Abstract. This study was conducted to determine the optimal vitrification conditions for immature bovine oocytes using the microdrop method. In experiment 1, the optimal pre-equilibration period for microdrop vitrification was examined. The maturation rate of vitrified oocytes with a 3 min first preequilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first preequilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first preequilibration period were intermediate. The value for a 1 min second pre-equilibration period (44.4%) was significantly higher (P<0.05) than those for a 0.5 (28.6%) and 2 min (21.4%) second preequilibration period. In experiment 2, the distribution of microtubules in matured oocytes was investigated. There was no difference among the first pre-equilibration times in terms of the rates of normal spindles in vitrified oocytes. However, this value was significantly higher (P<0.05) in the 1 min group (52.8%) compared with the 0.5 (16.7%) and 2 min groups (12.3%). In experiment 3, we investigated the developmental capacity of immature bovine oocytes vitrified under optimal preequilibration conditions (3 min and 1 min for the first and second pre-equilibrations, respectively). Although the total fertilization rates were significantly lower (P<0.05) in the vitrified oocytes (65.6%) compared with the control oocytes (92.4%), there was no difference in the rate of normal fertilization (2PN) between the vitrified (78.6%) and control (82.0%) oocytes. Cleavage and blastocyst rates were significantly lower (P<0.05) in vitrified oocytes (55.7 and 2.3%) than in control oocytes (84.4 and 34.7%). Thus, these results indicated that immature bovine oocytes can survive after microdrop vitrification and subsequently can be cultured to mature oocytes capable of undergoing fertilization in vitro and developing into blastocysts.

In order to test if nestin is a useful marker for various types of progenitor cells, we explored nestin expression in the retina during development. Nestin expression was co-evaluated with bromodeoxyuridine (BrdU) labeling and Griffonia simplicifolia isolectin B4 (GSIB4) histochemistry. Nestin immunoreactivity appears in cell soma of dividing neural progenitor cells and their leading processes in retinas from embryonic day (E) 13 to E20, in accordance with a BrdU-labeled pattern. At postnatal day (P) 5, it is restricted to the end feet of Müller cells. BrdU-labeled nuclei were mainly in the inner part of the inner nuclear layer in postnatal neonates. The retinal vessels demarcated with GSIB4-positive endothelial cells were first distributed in the nerve fiber layer from P3. Afterward the vascular branches sprouted and penetrated deeply into the retina. The endothelial cells positive for GSIB4 and the pericytes in the microvessels were additionally immunoreactive for nestin. Interestingly, the presumed migrating microglial cells showing only GSIB4 reactivity preceded the microvessels throughout the neuroblast layer during vascular sprouting and extension. These findings may suggest that nestin expression represents the proliferation and movement potential of the neural progenitor cells as well as the progenitor cells of the endothelial cell and the pericyte during retinal development. Thus, Müller glial cells might be potential neural progenitor cells of the retina, and the retinal microvasculature established by both the endothelial and the pericyte progenitor cells via vasculogenesis along microglia migrating routes sustains its angiogenic potential.

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The water extract of the fermented Rhus verniciflua stem bark (FRVSB) was prepared by hot water extracting at 100 o C for 8 h. The urushiol content of the FRVSB water extract was determined by HPLC. The urushiol was not contained in FRVSB water extract, whereas Rhus verniciflua stem bark (RVSB) water extract contained 3.4 mg%. At the lab scale size, suitable water extraction condition for a total solid, polyphenol and flavonoid from FRVSB was at over 100 o C for 6-8 h. The total solid contents was reduced in pilot scale processing system, with 5.7% of the extraction yield. The proximate composition (%) of FRVSB water extract obtained from industrial installation was moisture 4.34, crude fat 1.69, crude protein 10.21, and crude ash 15.80. Gallic acid (1,090.5 mg%) was the most abundant compound in phenolic acids, while fisetin (135.7 mg%) was the predominant flavonoid. The free sugar content was mannitol of 3.48%, glycerol of 0.19%, and glucose of 0.19%. Alanine (244 ppm), serine (231 ppm), and leucine (218 ppm) were predominant amino acids.