60 EFFECT OF CAKE COLLAPSE ON THE INTEGRITY OF FREEZE-DRIED BULL SPERMATOZOA

Abstract

During freezing, a solution changes into an amorphous phase at the glass transition temperature of the maximally freeze-concentrated phase (T′_g). The solution exhibits a cake-like porous structure under the optimal freeze-drying process. However, if the product temperature is higher than theT′_g during the drying phase, the glassy material will undergo viscous flow, resulting in loss of the porous structure. This is defined as the collapse phenomenon and may be related to instability of the freeze-dried products. The purpose of the present study was to investigate the effect of cake collapse on freeze-dried bull spermatozoa. One-way ANOVA was used for comparison of T′_g, DNA damage, and blastocyst yield. When the ANOVA was significant, differences among means were analysed by a Tukey test. In Experiment 1, factors affecting the T′_g were investigated. Using differential scanning calorimetry, theT′_g of an EGTA buffer (10 mM TRIS-HCl, 50 mM EGTA, and 50 mM NaCl, pH8.0) that has been conventionally used for sperm freeze-drying was determined to be –45.0°C. Modification of the EGTA buffer composition by complete removal of NaCl and addition of 0.5 M trehalose (referred to hereafter as mEGTA buffer) resulted in an increase in theT′_g up to –27.7°C. The T′_g of the mEGTA buffer cooled by direct immersing into liquid nitrogen (–29.4°C) was slightly lower (P < 0.05) than that cooled slowly at 20 and 1°C min^–1 (–27.6 and –27.2°C, respectively). In Experiment 2, the integrity of freeze-dried and rehydrated bull spermatozoa was investigated. Spermatozoa from a Japanese Black bull were suspended into mEGTA buffer (3 × 10⁷ cells mL^–1), cooled at 20°C min^–1, and then processed for drying for 6 h at 0, –15, and –30°C (ALPHA2-4; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Cakes were collapsed when the sperm suspension was dehydrated either at 0 or –15°C. In vitro-matured bovine oocytes were injected with rehydrated sperm, chemically activated (5 μM ionomycin, 7% ethanol, and 2 mM 6-DMAP), and then cultured for 8 days. Blastocyst yields after injection of sperm dried at 0 and –15°C, calculated from cleaved oocytes, were significantly lower than that of sperm dried at –30°C (0.7–3.7% v. 14.2%; P < 0.05). The level of DNA damage, assessed by the alkaline comet assay, was not different between the sperm populations dried at 0 and –30°C. Transmission electron microscopic observation revealed that the sperm membrane dried at 0°C was more damaged compared with that dried at –30°C (P < 0.05; chi-squared test with Bonferroni correction). In conclusion, incidence of collapse in freeze-dried cake may be a detrimental factor for maintenance of sperm integrity after freeze-drying, and can be inhibited by controlling the T′_g of the buffer and drying phase temperature.

H. Hara is Research Fellow of the Japan Society for the Promotion of Science (JSPS). This work was supported by a grant-in-aid for basic research from JSPS (no. 24580407) to S. Hochi.