Aniline by standard gradient isolation procedure conducted by the

Aniline blue and Toluidine blue staining  

blue staining was performed as our previous study (19). The air-dried smears were fixed in 3% buffered
glutaraldehyde in 0.2 m phosphate buffer (pH 7.2) for 30 min at room
temperature. Each smear was stained with 5% aqueous Aniline blue stain in 4% acetic acid (pH 3.5) for 5 min. Unstained or pale blue stained spermatozoa (Aniline
blue-) were normal and dark blue stained spermatozoa (Aniline blue+)
were considered as abnormal.

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Also, Toluidine blue staining was done as
described in our previous work (20). Pale blue sperm cells were considered as
normal (Toluidine blue-) and dark blue or violet/ purple spermatozoa
were categorized into abnormal cells (Toluidine blue+). For both
tests, at least 200 spermatozoa were checked in each slide and the normal and
abnormal spermatozoa were reported as a percentage.

Sperm preparation by discontinuous density gradients

preparation for each semen samples was performed by standard gradient isolation
procedure conducted by the WHO (17, 21) due to the isolation and purification of sperm
samples of each group. Briefly, a density gradient medium (In vitro, Denmark)
was prepared in a 15-mL test tube by layering 1 mL of 40% (v/v)
density-gradient medium over 1 mL of 80% (v/v) density gradient medium. About 1
mL of each well-mixed semen sample was placed on top of the density gradient
media and then centrifuged at 0.4 rcf for 15 min. Samples were collected in
sterile vessels, stored at -80ºC for further analysis.


Global DNA methylation analysis

The global
DNA methylation analysis was done using 5-mC DNA ELISA Kit Catalog Nos. D5326
(ZYMO RESEARCH CORP., USA). The global methylation assay for all samples was
performed in duplicate, showed in average as a final value with a generated
standard curve using negative and positive internal controls on the same plate.


2.6. RNA extraction
and cDNA synthesis

RNA was extracted from all of the samples using an RNeasy Plus Universal Mini
Kit (Qiagen) according to the manufacturer’s instructions. The RNA
concentration was determined by a NanoDrop spectrophotometry (Thermo
scientific, 2000c). The adjusted concentration of 100 ng/µl of purified total
RNAs were utilized for cDNA synthesis using RevertAid First Strand cDNA
Synthesis kit, Thermo Scientific according to the manufacturer’s guide.


2.7. Real-
Time Polymerase Chain Reaction

Quantitative RT-PCR was applied for evaluation
of gene expression using Fast SYBR®Green Master Mix, Applied Biosystems. All
evaluated genes (DNMT1, DNMT3A and DNMT3B) and control gene
beta-2-microglobulin (B2M) primers were listed
in table 1. The PCR mix in each well included the following: 12.5 ?l of
SYBR®Green Master Mix, 8.5 ?l dH2O, 1 ?l of the each forward and reverse
primers (10 pmol/µl), and 2 ?l of single strand cDNA in a final reaction volume
of 25 ?l. The thermal cycling program included an initial incubation at 95 ºC
for 20 seconds, followed by 40 cycles of 95 ºC for 3 seconds and 58 ºC for 30
seconds. A final 58 ºC to 95 ºC step was used to form the melt curve. Product
specificity was confirmed by dissociation curve analysis.  All steps of four genes expression process
for samples were performed duplicate. For verification of analysis and production
of the specific product, PCR final products were loaded on agarose gel 2%. The 2_?CT was calculated to represent
the levels of gene expression
after normalization to that of B2M. ?CT is the amount of (CT genes – CT B2M).


Statistical Analysis

The nonparametric
Mann-Whitney U test was used to analyse differences in sperm parameters, chromatin,
mRNA expression


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