Comparison of acrosome integrity of frozen-thawed spermatozoa among groups.

a<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Photographs showing DNA integrity of frozen-thawed spermatozoa in each group at a magnification of 400×.

<p>The symbol “−” and “+” represent cathode and anode respectively during electrophoresis of negatively charged DNA. <b>a.</b> pre-frozen spermatozoa; <b>b.</b> post-thawed spermatozoa of Group A; <b>c.</b> comet tail of post-thawed spermatozoa in group B; <b>d.</b> obvious long comet tail in post-thawed spermatozoa of Group C; <b>e.</b> post-thawed spermatozoa of Group D.</p

Comparison of viability and motility of frozen-thawed spermatozoa among the groups.

a<p>indicates a statistical difference when compared with post-thawing spermatozoa from Group A to Group D (P<0.05);</p>b<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Schematic illustration of the volume distribution of sperm suspension in each step when cryopreserved in 10 µm height channel.

<p>The density of spermatozoa sample was adjusted to 10⁵/µL before loading. 5×10⁻³ µL medium containing 1000 spermatozoa can be stored in 10 µm height channel and 800–900 spermatozoa can be finally transferred out in 1 mL fertilization medium.</p

Schema of spermatozoa store in PDMS chip and freezing tube.

<p><b>a.</b> spermatozoa cryopreserved in micro-channel of which height is 10 µm (group A) one by one; <b>b.</b> spermatozoa cryopreserved in micro-channel of which height is 50 µm (group B); <b>c.</b> spermatozoa cryopreserved in micro-channel of which height is 100 µm (group C); <b>d.</b> spermatozoa cryopreserved disorderly in a 1.8 ml freezing tube (group D).</p

Photographs of four categories of acrosome status evaluated by FITC-PNA staining at a magnification of 1000×.

<p><b>a.</b> I-intact acrosome; <b>b.</b> II-intermediate form of minimal acrosome reactivity; <b>c.</b> III-intermediate form of severe acrosome reactivity; <b>d.</b> IV-reacted acrosome.</p

Workflow of PDMS chip.

<p><b>a.</b> a PDMS chip compared with a coin; <b>b.</b> sample loading with a micro-injector; <b>c.</b> thawing of frozen spermatozoa; <b>d.</b> push the syringe and thawed spermatozoa in micro-channel is observed to be transferred into the tissue culture dish under the microscope.</p

Comparison of DNA integrity of frozen-thawed spermatozoa among groups.

a<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Characterization of fluid flow using microspheres.

<p>(A) Illustration of characterization experiment. Channels doped with microspheres were marked as yellow. The microsphere solution was added both in the inlet and outlet of the this channel. Other channels as well as the central hexagon were doped with SWM (marked as white). Red square indicated the region where (C) was captured. (B) The speed of microspheres in different region of the device (I: peripheral channel; II: interconnecting grooves; III: central hexagon) at 0, 15 min, 30 min after sample loading. P1, P2 and P3 were corresponded to the loading plans in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142555#pone.0142555.t001" target="_blank">Table 1</a>. Data are presented as Mean ± SD (n = 5). (C) The distribution of microspheres in the device when liquid level in each pool reached equilibrium in different loading plans (P1, P2 and P3, successively).</p

Chemotactic responses of spermatozoa to progesterone gradients.

<p>(A) An overview of concentration gradients generated in the hexagon. Progesterone solution was added in every other channel of the chip (red). Concentration gradients were generated in three regions in the hexagon corresponding to peripheral channels where progesterone solution was loaded. Blue square showed the field where sperm chemotaxis were observed. (B) A microscopic photograph of spermatozoa with several trajectories indicated (18 sperm). Each colored line represented a sperm trajectory within 3 s. (C-E) Comparisons of chemotactic parameters among three groups. Group A, 100 pM progesterone solution was added in peripheral channels; Group B, 1 mM progesterone solution was added; Group C, control. Data are presented as mean ± SD (n = 5). **: <i>p</i> < 0.05.</p