Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conven-tionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indi-cated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 +/- 1.9%), and 1.0 or 1.5 mm/s (56.5 +/- 1.5%, 56.7 +/- 1.7% respectively), and lowest survival at 0.5 (52.1 +/- 1.6%) and 3.0 mm/s (51.4 +/- 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and direc-tionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.