Room-temperature bonding of Al2O3 thin films deposited using atomic layer deposition.

In this study, room-temperature wafer bonding of Al2O3 thin films on Si thermal oxide wafers, which were deposited using atomic layer deposition (ALD), was realized using the surface-activated bonding (SAB) method. Transmission electron microscopy (TEM) observations indicated that these room-temperature-bonded Al2O3 thin films appeared to work well as nanoadhesives that formed strong bond between thermally oxidized Si films. The perfect dicing of the bonded wafer into dimensions of 0.5 mm × 0.5 mm was successful, and the surface energy, which is indicative of the bond strength, was estimated to be approximately 1.5 J/m2. These results indicate that strong bonds can be formed, which may be sufficient for device applications. In addition, the applicability of different Al2O3 microstructures in the SAB method was investigated, and the effectiveness of applying ALD Al2O3 was experimentally verified. This successful SAB of Al2O3 thin films, which is a promising insulator material, opens the possibility of future room-temperature heterogenous integration and wafer-level packaging.

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells.

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.

Bovine immature oocytes acquire developmental competence during meiotic arrest in vitro.

To test the hypothesis that oocytes require time to acquire developmental competence during meiotic arrest, we investigated the effects of butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinase, on the developmental competence of bovine oocytes after in vitro fertilization (IVF) following release from meiotic arrest. In the present study, 4 culture conditions were used: addition of BSA or fetal bovine serum (FBS) under 2 oxygen tensions (5% vs. 20%) during meiotic arrest with 100-microM BL I. The developmental competence to the blastocyst stage was higher (P < 0.01) in oocytes that were arrested in FBS-supplemented medium under 5% O2 (37%) than in oocytes that were arrested under other conditions (5%-24%) or that matured directly following follicle aspiration (23%). The time course of nuclear maturation of BL I-treated oocytes was also examined. The results demonstrated that oocytes treated with BL I start germinal vesicle (GV) breakdown and reach the metaphase II stage 5.5-6.0 h earlier than nonarrested oocytes. The developmental rates to the blastocyst stage of BL I-treated oocytes matured for 15.5 and 21 h were higher (P < 0.05) than those of nontreated oocytes matured for 21 and 26.5 h, respectively. These results demonstrate that bovine immature oocytes, which were arrested at the GV stage with BL I in FBS-supplemented medium under low oxygen tension, acquire higher developmental competence during meiotic arrest.

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air.

The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.