The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either L-ascorbic acid or a-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to a-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between a-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in a-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with a-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that a-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.
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 (-45.0ºC) 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 -27.7ºC. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15ºC) were significantly lower than those of sperm preserved in noncollapsed cake (drying temperature: -30ºC). 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. Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, 23 if the solution temperature is higher than the glass transition temperature of the maximally 24 freeze-concentrated phase (T g ') during drying phase, the glassy matrix undergoes viscous flow, 25 resulting in cake collapse. The purpose of the present study was to investigate the effect of cake 26 collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors 27 affecting the T g ' of conventional EGTA buffer (consisting of Tris-HCl, EGTA and NaCl) were 28 investigated in order to establish the main experimental protocol because EGTA buffer T g ' was 29too low (-45.0ºC) to suppress collapse. Modification of the EGTA buffer composition by 30 complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of 31 T g ' up to -27.7ºC. In the main experiment, blastocyst yields after ooplasmic injection of 32 freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15ºC) were 33 significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: 34 -30ºC). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm 35 functions to support embryonic development, and can be inhibited by controlling both T g ' of 36 freeze-drying buffer and temperature during the drying phase. 37 38
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.
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