting against oxidative damage and increasing the viability of spermatozoa 1,26,34 . Furthermore, we find the optimal plasma treatment reduces mRNA expression of NOX4, which in turn inhibits ROS production 35-37 , as well increases NRF2 level to maintain ROS at a relative low level by regulating the antioxidant defense system [38][39][40] , further improves the chicken sperm motility. On the other hand, inappropriate plasma treatments degrade the sperm quality as a result of excessive ROS accumulation and impair the antioxidant defense system through disruption of the NRF2-antioxidant signaling pathway. Sperm motility requires high ATP levels as the energy source 1 . Mitochondria have a crucial role in the energy metabolism and ATP generation 41 . Sperm motility is found to be directly and positively correlated with mitochondrial respiratory enzyme activity and ATP production in roosters 42 . Our results show plasma conditions can be optimized to enhance chicken sperm motility by increasing the activity of respiratory enzyme and ATP production through upregulation of the mRNA expression of ATP synthase subunits and ATP5A protein level in spermatozoa. Meanwhile, intracellular ROS generation is increased as byproducts in the mitochondrial respiratory chain 43 , but this do not induce an impairment of sperm quality because of the elevated scavenging activity of antioxidant enzyme which is mediated by an appropriate plasma exposure. However, inappropriate plasma treatment leads to mitochondrial enzymatic dysfunction 44,45 as a result of disruption of ATP production in the mitochondria 46 , resulting in axonemal damage, decreased sperm viability and motility, and more sever morphological defects in the midpiece, with corresponding deleterious effects on sperm capacitation and acrosome reaction 32,47 .   Therefore, appropriate plasma exposure conditions improve sperm energetic metabolism through mitochondrial ATP generation. In addition, our results reveal that the optimal plasma treatment decreases AMPK mRNA levels and AMPK phosphorylation, increases mTOR mRNA levels and mTOR phosphorylation in spermatozoa. These results can be explained by the fact that high ATP concentrations inactivate AMPK, which phosphorylates and activates the mTOR pathway 48 , further regulating intracellular energy metabolism 49 and sperm motility and survival 50,51 . However, the activation of AMPK and decrease of mTOR expression result in low sperm viabilities in the inappropriate plasma-treated groups because of the cell apoptosis effect regulated by AMPK-mTOR pathway 52,53 .
In this study, we examine DNA methylation levels of antioxidant defense-related and energetic metabolism-related genes. The results show exposure to 11.7 kV of plasma for 20 s increases the demethylation levels of NRF2, PRDX4, ATP5A1, and mTOR but decreases demethylation levels of KEAP1 and AMPKα2, resulting in the alteration of abovementioned gene expression in chicken spermatozoa because DNA demethylation has been associated with the gene transcriptional activation 54 , whereas hypermethylation is known to suppress gene expression 55,56 . Sperm DNA methylation levels are associated with sperm motility and DNA integrity, but not with sperm viability or morphology 18 . Sperm DNA hypermethylation results in impaired morphology 57 , low sperm motility 17,20 , infertility 58 , and even sperm apoptosis 59 . In the present study, optimal plasma-treated spermatozoa with DNA hypomethylation levels of NRF2, PRDX4, ATP5A1, and mTOR and hypermethylation levels of KEAP1 and AMPKα2 exhibit an increase of sperm motility, but no changes in the sperm viability, acrosome and DNA integrity, morphology, and total fertility. On the other hand, high-intensity plasma treatment shows significantly reversed methylation patterns  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.
SCiEntifiC REPORTS | (2018) 8:7576 | DOI:10.1038/s41598-018-26049-5 of the abovementioned genes and results in impaired sperm quality. Moreover, sperm DNA methylation influences DNA integrity and stable transmission of epigenetic information to the offspring 60 , which are crucial for embryonic development and post-natal growth 61 . These results demonstrate that plasma treatment of chicken spermatozoa may promote higher motility for next-generation poultry breeding.
In conclusion, exposure to 11.7 kV of plasma for 20 s enhances chicken sperm motility by regulating the demethylation levels of antioxidant defense-related and energetic metabolism-related genes in spermatozoa. Notably, optimal plasma treatment may potentially boost offspring production and thus warrant further investigation.

Materials and Methods
Plasma treatment. Fresh semen was collected from ten healthy cocks (Korean native chicken, approximately 60 weeks old; 10 cocks were used) that were raised in a chicken farm (Jeju National University, Jeju, Republic of Korea), following the method described by Blesbois, et al. 62 . Animal handling protocols were approved by the Institutional Committee for Ethics in Animal Experiments of Jeju National University and all experiments were performed in accordance with the institution guidelines. Individual ejaculates were then diluted at a 1:1 ratio with Beltsville Poultry Semen Extender 63 in a sterile petri dish, and this dilution was performed within 10 min of collection. The semen sample was then placed horizontally on the glass dielectric barrier of the plasma reactor (Fig. 1f), allowing the semen to be directly exposed to the plasma propagating from the tip of the electrode needles at varying durations and potentials following our previously described method 10 . Semen without plasma treatment was used as the control group. Plasma-treated semen samples were kept at 37 °C in an incubator for a few minutes until further analysis.

Sperm quality evaluation. Individual ejaculates were prepared at a 1:1 dilution in Beltsville Poultry Semen
Extender 63 and then a 1:30 dilution with 0.9% sodium chloride solution was performed prior to the sperm quality evaluation. Sperm count was measured using a hemocytometer according to World Health Organization criteria 64 . The evaluation of sperm motility was performed by two experienced individuals following World Health Organization criteria 64 . After liquefaction, 10 μl of diluted semen was pipetted onto a glass slide and covered with a cover slip. The analysis was performed at room temperature under a Leica DM 2500 microscope at a magnification of 400 ×. Sperm motility was calculated as the percentage of spermatozoa with linear and progressive motility 62 . Following liquefaction, 10 μl of diluted semen was spread onto a glass slide and allowed to air-dry at room temperature. Sperm vitality was analyzed by staining the smears with eosin Y. The evaluation was performed by counting the red-stained and unstained spermatozoa with brightfield optics at a magnification of 400 ×. Sperm vitality was expressed as the proportion (%) of unstained spermatozoa, and at least 100 spermatozoa were counted 64 . Semen smears were stained with Wright-Giemsa 65 , and sperm morphology was photographed using a Leica DM 2500 microscope equipped with a digital camera (DP71, Olympus) at a magnification of 1,000 ×. Acrosome integrity was calculated as the proportion (%) of acrosome covering 40% to 70% of the head area 64 . Semen smears were fixed in 96% ethanol-acetone (1:1) at 4 °C for 1 hour and air dried, then hydrolyzed in 0.1 N HCl at 4 °C for 5 min. Thereafter, the toluidine blue staining was performed as previously described by Kim, et al. 66 . Sperm nuclear DNA integrity was calculated as the percentage of spermatozoa with light blue-stained heads, and at least 200 spermatozoa were counted. Acrosome integrity and DNA integrity were analyzed under a Leica DM 2500 microscope using oil immersion with magnification of 1,000 ×.
Prior to artificial insemination (AI), healthy hens (Hyline brown chicken, raised at a chicken farm in Jeju National University, Jeju, Republic of Korea) that did not undergo AI nor naturally mated were randomly allocated into nine groups comprising ten hens per group. AI was performed twice a week with fresh non-plasma (control group) and plasma-treated semen at varying durations and potentials. To determine the total fertility for a five-day collection of fertilized eggs after AI, egg shells were wiped with 70% ethanol, incubated for 7 days at 37.5 °C under 45% to 65% relative humidity, and rotated 90 degrees every 2 h. Afterwards, breakout analysis of all incubated eggs was performed to check the number of embryos. Total fertility was calculated by dividing the number of embryos by the total number of incubated eggs in each group; values were expressed as percentages (%).

ROS, antioxidant enzyme, and ATP analyses.
Chicken semen was centrifuged at 600 × g for 10 min at 4 °C. Sperm pellets were washed with 0.9% sodium chloride solution, centrifuged again at 600 × g for 10 min at 4 °C, and suspended in 1 × phosphate buffered saline (PBS; 0.05 mol/l, pH 7.4) at a final concentration of 1 × 10 9 spermatozoa/ml. ROS levels were determined using the OxiSelect In Vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). The assay employed a proprietary quenched fluorogenic probe 2′,7′-dic hlorodihydrofluorescein DiOxyQ (DCFH-DiOxyQ), which is a specific ROS/RNS probe that is based on similar chemistry to 2′,7′-dichlorodihydrofluorescein diacetate. The DCFH-DiOxyQ probe is first primed with a quench removal reagent, and subsequently stabilized in the highly reactive DCFH form, which is rapidly oxidized to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF) by ROS/RNS. The amount of DCF in the sample is determined based on the relative fluorescence units obtained using a DCF standard curve and the fluorescence intensity of DCF is proportional to the total ROS/RNS level within the sample, using a GloMax Discover Multimode Detection System fluorescence plate reader (Promega, Madison, WI, USA) at 480 nm excitation/530 nm emission. ROS levels in the spermatozoa were expressed as nmole DCF/10 9 spz.
Spermatozoa were assayed for MDA, SOD, CAT, GPx, and ATP levels using kits from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA) following the manufacturer's instructions. The optical densities were determined using a GloMax Discover Multimode Detection System (Promega). The relative light unit value of ATP levels were measured using a luminometer (Sirius L Tube Luminometer, Titertek Berthold, Germany). SOD activity was expressed as U/10 9 spz, where one unit was defined as the amount of enzyme that reduces 1.0 µmole of superoxide to molecular oxygen and hydrogen peroxide per min at 25 °C. CAT activity was expressed as U/10 9 spz, wher U was defined as the amount of enzyme that decomposes 1.0 µmole of hydrogen peroxide to oxygen and water per min at 25 °C. GPx activity was expressed as mU/10 9 spz, where mU was defined as the amount of enzyme that will cause the oxidation of 1.0 nmole of NADPH to NADP + under the assay kit condition per min at 25 °C. MDA and ATP levels were expressed as nmole/10 9 spz.
Mitochondrial respiratory enzyme analysis. Mitochondria were isolated and purified from chicken spermatozoa (1 × 10 9 spermatozoa/ml) using Qproteome Mitochondria Isolation Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. The NADH levels and enzymatic activities of cytochrome c oxidase and ATP synthase in sperm mitochondria were measured using NAD+/NADH Quantitation Colorimetric Kit (BioVision, Milpitas, CA, USA), Cytochrome Oxidase Activity Colorimetric Assay Kit (BioVision), and ATP Synthase Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions, respectively. The optical densities of NADH, cytochrome c oxidase, and ATP synthase were measured using the GloMax Discover Multimode Detection System.
RT-PCR analysis. Total RNA isolation and purification from spermatozoa were performed as previously described by Shafeeque, et al. 67 . RT-PCR analysis was performed using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), 2× Prime Taq Premix (GENETBIO, Yuseong-gu, Daejeon, South Korea), and EvaGreen Dye (Biotium, Hayward, CA, USA). First-strand cDNA synthesis was performed using 1 μl of total RNA (1 μg/μl) following the manufacturer's protocol. RT-PCR was performed using a StepOne Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific). The following conditions were used for RT-PCR: 40 cycles of initial denaturation at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s and annealing for 1 min at the indicated temperatures shown in Supplementary Table S3. Melting curves were obtained at 95 °C for 15 s, 60 °C for 1 min, and increased to 95 °C for 15 s at increments of 0.3 °C. Primer sequences used for RT-PCR are shown in Supplementary Table S3.
The cycle threshold value for each sample was determined based on triplicate measurements. Equivalent dilutions were calculated according to the standard curve and then normalized against the housekeeping gene (β-actin). Reaction product sequences were confirmed by direct nucleotide sequencing using an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Relative expression levels were calculated using the 2 −ΔΔCT method.
Cloning and methylation sequencing. Purified BSP amplification products were ligated to the pGEM-T Easy Vector and transformed into competent JM109 cells using the pGEM-T Easy Vector system I (Promega). White colonies were selected from the ampicillin/X-Gal/IPTG plates, and colony PCR was performed using vector-directed primers to confirm the presence of inserts based on their expected fragment sizes. For plasmid isolation, ten positive colonies from each plate were inoculated into LB medium containing ampicillin. Plasmids containing the target DNA were extracted and purified using the Plasmid Midi Kit (QIAGEN) and then sequenced on an ABI Prism BioDye Terminator version 3.1 sequencing system. Cytosine methylation results were analyzed using the CyMATE online methylation sequence analysis software. Total methylation ratios were calculated by dividing the number of non-converted (methylated) cytosines by the total number of cytosines within the sequenced region; values were expressed as percentages (%). Average methylation levels were expressed as percentage (%) per site for each of the three types of cytosines (CG, CHG, and CHH), and were calculated by dividing the number of non-converted cytosines by the total number of cytosines of each type.

Statistical analysis.
All experiments were repeated three times, and data are presented as the mean ± standard error. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 16.0; SPSS, Chicago, IL, USA). Statistically significant difference among treatment groups were determined via one-way ANOVA and Fisher's least significant difference (LSD) test. P-values < 0.05 are considered significant. Data availability. The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.