Effect of AFPI and AFPIII at three different concentrations (0.1, 1 and 10 µg/ml) on motility rate and viability before freezing (fresh sperm) and after thawing.

<p>Values are expressed as means ± SD.</p><p>Within each row, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p

Representative 2-D gels of proteins extracted from the plasma membrane head isolated from fresh (A), cryopreserved with DMSO (B), with DMSO plus AFPI (C) and with DMSO plus AFPIII (D) gilthead sea bream spermatozoa.

<p>Extracted flagellar proteins (60 µg) were loaded onto non-linear IPG strips pH 3–10, and following isoelectric focusing were separated by 12.5% SDS–PAGE. Gels were silver stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#s2" target="_blank">Materials and Methods</a>. Spots that changed their expression after cryopreservation procedure are highlighted with a circle.</p

Effects of different cryoprotectants on different motility and viability parameters in sea bream spermatozoa before freezing (fresh sperm) and after different thawing conditions.

<p>Values are expressed as means ± SD for % motility, motility duration and % vitality and as means ± SEM for VCL and VSL. Within each column, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p><p>VSL: Velocity Straight Line.</p><p>VCL: curvilinear velocity.</p

Differences in relative abundance of 10 sperm head membrane spots in fresh and cryopreserved sea bream spermatozoa.

<p>Spots are highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#pone-0099992-g002" target="_blank">Figure 2</a> and the <i>x</i> axis identified the experimental conditions (fresh semen or cryopreserved with DMSO, with DMSO plus AFPI and with DMSO plus AFPIII). The <i>y</i>-axis corresponds to the intensity of spots expressed as normalized spot volume. Spot intensity is expressed as mean (dark bars) ± SD (light grey bars) of normalized spot volume (N = 18, that correspond to 6 different fish and 3 replicates for each). Different letters in the same graph indicate that mean values are significantly different by Mann–Whitney test (p<0.05). The spots showing a decrease of their expression after cryopreservation procedure are reported in the group 1, and those showing an increase are reported in the group 2.</p