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

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

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

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

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