Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa.

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg') during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg' of conventional EGTA buffer (consisting of Tris-HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg' was too low (-45.0°C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg' up to -27.7°C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15°C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: -30°C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg' of freeze-drying buffer and temperature during the drying phase.