Cryo-scanning electron microscopy discloses differences in dehydration of frozen boar semen stored in large containers.

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50 degrees C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from -5 degrees C to -100 degrees C, namely 2 degrees C/min, 50 degrees C/min and 1200 degrees C/min, the lattermost by plunging the samples into liquid nitrogen (LN(2)). The samples were thereafter fractured into LN(2) and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2 degrees C/min and 50 degrees C/min) but not at 1200 degrees C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.