Sperm membrane permeability evaluated through YO-PRO-1/PI assay.

<p>Bars show percentages (as mean±SEM) of viable spermatozoa without changes in permeability of plasma membrane (YO-PRO-1^−/PI⁻) before and after freeze/thawing when sperm is stored at 17°C either for 3 h or for 24 h. Different superscripts (<i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i>) mean significant differences (<i>P</i><0.05) among bars, each bar representing different holding times (3 h or 24 h), preservation (Ext vs. F-T) and post-thawing times (30 min or 240 min). Ext = Extended semen (17°C). FT-C = Frozen-thawed semen.</p

Sperm membrane lipid disorder evaluated through M540/YO-PRO-1 co-staining.

<p>The figure shows percentages (mean±SEM) of viable spermatozoa with low membrane lipid disorder (M540^−/YO-PRO-1⁻) before and after freeze/thawing when sperm is stored at 17°C either for 3 h or for 24 h. Different superscripts (<i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i>) mean significant differences (<i>P</i><0.05) among bars, each bar representing different holding times (3 h or 24 h), preservation (Ext vs. F-T) and post-thawing times (30 min or 240 min). Ext = Extended semen (17°C). FT-C = Frozen-thawed semen.</p

Effects of holding time prior to freeze-thawing on different sperm motility parameters after 30 and 240 min post-thawing at 37°C.

<p>Data are shown as mean ± SEM. Different superscripts (<i>a, b, c</i>, <i>d</i>) mean significant differences (<i>P</i><0.05) within rows (i.e. for a given motility parameter) among columns (i.e. comparing extended and frozen-thawed semen, and 30 and 240 min of post-thawing incubation at 37°C).</p

Intracellular peroxide levels (H₂DCFDA/PI staining), as percentages (as mean±SEM) of viable spermatozoa with high levels of intracellular peroxides (DCF⁺/PI⁻) before and after freeze/thawing when sperm is stored at 17°C either for 3 h or for 24 h.

<p>Different superscripts (<i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i>) mean significant differences (<i>P</i><0.05) among bars, each bar representing different holding times (3 h or 24 h), preservation (Ext vs. F-T) and post-thawing times (30 min or 240 min). Ext = Extended semen (17°C). FT-C = Frozen-thawed semen.</p

General scheme of the distribution of the proteins for the mini-array analysis.

<p>1A: AKT-1/AKT-2. 1B: CDK6. 1C: CYCLIN E. 1D: IRAK. 1E: PYK2/CAKb. 2A: CASPASE 9. 2B: C-KIT. 2C: CYCLIN H. 2D: PI3 KINASE/p85. 2E: C-RAF-1. 3A: CDC25. 3B: ERK-1. 3C: PKC. 3D: RAS. 3E: CDC6. 4A: CLUSTERIN. 4B: ERK-2. 4C: PP1, PP2A, PP2B, PPX. 4D: TRK A, B, C. 4E: CDK1/CDC2. 5A. CYCLIN A. 5B: GSK-3a. 5C: PTP1 (SH). 5D: CDK2. 5E: CYCLIN B. 6A: HSP70. 6B: PTP1 B. 6C: CDK4. 6D: CYCLIN D3. 6E: PTP2 (SH).</p

Regression equations between regression component 1 of PCA with cryotolerance indexes (<i>y</i>) and sperm parameters evaluated before freeze-thawing.

<p>From all the dependent variables submitted to the model, only the pSer-HSP70:total HSP70 ratio (<i>x</i>) was included, whereas the others were left out.</p

Percentage (as mean±SEM) of spermatozoa with intact plasma membrane (SYBR-14⁺/PI⁻, viable spermatozoa), before and after freeze/thawing when sperm is stored at 17°C either for 3 h or for 24 h.

<p>Different superscripts (<i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i>) mean significant differences (<i>P</i><0.05) among bars, each bar representing different holding times (3 h or 24 h), preservation (Ext vs. F-T) and post-thawing times (30 min or 240 min). Ext = Extended semen (17°C). FT-C = Frozen-thawed semen.</p

Mini-array analysis of the serine phosphorylation status of 30 sperm proteins after a HT of 3 h or 24 h.

<p>The mini-array analysis and the distribution of the proteins analysed are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090887#pone-0090887-g001" target="_blank">Figure 1</a>. The figure shows a representative image of two replicates coming from two different sperm samples and is representative of twelve separate experiments. Arrows (→) mark HSP70.</p

Comparison of serine-phosphorylation levels in 30 different sperm proteins after assessment through mini-array analyses.

<p>The values obtained for the HT of 3 h were transformed in order to obtain a basal arbitrary value of 100, from which the intensity values for the other samples (i.e. HT 24 h) were calculated. Results are given as mean ± SEM, and different superscripts (<i>a</i>, <i>b</i>) mean significant differences (<i>P</i><0.05) within rows (i.e. between the two holding times) for each protein.</p

Sperm DNA fragmentation (SDF) as percentage of spermatozoa with fragmented DNA (mean±SEM), before and after freeze/thawing when sperm is stored at 17°C either for 3 h or for 24 h.

<p>Different superscripts (<i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>) mean significant differences (<i>P</i><0.05) among bars, each bar representing different holding times (3 h or 24 h), preservation (Ext vs. FT) and post-thawing times (30 min or 240 min). Ext = Extended semen (15°C). FT = Frozen-thawed semen.</p